WO2024014697A1 - Corynebacterium sp. microbial variant producing l-glutamic-acid, and method for producing l-glutamic acid by using same - Google Patents
Corynebacterium sp. microbial variant producing l-glutamic-acid, and method for producing l-glutamic acid by using same Download PDFInfo
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- WO2024014697A1 WO2024014697A1 PCT/KR2023/007160 KR2023007160W WO2024014697A1 WO 2024014697 A1 WO2024014697 A1 WO 2024014697A1 KR 2023007160 W KR2023007160 W KR 2023007160W WO 2024014697 A1 WO2024014697 A1 WO 2024014697A1
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- glutamic acid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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
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- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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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.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/14—Glutamic acid; Glutamine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
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Definitions
- the present invention relates to a mutant microorganism of the genus Corynebacterium that produces L-glutamic acid and a method for producing L-glutamic acid using the same. More specifically, it relates to a new variant of the FAD-dependent oxidoreductase enzyme involved in the L-glutamic acid biosynthetic pathway, and a polynucleotide. and transformants, and a method for producing L-glutamic acid using the same.
- L-glutamic acid is a representative amino acid produced through microbial fermentation, and monosodium L-glutamate (MSG) helps maintain balance and harmony in the overall taste of food, making it useful in meat, fish, chicken, vegetables, sauces, soups, etc. It is widely used as a seasoning for home use and processed food production as it can increase preference for foods such as seasonings and improve the taste of low-salt foods with salt reduced by up to 30%.
- MSG monosodium L-glutamate
- glucose mainly passes through the glycolytic pathway, but some of it is metabolized into two molecules of pyruvic acid through the pentose phosphate pathway. Among them, one molecule fixes CO 2 to become oxaloacetic acid, and the other molecule combines with acetyl CoA from pyruvic acid to become citric acid. Oxaloacetic acid and citric acid again enter the citric acid cycle (TCA cycle) and become alpha-ketoglutaric acid.
- TCA cycle citric acid cycle
- the oxidative metabolic pathway oxidizing alpha-ketoglutarate to succinic acid is lacking, and isocitrate dehydrogenase and glutamate dehydrogenase are closely involved. Therefore, the reductive amino acidification reaction of alpha-ketoglutaric acid proceeds efficiently and L-glutamic acid is produced.
- L-glutamic acid For the production of L-glutamic acid, wild-type strains obtained in nature or mutant strains modified to improve their glutamic acid production ability can be used. Recently, in order to improve the production efficiency of L-glutamic acid, genetic recombination technology has been applied to microorganisms such as Escherichia coli and Corynebacterium, which are widely used in the production of useful substances such as amino acids and nucleic acids, to produce excellent L-glutamic acid production ability. Various recombinant strains or mutant strains and methods for producing L-glutamic acid using them are being developed.
- the purpose of the present invention is to provide novel FAD-dependent oxidoreductase variants.
- the present invention aims to provide a polynucleotide encoding the above variant.
- the present invention aims to provide a transformant containing the above variant or polynucleotide.
- the present invention aims to provide a method for producing L-glutamic acid using the above transformant.
- One aspect of the present invention provides a FAD-dependent oxidoreductase variant consisting of the amino acid sequence of SEQ ID NO: 2, in which the 200th alanine in the amino acid sequence of SEQ ID NO: 4 is substituted with threonine.
- FAD-dependent oxidoreductase used in the present invention is an enzyme that catalyzes a redox reaction to supply energy necessary for living organisms. It uses FAD to oxidize one compound while reducing another compound. It works.
- the FAD-dependent oxidoreductase may be a gene encoding a FAD-dependent oxidoreductase or a sequence substantially identical thereto.
- substantially identical means that when each gene sequence, that is, a base sequence or nucleotide sequence, and any other nucleotide sequence are aligned and analyzed to match as much as possible, the other nucleotide sequence is at least 70%, 80% identical to each nucleotide sequence. It means having sequence homology of more than 90% or more than 98%.
- the FAD-dependent oxidoreductase in the present invention includes the amino acid sequence of SEQ ID NO: 4.
- the amino acid sequence of SEQ ID NO: 4 may be derived from a wild-type microorganism of the genus Corynebacterium .
- the microorganism of the Corynebacterium genus may be Corynebacterium glutamicum .
- variant refers to a conservative substitution, deletion, modification or addition of one or more amino acids at the N-terminus, C-terminus and/or inside the amino acid sequence of a specific gene. refers to a polypeptide that is different from the amino acid sequence before mutation of the variant, but maintains its functions or properties.
- conservative substitution means replacing one amino acid with another amino acid with similar structural and/or chemical properties, and may have little or no effect on the activity of the protein or polypeptide.
- the amino acids include alanine (Ala), isoleucine (Ile), valine (Val), leucine (Leu), methionine (Met), asparagine (Asn), cysteine (Cys), glutamine (Gln), serine (Ser), Threonine (Thr), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), histidine (His), lysine (Lys), glycine (Gly) ) and proline (Pro).
- variants include those in which one or more portions, such as the N-terminal leader sequence or transmembrane domain, are removed, or portions are removed from the N- and/or C-terminus of the mature protein. .
- variants may have their abilities increased (enhanced), unchanged, or decreased (weakened) compared to the protein or polypeptide before the mutation.
- “increase or enhancement” means that when the activity of the protein itself increases compared to the protein before mutation, the overall degree of protein activity within the cell is increased due to increased expression or increased translation of the gene encoding the protein, etc. in the wild-type strain or the protein expressing the protein before mutation. Including cases where it is high compared to the strain, and combinations thereof.
- “reduction or weakening” refers to when the activity of the protein itself is reduced compared to the protein before the mutation, and the overall degree of protein activity within the cell is reduced due to inhibition of expression or translation of the gene encoding the protein, such as in the wild-type strain or the protein expressing the mutation before the mutation. It includes cases where it is low compared to the strain, and combinations thereof.
- variant may be used interchangeably with variant type, modification, variant polypeptide, mutated protein, mutation, etc.
- the variant in the present invention is a FAD-dependent oxidoreductase enzyme in which alanine, the 200th amino acid in the amino acid sequence of SEQ ID NO: 4, is replaced with threonine, and may be composed of the amino acid sequence of SEQ ID NO: 2.
- Another aspect of the invention provides a polynucleotide encoding the FAD-dependent oxidoreductase variant.
- Polynucleotide used in the present invention is a strand of DNA or RNA of a certain length or more, which is a polymer of nucleotides in which nucleotide monomers are connected in a long chain by covalent bonds, and more specifically, the variant. refers to a polynucleotide fragment encoding .
- the polynucleotide may include a base sequence encoding the amino acid sequence of SEQ ID NO: 2.
- the polynucleotide may include the base sequence represented by SEQ ID NO: 1.
- Another aspect of the present invention provides a vector containing a polynucleotide encoding the FAD-dependent oxidoreductase variant.
- Another aspect of the present invention provides a transformant comprising the FAD-dependent oxidoreductase variant or polynucleotide.
- vector refers to any type of nucleic acid sequence delivery structure used as a means to deliver and express a gene of interest in a host cell. Unless otherwise specified, the vector may refer to inserting and expressing the carried nucleic acid sequence into the host cell genome and/or allowing it to be expressed independently. These vectors contain essential regulatory elements that are operably linked so that the gene insert can be expressed, and “operably linked” means that the target gene and its regulatory sequences are functionally linked to each other to enable gene expression.
- regulatory elements include promoters for performing transcription, optional operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating termination of transcription and translation.
- the vector used in the present invention is not particularly limited as long as it can replicate within the host cell, and any vector known in the art can be used.
- the vectors 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 pBR series, pUC series, pBluescriptII series, These include, but are not limited to, pGEM-based, pTZ-based, pCL-based and pET-based.
