# Patent

## Title
Microorganisms having improved ornithine-producing ability and method for producing ornithine using the same

## Abstract

The present invention relates to a microorganism having an improved ornithine-producing ability, in which the biosynthetic pathway of arginine form ornithine is blocked, the intracellular glutamate level is increased, and the biosynthetic pathway of ornithine from glutamate is enhanced, and a method for producing ornithine using the microorganism.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a U.S. national phase application of International PCT Patent Application No. PCT/KR2011/009477, which was filed on Dec. 8, 2011, which claims priority to Korean Patent Application Nos. 10-2011-0130594, filed Dec. 7, 2011 and 10-2010-0124866, filed Dec. 8, 2010. These applications are incorporated herein by reference in their entireties.
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is HANO_022_00US_T25.txt. The text file is 39 KB, was created on, Aug. 28, 2013, and is being submitted electronically via EFS-Web.
TECHNICAL FIELD
The present invention relates to a microorganism having an improved ornithine-producing ability, and a method for producing ornithine using the same.
BACKGROUND ART
Ornithine is a substance widely found in plants, animals, and microorganisms and is used as a precursor in the biosynthesis of arginine, proline, and polyamines. As a non-essential amino acid, ornithine is not found in proteins, but is present in peptide antibiotics such as tyrosidine and gramicidine. Ornithine plays an important role in the pathway for excretion of urea produced from amino acid or ammonia via the ornithine cycle in the in vivo metabolism of higher animals.
Ornithine helps to build muscles and to reduce body fat, and thus is used as a nutritional supplement. Ornithine-alpha ketoglutarate (OKG) containing ornithine and alpha ketoglutaric acid at a ration of 2:1 is used as an immune enhancer. Ornithine is also used a drugs for improving liver cirrhosis and liver function disorders, because it helps to remove harmful ammonia from the liver. The known methods of producing ornithine are treatment of milk casein with digestive enzymes and use of transformed E. coli or industrial microorganisms belonging to Corynebacterium sp., which are widely used in the production of amino acids, nucleic acids, enzymes and antibiotic-like substances.
In the microorganisms belonging to Corynebacterium sp., L-arginine is synthesized from glutamate by an enzyme expressed from a gene on the arginine operon in the form of argCJBDFRGH. The arginine operon genes, which play the most important role in arginine biosynthesis, synthesize arginine using intracellular glutamate (L-glutamate) as a substrate, and ornithine is produced as an intermediate during the syntheses of arginine. Specifically, as in FIG. 2 schematically illustrating a synthetic pathway of arginine from glutamate in a microorganism belonging to Corynebacterium sp., it is known that argJ encodes an enzyme converting glutamate to N-acetyl glutamate, argB encodes an enzyme converting N-acetyl glutamate to N-acetylglutamyl phosphate, argC encodes an enzyme converting N-acetylglutamyl phosphate to N-acetyl glutamate semialdehyde, argD encodes an enzyme converting N-acetyl glutamate semialdehyde to N-acetyl ornithine, argJ encodes an enzyme converting N-acetyl ornithine to ornithine, argF encodes an enzyme converting ornithine to citrulline, argG encodes an enzyme converting citrulline to argininosuccinate, and argH encodes an enzyme converting argininosuccinate to arginine in the arginine synthetic pathway, and the ornithine synthetic pathway is included in the arginine synthetic pathway.
The known arginine-producing strains have been developed by introducing a mutation into the arginine operon or by promoter mutation to increase the expression levels of the enzymes involved in the arginine biosynthesis. Of them, argR controlling and suppressing the arginine operon expression and argB inhibited by arginine level have been widely studied as targets for increasing arginine production (Korean Patent Publication No. 2010-0060909)
To improve ornithine productivity, it is known that ornithine production is increased by the action of ornithine cyclodeaminase (ocd) by culturing the Corynebacterium microorganism in a medium supplemented with proline, or by modifying impellers and culturing conditions during the culture of the microorganism. Also, when a transformed E. coli is used, ornithine productivity is improved by culturing argF and argR-deleted strains in a medium supplemented with glutamate, or by using a transformed strain with the deletion of proB gene encoding γ-glutamulkinase involved in the first step of the synthetic pathway of proline from glutamate rather that the synthetic pathway of ornithine form glutamate.
Furthermore, Corynebacterium glutamicum have been consistently studied for the high-yield production of glutamate as an ornithine precursor. Glutamate excretion from Corynebacterium glutamicum is known to be increased by biotin limitation of treatment with penicillin G or a fatty acid ester surfactant. Since these treatments are correlated with damage in the cell wall, it had previously been thought that glutamate leaks passively through the damaged cell wall.
NCgl1221 protein derived from the wild type Corynebacterium glutamicum (Cgl 13032) facilities betaine efflux, and its amino acid sequence is similar to that of the E. coli mechanosensitive channel protein, yggB (Korean Patent Publication No. 2010-0017581).
DISCLOSURE
Technical Problem
In this background, the present inventors have made many efforts to develop a strain capable of producing the useful ornithine in a higher yield. As a result, they found that an ornithine-overproducing strain can be developed by blocking the biosynthetic pathway of arginine from ornithine, by blocking a protein involved in glutamate export to increase the intracellular glutamate level and by enhancing the biosynthetic pathway of ornithine from glutamate, thereby completing the present invention.
Technical Solution
An object of the present invention is to provide a microorganism having an improved ornithine-producing ability. Another object of the present invention is to provide a method for producing ornithine using the microorganism.
Advantageous Effect
The microorganism of the present invention having an improved ornithine-producing ability can be more effectively used in a wide variety of applications for ornithine production.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the ornithine biosynthetic pathway and related genes of the transformed Corynebacterium glutamicum of the present invention;
FIG. 2 shows the known arginine biosynthetic pathway of the Corynebacterium glutamicum; and
FIG. 3 shows a pDZ vector to be inserted into chromosome of the microorganism belonging to the Corynebacterium sp.



