Microorganism for producing glutamic acid-based amino acid and method for producing glutamic acid-based amino acid using same

Abstract

The present disclosure relates to a method for producing a glutamic acid-based amino acid, the method comprising culturing, in a culture medium, a Corynebacterium genus microorganism having a glutamic acid-based amino acid producing ability in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is weakened as compared to its endogenous activity; a composition for producing glutamic acid-based amino acids, comprising the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and to the use of said microorganism in the production of glutamic acid-based amino acids.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a method for producing a glutamate-based amino acid, the method comprising culturing in a culture medium a microorganism of the genus Corynebacterium having a glutamate-based amino acid production ability, an activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 in the microorganism being attenuated as compared to its endogenous activity; a composition for producing a glutamate-based amino acid, the composition comprising the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and a use of the microorganism in the production of a glutamate-based amino acid. BACKGROUND

[0002] Glutamate-derived L-amino acids, such as L-citrulline, are industrially useful because they have functions of dilating blood vessels and promoting ammonia excretion in vivo (K Takeda et al., 2013).

[0003] Microorganisms of the genus Corynebacterium, particularly C. glutamicum, are gram-positive microorganisms that are often used for the production of L-amino acids. For the production of L-amino acids, a target substance-specific approach, such as increasing the expression level of a gene encoding an enzyme mainly involved in the biosynthesis of L-amino acids, or deleting a gene not required for the biosynthesis of L-amino acids, in a Corynebacterium strain, is mainly used (US 9644009 B2). However, there is still a growing need for research on methods capable of efficiently producing L-amino acids.

[0004] Accordingly, there is still a need for research to efficiently increase glutamate-based amino acid production ability. SUMMARY

[0005] [TECHNICAL PROBLEM]

[0006] An object of the present disclosure is to provide a microorganism producing a glutamate-based amino acid and a method for producing a glutamate-based amino acid using the microorganism.

[0007] [TECHNICAL SOLUTION]

[0008] One aspect of the present disclosure provides a method for producing a glutamate-based amino acid, the method comprising culturing in a culture medium a microorganism of the genus Corynebacterium having a glutamate-based amino acid production ability, an activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 in the microorganism being attenuated as compared to its endogenous activity.

[0009] In one specific embodiment, the protein comprising the amino acid sequence of SEQ ID NO: 1 can be encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 2.

[0010] As for the microorganism according to any one of the foregoing embodiments, the microorganism of the genus Corynebacterium can be Corynebacterium glutamicum.

[0011] In another embodiment, the method can further include recovering the glutamic acid-based amino acid from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

[0012] In still another embodiment, the glutamic acid-based amino acid can be selected from the group consisting of citrulline, ornithine, arginine, glutamic acid, proline, and combinations thereof.

[0013] Another aspect of the present disclosure provides a composition for producing a glutamic acid-based amino acid, the composition comprising a microorganism of the genus Corynebacterium having glutamic acid-based amino acid production ability, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof, an activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 in the microorganism being attenuated as compared to its endogenous activity.

[0014] [Advantages]

[0015] The microorganism of the genus Corynebacterium having glutamic acid-based amino acid production ability of the present disclosure, in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is attenuated as compared to its endogenous activity, can produce a glutamic acid-based amino acid at a high yield, and thus is effectively applied to industrial production of a glutamic acid-based amino acid. DETAILED DESCRIPTION

[0016] The present disclosure will be described in detail hereinafter. Meanwhile, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific descriptions described below.

[0017] In addition, those skilled in the art will recognize or be able to ascertain only using routine experimentation many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be within the scope of the present disclosure. Furthermore, no limitation is intended to the scope of the present disclosure with respect to these specific descriptions.

[0018] As used in the specification and the appended claims of the present disclosure, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Singular terms shall include pluralities, and plural terms shall include the singular, unless the context dictates otherwise. As used in the specification and the appended claims of the present disclosure, the use of “or” can be used to mean “and / or” unless otherwise indicated.

[0019] As used herein, the term "about" can precede a particular value. The term "about" as used herein does not just include the precise number listed after the term "about", but also a range of numbers close to or approximate to the number. Whether any number is close or approximate to a particular number presented can be determined in view of the context in which the number is presented. In one example, the term "about" can refer to a range of -10% to +10% of an index value. In another example, the term "about" can refer to a range of -5% to +5% of a given numerical value, but is not limited thereto.

[0020] As used herein, descriptions such as the terms "first, second, third, …", "i), ii), iii), …" or "(a), (b), (c), (d), …" are used to distinguish similar constituents, and these terms do not mean that the constituents are continuous or sequential. For example, when the terms are used to refer to steps of a method, use, or assay, there can be no time interval between the steps, or they can be performed simultaneously, or they can be performed several seconds, minutes, hours, days, or months apart.

[0021] As used herein, the term "consisting essentially of can mean that, in the context of the subject matter claimed herein, unrecited components can be present.

[0022] As used herein, the term "consisting of refers to a total ratio of 100% of the specific components. The components or features recited after the term "consisting of can be essential or mandatory. In some embodiments, any other components or non-essential components can be excluded in addition to the components or features recited after the term "consisting of.

[0023] As used herein, the term "comprising" means the presence of the features, steps, or components recited after the term and does not exclude the presence or addition of one or more features, steps, or components. The components or features recited herein after the term "comprising" can be essential or mandatory. However, in some embodiments, the term can also include any other or non-essential components or features.

[0024] One aspect of the present disclosure provides a method of producing a glutamic acid-based amino acid, the method including culturing in a culture medium a microorganism of the genus Corynebacterium having a glutamic acid-based amino acid production ability, an activity of a protein including the amino acid sequence of SEQ ID NO: 1 in the microorganism being attenuated as compared to its endogenous activity.

[0025] As used herein, the term "protein comprising the amino acid sequence of SEQ ID NO: 1" refers to a hypothetical protein which is endogenously present in a microorganism of the genus Corynebacterium, the function of which is unknown.