- the vector can typically be constructed as a vector for cloning or as a vector for expression.
- Vectors for expression can be those commonly 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 transformed into a suitable host cell and then replicate independently of the host cell's genome or can be incorporated into the genome itself.
- the “suitable host cell” is a vector capable of replication and may include an origin of replication, which is a specific base sequence at which replication is initiated.
- a strong promoter capable of advancing transcription e.g., pL ⁇ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter
- the origin of replication operating in the eukaryotic cell included in the vector includes the f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adeno origin of replication, AAV origin of replication, and BBV origin of replication. It is not limited.
- promoters derived from the genome of mammalian cells e.g., metallothionine promoter
- promoters derived from mammalian viruses e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter
- tk promoter of HSV can be used and generally has a polyadenylation sequence as a transcription termination sequence.
- the recombinant vector may include a selection marker, which is used to select transformants (host cells) transformed with the vector and expresses the selection marker in the medium treated with the selection marker. Because only cells are viable, selection of transformed cells is possible.
- Representative examples of the selection marker include kanamycin, streptomycin, and chloramphenicol, but are not limited thereto.
- a transformant can be created by inserting a recombinant vector into a host cell, and the transformant may be obtained by introducing a recombinant vector into an appropriate host cell.
- the host cell is a cell capable of stably and continuously cloning or expressing the expression vector, and any host cell known in the art can be used.
- E. coli JM109 When transforming prokaryotic cells to produce recombinant microorganisms, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli coli strains such as E. coli XL1-Blue, Bacillus subtilis, Bacillus thuringiensis, Corynebacterium strains, Salmonella Typhimurium, Serratia marcescens and Pseudomonas species. The same variety of intestinal bacteria and strains may be used, but it is not limited to this.
- yeast e.g., Saccharomyces cerevisiae
- insect cells plant cells
- animal cells such as Sp2/0, CHO K1
- host cells e.g., CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc.
- CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc. can be used, but are not limited thereto.
- transformation refers to a phenomenon that artificially causes genetic changes by introducing foreign DNA into a host cell
- transformat refers to a phenomenon in which foreign DNA is introduced into a target gene. It refers to a host cell that stably maintains expression.
- an appropriate vector introduction technology is selected depending on the host cell to express the target gene or a recombinant vector containing the same within the host cell.
- vector introduction can be performed using electroporation, heat-shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, and DEAE.
- -It may be performed by the dextran method, the cationic liposome method, the lithium acetate-DMSO method, or a combination thereof, but is not limited thereto.
- the transformed gene can be expressed within the host cell, it can be included without limitation, whether it is inserted into the chromosome of the host cell or located outside the chromosome.
- the transformant includes cells transfected, transformed, or infected with the recombinant vector according to the present invention in vivo or in vitro, and may be used as the same term as recombinant host cell, recombinant cell, or recombinant microorganism.
- the transformant may be a microorganism of the genus Corynebacterium .
- the microorganisms of the Corynebacterium genus include Corynebacterium glutamicum , Corynebacterium crudilactis , Corynebacterium deserti , and Corynebacterium.
- Corynebacterium callunae Corynebacterium suranareeae , Corynebacterium lubricantis , Corynebacterium doosanense , Corynebacterium Corynebacterium efficiens , Corynebacterium uterequi, Corynebacterium stationis , Corynebacterium pacaense , Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium humireducens , Corynebacterium marinum , Corynebacterium halotolerans , Corynebacterium spheniscorum ), Corynebacterium grisburgense , Corynebacterium genus
- the transformant in the present invention is a strain containing the above-described FAD-dependent oxidoreductase variant or a polynucleotide encoding the same, or a vector containing the same, or a strain expressing the FAD-dependent oxidoreductase variant or polynucleotide. , or it may be a strain having activity against the FAD-dependent oxidoreductase variant, but is not limited thereto.
- the transformant may have the ability to produce L-glutamic acid.
- the transformant may naturally have the ability to produce L-glutamic acid, or may be artificially endowed with the ability to produce L-glutamic acid.
- the transformant may have improved L-glutamic acid production ability due to a change in the activity of FAD-dependent oxidoreductase.
- “Improved production capacity” as used in the present invention means increased productivity of L-glutamic acid compared to the parent strain.
- the parent strain refers to a wild type or mutant strain that is subject to mutation, and includes a subject that is directly subject to mutation or transformed with a recombinant vector, etc.
- the parent strain may be a wild type microorganism of the genus Corynebacterium or a microorganism of the genus Corynebacterium mutated from the wild type.
- the transformant according to the present invention exhibits increased L-glutamic acid production ability compared to the parent strain due to a change in the activity of the FAD-dependent oxidoreductase by introducing a FAD-dependent oxidoreductase variant. More specifically, the transformant has an L-glutamic acid production of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% compared to the parent strain.
- the transformant containing the FAD-dependent oxidoreductase variant may have an L-glutamic acid production increased by more than 5%, specifically 5 to 50% (preferably 7 to 30%), compared to the parent strain. .
- Another aspect of the present invention includes culturing the transformant in a medium; and recovering L-glutamic acid from the transformant or the medium in which the transformant was cultured.
- the culture can be carried out according to appropriate media and culture conditions known in the art, and a person skilled in the art can easily adjust the medium and culture conditions.
- the medium may be a liquid medium, but is not limited thereto.
- Cultivation methods may include, for example, batch culture, continuous culture, fed-batch culture, or combinations thereof, but are not limited thereto.
- the medium must meet the requirements of a specific strain in an appropriate manner and can be appropriately modified by a person skilled in the art.
- Culture media for microorganisms of the genus Corynebacterium may refer to known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981), but are not limited thereto.
- the medium may contain 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, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, It includes fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These substances may be used individually or in mixtures, but are not limited thereto.
- Nitrogen sources that may be used include peptone, yeast extract, broth, 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 can also be used individually or in 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 the corresponding sodium-containing salts. Additionally, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth, but is not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be included. Additionally, precursors appropriate for the culture medium may be used. The medium or individual components may be added to the culture medium in an appropriate manner in a batch or continuous manner during the culture process, but are not limited thereto.
- the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the microbial culture medium in an appropriate manner during cultivation. Additionally, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester during culture. Additionally, in order to maintain the aerobic state of the culture medium, oxygen or oxygen-containing gas (e.g., air) can be injected into the culture medium.
- the temperature of the culture medium may typically be 20 to 45°C, for example, 25 to 40°C. The culturing period may continue until the desired yield of useful material is obtained, for example, 10 to 160 hours.
- the step of recovering L-glutamic acid from the cultured transformant or the medium in which the transformant was cultured includes L-glutamic acid produced from the medium using a suitable method known in the art according to the culture method.
- -Glutamic acid can be collected or recovered. Examples include centrifugation, filtration, extraction, nebulization, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g. ammonium sulfate precipitation), chromatography (e.g. ion exchange, affinity, hydrophobic and Methods such as size exclusion) can be used, but are not limited to this.
- the step of recovering L-glutamic acid may be performed by centrifuging the culture medium at low speed to remove biomass and separating the obtained supernatant through ion exchange chromatography.
- the step of recovering L-glutamic acid may include a process of purifying L-glutamic acid.
- the FAD-dependent oxidoreductase mutant according to the present invention changes its enzyme activity by substituting one or more amino acids in the amino acid sequence constituting the FAD-dependent oxidoreductase, so that the recombinant microorganism containing it can efficiently produce L-glutamic acid.
- Figure 1 shows the structure of plasmid pK19msb according to an embodiment of the present invention.
- a vector expressing a mutant in which alanine (A) at position 200 in the amino acid sequence of the FAD-dependent oxidoreductase (SEQ ID NO: 4) was replaced with threonine (T) was constructed.