BEST MODE
In one aspect to achieve the above objects of the present invention, the present invention provides a microorganism having an improved ornithine-producing ability, in which the activities of ornithine carbamoyltransferase and a protein involved in glutamate export (NCgl1221) are modified to be attenuated, compared to their endogenous activities.
As used herein, “ornithine carbamoyltranferase (OCT)” refers to a catalytic enzyme that mediates the reaction between carbamoyl phosphate and ornithine to synthesize citrulline and phosphoric acid. OCT is present in a liver of urea-excreting animals as well as plant and microorganism, and in microorganism it is involved in arginine synthesis. The OCT enzyme comprises catalytic domain and regulatory domain, and when ornithine binds to the regulatory domain the enzyme activity is inhibited.
E. coli K12 strain has two types of OCT (ArgF and ArgI), and intestinal microorganism including E. coli B and W strains have OCT protein similar to ArgI. OCTs encoded by argF and argI have different amino acid sequences from each other, but they are considered as isoenzyme having the same function (EMBO J. (1982) 1:853857). Corynebacterium sp. strain only has OCT encoded by argF gene. OCT only acts in the synthetic pathway from ornithine to arginine, and thus if the OCT activity is weakened, the intracellular ornithine level can be increased.
For accumulation intracellular ornithine, the present invention provides a Corynebacterium microorganism in which the synthetic pathway of arginine from ornithine is blocked To achieve this, a transformed strain with a deletion of ornithine carbamoyltransferase-encoding gene was prepared. In this regard, the. ornithine carbamoyltransferase may be, but is not particularly limited to, a protein having an amino acid sequence of SEQ ID NO. 18, or a protein having 70% or more homology with the sequence, more preferably 80% or more homology with the sequence, much more preferably 90% or more homology with the sequence.
As used herein, “homology” refers to the similarity in nucleotide sequences or amino acid sequences of gene coding for a protein. When homology is sufficiently high, products of the corresponding gene may be the same or have a similar activity.
As used herein, “protein involved in glutamate export” refers to a type of mechanosensitive channels which function to export the intracellularly produced glutamate to extracellular environment. The present invention provides a Corynebacterium microorganism having improved ornithine productivity. For this work, a transformed strain capable of maintaining a high level of intracellular glutamate is prepared by deleting a gene encoding the protein that functions to excrete glutamate which is a raw material for ornithine synthesis.
By increasing the intracellular level of glutamate, i.e., a prescursor of ornithine, an ornithine biosynthetic pathway can be stimulated. In the present invention, glutamate exporting can be reduced or inhibited by diminishing the NCgl1221 activity.
The removed protein involved in glutamate export may be a protein having an amino acid sequence of SEQ ID No. 20 or an amino acid sequence having 70% or more homology thereto, and more preferably having 80% or more homology, even more preferably having 90% or more homology thereto, but is not limited thereto.
The activity of the ornithine carbamoyltransferase and the protein involved in glutamate export can be diminished by a method selected from the group consisting of (1) a partial or full deletion of a gene coding for the protein, (2) modification of an expression regulatory sequence for suppressing the gene expression, (3) modification of the nucleotide sequence on chromosome for diminishing the protein activity, and 4) a combination thereof, but is not limited thereto.
A partial or full deletion of a polynucleotide coding for the protein can be done by introducing a vector for chromosomal insertion into a microorganism, thereby replacing the polynucleotide coding for an endogenous target protein on chromosome with a partially removed polynucleotide or a marker gene. The length “partial” may vary depending on the type of polynucleotide, but specifically it refers to a length of 1 to 300 nucleotides, preferably 1 to 100 nucleotides, and more preferably 1 to 50 nucleotides.
Also, modification of an expression regulatory sequence for reducing expression of the polynucleotide can be done by inducing a modification on the expression regulatory sequence through deletion, insertion, non-conservative or conservative substitution of nucleotide sequence, or a combination thereof in order to diminish the activity of expression regulatory sequence, or by replacing the expression regulatory sequence with a nucleotide sequence having weaker activity. The expression regulatory sequence includes a promoter, an operator sequence, a sequence coding for ribosome-binding site, and a sequence regulating the termination of transcription and translation.
Furthermore modification of a polynucleotide sequence on chromosome, which codes for the enzyme of the present invention, can be done by inducing a mutation on the sequence through deletion, insertion, non-conservative or conservative substitution of polynucleotide sequence, or a combination thereof in order to diminish the enzymatic activity, or by replacing the sequence with a polynucleotide sequence which is modified to have weaker activity.
An used herein, “endogenous activity” refers to the activity of enzyme that a microorganism possesses in its native state. In the present invention, endogenous activity refers to the activity of ornithine carbamoyl transferase and a protein involved in glutamate export, i.e., NCgl1221 that a microorganism naturally possesses. Also, as used herein, “modified to have a weaker activity than an endogenous activity” refers to the state where an ornithine carbamoyl transferase and a protein involved in glutamate export, i.e., NCgl1221 do not function properly due to gene deletion or mutation and thus the activity of ornithine carbamoyl transferase and a protein involved in glutamate export, i.e., NCgl1221 that microorganism naturally possesses is weakened.
