WO2022231036A1

WO2022231036A1 - Novel variant, and method for producing l-glutamic acid using same

Info

Publication number: WO2022231036A1
Application number: PCT/KR2021/005454
Authority: WO
Inventors: 권나라; 이아름; 봉현주; 허란; 유혜련; 김비나; 손성광
Original assignee: 씨제이제일제당 (주)
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2022-11-03

Abstract

The present application relates to a novel variant, a Corynebacterium glutamicum strain comprising the variant, and a method for producing L-glutamic acid using the strain.

Description

Novel variant and L-glutamic acid production method using the same

In order to produce L-amino acids and other useful substances, various studies are being conducted for the development of high-efficiency production microorganisms and fermentation process technology. For example, a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in L-glutamic acid biosynthesis or removing a gene unnecessary for biosynthesis is mainly used (US 8206954 B2).

However, as the demand for L-glutamic acid increases, there is still a need for research to effectively increase the production capacity of L-glutamic acid.

The present inventors have completed the present application by developing a novel variant for increasing L-glutamic acid production, a Corynebacterium glutamicum strain including the variant, and a method for producing L-glutamic acid using the strain.

One object of the present application is to include (i) one or more variants of the present application, (ii) one or more polynucleotides encoding the variants, or (iii) a combination thereof, and having L-glutamic acid producing ability, It is to provide a strain Nebacterium glutamicum (Corynebacterium glutamicum).

Another object of the present application is to include (i) one or more variants of the present application, (ii) one or more polynucleotides encoding the variant, or (iii) a combination thereof, and having L-glutamic acid-producing ability, It is to provide a method for producing L-glutamic acid, comprising the step of culturing the Corynebacterium glutamicum strain in a medium.

In the case of culturing the Corynebacterium glutamicum strain, including the variant of the present application, it is possible to produce L-glutamic acid in a high yield compared to microorganisms having an existing unmodified polypeptide.

The present application will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below. In addition, a number of papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

One aspect of the present application is (i) any one or more variants selected from the group consisting of the following (a) to (l), (ii) one or more polynucleotides encoding the variant, or (iii) a combination thereof It provides a Corynebacterium glutamicum strain comprising a.

(a) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which glycine, an amino acid corresponding to position 32 of SEQ ID NO: 3, is substituted with aspartic acid;

(b) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which alanine, an amino acid corresponding to position 282 of SEQ ID NO: 7, is substituted with threonine;

(c) a D-alanine-D-alanine ligase A variant comprising the amino acid sequence set forth in SEQ ID NO: 9 in which glycine, an amino acid corresponding to position 256 of SEQ ID NO: 11, is substituted with serine;

(d) a glucosamine-6-phosphate deaminase variant consisting of the amino acid sequence shown in SEQ ID NO: 13, in which alanine, an amino acid corresponding to position 137 of SEQ ID NO: 15, is substituted with valine;

(e) an exinuclease ABC subunit A variant comprising the amino acid sequence set forth in SEQ ID NO: 17 in which glycine, an amino acid corresponding to position 575 of SEQ ID NO: 19, is substituted with aspartic acid;

(f) a ribonuclease P variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which histidine, an amino acid corresponding to position 32 of SEQ ID NO: 23, is substituted with tyrosine;

(g) an MFS transporter variant consisting of the amino acid sequence set forth in SEQ ID NO: 25, in which alanine, which is an amino acid corresponding to position 382 of SEQ ID NO: 27, is substituted with threonine;

(h) a galactoside O-acetyltransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 29, wherein leucine, an amino acid corresponding to position 106 of SEQ ID NO: 31, is substituted with phenylalanine;

(i) spermidine synthase consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which proline, an amino acid corresponding to position 453 of SEQ ID NO: 35, is substituted with serine, and alanine, an amino acid corresponding to position 396, is substituted with threonine variant;

(j) a spermidine synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 37, wherein proline, which is the amino acid corresponding to position 453 of SEQ ID NO: 35, is substituted with serine;

(k) a spermidine synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 39 in which alanine, an amino acid corresponding to position 396 of SEQ ID NO: 35, is substituted with threonine; and

(l) a glutamate synthase subunit alpha variant comprising the amino acid sequence shown in SEQ ID NO: 41 in which serine, an amino acid corresponding to position 1192 of SEQ ID NO: 43, is substituted with phenylalanine.

