KR20120007855A - A microorganism having enhanced l-ornithine productivity and a method of producing l-ornithine using the same - Google Patents

Abstract

PURPOSE: A L-ornitine-producing microorganism isp rovided to prepare L-ornitine of high yield and high efficiency and to be applied to medical, pharmaceutical, food, and feed industries. CONSTITUTION: A L-ornitine-producing microorganism has weakened intrinsic gluconate kinase(GntK) activity. The microorganism is Corynebacterium sp. The microorganism has enhanced argCJBD operon activity. A method for preparing L-ornitine comprises: a step of culturing the L-ornitine-producing microorganism; and a step of collecting L-ornitine.

Description

A microorganism having enhanced L-ornithine productivity and a method of producing L-ornithine using the same}

The present invention relates to L-ornithine producing microorganisms in which gluconate kinase (GntK) activity is attenuated compared to endogenous activity. The present invention also relates to a method for producing L-ornithine, comprising culturing the L-ornithine producing microorganism and recovering L-ornithine.

Ornithine is one of the amino acids present on the urea cycle in vivo. High levels of ammonia in the blood cause liver damage. Ornithine and citrulline combine with ammonia in the blood to form citrulline and arginine, respectively, to reduce the level of ammonia in the blood. L-ornithine has a detoxifying function against ammonia, such as citrulline and proline, and is used not only as a therapeutic agent for liver disease but also as a food material for preventing obesity. have.

On the other hand, Corynebacterium strain (Corynebacterium), in particular Corynebacterium glutamicum ( Corynebacterium glutamicum ) is a Gram-positive microorganism that is widely used for L-amino acid production. Thus, since the production of L-amino acid using the Corynebacterium strain is important, many attempts have been made to improve the production method thereof. Specifically, microorganisms used for ornithine production include micrococcus glutamicus , which is a citrulline or arginine nutritionally demanding mutant strain by ultraviolet light treatment, etc. (Kyowahako Co., Ltd., Japan, US Patent No. 2,988,489). , Brevibacterium lactofermentum (Brevibacterium lactofermentum), Brevibacterium Kawasaki (B. kawasaki), Brevibacterium Plastic boom (B. flavum) (Ajinomoto, Japan Patent Publication No. 68-8712, No. 68 8714 and No. 69-26910 call), Brevibacterium keto glutamicum (B. ketoglutamicum), bakteo as asparagus pinewooseu (Arthrobacter paraffineus), Corynebacterium hydrocarbyl carbonyl class tooth (Corynebacterium hydrocarboclastus ) (Kyowahako Industry Co., Ltd., Japan, US Patent No. 3,574,061) and the like are known. In addition, arginine or citrulline nutrient strains Asrobacter citruus ( A. citreus ), Brevibacterium lactofermentum ( B. Lactofermentum ), or Corynebacterium glutamicum ( C. glutamicum ) (Ajinomoto ( European Patent No. 0393708) is used to produce ornithine by selecting a nutritionally demanding mutant that is resistant to mycophenolic acid or ornithinol. US Patent No. 3,574,061 discloses a method for producing L-ornithine by culturing the nutritionally demanding mutant Brevibacterium ketoglutamcum ATCC 21092 in a medium containing hydrocarbons, for example, paraffin as the main carbon source. However, according to this method, the productivity of ornithine was not very high at 8.6 mg / ml in 4 days of culture. As such, studies on how to produce L-ornithine have been reported, but there is a problem that the production yield is not high. However, industrial mass production of L-ornithine is still required, and therefore, there is a need to develop a high-efficiency, high-yield production method of L-ornithine.

