Microorganism having enhanced l-glutamine producing ability, and l-glutamine producing method using the same

Abstract

To provide a microorganism having enhanced L-glutamine production ability and an L-glutamine production method using the same.SOLUTION: The present invention provides a microorganism of the genus Corynebacterium having phosphoenolpyruvate carboxykinase activity weakened and L-glutamine production ability. The microorganism has improved L-glutamine production ability compared to a parent or wild-type strain of the genus Corynebacterium in which phosphoenolpyruvate carboxykinase activity is not weakened. Also there is provided a method for producing L-glutamine, the method including the step of culturing a microorganism of the genus Corynebacterium having phosphoenolpyruvate carboxykinase activity weakened and L-glutamine production ability in a medium.SELECTED DRAWING: None

Description

Technical Field

[0001] This application relates to a microorganism with improved L-glutamine production ability and an L-glutamine production method using the same.

Background Art

[0002] L-glutamine is an amino acid widely used in pharmaceuticals such as digestive disease therapeutics, liver function enhancers, brain function enhancers, immune boosters, gastric ulcer therapeutics, alcoholism therapeutics, moisturizers for cosmetics, sports nutrition products, and nutritional products for patients, as well as in cosmetics and health foods.

[0003] For the production of L-glutamine by microorganisms, Corynebacterium glutamicum and Escherichia coli are typically used. In the L-glutamine biosynthetic pathway, α-ketoglutaric acid produced by the glycolysis process and the TCA cycle is used as a precursor, and L-glutamate is produced by glutamate dehydrogenase, and finally L-glutamine is produced by the reaction of glutamine synthetase (Non-Patent Document 1).

[0004] However, with the increasing demand for L-glutamine, research for effectively improving the production ability of L-glutamine is still required.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

[0006] [Non - Patent Document 1] Production of glutamate and glutamate - related aminoacids: Molecular Mechanism Analysis and Metabolic Engineering, Amino acid Biosynthesis - pathways, regulation and metabolic engineering pp1 - 38 [Non - Patent Document 2] Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]: 2444 [Non - Patent Document 3] Rice et al., 2000, Trends Genet. 16: 276 - 277 [Non - Patent Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443 - 453 [Non - Patent Document 5] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non - Patent Document 6] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) [Non - Patent Document 7] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994

Non - Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Non - Patent Document 11

Non - Patent Document 12

Non - Patent Document 13

Non - Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Summary of the Invention

Problems to be Solved by the Invention

[0007] As a result of intensive efforts to improve the L-glutamine production ability, the inventors have confirmed that L-glutamine is produced with high efficiency in microorganisms containing phosphoenolpyruvate carboxykinase, and have thus completed this application. reached.

Means for Solving the Problems

[0008] This application aims to provide a Corynebacterium microorganism with weakened phosphoenolpyruvate carboxykinase activity and the ability to produce L-glutamine.

[0009] In addition, this application aims to provide a method for producing L-glutamine, which includes culturing a Corynebacterium microorganism with weakened phosphoenolpyruvate carboxykinase activity and the ability to produce L-glutamine in a medium.

[0010] Furthermore, this application aims to provide a method for producing a microorganism for L-glutamine production, which includes a step of weakening phosphoenolpyruvate carboxykinase.

[0011] Furthermore, this application aims to provide a composition for L-glutamine production, which includes a microorganism with weakened phosphoenolpyruvate carboxykinase, a medium in which the microorganism is cultured, or a combination thereof.

[0012] Furthermore, this application aims to provide the use of a Corynebacterium microorganism with weakened phosphoenolpyruvate carboxykinase in the production of L-glutamine.

Advantages of the Invention

[0013] The microorganism containing the weakened phosphoenolpyruvate carboxykinase according to this application can produce L-glutamine with high efficiency. The produced L-glutamine can be applied to various products such as animal feed or animal feed additives, as well as human food or food additives, pharmaceuticals, etc.

Modes for Carrying Out the Invention

[0014] ​​The following will specifically describe these. Note that each description and embodiment disclosed in this application is applicable to other descriptions and embodiments respectively. That is, any combination of various elements disclosed in this application is included in this application. Also, this application is not limited to the following specific descriptions. Furthermore, those with ordinary knowledge in the relevant technical field will be able to recognize and confirm many equivalents of the specific aspects of this application described in this application through only ordinary experiments. Moreover, it is intended that such equivalents are also included in this application. Additionally, many papers and patent documents are referenced throughout this specification, and their citations are indicated. The entire disclosure content of the cited papers and patent documents is incorporated herein by reference, thereby more clearly explaining the level of the technical field to which this application belongs and the content of this application.

[0015] One aspect of this application for achieving the above object is to provide a Corynebacterium microorganism with weakened Phosphoenolpyruvate carboxykinase activity and having the ability to produce L-glutamine.