[0026] Specifically, the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present disclosure can be a protein having the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1, which is encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 2, but is not particularly limited in type, as long as it has an activity corresponding to the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1, while its activity in a microorganism of the genus Corynebacterium is attenuated compared to the endogenous activity, thereby improving the ability to produce glutamic acid-based amino acids. The protein comprising the amino acid sequence of SEQ ID NO: 1 encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 2 is known in the art, and the amino acid and polynucleotide sequences of the protein comprising the amino acid sequence of SEQ ID NO: 1 can be obtained from known databases (e.g., NCBI GenBank, etc.), but are not limited thereto.

[0027] In the present disclosure, the protein comprising the amino acid sequence of SEQ ID NO: 1 can be a protein comprising the amino acid sequence of SEQ ID NO: 1 derived from a microorganism of the genus Corynebacterium (Corynebacterium species), particularly Corynebacterium glutamicum, but is not limited thereto.

[0028] For example, the protein comprising the amino acid sequence of SEQ ID NO: 1 can comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto, as long as it has the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1. Specifically, the polypeptide having the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 can have or comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to SEQ ID NO: 1, or can consist of or can consist essentially of the amino acid sequence. For example, the protein comprising the amino acid sequence of SEQ ID NO: 1 can refer to a protein endogenously present in a microorganism of the genus Corynebacterium or Corynebacterium glutamicum, but is not limited thereto.

[0029] With respect to the amino acid sequences in the present disclosure, although it is described as a polypeptide "comprising" the amino acid sequence described by a particular sequence number, a polypeptide "consisting of" the amino acid sequence described by a particular sequence number, or a polypeptide or protein "having" the amino acid sequence described by a particular sequence number, it is obvious that any protein having an amino acid sequence in which part of the sequence is deleted, modified, substituted, conservatively substituted, or added can fall within the scope of the present disclosure, as long as it has the same or corresponding activity as the protein consisting of the amino acid sequence of the corresponding sequence number. For example, the addition of a sequence before or after the amino acid sequence, a naturally occurring mutation, a silent mutation or a conservative substitution thereof which does not change the function of the protein is not excluded as long as the protein has the same or corresponding activity as the modified protein, and it is obvious that even if the protein has such a sequence addition or mutation, it falls within the scope of the present disclosure.

[0030] For example, it can be the case where a sequence which does not change the function of the variant polypeptide of the present disclosure, a naturally occurring mutation, a silent mutation or a conservative substitution is added at the N-terminus, the C-terminus and / or the interior of the amino acid sequence. For example, the polypeptide can be conjugated with an N-terminal signal (or leader) sequence involved in co-translational or post-translational transfer of the protein. In addition, the polypeptide can be conjugated with other sequences or linkers so as to be able to identify, purify or synthesize the polypeptide.

[0031] As used herein, the term "conservative substitution" refers to the substitution of one amino acid for another amino acid having similar structure and / or chemical properties. Such substitution of an amino acid can generally occur based on similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids having nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; amino acids having polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For another example, amino acids can be classified into amino acids having charged side chains (charged amino acids), such as arginine, lysine, histidine, glutamic acid, and aspartic acid, and amino acids having uncharged side chains (uncharged amino acids; also referred to as neutral amino acids), such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. For another example, valine, leucine, and isoleucine can be classified as branched-chain amino acids. For another example, the 20 amino acids are classified according to size, starting from the group of relatively small amino acids, the amino acids can be classified into five groups; glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine. However, they are not necessarily limited thereto. Generally, a conservative substitution can hardly affect or not affect the activity of a polypeptide.

[0032] Further, the nucleotide sequence encoding the protein comprising the amino acid sequence of SEQ ID NO: 1 can be a nucleotide sequence encoding a protein showing the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 while the activity thereof in a coryneform microorganism is attenuated compared to the endogenous activity thereof, thereby improving the ability to produce a glutamic acid-based amino acid. For example, the nucleotide sequence can be the NCgl2914 gene, which is a gene encoding a hypothetical protein endogenously present in a coryneform microorganism, the function of which is unknown, but is not limited thereto.

[0033] For example, the protein comprising the amino acid sequence of SEQ ID NO: 1 can be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having 60% or more, 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, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 2, or consisting of or consisting essentially of the nucleotide sequence, but is not limited thereto. In addition, the nucleotide sequence of SEQ ID NO: 2 can be obtained from a known database, for example, NCBI GenBank, etc., but is not limited thereto.

[0034] In the present disclosure, the gene comprising the nucleotide sequence of SEQ ID NO: 2 can be used interchangeably with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, the gene or polynucleotide having the nucleotide sequence of SEQ ID NO: 2, the gene or polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, or the NCgl2914 gene.

[0035] Due to codon degeneracy or in consideration of preferred codons in an organism intended to express a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present disclosure, the polynucleotide of the present disclosure can undergo various modifications in the coding region without changing the amino acid sequence of the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present disclosure. Therefore, it is obvious that, due to codon degeneracy, a polynucleotide that can be translated into a polypeptide consisting of the amino acid sequence of the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present disclosure or a polypeptide having homology or identity thereto can also be included in the polynucleotide of the present disclosure. For example, the polynucleotide of the present disclosure can be SEQ ID NO: 2 or a degenerate sequence thereof.

[0036] In addition, the polynucleotide of the present disclosure can comprise a probe, which can be prepared from a known gene sequence, for example, any sequence without limitation, as long as it hybridizes to the complementary sequence of all or a part of the polynucleotide sequence of the present disclosure under stringent conditions to encode a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present disclosure.

[0037] As used herein, the term "homology" or "identity" refers to the degree of relatedness between two given amino acid sequences or nucleotide sequences and can be expressed in percentage. The terms homology and identity can be used interchangeably with each other in general.

[0038] Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms and can use the default gap penalties established by the program used. Essentially, homologous or identical sequences will typically hybridize to at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or full length under moderately or highly stringent conditions. Obviously, hybridization also encompasses polynucleotides that hybridize to polynucleotides containing universal or codon-degenerate codons, taking into account the degeneracy of the genetic code.

[0039] Whether any two polynucleotide or polypeptide sequences are substantially identical, similar or identical can be determined using known computer algorithms, such as the "FASTA" program, for example, using the default parameters in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used, as implemented in the Needleman program (version 5.0.0 or later) in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (including GCG programs package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [et al., J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ed.], Academic Press, San Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48: 1073). For example, homology, similarity or identity can be determined using the National Center for Biotechnology Information's BLAST or ClustalW.

