Corynebacterium glutamicum mutant strain with improved L-citrulline production ability and method for producing L-citrulline using the same

Abstract

The present invention relates to a Corynebacterium glutamicum mutant strain having improved L-citrulline production ability and a method for producing L-citrulline using the same. The Corynebacterium glutamicum mutant strain is capable of producing L-citrulline at a high yield and concentration by inhibiting the production of by-products through weakening or inactivating the activity of a protein expressed by the NCgl2657 gene.

Description

[Technical Field]

[0001] The present invention relates to a Corynebacterium glutamicum mutant strain having improved L-citrulline-producing ability and a method for producing L-citrulline using the same. [Background technology]

[0002] L-Citrulline is a non-essential amino acid known for its beneficial effects, including promoting ammonia metabolism, improving blood flow by dilating blood vessels, lowering blood pressure, promoting neurotransmission, enhancing immunity, and scavenging active oxygen species. Citrulline is synthesized as an intermediate product in the biosynthesis of arginine. Arginine biosynthesis in microorganisms involves eight enzymatic steps from L-glutamate via two distinct pathways: the linear and cyclic phases. In the linear phase, L-arginine is synthesized from L-glutamate via N-acetylglutamate, N-acetylornithine, ornithine, citrulline, and argininosuccinate. L-citrulline is typically produced by fermentation using microorganisms such as bacteria or yeast. This can be achieved using wild-type strains obtained naturally or mutant strains modified to enhance L-citrulline production. Recently, to improve L-citrulline production efficiency, genetic engineering has been applied to microorganisms such as Escherichia coli and Corynebacterium, which are commonly used to produce L-amino acids and other useful substances, to develop various recombinant strains or mutants with excellent L-citrulline production capabilities, as well as methods for producing L-citrulline using these strains. Korean Patent Nos. 10-1102263 and 10-1053429 confirmed that L-citrulline production can be improved by enhancing or attenuating the activity or expression of proteins, such as enzymes and transcription factors, involved in L-citrulline production. Therefore, it is expected that L-citrulline production can also be regulated by regulating the activity or expression of various proteins, including enzymes, that act on the L-citrulline production pathway. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Korean Patent No. 10-1102263 [Patent Document 2] Korean Patent No. 10-1053429 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a Corynebacterium glutamicum mutant strain with improved L-citrulline productivity.

[0005] Another object of the present invention is to provide a method for producing L-citrulline using the mutant strain. [Means for solving the problem]

[0006] The present inventors conducted research to develop a new mutant strain with improved L-citrulline production ability using a Corynebacterium glutamicum strain. When a mutation was introduced into the NCgl2657 gene, which encodes an enzyme involved in the production of 6-acetylornithine, a by-product produced in the L-citrulline biosynthetic pathway, or when the NCgl2657 gene was deleted, the activity of the enzyme was weakened or inactivated, resulting in a significant decrease in the amount of 6-acetylornithine produced, but also in an increase in the amount of L-citrulline produced, thereby completing the present invention.

[0007] One aspect of the present invention provides a Corynebacterium glutamicum mutant strain having improved L-citrulline-producing ability, in which the activity of a protein expressed by the NCgl2657 gene is weakened or inactivated.

[0008] The "NCgl2657 gene" used in the present invention refers to a gene encoding an enzyme (phosphate acetyltransferase) that produces acetyl phosphate, a precursor of acetylornithine, using acetyl-CoA and phosphate as substrates in the L-citrulline biosynthesis pathway.

[0009] According to one embodiment of the present invention, the NCgl2657 gene may be derived from a strain of the genus Corynebacterium. Specifically, the Corynebacterium strains include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uteri ... Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum striatum, Corynebacterium canis, Corynebacterium ammoniagenesThe bacterium may be, but is not limited to, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, or Corynebacterium flavescens.

[0010] According to one embodiment of the present invention, the NCgl2657 gene may be represented by the base sequence of SEQ ID NO:1.

[0011] As used herein, "attenuated activity" means that the expression level of a gene of interest is reduced compared to its original expression level. Such attenuation of activity includes cases where the activity of the protein itself is reduced compared to the activity of the protein originally possessed by the microorganism due to nucleotide substitution, insertion, deletion, or a combination thereof that encodes the gene, and cases where the overall enzyme activity in the cell is lower than that of a wild-type strain or a strain before transformation due to inhibition of expression or translation of the gene encoding the protein, etc., and combinations thereof.

