KR101285945B1 - Corynebacterium sp. Having Improved L-lysine Production and Process for Preparing the L-lysine Employing the Same - Google Patents

Abstract

The present invention relates to a microorganism of the genus Corynebacterium with improved L-lysine production capacity and a method for producing L-lysine using the same, and more specifically, the present invention reduces or inactivates the activity of endogenous glucose dehydrogenase (GDH). L-lysine comprising the step of culturing L-lysine-producing Corynebacterium microorganism, the microorganism and the microorganism using glucose as a carbon source, and recovering L-lysine from the culture. It relates to a manufacturing method of. By using the microorganism of the genus Corynebacterium with reduced activity of the endogenous glucose dehydrogenase enzyme of the present invention, not only the pentose phosphorylation pathway is activated but also the specific activity of G6PDH and 6PGD, enzymes involved in the pentose phosphorylation pathway. By increasing the NADPH production yield, and thus can improve the production yield of L- lysine using the NADPH, it will be widely used in the efficient and economical production of L- lysine.

Description

Microorganisms of the genus Corynebacterium with improved L-lysine production and a method for producing L-lysine using the same {Corynebacterium sp. Having Improved L-lysine Production and Process for Preparing the L-lysine Employing the Same}

The present invention relates to a microorganism of the genus Corynebacterium with improved L-lysine production capacity and a method for producing L-lysine using the same, and more specifically, the present invention reduces or inactivates the activity of endogenous glucose dehydrogenase (GDH). L-lysine comprising the step of culturing L-lysine-producing Corynebacterium microorganism, the microorganism and the microorganism using glucose as a carbon source, and recovering L-lysine from the culture. It relates to a manufacturing method of.

L-lysine is biosynthesized from oxal acetic acid through the lysine biosynthesis pathway. Aspartate semialdehyde dehydrogenase ( asd ) gene, dihydropicolinate reductase ( dap B) gene, among the enzymes constituting the sugar biosynthesis pathway, respectively And aspartate semialdehyde dehydrogenase, dihydropicolinate reductase and diaminopimelate dehydrogenase, encoded by the diaminopimelate dehydrogenase ( ddh ) gene, lysine biosynthesis as NADPH dependent reductase. Catalyzes the intermediate process for That is, three molecules of NADPH are directly consumed by sugar enzymes in one-molecule L-lysine biosynthesis process, and a direct relationship between the regeneration of NADPH and L-lysine biosynthesis in Corynebacterium glutamicum cells is known. (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). The main production pathways of NADPH in Corynebacterium are the TCA cycle and the pentose phosphate pathway. The main reducing power for L-lysine production is preferably by the oxidation process in the pentose phosphorylation pathway. The TCA cycle emits one molecule of NADPH and two molecules of CO ₂ per cycle, while the pentose phosphorylation pathway produces two molecules of NADPH and one molecule of CO ₂ per cycle, making it more economical in terms of carbon metabolic efficiency. Can be. In other words, as the yield of L-lysine producing strain increases, the demand for NADPH also increases, thereby increasing the dependence on the pentose phosphorylation pathway.

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.

Enzymes that catalyze the pentose phosphorylation pathway during L-lysine production include glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-phosphogluconate dehydrogenase, 6PGD) (Marx et al., Biotechnol Bioeng 56: 168-180, 1997; Wittmann and Heinzle, Microbiol 68: 5843-5849, 2002).

In addition, an effect of enhancing NADPH production-related gene expression in the pentose phosphorylation pathway of Corynebacterium L-lysine producing strains or an increase in L-lysine production capacity by using mutant enzymes has been reported. For example, J. Becker et al. Lysine upon enhanced expression through promoter replacement of the zwf gene encoding glucose 6-phosphate dehydrogenase in L-lysine producing Corynebacterium glutamicum. Increased production has been reported (J. Becker et al., J Biotechnol 132: 99-109, 2007), and European Patent No. 1302537 discloses a variant 6-phosphogluconate dehydrogenase (6-PGGluconate dehydrogenase, 6PGD). A method of producing L-amino acid by culturing L-amino acid-producing Corynebacteria into which a gene is introduced is disclosed.

A series of studies suggested that the conversion 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).

However, there has been no disclosure of Corynebacterium spp. Microorganisms with reduced activity of intrinsic glucose dehydrogenase (GDH) enzymes and increased NADPH biosynthesis in the pentose phosphorylation pathway.

Against this background, the inventors have made diligent efforts to develop a method for producing L-lysine more effectively. As a result, the activity of the endogenous glucose dehydrogenase enzyme is reduced to increase the production of NADPH through the pentose sugar phosphorylation pathway. It was confirmed that the production of lysine can be increased, the present invention was completed.

