WO2022196919A1 - L-시트룰린 생산능이 향상된 코리네박테리움 글루타미쿰 변이주 및 이를 이용한 l-시트룰린의 생산 방법 - Google Patents
L-시트룰린 생산능이 향상된 코리네박테리움 글루타미쿰 변이주 및 이를 이용한 l-시트룰린의 생산 방법 Download PDFInfo
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- WO2022196919A1 WO2022196919A1 PCT/KR2022/000971 KR2022000971W WO2022196919A1 WO 2022196919 A1 WO2022196919 A1 WO 2022196919A1 KR 2022000971 W KR2022000971 W KR 2022000971W WO 2022196919 A1 WO2022196919 A1 WO 2022196919A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- citrulline
- corynebacterium
- gene
- corynebacterium glutamicum
- ncgl2657
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/10—Citrulline; Arginine; Ornithine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01008—Phosphate acetyltransferase (2.3.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
Definitions
- the present invention relates to a Corynebacterium glutamicum mutant having improved L-citrulline production ability and a method for producing L-citrulline using the same.
- L-Citrulline is one of the non-essential amino acids, and it is known that citrulline has useful effects such as promoting ammonia metabolism or improving blood flow by dilating blood vessels, lowering blood pressure, neurotransmission, enhancing immunity, and scavenging free radicals.
- Citrulline is synthesized as an intermediate product during the biosynthesis of arginine. The biosynthesis of arginine in microorganisms is accomplished through eight enzymatic steps from L-glutamate through two different pathways, linear and cyclic.
- L-arginine is converted from L-glutamate to N-acetyl glutamate, N-acetyl ornithine, ornithine, citrulline, and arginine It is synthesized via argininosuccinate.
- L-citrulline production is generally produced by fermentation using microorganisms such as bacteria or yeast, and wild-type strains obtained in the natural state or mutants modified to improve L-citrulline-producing ability thereof can be used.
- microorganisms such as bacteria or yeast
- wild-type strains obtained in the natural state or mutants modified to improve L-citrulline-producing ability thereof can be used.
- genetic recombination technology is applied to microorganisms such as Escherichia coli and Corynebacterium, which are widely used for the production of L-amino acids and other useful substances, having excellent L-citrulline production ability.
- Various recombinant strains or mutants and methods for producing L-citrulline using the same are being developed. According to Korean Patent Nos.
- L-citrulline production ability was improved by enhancing or weakening the activity or expression of proteins such as enzymes and transcription factors related to L-citrulline production. Therefore, it can be expected that the production of L-citrulline can also be controlled by regulating the activity or expression of various proteins, including enzymes acting on the production pathway of L-citrulline.
- An object of the present invention is to provide a mutant Corynebacterium glutamicum having improved L-citrulline production ability.
- Another object of the present invention is to provide a method for producing L-citrulline using the mutant.
- the present inventors studied to develop a new mutant with improved L-citrulline production ability using a Corynebacterium glutamicum strain, and as a result, involved in the production of 6-acetylornithine produced as a by-product in the L-citrulline biosynthesis pathway.
- the activity of the enzyme is weakened or inactivated, so that the production of 6-acetylornithine is significantly reduced as well as the production of L-citrulline is increased.
- the present invention was completed.
- One aspect of the present invention provides a Corynebacterium glutamicum mutant with improved L-citrulline-producing ability, in which the activity of the protein expressed by the NCgl2657 gene is weakened or inactivated.
- NCgl2657 gene used in the present invention encodes an enzyme (phosphate acetyltransferase) that uses acetyl-CoA and phosphate as substrates in the L-citrulline biosynthesis pathway to produce acetyl phosphate, a precursor of acetylornithine. means a gene that
- the NCgl2657 gene may be derived from a genus strain of Corynebacterium .