Meanwhile, L-ornithine is biosynthesized from L-glutamic acid via cyclic pathway in Corynebacterium glutamicum (Cunin et al., Microbiol Rev 50: 314-352, 1986), and during the biosynthesis process The third step is catalyzed by NADPH dependent reductase encoded by the argC gene. A direct link between the regeneration of NADPH and the biosynthesis of certain metabolites was revealed through the biosynthesis of L-lysine in Corynebacterium glutamicum (Wittmann and Heinzle, Microbiol 68: 5843-5849, 2002; Marx et al. , J Biotechnol 104: 185-197, 2003; Ohnishi et al., Microbiol Lett 242: 265-274, 2005). NADPH is an enzyme that catalyzes the pentose phosphate pathway during the production of lysine in Corynebacterium glutamicum (glucose-6-phosphate dehydrogenase (G6PDH) and 6- It is mainly produced during oxidation by 6-phosphogluconate dehydrogenase (6PGD) (Marx et al., J Biotechnol 104: 185-197, 2003; Wittmann and Heinzle, Microbiol 68: 5843-5849 , 2002). A series of studies suggested that the shift of carbon flow to the pentose phosphorylation pathway could be a metabolic target for increasing the biosynthesis of certain metabolites that require NADPH supply. (Mascarenhas et al., Appl Environ Microbiol 57: 2995-2999, 1991; Sauer et al., Appl Environ Microbiol 62: 3687-3696, 1996; Marx et al., J Biotechnol 104: 185-197, 2003; Ohnishi et al., Microbiol Lett 242: 265-274, 2005).

In a previous study, we found that the production of L-ornithine is increased by homologous expression of argCJBD in mutant strains that lack citrulline and proline biosynthesis genes and block feedback control by an arginine inhibitor ( ArgR ). (Hwang et al., J Microbiol Biotechnol 18: 704-710, 2008). However, in order to maintain the maximum carbon flux in the biosynthetic pathway of L-ornithine, NADPH must be supplied sufficiently as well as the overexpression of argCJBD as well as the conversion of carbon flow to the pentose phosphorylation pathway.

Against this background, the inventors have made diligent efforts to find a method for obtaining L-ornithine with high efficiency and high yield. As a result, we have genetically modified the metabolic activity of the oxidative pentane phosphorylation pathway to increase NADPH production through the pentose phosphorylation pathway. The present invention was completed by confirming that L-ornithine production can be increased as a result.

One object of the present invention is to provide L-ornithine producing microorganisms in which gluconate kinase (GntK) activity is attenuated compared to endogenous activity.

Still another object of the present invention is to provide a method for producing L-ornithine, comprising 1) culturing the L-ornithine producing microorganism: and 2) recovering L-ornithine.

In one embodiment, the present invention provides an L-ornithine producing microorganism in which gluconate kinase (GntK) activity is attenuated compared to endogenous activity.

In the present invention, the term "L-ornithine" is a basic amino acid present in the urea circuit in a living body of a higher animal, and serves to protect the liver by converting ammonia, which is highly toxic to the liver, into a non-toxic urea and releasing it with urine. Refers to amino acids. L-ornithine is widely used as a food material for preventing obesity by increasing muscle synthesis and promoting basal metabolism by secreting growth hormone. Recently, it has been found that there are new functions such as skin beauty action such as wrinkle improvement. L-ornithine is in the form of L-ornithine alpha ketoglutarate (OKG: L-ornithine and alpha ketoglutaric acid in a 2: 1 ratio) for muscle enhancement, prevention of obesity and immunity. It is widely used as a material. L-ornithine is used as a medicine for improving liver disorders in Europe, in the form of L-ornithine salt in Japan, and as a food supplement in the United States.

As used herein, the term "gluconate kinase (GntK)" refers to an enzyme involved in the GntK pathway for biosynthesis of 6-phosphogluconic acid, which is an intermediate of the oxidation process of the pentose phosphorylation pathway of microorganisms. Microorganisms of Corynebacterium have two metabolic pathways, 6 G6PDH pathway and GntK pathway, for the biosynthesis of 6-phosphogluconic acid, an intermediate of the oxidation of the pentose phosphorylation pathway (Fig. 1). It means that it can metabolize at least part of glucose. Accordingly, the present inventors further block the carbon flow to 6-phosphogluconic acid through the blocking of the GntK pathway, and thus more carbon flow proceeds through oxidation of the pentose phosphorylation pathway, resulting in 6-phosphogluconate dehydrogena. It was predicted that NADPH regeneration was increased due to the specifically increased activity of (6PGD), and consequently, a mechanism for increasing the production of L-ornithine was proposed. Thus, it was intended to weaken the activity of enzymes that are believed to have gluconate kinase (GntK) activity in microorganisms. Preferably, the enzyme having gluconate kinase (GntK) activity in the present invention is NCgl2399 represented by SEQ ID NO: 1 or NCgl2905 represented by SEQ ID NO: 2.

