KR101622460B1 - Corynebacterium glutamicum having improved L-ornithine production and method for preparing L-ornithine using the same - Google Patents

Abstract

The present invention relates to a Corynebacterium glutamicum microorganism having improved L-ornithine producing ability, characterized in that the activity of a cell membrane protein is reduced or inactivated, a recombinant vector having reduced or inactivated cell membrane protein activity , A method for producing the Corynebacterium glutamicum microorganism and a method for producing L-ornithine using the same.

Description

Ornithine production method and a method for producing L-ornithine using the L-ornithine production method and a method for producing L-ornithine using the same,

The present invention relates to a Corynebacterium glutamicum microorganism having improved L-ornithine producing ability, characterized in that the activity of a cell membrane protein is reduced or inactivated, a recombinant vector having reduced or inactivated cell membrane protein activity , A method for producing the Corynebacterium glutamicum microorganism and a method for producing L-ornithine using the same.

L-ornithine is one of the amino acids present on elemental circuits in vivo and is known as a medicinal component that promotes liver function as an intermediate in arginine biosynthesis. L-ornithine has a detoxifying function against ammonia, such as citrulline and proline, and is used not only as a therapeutic agent for liver diseases, but also as a food material for preventing obesity, and has a novel function such as skin- .

On the other hand, the genus Corynebacterium sp .) microorganisms such as Corynebacterium glutamicum are gram-positive microorganisms that are widely used for the production of L-amino acids. Since the method of producing L-amino acid using microorganisms of the genus Corynebacterium is important, many attempts have been made to improve the production method thereof.

Specifically ornithine microorganisms used in the production of tin is required citrulline or arginine nutrition by ultraviolet treatment or the like configured mutant master micro Lactococcus glue Tommy kusu (Micrococcus glutamicus (Kyoho Kako Kogyo Co., Ltd., Japan, US Patent No. 2,988,489), Brevibacterium ( Brevibacterium lactofermentum , B. kawasaki , B. flavum (Ajinomoto, Japanese Patent Publication Nos. 68-8712, 68-8714 and 69-26,910), B. ketoglutamicum ), Arthrobacter paraffineus , C. hydrocarboclastus (Kyowa Hakko Kogyo Co., Ltd., U.S. Patent No. 3,574,061), and the like are known. It is also known that A. citreus , B. lactofermentum , C. glutamicum ( azurin ), which are auxotrophic strains of arginine and citrulline, It is known to use an auxotrophic mutant strain resistant to mycophenolic acid or ornithinol in the production of ornithine by screening.

The above-mentioned U.S. Patent No. 3,574,061 discloses a method for producing L-ornithine by culturing Brevibacterium keto glutamicum (ATCC 21092), an auxotrophic mutant, in a medium containing a hydrocarbon, for example, paraffin as a main carbon source However, according to this method, the productivity of ornithine was not as high as 8.6 mg / ml in the culture for 4 days.

However, since industrial mass production of L-ornithine is still required, there is a need to further develop the production method of L-ornithine.

On the other hand, L- ornithine is a genus Corynebacterium (Corynebacterium sp), one of the microorganisms Corynebacterium glutamicum (Corynebacterium glutamicum ) microorganism, and the biosynthetic process comprises the steps of: N-acetylglutamate kinase encoded by the argB gene; N-acetylglutamate kinase encoded by the argC gene; Acetylglutamate dehydrogenase, N-acetylglutamate dehydrogenase, N-acetylornithine aminotransferase encoded by the argD gene, and ornithine N-acetyltransferase (SEQ ID NO: ornithine N-acetyltransferase.

In a previous study, the present inventors found that the production of L-ornithine by the homologous expression of the argCJBD operon gene in a mutant strain in which citrulline and proline biosynthesis genes are defective and feedback control by arginine suppressor (ArgR) is blocked , But the increase in the production of L-ornithine was not dependent on the additional supply of glutamate (Hwang et al., (2008) J Microbiol Biotechnol 18: 704-710]. However, in order to maintain the maximum carbon flux in the biosynthesis pathway of L-ornithine, there is still a need for a method for producing L-ornithine through other methods as well as overexpression of the argCJBD gene.

Under such background, the present inventor has made intensive efforts to develop a method for more effectively producing L-ornithine. As a result, when a part of NCgl0929 ORF base sequence presumed to be a cell membrane protein coding sequence is derived from a chromosome, L - production of ornithine can be increased, and the present invention is completed.

An object of the present invention is to provide a method for producing L-ornithine, which is characterized in that the activity of an amino acid transporter, which is an intrinsic plasma membrane protein, is reduced or inactivated, and Corynebacterium glutamicum ) microorganisms.

It is still another object of the present invention to provide a method for producing Corynebacterium glutamicum microorganisms with improved L-ornithine production ability.

Another object of the present invention is to provide a method for producing L-ornithine using the Corynebacterium glutamicum microorganism with improved L-ornithine producing ability.

In one embodiment of the present invention for achieving the abovementioned objects is improved from Corynebacterium glutamicum (Corynebacterium ability intrinsic membrane protein of amino acid transport, characterized in that the decrease in activity or inactivation, L- ornithine Porter production glutamicum ) microorganisms.

Ornithine-producing microorganism by inducing the deletion of a part of the NCgl0929 gene base sequence, thereby inactivating the function of the cell membrane protein encoded by the NCgl0929 gene, thereby increasing the intracellular glutamate concentration and enhancing the L- It provides four bacterium glutamicum microorganisms.