- the Corynebacterium sp. strain is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium Corynebacterium callunae , Corynebacterium suranareeae , Corynebacterium lubricantis , Corynebacterium doosanense, Corynebacterium doosanense , Corynebacterium ipish Ens ( Corynebacterium efficiens ), Corynebacterium utereki ( Corynebacterium uterequi ), Corynebacterium stationis ( Corynebacterium stationis ), Corynebacterium stationis ( Cor
- the NCgl2657 gene may be represented by the nucleotide sequence of SEQ ID NO: 1.
- weakened activity means that the expression level of the gene as an object is reduced than the original expression level.
- the attenuation of this activity is when the activity of the protein itself is reduced compared to the activity of the protein possessed by the original microorganism through nucleotide substitution, insertion, deletion, or a combination thereof, and inhibition of the expression or translation of the gene encoding it For example, when the overall degree of enzymatic activity in the cell is low compared to the wild-type strain or the strain before modification, a combination thereof is also included.
- activation refers to a case in which the expression of a gene encoding a protein such as an enzyme, a transcription factor, a transport protein, etc. is not expressed at all compared to the wild-type strain or the strain before modification, or there is no activity even if it is expressed. it means.
- the attenuation or inactivation of the protein expressed by the NCgl2657 gene may be due to insertion, substitution, deletion, or a combination of all or part of the NCgl2657 gene.
- “some” means not all of the base sequence or polynucleotide sequence, and may be 1 to 300, preferably 1 to 100, more preferably 1 to 50, but is limited thereto. it is not
- Attenuation or inactivation of the protein expressed by the NCgl2657 gene may cause a mutation in the start codon of the NCgl2657 gene.
- the mutation in the start codon of the NCgl2657 gene may be to replace the start codon ATG of the NCgl2657 gene with another base, such as TTG or GTG.
- Leum glutamicum mutants were obtained.
- This Corynebacterium glutamicum mutant may include the NCgl2657 gene encoded by the nucleotide sequence of SEQ ID NO: 2.
- the attenuation or inactivation of the protein expressed by the NCgl2657 gene may be removal or deletion of the NCgl2657 gene.
- the NCgl2657 gene of the Corynebacterium glutamicum strain is deleted to the wild-type Corynebacterium glutamicum strain or the previously developed Corynebacterium glutamicum mutant to produce an excess of citrulline.
- improved productivity means that the productivity of L-citrulline is increased compared to the parent strain.
- the parent strain means a wild-type or mutant strain to be mutated, and includes a target to be directly mutated or transformed with a recombinant vector.
- the parent strain may be a wild-type Corynebacterium glutamicum strain or a strain mutated from the wild-type.
- the parent strain may be Corynebacterium glutamicum ATCC13032.
- the Corynebacterium glutamicum mutant strain with improved L-citrulline production ability increased L-citrulline production compared to the parent strain by introducing a mutation into the start codon of the NCgl2657 gene or removing the NCgl2657 gene.
- the L-citrulline concentration is increased by 5% or more, specifically 5 to 50%, and more specifically 10 to 30%, at this time as a by-product. 6-acetyl ornithine produced was reduced by at least 20%, specifically 20-100%, more specifically 30-100%.
- Corynebacterium glutamicum mutant strain according to one embodiment of the present invention can be implemented through a recombinant vector containing a mutant in the start codon of the NCgl2657 gene in the parent strain, or a recombinant vector that deletes the NCgl2657 gene.
- variant refers to a genetic variant in which the start codon of the NCgl2657 gene involved in the biosynthesis of L-citrulline is substituted from ATG to TTG.
- the term “vector” refers to an expression vector capable of expressing a target protein in a suitable host cell, and refers to a gene preparation comprising essential regulatory elements operably linked to express a gene insert.
- operably linked means that a gene requiring expression and its regulatory sequence are functionally linked to each other and linked in a way that enables gene expression
- regulatory element refers to a promoter for performing transcription, regulating transcription including any operator sequences for sequencing, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation.
- Such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like.
- the "recombinant vector” used in the present invention can be replicated independently of the genome of the host cell after transformation into a suitable host cell, or can be sutured into the genome itself.
- the "suitable host cell” may include a replication origin, which is a specific nucleotide sequence from which replication is initiated as a vector capable of replication.