As used herein, the term "intrinsic activity" refers to an active state of an enzyme possessed by a microorganism in a natural state, and in the present invention, means an active state of GntK naturally possessed by a microorganism.

In the present invention, "GntK activity is weakened compared to the intrinsic activity" means that GntK is not normally acted by the deletion or mutation of the gene on the chromosome encoding GntK, so that the GntK activity in which the microorganism has a natural state is weakened. do. Preferably, the gene encoding NCgl2399 or NCgl2905 or all of the genes is mutated by deletion, substitution or insertion, or its expression control sequence is mutated to mean that GntK does not function normally.

In the present invention, by introducing a recombinant vector containing a portion of the gluconate kinase gene into the microorganism, microorganisms with reduced GntK endogenous activity can be prepared by deleting or mutating the endogenous gluconate kinase gene. Insertion of the gene into the chromosome can be by any method known in the art, for example homologous recombination.

As used herein, the term "recombinant vector" refers to a gene construct that is capable of expressing a target protein or RNA of interest in a suitable host cell, and includes a gene construct comprising essential regulatory elements operably linked to express the gene insert. In the present invention, a recombinant vector represented by the cleavage map of FIG. 2 or 3 may be used.

As a parent strain introducing the recombinant vector, any microorganism capable of producing L-ornithine may be used without limitation, and preferably, a microorganism of the genus Corynebacterium or Brevibacterium may be used. For example, Corynebacterium hydrocarbyl carbonyl class tooth (Corynebacterium hydrocarboclastus), Corynebacterium glutamicum (C. glutamicum), Corynebacterium glutamicum ATCC13032, Corynebacterium thermoaminogenes amino to Ness (thermoaminogenes) FERM BP-1539, Brevibacterium Plastic boom (Brevibacterium flavum ) ATCC 14067, Brevibacterium lactofermentum ATCC 13869, and L-amino acid producing mutants or strains prepared therefrom such as Corynebacterium glutamicum KFCC10881, Corynebacterium glutami Kum KFCC11001, Corynebacterium glutamicum KCCM10770P may be included, and preferably, Corynebacterium glutamicum SJC8039 ( C. glutamicum ATCC13032, argF Δ, argR Δ). In a specific embodiment of the present invention, Corynebacterium glutamicum SJC8039 ( C. glutamicum ATCC13032, argF △, argR △) was used, but is not limited thereto.

According to one specific embodiment, the pK18mobsacB vector (Schafer et al., Gene 145: 69), which does not replicate in Corynebacterium glutamicum but has a function of performing marker-free deletion of the target gene -73, 1994) gene deletion was performed. That is, a recombinant vector is prepared to include a portion of a gene encoding NCgl2399 or NCgl2905 in the pK18mobsacB vector and an antibiotic marker and a Levansucrease gene ( SacB ), and then the recombinant vector is transferred to a microorganism by any method known in the art. By transformation and homologous recombination in chromosomes, Corynebacterium microorganisms for L-ornithine production with reduced GntK activity were produced.

In the present invention, a pK18mobsacB derivative including a part of the gene encoding NCgl2399 in the pK18mobsacB vector was prepared and named pSJ1034. The recombinant strain whose gene encoding NCgl2399 was destroyed by transforming it was named SJC8245. In addition, a pK18mobsacB derivative including a part of the gene encoding NCgl2905 in the pK18mobsacB vector was prepared and named as pSJ1035, which was transformed into the SJC8245 strain, and the recombinant strain which further destroyed the gene encoding NCgl2905 was named SJC8399. .

According to a specific embodiment, the culture of SJC8245, a Corynebacterium glutamicum strain lacking the NCgl2399 gene, resulted in a 41% specific activity of GntK and a 23.5% increase in NADPH concentration. The production of ornithine was found to increase by 24.8%. In addition, as a result of culturing SJC8399, a Corynebacterium glutamicum strain that additionally destroyed the NCgl2905 gene, which is assumed to be another gene of the enzyme having gluconate kinase activity in the SJC8245 strain, the activity of the GntK pathway was higher than that of SJC8245. It was confirmed that the decrease further, the concentration of NADPH was increased by 51.8%, the production of L- ornithine was increased by 63.8% (Tables 3 and 4). These results show that the L-ornithine production was increased by the microorganism of the present invention, and also showed that excessive NADPH is a factor influencing the increase of L-ornithine production.