[0012] As used herein, "inactivated" means that the expression of a gene encoding a protein such as an enzyme, transcription factor, or transport protein is completely absent compared to a wild-type strain or a strain before transformation, or that the gene is expressed but has no activity.

[0013] According to one embodiment of the present invention, the attenuation or inactivation of the activity of the protein expressed by the NCgl2657 gene may be due to the insertion, substitution, deletion, or a combination thereof of all or part of the NCgl2657 gene.

[0014] As used herein, the term "part" does not mean the entire base sequence or polynucleotide sequence, and may be, but is not limited to, 1 to 300, preferably 1 to 100, and more preferably 1 to 50.

[0015] According to one embodiment of the present invention, the attenuation or inactivation of the activity of the protein expressed by the NCgl2657 gene may be achieved by inducing a mutation in the start codon of the NCgl2657 gene.

[0016] More specifically, the start codon mutation of the NCgl2657 gene may be a substitution of the start codon ATG of the NCgl2657 gene with another codon, for example, TTG or GTG.

[0017] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant strain having a new start codon for the NCgl2657 gene was obtained by substituting the start codon ATG with TTG in the nucleotide sequence of SEQ ID NO: 1 encoding the NCgl2657 gene of the Corynebacterium glutamicum strain. Such a Corynebacterium glutamicum mutant strain may contain the NCgl2657 gene encoded by the nucleotide sequence of SEQ ID NO: 2.

[0018] According to one embodiment of the present invention, the attenuation or inactivation of the activity of the protein expressed by the NCgl2657 gene may be achieved by removing or deleting the NCgl2657 gene.

[0019] According to one embodiment of the present invention, the NCgl2657 gene of a Corynebacterium glutamicum strain was deleted to obtain a Corynebacterium glutamicum mutant strain that not only exhibits significantly reduced production of the by-product acetylornithine but also has improved L-citrulline production ability compared to a wild-type Corynebacterium glutamicum strain or a Corynebacterium glutamicum mutant strain that has already been developed to overproduce citrulline.

[0020] As used herein, "improved production ability" means increased L-citrulline productivity compared to a parent strain. The parent strain refers to a wild-type or mutant strain that can be mutated, including those that can be directly mutated or transformed with a recombinant vector. In the present invention, the parent strain may be a wild-type Corynebacterium glutamicum strain or a strain mutated from the wild-type.

[0021] According to one embodiment of the present invention, the parent strain may be Corynebacterium glutamicum ATCC13032.

[0022] According to one embodiment of the present invention, the Corynebacterium glutamicum mutant strain with improved L-citrulline production ability exhibits increased L-citrulline production ability compared to the parent strain by introducing a mutation into the start codon of the NCgl2657 gene or by deleting the NCgl2657 gene. In particular, the L-citrulline concentration is increased by 5% or more, specifically 5 to 50%, more specifically 10 to 30%, compared to the parent strain or a previously developed Corynebacterium glutamicum mutant strain, and the amount of 6-acetylornithine produced as a by-product is reduced by 20% or more, specifically 20 to 100%, more specifically 30 to 100%.

[0023] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant strain can be produced by a recombinant vector containing a mutant in which the initiation codon of the NCgl2657 gene in the parent strain is mutated, or a recombinant vector in which the NCgl2657 gene is deleted.

[0024] The term "mutant" as used herein refers to a gene mutant in which the start codon of the NCgl2657 gene involved in the biosynthesis of L-citrulline is substituted from ATG to TTG.

[0025] As used herein, the term "vector" refers to an expression vector capable of expressing a target protein in a suitable host cell, and refers to a gene construct containing the necessary regulatory elements operably linked to allow expression of a gene insert. Here, "operably linked" means that the gene to be expressed and its regulatory sequences are functionally linked to each other in a manner that allows expression of the gene. "Regulatory elements" include a promoter for transcription, an optional operator sequence for transcription control, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence for controlling the termination of transcription and translation. Examples of such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors.

[0026] The "recombinant vector" used in the present invention can replicate independently of the genome of a suitable host cell after being transformed into the host cell, or can be sewn into the genome itself. In this case, the "suitable host cell" is one in which the vector can replicate and which contains an origin of replication, which is a specific base sequence from which replication is initiated.