[0040] Homology, similarity or identity between polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., as disclosed in Needleman et al. (1970), J Mol Biol. 48:443, as implemented by Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines homology, similarity or identity as the value obtained by dividing the number of similar aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) unitary matrices (containing values of 1 for identical and 0 for non-identical), PAM matrices (described in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978)), and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty score of 3.0 for each gap, and an extra 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 end gaps.

[0041] Further, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing sequence information by performing Southern hybridization experiments under defined stringent conditions, and defined suitable hybridization conditions are within the skill in the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0042] As used herein, the term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. These conditions are described in detail in the literature (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). Examples thereof include conditions in which polynucleotides having a high degree of homology or identity, i.e., polynucleotides having 60% or more, 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, or 99% or more homology or identity, hybridize to each other, and polynucleotides having a low degree of homology or identity do not hybridize to each other, or conditions in which washing is performed once, in particular, twice to three times, at a salt concentration and temperature equivalent to 60°C, 1 x SSC, 0.1% SDS, specifically 60°C, 0.1 x SSC, 0.1% SDS, more specifically 68°C, 0.1 x SSC, 0.1% SDS, which are washing conditions of ordinary Southern hybridization.

[0043] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are allowed depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present disclosure can also include substantially similar nucleotide sequences as well as isolated nucleic acid fragments complementary to the entire sequence.

[0044] For example, polynucleotides having homology or identity to the polynucleotides of the present disclosure can be detected under the conditions described above using hybridization conditions including a hybridization step with a Tm value of 55°C. In addition, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by a person skilled in the art according to the purpose thereof.

[0045] The appropriate stringency of the hybridization polynucleotide depends on the length and degree of complementarity of the polynucleotide, and these variables are well known in the art (for example, J. Sambrook et al., supra).

[0046] In one specific embodiment, the glutamic acid-based amino acid of the present disclosure can be selected from the group consisting of citrulline, ornithine, arginine, glutamic acid, proline, and combinations thereof, but is not limited thereto.

[0047] For example, the glutamic acid-based amino acid of the present disclosure can be L-citrulline, but is not limited thereto.

[0048] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms, or microorganisms in which natural or artificial genetic modification has occurred, and it can be a microorganism in which a specific mechanism is weakened or enhanced due to insertion of an exogenous gene or enhancement or inactivation of an endogenous gene, and it can be a microorganism comprising a genetic modification for producing a desired polypeptide, protein, or product. As used herein, the terms "microorganism" and "strain" have the same meaning and can be used interchangeably without limitation.

[0049] As used herein, the term "microorganism having glutamic acid-based amino acid production ability" refers to a prokaryotic or eukaryotic microorganism strain capable of producing a glutamic acid-based amino acid in vivo, and can include a microorganism prepared by providing a parent strain not having glutamic acid-based amino acid production ability with glutamic acid-based amino acid production ability or a microorganism inherently having glutamic acid-based amino acid production ability. The glutamic acid-based amino acid production ability can be conferred or enhanced by species improvement.

[0050] For example, the microorganism having glutamic acid-based amino acid production ability of the present disclosure can be a microorganism (e.g., a recombinant strain) in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is weakened compared to its endogenous activity, but is not limited thereto.

[0051] As used herein, the term "unmodified microorganism" does not exclude a strain comprising a mutation that can occur naturally in a microorganism, and can refer to a wild-type strain or a natural strain itself, or can refer to a strain before a genetic variation changes a trait due to natural or artificial factors. The term "unmodified microorganism" can be used interchangeably with "strain before modification," "microorganism before modification," "unvaried strain," "unmodified strain," "unvaried microorganism," "parent strain before modification," "wild-type microorganism," "reference microorganism," or "standard microorganism." In the present disclosure, the unmodified microorganism can refer to a strain in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present disclosure is not weakened compared to its endogenous activity, or the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 has not yet been weakened compared to its endogenous activity, but is not limited thereto. In the present disclosure, the unmodified microorganism can be a microorganism comprising an amino acid sequence consisting of SEQ ID NO: 1 or a polynucleotide consisting of SEQ ID NO: 2, but is not limited thereto.

[0052] For the purposes of the present disclosure, the unmodified microorganism of the present disclosure can be a microorganism endogenously comprising the following:

[0053] i) a protein consisting of the amino acid sequence of SEQ ID NO: 1, or a protein consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequence of SEQ ID NO: 1; and / or

[0054] ii) a polynucleotide sequence capable of encoding a protein comprising an amino acid sequence having at least 80% homology with SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 2.

[0055] For the purpose of the present disclosure, the microorganism of the present disclosure can include any microorganism capable of producing a desired glutamic acid-based amino acid, in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is attenuated compared to its endogenous activity. For example, the microorganism of the present disclosure can be a genetically modified microorganism or a recombinant microorganism characterized by an increased glutamic acid-based amino acid production capacity by attenuating the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 compared to the endogenous activity, but is not limited thereto. Specifically, the recombinant strain in which the glutamic acid-based amino acid production capacity is increased can be a microorganism in which the glutamic acid-based amino acid production capacity is increased compared to a natural wild-type microorganism or an unmodified microorganism having the endogenous activity of a protein comprising the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0056] For example, the microorganism having a glutamic acid-based amino acid production capacity can be a prokaryotic or eukaryotic microorganism strain capable of producing a glutamic acid-based amino acid in vivo, and can include a microorganism inherently having a glutamic acid-based amino acid production capacity, or a microorganism prepared by providing a parent strain having no glutamic acid-based amino acid production capacity with a glutamic acid-based amino acid production capacity due to the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present disclosure being attenuated compared to its endogenous activity. The glutamic acid-based amino acid production capacity can be conferred or enhanced by species improvement.

[0057] The microorganism of the present disclosure can include any microorganism in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is attenuated compared to the endogenous activity by various known methods.

[0058] "Endogenous activity" refers to the activity of a particular polypeptide that the parent strain or unmodified microorganism originally has before the change in the trait due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification."