- Transformation as used in the present invention is to introduce a gene into a host cell so that it can be expressed in the host cell, and if the transformed gene can be expressed in the host cell, it can be inserted into the chromosome of the host cell or added to the chromosome other than the chromosome. It may be included without limitation whatever is located.
- the transformation method includes any method of introducing a nucleic acid into a cell, and can be performed by selecting a suitable standard technique as known in the art depending on the host cell.
- electroporation calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate -DMSO method, etc.
- CaPO4 calcium phosphate
- CaCl2 calcium chloride
- microinjection polyethylene glycol (PEG) method
- DEAE-dextran method cationic liposome method
- lithium acetate -DMSO method lithium acetate -DMSO method, etc.
- the host cells include cells transfected, transformed, or infected with the recombinant vector or polynucleotide of the present invention in vivo or in vitro.
- a host cell comprising the recombinant vector of the present invention is a recombinant host cell, a recombinant cell or a recombinant microorganism.
- the recombinant vector according to the present invention may include a selection marker
- the selection marker is for selecting transformants (host cells) transformed with the vector, in the medium treated with the selection marker. Since only cells expressing the selection marker can survive, selection of transformed cells is possible.
- Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, chloramphenicol, and the like.
- Genes inserted into the recombinant vector for transformation of the present invention may be substituted into host cells such as microorganisms such as Corynebacterium spp. and Escherichia coli due to homologous recombination crossover.
- the host cell may be a Corynebacterium sp. strain, for example, Corynebacterium glutamicum ( Corynebacterium glutamicum ) It may be a strain.
- another aspect of the present invention comprises: a) culturing the Corynebacterium glutamicum mutant in a medium; and b) recovering L-citrulline from the mutant or mutant culture medium.
- the culture may be performed according to an appropriate medium and culture conditions known in the art, and those skilled in the art may easily adjust the medium and culture conditions for use.
- the medium may be a liquid medium, but is not limited thereto.
- the culture method may include, for example, batch culture, continuous culture, fed-batch culture, or a combination culture thereof, but is not limited thereto.
- the medium should meet the requirements of a specific strain in an appropriate manner, and may be appropriately modified by a person skilled in the art.
- the culture medium for the Corynebacterium sp. strain may refer to the known literature (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, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, fatty acids such as 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 may be used include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
- the nitrogen source may also be used individually or as a mixture, but is not limited thereto.
- Sources of phosphorus that may be used include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt.
- the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth, but is not limited thereto.
- essential growth substances such as amino acids and vitamins may be included.
- precursors suitable for the culture medium may be used. The medium or individual components may be added batchwise or continuously by an appropriate method to the culture medium during the culturing process, but is not limited thereto.
- the pH of the culture medium by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the microorganism culture medium in an appropriate manner during culture.
- an antifoaming agent such as fatty acid polyglycol ester during culture can suppress the formation of bubbles.
- oxygen or oxygen-containing gas eg, air
- the temperature of the culture medium may be usually 20 °C to 45 °C, for example, 25 °C to 40 °C.
- the incubation period may be continued until a useful substance is obtained in a desired production amount, for example, it may be 10 to 160 hours.
- the step of recovering L-citrulline from the cultured mutant and the mutant culture medium is L-citrulline produced from the medium using a suitable method known in the art according to the culture method.
- a suitable method known in the art according to the culture method.
- the culture medium in the step of recovering L-citrulline, may be centrifuged at low speed to remove biomass, and the obtained supernatant may be separated through ion exchange chromatography.
- the step of recovering L-cittrulline may include a step of purifying L-cittrulline.
- the Corynebacterium glutamicum mutant according to the present invention can inhibit the production of by-products and produce L-citrulline in high yield and high concentration by weakening or inactivating the activity of the protein expressed by the NCgl2657 gene.
- Corynebacterium glutamicum mutant with weakened activity of the protein expressed by the NCgl2657 gene, Corynebacterium glutamicum ATCC13032 and E. coli DH5a (HIT Competent cellsTM, Cat No. RH618) was used.