In the present application, one or more may be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more, but is not limited thereto.

Specifically, the strain of the present application is (i) 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more selected from the group consisting of (a) to (l), 9 or more, 10 or more, 11 or more, or 12 or more variants, (ii) 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more encoding the variant , 11 or more or 12 or more polynucleotides, or (iii) a combination thereof, but is not limited thereto.

In the present application, in a strain comprising a combination of (i) any one or more variants selected from the group consisting of (a) to (l) and (ii) one or more polynucleotides encoding the variant, (i) The variant and the variant of (ii) above may be selected from the group consisting of (a) to (l), but may be the same or different, but is not limited thereto.

For example, the strain of the present application may include (a) a polynucleotide encoding the variant; and (b) a strain comprising a combination of variants, but is not limited thereto.

Variants of the present application are SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more of the amino acid sequence set forth in No. It may consist essentially of sequence.

In addition, the variant of the present application may include any one or more amino acid sequences selected from the group consisting of the following (aa) to (ll).

(aa) the amino acid corresponding to position 32 based on the amino acid sequence of SEQ ID NO: 3 in the amino acid sequence set forth in SEQ ID NO: 1 is aspartic acid, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 1 %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or an amino acid sequence having at least 99.9% homology or identity.

(bb) the amino acid corresponding to position 282 based on the amino acid sequence of SEQ ID NO: 7 in the amino acid sequence set forth in SEQ ID NO: 5 is threonine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 5 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;

(cc) the amino acid corresponding to position 256 based on the amino acid sequence of SEQ ID NO: 11 in the amino acid sequence set forth in SEQ ID NO: 9 is serine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 9 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;

(dd) in the amino acid sequence set forth in SEQ ID NO: 13, the amino acid corresponding to position 137 based on the amino acid sequence of SEQ ID NO: 15 is valine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 13 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;

(ee) the amino acid corresponding to position 575 based on the amino acid sequence of SEQ ID NO: 19 in the amino acid sequence set forth in SEQ ID NO: 17 is aspartic acid, and at least 70%, 75%, 80% with the amino acid sequence set forth in SEQ ID NO: 17 an amino acid sequence having at least %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;

(ff) the amino acid corresponding to position 32 based on the amino acid sequence of SEQ ID NO: 23 in the amino acid sequence set forth in SEQ ID NO: 21 is tyrosine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 21 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;

(gg) the amino acid corresponding to position 382 based on the amino acid sequence of SEQ ID NO: 27 in the amino acid sequence set forth in SEQ ID NO: 25 is threonine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 25 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;

(hh) in the amino acid sequence set forth in SEQ ID NO: 29, the amino acid corresponding to position 106 based on the amino acid sequence of SEQ ID NO: 31 is phenylalanine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 29 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;

(ii) in the amino acid sequence set forth in SEQ ID NO: 33, the amino acid corresponding to position 453 based on the amino acid sequence of SEQ ID NO: 35 is serine, the amino acid corresponding to position 396 is threonine, and the amino acid set forth in SEQ ID NO: 33 an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity to the sequence ;

(jj) in the amino acid sequence set forth in SEQ ID NO: 37, the amino acid corresponding to position 453 based on the amino acid sequence of SEQ ID NO: 35 is serine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 37 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;

(kk) In the amino acid sequence set forth in SEQ ID NO: 39, the amino acid corresponding to position 396 based on the amino acid sequence of SEQ ID NO: 35 is threonine, and at least 70%, 75%, 80% from the amino acid sequence set forth in SEQ ID NO: 39 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity; and

(ll) the amino acid corresponding to position 1192 based on the amino acid sequence of SEQ ID NO: 43 in the amino acid sequence set forth in SEQ ID NO: 41 is phenylalanine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 41 , 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or an amino acid sequence having at least 99.9% homology or identity.

In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

For example, sequence additions or deletions, naturally occurring mutations, silent mutations or conservation within the N-terminus, C-terminus and/or within the amino acid sequence that do not alter the function of the variants of the present application It is a case of having an enemy substitution.