[0016] "Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32)" in this application refers to an enzyme that is involved in the gluconeogenesis synthesis process and, in the presence of GTP, converts Oxaloacetate into Phosphoenolpyruvate and carbon dioxide. The Phosphoenolpyruvate carboxykinase of this application is used interchangeably with PEPCK.

[0017] The amino acid sequence of the aforementioned PEPCK is obtained from known databases such as Genbank of NCBI.

[0018] For example, the PEPCK of the present application may be a protein derived from a microorganism of the genus Corynebacterium. More specifically, the PEPCK of the present application may be derived from Corynebacterium glutamicum, Corynebacterium deserti, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium callunae, etc., but is not limited thereto.

[0019] The PEPCK of the present application may be an endogenous protein of the Corynebacterium genus microorganism of the present application.

[0020] In the present application, the PEPCK of the present application may have the amino acid sequence represented by SEQ ID NO: 1, may include the amino acid sequence, may consist of the amino acid sequence, or may be essentially consisting of the amino acid sequence.

[0021] In the present application, the amino acid sequence of SEQ ID NO: 1 includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity thereto. Also, it goes without saying that a protein having an amino acid sequence with such homology or identity and showing an efficacy corresponding to the protein containing the amino acid sequence of SEQ ID NO: 1 and having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added is also included in the present application.

[0022] For example, addition or deletion of a sequence that does not change the function of the protein of the present application at the N-terminus, C-terminus and / or inside of the amino acid sequence, naturally occurring mutations, silent mutations Those having a (silent mutation) or conservative substitution are exemplified.

[0023] The “conservative substitution” means that one amino acid is substituted with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on the similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues. Usually, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0024] The “homology” or “identity” in the present application means the degree to which two given amino acid sequences or base sequences are similar, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0025] The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard sequence algorithms, and the default gap penalty established by the program used may be used together. Substantially, homologous or identical sequences can generally hybridize with all or part of the sequence under moderately or highly stringent conditions. It goes without saying that hybridization also includes hybridization with a polynucleotide having a codon considering general codons or codon degeneracy in the polynucleotide.

[0026] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as default parameters like those in Non-Patent Document 2 and the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 4) as performed by the needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later versions) (including the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, FASTA (Non-Patent Documents 6, 7 and 8)). For example, homology, similarity or identity can be determined using BLAST or Clustal W of the National Center for Biotechnology Information.

[0027] The homology, similarity or identity of a polynucleotide or polypeptide can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 4, as disclosed in Non-Patent Document 9 for example. Briefly, the GAP program defines it as the value obtained by dividing the number of similar sequence symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program include: (1) a binary comparison matrix (with a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Non-Patent Document 11 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 10, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0028] The PEPCK of the present application may be encoded by the pck gene. The pck gene of the present application may be any gene known to encode a protein having PEPCK activity.

[0029] Specifically, the pck gene of the present application may be pck derived from a microorganism of the genus Corynebacterium. More specifically, the pck gene of the present application may be pck derived from the Corynebacterium glutamicum strain. For example, the pck gene of the present application may be a polynucleotide encoding WP_011015446.1 derived from Corynebacterium glutamicum ATCC13032.

[0030] The "polynucleotide" in the present application refers to a polymer of nucleotides in which nucleotide monomers are covalently linked in a long chain, meaning a DNA or RNA strand longer than a predetermined length, and more specifically refers to a polynucleotide fragment encoding the protein.

[0031] The polynucleotide encoding the PEPCK of the present application may include a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. As an example of the present application, the polynucleotide of the present application may have the base sequence of SEQ ID NO: 2, or may include the base sequence of SEQ ID NO: 2. Further, the polynucleotide of the present application may consist of the sequence of SEQ ID NO: 2, or may be essentially composed of the sequence of SEQ ID NO: 2. Specifically, the PEPCK may be encoded by a polynucleotide represented by the base sequence of SEQ ID NO: 2.

[0032] The polynucleotide of the present application can be variously modified in the coding region within the range where the amino acid sequence of PEPCK does not change, due to the degeneracy of codons or considering the preferred codons in the organism in which PEPCK of the present application is to be expressed. Specifically, the polynucleotide of the present application has a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more homology or identity with the sequence of SEQ ID NO: 2, or includes the base sequence, or consists of a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more homology or identity with the sequence of SEQ ID NO: 2, or is essentially composed of the base sequence, but is not limited thereto.

[0033] In addition, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or part of the polynucleotide sequence of the present application. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 12 and 13). For example, conditions for hybridizing polynucleotides having high homology or identity, i.e., polynucleotides having 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity, and not hybridizing polynucleotides having lower homology or identity, or washing conditions for ordinary Southern hybridization, such as washing once, specifically 2 to 3 times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0034] Hybridization requires that two nucleic acids have complementary sequences, even if base mismatches are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include not only nucleic acid sequences that are substantially similar, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0035] Specifically, polynucleotides having homology or identity with the polynucleotides of the present application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. The Tm value may be 60°C, 63°C or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art according to the purpose.