The main object of the present invention is to provide a microorganism of the genus Corynebacterium with improved L-lysine production, characterized in that the activity of endogenous glucose dehydrogenase (GDH) is reduced or inactivated.

Another object of the present invention is to provide a method for producing microorganisms of the genus Corynebacterium with improved L-lysine production capacity.

Still another object of the present invention is to provide a method for producing L-lysine using microorganisms of the genus Corynebacterium having improved L-lysine production capacity.

According to one embodiment for achieving the above object, the present invention is characterized in that the activity of the endogenous glucose dehydrogenase (GDH) reduced or inactivated, Corynebacterium genus microorganisms with improved L-lysine production capacity to provide.

The term "glucose dehydrogenase (GDH)" in the present invention means a dehydrogenase which converts D-glucose into D-glucono-1, 5-lactone in the pentose phosphate pathway and glucose dehydrogenase Or D-glucose: 1-redox enzyme.

The term "intrinsic" in the present invention means that the microorganism is in a natural state. In the present invention, it is used to indicate the activity of GDH or GDH naturally occurring in the genus Corynebacterium.

In the present invention, "decreased activity of glucose dehydrogenase enzyme" means that the activity of GDH is decreased by mutation of a gene encoding GDH. In the present invention, NCG0281, NCgl2582 and / or NCgl2053 encoding genes encoding GDH present on chromosomes of Corynebacterium sp. Microorganisms are mutated to show a state in which the activity of GDH expressed therefrom is reduced.

In the present invention, the term " inactivated "means that the expression of the gene encoding GDH is reduced to a low level or is not expressed at all, and when the expression is expressed, the activity is lost or decreased. May be accomplished by any inert method known in the art.

In the present invention, "reduce or inactivate the activity of glucose dehydrogenase (GDH)" is not particularly limited, but insertion mutation by insertion of one or more base pairs into the gene encoding the GDH, one or more base pairs in the gene Deletion mutations having a deletion of and nonsense codons or phases in the gene may be caused by one or more mutation methods selected from the group consisting of transition, transition mutations of base pairs introducing codons, preferably Is preferably used to delete the gene by homologous recombination.

In an embodiment of the present invention, a recombinant vector comprising a portion of a gene encoding GDH and an antibiotic marker is introduced into a microorganism to induce homologous recombination, thereby reducing or inactivating GDH activity. In this case, the antibiotic marker is not particularly limited, but it is preferable to use a kanamycin resistance gene, and the recombinant vector is not particularly limited thereto, but it is preferable to use one derived from a pK18mobsacB vector. More preferably, the recombinant vector is not particularly limited thereto, but pSJC3513 having a cleavage map shown in FIG. 2A, pSJC3515 having a cleavage map shown in FIG. 2B, pSJC3517 having a cleavage map shown in FIG. 2C, and the like may be used. Can be.

In addition, in order to further enhance the production capacity of L-lysine, the recombinant vector may further include a sacB gene, which is a gene encoding the glycan transfer enzyme Levansukraase.

The present inventors mutated a gene encoding GDH present on the chromosome of a microorganism of the genus Corynebacterium, thereby reducing the activity of GDH, resulting in glucose that is not used by GDH, depending on the pentose phosphorylation pathway, glucose-6-phosphate. In addition to producing NADPH through Glucose-6-phosphate dehydrogenase (G6PDH), the specific activities of G6PDH and 6PGD, enzymes involved in the production of NADPH in the pentose phosphorylation pathway, are increased. Was found to improve the production yield of L-lysine using NADPH.

According to the conventional pentose phosphorylation pathway, it is known that GDH converts glucose to gluconate, which is converted to 6-PG and then converted to ribulose 5-P while producing NADPH by 6PGD One). Figure 1 is a schematic diagram showing the central carbon metabolism process including the pentose phosphate (Pentose phosphate) and GDH pathway of Corynebacterium glutamicum.

However, the inventors have found that inactivating GDH in the pentose sugar phosphorylation pathway or decreasing its activity, glucose is converted to G6P, which is converted to 6-PG while generating NADPH by G6PDH and then to 6PGD. By converting to ribulose 5-P while producing NADPH, it was expected that the amount of biosynthesis of NADPH was increased. Increasing the amount of biosynthesis of NADPH means that the amount of biosynthesis of L-lysine, which requires a cofactor of NADPH, increases, resulting in increased biosynthesis of L-lysine by inactivation or deactivation of GDH. It was expected and proved experimentally.

The term "6-phosphategluconate dehydrogenase (6PGD)" as used herein refers to an oxidoreductase involved in the pentose phosphorylation pathway present in the cytoplasm, which converts 6-PG to ribulose 5-P (NADP +) is reduced by NADPH in addition to the reaction with nicotinamide adenine dinucleotide phosphate (NADP +).