[0059] As used herein, the term "attenuation" of a polypeptide activity has the concept of including all cases of attenuation of activity or loss of activity compared to the endogenous activity. Attenuation can be used interchangeably with terms such as lack (inactivation), deletion, disruption, down-regulation, reduction, weakening, inhibition, reduction, and the like.

[0060] For example, the attenuation of the activity of a polypeptide, which is a state in which the activity of a polypeptide is not completely inactivated even though the polypeptide activity is exhibited in a host cell (microorganism), can include a case in which the activity of the polypeptide is reduced compared to the endogenous activity or the activity before modification; or a case in which the polypeptide is not expressed at all or even if expressed, the activity is lost or reduced (inactivated) compared to the host cell (microorganism) before the change in the trait or the unmodified microorganism.

[0061] The attenuation can also include a case in which the activity of the polypeptide itself is attenuated or eliminated due to variation of the polynucleotide encoding the polypeptide or the like, compared to the activity of the polypeptide originally possessed by the microorganism; a case in which the total level of polypeptide activity in the cell is low due to inhibition of expression of the gene encoding the polypeptide or due to inhibition of translation into the polypeptide, compared to the natural strain; a case in which the gene is not expressed at all; and a case in which the polypeptide activity is lost even if the gene expresses.

[0062] The fact that the activity of the polypeptide is attenuated compared to the endogenous activity means that the activity of the polypeptide is reduced compared to the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before the change in the trait. The attenuation of the activity of the polypeptide can be confirmed by the degree of activity and the level of expression of the corresponding polypeptide or the amount of product released from the corresponding polypeptide.

[0063] For example, the attenuation indicates that the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 can be less than about 100%, about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, or 0% of the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 of the parent strain or unmodified microorganism before the change in the trait.

[0064] For example, inactivation can mean that the protein comprising the amino acid sequence of SEQ ID NO: 1 is not expressed at all or even if expressed, the activity is lost or attenuated compared to the unmodified microorganism.

[0065] The weakening of the activity of such a polypeptide can be performed by any method known in the art, but the method is not limited thereto, and the weakening can be achieved by applying various methods well known in the art (for example, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793; Sambrook et al., Molecular Cloning 2012, etc.).

[0066] Specifically, the weakening of the activity of the polypeptide of the present disclosure can be:

[0067] 1) deletion of all or part of the gene encoding the polypeptide;

[0068] 2) modification of the expression regulatory region (or expression regulatory sequence) to weaken the expression of the gene encoding the polypeptide;

[0069] 3) modification of the amino acid sequence constituting the polypeptide to eliminate or weaken the activity of the polypeptide (for example, deletion / substitution / addition of one or more amino acids in the amino acid sequence);

[0070] 4) modification of the polynucleotide sequence encoding the polypeptide to eliminate or weaken the activity of the polypeptide (for example, deletion / substitution / addition of one or more nucleotide bases in the nucleotide sequence of the polypeptide gene to encode a modified polypeptide, thereby eliminating or weakening the activity of the polypeptide);

[0071] 5) modification of the nucleotide sequence of the start codon or 5'-UTR region of the gene encoding the polypeptide;

[0072] 6) introduction of an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

[0073] 7) addition of a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide upstream of the Shine-Dalgarno sequence so as to form a secondary structure that makes ribosome attachment impossible;

[0074] 8) addition of a promoter to be reverse-transcribed to the 3' end of the open reading frame (ORF) of the polynucleotide sequence encoding the polypeptide (reverse transcription engineering, RTE);

[0075] 9) control of the intracellular localization of the protein (polypeptide); or

[0076] 10) a combination of two or more selected from 1) to 9), but not particularly limited thereto.

[0077] For example,

[0078] 1) The deletion of all or part of the gene encoding the polypeptide can be the removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, the replacement with a polynucleotide in which some nucleotide sequences are deleted, or the replacement with a marker gene.

[0079] Such deletion of part or all of the polynucleotide can be performed by a method of deleting the polynucleotide by homologous recombination using a vector for chromosomal insertion in microorganisms, or a method of inducing mutations with light (e.g., ultraviolet light) or chemicals, and then screening strains in which the target gene is deleted from the obtained mutants, but is not limited thereto. The method of deleting part or all of the gene can include a method using a DNA recombination technique. For example, deletion of part or all of the gene can be achieved by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to the target gene into a microorganism to cause homologous recombination. The nucleotide sequence or vector to be injected can include a dominant selection marker, but is not limited thereto.

[0080] In addition, 2) modification of the expression regulatory sequence can be: a variation in the expression regulatory region (or expression regulatory sequence) due to deletion, insertion, non-conservative substitution, or conservative substitution, or a combination thereof, or replacement with a sequence having weaker activity. The expression regulatory region includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination, but is not limited thereto.

[0081] In addition, 3) and 4) modification of the amino acid sequence or the polynucleotide sequence can be: a variation in the sequence due to deletion, insertion, non-conservative substitution, or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or replacement with an amino acid sequence or a polynucleotide sequence that is improved to have weaker activity or is improved to be an inactive amino acid sequence or a polynucleotide sequence, such that the activity of the polypeptide is weakened, but is not limited thereto. For example, the expression of the gene can be inhibited or weakened by introducing a variation in the polynucleotide sequence and forming a stop codon, but is not limited thereto.

[0082] In addition, 5) modification of the nucleotide sequence of the start codon or the 5'-UTR region of the gene encoding the polypeptide can be, for example, substitution with another start codon having a lower polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0083] 6) Introduction of antisense oligonucleotides (e.g. antisense RNA) complementary to the transcript of the gene encoding the polypeptide can be referred to the literature, for example [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0084] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide upstream of the Shine-Dalgarno sequence so as to form a secondary structure that makes ribosome attachment impossible, which can make mRNA translation impossible or slow down the mRNA translation rate.

[0085] 8) Addition of a promoter to be reverse-transcribed to the 3' end of the open reading frame (ORF) of the polynucleotide sequence encoding the polypeptide (reverse transcription engineering, RTE) can be weakening the activity by making antisense nucleotides complementary to the transcript of the gene encoding the polypeptide.