[0036] The appropriate stringency for hybridizing the polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the variables are known in the art (for example, Non-Patent Document 12).

[0037] The "microorganism (or strain)" in the present application includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially. Due to reasons such as the insertion of foreign genes or the enhancement or weakening of the activity of endogenous genes, the microorganism is one in which a specific mechanism is weakened or enhanced, and may be a microorganism in which genetic modification has been performed for the production of a target polypeptide, protein or product.

[0038] The polypeptides in the present application (for example, include proteins specified by the names of each enzyme. The "attenuation" of the activity of [[ID=]] means a concept that includes both a decrease in activity compared to the intrinsic activity and the disappearance of activity. The above-mentioned attenuation is used interchangeably with inactivation, deficiency, down-regulation, decrease, reduce, attenuation, etc. tion).

[0039] The above-mentioned attenuation includes cases where the activity of the polypeptide itself is decreased or removed compared to the activity of the polypeptide originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, etc., and the overall degree and / or concentration (expression level) of the polypeptide activity in the cell is decreased compared to the natural strain due to inhibition of the expression of the gene of the polynucleotide encoding it or inhibition of translation into the polypeptide, etc., and includes cases where the expression of the polynucleotide is completely absent, and / or cases where even if the polynucleotide is expressed, the polypeptide has no activity may also be included. Such attenuation of the activity of the polypeptide is not limited to these, and can be achieved by applying various methods well-known in the art (for example, Non-Patent Documents 14, 15, etc.).

[0040] This attenuation of the activity of the polypeptide is not limited to these, and can be achieved by applying various methods well-known in the art (for example, Non-Patent Documents 14, 15, etc.).

[0041] Specifically, the attenuation of the activity of the polypeptide of the present application can be achieved by: 1) deleting all or part of the gene encoding the polypeptide; 2) modifying the expression regulatory region (or expression regulatory sequence) so that the expression of the gene encoding the polypeptide is decreased; 3) modifying the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is deleted or attenuated (for example, deleting / substituting / adding one or more amino acids in the amino acid sequence); 4) modifying the nucleotide sequence encoding the polypeptide so that the activity of the polypeptide is deleted or attenuated (for example, deleting / substituting / adding one or more nucleotides in the nucleotide sequence of the gene encoding the polypeptide so that it encodes a polypeptide whose activity is deleted or attenuated); 5) modifying the base sequence of the start codon or 5'UTR region of the gene transcript encoding the polypeptide; 6) introducing an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide so that a secondary structure that makes it impossible for ribosomes to attach is formed; 8) adding a promoter so as to reverse transcribe to the 3' end of the open reading frame (ORF) of the nucleotide sequence encoding the polypeptide (Reverse transcription engineering, RTE); or 9) a combination thereof (for example, a combination of two or more selected from the above 1) to 8)), but is not particularly limited to the above examples.

[0042] For example, the above 1) deleting all or part of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, substituting it with a polynucleotide with some nucleotides deleted or a marker gene.

[0043] Modifying the expression regulatory region (or expression regulatory sequence) in step 2) may be performed by introducing mutations in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting it with a sequence having lower activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0044] Modifying the amino acid sequence or nucleotide sequence in steps 3) and 4) may be performed by deleting, inserting, non-conservative or conservative substitution, or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so that the activity of the polypeptide is weakened, or by substituting it with an amino acid sequence or polynucleotide sequence improved to have lower activity, or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, introducing a mutation into the polynucleotide sequence to form a stop codon can inhibit or weaken the expression of the gene, but is not limited thereto.

[0045] Modifying the start codon of the gene transcript encoding the polypeptide in step 5) or the nucleotide sequence encoding the 5'UTR region may be performed, for example, by substituting it with a nucleotide sequence encoding another start codon having a lower expression rate of the polypeptide compared to the endogenous start codon, but is not limited thereto.

[0046] Introducing an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide in step 6) may be performed, for example, as in Non-Patent Document 16.

[0047] Adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide so that a secondary structure that prevents the attachment of ribosomes is formed may be done by making mRNA translation impossible or weakening the rate.

[0048] Also, adding a promoter so as to reverse-transcribe at the 3'-end of the ORF of the nucleobase sequence encoding the polypeptide may be done by forming an antisense nucleotide complementary to the transcription product of the gene encoding the polypeptide and weakening the activity.

[0049] In the microorganism of the present application, partial or total modification of the polynucleotide can be induced by (a) homologous recombination using a chromosomal integration vector in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) treatment with light such as ultraviolet rays or radiation and / or chemical substances, but is not limited thereto. The method for partial or total modification of the gene includes methods by DNA recombination technology. For example, partial or total deletion of the gene is performed by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to cause homologous recombination. The introduced nucleotide sequence or vector may contain a dominant selection marker, but is not limited thereto.

[0050] As used herein, the vector of the present application means a DNA product containing the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression regulatory region (or expression regulatory sequence) so as to be able to express the target polypeptide in a suitable host. The expression regulatory region may include a promoter that initiates transcription, any operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate or function regardless of the host genome and may be integrated into the genome itself.