As used herein, the term “microorganism having an improved ornithine-producing ability” refers to a microorganism having a higher ornithine-producing ability than the parent strain, and the microorganism having an improved ornithine-producing ability may be, but is not particularly limited to, a microorganism that is further transformed to have higher activity acetylglutamate synthase (ArgJ) converting glutamate to acetyl glutamate (N-acetyl glutamate) or ornithine acetyltransferase (ArgJ) converting acetyl ornithine to ornithine, acetylglutamate kinase (ArgB) converting acetyl glutamate to acetylglutamyl phosphate (N-acetylglutamyl phosphate), acetyl gamma glutamyl phosphate reductase (ArgC) converting acetyl glutamyl phosphate to acetyl glutamate semialdehyde (N-acetyl glutamate semialdehyde), acetylornithine aminotransferase (ArgD) converting acetyl glutamate semialdehyde to acetylornithine (N-acetylornithine) or the like than the endogenous activity, in order to enhance the biosynthetic pathway of ornithine from glutamate.
The transformed microorganism was prepared by a method different from the known method of developing an ornithine-producing strain, that is, the known method is performed by eliminating or attenuating the function of ArgG that acts as a transciptional inhibitor in the arginine biosynthetic pathway to increase ornithine production and by further deleting the ornithine carbamoyltransferase gene and introducing feedback-resistant N-acetylglutamate synthase to increase ornithine production (Korean Publication No. 2010-0060909).
In this regard, the acetyl gamma phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), and acetylornithine aminotransferase (ArgD) may preferably have, but is not particularly limited to, the amino acid sequences of SEQ. ID NOs. 23, 25, 27, and 29, or 70% or more homology with the sequence, more preferably 80% or more homology with the sequence, and much more preferably 90% or more homology with the sequence, respectively. The increase in their activity may be performed by any one or more methods selected from the group consisting of 1) an increase in the copy number of a polynucleotide encoding the protein, and 2) an increase in the polynucleotide expression by modification of the expression control sequence, 3) enhancement of the enzyme activity by modification of the polynucleotide sequence on the chromosome, and 4) enhancement by a combination thereof.
To be specific, various methods can be used to increase the enzymatic in a microorganism in general. For example, the expression level of a polynucleotide can be increased by increasing the copy number of the polynucleotide transformation involving plasmid insertion, homologous recombination, conjugation, and translocation; modifying an expression regulatory sequence of the polynucleotide; amplifying a gene coding for a regulatory factor which stimulates the polynucleotide expression; or by deleting or inhibiting a gene coding for a regulatory factor which suppresses the polynucleotide expression. To be more specific, the expression level of a polynucleotide can be increased by operably linking a gene fragment comprising the polynucleotide to a multicopy vector which can be replicated in Corynebacterium sp. strains, by introducing single of multiple copies of the polynucleotide to the chromosome, or by replacing the expression regulatory sequence of polynucleotide with one having an improved activity including a strong promoter.
For instance, the argCJBD gene group may be transformed into a microorganism by using pHC139T vector to prepare a microorganism with significantly improved ornithine productivity. Alternatively, a microorganism in which ornithine biosynthetic pathway is enhanced may be prepared by improving a promoter region regulating the expression of argCJBD gene in the chromosome of microorganism or by replacing a promoter region by a promoter with more improved activity. In particular, a method for improving promoter region may involve, for replacing a promoter within the chromosome, preparing a gene fragment comprising nucleotide sequences of both terminal sites adjacent to the target site on the chromosome and a promoter sequence to be inserted in the same form as in the chromosome and following the same gene deletion method using a pDZ vector published by Korea Patent Publication No. 2009-0082702, but is not limited thereto. Here, the improved promoter may preferably be, but is not limited to, the pcj7 (or P(CJ7) promoter having a nucleotide sequence of SEQ ID No. 30 (Korea Patent Registration No. 0620092). The pDZ vector may preferably be, but is not limited to, a vector represented by a cleavage map of FIG. 3.
As used herein, “vector” reek to a DNA construct comprising a nucleotide sequence of gene which is operably linked to so appropriate expression regulatory sequence to express a target gene in a suitable host cell. The expression regulatory sequence comprises a promoter that can initiate transcription, an optional operator sequence for regulating the transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.
Examples of conventional vectors include a natural or recombinant plasmid, cosmid virus and bacteriophage. For instance, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt11, λgt11, Charon4A, and Charon21A can be used as a phage vector or cosmid vector. As a plasmid vector, pDZ vector, pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL type, and pET type may be used. A usable vector is not particularly limited, and any known expression vector preferably pDZ vector, can be used.
Meanwhile, the microorganism of the present invention may be, but is not particularly limited to, a microorganism prepared by transformation of a microorganism belonging to the Escherichia sp., the Shigella sp., the Citrobacter sp., the Salmonella sp., the Enterobacter sp., the Yersinia sp., the Klebsiella sp., the Erwinia sp., the Corynebacterium sp., the Brevibacterium sp., the Lactobacillus sp., the Selenomanas sp., or the Vibrio sp., which has the activities of ornithine carbamoyltransferase and a protein involved in glutamate export (NCgl1221).