[0086] 9) Controlling the intracellular localization of the protein (polypeptide) can mean targeting the protein (polypeptide) to a specific organelle or a specific intracellular space within the cell, for example, targeting the periplasm or cytoplasm by adding or removing a leader sequence that works in targeting the protein (polypeptide), but not limited thereto.

[0087] Such weakening of the activity of the polypeptide can mean that the activity or concentration of the corresponding polypeptide is weakened, or that the amount of the product produced by the corresponding polypeptide is reduced, based on the activity or concentration of the polypeptide expressed in the wild type or microbial strain before modification, but not limited thereto.

[0088] The modification of part or all of the polynucleotides in the microorganism of the present disclosure can be introduced by (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using an engineered nuclease (e.g. CRISPR-Cas9), and / or (b) treatment with light (e.g. ultraviolet light and radiation) and / or chemicals, but not limited thereto. The method of modifying part or all of the genes can include a method by DNA recombination technology. For example, a nucleotide sequence or a vector comprising a nucleotide sequence homologous to a target gene can be introduced into a microorganism to cause homologous recombination, resulting in deletion of part or all of the genes. The nucleotide sequence or vector to be introduced can comprise a dominant selection marker, but not limited thereto.

[0089] For example, the recombinant microorganism having glutamic acid-based amino acid production ability of the present disclosure can include any microorganism transformed with a vector to have an activity of a protein including the amino acid sequence of SEQ ID NO: 1 of the present disclosure weakened, thereby being able to produce a glutamic acid-based amino acid.

[0090] The vector of the present disclosure can include a DNA construct including a nucleotide sequence of a polynucleotide encoding a target polypeptide, which is operably linked to a suitable expression regulatory region (or expression regulatory sequence), so that the target polypeptide can be expressed in a suitable host. The expression regulatory region can include a promoter capable of initiating transcription, any operator sequence controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling transcription and translation termination. The vector can be transformed into a suitable host cell, and then replicate or function independently of the host genome, or can be integrated into the genome itself.

[0091] The vector used in the present disclosure is not particularly limited, but any vector known in the art can be used. Examples of commonly used vectors can include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. can be used as bacteriophage vectors or cosmid vectors; pDZ system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pSK system, pSKH system, and pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0092] For example, the polynucleotide encoding the target polypeptide can be inserted into a chromosome by a vector for intracellular chromosomal insertion. Inserting the polynucleotide into the chromosome can be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. The vector can also include a selection marker for checking chromosomal insertion. The selection marker is used to select cells transformed with the vector, i.e., for checking insertion of the target nucleic acid molecule, and a marker that confers a selectable phenotype (such as drug resistance, auxotrophy, cytotoxic agent resistance, or surface polypeptide expression) can be used. Only cells expressing the selection marker can survive or exhibit other phenotypic characteristics in an environment treated with a selection agent, and thus transformed cells can be selected.

[0093] As used herein, the term "transformation" refers to the introduction of a vector comprising a polynucleotide encoding a target polypeptide into a host cell or microorganism, such that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can be located by insertion into the chromosome of the host cell or located extrachromosomally, regardless of location, as long as it can be expressed in the host cell. Furthermore, the polynucleotide comprises DNA and / or RNA encoding a target polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct comprising all elements necessary for autonomous expression. The expression cassette can typically include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette can be in the form of an expression vector capable of self-replication. Furthermore, the polynucleotide can be introduced into the host cell in its own form and operably linked to sequences required for expression in the host cell, but is not limited thereto.

[0094] As used herein, the term "operably linked" refers to the configuration in which a regulatory sequence is placed in proper control phase with respect to the expression of a coding sequence. Thus, "operably linked" includes arrangements of regulatory regions (such as promoters, terminators, signal sequences, or enhancers) and targets (genes or polypeptides) in which the regions are functionally connected to each other, so that the expression, secretion, or function of the target (gene or polypeptide) can be modulated according to the known or desired activity of the regions. For example, the term refers to the functional linkage between a polynucleotide sequence and a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding a target variant polypeptide of the present disclosure.

[0095] As used herein, the term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion, but is not limited thereto.

[0096] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule comprising a coding sequence and operably linked regulatory sequences for its expression.

[0097] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence that is necessary for the expression of a coding sequence. Each regulatory sequence can be native (derived from the same source) or foreign (derived from a different gene) to the coding sequence. Examples of regulatory sequences can include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that terminates transcription and translation. The minimum unit of a regulatory sequence can include a promoter and a sequence that terminates transcription and translation.

[0098] The term "recombinant" as used herein with respect to a cell, polynucleotide, polypeptide, or vector refers to a cell, polynucleotide, polypeptide, or vector that has been modified by the introduction of a heterologous nucleic acid or polypeptide or alteration of a native polynucleotide or polypeptide, or a cell derived from the modified cell. Thus, for example, a recombinant cell can express genes that it does not normally express, or can express a native gene abnormally, under-express, or not express at all.

[0099] The microorganism of the present disclosure can include any microorganism in which the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 is attenuated as compared to its endogenous activity, through various known methods.

[0100] In one specific embodiment, the microorganism in which the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present disclosure is attenuated as compared to its endogenous activity can be a microorganism in which a polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO: 1 is deleted or modified, but is not limited thereto. Specifically, the microorganism can be a microorganism in which the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 is deleted due to deletion of the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

[0101] For example, the microorganism of the present disclosure having increased glutamic acid-based amino acid production ability can be a microorganism in which the glutamic acid-based amino acid production ability is increased as compared to an unmodified microorganism, but is not limited thereto. For example, the unmodified microorganism that is a test strain for comparing whether the glutamic acid-based amino acid production ability is increased can be a C. glutamicum argR* argG* strain, but is not limited thereto.

