KR102269637B1 - Mutant strain with enhanced L-citrulline or L-Arginine productivity and method for preparing L-citrulline or L-Arginine using the same - Google Patents

Abstract

The present invention relates to a mutant strain having improved L-citrulline or L-arginine production ability and a method for producing L-citrulline or L-arginine using the same, and the mutant strain according to one embodiment of the present invention comprises ornithine carbamoyltransferase and Since the activity of carbamoyl phosphate synthetase is enhanced and the production ability of L-citrulline or L-arginine is excellent, L-citrulline or L-arginine can be more effectively produced by using the mutant strain.

Description

Mutant strain with enhanced L-citrulline or L-Arginine productivity and method for preparing L-citrulline or L-Arginine using L-citrulline or L-arginine-producing ability improved mutant strain and method for producing L-citrulline or L-arginine using the same the same}

The present invention relates to a mutant strain having improved L-citrulline or L-arginine production ability and a method for producing L-citrulline or L-arginine using the same, and more particularly, ornithine carbamoyltransferase and carbamoyl Corynebacterium sp. mutant strain with enhanced L-citrulline or L-arginine production ability by enhancing the activity of carbamoyl phosphate synthetase and a method for producing L-citrulline or L-arginine using the same it's about

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. In the kidneys, citrulline is metabolized to arginine, producing nitric oxide (NO). That is, citrulline is not an amino acid constituting a protein in a living body, but as one of the intermediates of the urea cycle, it is generated from arginine together with NO, which is known as a substance having a vasodilatory action, and is further condensed with aspartic acid to be regenerated into arginine.

Arginine (arginine) is known to be contained in a free state in plants and the like. Arginine is one of the non-essential amino acids, but it is a semi-essential amino acid that must be supplied in case of special conditions such as growing children, stress, trauma, and cancer. It is widely used as a component of For medicine, it is used as a liver function enhancer, brain function enhancer, male infertility treatment, and general amino acid formulation. It is known that the concentration of plasma L-arginine sharply increases and NO bioavailability increases when L-citrulline and L-arginine are combined orally ingested.

On the other hand, biosynthesis of L-arginine in microorganisms is made through two different pathways, a linear step and a cyclic step, through eight enzymatic steps from L-glutamate. In the linear phase, L-arginine is converted from L-glutamate to N-acetylglutamate, N-acetylornithine, ornithine, citrulline, and argininosuccinate. It is synthesized through argininosuccinate. As an intermediate step in the biosynthesis of L-arginine, L-ornithine and carbamoyl phosphate are combined by ornithine carbamoyltransferase to catalyze the production of L-citrulline, and phosphate is generated as a by-product . The produced L-citrulline is finally synthesized into L-arginine by the enzyme reaction of argininosuccinic acid synthase (ArgG) and argininosuccinic acid lyase (ArgH). Among the biosynthetic steps of L-arginine, carbamoyl phosphate synthase refers to an enzyme that catalyzes the synthesis of carbamoyl phosphate from glutamine, and carbamoyl phosphate produced by carbamoyl phosphate synthase produces L-citrulline as described above. can be used to

Accordingly, the inventors of the present application prepared a mutant strain with enhanced activity of ornithine carbamoyltransferase and carbamoyl phosphate synthetase, and the mutant strain is L-citrulline or L-arginine The present invention was completed by confirming that the productivity was excellent.

Republic of Korea Patent No. 10-1138289

An object of the present invention is ornithine carbamoyltransferase (ornithine carbamoyltransferase) and carbamoyl phosphate synthetase (Carbamoyl phosphate synthetase) activity of the enhanced L-citrulline or L-arginine production ability is improved Corynebacterium genus ( Corynebacterium sp) ) to provide a mutant strain.

Another object of the present invention is

(a) culturing a mutant strain of the genus Corynebacterium in a medium; and

(b) to provide a method for producing L-citrulline or L-arginine, comprising the step of recovering L-citrulline or L-arginine from the cultured mutant strain or culture medium.

One aspect of the present invention is ornithine carbamoyltransferase (ornithine carbamoyltransferase) and carbamoyl phosphate synthetase (carbamoyl phosphate synthetase) activity of the enhanced L- citrulline or L- arginine production ability is improved Corynebacterium genus ( Corynebacterium) sp. ) provides a mutant strain.

According to one embodiment of the present invention, the mutant strain may have improved production ability of L-citrulline and L-arginine at the same time.

In the present invention, "ornithine carbamoyltransferase" refers to the reaction product of L-ornithine and carbamoyl phosphate by combining L-citrullline and phosphate. enzymes that catalyze the production of

In the present invention, "carbamoyl phosphate synthase" means an enzyme that catalyzes the synthesis of carbamoyl phosphate from glutamine or ammonia and bicarbonate. Carbamoyl phosphate can be combined with L-ornithine to synthesize L-citrulline.

In the present invention, the "improved production capacity" strain means that the productivity of L- citrulline and / or L- arginine is increased compared to the parent strain.

In the present invention, "parent strain" means a wild-type or mutated strain to be mutated, and includes a target to be directly mutated or transformed with a recombinant vector or the like. In the present invention, the parent strain may be a wild-type Corynebacterium glutamicum strain or a strain mutated from the wild-type. According to one embodiment of the present invention, the parent strain may be a Corynebacterium glutamicum ATCC13032 strain.

According to one embodiment of the present invention, the enhancement of the activity of ornithine carbamoyltransferase may be due to an increase in the expression of ornithine carbamoyltransferase ( argF) gene encoding ornithine carbamoyltransferase. According to one embodiment of the present invention, the argF gene is registered as NCgl1344 in the NCBI database. According to one embodiment of the present invention, the argF gene consists of the nucleotide sequence of SEQ ID NO: 19.