The term “conservative substitution” means substituting an amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

As used herein, the term "variant" means that one or more amino acids are conservatively substituted and/or modified so that they differ from the amino acid sequence before the mutation of the variant, but have functions or properties. refers to a polypeptide that is maintained. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein. The term "variant" may be used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutant and mutant (in English, modified, modified polypeptide, modified protein, mutant, mutein, divergent, etc.) and, as long as it is a term used in a mutated sense, it is not limited thereto.

For the purposes of the present application, the variant includes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein glycine, which is an amino acid corresponding to the 32nd position of SEQ ID NO: 3, is substituted with aspartic acid; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5 in which alanine, which is an amino acid corresponding to position 282 of SEQ ID NO: 7, is substituted with threonine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 9 in which glycine, an amino acid corresponding to position 256 of SEQ ID NO: 11, is substituted with serine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13 in which alanine, an amino acid corresponding to position 137 of SEQ ID NO: 15, is substituted with valine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17 in which glycine, an amino acid corresponding to position 575 of SEQ ID NO: 19, is substituted with aspartic acid; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 21 in which histidine, an amino acid corresponding to position 32 of SEQ ID NO: 23, is substituted with tyrosine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 25 in which alanine, which is an amino acid corresponding to position 382 of SEQ ID NO: 27, is substituted with threonine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 29 in which leucine, an amino acid corresponding to position 106 of SEQ ID NO: 31, is substituted with phenylalanine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 33 in which proline, an amino acid corresponding to position 453 of SEQ ID NO: 35, is substituted with serine, and alanine, an amino acid corresponding to position 396, is substituted with threonine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 37 in which proline, an amino acid corresponding to position 453 of SEQ ID NO: 35, is substituted with serine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 39 in which alanine, which is an amino acid corresponding to position 396 of SEQ ID NO: 35, is substituted with threonine; And it may be any one or more selected from the group consisting of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 41 in which serine, an amino acid corresponding to position 1192 of SEQ ID NO: 43, is substituted with phenylalanine.

In addition, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in protein translocation may be conjugated to the N-terminus of the mutant, either co-translationally or post-translationally. The variants may also be conjugated with other sequences or linkers for identification, purification, or synthesis.

As used herein, the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.

Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of a sequence under moderate or high stringent conditions. It is apparent that hybridization also includes hybridization with polynucleotides containing common codons or codons taking codon degeneracy into account in the polynucleotide.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the "FASTA" program. or, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine (GCG program 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 ETA/.] (1988) SIAM J Applied Math 48: 1073).For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity or identity.

Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

As an example of the present application, the variants of the present application include ABC transporter ATP-binding protein, D-alanine-D-alanine ligase A, glucosamine-6-phosphate deaminase, exinuclease ABC subunit A, ribonuclease. It may have the activity of any one or more of clease P, MFS transporter, galactoside O-acetyltransferase, spermidine synthase, and glutamate synthase subunit alpha. In addition, one or more variants or a combination thereof of the present application may have an activity to increase L-glutamic acid production capacity compared to the wild-type polypeptide.

As used herein, the term "ABC transporter ATP-binding protein" is a transmembrane protein and is a polypeptide that transports various substrates to the outside and the inside of the cell. Specifically, the ABC transporter ATP-binding protein of the present application may be used interchangeably as an ATP-binding cassette transporter or ABC-type transporter. In the present application, the sequence of the ABC transporter ATP-binding protein can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having an ABC transporter ATP-binding protein activity encoded by a gene of locus_tag="BBD29_01215" or locus_tag="BBD29_01305", but is not limited thereto.

As used herein, the term "D-alanine-D-alanine ligase A (D-alanine-D-alanine ligase A)" is a polypeptide forming a cell wall. Specifically, D-alanine-D-alanine ligase A of the present application may be used interchangeably with D-Ala-D-Ala ligase A or D-alanylalanine synthetase A. In the present application, the sequence of the D-alanine-D-alanine ligase A can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having D-alanine-D-alanine ligase A activity encoded by ddlA , but is not limited thereto.