In one embodiment of the present invention, the decrease or inactivation of the activity may be a Corynebacterium glutamicum microorganism which is obtained through site-specific deletion of a gene coding for the cell membrane protein of SEQ ID NO: 1, and more specifically, The gene coding for the cell membrane protein may be the NCg10929 gene of SEQ ID NO: 2.

In the present invention, the "reduction or inactivation of the activity" as referred to above means the insertion mutation by inserting one or more base pairs into the gene coding for the cell membrane protein, the deletion mutation which deletes one or more base pairs in the gene and the nonsense codon Or a transition of a base pair that introduces a different codon or a transversion mutation, and preferably a method of deleting the gene by homologous recombination is used But is not limited thereto.

&Quot; Inactivated " means that the expression of the gene encoding the cell membrane protein is decreased to a low level or is not expressed at all, and when the expression is expressed, the activity is lost or decreased. The mutation As well as by any inert method known in the art.

The inventors of the present invention have focused on the discharge and absorption pathway of glutamate corresponding to the precursor of L-ornithine or L-ornithine biosynthesis while carrying out various studies to develop microorganisms having excellent productivity of L-ornithine. It is known that the cell membrane protein expressed from the lysE gene is directly involved in the pathway of lysine and arginine in the Corynebacterium sp. Microorganism cells. In recent years, the cell membrane protein expressed from the NCgl1221 gene has been directly involved in the release pathway of glutamate (Nakamura et al., (2007) Appl Environ Microbiol 73: 4491-4498]. However, there is no known cell membrane protein directly involved in the excretion and absorption pathways of ornithine in Corynebacterium sp. Microorganisms, for example, Corynebacterium glutamicum cells. Ornithine production ability by changing the activity of a cell membrane protein having a similar function. The present invention has been completed based on this finding.

The term "L-ornithine" of the present invention is a basic amino acid present on the urea element in vivo of a higher animal, and protects the liver by converting ammonia, which is highly toxic to the liver, ≪ / RTI > L-ornithine is widely used as a food material to prevent obesity by promoting muscle metabolism and promoting basic metabolism by secretion of growth hormone. Recently, it has been found that there are new functions such as skin cosmetic action such as wrinkle improvement. L-ornithine is widely used as a material for muscle strengthening, obesity prevention and immunity enhancement in the form of L-ornithine alpha-keto glutamate salt (OKG: L-ornithine and alpha-keto glutamate are contained in a ratio of 2: 1) . L-ornithine is used as a medicinal product for improving hepatic disorder in Europe, and as a food material in the form of L-ornithine in Japan, and as a food supplement in the United States.

The term " L-ornithine production " in the present invention means a series of metabolic processes that produce ornithine from glutamate using a series of enzymes expressed from genes that are inherently present in microorganisms.

The term " Corynebacterium glutamicum microorganism having improved L-ornithine production ability "of the present invention means a microorganism capable of producing L-ornithine by the present invention on the chromosome of Corynebacterium glutamicum microorganism, Is deleted, and microorganisms capable of enhancing ornithine production ability by accumulating intracellular glutamate by deletion of NCgl0929 can be encompassed without limitation. The microorganism of the present invention is a microorganism of the genus Corynebacterium glutamicum.

The gene coding for the cell membrane protein is not particularly limited, but NCgl0929 of SEQ ID NO: 2 can be used. The microorganism of Corynebacterium glutamicum is not particularly limited, but preferably Corynebacterium glutamicum SJ9518 (Accession No. KACC91938P).

The term "NCgl0929 gene" of the present invention is also referred to as "Cgl0968 gene ", and the protein encoded by NCgl0929 gene in Corynebacterium glutamicum cells contains 28% or more of ArcD protein encoded by the ydgI gene of Escherichia coli Lt; / RTI > The NCgl0929 gene is a polynucleotide consisting of 1,506 nucleotides (SEQ ID NO: 2) and is presumed to produce an amino acid transporter comprising the amino acid sequence of SEQ ID NO: 1 consisting of 501 amino acids.

The term " homology " in the present invention means a base sequence or a similar degree of amino acid sequence of a gene encoding a protein. If the homology is sufficiently high, the expression product of the gene may have the same or similar activity.

In the present invention, it was confirmed that when the NCgl0929 gene was partially deleted in the chromosome of the L-ornithine producing microorganism, the productivity of L-ornithine was improved. In order to improve the productivity of L-ornithine, NCgl0929 gene May be partially or wholly deleted, and the productivity may be improved by using a Corynebacterium glutamicum microorganism.

The term "amino acid transporter" as used herein means a protein which is involved in the release or absorption of various kinds of amino acids as cell membrane proteins, and can be used as a substrate. Depending on the substrate used, A wide variety of transporter proteins may be present. In Example 4 of the present invention, it was confirmed that the production ability of L-ornithine was improved by deleting a part of the sequence of NCgl0929 gene presumed to encode an amino acid transporter (Table 4). Therefore, in the present invention, as long as it has the same activity as the above-mentioned amino acid transporter, not only the amino acid sequence of SEQ ID NO: 1 but also 80% or more, preferably 90% or more, more preferably 95% Or an amino acid sequence having a homology of 97% or more. As the sequence having such homology, any amino acid sequence which represents a protein exhibiting substantially the same or equivalent activity as the respective proteins can be included without limitation. It is also apparent that amino acid sequences in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention, provided that they have such homology.