The increase in accordance with one embodiment, the cover active strengthening together phosphate synthase cover carA (carbamoyl phosphate synthase small subunit) gene and carB (carbamoyl phosphate synthase large subunit) to encrypt together phosphate synthase gene expression may be due to According to one embodiment of the present invention, the carA gene is registered as NCgl1548 in the NCBI database, and the carB gene is registered as NCgl1547 in the NCBI database. According to one embodiment of the invention the carA is consisting of a nucleotide sequence of SEQ ID NO: 20, the carB consists of a nucleotide sequence of SEQ ID NO: 21.

In the present invention, "activity enhancement" or "enhanced activity" means that the activity of a protein itself is newly introduced or increased to derive an effect beyond its original function, as well as an increase in intrinsic gene activity, from internal or external factors. Manipulations such as endogenous gene amplification, deletion of inhibitory regulatory factors for gene expression, gene copy number increase, gene introduction from outside, modification of expression control sequences, in particular, promoter replacement or modification and increase in enzymatic activity by mutations in genes It refers to a state in which the activity of the microorganism after the operation is increased compared to the activity of the microorganism before the operation is made.

According to one embodiment of the present invention, the enhancement of ornithine carbamoyltransferase and carbamoyl phosphatase activity can be achieved by applying various methods well known in the art. The method for enhancing or increasing the activity of the enzyme can be applied to various methods well known in the art. Examples of the method include, but are not limited to, a base encoding enzymes by a method of additionally inserting a polynucleotide comprising a nucleotide sequence encoding the enzymes into a chromosome or a method of introducing the polynucleotide into a vector system. A method of increasing the copy number of a sequence, a method of replacing the promoter of enzymes with a strong promoter, a method of specifically introducing a mutation into the promoter, and a method of mutating into an enzyme with strong activity by gene mutation.

According to one embodiment of the present invention, enhancing ornithine carbamoyltransferase activity may change the promoter of the enzyme present in the arginine operon to a stronger promoter than the native promoter through mutation or substitution. An improved promoter having a base substitution mutation or a heterologous promoter may be linked instead of the promoter of the endogenous enzyme, and examples of the heterologous promoter include pcj7 promoter, lysCP1 promoter, efTu promoter, groEL promoter, aceA promoter, aceB promoter, etc., but are limited thereto. it is not

In the present invention, the "promoter" includes an untranslated nucleic acid sequence upstream of the coding region that includes a binding site for polymerase and has transcription initiation activity to mRNA of a gene downstream of the promoter, that is, the polymerase binds to Refers to a DNA region that initiates transcription of a gene, and is located at the 5' site of the mRNA transcription initiation site.

According to one embodiment of the present invention, enhancing ornithine carbamoyltransferase activity may be achieved by transforming the strain with an expression vector including a polynucleotide encoding ornithine carbamoyltransferase.

According to one embodiment of the present invention, enhancing the activity of carbamoyl phosphate synthase may be achieved by transforming the strain with an expression vector including a polynucleotide encoding carbamoyl phosphate synthase.

In the present invention, "vector" refers to any medium for cloning and/or transfer of a base to a host cell. A vector may be a replicator capable of binding other DNA fragments to bring about replication of the bound fragment. "Replication unit" refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as a self-unit of DNA replication in vivo, that is, is capable of replicating under its own control. . In the present invention, the vector is not particularly limited as long as it can replicate in a host, and any vector known in the art may be used. The vector used for the construction of the recombinant vector may be a plasmid, a cosmid, a virus or a bacteriophage in a natural state or a recombinant state. For example, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ vectors, pBR-based, pUC-based plasmid vectors may be used. , pBluescript II-based, pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used. The usable vector is not particularly limited and a known expression vector may be used, but is not limited thereto.

In the present invention, "transformation" refers to introducing 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 is inserted into the chromosome of the host cell or located outside the chromosome Anything may be included without limitation. According to one embodiment of the present invention, the transformation method includes any method of introducing a nucleic acid into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, 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 and the like may be used, but is not limited thereto.

According to one embodiment of the present invention, the increase in carbamoyl phosphate synthase expression is caused by the efTu promoter or superoxide dismutase (sod) promoter of a gene cluster present in the carAB operon including the carA and carB genes. It may be carried out by being substituted with the promoter of the gene encoding it.

According to one embodiment of the present invention, the strain may be Corynebacterium glutamicum.

According to one embodiment of the present invention, the Corynebacterium sp. strain is Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Corynebacterium acetoacidophilum ( Corynebacterium acetoacidophilum ), Corynebacterium acetoglutamicum ( Corynebacterium acetoglutamicum ), Corynebacterium alkanolyticum ), Corynebacterium callunae ), Corynebacterium lilium ( Corynebacterium lilium ), Corynebacterium melassecola Coryne ), Corynebacterium thermoaminogenes ( Corynebacterium thermoaminogenes ), Corynebacterium efficiens ( Corynebacterium efficiens ), or Corynebacterium herculis ( Corynebacterium herculis ) It may be, but is not limited thereto.

According to one embodiment of the present invention, the Corynebacterium sp. strain may be Corynebacterium glutamicum ATCC13032.

Another aspect of the present invention is

(a) culturing the mutant strain of the genus Corynebacterium in a medium; and

(b) provides a method for producing L-citrulline or L-arginine, comprising the step of recovering L-citrulline or L-arginine from the cultured mutant strain or culture medium.

According to one embodiment of the present invention, the culture can be made according to an appropriate medium and culture conditions known in the art, and those skilled in the art can easily adjust the medium and culture conditions for use. Specifically, 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.

According to one embodiment of the present invention, 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 Corynebacteria 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.

According to one embodiment of the present invention, the medium may include various carbon sources, nitrogen sources and trace element components. Carbon sources that can be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, 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 can 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. In addition, the culture medium may contain a metal salt 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. In addition, 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.