[0051] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant states. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0052] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker for confirming whether the polynucleotide has been introduced into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, that is, for confirming whether the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show different phenotypes, so that transformed cells can be selected.

[0053] In the present application, "transformation" means expressing a polypeptide encoded by a polynucleotide in a host cell by introducing a vector containing the polynucleotide encoding the target polypeptide into the host cell or microorganism. As long as the transformed polynucleotide is expressed in the host cell, it may be any one regardless of whether it is inserted into the host cell chromosome or located outside the chromosome. Further, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. As long as the polynucleotide is introduced and expressed in the host cell, it may be introduced in any form. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its own expression. Usually, the expression cassette contains the polynucleotide It includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked thereto. The expression cassette may be in the form of a self-replicating expression vector. Further, the polynucleotide may be introduced into a host cell in its own form and may be operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0054] Also, the “operably linked” means that a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding the target polypeptide of the present application is functionally linked to the polynucleotide sequence.

[0055] The microorganism of the present application may be a microorganism having improved L-glutamine production ability as compared with a parent strain or a wild-type Corynebacterium strain in which phosphoenolpyruvate carboxykinase activity is not weakened. That is, the microorganism of the present application is a microorganism that naturally has L-glutamine production ability, or a microorganism in which PEPCK or a polynucleotide encoding the same is weakened in a parent strain that does not have L-glutamine production ability, but is not limited thereto.

[0056] For example, a PEPCK-unmodified microorganism in which PEPCK is not weakened, which is a target strain for comparing whether or not the L-glutamine production ability is improved, is a wild-type Corynebacterium glutamicum ATCC13032 strain, a Corynebacterium glutamicum ATCC13032 strain in which the activity of glutamine synthetase (Glutamine synthetase, GlnA, EC 6.3.1.2) protein is enhanced, a microorganism in which the activity of the protein is enhanced in the microorganism by introducing a mutation (D401N) into the glnA gene encoding glutamine synthetase, a Corynebacterium glutamicum KCCM12645P (Patent Document 1) which is a glutamine-producing strain, or a Corynebacterium glutamicum KFCC-10680 (Patent Document 2) strain which is a glutamine-producing strain, but is not limited thereto.

[0057] As an example, the recombinant strain with improved production ability has an increase of about 1% or more, specifically about 2% or more, about 5% or more, about 6% or more, about 7% or more, about 7.2% or more, about 8% or more, about 9% or more, or about 9.2% or more (there is no particular limitation on the upper limit value, for example, about 200% or less, about 150% or less, about 100% or less, about 51% or less, about 40% or less, about 30% or less, about 20% or less, or about 15% or less) compared to the L-glutamine production ability of the parent strain before mutation, non-modified microorganism, or PEPCK non-modified microorganism. However, as long as it shows an increase in the + value compared to the production ability of the parent strain before mutation, non-modified microorganism, or PEPCK non-modified microorganism, any one can be used. As another example, the microorganism with improved L-glutamine production ability has an L-glutamine production ability that is about 1.01 times or more, about 1.02 times or more, about 1.05 times or more, about 1.06 times or more, about 1.07 times or more, about 1.072 times or more, about 1.08 times or more, about 1.09 times or more, or about 1.092 times or more (there is no particular limitation on the upper limit value, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) compared to the parent strain before mutation, non-modified microorganism, or PEPCK non-modified microorganism, but it is not limited thereto.

[0058] In the present application, the "non-modified microorganism" does not exclude strains containing mutations that can occur naturally in microorganisms, but means the wild-type strain or the natural strain itself, or the strain before genetic mutation and trait change due to natural factors or artificial factors. Also, in the present application, the "PEPCK non-modified microorganism" means a strain in which the PEPCK described in this specification or the polynucleotide encoding the same is not weakened or before being weakened. The PEPCK non-modified microorganism of the present application does not exclude strains containing modifications of other proteins or other genes other than the modification of PEPCK or the polynucleotide encoding the same.

[0059] In the present application, the "non-modified microorganism" is used interchangeably with "strain before modification", "microorganism before modification", "non-mutated strain", "non-modified strain", "non-mutated microorganism", or "reference microorganism".

[0060] The microorganism of the present application is a microorganism containing PEPCK with weakened intrinsic activity or a polynucleotide encoding the same, or a microorganism (e.g., a recombinant microorganism) genetically modified to contain PEPCK with weakened intrinsic activity or a polynucleotide encoding the same, but is not limited thereto. The "intrinsic activity" means the activity of a specific polypeptide originally possessed by the parental strain, wild type or unmodified microorganism before the trait change when the trait changes due to natural or artificial genetic mutations. This is used interchangeably with the "activity before modification". When the activity of a polypeptide is "weakened, inactivated, lacking, decreased, down-regulated, reduced, attenuated" compared to the intrinsic activity, it means that it is lower than the activity of the specific polypeptide originally possessed by the parental strain or unmodified microorganism before the trait change. When the activity of a polypeptide is "weakened, inactivated, lacking, decreased, down-regulated, reduced, attenuated" compared to the intrinsic activity, it means that it is lower than the activity of the specific polypeptide originally possessed by the parental strain or unmodified microorganism before the trait change.