- PCR was performed using the gDNA of wild-type Corynebacterium glutamicum ATCC13869 as a template, using primer pairs of primer 1 and primer 2 and primer pairs of primer 3 and primer 4, respectively. Afterwards, overlapping PCR was performed again using the mixture of the two PCR products as a template using primers 1 and 4 to obtain fragments.
- Takara PrimeSTAR Max DNA polymerase was used as the polymerase, and the PCR amplification conditions were denatured at 95°C for 5 minutes, then repeated 30 times at 95°C for 30 seconds, at 58°C for 30 seconds, and at 72°C for 1 minute and 30 seconds. Reaction was performed at 72°C for 5 minutes.
- the pK19msb vector was treated with smaI and ligated with the PCR product (fragment) obtained above, and the obtained plasmid was named pK_fprA(A200T).
- Primer name sequence number Primer sequence (5'-3') primer 1 6 TCATGGAGGGTTGATAGCAGGGC primer 2 7 GGAGTGAACTTCGTCTGAGCAGG primer 3 8 ACCTGCTCAGACGAAGTTCACTCC primer 4 9 CGTATTCTGGACGAGCATGGCG
- Corynebacterium glutamicum U3 (KCCM13218P) was used as the parent strain to introduce the FAD-dependent oxidoreductase mutant, and Electrocompy was modified based on the method of van der Rest et al. as a method for transformation of the U3 strain. An electrocompetent cell manufacturing method was used.
- a seed culture was prepared by primary culturing the U3 strain 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. Afterwards, isonicotinic acid hydrazine and 2.5% glycine at a concentration of 1 mg/ml were added to 100 ml of 2YT medium excluding glucose, and the seed culture was inoculated so that the OD610 value was 0.3, and incubated at 18°C. Cultured for 12 to 16 hours at 180 rpm to bring the OD610 value to 1.2 to 1.4.
- 2YT medium containing 16 g/l tryptone, 10 g/l yeast extract, and 5 g/l sodium chloride
- the culture was left on ice for 30 minutes and then centrifuged at 4°C and 4000 rpm for 15 minutes. Afterwards, the supernatant was discarded, and the precipitated U3 strain was washed four times with 10% glycerol solution and finally resuspended in 0.5 ml of 10% glycerol solution to prepare competent cells. Electroporation was performed using an electroporator from Bio-Rad. After adding the prepared competent cells and pK_fprA(A200T) vector to an electroporation cuvette (0.2 mm), an electric shock was applied under the conditions of 2.5 kV, 200 ⁇ , and 12.5 ⁇ F.
- Colonies generated by culturing at 30°C for 72 hours were cultured in BHI medium until stationary phase to induce secondary recombination, diluted to 10 -5 to 10 -7 and plated on antibiotic-free 2YT plate medium (containing 10% sucrose).
- a strain that had no kanamycin resistance and had the ability to grow on a medium containing 10% sucrose was selected, and it was named FPR1.
- the L-glutamic acid production capacity of the parent strain U3 and the mutant strain FPR1 into which the FAD-dependent oxidoreductase mutant was introduced was compared.
- Each strain (parent strain or mutant strain) was inoculated at 1% by volume in a 100 mL flask containing 10 mL of the medium for glutamic acid production shown in Table 2 below, and cultured with shaking at 30°C and 200 rpm for 48 hours. After completion of the culture, the concentration of L-glutamic acid in the medium was measured using HPLC (Agilent), and the results are shown in Table 3 below.
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Abstract
The present invention relates to a Corynebacterium sp. microbial variant producing L-glutamic acid and a method for producing L-glutamic acid by using same, and, more particularly, to a novel variant of FAD-dependent oxidoreductase involved in the L-glutamic acid biosynthetic pathway, a polynucleotide therefor, and a microorganism transformed therewith, and a method of producing L-glutamic acid by using same. The FAD-dependent oxidoreductase variant, according to the present invention, has one or more amino acids substituted in the amino acid sequence constituting the FAD-dependent oxidoreductase, and consequently, the activity of the enzyme is altered in such a way that a recombinant microorganism comprising the variant can efficiently produce L-glutamic acid.
Description
본 발명은 L-글루탐산을 생산하는 코리네박테리움 속 변이 미생물 및 이를 이용한 L-글루탐산의 생산 방법에 관한 것으로, 보다 구체적으로 L-글루탐산 생합성 경로에 관여하는 FAD 의존성 산화환원효소 신규 변이체, 폴리뉴클레오티드 및 형질전환체, 그리고 이를 이용한 L-글루탐산의 생산 방법에 관한 것이다.The present invention relates to a mutant microorganism of the genus Corynebacterium that produces L-glutamic acid and a method for producing L-glutamic acid using the same. More specifically, it relates to a new variant of the FAD-dependent oxidoreductase enzyme involved in the L-glutamic acid biosynthetic pathway, and a polynucleotide. and transformants, and a method for producing L-glutamic acid using the same.
L-글루탐산은 미생물 발효에 의해 생산되는 대표적인 아미노산으로, L-글루탐산 나트륨(monosodium L-glutamate, MSG)은 음식의 전체적인 맛에 균형과 조화를 이루도록 하여 고기, 생선, 닭, 야채, 소스, 수프, 양념 등 식품의 선호도를 높여주고 소금을 30%까지 줄인 저염 식품의 맛을 증진시켜줄 수 있어 가정용 및 가공식품 생산을 위한 조미료로 널리 이용되고 있다.L-glutamic acid is a representative amino acid produced through microbial fermentation, and monosodium L-glutamate (MSG) helps maintain balance and harmony in the overall taste of food, making it useful in meat, fish, chicken, vegetables, sauces, soups, etc. It is widely used as a seasoning for home use and processed food production as it can increase preference for foods such as seasonings and improve the taste of low-salt foods with salt reduced by up to 30%.
L-글루탐산의 발효 경로를 간단하게 살펴보면, 포도당은 주로 해당경로(glycolytic pathway)를 거치게 되지만 일부는 6탄당 인산경로(pentose phosphate pathway)를 거쳐서 2분자의 피루브산(pyruvic acid)으로 대사된다. 그 중 1분자는 CO2를 고정하여 옥살로아세트산(oxaloacetic acid)으로 되고 다른 1분자는 피루브산으로부터 아세틸코에이(acetyl CoA)와 결합하여 구연산(citric acid)으로 된다. 다시 옥살로아세트산과 구연산은 시트르산 회로(TCA cycle)로 들어가 알파-케토글루타르산(α-ketoglutaric acid)이 된다. 여기서, 알파-케토글루타르산으로부터 호박산(succinic acid)으로 산화되는 산화대사 경로가 결여되어 있고 또 이소시트레이트 디히드로게나제(isocitrate dehydrogenase)와 글루타메이트 디히드로게나제(glutamate dehydrogenase)가 밀접하게 관여하기 때문에 알파-케토글루타르산의 환원적 아미노산화 반응이 능률적으로 진행되어 L-글루탐산이 생성된다.Briefly looking at the fermentation pathway of L-glutamic acid, glucose mainly passes through the glycolytic pathway, but some of it is metabolized into two molecules of pyruvic acid through the pentose phosphate pathway. Among them, one molecule fixes CO 2 to become oxaloacetic acid, and the other molecule combines with acetyl CoA from pyruvic acid to become citric acid. Oxaloacetic acid and citric acid again enter the citric acid cycle (TCA cycle) and become alpha-ketoglutaric acid. Here, the oxidative metabolic pathway oxidizing alpha-ketoglutarate to succinic acid is lacking, and isocitrate dehydrogenase and glutamate dehydrogenase are closely involved. Therefore, the reductive amino acidification reaction of alpha-ketoglutaric acid proceeds efficiently and L-glutamic acid is produced.