According to one embodiment of the present invention, it is possible to adjust 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. In addition, the use of an antifoaming agent such as fatty acid polyglycol ester during culture can suppress the formation of bubbles. Additionally, in order to maintain the aerobic state of the culture medium, oxygen or oxygen-containing gas (eg, air) may be injected into the culture medium. 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 yield, for example, it may be 10 to 160 hours.

According to one embodiment of the present invention, the step of recovering L-citrulline or L-arginine from the cultured mutant strain and the culture medium is L- produced from the medium using a suitable method known in the art according to the culture method. Citrulline or L-arginine may be collected or recovered. e.g. centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g. ammonium sulfate precipitation), chromatography (e.g. ion exchange, affinity, hydrophobicity and size exclusion ) can be used, but is not limited thereto.

According to one embodiment of the present invention, in the step of recovering L-citrulline or L-arginine, the culture may be centrifuged at low speed to remove biomass, and the obtained supernatant may be separated through ion exchange chromatography.

According to one embodiment of the present invention, the step of recovering L-citrulline or L-arginine may include a step of purifying L-citrulline or L-arginine.

The mutant strain according to an embodiment of the present invention has excellent L-citrulline or L-arginine production ability due to enhanced activity of ornithine carbamoyltransferase and carbamoyl phosphate synthase. Citrulline or L-arginine can be prepared.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1. Confirmation of increase in L-citrulline productivity of strains with enhanced activity of ornithine carbamoyltransferase and/or carbamoyl phosphate synthase

Example 1-1. Construction of insertion vector for eftu promoter substitution

To enhance the carbamoyl phosphate synthase, an enzyme that produces carbamoyl phosphate required for the production of L-citrulline, by increasing the expression of carA and carB , these genes eftu the native promoter at the top of the operon. It was intended to be replaced with a promoter.

Genomic DNA was isolated from the Corynebacterium glutamicum ATCC13032 strain according to the manufacturer's manual using the Wizard ^® Genomic DNA Purification Kit (Promega, USA).

Primer name sequence (5'->3') SEQ ID NO: primer 1 5'- gcctggaaacctacgacgcg-3' SEQ ID NO: 1 primer 2 5'-taagtagggtattcattgcattatatg-3' SEQ ID NO: 2 primer 3 5'-tgcaatgaataccctacttagctgccaatt-3' SEQ ID NO: 3 primer 4 5'-ctttactcacgggtaaaaaatcctttcgta-3' SEQ ID NO: 4 primer 5 5' -ttttttacccgtgagtaaagacaccaccacc-3' SEQ ID NO: 5 primer 6 5'-cgtaggtttcatcagcagag-3' SEQ ID NO: 6

Tuf gene with a genomic DNA template; gene top 200bp and carA of (elongation factor Tu NCgl0480) (carbamoyl phosphate synthase small subunit; NCgl1548) and the parent gene pyrC gene; ORF (open reading frame) of (dihydroorotase NCgl1547) is The included DNA fragments 560bp and 569bp were ^{subjected to PCR using PrimeSTAR ®} Max DNA Polymerase (Takara, Japan).

PCR was performed by repeating 31 cycles under the conditions of 10 seconds at 98°C, 5 seconds at 55°C, and 1 minute at 72°C using primers 1-2, 3-4, and 5-6 sets, respectively, After electrophoresis of the obtained PCR product on a 1% agarose gel, bands having a size of 560, 220, and 569 bp, respectively, were eluted, and then PCR was performed under the same conditions using each PCR product as a template and 1-6 sets of primers. A PCR product containing carA and carB of 1309 bp size was obtained. The primer sequences used are shown in Table 1 above.

The pK19mobsacB plasmid (Gene, 145: 69-73, 1994) to be used as an insertion vector was digested with restriction enzymes Hind III and Xba I (Takara, Japan). The PCR product obtained above and the cleaved plasmid were ligated using the In-Fusion ^® HD Cloning Kit (Takara, Japan) according to the manufacturer's manual to construct an insertion vector and named it “pK19mobsacB-Peftu-carAB”. .

Example 1-2. argF Construction of overexpression vectors

Genomic DNA was isolated from the Corynebacterium glutamicum ATCC13032 strain according to the manufacturer's manual using the Wizard ^® Genomic DNA Purification Kit (Promega, USA).

SodA (NCgl2826) using the isolated genomic DNA as a template ^{PCR was performed using PrimeSTAR ®} Max DNA Polymerase (Takara, Japan) for 960 bp of the DNA fragment including the top 200 bp of the gene and the ORF of the argF gene.

Primer name sequence (5'-3') SEQ ID NO: primer 7 5'-GTCGACtagctgccaattattccgg-3', SEQ ID NO: 7 primer 8 5'-tggttgtgaagtcatatgaaaatcctttcg-3' SEQ ID NO: 8 primer 9 5'-cgaaaggattttcatatgacttcacaacca-3' SEQ ID NO: 9 primer 10 5'-GGATCCttacctcggctggttggccag-3' SEQ ID NO: 10

Using primers 7-8 and 9-10 sets, respectively, 31 cycles were repeated under the conditions of 98°C for 10 sec, 55°C for 5 sec, and 72°C for 1 min. PCR was performed, and the obtained PCR product was subjected to 1% agar After electrophoresis on a rose gel, bands having a size of 200 bp and 960 bp, respectively, were eluted to obtain a soda promoter and an argF fragment. The primer sequences used are shown in Table 2 above.

The amplified and isolated PCR product and pK19mobsacB plasmid were digested with restriction enzymes Sal I and BamHI restriction enzymes (Takara, Japan). The cleaved plasmid and PCR product were ligated using T4 ligase (Bioneer, Korea) to construct a recombinant vector for overexpressing argF, which was named “pA'-Psod-argF”.

