Microorganism of genus corynebacterium producing l-amino acids, and method for producing l-amino acids using the same

Abstract

To provide a microorganism of the genus Corynebacterium producing L-amino acid, a method for producing L-amino acid using the same, their use in L-amino acid production, and a composition for producing L-amino acid.SOLUTION: The present invention provides a microorganism of the genus Corynebacterium producing L-amino acid and having enhanced activity of glucose-6-phosphate dehydrogenase and fructokinase.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a Corynebacterium microorganism that produces L-amino acids, a method for producing L-amino acids using the same, use for L-amino acid production, and a composition for L-amino acid production.

Background Art

[0002] L-amino acids are the basic building blocks of proteins and are used as important materials such as pharmaceutical raw materials, food additives, animal feeds, nutritional agents, insecticides, and bactericides. Among them, L-lysine is an essential amino acid that is not biosynthesized in vivo and is known to be necessary for growth promotion, calcium metabolism, promotion of gastric juice secretion, and increase in resistance to diseases. The L-lysine is widely used in feeds, pharmaceuticals, foods, etc. Also, L-tryptophan is one of the essential amino acids and is used as a feed additive, infusion solution, pharmaceutical raw material, and health food material.

[0003] On the other hand, Corynebacterium strains, particularly Corynebacterium glutamicum, are gram-positive microorganisms that are widely used for the production of L-amino acids and other useful substances. For the production of the amino acids, various studies have been conducted for the development of highly efficient production microorganisms and fermentation process technologies. For example, an approach specific to the target substance such as increasing the expression of a gene encoding an enzyme involved in amino acid biosynthesis in Corynebacterium strains or removing a gene unnecessary for amino acid biosynthesis is mainly used (Korean Registered Patent Publication Nos. 10-0924065 and 10-1208480). In addition to such methods, methods of removing genes not involved in amino acid production and methods of removing genes whose specific functions are not known in amino acid production are also utilized. However, there is still a growing need for research on methods that can efficiently produce L-amino acids in high yields.

Prior Art Documents

Patent Documents

[0004] [Patent Document 1] Korean Patent Registration Gazette No. 10-0924065 [Patent Document 2] Korean Patent Registration Gazette No. 10-1208480 [Patent Document 3] U.S. Patent No. 7662943 B2 [Patent Document 4] Korean Patent No. 10-0159812 [Patent Document 5] Korean Patent No. 10-2035844 [Patent Document 6] U.S. Patent No. 10584338 B2 [Patent Document 7] U.S. Patent No. 8932861 B2 [Non-Patent Document]

[0005] [Non-Patent Document 1] Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873 (1993) [Non-Patent Document 2] Methods Enzymol., 183, 63, 1990 [Non-Patent Document 3] http: / / www.ncbi.nlm.nih.gov [Non-Patent Document 4] J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 [Non-Patent Document 5] F.M. Ausubel et al., Current Protocols in Molecular Biology [Non-Patent Document 6] Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Summary of the Invention

Problems to be Solved by the Invention

[0006] The problem to be solved by the present invention is to provide a Corynebacterium microorganism with enhanced activities of glucose-6-phosphate dehydrogenase and fructokinase.

Means for Solving the Problems

[0007] One object of the present invention is to provide a Corynebacterium microorganism that produces L - amino acids and has enhanced activities of glucose - 6 - phosphate dehydrogenase and fructokinase.

[0008] Another object of the present invention is to provide a method for producing L - amino acids using the above - mentioned microorganism.

[0009] Another object of the present invention is to provide the use of the above - mentioned microorganism for the production of L - amino acids.

[0010] Another object of the present invention is to provide a composition for producing L - amino acids containing the above - mentioned microorganism; and / or a culture of the above - mentioned microorganism.

Effects of the Invention

[0011] The microorganism of the present invention that produces L - amino acids and has enhanced activities of glucose - 6 - phosphate dehydrogenase and fructokinase can produce L - amino acids with high efficiency.

Modes for Carrying Out the Invention

[0012] Specifically explaining this, it is as follows. On the one hand, each explanation and embodiment disclosed in the present invention can be applied to each other explanation and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. Also, the scope of the present invention is not limited by the following specific description. Also, numerous papers and patent documents are referred to throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the level of the technical field to which the present invention belongs and the content of the present invention.

[0013] One aspect of the present invention provides a Corynebacterium microorganism that produces L - amino acids and has enhanced activities of Glucose - 6 - phosphate 1 - dehydrogenase and Fructokinase.

[0014] In the present invention, the term "L - amino acid" includes all L - amino acids that can be produced by a microorganism through a metabolic process from various carbon sources. Specifically, it may be basic amino acids such as L - lysine, L - arginine, L - histidine, non - polar amino acids such as L - valine, L - leucine, L - glycine, L - isoleucine, L - alanine, L - proline, L - methionine, polar amino acids such as L - serine, L - threonine, L - cysteine, L - aspartic acid, L - glutamine, aromatic amino acids such as L - phenylalanine, L - tyrosine, L - tryptophan, and acidic amino acids such as L - glutamic acid, L - aspartic acid. More specifically, in the present application, the L - amino acid may be, but is not limited to, L - lysine or L - tryptophan.

[0015] The term "Glucose - 6 - phosphate 1 - dehydrogenase (hereinafter referred to as 'Zwf')" in the present invention means that it is involved in the pentose phosphate pathway, which is a metabolic pathway, and plays a role in reducing NADP+ to NADPH while oxidizing glucose 6 - phosphate.

[0016] For the purpose of the present invention, the protein may also be named "G6PD", "G6PDH", "glucose-6-phosphate dehydrogenase" or "Zwf". The gene encoding the protein may be, for example, the zwf gene, but is not limited thereto. In the present invention, the "zwf gene" may be used interchangeably with the "gene encoding glucose-6-phosphate dehydrogenase". Further, the protein may be, for example, the same protein as that derived from Corynebacterium glutamicum, but is not limited thereto as long as it increases the production of L-amino acids.