The term "Glucose-6-phosphate dehydrogenase (G6PDH (EC 1.1.1.49))" in the present invention refers to glucose-6-phosphate dehydrogenase in the pentose- phosphate pathway, Means an enzyme involved in the reaction of converting a phosphate to a 6-phosphono-delta-lactone while reducing the nicotinamide adenine dinucleotide phosphate (NADP +) to NADPH.

As used herein, the term "L-lysine" refers to a kind of L-amino acid, which is an essential amino acid that is not synthesized in the body as one of the basic α-amino acids, and the chemical formula is NH ₂ (CH ₂ ) ₄ CH (NH ₂ ) COOH. L-lysine is biosynthesized from oxal acetic acid via a lysine biosynthetic pathway, and NADPH dependent reductase catalyzes the intermediate process for lysine biosynthesis. In one molecule of L-lysine biosynthesis, three molecules of NADPH are consumed directly by sugar enzymes and one molecule of NADPH is indirectly used.

As used herein, the term "microorganism of the genus Corynebacterium with improved L-lysine production capacity" means a microorganism of the genus Corynebacterium in which a gene encoding GDH present on a chromosome is mutated to decrease or inactivate GDH activity. The gene encoding the GDH is not particularly limited thereto, but NCgl0281 consisting of the nucleotide sequence of SEQ ID NO: 1, NCgl2582 consisting of the nucleotide sequence of SEQ ID NO: 2, or NCgl2053 consisting of the nucleotide sequence of SEQ ID NO: 3 is used. Preferably, the microorganism is not particularly limited, but preferably Corynebacterium glutamicum SJC8255 (KACC9 1636P), SJC8254 (KACC9 1635P) deposited at the National Institute of Agricultural Science on March 07, 2011. ) Or SJC8253 (KACC9 1634P).

In another embodiment of the invention, the invention comprises the step of mutating a gene encoding GDH present on the chromosome of a Corynebacterium genus by homologous recombination, thereby reducing GDH activity. It provides a method for producing microorganisms of the genus Corynebacterium improved productivity.

Specifically, the method for producing microorganisms of the genus Corynebacterium having improved productivity of L-lysine of the present invention comprises the steps of: (i) obtaining a polynucleotide fragment in which a part of a gene encoding GDH is deleted; (Ii) introducing the obtained polynucleotide fragment into a vector capable of introducing into the host cell and performing homologous recombination on a chromosome to obtain a recombinant vector; (Iii) introducing the obtained recombinant vector into a host cell to obtain a homologous recombination in which homologous recombination is induced on a chromosome of the host cell; And (iii) selecting a strain in which the deletion of the gene encoding GDH and the activity of the GDH is reduced or inactivated by the second homologous recombination on the chromosome of the homologous recombination.

At this time, the gene encoding the GDH is not particularly limited, but NCgl0281 consisting of the nucleotide sequence of SEQ ID NO: 1 having the open reading frame DNA sequence of Genbank accession number NC_003450, NCgl2582 consisting of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: It is preferable to use NCgl2053 which consists of 3 base sequences.

In addition, the host cell is not particularly limited thereto, but may use a microorganism that produces L-lysine, preferably a microorganism that produces NADPH by the pentose phosphorylation pathway including the GDH pathway and thereby produces L-lysine. Corynebacterium hydrocarboclastus, Corynebacterium glutamicum, Corynebacterium thermoaminogenes that produce L-lysine Microorganisms of the genus Corynebacterium, such as C. thermoaminogenes), and most preferably Corynebacterium glutamicum SJC8248 (KCTC 3027).

As used herein, the term "recombinant vector" is a vector that cannot be replicated separately from a chromosome of a host cell, wherein the gene construct includes an essential construct capable of homologous recombination with a particular gene on the chromosome of the host cell. In particular, it may include a selection gene such as an antibiotic resistance gene and levansucrase (levansucrase, sacB) gene. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. 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, pUC, pBluescriptII based , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used.

According to one embodiment of the present invention, the present inventors have found that a pK18 mobsacB vector (Schafer et al., 2002), which is capable of performing marker-free deletion of a target gene without being replicated in Corynebacterium glutamicum, , Gene 145: 69-73, 1994). Specifically, the pK18mobsacB vector comprises a polynucleotide fragment obtained by mutating, preferably deleting, a portion of NCgl0281, NCgl2582 and / or NCgl2053, which are genes encoding GDH, including an antibiotic marker and levansukraase gene (sacB). By constructing a recombinant vector and introducing the recombinant vector into a host cell capable of biosynthesizing L-lysine, including the gene encoding GDH on the chromosome by any method known in the art, homologous recombination within the chromosome Corynebacterium microorganisms for producing L-lysine with reduced or inactivated GDH activity were prepared.