[0102] For example, the microorganism having increased glutamic acid-based amino acid production ability can have an increased glutamic acid-based amino acid production ability of about 1% or more, specifically, about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 16% or more, or about 17% or more (the upper limit is not particularly limited, but can be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, or about 20% or less) compared to the parent strain before modification or the unmodified microorganism, but the increased amount is not limited thereto, as long as the production ability has an increased amount of + value compared to the production ability of the parent strain before modification or the unmodified microorganism. In another example, the recombinant strain having increased glutamic acid-based amino acid production ability can have an increased glutamic acid-based amino acid production ability of about 1.1-fold or more, about 1.11-fold or more, about 1.12-fold or more, about 1.13-fold or more, about 1.14-fold or more, about 1.15-fold or more, about 1.16-fold or more, or about 1.17-fold or more (the upper limit is not particularly limited, but can be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, about 2-fold or less, about 1.5-fold or less, about 1.4-fold or less, about 1.3-fold or less, or about 1.2-fold or less) compared to the parent strain before modification or the unmodified microorganism, but not limited thereto.

[0103] For example, the microorganism having an amino acid production ability based on glutamate can be a prokaryotic cell or a eukaryotic cell, specifically a prokaryotic cell. The prokaryotic cell can include, for example, a microbial strain of Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Corynebacteria sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacteria sp., Hypomononas sp., Chromobacterium sp., and Norcardia sp., or a microbial strain of a fungus or a yeast. Specifically, the microorganism can be a microbial strain of Escherichia sp., Corynebacteria sp., Leptospira sp., or a yeast. More specifically, the microorganism can be a microbial strain of Corynebacteria sp.

[0104] With regard to the microorganism according to any one of the foregoing specific embodiments, the microorganism of the present disclosure can be a microbial strain of Corynebacteria sp.

[0105] For example, the microorganism of the present disclosure can 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. Specifically, the microorganism of the present disclosure can be a Corynebacterium microorganism, more specifically, Corynebacterium glutamicum or Corynebacterium stationis, but is not limited thereto.

[0106] Meanwhile, the microorganism of the present disclosure having the glutamic acid-based amino acid production ability can include all natural wild-type microorganisms per se, microorganisms having improved glutamic acid-based amino acid production ability by enhancing or weakening the activity of a gene related to a glutamic acid-based amino acid production mechanism, or microorganisms having improved glutamic acid-based amino acid production ability by introducing or enhancing the activity of an exogenous gene.

[0107] As used herein, the term "culturing" refers to culturing the strain of the present disclosure under appropriately adjusted environmental conditions. The culturing procedure can be performed according to a suitable culture medium or culture condition known in the art. Such a culturing procedure can be easily adjusted by one skilled in the art according to the selected strain. Specifically, the culturing can be a batch type, a continuous type, and / or a fed-batch type, but is not limited thereto.

[0108] As used herein, "medium" refers to a mixture containing nutrients necessary for culturing the microorganism of the present disclosure as a main component, in which the medium provides nutrients including water, growth factors, etc. necessary for survival and growth. Specifically, as for the medium and other culture conditions for culturing the strain of the present disclosure, any medium used for the conventional culture of microorganisms can be used without particular limitation. However, the microorganism of the present disclosure can be cultured in a general medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin, etc. under aerobic conditions, while controlling the temperature, pH, etc. For example, the medium for the culture of a Corynebacterium strain can be found in the literature ["Manual of Methods for General Bacteriology", American Society for Bacteriology (Washington, D.C., USA, 1981)].

[0109] In the present disclosure, the carbon source can include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, sugar cane bagasse, and corn steep liquor can be used, and specifically, carbohydrates such as glucose and sterile pretreated molasses (i.e., molasses converted to reducing sugars) can be used, and appropriate amounts of other carbon sources can be variously used without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0110] As for 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, etc.; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0111] The phosphorus source can include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts. As for the inorganic compound, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. can be used, and in addition, amino acids, vitamins, and / or suitable precursors can be included. These components or precursors can be added to the medium in a batch or continuous manner. However, the present disclosure is not limited thereto.

[0112] Further, the pH of the culture medium can be adjusted by adding a compound (e.g., ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc.) to the culture medium in a suitable manner during the culturing of the microorganism of the present disclosure. Further, an antifoaming agent (e.g., fatty acid polyethylene glycol ester) can be used to suppress the formation of foam during the culturing. Further, oxygen or an oxygen-containing gas can be injected into the culture medium to maintain an aerobic state of the culture medium, or no gas can be injected, or nitrogen gas, hydrogen gas, or carbon dioxide gas can be injected to maintain an anaerobic or non-aerobic state, but not limited thereto.

[0113] In the culturing of the present disclosure, the culturing temperature can be maintained at 27°C to 37°C, specifically, 30°C to 33°C, and the culturing can be performed for about 20 hours to 120 hours, but not limited thereto.

[0114] As used herein, the term “culture” refers to a culture broth, a concentrated culture broth, a dried product of the culture broth, a culture filtrate, a concentrated culture filtrate, or a dried product of the culture filtrate obtained by culturing a specific microorganism in a culture medium, and the culture broth refers to those containing the specific microorganism, and the culture filtrate refers to those substantially not containing the specific microorganism (which means that the specific microorganism to be separated by filtration or the like is substantially excluded, but this does not mean that the microorganism is completely excluded from the filtrate). The dosage form of the culture is not limited, and can be, for example, a liquid, an emulsion, or a solid. Specifically, for the purpose of the present disclosure, the culture can contain a glutamic acid-based amino acid.

[0115] As used herein, the term “fermentation” refers to a process in which a microorganism decomposes organic matter using its own enzymes, and does not include putrefaction. Fermentation and putrefaction are carried out by similar processes. However, when the result of decomposition is to produce useful substances, it is called fermentation, and when it produces bad odor or harmful substances, it is called putrefaction.

[0116] In the present disclosure, the method of obtaining a fermentation product from a strain is not particularly limited, and the fermentation product can be obtained according to a method commonly used in the art or a similar field.

[0117] As used herein, the term “fermentation product” includes not only the fermentation substance itself, but also all kinds of substances, including a fermentation product produced by a strain, such as a strain culture medium in which the strain and the culture coexist, a fermentation product obtained by filtering the strain from the culture medium, a fermentation product obtained by sterilizing the strain from the culture medium and filtering the strain, an extract obtained by extracting the fermentation product or the culture medium containing the fermentation product, a dilution obtained by diluting the fermentation product or the extract thereof, a concentrate, a dried product obtained by drying the fermentation product or the extract thereof, a lysate obtained by collecting and lysing the strain cells, and the like.