[0017] The Zwf may have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, may consist of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or may include the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 3, but is not limited thereto. The sequences of SEQ ID NO: 1 or SEQ ID NO: 3 can be confirmed from the known database NCBI Genbank.

[0018] The Zwf may include a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4, but is not limited thereto. The sequences of SEQ ID NO: 2 or SEQ ID NO: 4 can be confirmed from the known database NCBI Genbank.

[0019] Specifically, the Zwf may be an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 1, SEQ ID NO: 3, and / or SEQ ID NO: 1, SEQ ID NO: 3. Further, it is obvious that a Zwf having such homology or identity and having an amino acid sequence showing a function corresponding to the Zwf, including an amino acid sequence in which some sequences are deleted, modified, substituted or added, is also included within the scope of the present invention.

[0020] In the terms of the present invention, the term "Fructokinase (hereinafter, referred to as 'CscK')" means an enzyme that transfers phosphate in the presence of ATP and catalyzes the reaction to produce D-fructose-6-phosphate and ADP.

[0021] For the purpose of the present invention, the said protein may also be named as "fructose phosphorylase", "Fructokinase" or "CscK". The gene encoding the said protein may be, for example, but not limited to, the csck gene. In the present application, the "csck gene" may be used interchangeably with the "gene encoding Fructokinase". Further, the said protein may be, for example, the same protein as that derived from Escherichia coli, but is not limited thereto as long as it can increase the production of L-amino acids.

[0022] The said CscK may have the amino acid sequence of SEQ ID NO: 5, consist of the amino acid sequence of SEQ ID NO: 5, or include the amino acid sequence described in SEQ ID NO: 5, but is not limited thereto. The sequence of SEQ ID NO: 5 can be confirmed from the known database NCBI Genbank.

[0023] The said CscK may include the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 6, but is not limited thereto. The sequence of SEQ ID NO: 6 can be confirmed from the known database NCBI Genbank.

[0024] Specifically, the said CscK may be an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 5 and / or the said SEQ ID NO: 5. Further, it is obvious that a CscK having such homology or identity and having an amino acid sequence showing the function corresponding to the said CscK and having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present invention.

[0025] The terms "homology" and "identity" in the present invention mean the degree related to two given amino acid sequences or nucleotide sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0026] The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard sequence algorithms, and the default gap penalties established by the programs used are both used. Substantially, homologous or identical sequences generally hybridize at least about 50%, 60%, 70%, 80% or 90% or more of the entire sequence or full length under moderately or highly stringent conditions. Hybridization also takes into account polynucleotides containing degenerate codons instead of codons in the polynucleotide.

[0027] Homology or identity to the polypeptide or polynucleotide sequence can be determined, for example, using the algorithm BLAST according to the literature [reference: Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)], or FASTA by Pearson [reference: Methods Enzymol., 183, 63, 1990]. Based on such an algorithm BLAST, programs called BLASTN and BLASTX have been developed (reference: http: / / www.ncbi.nlm.nih.gov). Also, whether any amino acid or polynucleotide sequence has homology, similarity or identity can be confirmed by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and the appropriate hybridization conditions defined are within the technical scope and are methods well known to those skilled in the art (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology).

[0028] Specifically, the "microorganism producing L - amino acid" includes all wild - type microorganisms and microorganisms that have undergone natural or artificial genetic modifications. Specifically, it is a microorganism in which a specific mechanism has been weakened or strengthened due to reasons such as insertion of an external gene, enhancement or inactivation of the activity of an endogenous gene, and may be a microorganism in which a genetic mutation has occurred or the L - amino acid production activity has been enhanced for the production of the target L - amino acid. For the purpose of the present invention, the microorganism producing the L - amino acid is characterized in that the expression or activity of the Zwf and Csck proteins is enhanced and the ability to produce the target L - amino acid is increased, and may be a genetically modified microorganism or a recombinant microorganism, but is not limited thereto.

[0029] The "enhancement of activity" of the term protein in the present invention means that the activity of the protein increases compared to the intrinsic activity. The "intrinsic activity" means the activity of a specific protein originally possessed by the parental strain or non-transformed microorganism before the trait change when the trait changes due to genetic mutations caused by natural or artificial factors. This can be used interchangeably with the "activity before transformation". The fact that the activity of a protein "increases" compared to the intrinsic activity means that it has improved compared to the activity of the specific protein originally possessed by the parental strain or non-transformed microorganism before the trait change.

[0030] The "increase in activity" can be achieved by introducing an exogenous protein or through enhancing the activity of an endogenous protein. Specifically, it may also be achieved through enhancing the activity of an endogenous protein. The presence or absence of the enhancement of the protein activity can be confirmed from the increase in the activity level, expression level of the protein, or the amount of the product produced from the protein.

[0031] The enhancement can be achieved by applying various methods well-known in the art and is not limited as long as the activity of the target protein can be enhanced compared to the microorganism before transformation. Specifically, it may be those using genetic engineering and / or protein engineering well-known to ordinary technicians in the art, which are routine methods in molecular biology, but are not limited thereto (for example, Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0032] In the present invention, the protein to be enhanced in activity, that is, the target protein, may be Zwf and Csck, but is not limited thereto.

[0033] Specifically, the enhancement of the protein activity of the present invention is 1) an increase in the intracellular copy number of the gene encoding the protein; 2) Replacing the gene expression regulatory sequence on the chromosome encoding the protein with a sequence having strong activity; 3) Modifying the nucleotide sequence of the start codon or 5'-UTR region of the gene transcript encoding the protein; 4) Modifying the amino acid sequence so as to enhance protein activity; 5) Modifying the polynucleotide sequence encoding the protein so as to enhance protein activity (for example, modifying the gene sequence encoding the protein so as to encode a protein modified to enhance activity); 6) Introducing a foreign polynucleotide showing the activity of the protein or a codon-optimized mutant polynucleotide of the polynucleotide; 7) Codon optimization of the polynucleotide encoding the protein; 8) Analyzing the tertiary structure of the protein, selecting an exposed site for modification or chemical modification; or 9) It may be, but is not limited to, a combination of two or more selected from the above 1) to 8).