Specifically, a recombinant vector pSJ3517 was prepared by inserting a polynucleotide fragment obtained by deleting a portion of NCgl0281 into a pK18mobsacB vector, and a recombinant vector pSJ3515 was prepared by inserting a polynucleotide fragment obtained by deleting a portion of NCgl2582 into a pK18mobsacB vector. The recombinant vector pSJ3513 was constructed by inserting a polynucleotide fragment obtained by deleting part of NCgl2053 in the pK18mobsacB vector.

Then, each of the recombinant vectors pSJ3517, pSJ3515 or pSJ3513 prepared above was introduced into Corynebacterium glutamicum SJC8248, a host cell capable of producing L-lysine, by electroporation, and the kanamycin resistance phenotype was selected to be gene-specific. Diagnostic PCR using a gene-specific primer confirmed that the plasmid was introduced into the chromosomal region of the gene by a single crossover. Subsequently, selection of secondary recombination in 10% sucrose medium and diagnostic PCR using gene-specific primers confirmed the excision of the plasmid from the chromosomal region of the gene. It was. As a result, the strains of Corynebacterium glutamicum, in which a part of NCgl0281, NCgl2582 or NCgl2053, which are genes encoding GDH, were deleted on the chromosome, were named SJC8255, SJC8254 or SJC8253, respectively. Deposited at the Genetic Resource Center, SJC8255 (Accession Number KACC9 1636P), SJC8254 (Accession Number KACC9 1635P), or SJC8253 (Accession Number KACC9 1634P), respectively, were assigned.

According to another embodiment of the present invention, the present invention is (i) inoculating and culturing the culture medium containing glucose as a part or all of the carbon source to the microorganism of the genus Corynebacterium with improved production capacity of L- lysine of the present invention Obtaining a culture; And, (ii) provides a method for producing L- lysine comprising recovering L- lysine from the culture.

The term "cultivation" in the present invention means cultivation of microorganisms under moderately artificially controlled environmental conditions. In the present invention, the method of culturing Corynebacterium sp. Microorganisms may be performed using a method well known in the art. Specifically, the culturing may be a batch process or an injection batch or repeated fed batch process. It can be cultured continuously in a batch process, but is not limited thereto.

The medium used for the culture should 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, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid Fatty acids such as linoleic acid, alcohols such as glycerol, 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 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. Potassium which 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 these materials, essential growth materials such as amino acids and vitamins can be used. 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.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable 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-lysine is obtained. For this purpose it is usually achieved in 10 to 160 hours. L-lysine may be excreted in culture medium or contained in cells.

In addition, the step of recovering L-lysine from the culture can be carried out by methods known in the art. Specifically, the known L-lysine recovery method is not particularly limited, but centrifugation, filtration, extraction, spraying, drying, thickening, precipitation, crystallization, electrophoresis, fractional dissolution (e.g. ammonium sulfate precipitation), chromatography Preference is given to using methods such as (eg ion exchange, affinity, hydrophobicity and size exclusion).

Meanwhile, in the present invention, in order to further increase L-lysine production, the activity of known enzymes related to the biosynthesis of L-lysine can be further regulated. Preferably, the present invention further provides asd, dapB, and ddh encoding the enzyme to modulate the activity of aspartate semialdehyde dehydrogenase, dihydropicolinate reductase and diaminopimelate dehydrogenase enzyme. Genes can be overexpressed to increase L-lysine production.

The present inventors cultured the microorganisms of the genus Corynebacterium with improved productivity of L-lysine prepared above in a medium containing glucose as a carbon source, and compared the level of increase in the production of L-lysine produced therefrom. As a result, as the activity of GDH was significantly reduced, the concentration of NADPH and the production of L-lysine increased.

In addition, the specific activity of G6PDH and 6PGD was further increased in the Corynebacterium glutamicum strain (SJC8255, SJC8254, or SJC8253) into which the recombinant vector pSJ3517, pSJ3515, or pSJ3513 of the present invention with reduced GDH activity was increased. Could.

Therefore, not only the pentose phosphorylation pathway is activated by the decrease of GDH activity, but also the specific activity of G6PDH and 6PGD, enzymes involved in the pentose phosphorylation pathway, increases the production of NADPH and the increase of NADPH production. It was once again confirmed that the amount of biosynthesis of L-lysine was increased.