In addition, the gene encoding each protein of the present invention may contain not only the nucleotide sequence coding for the amino acid sequence shown in the above SEQ ID NOs, but also the nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more , More preferably not less than 98%, and most preferably not less than 99%, of a protein having substantially the same or equivalent activity as the above-mentioned respective proteins. Also, it is obvious that base sequences having deletion, modification, substitution or addition of some sequences are also included within the scope of the present invention if they are base sequences having such homology.

More specifically, in one embodiment of the present invention, Corynebacterium glutamicum SJ8260 (ATCC13032, argFΔ, argRΔ, NCgl2053Δ, NCgl2582Δ, NCgl0281Δ) (KACC91800P) was used as the parent strain, and NCg10929 One example of the present invention is a mutant strain in which a part or all of the gene encoding glucose dehydrogenase (GDH) is additionally deleted. Glutamic acid.

Another embodiment of the present invention is a method for producing a cell membrane protein comprising the steps of: introducing an insertion mutation by inserting one or more base pairs into a gene encoding a cell membrane protein, a deletion mutation having deletion of one or more base pairs in the gene and a nonsense codon or a different codon Wherein the activity of the cell membrane protein is reduced or inactivated, including at least one mutation selected from the group consisting of a base pair transition or a transversion mutation.

Specifically, the gene coding for the cell membrane protein may be the NCg10929 gene of SEQ ID NO: 2.

As used herein, the term " recombinant vector " refers to a vector that can not be cloned separately from the chromosome of a host cell, and that includes a gene construct containing an essential component whose gene insert can be homologously recombined with a specific gene on the chromosome of the host cell Specifically, an antibiotic resistance gene may be contained as an antibiotic marker, and more specifically, a kanamycin resistance gene may be used as the antibiotic resistance gene.

Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in their natural or recombinant state. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as the phage vector or cosmid vector, and pBR type, pUC type, pBluescriptII type , pGEM system, pTZ system, pCL system, pET system and the like can be used. Specifically, those derived from pK18mobsacB vector can be used.

More specifically, the recombinant vector may further include a gene in which part or all of the NCgl0929 gene has been deleted, and the recombinant vector may be pSJC3501 having the cleavage map shown in FIG. 1, but is not limited thereto.

Another embodiment of the present invention includes a step of mutating a gene encoding a cell membrane protein present on the chromosome of a Corynebacterium glutamicum microorganism by a method of homologous recombination to reduce the activity of the cell membrane protein , And a method for producing Corynebacterium glutamicum microorganisms with improved productivity of L-ornithine.

Specifically,

I) obtaining a polynucleotide fragment in which a partial sequence of a gene encoding a cell membrane protein is mutated;

Ii) introducing the obtained polynucleotide fragment into a vector capable of being introduced into a host cell capable of producing L-ornithine and performing homologous recombination on the chromosome, to obtain a recombinant vector;

Iii) introducing the obtained recombinant vector into a host cell to obtain a transformant; And

And iv) selecting a strain that reduces or inactivates the activity of a cell membrane protein in the transformant.

Specifically, the gene coding for the cell membrane protein may be the NCg10929 gene of SEQ ID NO: 2, but is not limited thereto.

Alternatively, the host cell may be a microorganism that produces L-ornithine. However, microorganisms whose cell membrane protein activity is inhibited and intracellular glutamate excretion is inhibited may be used. Preferably, L-ornithine- Tincture, and most preferably Corynebacterium glutamicum SJ9518 (Accession No. KACC91938P).

According to one embodiment of the present invention, Escherichia NCgl0929 gene, which is presumed to be a cell membrane protein having an amino acid sequence that is 28% homologous with the amino acid sequence of the amino acid transporter of E. coli , was selected (Example 1), and a polynucleotide in which a part of the NCgl0929 gene sequence was deleted was selected as pK18mobsacB (Example 2 and FIG. 1), a part of NCgl0281, NCgl2582 and NCgl2053, which are genes encoding glucose dehydrogenase on the chromosome, can be deleted, while L-ornithine can be produced, and a recombinant vector pSJC3501 The recombinant vector pSJC3501 was transformed into the host microorganism Corynebacterium glutamicum SJ8260 to induce homologous recombination. Thus, the transformant Corynebacterium glue, in which some nucleotide sequences of the NCgl0929 gene coding for the cell membrane protein were deleted from the chromosome Tamikum SJ9518 was prepared (Example 3). In addition, it was confirmed that the concentration of glutamate and L-ornithine in the cells was increased in the Corynebacterium glutamicum SJ9518 (Table 3 and Table 4).

Accordingly, the present inventors have found that, in addition to the host microorganism Corynebacterium glutamicum SJ8260 in which all of NCgl0281, NCgl2582 and NCgl2053, which are genes encoding glucose dehydrogenase on the chromosome, can be produced while producing L-ornithine, A transformant strain in which some parts of the NCgl0929 gene of the Corynebacterium glutamicum genome chromosome was deleted by transforming the genome recombinant vector pSJC3501 to induce homologous recombination was referred to as " Corynebacterium glutamicum SJ9518 ""And deposited with the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology, on March 20, 2014, and received the accession number KACC91938P.