Preferably, the microorganism of the present invention may be may be Corynebacterium sp. strain, and more preferably Corynebacterium glutamicum. To be more specific, a wild-type strain Corynebacterium glutamicum ATTC 13032 f or a glutamate-overproducing strain KCCM-10735P (Korea Patent Publication No. 2008-0034334) may be used, but is not limited thereto. The KCCM-10785p strain is a glutamate-overproducing strain generated by deleting cg2624 (NCBI LOCUS ID YP_226636) and cg2115 (NCBI LOCUS ID YP_11074) which was generated by using mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG). Although glutamate overproduction by deletion of cg2624 and cg2115 have not been identified prior to the above publication, cg2624 is identified as pcaR, which is an IclR family regulatory protein, and the cg2115 is identified as sugR, which is a transcriptional regulator of sugar metabolism.
According to one embodiment of the present invention, a Corynebacterium glutamicum strain with a deletion of argF gene (ATTC 13032 ΔarF and KCCM-10785P ΔargF) (Example 1), a Corynebacterium glutamicum strain with deletions of argF and NCgl1221 genes (ATCC 13032 ΔargF ΔNCgl1221 and KCCM-10785P ΔargF ΔNCgl1221) (Example 2), a Corynebacterium glutamicum strain with deletions of argF and NCgl1221 genes and with an introduction of argCJBD gene (ATCC 13032 ΔargF ΔNCgl1221/pHC139T-argCJBD (Cgl) and KCCM-10785P ΔargF ΔNCgl1221/pHC139T-argCJBD (Cgl)) (Example 3-1), and a Corynebacterium glutamicum strain with deletions of argF and NCgl1221 genes and with replacement of the promoter of the argCJBD gene cluster in the chromosome (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD and KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD) (Example 3-2) were prepared. The result of comparing their ornithine productivities showed that the Corynebacterium glutamicum strain with deletions of argF and NCgl1221 genes and with replacement of the promoter of the argCJBD gene cluster in the chromosome (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD and KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD) has excellent ornithine productivity (Tables 5 and 6).
Therefore, the ornithine-producing strain having an improved ornithine-producing ability was designated as “CC01-0061 (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD)”, and deposited under the Budapest Treaty to the Korean Culture Center of Microorganisms whose address is Hongje-1-dong, Seodaemun-gu, Seoul on Nov. 24, 2010, with Accession No. KCCM11137P.
In another aspect of the present invention to achieve the above objects, the present invention provides a method for producing ornithine, including the steps of (i) culturing the microorganism having an improved ornithine-producing ability to obtain a culture; and (ii) recovering ornithine from the cultured microorganism or the culture.
In the method, culturing the microorganism may preferably be done by batch culture, continuous culture, and fed-batch culture known in the art, but is not limited thereto. Furthermore, as for the culturing condition, and optimal pH of 5 to 9, preferably pH 6 to 8, and most preferably pH6.8 can be maintained by using a basic chemical (for example: sodium hydroxide, potassium hydroxide or ammonia) or acidic chemical (for example: phosphoric acid or sulfuric acid). Also, an aerobic condition can be maintained by adding oxygen or oxygen-containing gas mixture to a cell culture. The culturing temperature may be maintained at 20° C. to 45° C., and preferably at 25° C. to 40° C. In addition, it is preferable to culture for about 10 to 160 hours. The ornithine produced by the above culturing may be excreted to a culture medium or remain inside the cell.
Furthermore, the medium for culturing may comprise sugar and carbohydrate (for example: glucose, sucrose, lactose, fructose, maltose, molasse, starch and cellulose), oil and fat (for example: soybean oil, sunflower seed oil, peanut oil and coconut oil), fatty acid (for example: palmitic acid, stearic acid and linoleic acid), alcohol (for example: glycerol and ethanol), and organic acid (for example: acetic acid) individually or in combination as a carbon source; nitrogen-containing organic compound (for example: peptone, yeast extract, meat juice, malt extract, corn solution, soybean meal powder and urea), or inorganic compound (for example: ammonium sulfate, ammonium chloride, phosphate or ammonium, ammonium carbonate, and ammonium nitrate) individually or in combination as a nitrogen source; potassium dihydrogen phosphate, dipotassium phosphate, or sodium-containing salt corresponding thereto individually or in combination as a phosphorus source; other essential growth-stimulating substances including metal salts (for example: magnesium sulfate or iron sulfate), amino acids, and vitamins.
[Mode for Invention]
Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
EXAMPLE 1
Preparation of argF-Deleted Corynebacterium Glutamicum Strain
In this example, and argF-deleted strain was prepared from a wild-type Corynebacterium glutamicum strain ATCC 13032 and a glutamate-overproducing strain KCCM-10785P which was generated by deleting cg2624 and cg2115 genes in a glutamate-producing strain KFCC-11074 generated by using mutagen such as NTG (Korea Patent Publication No. 2008-0034334) in order to block a synthetic pathway or arginine from ornithine. The arginine biosynthetic genes of Corynebacterium glutamicum ATCC 13032 are organized in an operon having a form of argCJBDFRGH, and a deletion target argF gene (SEQ ID No. 17) is present adjacent to the genes coding for enzymes involved in ornithine synthetic pathway on the chromosome. Thus, a plasmid for deleting argF gene was prepared based on the nucleotide sequence of argD and argR which are located adjacent to the deletion target argF gene.
To be specific, based on the nucleotide sequence of argD and argR of the ATCC 13032 strain, a homologous recombination fragment adjacent to the N-terminal sequence of argF and a homologous recombination fragment adjacent to the C-terminal sequence of argF were constructed. For this, the fragment adjacent to the N-terminal sequence of argF was obtained by PCR using the genomic DNA from ATCC 13032 strain as a template, and primers (SEQ ID Nos. 1 and 2) (28 cycle of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 30 seconds at 72° C.). Likewise, the fragment adjacent to the C-terminal sequence of argF was obtained by PCR using the genomic DNA from ATCC 13032 strain as a template, and primers (SEQ ID Nos. 3 and 4) under same PCR condition (Table1)