G6PDH and 6PGD are allosteric and are known to be influenced by feedback control modulation in Corynebacterium glutamicum (Moritz et al. 2000). Therefore, it is expected that the release of feedback control regulation in the SJC8253, SJC8254 and SJC8255 of these enzymes may have a greater effect on the production of L-lysine.

By using microorganisms of the genus Corynebacterium with reduced activity of the endogenous glucose dehydrogenase enzyme of the present invention, not only the pentose phosphorylation pathway is activated, but also the specific activities of G6PDH and 6PGD, enzymes involved in the pentose phosphorylation pathway By increasing the NADPH production yield, and thus can improve the production yield of L- lysine using the NADPH, it will be widely used for efficient and economical production of L- lysine.

Figure 1 is a schematic diagram showing the central carbon metabolism process including the pentose phosphate (Pentose phosphate) and GDH pathway of Corynebacterium glutamicum.
2A is a schematic diagram showing a gene map of a vector pSJ3513 for mutating NCgl2053, a GDH gene existing in the chromosome of Corynebacterium glutamicum.
2B is a schematic diagram showing a gene map of vector pSJ3515 for mutating NCgl2582, a GDH gene existing in the chromosome of Corynebacterium glutamicum.
2C is a schematic diagram showing a gene map of vector pSJ3517 for mutating NCgl0281, a GDH gene existing in the chromosome of Corynebacterium glutamicum.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not limited by these embodiments.

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

Example  1-1: Mutated GDH Obtain genes encoding

In order to prepare a transformant having reduced GDH activity, a recombinant vector comprising a polynucleotide fragment in which an open reading frame of the genes encoding GDH, NCgl0281, NCgl2582 or NCgl2053, was internally deleted was prepared. .

First, primers 0281F1, 0281F2, 0281R1, 0281R2, 2582F1, 2582F2, 2582R1, 2582R2, based on the sequences of NCgl0281, NCgl2582 and NCgl2053, to prepare polynucleotide fragments in which the nucleotide sequences of NCgl0281, NCgl2582 and NCgl2053 are deleted internally. 2053F1, 2053F2, 2053R1 and 2053R2 were prepared (Table 1).

Base sequence of primer Name of the primer Base sequence SEQ ID NO: 2053F1 ccc aagctt AAGACACCGGTCATGGTG (HindIII) 4 2053R1 tcc cccggg TGCTACGGCAGCTCCAAT (SmaI) 5 2053F2 tcc cccggg GACGAAGCCAGCTATGTG (SmaI) 6 2053R2 ccc aagctt ACTCCTGTGCTTCCTTCC (HindIII) 7 2582F1 ccc aagctt CCTCCGCCCCAATTATTC (HindIII) 8 2582R1 cgg ggtacc GGTCTCTGCAGCTTGTTC (KpnI) 9 2582F2 cgg ggtacc GGTCTGGTTTCGTTCCTG (KpnI) 10 2582R2 ccc aagctt CCATCCTGCACTTCTCAG (HindIII) 11 0281F1 ccc aagctt GTCCAAGCCGATTGACTC (HindIII) 12 0281R1 cgg ggtacc GATTGCTGCGACAGTCTC (KpnI) 13 0281F2 cgg ggtacc CAGGAACGTGGCAAGAAC (KpnI) 14 0281R2 ccc aagctt CGGCTTCGGTGAGAACTT (HindIII) 15

In Table 1, the underlined sequences indicate restriction positions for restriction enzymes as shown in parentheses, and the uppercase letters indicate sequences of bacterial genes.

Specific DNA sequences were amplified by PCR from genomic DNA of C. glutamicum ATCC13032 cells using 2053 F1 primer (SEQ ID NO: 4) and 2053 R1 primer (SEQ ID NO: 5). The PCR reaction was performed with 2 µl of 250ng C. glutamicum ATCC13032 g-DNA, 2µl of 20pm F1 primer and 2053 R1 primer of 10pmole, 20µl of 2unit taq DNA polymerase (Bioeseang, Co., Inc), 24µl of sterile distilled water. 1,175 bp DNA fragment was obtained using the conditions of repeating denaturation (94 DEG C for 1 minute), annealing (58 DEG C for 1 minute), and elongation (70 DEG C for 1 minute and 30 seconds) 30 times.

Specific DNA sequences were amplified by PCR from genomic DNA of C. glutamicum ATCC13032 cells using 2053 F2 primer (SEQ ID NO: 6) and 2053 R2 primer (SEQ ID NO: 7). The PCR reaction was performed with 250 μl of C. glutamicum ATCC13032 g-DNA, 2 μl of 10 pmole 2053 F2 primer and 2053 R2 primer, 20 μl of 2 unit taq DNA polymerase (Bioeseang, Co., Inc), 24 μl of sterile distilled water. The DNA fragment of 1,237bp was obtained using the conditions which repeat 30 times of denaturation (94 degreeC 1 minute), annealing (58 degreeC 1 minute), and elongation (70 degreeC 1 minute 30 second) 30 times.