[0034] Specifically, the increase in the intracellular copy number of the gene encoding the protein in 1) above can be achieved by any method known in the art, for example, by introducing into the host cell a vector that replicates and functions regardless of the host and to which the gene encoding the protein is operably linked. Or it can be carried out by introducing into the host cell a vector for inserting the gene into the chromosome in the host cell to which the gene is operably linked, but is not limited thereto.

[0035] In the present invention, the term "vector" means a DNA construct containing a polynucleotide sequence encoding a target protein in a form operably linked to regulatory sequences suitable for expressing the target protein in a suitable host. The expression regulatory sequences can include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences for regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector can be replicated or function independently of the host genome and can integrate into the genome itself.

[0036] The vectors used in the present invention are not particularly limited, and any vector known in the art can be utilized. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0037] In the present invention, the term "transformation" means introducing a recombinant vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, all of these can be included regardless of whether it is inserted into the host cell chromosome or located extrachromosomally. The method of transformation includes any method of introducing nucleic acid into a cell, and appropriate standard techniques can be selected and carried out as known in the art by the host cell. For example, there are, but are not limited to, electroporation, precipitation of calcium phosphate (CaPO4), precipitation of calcium chloride (CaCl2), microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, etc.

[0038] Also, in the above, the term "operably linked" means that a promoter sequence or an expression regulatory region that initiates and mediates the transcription of a polynucleotide encoding the target protein of the present invention is functionally linked to the polynucleotide sequence. An operable linkage can be produced using genetic recombination techniques known in the art, and site-specific DNA cleavage and ligation can be produced using cleavage and ligation enzymes in the art, etc., but are not limited thereto.

[0039] The method of replacing the gene expression regulatory sequence on the chromosome encoding the protein in 2) with a sequence having strong activity can be any method known in the art. For example, nucleic acid sequences can be deleted, inserted, non-conservatively or conservatively substituted, or mutations in the sequence can be induced by a combination thereof to further enhance the activity of the expression regulatory sequence, or it can be performed by replacing it with a nucleic acid sequence having stronger activity. The expression regulatory sequence can include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like. Specifically, the method may be, but is not limited to, ligating a strong heterologous promoter instead of the original promoter.

[0040] Examples of known strong promoters can include, but are not limited to, the CJ1 - CJ7 promoters (U.S. Patent No. US7662943 B2), the lac promoter, the Trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, and the rmf promoter, and all those that replace with a promoter stronger than the intrinsic activity are included.

[0041] The modification of the nucleotide sequence of the start codon or the 5'-UTR region of the gene transcript encoding the protein in 3) can be any method known in the art. For example, it may be, but is not limited to, replacing the intrinsic start codon of the protein with another start codon having a higher protein expression rate compared to the intrinsic start codon.

[0042] The modifications of the amino acid sequences or polynucleotide sequences in 4) and 5) above can be carried out by any method known in the art. For example, the polynucleotide sequence can be deleted, inserted, non-conservatively or conservatively substituted, or mutations on the expression regulatory sequence can be induced by these combinations to further enhance the activity of the polynucleotide sequence, or it can be carried out by replacing it with a polynucleotide sequence improved to have stronger activity. The replacement may specifically be inserting the gene into the chromosome by homologous recombination, but is not limited thereto. The vector used at this time can further contain a selection marker for confirming the presence or absence of chromosomal insertion. The selection marker is for selecting cells transformed with the vector, that is, for confirming the presence or absence of the gene insertion to be introduced. Markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins are used, but are not limited thereto. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other phenotypes, so that transformed cells can be selected.

[0043] The introduction of a foreign polynucleotide showing the activity of the protein in 6) above can be carried out by any method known in the art. For example, a foreign polynucleotide encoding a protein showing the same / similar activity as the protein, or its codon-optimized mutant polynucleotide can be introduced into the host cell. The foreign polynucleotide can be used without limitation to its origin and sequence as long as it shows the same / similar activity as the protein. Also, the codon can be optimized and introduced into the host cell so that the introduced foreign polynucleotide is optimally transcribed and translated in the host cell. The introduction is appropriately selected and executed by those skilled in the art using known transformation methods. When the introduced polynucleotide is expressed in the host cell, a protein can be generated and its activity can be increased.

[0044] The codon optimization of the polynucleotide encoding the aforementioned 7) protein may be such that the endogenous polynucleotide is codon-optimized to increase transcription or translation in the host cell, or the codons of the exogenous polynucleotide are optimized so that optimized transcription and translation occur in the host cell.

[0045] The analysis of the tertiary structure of the aforementioned 8) protein and the selection of exposed sites for modification or chemical modification may be, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins according to the degree of sequence similarity to determine candidate template proteins, and based on this, confirming the structure and selecting exposed sites for modification or chemical modification to modify or chemically modify them.

[0046] The enhancement of the activity of such a protein may be an increase in the activity or concentration of the corresponding protein based on the activity or concentration of the protein expressed in the wild-type or pre-modified microbial strain, or an increase in the amount of the product produced from the protein, but is not limited thereto. In the present invention, the term "pre-modified strain" or "pre-modified microorganism" does not exclude strains containing mutations that can occur naturally in microorganisms, but means either the natural-type strain itself or the strain before the traits change due to genetic mutations caused by artificial factors. In the present invention, the trait change may be an enhancement of the activity of PheA. The "pre-modified strain" or "pre-modified microorganism" may be used interchangeably with "non-mutant strain", "non-modified strain", "non-mutant microorganism", "non-modified microorganism" or "reference microorganism".

[0047] In the present invention, the reference microorganism is not particularly limited as long as it is a microorganism that produces L-amino acids, and mutant strains with enhanced L-amino acid productivity compared to the wild type are also included without limitation. Examples thereof include Corynebacterium glutamicum KCCM11016P (Korean Registered Patent No. 10-0159812), KCCM10770P (Korean Registered Patent No. 10-0924065), CJ3P (Binder et al. Genome Biology 2012, 13:R40) strain that produce L-lysine, or Corynebacterium glutamicum KCCM12218P (Korean Registered Patent No. 10-2035844) strain that produce L-tryptophan, and strains in which one or more genetic modifications are added to the above strains to enhance the biosynthetic pathway may be included, but are not limited thereto.