- the Corynebacterium glutamicum ATCC13032 is a CM- composition of 5 g of glucose, 2.5 g of NaCl, 5.0 g of yeast extract, 1.0 g of urea, 10.0 g of polypeptone and 5.0 g of beef extract in 1 L of distilled water. Incubated in broth medium (pH 6.8) at a temperature of 30 °C.
- the E. coli DH5a was cultured at a temperature of 37° C. on LB medium having a composition of 10.0 g of tryptone, 10.0 g of NaCl and 5.0 g of yeast extract in 1 L of distilled water.
- PCR was performed with a combination of primers 1 and 2 and primers 3 and 4 using this as a template, respectively.
- the PCR product thus obtained was again amplified by crossover PCR using a combination of primers 1 and 4, and then inserted into the site of restriction enzymes HindIII and XbaI of the recombinant vector pK19mobSacB. This was named vector pK19ms/NCgl2657(A1T).
- the primers in Table 1 below were used to construct the vector.
- primer Primer sequence (5' ⁇ 3') SEQ ID NO: Primer 1 ctatgaccatgattacgccacagtggagaacacgatcgcg 3 Primer 2 gaggtcggtgtgtcagacaaacatcgcctttctaatttca 4 Primer 3 tgaaattagaaaggcgatgtttgtctgacacaccgacctc 5 Primer 4 gctcggtacccggggatcctcggcgtcgttgtggacc 6
- PCR was performed under the following conditions using the above primers.
- a Thermocycler TP600, TAKARA BIO Inc., Japan
- each deoxynucleotide triphosphate dATP, dCTP, dGTP, dTTP
- 100 ⁇ M was added to the reaction solution with 1 pM of oligonucleotides, Corynebacterium glutamicum ( C. glutamicum )
- C. glutamicum Corynebacterium glutamicum
- PrimeSTAR Max DNA Polymerase (Takara, Japan) 25 to 30 cycles were performed in the presence of 1 unit.
- PCR conditions were (i) denaturation step: 94°C for 30 seconds, (ii) annealing step: 58°C for 30 seconds, and (iii) extension step: 1-2 at 72°C. It was carried out under the conditions of minutes (providing a polymerization time of 2 minutes per 1 kb).
- the gene fragment thus prepared was cloned into the pK19mobSacB vector using self-assembly cloning.
- the vector was transformed into E. coli DH5a, spread on an LB-agar plate containing 50 ⁇ g/ml kanamycin, and cultured at 37° C. for 24 hours. After isolating the finally formed colony and confirming that the insert is exactly present in the vector, this vector was isolated and used for recombination of the Corynebacterium glutamicum strain.
- the amplification of the corresponding genes is amplified by PCR from Corynebacterium glutamicum ATCC13032 genomic DNA and inserted into the pK19mobSacB vector by self-assembled cloning method according to the strategy and selected from E. coli DH5a did.
- a DNA ligation kit (Takara, Japan) and restriction enzymes HindIII, XbaI (NEB, USA) were used according to the supplied buffer and protocol.
- colonies induced by the primary homologous recombination using primers 1 and 4 in Table 1, colonies confirmed for amplification by PCR under the same conditions as in 1-1 above were selected as the primary recombinant strain, and 2YT liquid medium (Tryptone 16 g/ After 12 hours incubation in L, yeast extract 10 g/L and NaCl 5 g/L), 2YT-10% sucrose solid medium (Tryptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L, agar 1.5) % and sucrose 100 g/L) to induce secondary homologous recombination to remove antibiotic markers.
- 2YT liquid medium Teryptone 16 g/ After 12 hours incubation in L, yeast extract 10 g/L and NaCl 5 g/L
- 2YT-10% sucrose solid medium Typtone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L, agar 1.5
- the Corynebacterium glutamicum ATCC13032, E. coli DH5a and antibiotics were the same as those used in Example 1.
- a vector was prepared in the same manner as in Example 1, except that primers 5 and 6 were used instead of primers 1 and 2, and primers 7 and 8 were used instead of primers 3 and 4.