[0061] As another example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Co rynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Coryneb acterium singulare, Corynebacterium halotolerans, Coryneb acterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Coryneb Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, specifically Corynebacterium glutamicum, but not limited thereto.

[0062] As another example, the recombinant microorganism of the present application may be a microorganism in which the production ability of L-glutamine is enhanced by further enhancing the activity of some proteins in the L-glutamine biosynthetic pathway or further weakening the activity of some proteins in the L-glutamine degradation pathway.

[0063] Specifically, in the present application, the Corynebacterium genus microorganism is a microorganism into which the above-mentioned glnA enhancement (for example, increase in copy number, change in promoter, release of activity regulation by adenylation, weakening of glnE, or weakening of the activity of PII protein; Patent Document 3) is further introduced, a microorganism into which GDH (glutamate dehydrogenase) enhancement (for example, increase in copy number, or modification of expression regulatory sequence) is further introduced (Patent Document 3), a microorganism into which a mutation of glnA enhancement is further introduced (Patent Document 1), but not limited thereto.

[0064] The "enhancement" of the polypeptide activity in the present application means improving the activity of the polypeptide compared to the endogenous activity. The above-mentioned enhancement is used interchangeably with activation, up-regulation, overexpression, increase, etc. Here For activation, enhancement, upregulation, overexpression, and improvement, it all includes showing an activity that was not originally present, or having an improved activity compared to the intrinsic activity or the activity before modification. The "intrinsic activity" means the activity of a specific polypeptide that the parental strain or unmodified microorganism had before the trait change when the trait changes due to genetic mutation by natural or artificial factors. This is used interchangeably with the "activity before modification". When the activity of a polypeptide is "enhanced", "upregulated", "overexpressed", or "improved" compared to the intrinsic activity, it means an improvement compared to the activity and / or concentration (expression level) of the specific polypeptide that the parental strain or unmodified microorganism had before the trait change.

[0065] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of the intrinsic polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the degree of activity of the polypeptide, the expression level, or the amount of the product produced from the polypeptide.

[0066] For the enhancement of the polypeptide activity, various methods well-known in the art can be applied, and any method can be used as long as it can enhance the activity of the target polypeptide more than that of the microorganism before modification. Specifically, it is a usual method in molecular biology, using genetic engineering and / or protein engineering well-known to those with ordinary knowledge in the technical field, but it is not limited thereto (for example, Non-Patent Documents 15, 17, etc.).

[0067] Specifically, the enhancement of the polypeptide activity of the present application is achieved by: 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence having strong activity; 3) modifying the nucleotide sequence encoding the start codon or 5' UTR region of the gene transcript encoding the polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying the polynucleotide sequence encoding the polypeptide so that the polypeptide activity is enhanced (for example, modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so that the polypeptide activity is enhanced); 6) introducing a foreign polypeptide showing the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) optimizing the codons of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide, selecting and modifying the exposed portion, or chemically modifying it; or 9) by a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0068] More specifically, the above-mentioned 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide is carried out by introducing into the host cell a vector that replicates and functions regardless of the host and to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be carried out by introducing one copy or two or more copies of the polynucleotide encoding the polypeptide into the chromosome in the host cell. The introduction into the chromosome is carried out by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0069] Replacing the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide in 2) with a sequence having strong activity can be achieved, for example, by causing mutations in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof so that the activity of the expression regulatory region is further enhanced, or by replacing it with a sequence having higher activity. The expression regulatory region includes, but is not particularly 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. For example, it can be achieved by replacing the original promoter with a strong promoter, but is not limited thereto.

[0070] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (Patent Document 4), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 5), the O2 promoter (Patent Document 6), the tkt promoter, the yccA promoter, and the like.

[0071] Modifying the start codon of the gene transcript encoding the polypeptide in 3) or the base sequence encoding the 5'UTR region can be achieved, for example, by replacing it with a base sequence encoding another start codon with a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0072] Modifying the amino acid sequence or polynucleotide sequence of the above (4) and (5) may involve introducing sequence mutations by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to enhance the activity of the polypeptide, or substituting with an improved amino acid sequence or polynucleotide sequence having higher activity, or an amino acid sequence or polynucleotide sequence improved to enhance the activity, but is not limited thereto. Specifically, the substitution may be performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. Here, the vector used may further contain a selection marker for confirming whether it has been inserted into the chromosome.