[0048] For the purpose of the present invention, any microorganism that produces L-amino acids and whose Zwf and Csck activities are enhanced by the method described above is possible. In the present application, the "microorganism that produces L-amino acids" may be used interchangeably with "L-amino acid-producing microorganism" and "microorganism having L-amino acid productivity", and specifically, it may be a microorganism belonging to the genus Corynebacterium, but is not limited thereto.

[0049] In the present application, the term "Corynebacterium microorganism" can include all Corynebacterium microorganisms. Specifically, it may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, and more specifically, it may be Corynebacterium glutamicum.

[0050] On the one hand, it has already been known that Corynebacterium microorganisms can produce L-amino acids, but their production ability is significantly low, and not all of the genes and the principles of the mechanisms acting on the production mechanism have been clarified. Therefore, the "Corynebacterium microorganism that produces L-amino acids" of the present invention is the natural wild-type microorganism itself, a Corynebacterium microorganism in which the activity of genes related to the mechanism of L-amino acid production is enhanced, inactivated, or has an improved L-amino acid production ability, or a Corynebacterium microorganism in which the activity of an external gene is introduced or enhanced and has an improved L-amino acid production ability.

[0051] As another aspect, the present invention provides a method for producing L-amino acids, including the steps of culturing the microorganism according to the present invention in a medium; and recovering L-amino acids from the microorganism or the medium.

[0052] The microorganism according to the present invention is as described above.

[0053] In the method of the present invention, any culture conditions and culture methods known in the art are used for culturing the Corynebacterium microorganism.

[0054] In the present invention, the term "cultivation" means growing microorganisms under appropriately artificially controlled environmental conditions. The method for cultivating L-amino acids using microorganisms that produce L-amino acids in the present invention can be carried out using methods widely known in the art. Specifically, the cultivation can be carried out continuously in a batch process, a fed batch or a repeated fed batch process (fed batch or repeated fed batch process), but is not limited thereto. The medium used for cultivation must meet the requirements of a specific strain in an appropriate manner. Culture media for Corynebacterium strains are known (for example, Manual of Methods for General Bacteriology by the American Society for Bacteriology, Washington D.C., USA, 1981).

[0055] Carbon sources used in the medium 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, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These substances can be used individually or as a mixture and are not limited thereto.

[0056] Nitrogen sources 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. Nitrogen sources can also be used individually or as a mixture and are not limited thereto.

[0057] As the phosphorus source contained in the medium, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a salt containing the corresponding sodium may be included. Further, the culture medium can contain metal salts such as magnesium sulfate or iron sulfate necessary for growth. Finally, essential growth substances such as amino acids and vitamins are used in addition to the above substances. Also, appropriate precursors are used in the culture medium. The above raw materials may be added batchwise or continuously to the culture in an appropriate manner during the culture process, but are not limited thereto.

[0058] During the culture of the microorganism, the pH of the culture can be adjusted by using basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid in an appropriate manner. Also, the generation of bubbles can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. To maintain an aerobic state, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture. The temperature of the culture water is usually 20°C to 45°C, specifically 25°C to 40°C. The culture time can be continued until the production amount of the target L-amino acid is obtained, specifically, it may be 10 to 160 hours. However, it is not limited to these examples.

[0059] The present invention may further include, but is not limited to, a step of preparing the medium before the culturing step by the method of the present invention.

[0060] The separation of L-amino acid from the culture can be carried out by conventional methods known in the art. Such separation methods include methods such as centrifugation, filtration, chromatography, and crystallization. For example, the culture can be centrifuged at a low speed to remove biomass, and the obtained supernatant can be separated through ion exchange chromatography, but it is not limited thereto.

[0061] Also, the recovery step can include a purification step and may be carried out using an appropriate method known in the art.

[0062] The present invention, in another aspect, provides the use of the microorganism according to the present invention for L - amino acid production.

[0063] The microorganism and L - amino acid according to the present invention are as described above.

[0064] The present invention, in another aspect, provides a composition for L - amino acid production comprising the microorganism according to the present invention; and / or a culture of the microorganism.

[0065] The microorganism and L - amino acid according to the present invention are as described above.

Examples

[0066] Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0067] Example 1. Preparation of an enhanced vector Example 1 - 1. Preparation of a vector in which the glucose - 6 - phosphate dehydrogenase gene (zwf) derived from Corynebacterium glutamicum ATCC13032 is enhanced For the enhancement of the gene, zwf ligated with the SPL13 promoter (U.S. Patent No. US10584338 B2) and the CJ7 promoter derived from Corynebacterium ammoniagenes U.S. Registered Patent US7662943 B2 was inserted into the transposon of Corynebacterium glutamicum ATCC13032 to confirm the effect.

[0068] Using Corynebacterium glutamicum ATCC13032 genomic DNA as templates respectively, primers containing the polynucleotides of SEQ ID NO: 9, 10 (Table 1) and SEQ ID NO: 13, 14 (Table 2) were prepared based on the NCBI nucleotide sequence (NC_003450.3) information, and PCR (SolgTM Pfu-X DNA polymerase) was performed under the conditions in Table 4 to obtain the zwf gene fragment for constructing the vector.