L-글루탐산의 생산은 자연상태에서 수득된 야생형 균주나 이의 글루탐산 생산능이 향상되도록 변형된 변이주를 이용할 수 있다. 최근에는 L-글루탐산의 생산 효율을 개선시키기 위해 아미노산, 핵산과 같은 유용물질 생산에 많이 이용되는 대장균, 코리네박테리움 등의 미생물을 대상으로 유전자 재조합 기술을 적용하여 우수한 L-글루탐산 생산능을 갖는 다양한 재조합 균주 또는 변이주 및 이를 이용한 L-글루탐산 생산 방법이 개발되고 있다. 특히 L-글루탐산의 생합성 경로에 관여하는 효소, 전사인자, 수송 단백질 등의 유전자를 대상으로 하거나, 또는 이들의 발현을 조절하는 프로모터에 변이를 유도하여 L-글루탐산의 생산량을 증대시키려는 시도가 있었다. 그러나 L-글루탐산 생산에 직간접적으로 연관된 효소, 전사인자, 수송 단백질 등 단백질의 종류가 수십여 종에 이르기 때문에 이러한 단백질의 활성 변화에 따른 L-글루탐산 생산능 증가 여부에 관해 여전히 많은 연구가 필요한 실정이다.For the production of L-glutamic acid, wild-type strains obtained in nature or mutant strains modified to improve their glutamic acid production ability can be used. Recently, in order to improve the production efficiency of L-glutamic acid, genetic recombination technology has been applied to microorganisms such as Escherichia coli and Corynebacterium, which are widely used in the production of useful substances such as amino acids and nucleic acids, to produce excellent L-glutamic acid production ability. Various recombinant strains or mutant strains and methods for producing L-glutamic acid using them are being developed. In particular, there have been attempts to increase the production of L-glutamic acid by targeting genes such as enzymes, transcription factors, and transport proteins involved in the biosynthetic pathway of L-glutamic acid, or by inducing mutations in promoters that control their expression. However, since there are dozens of types of proteins, such as enzymes, transcription factors, and transport proteins, that are directly or indirectly related to L-glutamic acid production, much research is still needed on whether L-glutamic acid production capacity increases due to changes in the activity of these proteins. am.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Document]
미국등록특허 제6,852,516호US Patent No. 6,852,516
미국등록특허 제6,962,805호US Patent No. 6,962,805
본 발명은 신규한 FAD 의존성 산화환원효소 변이체를 제공하는 것을 목적으로 한다.The purpose of the present invention is to provide novel FAD-dependent oxidoreductase variants.
또한, 본 발명은 상기 변이체를 암호화하는 폴리뉴클레오티드를 제공하는 것을 목적으로 한다.Additionally, the present invention aims to provide a polynucleotide encoding the above variant.
또한, 본 발명은 상기 변이체 또는 폴리뉴클레오티드를 포함하는 형질전환체를 제공하는 것을 목적으로 한다.Additionally, the present invention aims to provide a transformant containing the above variant or polynucleotide.
또한, 본 발명은 상기 형질전환체를 이용한 L-글루탐산의 생산 방법을 제공하는 것을 목적으로 한다.Additionally, the present invention aims to provide a method for producing L-glutamic acid using the above transformant.
본 발명의 일 양상은 서열번호 4의 아미노산 서열에서 200번째 알라닌이 트레오닌으로 치환된, 서열번호 2의 아미노산 서열로 이루어진 FAD 의존성 산화환원효소 변이체를 제공한다.One aspect of the present invention provides a FAD-dependent oxidoreductase variant consisting of the amino acid sequence of SEQ ID NO: 2, in which the 200th alanine in the amino acid sequence of SEQ ID NO: 4 is substituted with threonine.
본 발명에서 사용된 “FAD 의존성 산화환원효소(FAD-dependent oxidoreductase)”는 생체에 필요한 에너지를 공급하기 위해 산화환원 반응을 촉매하는 효소로, FAD를 이용하여 다른 화합물을 환원시키면서 한 화합물을 산화시키는 작용을 한다. 상기 FAD 의존성 산화환원효소는 FAD 의존성 산화환원효소를 암호화하는 유전자 또는 이와 실질적 동일성을 가지는 서열일 수 있다. 여기서 “실질적 동일성”이란 각각의 유전자 서열, 즉 염기서열 또는 뉴클레오티드 서열과 임의의 다른 뉴클레오티드 서열을 최대한 대응되도록 정렬하여 분석하였을 때 상기 임의의 다른 뉴클레오티드 서열이 각각의 뉴클레오티드 서열과 70% 이상, 80% 이상, 90% 이상 또는 98% 이상의 서열 상동성을 가지는 것을 의미한다.“FAD-dependent oxidoreductase” used in the present invention is an enzyme that catalyzes a redox reaction to supply energy necessary for living organisms. It uses FAD to oxidize one compound while reducing another compound. It works. The FAD-dependent oxidoreductase may be a gene encoding a FAD-dependent oxidoreductase or a sequence substantially identical thereto. Here, “substantial identity” means that when each gene sequence, that is, a base sequence or nucleotide sequence, and any other nucleotide sequence are aligned and analyzed to match as much as possible, the other nucleotide sequence is at least 70%, 80% identical to each nucleotide sequence. It means having sequence homology of more than 90% or more than 98%.
본 발명에서의 FAD 의존성 산화환원효소는 서열번호 4의 아미노산 서열을 포함한다. The FAD-dependent oxidoreductase in the present invention includes the amino acid sequence of SEQ ID NO: 4.
본 발명의 일 구체예에 따르면, 상기 서열번호 4의 아미노산 서열은 야생형 코리네박테리움(Corynebacterium) 속 미생물에서 유래한 것일 수 있다.According to one embodiment of the present invention, the amino acid sequence of SEQ ID NO: 4 may be derived from a wild-type microorganism of the genus Corynebacterium .
보다 구체적으로, 상기 코리네박테리움 속 미생물은 코리네박테리움 글루타미컴(Corynebacterium glutamicum)일 수 있다.More specifically, the microorganism of the Corynebacterium genus may be Corynebacterium glutamicum .
본 발명에서 사용된 “변이체”는 특정 유전자의 아미노산 서열 중 N-말단, C-말단 및/또는 내부에서 하나 이상의 아미노산이 보존적 치환(conservative substitution), 결실(deletion), 변형(modification) 또는 부가되어 상기 변이체의 변이 전 아미노산 서열과 상이하나 기능(functions) 또는 특성(properties)이 유지되는 폴리펩티드를 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. 상기 아미노산으로는 알라닌(Ala), 이소루신(Ile), 발린(Val), 루신(Leu), 메티오닌(Met), 아스파라긴(Asn), 시스테인(Cys), 글루타민(Gln), 세린(Ser), 트레오닌(Thr), 페닐알라닌(Phe), 트립토판(Trp), 티로신(Tyr), 아스파트산(Asp), 글루탐산(Glu), 아르기닌(Arg), 히스티딘(His), 라이신(Lys), 글리신(Gly) 및 프롤린(Pro)으로부터 선택된 것이다.As used in the present invention, “variant” refers to a conservative substitution, deletion, modification or addition of one or more amino acids at the N-terminus, C-terminus and/or inside the amino acid sequence of a specific gene. refers to a polypeptide that is different from the amino acid sequence before mutation of the variant, but maintains its functions or properties. Here, “conservative substitution” means replacing one amino acid with another amino acid with similar structural and/or chemical properties, and may have little or no effect on the activity of the protein or polypeptide. The amino acids include alanine (Ala), isoleucine (Ile), valine (Val), leucine (Leu), methionine (Met), asparagine (Asn), cysteine (Cys), glutamine (Gln), serine (Ser), Threonine (Thr), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), histidine (His), lysine (Lys), glycine (Gly) ) and proline (Pro).