[0027] As used herein, "transformation" refers to the introduction of a gene into a host cell so that it can be expressed within the host cell. The transformed gene may be either intrachromosomally or extrachromosomally located, as long as it can be expressed in the host cell. According to one embodiment of the present invention, the transformation method may include any method for introducing nucleic acid into a cell, and may be performed using a suitable standard technique known in the art depending on the host cell. Examples of suitable methods include, but are not limited to, electroporation, calcium phosphate (CaPO) precipitation, calcium chloride (CaCl) precipitation, microinjection, polyethylene glycol (PEG) precipitation, DEAE-dextran precipitation, cationic liposome precipitation, and lithium acetate-DMSO precipitation. According to one embodiment of the present invention, electroporation (van der Rest et al., Appl. Microbiol. Biotechnol., 52, 541-545, 1999) may be used as the transformation method.

[0028] The host cell includes cells transfected, transformed, or infected in vivo or in vitro with a recombinant vector or polynucleotide of the invention. A host cell containing a recombinant vector of the invention may be a recombinant host cell, recombinant cell, or recombinant microorganism.

[0029] Furthermore, the recombinant vector of the present invention may contain a selection marker, which is used to select transformants (host cells) transformed with the vector, and only cells expressing the selection marker can survive in a medium treated with the selection marker, allowing for the selection of transformed cells. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, and chloramphenicol.

[0030] The gene inserted into the recombinant vector for transformation of the present invention is transposed into host cells such as microorganisms such as Corynebacterium sp. strains and Escherichia coli by homologous crossover.

[0031] According to one embodiment of the present invention, the host cell may be a strain of the genus Corynebacterium, for example a strain of Corynebacterium glutamicum.

[0032] Another aspect of the present invention provides a method for producing L-citrulline, comprising: a) culturing the Corynebacterium glutamicum mutant strain in a medium; and b) recovering L-citrulline from the mutant strain or the medium in which the mutant strain has been cultured.

[0033] The culture may be performed using an appropriate medium 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, but is not limited to, a liquid medium. The culture method may include, but is not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0034] According to one embodiment of the present invention, the medium should be appropriately adapted to meet the requirements of a specific strain and can be modified by a person skilled in the art. Culture media for Corynebacterium strains can be found in known literature (Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington DC, USA, 1981), but are not limited thereto.

[0035] According to one embodiment of the present invention, the medium can contain a variety of carbon sources, nitrogen sources, and trace element components. Usable carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used individually or in mixtures, but are not limited to these. Usable nitrogen sources include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean malt, 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 mixtures, but are not limited to these. Usable phosphorus sources can include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium may also contain, but is not limited to, metal salts necessary for growth, such as magnesium sulfate or iron sulfate. Other essential growth substances, such as amino acids and vitamins, may also be included. Appropriate precursors can also be used for the culture medium. The medium or individual components may be added to the culture solution in an appropriate manner during the culture process, either batchwise or continuously, but are not limited to these.

[0036] 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. Furthermore, foam formation can be suppressed during cultivation using an antifoaming agent such as a fatty acid polyglycol ester. Additionally, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture medium to maintain an aerobic state in the culture medium. The temperature of the culture medium can generally be 20°C to 45°C, for example, 25°C to 40°C. The cultivation period can be continued until a desired amount of useful substance is produced, and can be, for example, 10 to 160 hours.

[0037] According to one embodiment of the present invention, the step of recovering L-citrulline from the cultured mutant strain and the medium in which the mutant strain was cultured can be carried out by using a suitable method known in the art depending on the culture method, such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion), etc., but is not limited thereto.

[0038] According to one embodiment of the present invention, the step of recovering L-citrulline can be carried out by centrifuging the culture medium at low speed to remove the biomass, and separating the resulting supernatant by ion exchange chromatography.

[0039] According to one embodiment of the present invention, the step of recovering L-citrulline may include a step of purifying L-citrulline. [Effects of the Invention]

[0040] The Corynebacterium glutamicum mutant strain according to the present invention inhibits the production of by-products by weakening or inactivating the activity of the protein expressed by the NCgl2657 gene, thereby enabling the production of L-citrulline at a high yield and high concentration. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention will be described in more detail below. However, such description is merely provided as an example for understanding the present invention, and the scope of the present invention is not limited by such exemplary description.