[0069]

Table 1

[0070]

Table 2

[0071]

Table 3

[0072]

Table 4

[0073] To obtain the SPL13 promoter and the CJ7 promoter derived from Corynebacterium ammoniagenes, PCR (SolgTM Pfu-X DNA polymerase) was performed using the primers of SEQ ID NO: 7 and 8 (Table 1), SEQ ID NO: 11 and 12 (Table 2) (Table 4). The amplified SPL13, CJ7 promoter sites, the zwf gene fragment, and the vector pDZTn (US Patent Registration US8932861 B2) digested with ScaI restriction enzyme were ligated using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method, then transformed into Escherichia coli DH5α, and spread on an LB solid medium conjugated with kanamycin (25 mg / l). To select colonies transformed with the vector ligated with the target gene and pDZTn, PCR was performed using the primers of SEQ ID NO: 47 and 48 (Table 3). Plasmids were obtained from the selected colonies using the commonly known plasmid extraction method, and the plasmids were named pDZTn-Pspl13-zwf (C.gl13032) and pDZTn-Pcj7-zwf (C.gl13032).

[0074] Example 1-2. Preparation of a vector with enhanced glucose-6-phosphate dehydrogenase gene (zwf) derived from Corynebacterium glutamicum ATCC13869 To confirm the enhancing effect of zwf derived from Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC13869 genomic DNA was used as a template respectively, and primers containing the polynucleotides of SEQ ID NO: 17, 18 (Table 5) and SEQ ID NO: 21, 22 (Table 6) were prepared based on the information on the Corynebacterium glutamicum ATCC13869 gene and its surrounding base sequences registered in the National Institutes of Health GenBank (NIH GenBank), and PCR (SolgTM Pfu-X DNA polymerase) was performed under the conditions of Table 4 to obtain a zwf gene fragment for preparing a vector.

[0075]

Table 5

[0076]

Table 6

[0077] To obtain the SPL13 promoter and the CJ7 promoter derived from Corynebacterium ammoniagenes, PCR (SolgTM Pfu-X DNA polymerase) was performed under the conditions of Table 4 using the primers of SEQ ID NO: 15 and 16 (Table 5), SEQ ID NO: 19 and 20 (Table 6).

[0078] The amplified SPL13, CJ7 promoter sites, the zwf gene fragment, and the vector pDZTn (U.S. Patent Registration No. US8932861 B2) digested with ScaI restriction enzyme were ligated using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method, then transformed into Escherichia coli DH5α, and spread on an LB solid medium conjugated with kanamycin (25 mg / l). To select colonies transformed with the vector ligated with the target gene and pDZTn, PCR was performed using the primers of SEQ ID NO: 47 and 48 (Table 3). Plasmids were obtained from the selected colonies using a commonly known plasmid extraction method, and these plasmids were named pDZTn-Pspl13-zwf (C.gl13869) and pDZTn-Pcj7-zwf (C.gl13869).

[0079] Example 1-3. Preparation of a vector with enhanced fructokinase gene (cscK) derived from Escherichia coli The nucleotide sequence of the fructokinase gene derived from Escherichia has already been clarified and published. The cscK gene information was obtained from Escherichia coli W (CP002967) of NCBI, and primers containing the polynucleotides of SEQ ID NO: 25 and 26 (Table 7), SEQ ID NO: 29 and 30 (Table 8) were prepared, and PCR (SolgTM Pfu-X DNA polymerase) was performed under the conditions of Table 4 to obtain a cscK gene fragment for constructing a vector.

[0080] To obtain the SPL13 promoter and the CJ7 promoter, PCR (SolgTM Pfu-X DNA polymerase) was performed under the conditions of Table 4 using the primers of SEQ ID NO: 23 and 24 (Table 7), SEQ ID NO: 27 and 28 (Table 8).

[0081] After ligating the amplified SPL13, CJ7 promoter sites, the cscK (E.co) gene fragment, and the vector pDZTn (U.S. Patent Registration US8932861 B2) digested with ScaI restriction enzyme using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method, it was transformed into Escherichia coli DH5α and spread on an LB solid medium conjugated with kanamycin (25 mg / l). In order to select colonies transformed with the vector ligated with the target gene and pDZTn, PCR was performed with the primers of SEQ ID NO: 47 and 48 (Table 3). Plasmids were obtained from the selected colonies using the commonly known plasmid extraction method, and the plasmids were named pDZTn-Pspl13-cscK (E.co) and pDZTn-Pcj7-cscK (E.co).

[0082]

Table 7

[0083]

Table 8

[0084] Example 1-4. Preparation of a vector in which zwf derived from Corynebacterium glutamicum ATCC13032 and cscK derived from Escherichia coli are enhanced To confirm the effects of individual gene enhancement in Example 1-1 and Example 1-3 and to confirm the simultaneous enhancement effect of both genes, a vector was prepared.

[0085] Using pDZTn-Pspl13-zwf(C.gl13032) of Example 1-1 and pDZTn-Pcj7-cscK(E.co) of Example 1-3 as templates respectively, primers containing the polynucleotides of SEQ ID NO: 31, 32 and SEQ ID NO: 33, 34 (Table 9) were prepared, and PCR (SolgTM Pfu-X DNA polymerase) was performed under the conditions of Table 4 to obtain Pspl13-zwf(C.gl13032) and Pcj7-cscK(E.co). The amplified Pspl13-zwf(C.gl13032) and Pcj7-cscK(E.co) gene fragments and the vector pDZTn (U.S. Registered Patent US8932861 B2) cleaved with ScaI restriction enzyme were ligated using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method, then transformed into Escherichia coli DH5α and spread on an LB solid medium conjugated with kanamycin (25 mg / l). To select colonies transformed with the vector in which the target gene and pDZTn were ligated, PCR was performed with the primers of SEQ ID NO: 47 and 48 (Table 3). A plasmid was obtained from the selected colonies using a commonly known plasmid extraction method, and the plasmid was named pDZTn-Pspl13-zwf(C.gl13032)_Pcj7-cscK(E.co).