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 transmembrane domain, are removed, or portions are removed from the N- and/or C-terminus of the mature protein. .
이러한 변이체는 그 능력이 변이 전 단백질 또는 폴리펩티드에 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.These variants may have their abilities increased (enhanced), unchanged, or decreased (weakened) compared to the protein or polypeptide before the mutation. Here, “increase or enhancement” means that when the activity of the protein itself increases compared to the protein before mutation, the overall degree of protein activity within the cell is increased due to increased expression or increased translation of the gene encoding the protein, etc. in the wild-type strain or the protein expressing the protein before mutation. Including cases where it is high compared to the strain, and combinations thereof. In addition, “reduction or weakening” refers to when the activity of the protein itself is reduced compared to the protein before the mutation, and the overall degree of protein activity within the cell is reduced due to inhibition of expression or translation of the gene encoding the protein, such as in the wild-type strain or the protein expressing the mutation before the mutation. It includes cases where it is low compared to the strain, and combinations thereof. In the present invention, variant may be used interchangeably with variant type, modification, variant polypeptide, mutated protein, mutation, etc.
본 발명에서의 변이체는 서열번호 4의 아미노산 서열에서 200번째 위치한 아미노산인 알라닌이 트레오닌으로 치환된 FAD 의존성 산화환원효소로, 서열번호 2의 아미노산 서열로 이루어진 것일 수 있다.The variant in the present invention is a FAD-dependent oxidoreductase enzyme in which alanine, the 200th amino acid in the amino acid sequence of SEQ ID NO: 4, is replaced with threonine, and may be composed of the amino acid sequence of SEQ ID NO: 2.
본 발명의 다른 양상은 상기 FAD 의존성 산화환원효소 변이체를 암호화하는 폴리뉴클레오티드를 제공한다.Another aspect of the invention provides a polynucleotide encoding the FAD-dependent oxidoreductase 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.“Polynucleotide” used in the present invention is a strand of DNA or RNA of a certain length or more, which is a polymer of nucleotides in which nucleotide monomers are connected in a long chain by covalent bonds, and more specifically, the variant. refers to a polynucleotide fragment encoding .
상기 폴리뉴클레오티드는 서열번호 2의 아미노산 서열을 암호화하는 염기서열을 포함할 수 있다.The polynucleotide may include a base sequence encoding the amino acid sequence of SEQ ID NO: 2.
본 발명의 일 구체예에 따르면, 상기 폴리뉴클레오티드는 서열번호 1로 표시되는 염기서열을 포함하는 것일 수 있다.According to one embodiment of the present invention, the polynucleotide may include the base sequence represented by SEQ ID NO: 1.
본 발명의 다른 양상은 상기 FAD 의존성 산화환원효소 변이체를 암호화하는 폴리뉴클레오티드를 포함하는 벡터를 제공한다.Another aspect of the present invention provides a vector containing a polynucleotide encoding the FAD-dependent oxidoreductase variant.
또한, 본 발명의 다른 양상은 상기 FAD 의존성 산화환원효소 변이체 또는 폴리뉴클레오티드를 포함하는 형질전환체를 제공한다.Additionally, another aspect of the present invention provides a transformant comprising the FAD-dependent oxidoreductase 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.As used in the present invention, “vector” refers to any type of nucleic acid sequence delivery structure used as a means to deliver and express a gene of interest in a host cell. Unless otherwise specified, the vector may refer to inserting and expressing the carried nucleic acid sequence into the host cell genome and/or allowing it to be expressed independently. These vectors contain essential regulatory elements that are operably linked so that the gene insert can be expressed, and “operably linked” means that the target gene and its regulatory sequences are functionally linked to each other to enable gene expression. means, “regulatory elements” include promoters for performing transcription, optional operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating 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 can replicate within the host cell, and any vector known in the art can be used. Examples of the vectors 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 pBR series, pUC series, pBluescriptII series, These include, but are not limited to, pGEM-based, pTZ-based, pCL-based and pET-based.
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 vector can typically be constructed as a vector for cloning or as a vector for expression. Vectors for expression can be those commonly 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 transformed into a suitable host cell and then replicate independently of the host cell's genome or can be incorporated into the genome itself. At this time, the “suitable host cell” is a vector capable of replication and may include an origin of replication, which is a specific base sequence at which replication is initiated. For example, when the vector used is an expression vector and a prokaryotic cell is used as the host, a strong promoter capable of advancing transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter), It typically includes a ribosome binding site for initiation of translation and a transcription/translation termination sequence. In the case of using a eukaryotic cell as a host, the origin of replication operating in the eukaryotic cell included in the vector includes the f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adeno origin of replication, AAV origin of replication, and BBV origin of replication. It is not limited. Additionally, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, 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 recombinant vector may include a selection marker, which is used to select transformants (host cells) transformed with the vector and expresses the selection marker in the medium treated with the selection marker. Because only cells are viable, selection of transformed cells is possible. Representative examples of the selection marker include kanamycin, streptomycin, and chloramphenicol, but are not limited thereto.
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 may be obtained by introducing a recombinant vector into an appropriate host cell. The host cell is a cell capable of stably and continuously cloning or expressing the expression vector, and any host cell known in the art can be used.
재조합 미생물을 제작하기 위하여 원핵세포에 형질전환시키는 경우에는 숙주세포로서 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 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, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli coli strains such as E. coli XL1-Blue, Bacillus subtilis, Bacillus thuringiensis, Corynebacterium strains, Salmonella Typhimurium, Serratia marcescens and Pseudomonas species. The same variety of intestinal bacteria and strains may be used, but it is not limited to this.
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, are used as host cells. , CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc. can be used, 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.As used in the present invention, “transformation” refers to a phenomenon that artificially causes genetic changes by introducing foreign DNA into a host cell, and “transformat” refers to a phenomenon in which foreign DNA is introduced into a target gene. It refers to a host cell that stably maintains expression.
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.For the transformation, an appropriate vector introduction technology is selected depending on the host cell to express the target gene or a recombinant vector containing the same within the host cell. For example, vector introduction can be performed using electroporation, heat-shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, and DEAE. -It may be performed by the dextran method, the cationic liposome method, the lithium acetate-DMSO method, or a combination thereof, but is not limited thereto. As long as the transformed gene can be expressed within the host cell, it can be included without limitation, whether it is inserted into the chromosome of the host cell or located outside the chromosome.
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 transformant includes cells transfected, transformed, or infected with the 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 일 구체예에 따르면, 상기 형질전환체는 코리네박테리움(Corynebacterium) 속 미생물인 것일 수 있다.According to one embodiment of the present invention, the transformant may be a microorganism of the genus Corynebacterium .
보다 구체적으로, 상기 코리네박테리움 속 미생물로는 코리네박테리움 글루타미컴(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) 등이 있으나, 이에 한정되는 것은 아니다.More specifically, the microorganisms of the Corynebacterium genus include Corynebacterium glutamicum , Corynebacterium crudilactis , Corynebacterium deserti , and Corynebacterium. Corynebacterium callunae , Corynebacterium suranareeae , Corynebacterium lubricantis , Corynebacterium doosanense , Corynebacterium Corynebacterium efficiens , Corynebacterium uterequi, Corynebacterium stationis , Corynebacterium pacaense , Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium humireducens , Corynebacterium marinum , Corynebacterium halotolerans , Corynebacterium spheniscorum ), Corynebacterium freiburgense , Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes , Corynebacterium Corynebacterium renale , Corynebacterium pollutisoli, Corynebacterium imitans , Corynebacterium caspium , Corynebacterium testudino These include, but are not limited to, Corynebacterium testudinoris, Corynebacaterium pseudopelargi , or Corynebacterium flavescens .