As a result, the microorganism of the present invention attenuated the GntK activity, specifically increased the activity of 6GDP, increased the oxidation process of the pentose phosphorylation pathway, and significantly increased the concentration of NADPH in the cells (Table 4). Since the biosynthetic pathway of L-ornithine in the microorganism of the genus Corynebacterium is catalyzed by NADPH dependent reductase, it was found that the microorganism of the present invention increased L-ornithine production.

In addition, in the present invention, in order to further increase the L-ornithine production amount, the activity of known enzymes related to the biosynthesis of L-ornithine can be further controlled. Preferably, the present invention may further increase the L-ornithine production by overexpressing the argCJBD gene by regulating argCJBD operon activity (Hwang et al., J Microbiol Biotechnol 18: 704-710, 2008).

As another aspect, the present invention, 1) culturing the L- ornithine producing microorganism; And 2) to provide a method for producing L- ornithine comprising the step of recovering L- ornithine.

The term "culture" in the present invention means to grow microorganisms under environmental conditions that are appropriately artificially controlled. In the present invention, the method of culturing L-ornithine using Corynebacteria may be performed using a method well known in the art. Specifically, the culture may be continuously cultured in a batch process or in a fed batch or repeated fed batch process, but is not limited thereto.

The medium used for cultivation must meet the requirements of the particular strain in an appropriate manner. Culture media for Corynebacteria strains are known (eg, Manual of Methods for General Bacteriology. American Society for Bacteriology.Washington D.C., USA, 1981). Sugar sources that can be used include sugars and carbohydrates such as glucose, saccharose, 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, ethanol, and organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used include peptone, yeast extract, gravy, 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. Nitrogen sources can also be used individually or as a mixture. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, the culture medium should contain metal salts such as magnesium sulfate or iron sulfate required for growth. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used. In addition, suitable precursors to the culture medium may be used. The above-mentioned raw materials may be added batchwise or continuously by a suitable method for the culture during the culture.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture. In addition, antifoaming agents such as fatty acid polyglycol esters can be used to inhibit bubble generation. Inject oxygen or oxygen-containing gas (eg, air) into the culture to maintain aerobic conditions. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. Incubation is continued until the maximum amount of L-ornithine production is obtained. For this purpose it is usually achieved in 10 to 160 hours. L-ornithine may be excreted in culture medium or contained in cells.

The method for producing L-ornithine of the present invention includes recovering ornithine from cells or culture medium. Methods of recovering L-ornithine from cells or culture media are well known in the art. Filtration, anion exchange chromatography, crystallization and HPLC may be used for the L-ornithine recovery method, but is not limited to these examples.

The present invention is capable of producing L-ornithine with high yield and high efficiency as the gluconate kinase (GntK) activity provides an attenuated L-ornithine producing microorganism compared to intrinsic activity, and thus the L-ornithine It can be usefully used in human medicine and pharmaceutical industry, food industry, feed industry and chemical industry.

1 is a schematic diagram of the metabolism of the pentose phosphorylation pathway of Corynebacterium glutamicum.
2 is a diagram showing a marker-free deletion vector pSJ1034 of the NCgl2399 gene in the Corynebacterium chromosome.
3 is a diagram showing a marker-free deletion vector pSJ1035 of the NCgl2905 gene in the Corynebacterium chromosome.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  1: Preparation of Mutant Wines Deleting Site-Specific Genes

In the present invention, the parent strain Corynebacterium glutamicum SJC8039 ( C. glutamicum ATCC13032, argF △, argR △) (Hwang et al., J Microbiol Biotechnol 18: 704-710, 2008) was used. Primers were prepared to prepare recombinant strains SJC8399 and SJC8245 of the present invention from the parent strain. First, primers 2399F1, 2399F2, 2399R1, 2399R2, 2905F1, 2905F2 and 2905R1, respectively, based on the sequences of NCgl2399 (SEQ ID NO: 1) and NCgl2905 (SEQ ID NO: 2) to construct inactivated fragments of genes encoding NCgl2905 or NCgl2399. , 2905R2 was produced (Table 1). Underlined sequences indicate restriction positions for restriction enzymes as shown in parentheses, and uppercase letters indicate sequences of microbial genes.