[0073] Introducing a foreign polynucleotide that exhibits the activity of the above (6) polypeptide is performed by introducing into a host cell a foreign polynucleotide encoding a polypeptide that exhibits the same / similar activity as the above polypeptide. The origin and sequence of the foreign polynucleotide may be any as long as it exhibits the same / similar activity as the above polypeptide. The introduction can be appropriately selected and performed by those skilled in the art using known transformation methods. The polypeptide may be generated and its activity may be enhanced by the expression of the polynucleotide introduced as described above in the host cell.

[0074] Optimizing the codons of the polynucleotide encoding the above (7) polypeptide is performed by optimizing the codons of the endogenous polynucleotide so that transcription or translation increases in the host cell, or by optimizing the codons of the foreign polynucleotide so that optimized transcription and translation are performed in the host cell.

[0075] Analyzing the tertiary structure of the polypeptide described in 8) above, selecting and modifying the exposed portions, or chemically modifying them may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which the sequence information of known proteins is stored, determining candidates for the template protein according to the degree of sequence similarity, confirming the structure based on this, and selecting and modifying or chemically modifying the exposed portions to be modified.

[0076] Such enhancement of polypeptide activity is achieved by the activity or concentration of the corresponding polypeptide, the expression level being improved compared to the activity or concentration of the polypeptide expressed in the wild-type or unmodified microbial strain, or the amount of the product produced from the polypeptide being increased, but is not limited thereto.

[0077] Another aspect of the present application provides a method for producing L-glutamine, which includes culturing a Corynebacterium microorganism having weakened phosphoenolpyruvate carboxykinase activity and having the ability to produce L-glutamine in a medium.

[0078] The method for producing L-glutamine of the present application may include culturing a microorganism containing PEPCK weakened from its endogenous activity or a polynucleotide encoding the same, or a microorganism genetically modified to contain PEPCK weakened from its endogenous activity or a polynucleotide encoding the same in a medium.

[0079] "Culturing" in the present application means growing the microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culturing conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art according to the selected microorganism. Specifically, the culturing is, but is not limited to, batch, continuous, and / or fed-batch culture.

[0080] In this application, the "medium" refers to a substance obtained by mixing, as the main components, the nutrients necessary for culturing the microorganisms of this application, and supplies nutrients such as water, which is essential for survival and growth, and growth factors. Specifically, the medium and other culture conditions used for culturing the microorganisms of this application may be any that are used for culturing ordinary microorganisms. The microorganisms of this application can be cultured under aerobic conditions by adjusting the temperature, pH, etc. in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin, etc.

[0081] Specifically, the culture medium for microorganisms of the genus Corynebacterium is disclosed in Non-Patent Document 18.

[0082] Examples of the carbon source in this application include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, sugar alcohols such as mannitol and sorbitol, organic acids such as pyruvic acid, lactic acid, and citric acid, and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) can be used, and any other appropriate amount of carbon source can also be used. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0083] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0084] Examples of the phosphorus source include potassium dihydrogen phosphate, dipotassium hydrogen phosphate or their corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. can be used. In addition, amino acids, vitamins and / or suitable precursors, etc. can be used. These components or precursors can be added to the medium in a batch or continuous manner. However, it is not limited thereto.

[0085] Also, during the culture of the microorganism of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in a suitable manner. Furthermore, during the culture, an antifoaming agent such as a fatty acid polyglycol ester can be used to suppress the generation of bubbles. Furthermore, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium. In order to maintain the anaerobic and microaerobic states, it may not be necessary to inject a gas, and nitrogen, hydrogen or carbon dioxide gas may be injected, but it is not limited thereto.

[0086] In the culture of the present application, the culture temperature is maintained at 20 to 45 °C, specifically 25 to 40 °C, and cultured for about 10 to 160 hours, but is not limited thereto.

[0087] The L-glutamine produced by culturing in this application may be secreted into the culture medium or remain intracellularly.

[0088] The method for producing L-glutamine in this application may further include, for example, before the step of culturing, the step of preparing the microorganism of this application, the step of preparing a culture medium for culturing the microorganism, or a combination thereof (in any order).

[0089] The method for producing L-glutamine in this application may further include the step of recovering L-glutamine from the culture medium (the medium in which the culture was performed) or the cultured microorganism used in the culture. The step of recovering may further be included after the step of culturing.

[0090] The recovery may be to recover the target L-glutamine using a suitable method known in the art according to the method for culturing the microorganism of this application, for example, batch, continuous, fed-batch culture methods, etc. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and other various chromatographies, HPLC, or a combination thereof may be used, and the target L-glutamine can be recovered from the culture medium or the microorganism using a suitable method known in the art.

[0091] Also, the method for producing L-glutamine in this application may further include a purification step. The purification can be performed by a suitable method known in the art. For example, when the method for producing L-glutamine in this application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, simultaneously, or integrated as one step, but are not limited thereto.

[0092] As described above, the PEPCK, polynucleotide, vector, microorganism, etc. in the method of the present application are as described above.

[0093] Still another aspect of the present application provides a composition for L-glutamine production comprising a microorganism with weakened PEPCK, a medium in which it is cultured, or a combination thereof.