본 발명에서의 형질전환체는 전술한 FAD 의존성 산화환원효소 변이체 또는 이를 암호화하는 폴리뉴클레오티드를 포함하거나, 또는 이를 포함하는 벡터를 포함하는 균주, 상기 FAD 의존성 산화환원효소 변이체 또는 폴리뉴클레오티드를 발현하는 균주, 또는 상기 FAD 의존성 산화환원효소 변이체에 대한 활성을 가지는 균주일 수 있으나, 이에 한정되는 것은 아니다.The transformant in the present invention is a strain containing the above-described FAD-dependent oxidoreductase variant or a polynucleotide encoding the same, or a vector containing the same, or a strain expressing the FAD-dependent oxidoreductase variant or polynucleotide. , or it may be a strain having activity against the FAD-dependent oxidoreductase variant, but is not limited thereto.
본 발명의 일 구체예에 따르면, 상기 형질전환체는 L-글루탐산 생산능을 가지는 것일 수 있다.According to one embodiment of the present invention, the transformant may have the ability to produce L-glutamic acid.
상기 형질전환체는 자연적으로 L-글루탐산 생산능을 가지고 있거나, 또는 인위적으로 L-글루탐산 생산능이 부여된 것일 수 있다.The transformant may naturally have the ability to produce L-glutamic acid, or may be artificially endowed with the ability to produce L-glutamic acid.
본 발명의 일 구체예에 따르면, 상기 형질전환체는 FAD 의존성 산화환원효소의 활성이 변화되어 L-글루탐산 생산능이 향상된 것일 수 있다.According to one embodiment of the present invention, the transformant may have improved L-glutamic acid production ability due to a change in the activity of FAD-dependent oxidoreductase.
본 발명에서 사용된 “생산능이 향상된”은 모균주에 비해 L-글루탐산의 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 미생물 또는 야생형으로부터 변이된 코리네박테리움 속 미생물일 수 있다.“Improved production capacity” as used in the present invention means increased productivity of L-glutamic acid compared to the parent strain. The parent strain refers to a wild type or mutant strain that is subject to mutation, and includes a subject that is directly subject to mutation or transformed with a recombinant vector, etc. In the present invention, the parent strain may be a wild type microorganism of the genus Corynebacterium or a microorganism of the genus Corynebacterium mutated from the wild type.
본 발명에 따른 형질전환체는 FAD 의존성 산화환원효소 변이체가 도입됨으로써 FAD 의존성 산화환원효소의 활성이 변화하여 모균주에 비해 증가된 L-글루탐산 생산능을 나타낸다. 보다 구체적으로, 상기 형질전환체는 모균주에 비해 L-글루탐산 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 FAD 의존성 산화환원효소 변이체를 포함한 형질전환체는 모균주에 비해 L-글루탐산 생산량이 5% 이상, 구체적으로는 5 내지 50% (바람직하게는 7 내지 30%) 증가된 것일 수 있다.The transformant according to the present invention exhibits increased L-glutamic acid production ability compared to the parent strain due to a change in the activity of the FAD-dependent oxidoreductase by introducing a FAD-dependent oxidoreductase variant. More specifically, the transformant has an L-glutamic acid production of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% compared to the parent strain. %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% increase, or 1.1x, 1.5x, 2x, 2.5x, 3x , it may be increased by 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, or 10 times. It is not limited. For example, the transformant containing the FAD-dependent oxidoreductase variant may have an L-glutamic acid production increased by more than 5%, specifically 5 to 50% (preferably 7 to 30%), compared to the parent strain. .
본 발명의 다른 양상은 상기 형질전환체를 배지에서 배양하는 단계; 및 상기 형질전환체 또는 형질전환체가 배양된 배지로부터 L-글루탐산을 회수하는 단계를 포함하는 L-글루탐산의 생산 방법을 제공한다.Another aspect of the present invention includes culturing the transformant in a medium; and recovering L-glutamic acid from the transformant or the medium in which the transformant was 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 culture can be carried out according to appropriate media and culture conditions known in the art, and a person skilled in the art can easily adjust the medium and culture conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. Cultivation methods may include, for example, batch culture, continuous culture, fed-batch culture, or combinations thereof, but are not limited thereto.
본 발명의 일 구체예에 따르면, 상기 배지는 적절한 방식으로 특정 균주의 요건을 충족해야 하며, 통상의 기술자에 의해 적절하게 변형될 수 있다. 코리네박테리움 속 미생물에 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 embodiment of the present invention, the medium must meet the requirements of a specific strain in an appropriate manner and can be appropriately modified by a person skilled in the art. Culture media for microorganisms of the genus Corynebacterium may refer to known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981), but are 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 embodiment of the present invention, the medium may contain 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, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, It includes fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These substances may be used individually or in mixtures, but are not limited thereto. Nitrogen sources that may be used include peptone, yeast extract, broth, 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 can also be used individually or in 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 the corresponding sodium-containing salts. Additionally, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth, but is not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be included. Additionally, precursors appropriate for the culture medium may be used. The medium or individual components may be added to the culture medium in an appropriate manner in a batch or continuous manner during the culture process, 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 embodiment of the present invention, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the microbial culture medium in an appropriate manner during cultivation. Additionally, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester during culture. Additionally, in order to maintain the aerobic state of the culture medium, oxygen or oxygen-containing gas (e.g., air) can be injected into the culture medium. The temperature of the culture medium may typically be 20 to 45°C, for example, 25 to 40°C. The culturing period may continue until the desired yield of useful material is obtained, for example, 10 to 160 hours.
본 발명의 일 구체예에 따르면, 상기 배양된 형질전환체 또는 형질전환체가 배양된 배지에서 L-글루탐산을 회수하는 단계는 배양 방법에 따라 당해 분야에 공지된 적합한 방법을 이용하여 배지로부터 생산된 L-글루탐산을 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 embodiment of the present invention, the step of recovering L-glutamic acid from the cultured transformant or the medium in which the transformant was cultured includes L-glutamic acid produced from the medium using a suitable method known in the art according to the culture method. -Glutamic acid can be collected or recovered. Examples include centrifugation, filtration, extraction, nebulization, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g. ammonium sulfate precipitation), chromatography (e.g. ion exchange, affinity, hydrophobic and Methods such as size exclusion) can be used, but are not limited to this.
본 발명의 일 구체예에 따르면, 상기 L-글루탐산을 회수하는 단계는 배양 배지를 저속 원심분리하여 바이오매스를 제거하고 얻어진 상등액을 이온교환 크로마토그래피를 통하여 분리할 수 있다.According to one embodiment of the present invention, the step of recovering L-glutamic acid may be performed by centrifuging the culture medium at low speed to remove biomass and separating the obtained supernatant through ion exchange chromatography.
본 발명의 일 구체예에 따르면, 상기 L-글루탐산을 회수하는 단계는 L-글루탐산을 정제하는 공정을 포함할 수 있다.According to one embodiment of the present invention, the step of recovering L-glutamic acid may include a process of purifying L-glutamic acid.
본 발명에 따른 FAD 의존성 산화환원효소 변이체는 FAD 의존성 산화환원효소를 구성하는 아미노산 서열 중 하나 이상의 아미노산이 치환됨으로써 효소 활성이 변화되어, 이를 포함하는 재조합 미생물은 L-글루탐산을 효율적으로 생산할 수 있다.The FAD-dependent oxidoreductase mutant according to the present invention changes its enzyme activity by substituting one or more amino acids in the amino acid sequence constituting the FAD-dependent oxidoreductase, so that the recombinant microorganism containing it can efficiently produce L-glutamic acid.