- the constructed vector was named pK19ms/ ⁇ NCgl2657, and the primers in Table 2 below were used to construct it.
- primer primer sequence (5' ⁇ 3') SEQ ID NO: Primer 5 ctatgaccatgattacgccacagtggagaacacgatcgcg 7 Primer 6 caaaacaagtgccaatgccaacatcgcctttctaatttca 8 Primer 7 tgaaattagaaaggcgatgttggcattggcacttgttttg 9 Primer 8 gctcggtacccggggatcctatcgatgctcatgccagcgg 10
- a CT3 strain a Corynebacterium glutamicum mutant strain, was prepared in the same manner as in Example 1, except that pK19ms/ ⁇ vector was used instead of pK19ms/NCgl2657(A1T) vector. After finally selecting a strain that is not resistant to kanamycin and can grow in 10% sucrose medium, PCR was performed under the same conditions as in Example 1 using primers 5 and 8 in Table 2 to confirm that the NCgl2657 gene was removed. The strain in which the NCgl2657 gene was deleted was named CT3.
- Corynebacterium glutamicum mutant strain with enhanced activity of ornithine carbamoyltransferase and carbamoyl phosphate synthase to produce excessive citrulline (refer to Korean Patent Application No. 10-2019-0151321, hereinafter referred to as 'CT1') was used.
- the CT1 strain was confirmed to have increased L-production amount and fermentation yield compared to Corynebacterium glutamicum ATCC13032 used as the parent strain.
- Each strain was inoculated into a citrulline seed medium and cultured at 30° C. for 10 hours, and then 250 mL of the culture solution was inoculated into a 5 L incubator medium. When all the sugars contained in the initial medium were exhausted, an additional medium was added.
- the composition of the medium used in this experiment is shown in Table 3 below.
- the culture solution was diluted 100 times with distilled water, filtered with a 0.45 um filter, and then the column (DionexIonPac TM CS12A) and high-performance liquid chromatography (HPLC) equipped with a UV detector (195 mm) were used in the culture solution produced in the strain. The concentrations of L-citrulline and 6-acetylornithine, a byproduct, were measured, and the results are shown in Table 4 below.
- composition (/L) Citrulline seed medium Glucose 2.67%, YPA 3.14%, KH 2 PO 4 0.1%, K 2 HPO 4 0.1%, MnSO 4 10 ppm, FeSO 4 10 ppm, Biotin 100 ug/L, Thiamin-HCl 200 ug/L 5L incubator medium Glucose 8%, MgSO 4 0.08%, FeSO 4 16 ppm, MnSO 4 8 ppm, CuSO 4 1.6 ppm, ZnSO 4 1.6 ppm, CaCl 2 32 ppm, Biotin 160 ppb, Thiamine-HCl 8 ppm, HVP 0.4%, NaCl 0.08 %, KH 2 PO 4 0.22%, (NH 4 ) 2 SO 4 2.4% additional badges Glucose 52%, MgSO 4 0.1%, CaCl 2 120 ppm, Biotin 160 ppb, Thiamine-HCl 16 ppm, NH 4 H 2 PO 4 0.08%, (NH
- the Corynebacterium glutamicum mutant CT2 in which the activity of the enzyme encoded by the NCgl2657 gene is weakened and the Corynebacterium glutamicum mutant CT3 in which the NCgl2657 gene is deleted are present in the previously developed mutant CT1.
- the production of by-product 6-acetylornithine was decreased by about 69% and 99%, respectively, and the productivity of L-citrulline was increased by about 17.8% and 12.2%, respectively.