Examples 1-3. Production of mutant strains of Corynebacterium glutamicum

Corynebacterium glutamicum ( Corynebacterium glutamicum ) pK19mobsacB-Peftu-carAB prepared in Example 1-1 and pA'-Psod-argF prepared in Example 1-2 to ATCC13032 strain (hereinafter, ATCC13032 strain) It was introduced by electroporation.

Specifically, the ATCC13032 strain was first cultured in 100 ml of RG medium (Beef extract 10 g/L, BHI 40 g/L, sorbitol 30 g/L), and 2.5 g/L glycine, 400 mg/L isoniazid, 0.1 ml in the same medium. A medium to which /L Tween80 was added was prepared. Then, the _{seed culture was inoculated so that the OD 610} value was 0.3, and then incubated at 30° C. and 180 rpm for 3 to 5 hours so that the OD ₆₁₀ value was 1.6 to 1.8. After standing on ice for 30 minutes, centrifugation was performed at 4°C and 3500 rpm for 15 minutes. Thereafter, the supernatant was discarded and the precipitated ATCC13032 strain was washed 4 times with a 10% glycerol solution, and finally resuspended in 0.5 ml of a 10% glycerol solution. Electroporation was performed using a Bio-Rad electroporator. The competent cell prepared by the above method was put into an electroporation cuvette (0.2 mm) and the pK19mobsacB-Peftu-carAB vector prepared in Example 1-1 or the pA′-Psod prepared in Example 1-2 After adding -argF, an electric shock was applied under the conditions of 2.5 kV, 200 Ω and 12.5 μF. Immediately after the electric shock, 1 ml of regeneration medium (Brain Heart infusion 18.5 g/l sorbitol 0.5 M) was added, and heat treatment was performed at 46° C. for 6 minutes. After cooling at room temperature, transfer to a 15ml cap tube, incubate at 30℃ for 2 hours, and a solid medium containing 30㎍/ml of kanamycine (Brainheart infusion 40g/L, D-sorbitol 30g/L, Beef extract 10g /L, Agar 20g/L). The primary recombinant strain was selected by culturing at 30° C. for 3 to 4 days, and after 12 hours incubation in 2YT liquid medium (Tryptone 16g/L, Yeast extract 10g/L, NaCl 5g/L), 2YT-10% sucrose solid medium (Tryptone 16g/L, Yeast extract 10g/L, NaCl 5g/L, sucrose 100g/L) to induce secondary recombination. Colonies cultured in 2YT-10% sucrose solid medium were patched in 2YT-Km medium (Tryptone 16g/L, Yeast extract 10g/L, NaCl 5g/L, kanamycin 30㎍/㎖ and 2YT-10%sucrose) by patching. A strain that has resistance to kanamycin and can grow in 10% sucrose medium was finally selected.

The ATCC13032 strain used as the parent strain was named "CIT 1" strain. By confirming whether Peftu-carAB was normally inserted into the chromosome of the recombinant strain through PCR and sequencing, it was confirmed whether the native promoter at the top, in which the carA and carB genes were present as operons, was replaced with the eftu promoter, and this 2" strain.

In addition, in order to overexpress argF, a strain transformed with the pA'-Psod-argF vector was selected and named "CIT-1/pA'-Psod-argF" strain.

In order to overexpress argF in the CIT-2 strain, the CIT-2 strain was transformed in the same manner as above using the pA'-Psod-argF vector, and this was transformed into a "CIT-2/pA'-Psod-argF" strain. named.

Examples 1-4. Analysis of argF, carA, and carB expression levels in a mutant strain of Corynebacterium glutamicum

Corynebacterium glutamicum ATCC13032 (hereinafter, CIT-1) as the parent strain, CIT-2, CIT-1/pA'-Psod-argF and CIT-2/pA'- For the Psod-argF strain, RNA expression levels of argF , carA, and carB were analyzed by RT-qPCR.

CIT-1, CIT-2, CIT-1/pA'-Psod-argF or CIT-2/pA'-Psod-argF strains were each prepared in a flask of 10 ml of seed medium ((/L): 95% Glucose 10.5 g, Beef extract 10g, yeast extract 10g, Polypeptone 10g, NaCl 2.5g, Arginine 100mg) was cultured at 30°C and 160rpm for 24 hours. Each strain cultured in a flask of 50 ml titer medium ((/L): 95% Glucose 105.3 _{g, MgSO 4} 1 g, YPA 4 g, KH ₂ PO ₄ 0.8 g, Na ₂ HPO ₄ 1.2 g, (NH ₄ ) ₂ SO ₄ 30g, FeSO ₄ 20mg, MnSO ₄ 20mg, ZnSO ₄ 10mg, Arginine 100mg, Biotin 10㎍, Thiamine 200㎍) was inoculated and cultured at 30℃, 140rpm, for 20 hours.

RNA was extracted according to the instruction manual using the Rneasy Mini Kit (QIAGEN, Germany) by collecting the culture termination solution at the same concentration.

After synthesizing cDNA with 500 ng of RNA as a template using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, USA), ^{real-time quantitative analysis using iQ TM} SYBR Green Supermix (Bio-Rad Laboratories, Hercules, USA) PCR (RT-qPCR; CFX96 Real-Time PCR, Bio-Rad, USA) was performed. After pre-denaturation of cDNA at 95°C for 1 minute, 40 cycles were repeated while changing the temperature and time to 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 45 seconds. Information was collected at 72°C, and the temperature was increased from 65°C to 95°C by 0.5°C to confirm the melt curve of the primer used, and information was collected whenever the temperature was changed. All of the primers were selected and used to show an efficiency of 95 to 100%. The expression of argF, carA, and carB was normalized using the 16s rRNA gene as an endogenous control, and the results are shown in Table 3. In Table 3, the expression level of car AB represents the average value of the expression level of carA and the expression level of carB. The primers used to amplify the gene are shown in Table 4 below.