Specific DNA sequences were amplified by PCR from genomic DNA of C. glutamicum ATCC13032 cells using 2582 F1 primer (SEQ ID NO: 8) and 2582 R1 primer (SEQ ID NO: 9). The PCR reaction was performed with 250 μl of C. glutamicum ATCC13032 g-DNA, 2 μl of 10pmole 2582 F1 primer and 2582 R1 primer, 20 μl of 2 units of taq DNA polymerase (Bioeseang, Co., Inc), 24 μl of sterile distilled water. 981 bp DNA fragment was obtained using the conditions of repeating denaturation (94 DEG C for 1 minute), annealing (58 DEG C for 1 minute), and elongation (70 DEG C for 1 minute) 30 times.

Specific DNA sequences were amplified by PCR from genomic DNA of C. glutamicum ATCC13032 cells using 2582 F2 primer (SEQ ID NO: 10) and 2582 R2 primer (SEQ ID NO: 11). The PCR reaction was performed with 250 μl of C. glutamicum ATCC13032 g-DNA, 2 μl of 10 pmole 2582 F2 primer and 2582 R2 primer, 20 μl of 2 units of taq DNA polymerase (Bioeseang, Co., Inc), 24 μl of sterile distilled water. A DNA fragment of 962 bp was obtained using conditions of denaturation (94 ° C. 1 minute), annealing (58 ° C. 1 minute) and elongation (70 ° C. 1 minute) for 30 times.

Specific DNA sequences were amplified by PCR from genomic DNA of C. glutamicum ATCC13032 cells using 0281 F1 primer (SEQ ID NO: 12) and 0281 R1 primer (SEQ ID NO: 13). The PCR reaction product was prepared with 2 µl of 250ng C. glutamicum ATCC13032 g-DNA, 2 µl of 10pmole 0281 F1 primer and 0281 R1 primer, 20 µl of 2unit taq DNA polymerase (Bioeseang, Co., Inc), 24 µl of sterile distilled water. 1,185 bp DNA fragment was obtained using the conditions of repeating denaturation (94 ° C. 1 minute), annealing (58 ° C. 1 minute) and elongation (70 ° C. 1 minute 30 seconds) 30 times.

Specific DNA sequences were amplified by PCR from genomic DNA of C. glutamicum ATCC13032 cells using 0281 F2 primer (SEQ ID NO: 14) and 0281 R2 primer (SEQ ID NO: 15). The PCR reaction was performed with 250 μl of C. glutamicum ATCC13032 g-DNA, 2 μl of 10 pmole 0281 F2 primer and 0281 R2 primer, 20 μl of 2 unit taq DNA polymerase (Bioeseang, Co., Inc), 24 μl of sterile distilled water. 1,145 bp DNA fragment was obtained using the conditions of denaturation (94 ° C. 1 minute), annealing (58 ° C. 1 minute) and elongation (70 ° C. 1 minute 30 seconds) for 30 times.

Example  1-2: Obtaining Recombinant Vectors and Transformants

Recombinant vectors pSJ3513, pSJ3515 and pSJ3517 were constructed using pK18mobsacB, a non-replicable vector in Corynebacterium glutamicum for site specific gene disruption.

The pSJ3513 is a recombinant vector containing the HindIII terminus of 2,938-bp NCgl2053 and includes internal gene loss of the SmaI fragment of NCgl2053. The SmaI fragment was generated by cross PCR with a pair of Corynebacterium glutamicum ATCC13032 genomic DNA as 2053F1-2053R1 primer and 2053F2-2053R2 primer.

PSJ3515 is a recombinant vector containing the HindIII terminus of 1,943-bp NCgl2582 and includes an internal gene loss of the KpnI fragment of NCgl2582. The KpnI fragment was generated by cross PCR using a pair of Corynebacterium glutamicum ATCC13032 genomic DNA as a template with 2582F1-2582R1 primer and 2582F2-2582R2 primer.

The pSJ3517 is a recombinant vector containing the HindIII terminus of 2,330-bp NCgl0281 and includes internal gene loss of KpnI fragment of NCgl0281. The KpnI fragment was generated by cross PCR using a pair of Corynebacterium glutamicum ATCC13032 genomic DNA as a template with a 0281F1-0281R1 primer and a 0281F2-0281R2 primer.