As used herein, the term "glucosamine-6-phosphate deaminase" is a polypeptide that converts acetyl glucosamine 6-phosphate to glucosamine 6-phosphate. Specifically, glucosamine-6-phosphate deaminase of the present application may be used in combination with glucosamine-6-phosphate isomerase, GlcN6P deaminase or GNPDA. In the present application, the sequence of the glucosamine-6-phosphate deaminase can be obtained from GenBank of NCBI, which is a known database. Specifically, it may be a polypeptide having glucosamine-6-phosphate deaminase activity encoded by nagB , but is not limited thereto.

As used herein, the term "Excinuclease ABC subunit A" is a polypeptide that repairs DNA caused by UV damage. Specifically, exinuclease ABC subunit A of the present application may be used interchangeably as exciton nuclease subunit A, UvrABC endonuclease subunit A, or UvrA. In the present application, the sequence of the exinuclease ABC subunit A can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having exinuclease ABC subunit A activity encoded by uvrA , but is not limited thereto.

As used herein, the term "ribonuclease P (ribonuclease P)" is a type of ribonuclease that cuts RNA. It is distinct from other RNases. Specifically, ribonuclease P of the present application may be used in combination with a ribonuclease P protein component. In the present application, the ribonuclease P sequence can be obtained from GenBank of NCBI, which is a known database. Specifically, it may be a polypeptide having ribonuclease P activity encoded by rnpA, but is not limited thereto.

As used herein, the term "MFS transporter (MFS transporter)" is one of the membrane transport proteins belonging to the MFS (Major facilitator superfamily), a protein that promotes the movement of small solutes across the cell membrane in response to a chemical osmotic gradient to be. Specifically, it may be used interchangeably with terms such as MFS transporter and MFS transporter. In the present application, the sequence of the MFS transporter can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having an MFS transporter activity encoded by iolT2, but is not limited thereto.

As used herein, the term "galactoside O-acetyltransferase" is an enzyme that transfers an acetyl group from acetyl-CoA to galactoside, glucoside and lactoside. Specifically, the galactoside O-acetyltransferase of the present application may be used in combination with galactoside acetyltransferase, thiogalactoside transacetylase, or GAT. In the present application, the galactoside O-acetyltransferase sequence can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having a galactoside O-acetyltransferase activity encoded by maa, but is not limited thereto.

As used herein, the term "spermidine synthase" is a polypeptide that biosynthesizes spermidine. Specifically, spermidine synthase of the present application may be used in combination with polyamine aminopropyltransferase, putrescine aminopropyltransferase, PAPT, SPDS or SPDSY. In the present application, the spermidine synthase sequence can be obtained from GenBank of NCBI, which is a known database. Specifically, it may be a polypeptide having spermidine synthase activity encoded by speE, but is not limited thereto.

As used herein, the term "glutamate synthase subunit alpha" catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate. Specifically, the glutamate synthase subunit alpha of the present application may be used in combination with GltB. In the present application, the sequence of the glutamate synthase subunit alpha can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having a glutamate synthase subunit alpha activity encoded by gltB , but is not limited thereto.

As used herein, the term "corresponding to" refers to an amino acid residue at a listed position in a polypeptide, or to an amino acid residue that is similar, identical or homologous to a listed residue in a polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that refers to a specific sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

For example, any amino acid sequence is aligned with SEQ ID NO: 3, and based on this, each amino acid residue of the amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 3 . For example, a sequence alignment algorithm such as that described in this application can identify the position of an amino acid, or a position at which modifications, such as substitutions, insertions, or deletions, occur compared to a query sequence (also referred to as a "reference sequence").

Such alignments include, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000), Trends Genet. 16: 276-277), etc., but is not limited thereto, and a sequence alignment program known in the art, a pairwise sequence comparison algorithm, etc. may be appropriately used.

As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, encoding the variant. polynucleotide fragments.

Polynucleotides encoding the variants of the present application are SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, It may include one or more base sequences encoding any one or more of the amino acid sequences set forth in SEQ ID NO: 39 and SEQ ID NO: 41. As an example of the present application, the polynucleotide of the present application is SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42 may have, consist of, or consist essentially of any one or more sequences.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application is SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, 98 % or more and less than 100% of the base sequence, or SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96% homology or identity to any one or more sequences or more, 97% or more, 98% or more, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto.