[0086] Similar to the above method, using pDZTn-Pcj7-zwf(C.gl13032) of Example 1-1 and pDZTn-Pspl13-cscK(E.co) of Example 1-3 as templates respectively, primers containing the polynucleotides of SEQ ID NO: 35, 36 (Table 10) and SEQ ID NO: 37, 38 (Table 10) were prepared, and PCR (SolgTM Pfu-X DNA polymerase) was performed under the conditions shown in Table 4 to obtain Pcj7-zwf(C.gl13032) and Pspl13-cscK(E.co). The amplified Pcj7-zwf(C.gl13032) and Pspl13-cscK(E.co) gene fragments and the vector pDZTn (U.S. Registered Patent US8932861 B2) digested with ScaI restriction enzyme were ligated using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method, then transformed into Escherichia coli DH5α, and spread on an LB solid medium conjugated with kanamycin (25 mg / l). In order to select colonies transformed with the vector in which the target gene and pDZTn were ligated, PCR was performed with the primers of SEQ ID NO: 47 and 48 (Table 3). Plasmids were obtained from the selected colonies using a commonly known plasmid extraction method, and the plasmid was named pDZTn-Pcj7-zwf(C.gl13032)_Pspl13-cscK(E.co).

[0087]

Table 9

[0088]

Table 10

[0089] Example 1-5. Preparation of a vector with enhanced zwf from Corynebacterium glutamicum ATCC13869 and cscK from Escherichia coli To confirm the individual gene enhancement effects of Example 1-2 and Example 1-3 and to confirm the simultaneous enhancement effect of both genes, a vector was prepared.

[0090] Using pDZTn-Pspl13-zwf(C.gl13869) of Example 1-2 and pDZTn-Pcj7-cscK(E.co) of Example 1-3 as templates respectively, primers containing the polynucleotides of SEQ ID NO: 39, 10 and SEQ ID NO: 41, 42 (Table 11) were prepared, and PCR (SolgTM Pfu-X DNA polymerase) was performed under the conditions of Table 4 to obtain Pspl13-zwf(C.gl13869) and Pcj7-cscK(E.co). The amplified Pspl13-zwf(C.gl13869) and Pcj7-cscK(E.co) gene fragments and the vector pDZTn (U.S. Registered Patent US8932861 B2) cleaved with ScaI restriction enzyme were ligated using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method, then transformed into Escherichia coli DH5α and spread on an LB solid medium conjugated with kanamycin (25 mg / l). To select colonies transformed with the vector in which the target gene and pDZTn were ligated, PCR was performed with the primers of SEQ ID NO: 49 and 50 (Table 3). A plasmid was obtained from the selected colonies using the commonly known plasmid extraction method, and the plasmid was named pDZTn-Pspl13-zwf(C.gl13869)_Pcj7-cscK(E.co).

[0091] Similar to the above method, using pDZTn-Pcj7-zwf(C.gl13869) of Example 1-2 and pDZTn-Pspl13-cscK(E.co) of Example 1-3 as templates respectively, primers containing the polynucleotides of SEQ ID NO: 43, 44 and SEQ ID NO: 45, 46 (Table 12) were prepared, and PCR (SolgTM Pfu-X DNA polymerase) was performed under the conditions in Table 4 to obtain Pcj7-zwf(C.gl13869) and Pspl13-cscK(E.co). The amplified Pcj7-zwf(C.gl13869) and Pspl13-cscK(E.co) gene fragments and the vector pDZTn (U.S. Registered Patent US8932861 B2) cleaved with ScaI restriction enzyme were ligated using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method, then transformed into Escherichia coli DH5α, and spread on an LB solid medium conjugated with kanamycin (25 mg / l). In order to select colonies transformed with the vector in which the target gene and pDZTn were ligated, PCR was performed with the primers of SEQ ID NO: 47 and 48 (Table 3). Plasmids were obtained from the selected colonies using the commonly known plasmid extraction method, and this plasmid was named pDZTn-Pcj7-zwf(C.gl13869)_Pspl13-cscK(E.co).

[0092]

Table 11

[0093]

Table 12

[0094] Example 2. Preparation of an enhanced strain Example 2-1. Preparation of a lysine-producing strain with enhanced zwf derived from Corynebacterium glutamicum ATCC13032 Using the pDZTn-Pspl13-zwf(C.gl13032) and pDZTn-Pcj7-zwf(C.gl13032) vectors prepared in Example 1-1, Corynebacterium glutamicum KCCM11016P (Korean Registered Patent No. 10-0159812), KCCM10770P (Korean Registered Patent No. 10-0924065), and CJ3P (Binder et al. Genome Biology 2012, 13:R40) strains that produce L-lysine were transformed by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). After that, strains in which Pspl13-zwf(C.gl13032) and Pcj7-zwf(C.gl13032) were inserted between transposon genes were obtained through a secondary crossing process. PCR and nucleotide sequence analysis were performed using primers with SEQ ID NO: 47 and SEQ ID NO: 48 (Table 3) that can amplify the adjacent sites containing the positions where the genes were inserted to confirm the genetic manipulation. The strains thus obtained were named Corynebacterium glutamicum KCCM11016P_Pspl13-zwf(C.gl13032), KCCM11016P_Pcj7-zwf(C.gl13032), KCCM10770P_Pspl13-zwf(C.gl13032), KCCM10770P_Pcj7-zwf(C.gl13032), CJ3P_Pspl13-zwf(C.gl13032), and CJ3P_Pcj7-zwf(C.gl13032).

[0095] Example 2-2. Preparation of a Tryptophan-Producing Strain with Enhanced zwf Derived from Corynebacterium glutamicum ATCC13869 Using the pDZTn-Pspl13-zwf(C.gl13869) and pDZTn-Pcj7-zwf(C.gl13869) vectors prepared in Example 1-2, the Corynebacterium glutamicum KCCM12218P (Korean Registered Patent No. 10-2035844) strain that produces L-tryptophan was transformed by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). After that, through a secondary crossing process, strains in which Pspl13-zwf(C.gl13869) and Pcj7-zwf(C.gl13869) were inserted between transposon genes were obtained. PCR and nucleotide sequence analysis were performed using the primers of SEQ ID NO: 47 and SEQ ID NO: 48 (Table 3) that can amplify the adjacent sites including the positions where the genes were inserted to confirm the genetic manipulation. The strains thus obtained were named Corynebacterium glutamicum KCCM12218P_Pspl13-zwf(C.gl13869) and KCCM12218P_Pcj7-zwf(C.gl13869).