[0118] With regard to the method of the present disclosure, the culturing of the microorganism can be performed using any culture conditions and culture methods known in the art. Such a culturing procedure can be easily adjusted for use by those skilled in the art depending on the strain selected.

[0119] The glutamic acid-based amino acid produced by the culturing of the present disclosure can be released into the culture medium or can remain in the cell.

[0120] In one specific embodiment, the method of producing a glutamic acid-based amino acid of the present disclosure can further include the step of preparing the microorganism of the present disclosure, the step of preparing a culture medium for culturing the strain, or a combination of these steps (regardless of the order, in any order), for example, prior to the culturing step.

[0121] The method of producing a glutamic acid-based amino acid of the present disclosure can further include recovering the desired substance (particularly, the glutamic acid-based amino acid) from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium. The recovery step can also be included after the culturing step.

[0122] The recovery can be the method of culturing the microorganism according to the present disclosure, for example, batch, continuous, or fed-batch type culturing, by using a suitable method known in the art to collect the desired glutamic acid-based amino acid. For example, centrifugation, filtration, treatment with a crystallization protein precipitant (salting out), extraction, sonication, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography), HPLC, and combinations of these methods can be used, and the desired substance, particularly the glutamic acid-based amino acid, can be recovered from the culture medium or the microorganism by using a suitable method known in the art.

[0123] In addition, the method of producing a glutamic acid-based amino acid of the present disclosure can further include a purification step. The purification can be performed by using a suitable method known in the art. In one exemplary embodiment, when the method of producing a glutamic acid-based amino acid of the present disclosure includes both the recovery step and the purification step, the recovery step and the purification step can be performed continuously or discontinuously regardless of the order, or can be performed simultaneously or integrated into one step, but are not limited thereto.

[0124] With regard to the method of the present disclosure, the attenuation of the protein activity comprising the amino acid sequence of SEQ ID NO: 1 and the glutamic acid-based amino acid or the like are as described in other aspects.

[0125] Another aspect of the present disclosure provides a composition for producing glutamic acid-based amino acids, the composition comprising a Corynebacterium microorganism having glutamic acid-based amino acid production ability, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof, in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present disclosure is attenuated as compared to its endogenous activity.

[0126] The composition of the present disclosure can further comprise any suitable excipient generally used in a composition for producing glutamic acid-based amino acids, examples of which can include a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, an isotonic agent, etc., but are not limited thereto.

[0127] In a specific embodiment, each component present in the composition of the present disclosure can be included in a microorganism effective amount, or in an amount that can be appropriately present in a composition for production.

[0128] As to the composition of the present disclosure, the attenuation of the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 and glutamic acid-based amino acids, etc. are as described in other aspects.

[0129] Another aspect of the present disclosure provides use of a Corynebacterium microorganism having glutamic acid-based amino acid production ability, in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present disclosure is attenuated as compared to its endogenous activity in the production of glutamic acid-based amino acids.

[0130] As to the use of the present disclosure, the attenuation of the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 and glutamic acid-based amino acids, etc. are as described in other aspects.

[0131] [Means of carrying out the present invention]

[0132] The present disclosure will be described in greater detail by way of example embodiments hereinafter. However, the following example embodiments are merely for the purpose of illustrating preferred embodiments of the present disclosure, and thus are not intended to limit the scope of the present disclosure thereto. Meanwhile, those skilled in the technical field of the present disclosure or a similar technical field can fully understand and easily implement technical matters not described in the present specification.

[0133] Example 1: Construction of a Recombinant Vector for NCgl2914 Gene Deletion

[0134] To detect the relationship between the inactivation of the protein comprising the amino acid sequence of SEQ ID NO: 1 and the production of L-citrulline (a representative exemplary amino acid based on glutamic acid), a recombinant plasmid was constructed to delete the gene comprising the nucleotide sequence of NCgl2914 encoding the protein comprising the amino acid sequence of SEQ ID NO: 1 on the chromosome of the coryneform bacterial strain producing L-citrulline.

[0135] First, to construct a recombinant vector capable of deleting a gene on the chromosome of a coryneform microbial microorganism, four primers corresponding to SEQ ID NOs: 3, 4, 5, and 6 were synthesized.

[0136] In detail, to delete the ORF region of the NCgl2914 gene (SEQ ID NO: 2), primers of SEQ ID NOs: 3, 4, 5, and 6 were synthesized to have BamHI and XbaI restriction enzyme sites at the 5' and 3' ends. PCR was performed using the primers of SEQ ID NOs: 3, 4, 5, and 6 and the chromosomal DNA of Corynebacterium glutamicum ATCC13869 as a template. As a result, it was confirmed that each of the 1000 bp DNA fragments corresponding to the upstream and downstream portions of the gene encoding the protein was amplified. The PCR reaction conditions were denaturation at 95°C for 10 minutes, followed by 30 cycles each comprising denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, and then polymerization at 72°C for 10 minutes. Thereafter, the PCR fragments were extracted using a gel purification kit (QIAGEN). The obtained recombinant arm gene fragments and the vector pDCM2 (WO WO2021-187781 A1) digested with BamHI and XbaI restriction enzymes were ligated using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix), and then transformed into Escherichia coli DH5α and plated on LB solid medium containing kanamycin (25 mg / l).

[0137] To select colonies transformed with the vector in which the target gene and pDCM2 were ligated, PCR was performed using primers of SEQ ID NOs: 7 (pDCM2_F) and 8 (pDCM2_R). Using a well-known plasmid extraction method, a plasmid was obtained from the selected colonies, designated pDCM2-ΔNCgl2914.

[0138] Example 2: Preparation of a microorganism producing L-citrulline

[0139] To prepare a microorganism producing L-citrulline, a vector for replacing the 47th glutamic acid of the argR (ANU33619.1) protein sequence with a stop codon was constructed. The genome of the wild-type C. glutamicum ATCC13869 was used as a template, and a primer pair of SEQ ID NOs: 9 and 10 was used to amplify the homologous recombination arm A, and a primer pair of SEQ ID NOs: 11 and 12 was used to amplify the homologous recombination arm B. Thereafter, a plasmid was obtained in the same manner as in Example 1, and the plasmid was named pDCM2-argR (E47*).