Flask Relative carAB expression level Relative argF expression level CIT-1 1.00 1.00 CIT-2 4.51 1.22 CIT-1/pA’-Psod-argF 0.96 15.05 CIT-2/pA’-Psod-argF 5.12 17.84

As shown in Table 3, in CIT-2, a strain in which the upper native promoter in which the carA and carB genes exist as operons is substituted with the eftu promoter, carAB expression levels ( average) was significantly increased by 4.5 times in the strain overexpressing the argF CIT-1 / pA'-Psod- argF was the expression of argF prepare CIT-1 significantly increased by 15 times, the carA and carB genes operon In the CIT-2/pA'-Psod-argF strain in which the existing upper native promoter was substituted with the eftu promoter and argF was overexpressed, it was confirmed that the expression of carAB increased by 5.12 times compared to CIT-1 and the expression amount of argF increased by 17.84 times. did. Therefore, since it was confirmed that the expression levels of carAB and argF were increased by performing RT-qPCR, the strains generated in Examples 1-1 and 1-2 were obtained by increasing carA, carB gene expression and/or argF gene expression. It can be seen that the activity of carbamoyl phosphate synthase and/or ornithine carbamoyltransferase is enhanced.

Primer name sequence (5'->3') SEQ ID NO: carA Forward 5'-acgtaccttcaccggatttg-3' SEQ ID NO: 11 Reverse 5'-gaaggatcggtcatggtttct-3' SEQ ID NO: 12 carB Forward 5'-gccggaatacatcgacaaga-3' SEQ ID NO: 13 Reverse 5'-agctggatagctgcgttaag-3' SEQ ID NO: 14 argF Forward 5'-cttcaactcgtactcgcttctc-3' SEQ ID NO: 15 Reverse 5'-actcgcccttacccatct-3' SEQ ID NO: 16 16s rRNA Forward 5'-acccttgtcttatgttgccag-3' SEQ ID NO: 17 Reverse 5'-tgtaccgaccattgtagcatg-3' SEQ ID NO: 18

Examples 1-5. Confirmation of L-citrulline productivity of a mutant strain of Corynebacterium glutamicum.

As the parent strain, Corynebacterium glutamicum ATCC13032 (hereinafter, CIT-1), CIT-2 prepared in Examples 1-3, or CIT-1/pA'-Psod-argF strains were used in flask seed medium solid medium, respectively. ((/L): 95% Glucose 10.5g, Beef extract 10g, Yeast extract 10g, Polypeptone 10g, NaCl 2.5g, Arginine 100mg) was patched and cultured at 30° C. for 24 hours. The CIT-2/pA'-Psod-argF strain was patched into a flask seed medium containing 30 mg/ml of kanamycin and cultured at 30° C. for 24 hours. Cultured colonies in a flask of 10 ml titer medium ((/L): 95% Glucose 105.3 _{g, MgSO 4} 1 g, YPA 4 g, KH ₂ PO ₄ 0.8 g, Na ₂ HPO ₄ 1.2 g, (NH ₄ ) ₂ SO ₄ 30g, FeSO ₄ 20mg, MnSO ₄ 20mg, ZnSO ₄ 10mg, Arginine 100mg, Biotin 100㎍, Thiamine 200㎍) was inoculated and cultured at 30℃, 160rpm, for 30 hours.

After completion of the culture, the culture solution in the culture solution was diluted 100 times with distilled water and filtered through a 0.45um filter. Then, high-performance liquid chromatography (HPLC; agilent technologies 1260 infinity, agilent technologies, USA) was used to analyze L-citrulline production. The L-citrulline production amount produced by the CIT-1, CIT-2, CIT-1/pA'-Psod-argF or CIT-2/pA'-Psod-argF strains is shown in Table 5 below. In Table 5, CIT (%) means the percentage of the production of citrulline produced by each strain, and the fermentation yield (Yp/s) indicates the amount of L-citrulline produced relative to the consumed sugar.

strain CIT (%) Fermentation yield (Yp/s) CIT-1 1.28 28.3 CIT-2 1.38 32.2 CIT-1/pA’-Psod-argF 1.38 33.2 CIT-2/pA’-Psod-argF 1.62 38.7

As shown in Table 5, L-citrulline production of CIT-1/pA'-Psod-argF with increased expression of CIT-2 and argF with increased expression of carA and carB compared to the parent strain CIT-1 ( %) and it can be seen that the fermentation yield increased.

On the other hand, in the CIT-2/pA'-Psod-argF strain with increased expression of argF, carA and carB, the L-citrulline production (%) and fermentation yield compared to CIT-2 and CIT-1/pA'-Psod-argF. was increased, and from this, it can be seen that the production of L-citrulline synergistically increased due to the increase in the expression of carA, carB gene, and the expression of argF gene.

As described above, L-citrulline is a precursor of L-arginine, and in the strain having improved L-citrulline production ability, it can be reasonably expected that the production ability of L-arginine is also improved. Since the mutant strain of the above example increased the production of L-citrulline than the parent strain, an improvement in the production ability of L-arginine can also be expected.

Example 2. Confirmation of increased L-arginine productivity of strains with enhanced activity of ornithine carbamoyltransferase and/or carbamoyl phosphate synthase

Example 2-1. Construction of insertion vector for sodA promoter substitution

To increase the expression of carA and carB genes expressing carbamoyl phosphate synthase, the native promoter was replaced with the sodA promoter (promoter of the gene encoding superoxide dismutase; sod). In the same manner as in Example 1-1, except for using the upper 200 bp of the sodA gene instead of the upper 200 bp of the tuf gene and obtaining a PCR product using the primers 1-11, 3-12, and 5-13 sets of Table 6 A vector was constructed. The prepared insertion vector was named “pK19mobsacB-Psod-carAB”.