In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 32nd position of SEQ ID NO: 1 is one of the codons encoding aspartic acid; The codon encoding the amino acid corresponding to position 282 of SEQ ID NO: 1 is one of the codons encoding threonine; The codon encoding the amino acid corresponding to position 256 of SEQ ID NO: 1 is one of the codons encoding serine; The codon encoding the amino acid corresponding to position 137 of SEQ ID NO: 1 is one of the codons encoding valine; The codon encoding the amino acid corresponding to position 575 of SEQ ID NO: 1 is one of the codons encoding aspartic acid; The codon encoding the amino acid corresponding to position 32 of SEQ ID NO: 1 is one of the codons encoding tyrosine; The codon encoding the amino acid corresponding to position 382 of SEQ ID NO: 1 is one of the codons encoding threonine; The codon encoding the amino acid corresponding to position 106 of SEQ ID NO: 1 is one of the codons encoding phenylalanine; The codon encoding the amino acid corresponding to position 453 of SEQ ID NO: 41 is one of the codons encoding serine, the codon encoding the amino acid corresponding to position 396 is one of the codons encoding threonine; The codon encoding the amino acid corresponding to position 453 of SEQ ID NO: 45 is one of the codons encoding serine; The codon encoding the amino acid corresponding to position 396 of SEQ ID NO: 47 is one of the codons encoding threonine; And the codon encoding the amino acid corresponding to position 1192 of SEQ ID NO: 1 may be one of the codons encoding phenylalanine.

In addition, the polynucleotide of the present application may be included without limitation as long as it can hybridize under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application. can The "stringent condition" means a condition that enables specific hybridization between polynucleotides. These conditions are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, polynucleotides with high homology or identity, 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 a condition in which polynucleotides having 99% or more homology or identity hybridize with each other and polynucleotides with lower homology or identity do not hybridize, or a washing condition of conventional Southern hybridization at 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS at a salt concentration and temperature equivalent to once, specifically twice The conditions for washing to 3 times can be enumerated.

Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the overall sequence.

Specifically, a polynucleotide having homology or identity to the polynucleotide of the present application can be detected using the hybridization conditions including a hybridization step at a Tm value of 55°C and using the above-described conditions. In addition, the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.

The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (eg, J. Sambrook et al., supra).

The vector of the present application may include a DNA preparation comprising the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. can The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.

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

For example, a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted. The selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. Markers to be given can be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.

As used herein, the term "transformation" refers to introducing a vector including a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and/or RNA encoding a target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term “operably linked” as used herein means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target variant of the present application and the polynucleotide sequence are functionally linked.

The strain of the present application is one or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more variant polypeptides of the present application, the polypeptide 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more polynucleotides encoding, or 1 comprising a polynucleotide of the present application or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more vectors, or a combination thereof.

As used herein, the term "strain (or microorganism)" includes both wild-type microorganisms and microorganisms in which genetic modification has occurred naturally or artificially. As a result, a specific mechanism is weakened or enhanced as a microorganism, and may be a microorganism including genetic modification for the production of a desired polypeptide, protein or product.

The strain of the present application includes a strain comprising any one or more of one or more variants of the present application, one or more polynucleotides of the present application, and one or more vectors comprising the polynucleotides of the present application; strains modified to express one or more variants of the present application or one or more polynucleotides of the present application; a strain expressing one or more variants of the present application, or one or more polynucleotides of the present application (eg, a recombinant strain); Or it may be a strain (eg, a recombinant strain) having one or more variant activities of the present application, but is not limited thereto.

The strain of the present application may be a strain having the ability to produce L-glutamic acid.

The strains of the present application naturally contain ABC transporter ATP-binding protein, D-alanine-D-alanine ligase A, glucosamine-6-phosphate deaminase, exinuclease ABC subunit A, ribonuclease P, MFS transporter, galactoside O-acetyltransferase, spermidine synthase, glutamate synthase subunit alpha or L-glutamic acid producing ability, or ABC transporter ATP-binding protein, D-alanine-D- Alanine ligase A, glucosamine-6-phosphate deaminase, exinuclease ABC subunit A, ribonuclease P, MFS transporter, galactoside O-acetyltransferase, spermidine synthase, glutamate synthase One or more variants of the present application, one or more polynucleotides encoding the same (or a vector comprising the polynucleotides), or a combination thereof are introduced into a parent strain lacking subunit alpha or L-glutamic acid production ability, and/or L- It may be a microorganism to which the ability to produce glutamic acid is granted, but is not limited thereto.