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Abstract
Description
프라이머 | 프라이머 서열 (5'→3') | 서열번호 |
프라이머 1 | ctatgaccatgattacgccacagtggagaacacgatcgcg | 3 |
프라이머 2 | gaggtcggtgtgtcagacaaacatcgcctttctaatttca | 4 |
프라이머 3 | tgaaattagaaaggcgatgtttgtctgacacaccgacctc | 5 |
프라이머 4 | gctcggtacccggggatcctcggcgtcgccgttgtggacc | 6 |
프라이머 | 프라이머 서열 (5'→3') | 서열번호 |
프라이머 5 | ctatgaccatgattacgccacagtggagaacacgatcgcg | 7 |
프라이머 6 | caaaacaagtgccaatgccaacatcgcctttctaatttca | 8 |
프라이머 7 | tgaaattagaaaggcgatgttggcattggcacttgttttg | 9 |
프라이머 8 | gctcggtacccggggatcctatcgatgctcatgccagcgg | 10 |
조성 (/L) | |
시트룰린 종배지 | 포도당 2.67%, YPA 3.14%, KH2PO4 0.1%, K2HPO4 0.1%, MnSO4 10 ppm, FeSO4 10 ppm, Biotin 100 ug/L, Thiamin-HCl 200 ug/L |
5L 배양기 배지 | 포도당 8%, MgSO4 0.08%, FeSO4 16 ppm, MnSO4 8 ppm, CuSO4 1.6 ppm, ZnSO4 1.6 ppm, CaCl2 32 ppm, Biotin 160 ppb, Thiamine-HCl 8 ppm, HVP 0.4%, NaCl 0.08%, KH2PO4 0.22%, (NH4)2SO4 2.4% |
추가 배지 | 포도당 52%, MgSO4 0.1%, CaCl2 120 ppm, Biotin 160 ppb, Thiamine-HCl 16 ppm, NH4H2PO4 0.08%, (NH4)2SO4 4.4% |
균주 | L-시트룰린(%) | 6-아세틸오르니틴 (%) |
CT1 | 9.73 | 1.21 |
CT2 | 11.46 | 0.38 |
CT3 | 10.92 | 0.01 |
Claims (4)
- NCgl2657 유전자에 의해 발현되는 단백질의 활성이 약화 또는 불활성화된, L-시트룰린 생산능이 향상된 코리네박테리움 글루타미쿰(Corynebacterium glutamicum) 변이주.
- 청구항 1에 있어서,상기 NCgl2657 유전자는 서열번호 1의 염기서열로 표시되는 것인 코리네박테리움 글루타미쿰 변이주.
- 청구항 1에 있어서,상기 NCgl2657 유전자에 의해 발현되는 단백질의 활성 약화 또는 불활성화는 NCgl2657 유전자의 전부 또는 일부가 삽입, 치환 또는 결실된 것인 코리네박테리움 글루타미쿰 변이주.
- a) 청구항 1의 변이주를 배지에서 배양하는 단계; 및b) 상기 변이주 또는 변이주가 배양된 배지로부터 L-시트룰린을 회수하는 단계를 포함하는 L-시트룰린의 생산 방법.
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US18/280,846 US20240150801A1 (en) | 2021-03-17 | 2022-01-19 | Mutant of corynebacterium glutamicum with enhanced l-citrulline productivity and method for preparing l-citrulline using the same |
JP2023557193A JP2024511393A (ja) | 2021-03-17 | 2022-01-19 | L-シトルリン生産能が向上したコリネバクテリウム・グルタミカム変異株およびこれを用いたl-シトルリンの生産方法 |
EP22771585.1A EP4310189A1 (en) | 2021-03-17 | 2022-01-19 | Corynebacterium glutamicum variant having improved l-citrulline production ability, and method for producing l-citrulline by using same |
CN202280021948.8A CN116997655A (zh) | 2021-03-17 | 2022-01-19 | L-瓜氨酸生产能力得到提高的谷氨酸棒状杆菌变异株及利用其的l-瓜氨酸的生产方法 |
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KR1020210034544A KR20220129797A (ko) | 2021-03-17 | 2021-03-17 | L-시트룰린 생산능이 향상된 코리네박테리움 글루타미쿰 변이주 및 이를 이용한 l-시트룰린의 생산 방법 |
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KR101102263B1 (ko) | 2005-09-27 | 2012-01-03 | 아지노모토 가부시키가이샤 | L-아미노산 생산 세균 및 l-아미노산 생산 방법 |
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