Primer name sequence (5'->3') SEQ ID NO: primer 1 5'- gcctggaaacctacgacgcg-3' SEQ ID NO: 1 primer 11 5'-catataatgcaatgaataccctactta-3' SEQ ID NO: 22 (complementary sequence of SEQ ID NO: 2) primer 3 5'-tgcaatgaataccctacttagctgccaatt-3' SEQ ID NO: 3 primer 12 5'-tacgaaaggattttttacccgtgagtaaag-3' SEQ ID NO:23
(Complementary sequence of SEQ ID NO: 4) primer 5 5' -ttttttacccgtgagtaaagacaccaccacc-3' SEQ ID NO: 5 primer 13 5'-ctctgctgatgaaacctacg -3' SEQ ID NO: 24
(Complementary sequence of SEQ ID NO: 6)

Example 2-2. argF Construction of overexpression vectors

An argF overexpression vector linked to the soda promoter was prepared in the same manner as in Example 1-2, and was named “pA'-Psod-argF”.

Example 2-3. Production of mutant strains of Corynebacterium glutamicum

Corynebacterium glutamicum ( Corynebacterium glutamicum ) pK19mobsacB-Psod-carAB prepared in Example 2-1 and pA'-Psod-argF prepared in Example 2-2 to ATCC13032 strain (hereinafter, ATCC13032 strain) It was introduced by electroporation. The method and medium for use and selection for mutant strain production are the same as in Examples 1-3.

The ATCC13032 strain used as the parent strain was named "AXR-5" strain. By confirming whether Psod-carAB was normally inserted into the chromosome of the recombinant strain through PCR and sequencing, it was confirmed whether the native promoter at the top of which carA and carB genes were present as operons was replaced with the sodA promoter, and this It was designated as a 6" strain.

In addition, a strain transformed with the parent strain AXR-5 with the pA'-Psod-argF vector was selected and named "AXR-5/pA'-Psod-argF" strain. The strain transformed by the same method using the pA'-Psod-argF vector to the AXR-6 strain was named "AXR-6/pA'-Psod-argF" strain.

Example 2-4. Analysis of argF, carA, and carB expression levels in a mutant strain of Corynebacterium glutamicum

Corynebacterium glutamicum ATCC13032 (hereinafter, AXR-5) as a parent strain, AXR-6, AXR-5/pA'-Psod-argF and AXR-6/pA'- For the Psod-argF strain, RNA expression levels of argF , carA, and carB were analyzed by RT-qPCR.

AXR-5, AXR-6, AXR-5/pA'-Psod-argF, and AXR-6/pA'-Psod-argF strains were each prepared in a flask of 10 ml of seed medium ((/L): 95% Glucose 36.8 g, MgSO ₄ 1 g, FeSO ₄ 10 mg, MnSO ₄ 10 mg, KH ₂ PO ₄ 1 g, K ₂ HPO ₄ 1 g, YSP 20 g, Urea 1.5 g, (NH ₄ )SO ₄ 5 g, Biotin 100 μg, Thiamine 100 μg, CPN 100 mg, Cysteine 50mg) incubated for 20 hours at 30°C and 160rpm. Each strain cultured in a flask of 50 ml titer medium ((/L): 95% Glucose 52.6g, Sucrose 50g, MgSO ₄ 1g, KH ₂ PO ₄ 2g, (NH ₄ ) ₂ SO ₄ 40g, FeSO ₄ 20mg, MnSO ₄ 20mg, Biotin 100ug, Thiamine 100ug, YSP 4g, Urea 2g, Arginine 40g) was inoculated and cultured at 30℃, 140rpm, for 20 hours.

RNA was extracted according to the instruction manual using the Rneasy Mini Kit (QIAGEN, Germany) by collecting the culture termination solution at the same concentration. cDNA production and RT-qPCR are the same as in Examples 1-4.

The expression of argF, carA, and carB was normalized using the 16s rRNA gene as an endogenous control, and the results are shown in Table 7. In Table 7, the expression level of car AB represents the average value of the expression level of carA and carB. The primers used to amplify the gene are the same as in Table 4 above.

Flask Relative value of carAB expression level Relative value of argF expression level AXR-5 1.00 1.00 AXR-6 3.62 0.99 AXR-5/pA’-Psod-argF 1.10 20.13 AXR-6/pA’-Psod-argF 4.68 15.22

As shown in Table 7, in AXR-6, a strain in which the upper native promoter in which the carA and carB genes are present as an operon is substituted with the sodA promoter, the carAB expression level (carA and carB expression levels of the parent strain AXR-5) avg) was significantly increased by the 3.62-fold, was argF is the expression of the overexpressed strain AXR-5 / pA'-Psod- argF is compared AXR argF-5 significantly increased by 20.13 times, the carA and carB genes operon In the AXR-6/pA'-Psod-argF strain in which the existing upper native promoter was substituted with the sodA promoter and argF was overexpressed, it was confirmed that the expression of carAB increased by 4.68 times compared to AXR-5, and the expression amount of argF increased by 15.22 times. did. Therefore, since it was confirmed that the expression levels of carAB and argF were increased by performing RT-qPCR, the strains prepared in Examples 2-1 to 2-3 showed an increase in carA, carB gene expression and/or argF gene expression. It can be seen that the activity of carbamoyl phosphate synthetase and/or ornithine carbamoyltransferase is enhanced.