For example, the strain of the present application is a cell or microorganism that is transformed with one or more polynucleotides encoding one or more variants of the present application or one or more vectors comprising the same, and expresses one or more variants of the present application, For the purpose, the strain of the present application may include all microorganisms capable of producing L-glutamic acid, including one or more variants of the present application. For example, the strain of the present application may be a recombinant strain having an increased ability to produce L-glutamic acid by introducing a polynucleotide encoding one or more variants of the present application into a natural wild-type microorganism or a microorganism producing L-glutamic acid. The recombinant strain having an increased L-glutamic acid production ability may be a microorganism having an increased L-glutamic acid production ability compared to a natural wild-type microorganism or an unmodified microorganism (ie, a microorganism that does not express the mutant), but is not limited thereto . For example, the strain with increased L-glutamic acid production capacity of the present application is SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35 Or it may be a microorganism having an increased L-glutamic acid production ability compared to Corynebacterium glutamicum comprising the polypeptide of SEQ ID NO: 43 or a polynucleotide encoding the same, but is not limited thereto. For example, the non-modified microorganism, which is the target strain for comparing the increase in L-glutamic acid production ability, is the ATCC13869 strain or the ATCC13869 ΔodhA strain (Appl Environ Microbiol. 2007 Feb; 73(4): 1308-19. Epub 2006 Dec 8.), but is not limited thereto.

For example, the recombinant strain with increased production capacity is about 1% or more, about 5% or more, about 10% or more, about 15% or more, or about 20% of the L-glutamic acid production capacity of the parent strain or unmodified microorganism before mutation. Above (the upper limit is not particularly limited, for example, it may be about 200% or less, about 150% or less, about 100% or less, or about 50% or less) may be increased, but the production of the parent strain or unmodified microorganism before mutation It is not limited thereto, as long as it has an increase in the + value compared to the performance. In another example, the recombinant strain with increased production capacity has an L-glutamic acid production capacity of about 1.01 times or more, about 1.05 times or more, about 1.1 times or more, 1.15 times or more, or about 1.2 times compared to the parent strain or unmodified microorganism before mutation. It may be increased by more than twice (the upper limit is not particularly limited, for example, it may be about 20 times or less), but is not limited thereto. The term “about” is a range including all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc. not limited

As used herein, the term "unmodified microorganism" does not exclude a strain containing a mutation that can occur naturally in a microorganism, it is a wild-type strain or a natural-type strain itself, or a genetic variation caused by natural or artificial factors. It may mean the strain before being changed. For example, the unmodified microorganism may refer to a strain into which one or more variants described herein have not been introduced or have been introduced. The "unmodified microorganism" may be used interchangeably with "strain before modification", "microbe before modification", "unmodified strain", "unmodified strain", "unmodified microorganism" or "reference microorganism".

In another example of the present application, the microorganism of the present application is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Cory Nebacterium efficiens ( Corynebacterium efficiens ), Corynebacterium callunae ), Corynebacterium stationis , Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium halo Tolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans imitans ), Corynebacterium testudinoris ) or Corynebacterium flavescens ).

The microorganism of the present application may be a microorganism in which the activity of the OdhA protein is further weakened or inactivated.

As used herein, the term “attenuation” of polypeptide activity is a concept that includes both reduced or no activity compared to intrinsic activity. The attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, attenuation, and the like.

The attenuation is when the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide possessed by the original microorganism due to mutation of the polynucleotide encoding the polypeptide, etc. When the overall polypeptide activity level and/or concentration (expression amount) in the cell is lower than that of the native strain due to (translation) inhibition, etc., when the expression of the polynucleotide is not made at all, and/or when the expression of the polynucleotide is Even if there is no activity of the polypeptide, it may also be included. The "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by the parent strain, wild-type or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification". "Inactivation, deficiency, reduction, downregulation, reduction, attenuation" of the activity of the polypeptide compared to the intrinsic activity means that the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation is lowered.