In another embodiment of the present invention,

i) Inoculating and culturing the Corynebacterium glutamicum microorganism in a culture medium containing a carbon source or a nitrogen source to obtain a culture; And

ornithine, and ii) recovering L-ornithine from the culture.

The term "cultivation" in the present invention means cultivation of microorganisms under moderately artificially controlled environmental conditions. The method for culturing the Corynebacterium glutamicum microorganism in the present invention can be carried out by a method well known in the art. Specifically, the culturing can be carried out continuously in a batch process or in an injection batch or a repeated batch batch process (but not limited thereto).

The culture conditions include, but are not limited to, a basic compound (for example, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (for example, phosphoric acid or sulfuric acid) pH 6 to 8, most preferably pH 6.8), and oxygen or an oxygen-containing gas mixture can be introduced into the culture to maintain aerobic conditions. The culture temperature can be maintained at 20 to 45 캜, preferably 25 to 40 캜, and bubble formation can be suppressed by using a defoaming agent such as fatty acid polyglycol ester, and the culture is preferably performed for 10 to 160 hours . The ornithine produced by the above culture may be secreted into the culture medium or left in the cells.

The medium used for the culture should meet the requirements of the particular strain in an appropriate manner. Culture media for Corynebacterium glutamicum microorganisms are known (see, for example, the Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington, D.C., USA, 1981). Sugars which may be used 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, coconut oil, Stearic acid, fatty acids such as linoleic acid, glycerol, alcohols such as ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Examples of nitrogen sources that may be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used individually or as a mixture. The number of people that can be used may include potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth.

In addition, essential growth materials such as amino acids and vitamins can be used in addition to the above materials. In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method.

In addition, the step of recovering L-ornithine from the culture can be carried out by methods known in the art.

Specifically, known L-ornithine recovery methods include, but are not limited to, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (for example, ammonium sulfate precipitation) (For example, ion exchange, affinity, hydrophobicity and size exclusion).

In one embodiment of the present invention, the control (host cell Corynebacterium glutamicum SJ8260) and the transformant of the present invention (Corynebacterium glutamicum SJ9518) are cultured, and the intrinsic glutamate The concentration of the intrinsic glutamate of the transformant provided by the present invention was increased by about 71% as compared to the control (Table 3). As a result of measuring and comparing the concentrations of L-ornithine produced in the control and the transformant of the present invention, the concentration of L-ornithine produced in the transformant provided in the present invention was about (Table 4).

The Corynebacterium glutamicum microorganism having reduced intrinsic plasma membrane protein activity of the present invention is mutated to increase the intracellular glutamate concentration and to enhance the L-ornithine production ability. The specificity of the L-ornithine biosynthetic pathway Ornithine production can be improved by increasing the total carbon flow, so that it can be widely used for efficient and economical production of L-ornithine.

1 is a schematic diagram showing a gene map of vector pSJC3501 for mutating NCg10929, a gene encoding a cell membrane protein existing in the chromosome of Corynebacterium glutamicum.

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

Example  One. Corynebacterium Glutamicum  Intrinsic amino acid Transporter  Screening of polynucleotides encoding proteins

In order to select a gene encoding an L-ornithine importer protein, which is a type of endogenous amino acid transporter of Corynebacterium glutamicum, which is a representative example of a microorganism belonging to the genus Corothem bacteria, NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to Escherichia coli (Basic Local Alignment Search Tool) using the amino acid sequence of ydgI, which is a gene coding for the amino acid transporter derived from the mouse, as a query sequence. As a result, the ORF sequence (SEQ ID NO: 2) of NCgl0929 gene showing homology of 28% Respectively. The selected NCgl0929 gene was confirmed to be a gene encoding an amino acid sequence (SEQ ID NO: 1) presumed to be a cell membrane protein. In order to confirm the function of SEQ ID NO: 2 thus newly identified, the following experiment was carried out.

Example  2. Corynebacterium Glutamicum NCgl0929  Identification of gene-coding cell membrane proteins

2-1. L-ornithine You need to  representative Corynebacterium Glutamicum SJ9500  Production of strain

In order to investigate whether the cell membrane protein encoded by NCgl0929 gene of Corynebacterium glutamicum identified in Example 1 had a function as L-ornithine importer, wild type Corynebacterium glutamicum strain (ATCC 13032 Ornithine, and a gene-disrupted mutant strain in which the nucleotide sequence of NCgl0929 ORF was deleted in the genetic background of L-ornithine requirement was prepared.

First, 0929F1, 0929R1, 0929F2 and 0929F2 primers were prepared based on the sequence of an argCF primer and an argDR primer, NCg10929, which are based on genomic DNA of Corynebacterium glutamicum ATCC 13032 (Table 1).

SEQ ID NO: Name of the primer The base sequence (5'-3 ') Restriction enzyme 3 argCF gc tctaga TCCAGTTCAGGAAGCACC XbaI 4 argDR gc tctaga ACTTTGGTCCCTCGAGTG XbaI 5 0929F1 ccc aagctt CATCGCCACCATGAATCG HindIII 6 0929R1 gg ggtacc TTGGGCGATGACTGAACC KpnI 7 0929F2 gg ggtacc TTCCTCAACGAGACCACC KpnI 8 0929R2 gc tctaga TGGCAGTTGTAGGGAAGG XbaI

In Table 1, the underlined sequence indicates the restriction site for the restriction enzyme as shown in parentheses, and the upper case means the sequence of the bacterial gene. The open reading frame DNA sequence of NCgl0929 has the Genbank accession number NC_003450.