[0140] To prepare a microorganism having an increased L-citrulline production ability, a vector for replacing the 68th phenylalanine of the argG (ANU33620.1) protein sequence with a stop codon was constructed. The genome of C. glutamicum ATCC13869 was used as a template, and a primer pair of SEQ ID NOs: 13 and 14 was used to amplify the homologous recombination arm A, and a primer pair of SEQ ID NOs: 15 and 16 was used to amplify the homologous recombination arm B. Thereafter, a plasmid was obtained using the above-described method, and the plasmid was named pDCM2-argG (F68*).

[0141] The wild-type C. glutamicum ATCC13869 was transformed with the constructed pDCM2-argR (E47*) vector by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and then a strain in which the 139th base sequence of argR was replaced from guanine (G) to thymine (T) and the 47th base sequence was replaced with a stop codon was obtained by a double crossover process. PCR and base sequence analysis were performed using a primer pair of SEQ ID NOs: 9 and 12 capable of amplifying the adjacent regions including the insertion position of the corresponding gene, and the corresponding genetic manipulation was confirmed. The microorganism thus obtained was named C. gl::argR*.

[0142] To prepare a microorganism having a further enhanced citrulline production ability in C. gl::argR*, the microorganism was obtained using the pDCM2-argG (F68*) vector by the above-described method. PCR and base sequence analysis were performed using a primer pair of SEQ ID NOs: 13 and 16 capable of amplifying the adjacent regions including the insertion position of the corresponding gene, and the corresponding genetic manipulation was confirmed. The microorganism thus obtained was named C. gl::argR*_argG*.

[0143] The primer sequences used are shown in Table 1 below.

[0144] [Table 1]

[0145]

[0146] Example 3: Preparation of NCgl2914 gene-deficient strain

[0147] Based on the L-citrulline-producing strain, a strain in which a gene comprising the nucleotide sequence of SEQ ID NO: 2 was deleted was prepared.

[0148] Specifically, the recombinant plasmid pDCM2-ΔNCgl2914 constructed in Example 1 was transformed into the L-citrulline-producing strain (C. gl::argR*_argG*) prepared in Example 2 by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999).

[0149] Thereafter, secondary recombination was performed on a solid plate medium containing sucrose. For the C. glutamicum transformant in which secondary recombination was completed, PCR was performed using primer 3 and primer 6, thereby identifying a strain in which a gene was deleted. The recombinant strain was named C. glutamicum C. gl::argR*_argG*_ΔNCgl2914.

[0150] Example 4: Evaluation of L-citrulline production capacity of NCgl2914 gene-deficient strain

[0151] In order to analyze the L-citrulline production capacity, the following method was used to culture the parent strain C. glutamicum C. gl::argR*_argG* strain and the prepared C. glutamicum strain.

[0152] Each strain was inoculated into a 250 mL corner-baffled flask containing 25 mL of the following production medium and cultured at 33°C at a speed of 200 rpm for 48 hours. After completion of the culture, the cell mass and L-citrulline yield were measured using HPLC.

[0153] <Production medium (pH 7.2)>

[0154] Crude sugar 50 g, (NH4)2SO4 30 g, yeast extract 1 g, KH2PO4 1.1 g, MgSO4·7H2O 1.2 g, L-arginine 0.2 g, biotin 1 mg, thiamine hydrochloride 5 mg, calcium pantothenate 5 mg, nicotinamide 15 mg, MnSO4 10 mg, FeSO4 10 mg, ZnSO4 0.5 mg, CuSO4 0.5 mg, CaCO3 30 g (based on 1 liter of distilled water)

[0155] The above experiment was repeated twice, and the culture results (average values) are shown in Table 2 below.

[0156] [Table 2]

[0157]

[0158] As shown in Table 2, the L-citrulline productivity of C. gl::argR*_argG*_ΔNCgl2914 obtained by deleting the NCgl2914 gene from the L-citrulline-producing strain C. glutamicum C.gl::argR*_argG* was increased by an average of 17% compared to the parent strain, without affecting the cell mass.

[0159] Accordingly, it was confirmed that the productivity of L-citrulline can be improved by deleting a gene comprising the nucleotide sequence of SEQ ID NO: 2 in a Corynebacterium microorganism.

[0160] Based on the above description, those skilled in the art will understand that the present disclosure can be implemented in different specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are not restrictive but are illustrative in all aspects. The scope of the present disclosure is defined by the appended claims rather than by the specification following them, and thus all changes and modifications falling within the boundaries and scope of the claims, or equivalent replacements of these boundaries and scope, are intended to be covered by the claims.

Claims

1. A method of producing a glutamic acid-based amino acid, the method comprising culturing in a culture medium a microorganism of the genus Corynebacterium having a glutamic acid-based amino acid production ability, an activity of a protein comprising an amino acid sequence of SEQ ID NO: 1 in the microorganism being attenuated as compared to its endogenous activity.

2. The method according to claim 1, wherein the protein comprising an amino acid sequence of SEQ ID NO: 1 is encoded by a gene comprising a nucleotide sequence of SEQ ID NO:

2.

3. The method according to claim 1, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

4. The method according to claim 1, further comprising recovering the glutamic acid-based amino acid from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

5. The method according to claim 1, wherein the glutamic acid-based amino acid is selected from the group consisting of citrulline, ornithine, arginine, glutamic acid, proline, and combinations thereof.

6. A composition for producing a glutamic acid-based amino acid, the composition comprising a microorganism of the genus Corynebacterium having a glutamic acid-based amino acid production ability, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof, an activity of a protein comprising an amino acid sequence of SEQ ID NO: 1 in the microorganism being attenuated as compared to its endogenous activity.

7. Use of a microorganism of the genus Corynebacterium having a glutamic acid-based amino acid production ability, an activity of a protein comprising an amino acid sequence of SEQ ID NO: 1 in the microorganism being attenuated as compared to its endogenous activity, in the production of a glutamic acid-based amino acid.

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