도 1은 본 발명의 일 실시예에 따른 플라스미드 pK19msb의 구조를 나타낸 것이다.Figure 1 shows the structure of plasmid pK19msb according to an 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 merely provided as an example to aid understanding of the present invention, and the scope of the present invention is not limited by this example description.
실시예 1. FAD 의존성 산화환원효소 변이체 발현을 위한 벡터 제작Example 1. Construction of vector for expression of FAD-dependent oxidoreductase variant
FAD 의존성 산화환원효소의 아미노산 서열 (서열번호 4)에서 200번째 위치한 알라닌(A)이 트레오닌(T)으로 치환된 변이체를 발현하는 벡터를 제작하였다. A vector expressing a mutant in which alanine (A) at position 200 in the amino acid sequence of the FAD-dependent oxidoreductase (SEQ ID NO: 4) was replaced with threonine (T) was constructed.
야생형 코리네박테리움 글루타미컴(Corynebacterium glutamicum) ATCC13869의 gDNA를 주형으로 프라이머 1 및 프라이머 2의 프라이머 쌍과 프라이머 3 및 프라이머 4의 프라이머 쌍을 이용하여 각각 PCR을 수행하였다. 이후, 두 개의 PCR 산물을 혼합한 혼합물을 주형으로 프라이머 1 및 프라이머 4를 이용하여 다시 오버랩핑 PCR을 수행하여 단편을 획득하였다. 여기서 중합효소는 Takara PrimeSTAR Max DNA polymerase를 사용하였으며, PCR 증폭 조건은 95℃에서 5분 변성한 후, 95℃에서 30초, 58℃에서 30초, 72℃에서 1분 30초를 30회 반복하고 72℃에서 5분간 반응하였다다. pK19msb 벡터에 smaI을 처리하고 상기에서 수득한 PCR 산물 (단편)과 연결(ligation)하였으며, 여기서 얻은 플라스미드를 pK_fprA(A200T)라 명명하였다.PCR was performed using the gDNA of wild-type Corynebacterium glutamicum ATCC13869 as a template, using primer pairs of primer 1 and primer 2 and primer pairs of primer 3 and primer 4, respectively. Afterwards, overlapping PCR was performed again using the mixture of the two PCR products as a template using primers 1 and 4 to obtain fragments. Here, Takara PrimeSTAR Max DNA polymerase was used as the polymerase, and the PCR amplification conditions were denatured at 95°C for 5 minutes, then repeated 30 times at 95°C for 30 seconds, at 58°C for 30 seconds, and at 72°C for 1 minute and 30 seconds. Reaction was performed at 72°C for 5 minutes. The pK19msb vector was treated with smaI and ligated with the PCR product (fragment) obtained above, and the obtained plasmid was named pK_fprA(A200T).
벡터 제작에 사용된 프라이머 서열은 하기 표 1과 같다.Primer sequences used for vector construction are shown in Table 1 below.
| 프라이머 명칭Primer name | 서열번호sequence number | 프라이머 서열 (5'-3')Primer sequence (5'-3') |
| 프라이머 1primer 1 | 66 | TCATGGAGGGTTGATAGCAGGGC TCATGGAGGGTTGATAGCAGGGC |
| 프라이머 2primer 2 | 77 | GGAGTGAACTTCGTCTGAGCAGG GGAGTGAACTTCGTCTGAGCAGG |
| 프라이머 3primer 3 | 88 | ACCTGCTCAGACGAAGTTCACTCCACCTGCTCAGACGAAGTTCACTCC |
| 프라이머 4primer 4 | 99 | CGTATTCTGGACGAGCATGGCG CGTATTCTGGACGAGCATGGCG |
실시예 2. FAD 의존성 산화환원효소 변이체가 도입된 변이주 제작Example 2. Production of mutant strains into which FAD-dependent oxidoreductase mutants are introduced
FAD 의존성 산화환원효소 변이체를 도입하기 위한 모균주로 코리네박테리움 글루타미컴 U3 (KCCM13218P)를 사용하였고, U3 균주의 형질전환을 위한 방법으로 van der Rest 등의 방법을 기본으로 수식한 일렉트로컴피턴트 셀(electrocompetent cell) 제조법을 사용하였다. Corynebacterium glutamicum U3 (KCCM13218P) was used as the parent strain to introduce the FAD-dependent oxidoreductase mutant, and Electrocompy was modified based on the method of van der Rest et al. as a method for transformation of the U3 strain. An electrocompetent cell manufacturing method was used.
먼저, 2% 포도당이 첨가된 2YT 배지 (트립톤 16 g/ℓ, 효모추출물 10 g/ℓ 및 염화나트륨 5 g/ℓ 함유) 10 ㎖에서 U3 균주를 1차 배양하여 종배양액을 준비하였다. 이후 포도당을 제외한 2YT 배지 100 ㎖에 1 mg/㎖ 농도의 이소니코틴산 히드라진(isonicotinic acid hydrazine) 및 2.5% 글리신(glycine)을 첨가하고, OD610 값이 0.3이 되도록 종배양액을 접종한 후, 18℃, 180 rpm으로 12 ~ 16시간 배양하여 OD610 값이 1.2 ~ 1.4가 되도록 하였다. 배양액을 얼음에서 30분간 방치한 후, 4℃, 4000 rpm으로 15분간 원심분리하였다. 그 뒤 상등액을 버리고 침전된 U3 균주를 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 pK_fprA(A200T) 벡터를 첨가한 후, 2.5 kV, 200 Ω 및 12.5 ㎌의 조건으로 전기충격을 가하였다. 전기충격이 끝난 즉시 재생(regeneration) 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 BHI 배지에서 정지기까지 배양하여 2차 재조합을 유도했으며 10-5 ~ 10-7까지 희석하여 항생제가 없는 2YT 평판 배지 (10% sucrose 함유)에 도말하여 카나마이신 내성도가 없고 10% 수크로스가 포함된 배지에서 성장성이 있는 균주를 선별하였으며, 이를 FPR1이라 명명하였다.First, a seed culture was prepared by primary culturing the U3 strain 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. Afterwards, isonicotinic acid hydrazine and 2.5% glycine at a concentration of 1 mg/ml were added to 100 ml of 2YT medium excluding glucose, and the seed culture was inoculated so that the OD610 value was 0.3, and incubated at 18°C. Cultured for 12 to 16 hours at 180 rpm to bring the OD610 value to 1.2 to 1.4. The culture was left on ice for 30 minutes and then centrifuged at 4°C and 4000 rpm for 15 minutes. Afterwards, the supernatant was discarded, and the precipitated U3 strain was washed four times with 10% glycerol solution and finally resuspended in 0.5 ml of 10% glycerol solution to prepare competent cells. Electroporation was performed using an electroporator from Bio-Rad. After adding the prepared competent cells and pK_fprA(A200T) vector to an electroporation cuvette (0.2 mm), an electric shock was applied under the conditions of 2.5 kV, 200 Ω, and 12.5 ㎌. Immediately after the electric shock, 1 ml of regeneration medium (containing Brain Heart infusion 18.5 g/l and sorbitol 0.5 M) was added and heat treated at 46°C for 6 minutes. After cooling to room temperature, transfer to a 15 ㎖ cap tube, incubate for 2 hours at 30℃, and add selection medium (tryptone 5 g/ℓ, NaCl 5 g/ℓ, yeast extract 2.5 g/ℓ, Brain Heart infusion powder 18.5 g/ℓ). , containing 15 g/l agar, 91 g/l sorbitol, and 20 ㎍/l kanamycin). Colonies generated by culturing at 30°C for 72 hours were cultured in BHI medium until stationary phase to induce secondary recombination, diluted to 10 -5 to 10 -7 and plated on antibiotic-free 2YT plate medium (containing 10% sucrose). A strain that had no kanamycin resistance and had the ability to grow on a medium containing 10% sucrose was selected, and it was named FPR1.