primer Base sequence SEQ ID NO: 2399F1 gc tctaga CCCCAGAACATGCTGACG (XbaI) 3 2399R1 gg ggtacc CGCTGCTAGGGCTTTACC (KpnI) 4 2399F2 gg ggtacc GGAACCGTCTTCGTCCACC (KpnI) 5 2399R2 gc tctaga GTCACCGCTGGTACATCC (XbaI) 6 2905F1 gc tctaga CATTGGAGCATCCGTAGC (XbaI) 7 2905R1 tcc cccggg GCCTTCACCATCGACCAA (SmaI) 8 2905F2 tcc cccggg GTTCCCGAACAGATCCCC (SmaI) 9 2905R2 gc tctaga CGCTCTGACCTGCCTAAC (XbaI) 10

PK18mobsacB vector (Schafer et al.) With the ability to perform marker-free deletion of the target gene without replication in Corynebacterium glutamicum for site specific gene disruption al., Gene 145: 69-73, 1994), in which the open reading frames of NCgl2399 and NCgl2905 were internally deleted to create gene-disrupted mutant strains. pK18mobsacB derivatives were made. The pK18mobsacB derivatives were named pSJ1034 and pSJ1035, respectively.

pSJ1034 is a pK18mobsacB derivative containing the Xba I terminus of 2,267 bp NCgl2399, which includes the internal gene loss of NCgl2399. The NCgl2399 internal gene loss was generated by cross-PCR using 2399F1-2399R1 primer and 2399F2-2399R2 primer pair as a template of Corynebacterium glutamicum SJC8039 genomic DNA. pSJ1035 is a pK18mobsacB derivative with the Xba I terminus of 2,819 bp NCgl2905, which includes the internal gene loss of NCgl2905. The NCgl2905 internal gene loss was generated by cross PCR with 2905F1-2905R1 primer pair and 2905F2-2905R2 primer pair as a template of Corynebacterium glutamicum SJC8039 genomic DNA.

The recombinant plasmid was transformed into wild Corynebacterium glutamicum by electroporation (van der Rest et al., Appl Microbiol Biotechnol 52: 541-545, 1999), and the plasmid was first recombinant (crossed out). ) Into the chromosome. The plasmid was then excised from the chromosome via secondary recombination (crossing) in a solid plate medium containing 10% sucrose.

Corynebacterium glutamicum transformants having completed the second recombination through diagnostic PCR using pairs of gene-specific primers 2905F1-2905R2 and 2399F1-2399R2 The deletion of the NCgl2905 and NCgl2399 genes, respectively, was confirmed. The Genbank accession numbers of the open reading frame DNA sequences of NCgl2905 and NCgl2399 are NC_003450.

Example  2: NCgl2905  And / or NCgl2399  Gene deleted Corynebacterium Glutamicum  culture

Corynebacterium glutamicum SJC8399 and Corynebacterium SJC8245, L-ornithine producing strains, were cultured in the following manner for L-ornithine production. The seed medium was incubated for 24 hours using Recovery Glucose medium (80 g BHI, 20 g glucose, 60 g sorbitol / L). The production medium was MMY medium [0.8 g KH ₂ PO ₄ , 10 g (NH ₄ ) ₂ SO _4, 1 g MgSO ₄ ㆍ 7H ₂ O, 1.2 g Na ₂ HPO ₄ , 20 mg MnSO ₄ ㆍ H ₂ O, 20 mg FeSO ₄ 7H ₂ O, 10 mg, ZnSO ₄ 7H ₂ O, 10 g yeast extract, and 60 g glucose / L, pH 7.0] were used. A 100 ml baffle flask containing a seed medium was inoculated with Corynebacterium glutamicum mother strain SJC8039 and the strain Corynebacterium glutamicum SJC8399 and Corynebacterium glutamicum SJC8245, respectively, and shaken (200 rpm). ). After separating and harvesting the glutamicum cultured in the seed medium, the glutamicum was resuspended in a 100 ml baffle flask containing 10 ml of the production medium until the absorbance at 600 nm was 0.4-0.5. Thereafter, 0.2 g of sterile CaCO ₃ per 10 ml was added, and kanamycin was added to a final concentration of 50 μg / ml if necessary. The incubation was carried out in a 200 rpm rotary stirrer at 30 ° C.