[0096] Example 2-3. Preparation of Lysine and Tryptophan Producing Strains with Enhanced Escherichia coli-derived cscK Using the pDZTn-Pspl13-cscK(E.co) and pDZTn-Pcj7-cscK(E.co) vectors prepared in Examples 1-3, Corynebacterium glutamicum KCCM11016P (Korean Registered Patent No. 10-0159812), KCCM10770P (Korean Registered Patent No. 10-0924065), CJ3P (Binder et al. Genome Biology 2012, 13:R40) strains that produce L-lysine and Corynebacterium glutamicum KCCM12218P (Korean Registered Patent No. 10-2035844) strain that produces L-tryptophan were transformed by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). After that, strains in which Pspl13-cscK(E.co) and Pcj7-cscK(E.co) were inserted between transposon genes were obtained through a secondary crossing process. PCR and nucleotide sequence analysis were performed using primers of SEQ ID NO: 47 and SEQ ID NO: 48 (Table 3) that can amplify the adjacent site containing the position where the gene was inserted to confirm the genetic manipulation. The lysine-producing strains thus obtained were named Corynebacterium glutamicum KCCM11016P_Pspl13-cscK(E.co), KCCM11016P_Pcj7-cscK(E.co), KCCM10770P_Pspl13-cscK(E.co), KCCM10770P_Pcj7-cscK(E.co), CJ3P_Pspl13-cscK(E.co), CJ3P_Pcj7-cscK(E.co), and the tryptophan-producing strains were named KCCM12218P_Pspl13-cscK(E.co), KCCM12218P_Pcj7-cscK(E.co).

[0097] Example 2-4. Preparation of Lysine-Producing Strains with Enhanced zwf Derived from Corynebacterium glutamicum ATCC13032 and cscK Derived from Escherichia coli Using the pDZTn-Pspl13-zwf(C.gl13032)_Pcj7-cscK(E.co) and pDZTn-Pcj7-zwf(C.gl13032)_Pspl13-cscK(E.co) vectors prepared in Examples 1-4, Corynebacterium glutamicum KCCM11016P (Korean Registered Patent No. 10-0159812), KCCM10770P (Korean Registered Patent No. 10-0924065), and CJ3P (Binder et al. Genome Biology 2012, 13:R40) strains that produce L-lysine were transformed by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). After that, through a secondary crossing process, strains in which Pspl13-zwf(C.gl13032)_Pcj7-cscK(E.co) and Pcj7-zwf(C.gl13032)_Pspl13-cscK(E.co) were inserted between transposon genes were obtained. PCR and nucleotide sequence analysis were performed using the primers of SEQ ID NO: 47 and SEQ ID NO: 48 (Table 3) that can amplify the adjacent sites including the positions where the genes were inserted to confirm the genetic manipulation. The strains thus obtained were named Corynebacterium glutamicum KCCM11016P_Pspl13-zwf(C.gl13032)_Pcj7-cscK(E.co), KCCM11016P_Pcj7-zwf(C.gl13032)_Pspl13-cscK(E.co), KCCM10770P_Pspl13-zwf(C.gl13032)_Pcj7-cscK(E.co), KCCM10770P_Pcj7-zwf(C.gl13032)_Pspl13-cscK(E.co), CJ3P_Pspl13-zwf(C.gl13032)_Pcj7-cscK(E.co), and CJ3P_Pcj7-zwf(C.gl13032)_Pspl13-cscK(E.co).

[0098] Example 2-5. Preparation of Tryptophan-Producing Strains with Enhanced zwf Derived from Corynebacterium glutamicum ATCC13869 and cscK Derived from Escherichia coli Using the pDZTn-Pspl13-zwf(C.gl13869)_Pcj7-cscK(E.co) and pDZTn-Pcj7-zwf(C.gl13869)_Pspl13-cscK(E.co) vectors prepared in Examples 1-5 respectively, the Corynebacterium glutamicum KCCM12218P strain (Korean Registered Patent No. 10-2035844) that produces L-tryptophan was transformed by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). After that, through a secondary crossing process, strains in which Pspl13-zwf(C.gl13869)_Pcj7-cscK(E.co) and Pcj7-zwf(C.gl13869)_Pspl13-cscK(E.co) were inserted between transposon genes were obtained respectively. PCR and nucleotide sequence analysis were performed using the primers of SEQ ID NO: 47 and SEQ ID NO: 48 (Table 3) that can amplify the adjacent sites including the positions where the genes were inserted to confirm the genetic manipulation. The tryptophan-producing strains thus obtained were named Corynebacterium glutamicum KCCM12218P_Pspl13-zwf(C.gl13869)_Pcj7-cscK(E.co) and KCCM12218P_Pcj7-zwf(C.gl13869)_Pspl13-cscK(E.co).

[0099] Example 3. Comparison of the L-lysine or L-tryptophan production ability of Zwf and cscK enhanced strains Example 3-1. Comparison of L-lysine production ability of zwf derived from Corynebacterium glutamicum ATCC13032 and cscK enhanced strains derived from Escherichia coli The KCCM11016P, KCCM10770P, and CJ3P-based zwf(C.gl13032), cscK(E.co) co-enhanced strains prepared in Example 2-4, the KCCM11016P, KCCM10770P, and CJ3P-based zwf(C.gl13032) enhanced strains prepared in Example 2-1, and the KCCM11016P, KCCM10770P, and CJ3P-based cscK(E.co) enhanced strains prepared in Example 2-3 were cultured by the following methods respectively, and the cell mass, sugar consumption ability, and lysine production ability were compared.

[0100] First, each strain was inoculated into a 250-ml conical baffle flask containing 25 ml of seed medium and cultured with shaking at 200 rpm at 30°C for 20 hours. 1 ml of the seed culture solution was inoculated into a 250-ml conical baffle flask containing 24 ml of production medium and cultured with shaking at 200 rpm at 37°C for 42 hours. After the culture was completed, the production amount of L-lysine was measured by HPLC. The above experiment was repeated three times, and the culture results (average values) are shown in Tables 13, 14, and 15.