실험예 1. FAD 의존성 산화환원효소 변이체가 도입된 변이주의 L-글루탐산 생산능 평가Experimental Example 1. Evaluation of L-glutamic acid production capacity of mutant strains introduced with FAD-dependent oxidoreductase mutants
모균주 U3와 FAD 의존성 산화환원효소 변이체가 도입된 변이주 FPR1의 L-글루탐산 생산능을 비교하였다.The L-glutamic acid production capacity of the parent strain U3 and the mutant strain FPR1 into which the FAD-dependent oxidoreductase mutant was introduced was compared.
하기 표 2의 글루탐산 생산용 배지 10 mL가 담긴 100 mL 플라스크에 각 균주 (모균주 또는 변이주)를 부피 기준으로 1%씩 접종하여 30℃, 200 rpm의 조건으로 48시간 진탕 배양하였다. 배양 종료 후 HPLC (Agilent)를 사용하여 배지 내 L-글루탐산의 농도를 측정하였고, 그 결과를 각각 하기 표 3에 나타내었다.Each strain (parent strain or mutant strain) was inoculated at 1% by volume in a 100 mL flask containing 10 mL of the medium for glutamic acid production shown in Table 2 below, and cultured with shaking at 30°C and 200 rpm for 48 hours. After completion of the culture, the concentration of L-glutamic acid in the medium was measured using HPLC (Agilent), and the results are shown in Table 3 below.
| 조성Furtherance | 함량content |
| GlucoseGlucose | 70 g/L70g/L |
| (NH4)2SO4 (NH 4 ) 2 SO 4 | 5 g/L5g/L |
| MgSO4 MgSO 4 | 0.4 g/L0.4g/L |
| UreaUrea | 2 g/L2g/L |
| 대두가수분해물Soybean hydrolyzate | 1.5% v/v1.5% v/v |
| KH2PO4 KH 2 PO 4 | 1.0 g/L1.0g/L |
| FeSO4 FeSO4 | 10 mg/L10mg/L |
| MnSO4 MnSO 4 | 10 mg/L10mg/L |
| Thiamine_HClThiamine_HCl | 200 ug/L200ug/L |
| BiotinBiotin | 2 ug/L2ug/L |
| CaCO3 CaCO 3 | 5%5% |
| 균주strain | L-글루탐산 생산량 (g/L)L-glutamic acid production (g/L) |
| U3U3 | 11.311.3 |
| FPR1FPR1 | 12.7512.75 |
상기 표 3에 나타낸 바와 같이, FAD 의존성 산화환원효소 변이체가 도입된 변이주에서는 모균주에 비해 L-글루탐산 생산량이 약 12.8% 향상된 것으로 확인되었다. 이러한 결과는 FAD 의존성 산화환원효소 변이에 의해 글루탐산 생산 경로의 탄소원 흐름이 증가함으로써 L-글루탐산 생산성이 증가함을 시사한다.As shown in Table 3, in the mutant strain into which the FAD-dependent oxidoreductase mutant was introduced, L-glutamic acid production was confirmed to be improved by about 12.8% compared to the parent strain. These results suggest that L-glutamic acid productivity increases by increasing carbon source flow in the glutamic acid production pathway due to FAD-dependent oxidoreductase mutation.
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.So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form 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 in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.
[수탁번호][Accession number]
기탁기관명 : 한국미생물보존센터(KCCM)Name of depository organization: Korea Center for Microbial Conservation (KCCM)
수탁번호 : KCCM13218PAccession number: KCCM13218P
수탁일자 : 20220629Trust date: 20220629
Claims (6)
- 서열번호 4의 아미노산 서열에서 200번째 알라닌이 트레오닌으로 치환된, 서열번호 2의 아미노산 서열로 이루어진 FAD 의존성 산화환원효소 변이체.A FAD-dependent oxidoreductase variant consisting of the amino acid sequence of SEQ ID NO: 2, in which alanine at position 200 in the amino acid sequence of SEQ ID NO: 4 is substituted with threonine.
- 청구항 1의 변이체를 암호화하는 폴리뉴클레오티드.A polynucleotide encoding the variant of claim 1.
- 청구항 1의 변이체 또는 청구항 2의 폴리뉴클레오티드를 포함하는 형질전환체.A transformant comprising the variant of claim 1 or the polynucleotide of claim 2.
- 청구항 3에 있어서,In claim 3,상기 형질전환체는 코리네박테리움(Corynebacterium) 속 미생물인 것인 형질전환체.The transformant is a transformant that is a microorganism of the genus Corynebacterium .
- 청구항 3에 있어서,In claim 3,상기 형질전환체는 L-글루탐산 생산능을 가지는 것인 형질전환체.The transformant is a transformant having the ability to produce L-glutamic acid.
- 청구항 3의 형질전환체를 배지에서 배양하는 단계; 및Culturing the transformant of claim 3 in a medium; and상기 형질전환체 또는 형질전환체가 배양된 배지로부터 L-글루탐산을 회수하는 단계를 포함하는 L-글루탐산의 생산 방법.A method for producing L-glutamic acid comprising the step of recovering L-glutamic acid from the transformant or the medium in which the transformant was cultured.
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| KR1020220085238A KR20240008454A (en) | 2022-07-11 | 2022-07-11 | Mutant microorganism of Corynebacterium genus producing L-glutamic acid and method for producing L-glutamic acid using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100837844B1 (en) * | 2006-12-14 | 2008-06-13 | 씨제이제일제당 (주) | - - Microoragnism of Corynbacterium genus with an enhanced L-glutamic acid productivity and method of producing L-glutamic acid using the microorganism |
| JP2014036576A (en) * | 2010-12-10 | 2014-02-27 | Ajinomoto Co Inc | Method for producing l-amino acids |
| KR102269639B1 (en) * | 2020-02-12 | 2021-06-25 | 대상 주식회사 | Mutant of Corynebacterium glutamicum with enhanced L-glutamic acid productivity and method for preparing L-glutamic acid using the same |
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- 2022-07-11 KR KR1020220085238A patent/KR20240008454A/en unknown
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100837844B1 (en) * | 2006-12-14 | 2008-06-13 | 씨제이제일제당 (주) | - - Microoragnism of Corynbacterium genus with an enhanced L-glutamic acid productivity and method of producing L-glutamic acid using the microorganism |
| JP2014036576A (en) * | 2010-12-10 | 2014-02-27 | Ajinomoto Co Inc | Method for producing l-amino acids |
| KR102269639B1 (en) * | 2020-02-12 | 2021-06-25 | 대상 주식회사 | Mutant of Corynebacterium glutamicum with enhanced L-glutamic acid productivity and method for preparing L-glutamic acid using the same |
Non-Patent Citations (2)
| Title |
|---|
| DATABASE Protein 20 May 2022 (2022-05-20), ANONYMOUS: "FAD-dependent oxidoreductase [Corynebacterium glutamicum]", XP093128413, retrieved from NCBI Database accession no. WP_216312814.1 * |
| VAN DEN HEUVEL R. H. H., CURTI B., VANONI M. A., MATTEVI A.: "Glutamate synthase: a fascinating pathway from L-glutamine to L-glutamate", CMLS CELLULAR AND MOLECULAR LIFE SCIENCES., BIRKHAUSER VERLAG, HEIDELBERG., DE, vol. 61, no. 6, 1 March 2004 (2004-03-01), DE , pages 669 - 681, XP093128416, ISSN: 1420-682X, DOI: 10.1007/s00018-003-3316-0 * |
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