In-frame site-specific gene deletion of the argCJBD operon encoding the enzymes of ornithine biosynthetic pathway to produce the ornithine requiring strain from the wild-type Corynebacterium glutamicum strain (ATCC13032) specific gene disruption). For this, pK18mobsacB, which is not replicable in Corynebacterium glutamicum, was used, and a pK18mobsacB derivative pSJC3509 having an internally imprecipitated polynucleotide fragment of the argCJBD operon was constructed.

PSJC3509 is a pK18mobsacB derivative containing an internal gene deletion of 3,447 bp NcoI fragment from an argCJBD operon DNA fragment containing 4,838 bp of XbaI end, and the argCJBD operon nucleotide fragment containing the XbaI end is a Corynebacterium glutamicum ATCC 13032 genomic DNA was amplified by PCR using a pair of argCF primer (SEQ ID NO: 3) and argDR primer (SEQ ID NO: 4) as a template.

PCR products were prepared by adding 2 μl (10 pmoles) of the Corynebacterium glutamicum ATCC13032 genomic DNA (250 ng), 10 pmole of the argCF primer, and the argDR primer, respectively, to the taq DNA polymerase (Bioeseang, Co., (2 units) and sterilized distilled water (24 μl), and the conditions of denaturation (95 ° C., 30 seconds), annealing (55 ° C., 30 seconds) and polymerization (68 ° C., 1 minute) .

The recombinant vector pSJC3509 was transformed into Corynebacterium glutamicum wild-type strain (ATCC 13032) by electroporation (van der Rest et al. 1999), and the recombinant vector was introduced into the chromosome by a single crossover Respectively. Thereafter, excision of the plasmid was induced from the chromosome through secondary recombination in 10% sucrose.

Thereafter, all of the argCJBD genes were confirmed to be deleted by diagnostic PCR using a gene-specific primer and named as SJ9500. In the minimal medium, the Corynebacterium glutamicum SJ9500 strain Of ornithine.

2-2. Corynebacterium Glutamicum SJ9500  In the strain NCg10929  ORF ( open  reading frame ) ≪ / RTI > and NCg10929  Identification of function of coding protein

In order to prepare a mutant strain in which the nucleotide sequence of NCgl0929 ORF was specifically deleted in the Corynebacterium glutamicum SJ9500 strain prepared in the above 2-1, the pK18mobsacB vector which is not replicable in Corynebacterium glutamicum , A pK18mobsacB derivative, pSJC3501, containing a nucleotide fragment in which a part of the NCgl0929 ORF base sequence was deleted as an in-frame, was prepared.

PSJC3501 is a pK18mobsacB derivative containing the HindIII-XbaI terminus of the NCgl0929 ORF corresponding to 2,108 bp and contains a deletion of the internal gene sequence of the KpnI fragment of the NCgl0929 ORF. The NCgl0929 ORF fragment containing the disappearance of the internal gene sequence of the KpnI fragment was amplified using the Corynebacterium glutamicum ATCC13032 genomic DNA as a template with the 0929F1-0929R1 primer (SEQ ID NOS: 5 and 6) and the 0929F2-0929R2 primer 8) pairs.

At this time, 2 μl (10 pmoles) of each of the genomic DNA of Corynebacterium glutamicum ATCC13032 (250 ng), 0929F1-0929R1 primer and 0929F2-0929R2 primer, respectively, and taq DNA polymerase (Bioeseang, Inc.) And 20 μl of sterile distilled water and incubating for 30 cycles of denaturation (94 ° C. for 1 min), annealing (58 ° C. for 1 min) and elongation (70 ° C. for 1 min) 1,009 bp and 1,099 bp, respectively, and these were introduced into pK18 mobsacB to obtain a vector pK18mob0929 containing the polynucleotide corresponding to 2,108 bp including the HindIII-XbaI end (Fig. 1). Brief Description of the Drawings Fig. 1 is a schematic diagram showing a cleavage map of a recombinant vector pSJC3501.

The recombinant vector pSJC3501 was transformed into Corynebacterium glutamicum SJ9500 strain by electroporation, and the plasmid was introduced into the chromosome by single crossover. Subsequently, plasmid resection was induced from the chromosome by secondary recombination in 10% sucrose.

Then, it was confirmed that the site-specific base sequence of the NCgl0929 ORF was deleted from the chromosome through diabetic PCR using a gene-specific primer and named as SJ9504. The Corynebacterium glutamicum SJ9504 strain was found to be unable to grow cells in the minimal medium but recovered in the minimal medium containing ornithine (Table 2). From this, it was found that the cell membrane protein encoded by NCgl0929 gene of Corynebacterium glutamicum does not have the function of ornithine importer.

Strain Colony formation of minimal media Ornithine
When present Ornithine
Absent SJ9500 + - SJ9504 + -

Example  3. Corynebacterium Glutamicum  Ornithine From production strains NCgl0929  Production of site-specific mutants of the base sequence

The nucleotide sequence of the NCgl0929 ORF was deleted locally in Corynebacterium glutamicum SJ8260 (ATCC13032, argFΔ, argRΔ, NCgl2053Δ, NCgl2582Δ, NCgl0281Δ) (KACC91800P) which is the genome of the recombinant genome of the present invention Mutants were prepared.