TABLE 1







Primers for preparation of argF-deleted 


strain (ΔargF)










SEQ. 



Name
ID NO.
Sequence(5′-3′)





argF-del-
1
CGGGATCCTGGCCGTACCGGCGATTTCT


F1_BamHI




 


argF-del-
2
CGCGTCGACAAGTTTGAGTCCTTTATGCG


R1_SalI




 


argF-del-
3
CGCGTCGACGACATGTCCCTTGGCTCAAC


F2_SalI




 


argE-del-
4
TGCTCTAGAAGTAATTCACCTAGTTCTTTACC


R2_XbaI









The above-prepared homologous recombination fragment adjacent to the N-terminal sequence of argF was digested with BamHI and SalI, and the homologous recombination fragment adjacent to the C-terminal sequence of argF was digested with SalI and XbaI. Then each of the cleaved fragments were inserted into the pDZ vector which was also digested with BamHI and XbaI, thereby producing a plasmid pDZ-argF(K/O).
The above-prepared plasmid pDZ-argF(K/O) was transformed into the ATCC 13032 strain and KCCM-10785P strain. Then, the transformed strains were plated and cultured on BHIS plate Braine heart infusion 37 g/l, sorbitol 91 g/l, agar 2%) which contains kanamycin (25 μg/ml) and X-gal (5-bromo-4-chloro-3-indolin-β-D-galactoside), while letting the colonies to grow on the plate. Among the colonies formed on the plate, colonies with blue colour was collected to select for the strain inserted with the plasmid pDZ-argF(K/O).
The above-selected strains were cultured with shaking in CM medium (glucose 10 g/l, polypeptone 10 g/l, yeast extract 5 g/l, beef extract 5 g/l, NaCl 2.5 g/l, urea 2 g/l, pH 6.8) at 30° C. for 8 hours. Subsequently, each cell culture was serially diluted from 10−4 to 10−10. Then the diluted samples were plated and cultured on an X-gal-containing solid medium letting the colonies to grow. Among the colonies formed on the plate, only the white colonies which appear at relatively low frequency were collected to select for the argF-deleted strains.
Successful insertion of the plasmid pDZargF(K/O) into the above-selected strains was confirmed by performing PCR using the chromosomal DNA from the above-selected strain as a template, and primers of SEQ ID Nos. 1 and 4. Through this PCR confirmation, it was confirmed that the above-selected strain is the argF-deleted strain (i.e., ATCC 13032 ΔargF and KCCM-10785P ΔargF).
EXAMPLE 2
Preparation of argF- and NCgl1221-deleted Corynebacterium Glutamicum Strain
NCgl1221 gene encoding the protein involved in glutamate export was further deleted in ATCC 13032 ΔargF strain and KCCM-10785P ΔargF strain obtained in Example 1 in order to increase the intracellular of glutamate which is an ornithine precursor.
To be specific, based on the nucleotide sequence (SEQ ID No. 19) of NCgl1221 the ATCC 13032 strain, a homologous recombination fragment adjacent to the N-terminal sequence of NCgl1221 and a homologous recombination fragment adjacent to the C-terminal sequence of NCgl1221 were constructed. For this, the fragment adjacent to the N-terminal adjacent sequence of NCgl1221 was generated by PCR using the genomic DNA from ATCC13032 strain as a template and primers (SEQ ID Nos. 5 and 6), and the fragment adjacent to the C-terminal sequence of NCgl1221 was generated by PCR using the genomic DNA from ATCC 13032 strain as a template and primers (SEQ ID Nos. 7 and 8) under the same PCR condition as in Example 1 (Table 2).