Example 2-5. Confirmation of L-arginine productivity of mutant strains of Corynebacterium glutamicum

Corynebacterium glutamicum ATCC13032 (hereinafter, AXR-5) as a parent strain, AXR-6, AXR-5/pA'-Psod-argF, and AXR-6/pA'- Each of the Psod-argF strains was treated with a flask seed medium solid medium ((/L): 95% Glucose 36.8 _{g, MgSO 4} 1 g, FeSO ₄ 10 mg, MnSO ₄ 10 mg, KH ₂ PO ₄ 1 g, K ₂ HPO ₄ 1 g, YSP 20 g, Urea 1.5g, (NH ₄ )SO ₄ 5g, Biotin 100μg, Thiamine 100μg, CPN 100mg, Cysteine 50mg) was patched (patching) and incubated at 30 ℃ for 24 hours. Cultured colonies in a flask of 10 ml titer medium ((/L): 95% Glucose 52.6 g, Sucrose 50 g, MgSO ₄ 1 g, KH ₂ PO ₄ 2 g, (NH ₄ ) ₂ SO ₄ 40 g, FeSO ₄ 20 mg, MnSO ₄ 20mg, Biotin 100μg, Thiamine 100μg, YSP 4g, Urea 2g, Arginine 40g) and incubated at 30℃ 160rpm, 30 hours.

After completion of the culture, the culture solution in the culture solution was diluted 100 times with distilled water, filtered through a 0.45 μm filter, and then high-performance liquid chromatography (HPLC; agilent technologies 1260 infinity, agilent technologies, USA) was used to analyze L-arginine production.

Table 8 below shows the L-citrulline production produced by the AXR-5, AXR-6, AXR-5/pA'-Psod-argF, and AXR-6/pA'-Psod-argF strains. In the table, ARG (%) means the percentage of the production of arginine produced by each strain, and the fermentation yield (Yp/s) indicates the amount of L-arginine produced relative to the consumed sugar.

strain ARG (%) Yield (Yp/s) AXR-5 0.82 16.36 AXR-6 0.90 17.99 AXR-5/pA’-Psod-argF 0.95 18.55 AXR-6/pA’-Psod-argF 1.25 20.23

As shown in Table 8, L-arginine production of AXR-5/pA'-Psod-argF with increased expression of AXR-6 and argF with increased expression of carA and carB compared to parent strain AXR-5 ( %) and it can be seen that the fermentation yield increased.

On the other hand, in the AXR-6/pA'-Psod-argF strain with increased expression levels of argF, carA and carB, L-arginine production (%) and fermentation yield compared to AXR-5 and AXR-5/pA'-Psod-argF was increased, and from this, it can be seen that the production of L-arginine was synergistically increased by the increase in the expression of the carA, carB gene and the expression of the argF gene.