For the site-specific gene disruption, the recombinant vector pSJC3501, which is the pK18mbsacB derivative prepared in Example 2-2, was transformed into Corynebacterium glutamicum SJ8260 strain by electroporation method , And the recombinant vector was introduced into the chromosome by a single crossover. Subsequently, plasmid resection was induced from the chromosome by secondary recombination in 10% sucrose.

It was confirmed that the site-specific base sequence of NCg10929 ORF was deleted from the chromosome through diabe- nastic PCR using gene-specific primers and named as SJ9518 and deposited on March 20, 2014 at the National Institute of Agricultural Science And received grant number KACC91938P.

Example  4. NCg10929 ORF Lt; RTI ID = 0.0 > Corynebacterium  Confirmation of increased production of L-ornithine in glutamicum

Ornithine was produced from L-ornithine producing strain Corynebacterium glutamicum SJ9518 prepared in Example 3 by the following method. At this time, Corynebacterium glutamicum SJ8260 was cultured as a control.

And cultured for 24 hours using Recovery Glucose medium (80 g BHI, 20 g glucose, 60 g sorbitol / L) as the seed medium. The production medium was MMY medium [0.8 g KH ₂ PO ₄ , 10 g (NH ₄ ) ₂ SO ₄ , 1 g MgSO ₄ 7H ₂ O, 1.2 g Na ₂ HPO ₄ , 2 mg MnSO ₄ H ₂ O, 2 mg FeSO ₄ 7H ₂ O, 1 mg ZnSO ₄ 7H ₂ O, 10 g yeast extract, and 60 g glucose / L, pH 7.0].

Corynebacterium glutamicum SJ8260 (control group) and the mutant SJ9518 prepared above were each inoculated into a 100-ml baffle flask containing seed medium and shake cultured (200 rpm) for 24 hours. Cultures obtained from the seed culture were obtained and the obtained cells were resuspended at 100 nm in a 100 ml baffle flask containing 10 ml of MMY medium to exhibit an absorbance of 0.4-0.5 and 0.2 g of sterilized CaCO ₃ was added and cultured in a 30 rpm rotary stirrer at 200 rpm.

From the above cultures, the growth of each strain, the concentration of glutamate in the cell culture extract and the culture supernatant, and the concentration of L-ornithine in the culture supernatant were measured and compared.

4-1. Glutamate  Concentration measurement

Cell viability was measured by spectrophotometry at 600 nm from the culture, and the concentration of glutamate was measured using a color reaction using a Glutamate Assay kit (Abcam Inc., MA, USA). At this time, the experimental results are based on the results obtained from at least three independent cultures, and the standard deviation in all cases was less than 10%. The measured glutamate concentrations are shown in Table 3 below.

Strain Glutamate concentration (nmol) Growth rate (OD ₆₀₀ ) Intracellular Extracellular SJ8260 2.79 7.84 47.6 SJ9518 4.78 4.13 46.7

As shown in Table 3, the mutant Corynebacterium glutamicum SJ9518 in which the site-specific base sequence of the NCgl0929 ORF was deleted from the chromosome was increased by about 71% in the intracellular glutamate compared to the control SJ8260, And the concentration of glutamate was decreased by about 47%. In addition, there was no difference in the degree of growth between the mutant strain and the control strain. This fact supports the fact that the cell membrane protein encoded by the NCgl0929 ORF has a function that is involved in the release of glutamate rather than involving cell membrane movement of ornithine.

As a result, the intracellular glutamate concentration was increased in SJ9518 (mutant strain in which the site-specific base sequence of NCgl0929 ORF was deleted from the chromosome) of the present invention, and the increase of intracellular glutamate concentration, which is a precursor of ornithine biosynthesis, Ornithine, and thus it has been confirmed whether or not the transformant of the present invention actually shows an increase in the production amount of L-ornithine.

4-2. Determination of the concentration of L-ornithine

The control (SJ8260) and the transformant (SJ9518) were cultured in the same manner as described above, and the concentration of L-ornithine in each strain was measured and compared. At this time, the growth of the strain was measured by spectroscopy at 600 nm, and the concentration of L-ornithine was measured using Agilent 1100 series HPLC and Zorbax Eclipse C18 column. The concentration of L-ornithine was quantified using an Agilent 1100 series HPLC and a Zorbax Eclipse C18 column. At this time, the experimental results are based on the results obtained from at least three independent cultures, and the standard deviation in all cases was less than 10%.

Strain L-ornithine concentration
(g L ^-1 ) Growth rate (OD ₆₀₀ ) SJ8260 14.1 47.6 SJ9518 23.8 46.7

As shown in Table 4, it was confirmed that the concentration of L-ornithine was increased by about 68.8% in the case of SJ9518 as compared with the control (SJ8260). In addition, there was no difference in the degree of growth between the mutant strain and the control strain. These results indicate that the intracellular glutamate concentration was increased in SJ9518, a mutant strain in which the site-specific base sequence of Corynebacterium glutamicum NCgl0929 ORF was deleted from the chromosome, and the increase in intracellular glutamate concentration, which is a precursor of ornithine biosynthesis, L-ornithine production.