[0101] <Seed medium (pH 7.0)> Raw sugar 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4·7H2O 0.5 g, biotin 0.1 mg, thiamine HCl 1 mg, calcium pantothenate 22 mg, nicotinamide 2 g (based on 1 liter of distilled water)

[0102] <Production medium (pH 7.0)> Raw sugar 45 g, (NH4)2SO4 15 g, soybean protein 10 g, molasses 10 g, KH2PO4 0.55 g, MgSO4·7H2O 0.6 g, biotin 0.9 mg, thiamine hydrochloride 4.5 mg, calcium pantothenate 4.5 mg, nicotinamide 30 mg, MnSO4 9 mg, FeSO4 9 mg, ZnSO4 0.45 mg, CuSO4 0.45 mg, CaCO3 30 g (based on 1 liter of distilled water).

[0103] The zwf (C.gl13032), cscK (E.co) single or co-enhanced lysine-producing strains prepared above showed a 1.2 - 2.6% improvement in yield compared to the parent strain KCCM11016P as shown in Table 13 below. Since 4 mol of NADPH is required for the production of 1 mol of lysine from Corynebacterium, an increase in the NADPH supply capacity in lysine-producing strains is closely related to an immediate increase in yield (Kjeld Raunkjaer Kjeldsen et al., Biotechnol. Bioeng., 2009 Feb 1;102(2):583 - 97.). In the present invention, the zwf gene was singly enhanced in the KCCM1016P strain to improve the NADPH supply capacity, and as a result, an improvement effect of 0.9 g / L in production was confirmed. Furthermore, the cscK (E.co) gene was introduced so that intracellular fructose generated during the utilization of raw sugar by Corynebacterium could be immediately utilized, and an improvement effect of 2.1 / L, which was 15.2% higher than that of the parent strain KCCM11016P, was confirmed. Finally, by co-enhancing cscK and zwf, it was confirmed by the evaluation of the KCCM11016P_Pspl13-zwf (C.gl13032)_Pcj7-cscK (E.co) strain that the reducing power supply capacity through the zwf gene was maximized and the improvement effect of lysine yield increased significantly compared to the single enhancement ratio.

[0104] The enhancement effect of the said gene showed a similar effect in other lysine-producing strains KCCM10770P and CJ3P as shown in Tables 14 and 15. In all cases, when co-enhanced compared to the zwf (C.gl13032) and cscK (E.co) single-enhanced strains, the production amounts of 1.9 g / L and 1.6 g / L, which were increased by 22.1% and 27.1% respectively compared to the parent strain, were improved.

[0105]

Table 13

[0106]

Table 14

[0107]

Table 15

[0108] Example 3-2. Comparison of L-tryptophan production ability between zwf derived from Corynebacterium glutamicum ATCC13869 and cscK-enhanced strain derived from Escherichia coli The KCCM12218P-based zwf (C.gl13869), cscK (E.co) co-enhanced strain prepared in Example 2-5, the KCCM12218P-based zwf (C.gl13869) enhanced strain prepared in Example 2-2, and the KCCM12218P-based cscK (E.co) enhanced strain prepared in Example 2-3 were each cultured in the following manner, and the cell mass, sugar consumption ability, and tryptophan production ability were compared.

[0109] First, each strain was inoculated into a 250 ml conical baffle flask containing 25 ml of the seed medium and cultured with shaking at 30 °C for 20 hours at 200 rpm. 1 ml of the seed culture solution was inoculated into a 250 ml conical baffle flask containing 24 ml of the production medium and cultured with shaking at 37 °C for 42 hours at 200 rpm. After the culture was completed, the production amount of L-tryptophan was measured by HPLC. The above experiment was repeated 3 times, and the culture results (average values) are shown in Table 16.

[0110] <Seed medium (pH 7.0)> Raw sugar 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4·7H2O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium pantothenate 2000 μg, nicotinamide 2000 μg (based on 1 liter of distilled water)

[0111] <Production medium (pH 7.0)> Raw sugar 30 g, (NH4)2SO4 15 g, MgSO4·7H2O 1.2 g, KH2PO4 1 g, yeast extract 5 g, biotin 900 μg, thiamine hydrochloride 4500 μg, calcium pantothenate 4500 μg, CaCO3 30 g (based on 1 liter of distilled water).

[0112] In the tryptophan-producing strains in which the prepared zwf (C.gl13869) and cscK (E.co) genes were individually enhanced, the yield of tryptophan was improved by 0.58 - 1.67% compared to the parent strain KCCM12218P as shown in Table 16 below. In particular, the enhancement of the expression of the cscK gene had a greater impact on the improvement of the tryptophan yield than the enhancement of the zwf gene. This is because a part of the fructose generated by the decomposition of the original sugar inside the cell was not discharged outside the cell, but was converted into fructose-6-phosphate and flowed into the glycolysis process. It is judged that the fructose discharged by such a phenomenon was used as a tryptophan precursor without consuming the PEP pool into which it flowed. In addition, the enhancement of the tryptophan yield by the expression of the zwf gene is judged to have improved the intracellular concentrations of PRPP and E4P used as tryptophan precursors. In the strain in which both the zwf and cscK genes were simultaneously enhanced, the tryptophan yield improvement factor was integrated, and the production amount of tryptophan was improved by 34% - 45% compared to the parent strain KCCM12218P. A significantly improved tryptophan yield was confirmed compared to when the two genes were individually enhanced.

[0113]

Table 16

[0114] From the above description, those skilled in the technical field to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. In this regard, it should be understood that the embodiments described above are merely exemplary and not restrictive. The scope of the present invention should be interpreted as including the meaning and scope of the claims described below, and all changes or modified forms derived from the equivalent concepts thereof within the scope of the present invention.

Claims

[Claim 1] A Corynebacterium microorganism which produces L-amino acids and has enhanced activities of glucose-6-phosphate 1-dehydrogenase and fructokinase.

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