Each recombinant vector was introduced and transformed by electroporation into corynebacterium glutamicum SJC8248, a host cell capable of producing L-lysine (van der Rest et al. 1999), and a kanamycin resistance phenotype was selected for gene specificity. Diagnostic PCR using a gene-specific primer confirmed that the plasmid was introduced into the chromosomal region of the gene by a single crossover. Subsequently, selection of secondary recombination in 10% sucrose medium and diagnostic PCR using gene-specific primers confirmed the excision of the plasmid from the chromosomal region of the gene. It was. As a result, a mutant strain having a specific region of the NCgl0281, NCgl2582 or NCgl2053 gene was deleted, and these were named Corynebacterium glutamicum SJC8255, Corynebacterium glutamicum SJC8254, and Corynebacterium glutamicum SJC8253, respectively. On March 07, 2011, they were deposited with the National Institute of Agricultural Science, Agricultural Genetic Resource Center, and were given accession numbers KACC9 1636P, KACC9 1635P, and KACC9 1634P, respectively.

Example  2 : NCgl0281 , NCgl2582  And / or NCgl2053  Gene deleted Corynebac Terium Glutamicum  L-lysine production

Each of the L-lysine producing strains prepared in Example 1 was cultured in the following manner for L-lysine production from Corynebacterium glutamicum SJC8253, SJC8254 and SJC8255. At this time, as a control, Corynebacterium glutamicum SJC8248, which is a host cell, was cultured and used.

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 [0.8g KH ₂ PO ₄ , 10g (NH ₄ ) ₂ SO ₄ , 1g MgSO ₄ 7H ₂ O, 1.2g Na ₂ HPO ₄ , 2mg MnSO ₄ H ₂ O, 2mg FeSO ₄ 7H ₂ O, 1 mg ZnSO ₄ 7H ₂ O, 10 g yeast extract, and 60 g glucose / L, pH 7.0].

100 ml baffle flasks containing seed medium were inoculated with control and strain Corynebacterium glutamicum SJC8253, SJC8254 or SJC8255, respectively, and shaken (200 rmp). After harvesting the isolated glutamicum incubated 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. The incubation was carried out in a 200 rpm rotary stirrer at 30 ° C.

The growth rate, concentration of NADPH and concentration of L-lysine of each strain were measured and compared from each of the above cultures (Tables 2 and 3). At this time, the cell growth rate was measured by spectroscopy at 600 nm, the concentration of L- lysine was quantified using Agilent 1100 series HPLC and Zorbax Eclipse C18 column. The dry cell mass was measured based on a point corresponding to 0.3 g of dry weight per 1 L with an absorbance value of 600 nm as 1. The concentration of NADPH was measured by an enzymatic cycling reaction using an EnzyChrom ™ NADP ⁺ / NADPH assay kit (BioAssay Systems, CA, USA).

Growth rate of strain Corynebacterium glutamicum Growth rate (μ _max ) SJC8248 0.69 SJC8253 0.62 SJC8254 0.70 SJC8255 0.61

Concentration of NADPH and Concentration of L-Lysine Corynebacterium glutamicum NADPH (μM) L-lysine (gl ^-1 ) SJC8248 2.10 4.7 SJC8253 2.91 5.6 SJC8254 2.23 5.2 SJC8255 2.99 6.1

As shown in Table 2 and Table 3, the concentration of NADPH was increased by 6.2% (2.1 vs. 2.23 μM) when the SJC8254 strain lacking NCgl2582 was cultured in the S.C. strain SJC8248 in Corynebacterium glutamicum. The production increased by 10.6% (4.7 vs. 5.2g / l), but the NADPH concentration increased by 38.6% (2.1 vs. 2.91μM) in the SJC8253 strain lacking NCgl2053, and the production of L-lysine was 19.1%. NADPH concentration was increased by 42.4% (2.1 vs. 2.99μM), and L-lysine production was increased by 29.8% when the SJC8255 strain lacking NCgl0281 was cultured (4.7 vs. 5.6 g / l). 4.7 vs. 6.1 g / l).

These results indicate that excess NADPH is required to increase the biosynthesis of L-lysine in Corynebacterium glutamicum.

In addition, SJC8253 (deleted NCgl2053), SJC8254 (deleted NCgl2582), and SJC8255 (deleted NCgl0281) showed almost similar or slightly decreased growth rates compared to SJC8248 (parent strain), but intracellular NADPH Concentrations and L-lysine production increased significantly.

Example  3: GDH Compare activity

Corynebacterium glutamicum cultured in the production medium was harvested using centrifugation during the expontial phase and resuspended in 100 mM Tris / HCl buffer (pH 7.5). Cells were destroyed 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 in the method for enzymatic analysis was determined by the Bradford method (Bradford 1976), and the activities of G6PDH, 6PGD and GDH were determined by NADPH spectroscopic formation at 340 nm (Frunzke et al. 2008) (Table 4 ).