As a result, the intracellular glutamate concentration of the Corynebacterium sp. Strain in which the activity of the cell membrane protein of the present invention was decreased or inactivated was increased and the increase of the glutamate concentration, which is the precursor of intracellular ornithine biosynthesis, Suggesting an increase in the production of ornithine.

National Institute of Agricultural Science KACC91938P 20140320

<110> SANGJI UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Corynebacterium glutamicum having improved L-ornithine production          and method for preparing L-ornithine using the same <130> KPA140341-KR <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 501 <212> PRT <213> Corynebacterium glutamicum <220> <221> PEPTIDE &Lt; 222 > (1) <223> L-ornithine importer <400> 1 Met Asn Thr Gln Ser Asp Ser Ala Gly Ser Gln Gly Ala Ala Ala Thr   1 5 10 15 Ser Arg Thr Val Ser Ile Arg Thr Leu Ile Ala Leu Ile Ile Gly Ser              20 25 30 Thr Val Gly Ala Gly Ile Phe Ser Ile Pro Gln Asn Ile Gly Ser Val          35 40 45 Ala Gly Pro Gly Ala Met Leu Ile Gly Trp Leu Ile Ala Gly Val Gly      50 55 60 Met Leu Ser Val Ala Phe Val Phe His Val Leu Ala Arg Arg Lys Pro  65 70 75 80 His Leu Asp Ser Gly Val Tyr Ala Tyr Ala Arg Val Gly Leu Gly Asp                  85 90 95 Tyr Val Gly Phe Ser Ser Ala Trp Gly Tyr Trp Leu Gly Ser Val Ile             100 105 110 Ala Gln Val Gly Tyr Ala Thr Leu Phe Phe Ser Thr Leu Gly His Tyr         115 120 125 Val Pro Leu Phe Ser Gln Asp His Pro Phe Val Ser Ala Leu Ala Val     130 135 140 Ser Ala Leu Thr Trp Leu Val Phe Gly Val Val Ser Arg Gly Ile Ser 145 150 155 160 Gln Ala Phe Leu Thr Thr Val Thr Thr Val Ala Lys Ile Leu Pro                 165 170 175 Leu Leu Cys Phe Ile Leu Val Ala Phe Leu Gly Phe Ser Trp Glu             180 185 190 Lys Phe Thr Val Asp Leu Trp Ala Arg Asp Gly Gly Val Gly Ser Ile         195 200 205 Phe Asp Gln Val Arg Gly Ile Met Val Tyr Thr Val Trp Val Phe Ile     210 215 220 Gly Ile Glu Gly Ala Ser Val Tyr Ser Arg Gln Ala Arg Ser Ser Ser 225 230 235 240 Asp Val Ser Arg Ala Thr Val Ile Gly Phe Val Ala Val Leu Leu Leu                 245 250 255 Leu Val Ser Ile Ser Ser Leu Ser Phe Gly Val Leu Thr Gln Gln Glu             260 265 270 Leu Ala Ala Leu Pro Asp Asn Ser Ala Ser Val Leu Glu Ala Val         275 280 285 Val Gly Pro Trp Gly Ala Leu Ile Ser Leu Gly Leu Cys Leu Ser     290 295 300 Val Leu Gly Ala Tyr Val Ser Trp Gln Met Leu Cys Ala Glu Pro Leu 305 310 315 320 Ala Leu Met Ala Met Asp Gly Leu Ile Pro Ser Lys Ile Gly Ala Ile                 325 330 335 Asn Ser Arg Gly Ala Ala Trp Met Ala Gln Leu Ile Ser Thr Ile Val             340 345 350 Ile Gln Ile Phe Ile Ile Phe Phe Leu Asn Glu Thr Thr Tyr Val         355 360 365 Ser Met Val Gln Leu Ala Thr Asn Leu Tyr Leu Val Pro Tyr Leu Phe     370 375 380 Ser Ala Phe Tyr Leu Val Met Leu Ala Thr Arg Gly Lys Gly Ile Thr 385 390 395 400 His Pro His Ala Gly Thr Arg Phe Asp Asp Ser Gly Pro Glu Ile Ser                 405 410 415 Arg Arg Glu Asn Arg Lys His Leu Ile Val Gly Leu Val Ala Thr Val             420 425 430 Tyr Ser Val Trp Leu Phe Tyr Ala Ala Glu Pro Gln Phe Val Leu Phe         435 440 445 Gly Ala Met Ala Met Leu Pro Gly Leu Ile Pro Tyr Val Trp Thr Arg     450 455 460 Ile Tyr Arg Gly Glu Gln Val Phe Asn Arg Phe Glu Ile Gly Val Val 465 470 475 480 Val Val Leu Val Val Ala Ala Ser Ala Gly Val Ile Gly Leu Val Asn                 485 490 495 Gly Ser Leu Ser Leu             500 <210> 2 <211> 1506 <212> DNA <213> Corynebacterium glutamicum <220> <221> gene &Lt; 222 > (1) .. (1506) <223> NCg10929 ORF <400> 2 gtgaatactc aatcagattc tgcggggtct caaggtgcag cggccacaag tcgtactgta 60 tctattagaa ccctcatcgc gctgatcatc ggatcgaccg tcggcgcggg aattttctcc 120 atccctcaaa acatcggctc agtcgcaggt cccggcgcga tgctcatcgg ctggctgatc 180 gccggtgtgg gcatgttgtc cgtagcgttc gtgttccatg ttcttgcccg ccgtaaacct 240 cacctcgatt ctggcgtcta cgcatatgcg cgtgttggat tgggcgatta tgtaggtttc 300 tcctccgctt ggggttattg gctgggttca gtcatcgccc aagttggcta cgcaacgtta 360 tttttctcca cgttgggcca ctacgtaccg ctgttttccc aagatcatcc atttgtgtca 420 gcgttggcag ttagcgcttt gacctggctg gtgtttggag ttgtttcccg aggaattagc 480 caagctgctt tcttgacaac ggtcaccacc gtggccaaaa ttctgcctct gttgtgcttc 540 atcatccttg ttgcattctt gggctttagc tgggagaagt tcactgttga tttatgggcg 600 cgtgatggtg gcgtgggcag catttttgat caggtgcgcg gcatcatggt gtacaccgtg 660 tgggtgttca tcggtatcga aggtgcatcg gtatattccc gccaggcacg ctcacgcagt 720 gatgtcagcc gagctaccgt gattggtttt gtggctgttc tccttttgct ggtgtcgatt 780 tcttcgctga gcttcggtgt actgacccaa caagagctcg ctgcgttacc agataattcc 840 atggcgtcgg tgctcgaagc tgttgttggt ccatggggtg ccgcattgat ttcgttgggt 900 ctgtgtcttt cggttcttgg ggcctatgtg tcctggcaga tgctctgcgc agaaccactg 960 gcgttgatgg caatggatgg cctcattcca agcaaaatcg gggccatcaa cagccgcggt 1020 gctgcctgga tggctcagct gatctccacc atcgtgattc agattttcat catcattttc 1080 ttcctcaacg agaccaccta cgtctccatg gtgcaattgg ctaccaacct atacttggtg 1140 ccttacctgt tctctgcctt ttatctggtc atgctggcaa cacgtggaaa aggaatcacc 1200 ccccacatg ccggcacacg ttttgatgat tccggtccag agatatcccg ccgagaaaac 1260 cgcaaacacc tcatcgtcgg tttagtagca acggtgtatt cagtgtggct gttttacgct 1320 gcagaaccgc agtttgtcct cttcggagcc atggcgatgc ttcccggctt aatcccctat 1380 gtgtggacaa ggatttatcg tggcgaacag gtgtttaacc gctttgaaat cggcgtggtt 1440 gttgtcctgg tcgttgctgc cagcgcgggc gttattggtt tggtcaacgg atcactatcg 1500 ctttaa 1506 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> ArgCF primer <400> 3 gctctagatc cagttcagga agcacc 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> argDR primer <400> 4 gctctagaac tttggtccct cgagtg 26 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 0929F1 primer <400> 5 cccaagcttc atcgccacca tgaatcg 27 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 0929R1 primer <400> 6 ggggtacctt gggcgatgac tgaacc 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 0929F2 primer <400> 7 ggggtacctt cctcaacgag accacc 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 0929R2 primer <400> 8 gctctagatg gcagttgtag ggaagg 26