TABLE 2







Primers for preparation of NCgl1221-deleted strain










SEQ 



Name
ID NO.
Sequence (5′-3′)





NCgl1221-del-
5
CGGGATCCGTCCAAGCCAAGCCGATTTCAAC


F1_BamHI




 


NCgl1221-del-
6
ACGCGTCGACCCACTCGGCGCTTGATAATAC


R1_SalI




 


Ncgl1221-del-
7
ACGCGTCGACCTGGAACAAGAACTCTCCAGC


F2_SalI




 


NCgl1221-del-
8
CTAGTCTAGA GGTTGGTGCTTCCACTGCTG


R2_XbaI









The above-prepared homologous recombination fragment adjacent to the N-terminal sequence of NCgl1221 was digested with BamHI and SalI. Likewise, the homologous recombination fragment adjacent to the C-terminal sequence of NCgl1221 was digested with SalI and XbaI. Then each of the cleaved fragments was inserted into the pDZ vector that was cleaved with BamHI and XbaI, thereby producing a plasmid pDZ-NCgl1221(K/O).
The above-prepared plasmid pDZ-NCgl1221(K/O) was transformed into ATCC 13032 ΔargF strain and KCCM-10785P ΔargF strain. Then, the transformed strains were plated and cultured on BHIS plate (Braine heart infusion 37 g/l, sorbitol 91 g/l, agar 2%) which contains kanamycin (25 μg/ml) and X-gal (5-bromo -4-chloro-3-indolin-β-D-galactoside), while letting the colonies to grow on the plate. Among the colonies formed on the plate, colonies with blue colour was collected to select for the strain inserted with the plasmid pDZ-NCgl1221(K/O).
The above-selected strains were cultured with shaking in CM medium at 30° C. for 8 hours. Subsequently, each cell culture was serially diluted from 10−to 10−10. then the diluted samples were plated and cultured on an X-gal-containing solid medium, letting the colonies to grow. Among the colonies formed on the plate, only the white colonies which appear relatively low frequency were collected to select for the NCgl1122l-deleted strains.
Successful insertion of the plasmid pDZ-NCgl1221(K/O) into the above-selected strains was confirmed by performing PCR using the chromosomal DNA from the above-selected strain as a template, and primers of SEQ ID Nos. 5 and 8. The selected NCgl1221-deleted strains were named as ATCC 13032 ΔargF ΔNCgl1221 or KCCM-10785P ΔargF ΔNCgl1221 accordingly.
EXAMPLE 3
Preparation of argCJBD-introduced Corynebacterium Glutamate Strain
EXAMPLE 3-1
Cloning of argCJBD Gene and Preparation of Transformant
In this example, a vector inserted with argC, argJ, argB, and argD genes (SEQ ID Nos. 22, 24, 26, and 28 and respectively) was prepared and a transformant was prepared by introducing the same, in order to enhance the ornithine synthetic pathway by increasing the copy number of argCJBD (SEQ ID No. 21, comprising the promoter region) which codes for the enzymes involved in a synthetic pathway of ornithine from glutamate.
First, PCR was performed to obtain argCJBD gene by the chromosome of ATCC 13032 strain as a template and primers (SEQ ID Nos. 9 and 10, Table 3) (30 cycles of denaturation for 40 seconds at 95° C., annealing for 40 seconds at 55° C., and extension for 150 seconds at 72° C.), thereby obtaining a gene fragment having a size of 4,900 bp.







TABLE 3







Primiers for acquisition of argCJBD gene 


fragment of ATCC 13032










SEQ



Name
 ID NO.
Sequence (5′-3′)





P_argC-5-
 9
CGGGGTACCCTCCTCCAGCAGCTCTAGCTC


KpnI




 


argD-3_XbaI
10
TGCTCTAGAAAGTTTGAGTCCTTTATGCG









The above-prepared gene fragment was run through gel electrophoresis on 0.8% agarose gel, and a band of the target size was cut and DNA sample was isolated therefrom. The isolated DNA was digested with KpnI and XbaI to obtain a fragment, then the cleaved fragment was cloned into a pHC139t-gfp vector (Korea Patent Publication No. 2008-0074286), thereby producing an expression vector pHC139T-argCJBD(Cgl).
Subsequently, the expression vector pHC139T-argCJBD(Cgl) prepared for increasing the production level of ornithine in the cell was introduced into ATCC 13032 ΔargF NCgl1221 strain and KCCM-10785P and ΔargF ΔNCgl1221 strain through electroporation. Then, a successful transformant was selected by plating the transformed cells on BHIS plate containing 25 μg/ml kanamycin. Finally, each of the selected transformants was named as ATCC 13032 ΔargF ΔNCgl1221/pHC139T-argCJBF(Cgl) and KCCM-10785P ΔargF ΔNCgl1221/pHC139T-argCJBD(Cgl) accordingly.
EXAMPLE 3-2
Substitution of the Promoter of argeCJBD Gene in the Chromosome
In this example, a promoter of argCJBD was substituted with CJ7 promoter which was newly developed by the present applicant in the chromosome, in order to increase the expression level by removing the regulation of the argCJBD gene which codes for the enzymes involved in a synthetic pathway ornithine from glutamate.
First, a homologous recombination fragment comprising a CJ7 promoter and a nucleotide sequence of both terminal sites of the promoter was prepared.
To be specific, the nucleotide sequence of 5′-terminal site of CJ7 promoter was obtained by performing PCR using the genomic DNA from ATTC 13032 strain as a template and primers (SEQ ID Nos. 11 and 12) (28 cycles of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 30 seconds at 72° C.). Likewise, the nucleotide sequence of CJ7 promoter region was obtained by PCR using primers (SEQ ID Nos. 13 and 14) under same PCR condition, and the nucleotide sequence of 3′-terminal site of CJ7 promoter was obtained by PCR using the genomic DNA from ATCC 13032 strains as a template and primers (SEQ ID Nos. 15 and 16) under same PCR condition.