[0042] Example 1. Preparation of Corynebacterium glutamicum mutant strains To generate a mutant strain of Corynebacterium glutamicum with reduced activity of the protein expressed by the NCgl2657 gene, Corynebacterium glutamicum ATCC13032 and E. coli DH5a (HIT Competent Cells) were used. TM , Cat No. RH618) was used.

[0043] The Corynebacterium glutamicum ATCC13032 was cultured at 30°C in CM-broth medium (pH 6.8) containing 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.

[0044] The E. coli DH5a was cultured at 37°C on LB medium containing 10.0 g of tryptone, 10.0 g of NaCl, and 5.0 g of yeast extract in 1 L of distilled water.

[0045] The antibiotic kanamycin was a product of Sigma, and DNA sequencing analysis was performed by Macrogen Corporation.

[0046] 1-1. Construction of recombinant vectors Chromosomal DNA was extracted from Corynebacterium glutamicum ATCC13032 using the Wizard Genomic DNA Purification Kit (Promega, USA). PCR was then performed using this as a template with primers 1 and 2, and primers 3 and 4. The resulting PCR product was amplified by crossover PCR using primers 1 and 4, and then inserted into the HindIII and XbaI restriction enzyme sites of the recombinant vector pK19mobSacB. This vector was designated pK19ms / NCgl2657(A1T). The primers listed in Table 1 below were used to construct this vector.

[0047] [Table 1]

[0048] PCR was performed using the above primers under the following conditions. Using a thermocycler (TP600, Takara Bio Inc., Japan), 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) was added to the reaction mixture. 1 pM of oligonucleotides was used, and 10 ng of chromosomal DNA from Corynebacterium glutamicum (C. glutamicum) ATCC 13032 was used as the template. PCR was performed for 25–30 cycles in the presence of 1 unit of PrimeSTAR Max DNA Polymerase (Takara, Japan). The PCR conditions were: (i) denaturation step: 94°C for 30 seconds; (ii) annealing step: 58°C for 30 seconds; and (iii) extension step: 72°C for 1–2 minutes (allowing 2 minutes of polymerization time per 1 kb fragment).

[0049] The gene fragment thus prepared was cloned into the pK19mobSacB vector using self-assembly cloning. The vector was transformed into E. coli DH5a, spread onto an LB-agar plate containing 50 μg / ml kanamycin, and cultured at 37°C for 24 hours. The resulting colonies were isolated to confirm that the insert was correctly present in the vector. The vector was then isolated and used to recombine Corynebacterium glutamicum.

[0050] A common step in these methods was to amplify the gene of interest from Corynebacterium glutamicum ATCC 13032 genomic DNA using PCR, insert it into the pK19mobSacB vector using a self-assembled cloning strategy, and then select it from E. coli DH5a. The amplified gene was inserted into the pK19mobSacB vector using a DNA ligation kit (Takara, Japan) and the restriction enzymes HindIII and XbaI (NEB, USA) according to the supplied buffer and protocol.

[0051] 1-2. Production of mutant strains The pK19ms / NCgl2657(A1T) vector was used to construct the Corynebacterium glutamicum mutant strain CT2.

[0052] The vector was prepared to a final concentration of 1 μg / μl or higher, and primary recombination was induced in Corynebacterium glutamicum ATCC 13032 using electroporation (see Tauch et al., FEMS Microbiology Letters 123 (1994) 343-347). The electroporated strain was then plated onto 2YT-Km solid medium (containing 16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, 1.5% agar, and 30 μg / mL kanamycin). Colonies were obtained by culturing at 30°C for 2 days. Among the colonies in which primary homologous recombination was induced, those that showed PCR amplification using primers 1 and 4 in Table 1 under the same conditions as in 1-1 were selected as primary recombinant strains. These were then cultured in 2YT liquid medium (containing 16 g / L tryptone, 10 g / L yeast extract, and 5 g / L NaCl) for 12 hours and then plated on 2YT-10% sucrose solid medium (containing 16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, 1.5% agar, and 100 g / L sucrose) to induce secondary homologous recombination and remove the antibiotic marker. Strains that were not kanamycin-resistant and could grow on 10% sucrose medium were finally selected, and the introduction of a mutation into the start codon of the NCgl2657 gene was confirmed by nucleotide sequence analysis (see Schafer et al., Gene 145 (1994) 69-73). Finally, a Corynebacterium glutamicum mutant strain (CT2) carrying the mutant NCgl2657 gene was obtained.