[0094] The composition of the present application may further contain any suitable excipient commonly used in compositions for L-glutamine production. Such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffering agents, stabilizing agents, isotonic agents, etc.

[0095] Still another aspect of the present application provides a method for producing a microorganism for L-glutamine production, which includes the step of weakening PEPCK.

[0096] Still another aspect of the present application provides the use of a Corynebacterium microorganism with weakened PEPCK for L-glutamine production.

[0097] As described above, the PEPCK, weakening, Corynebacterium microorganism, etc. are as described above.

Examples

[0098] Hereinafter, the present application will be described in more detail with reference to examples. However, these examples are only preferred embodiments for illustrating the present application, and the present application is not limited thereto. Technical matters not described in this specification can be fully understood and easily implemented by those skilled in the technical field of the present application or similar technical fields.

Examples

[0099] Preparation of a vector for weakening the pck gene To weaken the pck gene encoding phosphoenolpyruvate carboxykinase, a vector for gene deletion was constructed.

[0100] Specifically, to prepare a strain lacking the pck gene portion, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and the primers of SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The primer sequences used here are shown in Table 1.

[0101] [Table 1]

[0102] As the polymerase for the PCR reaction, PfuUltra TM High-fidelity DNA polymerase (Stratagene) was used. The PCR conditions were set to perform 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization reaction at 72°C for 1 minute. As a result, a 929-bp DNA fragment upstream of the 5' end centered on the pck gene start codon and a 1000-bp DNA fragment downstream of the 3' end centered on the pck gene stop codon were obtained respectively. Using a PCR purification kit (QUIAGEN), the two amplified DNA fragments were purified and used as the inserted DNA fragments for plasmid preparation. On the other hand, the pDZ vector (Patent Document 7) treated with the restriction enzyme salI and then heat-treated at 65°C for 20 minutes was cloned according to the provided manual using an Infusion Cloning Kit (TaKaRa) so that the molar concentration (M) ratio of the inserted DNA fragment amplified by the above PCR was 1:2, thereby constructing a vector pDZ-ΔPEPCK for pck gene deletion.

Example

[0103] Preparation of a Corynebacterium strain having wild-type-based L-glutamine production ability An L-glutamine-producing strain was found from wild-type Corynebacterium glutamicum ATCC13032. Specifically, in order to improve the activity of glutamine synthetase, which is the last enzyme in the biosynthetic pathway, a strain (deposit number KCCM12645P) was prepared by introducing the glnA (D401N) mutant (SEQ ID NO: 11), which is the gene encoding glutamine synthetase.

[0104] More specifically, in order to prepare a strain into which the glnA (D401N) mutation was introduced, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and the primers of SEQ ID NO: 7 and SEQ ID NO: 8, or SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The primer sequences used here are shown in Table 2.

[0105] [Table 2]

[0106] As the polymerase for the PCR reaction, PfuUltra TM high-fidelity DNA polymerase was used. The PCR conditions were set to perform 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization reaction at 72°C for 1 minute. As a result, a DNA fragment upstream of the 5'-end centered on the glnA gene D401N mutation and a DNA fragment downstream of the 3'-end were obtained respectively. Using a PCR purification kit, the two amplified DNA fragments were purified and used as the inserted DNA fragments for plasmid preparation. On the other hand, the pDZ vector (Patent Document 7) treated with the restriction enzyme speI and then heat-treated at 65°C for 20 minutes, and the molar concentration (M) ratio of the inserted DNA fragment amplified by the above PCR was 1:2. Using an In-Fusion cloning kit and cloning according to the provided manual, pDZ-glnA (D401N) for the insertion of the glnA (D401N) gene mutation was prepared.

[0107] The prepared vector was transformed into Corynebacterium glutamicum ATCC13032 by electroporation and the electric pulse method (Non-Patent Document 19), and a strain containing the glnA (D401N) mutation on the chromosome was obtained by homologous chromosomal recombination. The strain was named Corynebacterium glutamicum CA11-4021 and deposited with the Korean Culture Center of Microorganisms (KCCM), an international depository authority under the Budapest Treaty, under the accession number KCCM12645P on December 19, 2019.

Example

[0108] Preparation and evaluation of an L-glutamine-producing strain with a weakened pck gene In Corynebacterium glutamicum CA11-4021, an L-glutamine-producing strain prepared in Example 2, the pck gene was weakened. More specifically, the vector prepared in Example 1 was transformed into Corynebacterium glutamicum CA11-4021 by electroporation, and through a secondary crossing process, an L-glutamine-producing strain with a defective pck gene on the chromosome was obtained. This was named Corynebacterium glutamicum CA11-4023.

[0109] Corynebacterium glutamicum CA11-4023 was cultured by the following method, and its glutamine-producing ability was measured.