TABLE 4







Primers for substitution of argCJBD gene promoter










SEQ



Name
 ID NO.
Sequence (5′-3′)





argC-L-
11
CGGGATCCGCAACGCTTGCGGTGAGAGA


5-BamHI




 


arC-L-
12
CCGGAATTCCTGGAAGTGGTCGAAGAAGA


3-EcoRI




 


CJ7-5-
13
CCGGAATTCGCCGGCATAGCCTACCGATG







EcoRI









CJ7-3-
14
TGCTCTAGAGATATCAGTGTTTCCTTTCG


XbaI




 


artC-R-
15
TGCTCTAGAATGATAATGCATAACGTGTA


5-XbaI




 


argC-R-
16
ACGCGTCGACGCTTTCCGGAGGTGTTGTAC


3-SalI









The above-prepared 5′-terminal site fragment of promoter (argC-L) was digested with BamHI and EcoRI, the CJ7 promoter region fragment was digested with EcoRI and XbaI, and the 3′-terminal site fragment of promoter (argC-R) was digested with XbaI and SalI. then each of the cleaved PCR products was cloned into the pDZ vector which was also digested with BamHI and SalI, thereby producing an expression vector pDZ-CJ7(arg) in which the promoter of argCJBD was substituted with CJ7 promoter.
The above-prepared expression vector pDZ-CJ7(arg) was transformed into ATCC 13032 ΔargF ΔNCgl1221 strain and KCCM-10785P ΔargF ΔNCgl1122I strain through electroporation. Then, the transformants were cultured with shaking in CM medium (30° C., 8 hours), and the cell culture was serially diluted from 10−4 to 10−10. Then, the diluted samples were placed and cultured on BHIS plate containing 25 μg/ml kanamycin and X-gal, letting the colonies to grow.
The white colonies which appear at low frequency were isolated from most of the blue colonies, thereby selecting only the strain where the arg promoter was successfully substituted with CJ7 promoter through double crossover.
Successful substitution of argCJBD promoter in chromosome by the introduced expression vector pDZ-CJ7(arg) was confirmed by performing PCR using the genomic DNA from the above-selected strains as a template and primers (SEQ ID Nos. 13 and 16) (28 cycles of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 60 seconds at 72° C.). Finally, the confirmed strains were named as ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD and KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD accordingly.
EXAMPLE 4
Improvement of Ornithine Productivity by Deletion of argF and NCgl1221 Genes and Enhancement of argCJBD Expression Level
EXAMPLE 4-1
Ornithine Productivity of ATCC 13032 Corynebacterium Glutamicum-cerived Strain
In order to examine whether ornithine productivity is affected by deletion of argF and NCgl1221 genes and enhancement of argCJBD expression level in the ATCC 13032 Corynebacterium glutamicum-derived strains, the ornithine-producing ability between the strains prepared in Examples 2 and 3 was compared.
In detail, each of the strains prepared in Examples 2 and 3 (ATCC 13032 ΔargF ΔNCgl1221, ATTC 13032 ΔargF ΔNCGl1221/pHC139T-argCJBD(Cgl), ATTC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCNBD) was spread on a CMA plate containing 1 mM arginine, and cultured at 37° C. for 24 hours. Each of the strains cultured was inoculated in 25 ml of titration medium (2% (w/v) glucose, 1% (w/v) polypeptone, 0.5% (w/v) yeast extract, 0.5% (w/v) (NH4)2SO4, 0.15% (w/v) urea, 0.4% (w/v) KH2PO4, 0.8% (w/v) K2HPO4, 0.05% (w/v) MgSO4, 100 μg/l biotin and 1 mg/l thiamine) containing 1 mM arginine, and then cultured with shaking at 30° C. and 200 rpm for 48 hours and the concentration of ornithine produced in the each culture was determined and compared to each other (Table 5). At this time, strain ATCC13032 with no genomic modification was used as a control group.