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aacatggcca actcctacat gattggcttt 540 gccaccgcgg gcatggatat ttccatcatc gctcctgaag ggttccagcc tcgtgcggaa 600 ttcgtggagc gcgcggaaaa gcgtggccag gaaaccggcg cgaaggttgt tgtcaccgac 660 agcctcgacg aggttgccgg cgccgatgtt gtcatcaccg atacctgggt atccatgggt 720 atggaaaacg acggcatcga tcgcaccaca cctttcgttc cttaccaggt caacgatgag 780 gtcatggcga aagctaacga cggcgccatc ttcctgcact gccttcctgc ctaccgtggc 840 aaagaagtgg cagcctccgt gattgatgga ccagcgtcca aagttttcga tgaagcagaa 900 aaccgcctcc acgctcagaa agcactgctg gtgtggctgc tggccaacca gccgaggtaa 960 960 <210> 20 <211> 1182 <212> DNA <213> Corynebacterium glutamicum <400> 20 gtgagtaaag acaccaccac ctaccaggga gtcaccgaga tcggatccgt tccggcatac 60 ctggttcttg cagacggacg taccttcacc ggatttggct ttggagctat cggcaccacc 120 cttggtgagg cagtgttcac taccgccatg accggttacc aagaaaccat gaccgatcct 180 tcctatcacc gccagattgt tgtggctacc gcaccacaga tcggcaacac cggctggaac 240 gatgaggaca acgagtcccg cgacggcaag atttgggttg caggccttgt tatccgcgac 300 ctcgcagcac gtgtgtccaa ctggcgcgcc accacctcct tgcagcagga aatggcaggc 360 cagggcatcg tcggcatcgg cggaatcgac acccgcgcac tggttcgcca cctgcgcaat 420 gaaggttcca ttgcagcggg catcttctcc ggcgctgacg cacagcgccc agttgaagaa 480 ctcgtagaga tcgtcaagaa tcagccagca atgaccggcg caaacctctc cgttgaggtc 540 tctgctgatg aaacctacgt catcgaagct gaaggcgaag agcgccacac cgtcgtggcc 600 tacgacctgg gcattaagca aaacacccca cgtcgtttct ctgcacgcgg tgttcgcacc 660 gtcatcgtgc ctgctgaaac cccattcgag gatatcaagc agtacaaccc atcaggcgtg 720 ttcatctcca acggccctgg cgatcctgca gcagcagacg tcatggttga tatcgtccgc 780 gaagttcttg aagccgacat tccattcttt ggcatctgct tcggcaacca gattcttggc 840 cgcgcattcg gcatggagac ctacaagctg aagttcggcc accgcggcat caacgttcca 900 gtgaagaacc acatcaccgg caagatcgac atcaccgccc agaaccacgg cttcgcactc 960 aagggtgaag caggccagga attcgagacc gatttcggca ctgcaattgt cacccacacc 1020 tgcctcaacg acggcgtcgt tgaaggtatt gcgctgaagt ccggacgcgc atactccgtt 1080 cagtaccacc cagaggccgc tgccggccca aatgatgcaa gccccctgtt tgaccagttt 1140 gttgagctga tggatgcaga cgctcagaag aaaggcgcat aa 1182 <210> 21 <211> 3342 <212> DNA <213> Corynebacterium glutamicum <400> 21 atgccaaagc gttcagatat taaccacgtc ctcgtcatcg gttccggccc catcgtcatt 60 ggccaggcat gtgaattcga ctactccggc acccaggctt gccgcgtgct gaaggaagag 120 ggactgcgcg tcaccctcat caactccaac ccagcaacga tcatgaccga cccagaaatg 180 gctgaccaca cctacgtgga gccaatcgag ccggaataca tcgacaagat tttcgctaag 240 gaaatcgagc agggccaccc aatcgacgcc gtcctggcaa cccttggtgg ccagactgca 300 cttaacgcag ctatccagct ggatcgcctc ggcatcctgg aaaagtacgg cgttgaactc 360 atcggtgcag acatcgatgc cattgagcgc ggcgaagatc gccagaagtt caaggatatt 420 gtcaccacca tcggtggcga atccgcgcgt tcccgcgtct gccacaacat ggaagaagtc 480 cacgagactg tcgcagaact cggccttcca gtagtcgtgc gtccatcctt cactatgggt 540 ggcctgggct ccggtcttgc atacaacacc gaagaccttg agcgcatcgc tggtggcgga 600 cttgctgcat ctcctgaagc aaacgtcttg atcgaagaat ccatccttgg ttggaaggaa 660 ttcgagctcg agctcatgcg cgataccgca gacaacgttg tggttatctg ctccattgaa 720 aacgtcgacg cactgggcgt gcacaccggc gactctgtca ccgtggcacc tgccctgacc 780 ctgactgacc gtgaattcca 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gactccgtac cactactccg catacgagct ggatccagca 1680 gctgagtctg aggtcgcacc acagactgag cgtgaaaagg tcctgatctt gggctccggt 1740 ccaaaccgca tcggccaggg catcgagttc gactactcct gtgttcacgc agctcttgag 1800 ctctcccgcg tcggctacga aactgtcatg gtcaactgca acccagagac cgtgtccacc 1860 gactacgaca ccgctgaccg cctgtacttc gagccactga ccttcgaaga cgtcatggag 1920 gtctaccacg ctgaggcgca gtccggcacc gtcgcaggtg ttatcgtcca gcttggtggc 1980 cagactcctc tgggcttggc agatcgtttg aagaaggctg gcgtccctgt cattggtacc 2040 tccccagagg caatcgacat ggctgaggac cgtggcgagt tcggtgcact gctgaaccgc 2100 gagcagcttc ctgctccagc attcggcacc gcaacctctt tcgaagaggc tcgcacagta 2160 gccgatgaga tcagctaccc agtgctggtt cgcccttcct acgtcttggg tggccgtggc 2220 atggagattg tctacgatga ggcttccctc gaggattaca tcaaccgcgc aactgagttg 2280 tcttctgacc acccagtgct ggttgaccgc ttcctagaca acgctattga gatcgacgtc 2340 gacgcactgt gcgacggcga cgaggtctac ctggcaggcg tcatggagca catcgaggaa 2400 gccggcattc actccggtga ctccgcatgt gcacttcctc caatgacttt gggcgcacag 2460 gacatcgaga aggtccgcga agcaaccaag aagctggctc tgggcatcgg tgtacagggc 2520 ctgatgaacg tccagtacgc actcaaggac gacatcctct acgtcatcga ggcaaaccca 2580 cgtgcatccc gcaccgtgcc gttcgtctcc aaggcaacgg gcgtcaacct ggccaaggca 2640 gcatcccgta tcgcagtggg cgccaccatc aaggatctcc aagatgaggg catgattcct 2700 accgagtacg acggcggctc cttgccactg gacgctccaa tcgctgtgaa ggaagcagtg 2760 ttgccgttca accgcttccg tcgcccagat ggaaagaccc tggacaccct gctttcccca 2820 gagatgaagt ccactggcga ggtcatgggc ttggccaaca acttcggcgc tgcatatgca 2880 aaggctgaag ctggcgcgtt tggtgcattg ccaaccgaag gcaccgtctt cgtgaccgtg 2940 gctaaccgcg acaagcgcac cctgatcctg ccaatccagc gcctggcgtt gatgggctac 3000 aagatcctcg ccaccgaagg caccgcaggc atgctgcgcc gcaacggcat tgagtgtgaa 3060 gttgtgctca aggcttccga catccgcgaa ggtgtagagg gcaagtccat cgtggatcgt 3120 atccgcgaag gcgaagttga cctcatcctc aacaccccag ctggttctgc tggcgctcgc 3180 cacgatggct acgatatccg cgcagcagca gtgaccgtgg gtgttccgct gatcaccact 3240 gttcagggtg tcaccgcagc tgtccagggc atagaggccc tgcgtgaggg cgttgtcagc 3300 gtccgcgcgc tgcaggaact cgaccacgca gtcaaggctt aa 3342 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 11 <400> 22 catataatgc aatgaatacc ctactta 27 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 12 <400> 23 tacgaaagga ttttttaccc gtgagtaaag 30 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 13 <400> 24 ctctgctgat gaaacctacg 20

Claims (5)

argF (ornithine carbamoyltransferase), carA (carbamoyl phosphate synthase small subunit) and carB (carbamoyl phosphate synthase large subunit) gene expression is increased, so that ornithine carbamoyltransferase Corynebacterium sp. mutant strain with improved activity of L-citrulline or L-arginine with enhanced activity.
The mutant strain of the genus Corynebacterium according to claim 1, wherein the increase in expression of the argF gene is due to introduction of an overexpression vector.
The method according to claim 1, wherein the increase in carA and carB gene expression is increased by a promoter replacement, Corynebacterium mutant strain.
The mutant strain of any one of claims 1 to 3, wherein the strain is Corynebacterium glutamicum .
(a) culturing the mutant strain of the genus Corynebacterium according to any one of claims 1 to 3 in a medium; and
(b) a method for producing L-citrulline or L-arginine, comprising the step of recovering L-citrulline or L-arginine from the cultured mutant strain or culture medium.