Example  3: Determination of enzyme activity and thus L-ornithine production

Corynebacterium glutamicum cultured in the production medium was harvested by centrifugation during the expontial phase and resuspended in 100 mM Tris / HCl buffer (pH 7.5). Cell walls were broken down using glass beads and centrifuged to obtain cell extracts. All these treatments were performed at 4 ° C., and the supernatants were harvested for specific enzyme activity analysis.

The amount of protein for enzymatic analysis was determined by the Bradford method (Bradford 1976). The activity of G6PDH and 6PGD was measured by NADPH spectroscopic formation at 340 nm (Frunzke et al., Mol Microbiol 67: 305-322, 2008). GntK activity was determined by coupled enzymatic assay of 6PGD (Frunzke et al., Mol Microbiol 67: 305-322, 2008). Cell growth rate was measured spectroscopically at 600 nm, and L-ornithine concentration was determined by colorimetry using ninhydrin (Chinard et al., J Biol Chem 199: 91-95, 1952). Dry cell mass was measured based on the absorbance value of 1 at 600 nm corresponds to a dry weight of 0.3g per 1L. The concentration of NADPH was determined by an enzymatic cycling reaction using the EnzyChrom ^™ NADP ⁺ / NADPH Assay Kit (BioAssay Systems, CA, USA).

As a result, SJC8245, a Corynebacterium glutamicum strain lacking the gene encoding NCgl2399 of the present invention, was found to retain 41% of GntK specific activity (0.51 vs 0.21 U mg / protein). In addition, by additionally destroying NCgl2905, another gene of the enzyme also possessing gluconate kinase (EC: 2.7.1.12) activity in the SJC8245 strain, Corynebacterium glutamicum named SJC8399 was obtained. It was confirmed that the activity of the GntK pathway was further reduced compared to the SJC8245 (0.11 U mg / protein).

In the case of inactivation of NCgl2399, NADPH concentration was increased by 23.5%, and L-ornithine production was increased by 24.8%. In addition, when both NCgl2399 and NCgl2905 were inactivated, NADPH concentration was increased by 51.8%, and L-ornithine production was increased by 63.8%. These results indicate that excess NADPH is required to increase the concentration of L-ornithine in Corynebacterium glutamicum.

Table 2 shows the growth rate of Corynebacterium glutamicum, Table 3 shows the accumulation concentration of NADPH and L-ornithine, and Table 4 shows the specific activity of the core enzymes of the core carbon metabolism process. As shown in Tables 2 and 3, SJC8245 (which NCgl2399 is inactivated) and SJC8399 (which both NCgl2399 and NCgl2905 are inactivated) have a similar or slightly reduced growth rate compared to SJC8039 (parent strain). Intracellular NADPH levels and L-ornithine production increased significantly.

Corynebacterium glutamicum Growth rate (μ _max ) SJC8039 0.69 SJC8245 0.70 SJC8399 0.61

Corynebacterium glutamicum NADPH (mmol / g) L-ornithine (mg / L) SJC8039 2.55 141 SJC8245 3.15 176 SJC8399 3.87 231

Specifically, when comparing the specific activities of G6PDH and 6PGD, as shown in Table 4, the specific activity of 6PGD was significantly increased in SJC8245 and SJC8399 compared to SJC8039. However, little change was observed in G6PDH activity.

Corynebacterium Glutamicum Activity (U mg / protein) G6PDH 6PGD GntK SJC8039 0.12 0.25 0.51 SJC8245 0.18 0.52 0.21 SJC8399 0.12 0.69 0.11

The above values represent mean values based on the results obtained in at least three independent cultures and in all cases the standard deviation was less than 10%.