[0053] Example 2. Preparation of Corynebacterium glutamicum mutant strains To generate a Corynebacterium glutamicum mutant strain lacking the NCgl2657 gene, wild-type Corynebacterium glutamicum ATCC13032 and E. coli DH5a were used.

[0054] The Corynebacterium glutamicum ATCC13032, E. coli DH5a, and antibiotics were the same as those used in Example 1.

[0055] 2-1. Vector construction A vector was constructed 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 / ΔNCgl2657, and the primers listed in Table 2 below were used to construct it.

[0056] [Table 2]

[0057] 2-2. Production of mutant strains A Corynebacterium glutamicum mutant strain, CT3, was prepared in the same manner as in Example 1, except that the pK19ms / β vector was used instead of the pK19ms / NCgl2657(A1T) vector. Strains that were not resistant to kanamycin and could grow in 10% sucrose medium were finally selected, and PCR was performed using primers 5 and 8 in Table 2 under the same conditions as in Example 1 to confirm whether the NCgl2657 gene had been removed. The strain lacking the NCgl2657 gene was designated CT3.

[0058] Comparative Example 1. Corynebacterium glutamicum mutant strain To overproduce citrulline, a Corynebacterium glutamicum mutant strain (see Korean Patent Application No. 10-2019-0151321, hereinafter referred to as "CT1") was used, in which the activities of ornithine carbamoyltransferase and carbamoylphosphate synthase were enhanced.

[0059] The CT1 strain was confirmed to have increased L-citrulline production and fermentation yield compared to the parent strain, Corynebacterium glutamicum ATCC13032.

[0060] Experimental Example 1: Confirmation of L-citrulline productivity in mutant strains The L-citrulline productivity of the NCgl2657 gene mutant strains (CT2 and CT3 strains) prepared in Examples 1 and 2 was compared with that of the CT1 strain of Comparative Example 1.

[0061] Each strain was inoculated into citrulline seed medium and cultured at 30°C for 10 hours, after which 250 mL of the culture medium was inoculated into the medium in a 5 L incubator. When all the sugar contained in the initial medium was used up, additional medium was added. The composition of the medium used in this experiment is shown in Table 3 below. After the culture was completed, the culture medium was diluted 100 times with distilled water, filtered through a 0.45 μm filter, and then passed through a column (Dionex IonPac TM The concentrations of L-citrulline and the by-product 6-acetylornithine in the culture medium produced by the strain were measured using a high performance liquid chromatograph (HPLC) equipped with a CS12A and an ultraviolet detector (195 mm), and the results are shown in Table 4 below.

[0062] [Table 3]

[0063] [Table 4]

[0064] As shown in Table 4, the Corynebacterium glutamicum mutant strain CT2, in which the activity of the enzyme encoded by the NCgl2657 gene was weakened, and the Corynebacterium glutamicum mutant strain CT3, in which the NCgl2657 gene was deleted, showed approximately 69% and 99% reductions in production of the by-product 6-acetylornithine, respectively, and approximately 17.8% and 12.2% increases in L-citrulline productivity, respectively, compared to the previously developed mutant strain CT1. These results demonstrate that weakening or inactivating the activity of the protein expressed by the NCgl2657 gene not only significantly reduces the production of by-products, but also improves the strain's L-citrulline-producing ability, enabling the production of highly pure L-citrulline.

[0065] The present invention has been described above with a focus on its preferred embodiments. Those skilled in the art will understand that the present invention can be realized in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is defined by the claims, not the above description, and all variations within the scope of the claims should be construed as being within the scope of the present invention.

Claims

1. A mutant Corynebacterium glutamicum strain in which the start codon of the NCgl2657 gene is mutated.

2. The Corynebacterium glutamicum mutant according to claim 1, wherein the NCgl2657 gene is represented by the base sequence of SEQ ID NO:

1.

3. 2. The Corynebacterium glutamicum mutant strain of claim 1, wherein the mutation in the start codon of the NCgl2657 gene is a substitution of ATG with TTG or GTG.

4. a) culturing the Corynebacterium glutamicum mutant strain according to claim 1 in a medium; and b) recovering L-citrulline from the cultured mutant strain or the medium in which the mutant strain was cultured; A method for producing L-citrulline, comprising:

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