TABLE 5







Comparison of ornithine productivity in ATCC 13032-derived


strains









Ornithine



content


Strain
(g/l)





ATCC 13032
0.0


ATCC 13032 ΔargF ΔNCgl1221
6.0


ATCC 13032 ΔargF ΔNCgl1221/pHC139T-argCJBD(Cgl)
6.4


ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD
7.7









As shown in Table 5, the argF and NCgl1221-deleted strain produced 6.0 g/l of ornithine, which was not produced by the wild-type strain. With respect to the increase of argCJBD gene expression level, when the argCJBD gene was introduced in the form of vector, the concentration of the produced ornithine was 6.4 g/l, and when the argCJBD promoter on the chromosome was replaced by CJ7, the concentration of the produced ornithine was slightly increased to 7.7 g/l.
EXAMPLE 4-2
Ornithine Productivity of Glutamate-producing Corynebacterium Glutamicum KCCM-10785P-derived Strain
In order to examine whether ornithine productivity is affected by deletion of argF and NCgl1221 genes and enhancement of argCJBD expression level in the strain Corynebacterium glutamicum strain KCCM-10785P overproducing glutamate, precursor of ornithine, the ornithine-producing ability between the strains prepared in Examples 2 and 3 was compared.
In detail, each of the strains prepared in Examples 2 and 3 (KCCM-10785P ΔargF ΔNCgl1221, KCCM-10785P ΔargF ΔNCgl1221/pHC139T-argCJBD(Cgl), KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD) was inoculated in the same manner as in Example 4-1, and then cultured with shaking at 30° C. and 200 rpm for 48 hours, and the concentration of ornithine produced in each culture was determined and compared to each other (Table 6). At this time, a strain KCCM-10785P with no genetic modification was used as a control group.







TABLE 6







Comparison of ornithine productivity in KCCM-10785P-derived


strains












Glutamate
Ornithine




content
content



strain
(g/l)
(g/l)







KCCM-10785P
15.5 
0.0



KCCM-10785P ΔargF ΔNCgl1221
5.2
7.6



KCCM-10785P ΔargF ΔNCgl1221/
4.8
7.9



phC139T-argCJBD(Cgl)





KCCM-10785P ΔargF





ΔNCgl1221 P(CJ7)-argCJBD
2.0
9.0










As shown in Table 6, argF and NCgl1221 deletions in the glutamate-overproducing strain showed 7.6 g/l ornithine production, which was not produced by the wild-type strain. With respect to the increase of argCJBD gene expression level, when the argCJBD gene was introduced in the form of vector, the concentration of the produced ornithine was 7.9 g/l, and when the argCJBD promoter on the chromosome was replaced by CJ7, the concentration of the produced ornithine was slightly increased to 9.0 g/l.
Accordingly, it can be seen that ornithine production can be increased through enhancement of the synthetic pathway of ornithine by increasing the argCJBD gene expression level.
Therefore, the present inventor designated the strain having the most excellent ornithine productivity prepared in Example 3-2 as “CC01-0061 (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD)”, and deposited it under the Budapest Treaty to the Korean Culture Center of Microorganisms whose address is Hongje-1-dong, Seodaemun-gu, Seoul on Nov. 24, 2010, with the Accession No. KCCM11137P.
It will be apparent to those skilled in the art that various modifications and changes may be made without departing from the scope and spirit of the invention. Therefore, it should be understood that the above embodiments are not limitative, but illustrative in all aspects. Therefore all changes and modifications that fall within metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the claims.

## Claims

1. A microorganism belonging to Corynebacterium glutamicum having an ornithine-producing ability, wherein the activities of ornithine carbamoyltransferase and a protein involved in glutamate export are modified to be attenuated, compared to their endogenous activities;
wherein the protein involved in glutamate export has an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having 70% or more homology with SEQ ID NO: 20.

2. The microorganism according to claim 1, wherein the ornithine carbamoyltransferase has an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having 70% or more homology with the sequence.

3. The microorganism according to claim 1, wherein the activity of the ornithine carbamoyltransferase and the protein involved in glutamate export is attenuated by a method selected from the group consisting of (1) a partial or full deletion of a gene coding for the protein, (2) modification of an expression regulatory sequence for suppressing the gene expression, (3) modification of the gene sequence on chromosome for diminishing the protein activity, and (4) a combination thereof.

4. The microorganism according to claim 1, wherein activities of acetyl gamma glutamyl phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB) and acetylornithine aminotransferase (ArgD) are further enhanced, compared to their endogenous activities.

5. The microorganism according to claim 4, wherein each of ArgC, ArgJ, ArgB, and ArgD has an amino acid sequence of SEQ ID NOs: 23, 25, 27, and 29, or an amino acid sequence having 70% or more homology with the sequence.

6. The microorganism according to claim 4, wherein the activity of proteins is enhanced by a method selected from the group consisting of (1) increase of the copy number of a polynucleotide coding for the protein, (2) modification of an expression regulatory sequence for increasing the polynucleotide expression, (3) modification of the polynucleotide sequence on a chromosome for enhancing an activity of the enzyme, and (4) a combination thereof.

7. The microorganism according to claim 1, wherein the microorganism is Corynebacterium glutamicum.

8. A method for producing ornithine, comprising the steps of:
(i) culturing the microorganism according to claim 1; and
(ii) recovering ornithine from the cultured microorganism or the culture.