Claims (9)

A Corynebacterium glutamicum microorganism improved in the ability to produce L-ornithine, wherein the activity of an endogenous plasma membrane protein consisting of the amino acid sequence of SEQ ID NO: 1 is reduced or inactivated.
2. The Corynebacterium glutamicum microorganism according to claim 1, wherein the gene encoding the cell membrane protein is the NCgl0929 gene consisting of the nucleotide sequence of SEQ ID NO: 2.
2. The Corynebacterium glutamicum microorganism according to claim 1, wherein the reduction or inactivation of the activity is achieved through site-specific deletion of the NCgl0929 gene consisting of the nucleic acid sequence of SEQ ID NO: 2.
2. The method of claim 1, wherein the decrease or inactivation of the activity is caused by an insertion mutation by insertion of one or more base pairs into a gene encoding the cell membrane protein, a deletion mutation having deletion of one or more base pairs in the gene, Or a transition of a base pair that introduces a different codon or a transversion mutation. &Lt; RTI ID = 0.0 &gt; [0031] &lt; / RTI &gt;
The microorganism of Claim 1, wherein the Corynebacterium glutamicum microorganism is further deficient in part or all of the gene encoding glucose dehydrogenase (GDH).
The corynebacterium glutamicum microorganism according to claim 1, wherein said Corynebacterium glutamicum microorganism is Corynebacterium glutamicum SJ9518 deposited under accession number KACC91938P.
i) obtaining a polynucleotide fragment in which a partial sequence of the NCgl0929 gene of SEQ ID NO: 2 encoding a cell membrane protein has been mutated;
ii) introducing the obtained polynucleotide fragment into a vector capable of being introduced into a host cell capable of producing L-ornithine and performing homologous recombination on the chromosome, to obtain a recombinant vector;
Iii) introducing the obtained recombinant vector into a host cell to obtain a transformant; And
The method of any one of claims 1 to 6, comprising the step of iv) selecting a strain that has decreased or inactivated the activity of the plasma membrane protein in the transformant.
i) inoculating and culturing the Corynebacterium glutamicum microorganism of any one of claims 1 to 6 in a culture medium containing a carbon source or a nitrogen source to obtain a culture; And
ii) recovering L-ornithine from the culture.
9. The method of claim 8,
Ornithine is carried out by a method selected from the group consisting of centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution, chromatography, Way.

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