[0110] First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of the seed medium and cultured with shaking at 30 °C and 200 rpm for 20 hours. Next, 1 ml of the seed culture solution was inoculated into a 250 ml corner baffle flask containing 24 ml of the production medium and cultured with shaking at 32 °C and 200 rpm for 48 hours. The compositions of the seed medium and the production medium are as follows. After the culture was completed, the concentration of L-glutamine was measured using HPLC (Waters 2478). The measurement results of the glutamine-producing ability and the sugar consumption rate are shown in Table 3. <Seed medium (pH 7.0)> 20 g of glucose, 10 g of peptone, 5 g of yeast extract, 1.5 g of urea, 4 g of KH2PO4, 8 g of K2HPO4, 0.5 g of MgSO4·7H2O, 100 μg of biotin, 1000 μg of thiamine HCl, 2000 μg of calcium pantothenate, 2000 μg of nicotinamide (in 1 liter of distilled water) <Glutamine production medium (pH 8.0)> 60 g of raw sugar, 45 g of (NH4)2SO4, 0.48 g of soy protein, 50 g of CaCO3, 0.4 g of MgSO4·7H2O, 1 g of KH2PO4, 0.2 mg of thiamine hydrochloride, 0.3 mg of biotin, 60 mg of nicotinamide, 10 mg of FeSO4·7H2O, 10 mg of MnSO4·H2O (in 1 liter of distilled water)

[0111]

Table 3

[0112] As a result, it was confirmed that in CA11-4023 in which the pck gene was weakened, the glutamine production ability was improved by 7.2% compared to the parent strain CA11-4021.

[0113] The CA11-4023 strain was deposited with the Korea Culture Center of Microorganisms (KCCM), an international depositary authority under the Budapest Treaty, under the accession number KCCM12916P on December 22, 2020.

Example

[0114] Preparation and evaluation of a high-concentration L-glutamine-producing strain with a weakened pck gene Using the Corynebacterium glutamicum KFCC-10680 (Patent Document 2) strain, which is a well-known glutamine-producing strain, the pck gene was attenuated in the same manner as in Example 3. More specifically, the pDZ-ΔPEPCK vector prepared in Example 1 was transformed into Corynebacterium glutamicum KFCC-10680 by electroporation, and through a secondary crossing process, a strain with a defective pck gene on the chromosome was obtained. This was named Corynebacterium glutamicum KFCC-10680Δpck and cultured by the following method to measure its glutamine-producing ability.

[0115] First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of the seed medium and cultured with shaking at 30 °C and 200 rpm for 20 hours. Next, 1 ml of the seed culture solution was inoculated into a 250 ml corner baffle flask containing 24 ml of the production medium and cultured with shaking at 32 °C and 200 rpm for 48 hours. The compositions of the seed medium and the production medium are as follows. After the culture was completed, the concentration of L-glutamine was measured using HPLC (Waters 2478). The measurement results of the glutamine-producing ability and the sugar consumption rate are shown in Table 4. <Seed medium (pH 7.0)> Glucose 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 (in 1 liter of distilled water) <Glutamine production medium (pH 8.0)> Raw sugar 60 g, (NH4)2SO4 45 g, soybean protein 0.48 g, CaCO3 50 g, MgSO4·7H2O 0.4 g, KH2PO4 1 g, thiamine hydrochloride 0.2 mg, biotin 0.3 mg, nicotinamide 60 mg, FeSO4·7H2O 10 mg, MnSO4·H2O 10 mg (in 1 liter of distilled water)

[0116]

Table 4

[0117] As a result, it was confirmed that in KFCC-10680Δpck lacking the pck gene, the glutamine-producing ability was improved by 9.2% compared to the parent strain KFCC-10680.

[0118] From the above description, those skilled in the technical field to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical idea and essential features. It should be understood that the above embodiments are merely illustrative and not restrictive. The present application should be construed as including all changes and modifications derived from the meaning and scope of the claims rather than the specification and their equivalent concepts.

[0119] JPEG2025108542000005.jpg214170

[0120] JPEG2025108542000006.jpg214170

Claims

1. A Corynebacterium microorganism having weakened phosphoenolpyruvate carboxykinase activity and the ability to produce L-glutamine.

2. The microorganism according to claim 1, wherein the microorganism has an improved ability to produce L-glutamine as compared to a parent strain or a wild-type Corynebacterium strain in which the phosphoenolpyruvate carboxykinase activity has not been weakened.

3. The microorganism according to claim 1, wherein the phosphoenolpyruvate carboxykinase is an endogenous protein.

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

5. The microorganism according to claim 1, wherein the phosphoenolpyruvate carboxykinase consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more identity thereto.

6. A method for producing L-glutamine, comprising culturing the microorganism according to any one of claims 1 to 5 in a medium.

7. The method for producing L-glutamine according to claim 6, further comprising recovering L-glutamine from the medium or the microorganism after the culturing step.

8. Use of a Corynebacterium microorganism with weakened phosphoenolpyruvate carboxykinase in the production of L-glutamine.

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