<110> SANGJI UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> A microorganism having enhanced L-ornithine productivity and a          method of producing L-ornithine using the same <130> PA100296 / KR <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 167 <212> PRT <213> Corynebacterium glutamicum ATCC 13032 <220> <221> PEPTIDE (222) (1) .. (167) <223> NCgl2399 gluconate kinase <400> 1 Met Ser Ala Ala Glu Gly Leu His Ile Val Val Met Gly Val Ser Gly   1 5 10 15 Cys Gly Lys Ser Ser Val Gly Lys Ala Leu Ala Ala Glu Leu Gly Ile              20 25 30 Glu Tyr Lys Asp Gly Asp Glu Leu His Pro Gln Glu Asn Ile Asp Lys          35 40 45 Met Ala Ser Gly Gln Ala Leu Asp Asp Asp Asp Arg Ala Trp Trp Leu      50 55 60 Val Gln Val Gly Lys Trp Leu Arg Asp Arg Pro Ser Gly Val Ile Ala  65 70 75 80 Cys Ser Ala Leu Lys Arg Ser Tyr Arg Asp Leu Leu Arg Thr Lys Cys                  85 90 95 Pro Gly Thr Val Phe Val His Leu His Gly Asp Tyr Asp Leu Leu Leu             100 105 110 Ser Arg Met Lys Ala Arg Glu Asp His Phe Met Pro Ser Thr Leu Leu         115 120 125 Asp Ser Gln Phe Ala Thr Leu Glu Pro Leu Glu Asp Asp Glu Asp Gly     130 135 140 Lys Val Phe Asp Val Ala His Thr Ile Ser Glu Leu Ala Ala Gln Ser 145 150 155 160 Ala Glu Trp Val Arg Asn Lys                 165 <210> 2 <211> 494 <212> PRT <213> Corynebacterium glutamicum ATCC 13032 <220> <221> PEPTIDE (222) (1) .. (494) <223> NCgl2905 gluconate kinase <400> 2 Met Gly Ser Ile Pro Thr Met Ser Ile Pro Phe Asp Asp Ser Arg Gly   1 5 10 15 Pro Tyr Val Leu Ala Met Asp Ile Gly Ser Thr Ala Ser Arg Gly Gly              20 25 30 Leu Tyr Asp Ala Ser Gly Cys Pro Ile Lys Gly Thr Lys Gln Arg Glu          35 40 45 Ser His Glu Phe Thr Thr Gly Glu Gly Val Ser Thr Ile Asp Ala Asp      50 55 60 Gln Val Val Ser Glu Ile Thr Ser Val Ile Asn Gly Ile Leu Asn Ala  65 70 75 80 Ala Asp His His Asn Ile Lys Asp Gln Ile Ala Ala Val Ala Leu Asp                  85 90 95 Ser Phe Ala Ser Ser Leu Ile Leu Val Asp Gly Glu Gly Asn Ala Leu             100 105 110 Thr Pro Cys Ile Thr Tyr Ala Asp Ser Arg Ser Ala Gln Tyr Val Glu         115 120 125 Gln Leu Arg Ala Glu Ile Asp Glu Glu Ala Tyr His Gly Arg Thr Gly     130 135 140 Val Cys Leu His Thr Ser Tyr His Pro Ser Arg Leu Leu Trp Leu Lys 145 150 155 160 Thr Glu Phe Glu Glu Glu Phe Asn Lys Ala Lys Tyr Val Met Thr Ile                 165 170 175 Gly Glu Tyr Val Tyr Phe Lys Leu Ala Gly Ile Thr Gly Met Ala Thr             180 185 190 Ser Ile Ala Ala Trp Ser Gly Ile Leu Asp Ala His Thr Gly Glu Leu         195 200 205 Asp Leu Thr Ile Leu Glu His Ile Gly Val Asp Pro Ala Leu Phe Gly     210 215 220 Glu Ile Arg Asn Pro Asp Glu Pro Ala Thr Asp Ala Lys Val Val Asp 225 230 235 240 Lys Lys Trp Lys His Leu Glu Glu Ile Pro Trp Phe His Ala Ile Pro                 245 250 255 Asp Gly Trp Pro Ser Asn Ile Gly Pro Gly Ala Val Asp Ser Lys Thr             260 265 270 Val Ala Val Ala Ala Ala Thr Ser Gly Ala Met Arg Val Ile Leu Pro         275 280 285 Ser Val Pro Glu Gln Ile Pro Ser Gly Leu Trp Cys Tyr Arg Val Ser     290 295 300 Arg Asp Gln Cys Ile Val Gly Gly Ala Leu Asn Asp Val Gly Arg Ala 305 310 315 320 Val Thr Trp Leu Glu Arg Thr Ile Ile Lys Pro Glu Asn Leu Asp Glu                 325 330 335 Val Leu Ile Arg Glu Pro Leu Glu Gly Thr Pro Ala Val Leu Pro Phe             340 345 350 Phe Ser Gly Glu Arg Ser Ile Gly Trp Ala Ala Ser Ala Gln Ala Thr         355 360 365 Ile Thr Asn Ile Gln Glu Gln Thr Gly Pro Glu His Leu Trp Arg Gly     370 375 380 Val Phe Glu Ala Leu Ala Leu Ser Tyr Gln Arg Val Trp Glu His Met 385 390 395 400 Gly Lys Ala Gly Ala Ala Pro Glu Arg Val Ile Ala Ser Gly Arg Val                 405 410 415 Ser Thr Asp His Pro Glu Phe Leu Ala Met Leu Ser Asp Ala Leu Asp             420 425 430 Thr Pro Val Ile Pro Leu Glu Met Lys Arg Ala Thr Leu Arg Gly Thr         435 440 445 Ala Leu Ile Val Leu Glu Gln Leu Glu Pro Gly Gly Thr Arg Ala Thr     450 455 460 Pro Pro Phe Gly Thr Thr His Gln Pro Arg Phe Ala His His Tyr Ser 465 470 475 480 Lys Ala Arg Glu Leu Phe Asp Ala Leu Tyr Leu Lys Leu Val                 485 490 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer 2399F1 <400> 3 gctctagacc ccagaacatg ctgacg 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer 2399R1 <400> 4 ggggtacccg ctgctagggc tttacc 26 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 2399F2 <400> 5 ggggtaccgg aaccgtcttc gtccacc 27 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer 2399R2 <400> 6 gctctagagt caccgctggt acatcc 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer 2905F1 <400> 7 gctctagaca ttggagcatc cgtagc 26 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 2905R1 <400> 8 tcccccgggg ccttcaccat cgaccaa 27 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 2905F2 <400> 9 tcccccgggg ttcccgaaca gatcccc 27 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer 2905R2 <400> 10 gctctagacg ctctgacctg cctaac 26