Specific Activity of Core Enzymes in Central Carbon Metabolism Corynebacterium
Glutamicum Specific activity (U mg ^-1 protein) G6PDH 6PGD GDH SJC8248 0.10 0.70 1.82 SJC8253 0.23 1.03 0.25 SJC8254 0.13 0.96 1.15 SJC8255 0.16 1.56 0.03

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

As shown in Table 4, the specific activity of GDH remained about 63.2% for SJC8254 (1.82 vs. 1.15 U / mg protein) and 13.7% (1.82 vs. 0.25 U / mg protein) for SJC8253. And SJC8255 showed 1.6% (1.82 vs. 0.03 U / mg protein) of GDH specific activity.

Specifically, when comparing the specific activities of G6PDH and 6PGD, the specific activities of G6PDH and 6PGD were significantly increased in SJC8253, SJC8254 and SJC8255 compared to SJC8248.

The increased production of L-lysine by blocking the GDH pathway was probably the result of an increase in NADPH regeneration due to the increased specific activity of G6PDH and 6PGD, and from these results were estimated to be the NCgl0281, NCgl2582 and / or GDH genes. The mechanism of the method of producing L-lysine from glucose using a mutant that inactivated NCgl2053 can be proposed. Increasing NADPH regeneration due to increased specific enzymatic activity of G6PDH and 6GPD resulted in increased production of L-lysine.

Agricultural Genetic Resource Center KACC91634P 20110307 Agricultural Genetic Resource Center KACC91635P 20110307 Agricultural Genetic Resource Center KACC91636P 20110307

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Claims (11)

L- characterized in that the activity of the endogenous glucose dehydrogenase (GDH) encoded by the NCg10281 gene consisting of the nucleotide sequence of SEQ ID NO: 1 or the NCg12582 gene consisting of the nucleotide sequence of SEQ ID NO: 2 is reduced or inactivated. Microorganisms of the genus Corynebacterium with improved lysine production.
The method of claim 1,
The reduction or inactivation may be due to insertion mutations by insertion of one or more base pairs into the gene encoding the GDH, deletion mutations with deletion of one or more base pairs in the gene, and base pairs that introduce nonsense codons or different codons in the gene. A microorganism of the genus Corynebacterium with improved L-lysine production capacity, which is caused by one or more mutation methods selected from the group consisting of transition or transmutation mutations.
The method of claim 1,
Said reduction or inactivation is a microorganism of the genus Corynebacterium with improved L-lysine production capacity that is caused by transformation by a recombinant vector comprising a portion of the gene encoding the GDH and an antibiotic marker.
The method of claim 3,
The antibiotic marker is a kanamycin resistance gene is a gene of Corynebacterium genus L- lysine is improved.
The method of claim 3,
The recombinant vector is a microorganism of the genus Corynebacterium improved L- lysine production capacity is derived from the pK18mobsacB vector.
The method of claim 3,
The recombinant vector is a microorganism of the genus Corynebacterium improved L- lysine production capacity further comprises sacB gene.
The method according to claim 6,
The recombinant vector is pSJC3515 having a cleavage map shown in FIG. 2B or pSJC3517 having a cleavage map shown in FIG. 2C.
delete The method of claim 1,
The microorganism of the genus Corynebacterium is corynebacterium glutamicum SJC8254 (KACC9 1635P) or Corynebacterium glutamicum SJC8255 (KACC9 1636P) Corynebacterium genus microorganisms with improved production capacity.
(Iii) obtaining a polynucleotide fragment in which the NCg10281 gene consisting of the nucleotide sequence of SEQ ID NO: 1 encoding the endogenous glucose dehydrogenase (GDH) or the partial sequence of the NCg12582 gene consisting of the nucleotide sequence of SEQ ID NO: 2 is mutated; step;
(Ii) introducing the obtained polynucleotide fragment into a vector capable of introducing into a host cell capable of producing L-lysine and carrying out homologous recombination on a chromosome to obtain a recombinant vector;
(Iii) introducing the obtained recombinant vector into a host cell to obtain a homologous recombination product; And
(Iii) improving the L-lysine production capacity according to any one of claims 1 to 7, and 9, comprising selecting a strain in which the activity of GDH is reduced or inactivated in the homologous recombination product. Method for producing microorganisms of the genus Corynebacterium.
(Iii) inoculating and culturing the culture medium containing glucose as part or all of the carbon source, incubated with microorganisms of Corynebacterium having improved L-lysine production capacity of any one of claims 1 to 7, and Obtaining water; And
(Ii) recovering L-lysine from the culture.

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