Claims (10)

L-ornithine producing microorganism, wherein gluconate kinase (GntK) activity is attenuated relative to intrinsic activity.
The microorganism of claim 1, wherein the activity of at least one of a gluconate kinase having an amino acid sequence of SEQ ID NO: 1 and a gluconate kinase having an amino acid sequence of SEQ ID NO: 2 is attenuated relative to intrinsic activity.
The microorganism of claim 1, wherein the microorganism is a genus Corynebacterium.
The microorganism of claim 1, wherein the endogenous activity of the protein is attenuated by deletion, substitution, or insertion of a gene on a chromosome that encodes a protein having gluconate kinase activity.
2. The microorganism according to claim 1, wherein the expression control sequence of a gene on a chromosome encoding a protein having gluconate kinase activity is mutated to attenuate the endogenous activity of the protein.
The microorganism of claim 1, wherein a recombinant vector comprising a portion of the gluconate kinase is introduced to attenuate intrinsic activity.
The microorganism of claim 6, wherein the microorganism is introduced with at least one of the recombinant vector of FIG. 2 and the recombinant vector of FIG. 3 such that the activity of the gluconate kinase is weakened relative to the endogenous activity.
The microorganism of claim 1, wherein the microorganism further has increased activity of argCJBD operon.
The microorganism of claim 1, wherein the microorganism is a parent strain of Corynebacterium glutamicum ATCC13032.
A method of producing L-ornithine, comprising the steps of: 1) culturing the microorganism according to any one of claims 1 to 9; and 2) recovering L-ornithine.

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