Attenuation of the activity of such a polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by application of various methods well known in the art (eg, 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.).

Specifically, the attenuation of the polypeptide activity of the present application is

1) deletion of all or part of a gene encoding a polypeptide;

2) modification of the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

3) modification of the amino acid sequence constituting the polypeptide such that the activity of the polypeptide is eliminated or attenuated (eg, deletion/substitution/addition of one or more amino acids on the amino acid sequence);

4) modification of the gene sequence encoding the polypeptide such that the activity of the polypeptide is eliminated or attenuated (eg, one or more nucleobases on the nucleotide sequence of the polypeptide gene to encode a polypeptide modified such that the activity of the polypeptide is eliminated or attenuated) deletion/replacement/addition of);

5) modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide;

6) introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of said gene 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 to form a secondary structure that cannot be attached to the ribosome;

8) addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE); or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

for example,

1) The deletion of a part or all of the gene encoding the polypeptide may be the removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

In addition, the above 2) modification of the expression control region (or expression control sequence), deletion, insertion, non-conservative or conservative substitution, or a combination thereof, mutation in the expression control region (or expression control sequence) occurs, or weaker replacement with an active sequence. The expression control 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.

In addition, 5) the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another start codon having a lower expression rate of the polypeptide compared to the intrinsic start codon It may be substituted with a sequence, but is not limited thereto.

In addition, the modification of the amino acid sequence or polynucleotide sequence of 3) and 4) above deletes, inserts, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to weaken the activity of the polypeptide. Or a combination thereof may result in a mutation in sequence, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, by introducing a mutation in the polynucleotide sequence to form a stop codon, the expression of a gene may be inhibited or attenuated, but is not limited thereto.

6) The introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide is described, for example, in Weintraub, H. et al., Antisense-RNA as a molecular tool. for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

7) Addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot be attached to the ribosome is mRNA translation It may make it impossible or slow it down.

8) the addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) is an antisense complementary to the transcript of the gene encoding the polypeptide It may be to attenuate activity by making nucleotides.

As used herein, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The reinforcement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Herein, activation, enhancement, up-regulation, overexpression, and increase may include all of those exhibiting an activity that was not originally possessed, or exhibiting an improved activity compared to an intrinsic activity or an activity prior to modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. "Enhance", "up-regulation", "overexpression" or "increase" in the activity of a polypeptide compared to its intrinsic activity means the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation. And / or concentration (expression amount) means improved compared to.

The enrichment can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and/or concentration (expression amount) of the endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from the increase in the level of activity, expression level, or the amount of product excreted from the polypeptide.

The enhancement of the activity of the polypeptide can be applied by various methods well known in the art, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before modification. Specifically, it may be one using genetic engineering and/or protein engineering well known to those skilled in the art, which is a routine method of molecular biology, but is not limited thereto (eg, Sitnicka et al. Functional Analysis of Genes. Advances in Cell). Biology 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, the enhancement of the polypeptide activity of the present application is

1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide;

2) replacing the gene expression control region on the chromosome encoding the polypeptide with a sequence with strong activity;

3) modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide;

4) modification of the amino acid sequence of said polypeptide to enhance polypeptide activity;

5) modification of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide (eg, modification of the polynucleotide sequence of the polypeptide gene to encode a polypeptide that has been modified to enhance the activity of the polypeptide);

6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) codon optimization of the polynucleotide encoding the polypeptide;

8) by analyzing the tertiary structure of the polypeptide to select an exposed site for modification or chemical modification; or

More specifically,

1) The increase in the intracellular copy number of the polynucleotide encoding the polypeptide is achieved by introduction into the host cell of a vector to which the polynucleotide encoding the polypeptide is operably linked, which can replicate and function independently of the host it may be Alternatively, the polynucleotide encoding the polypeptide may be achieved by introducing one copy or two or more copies into a chromosome in a host cell. The introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome in the host cell into the host cell, but is not limited thereto. The vector is the same as described above.

2) Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence with strong activity is, for example, deletion, insertion, non-conservative or Conservative substitution or a combination thereof may result in a mutation in the sequence, or replacement with a sequence having a stronger activity. The expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but is not limited thereto.

Examples of known strong promoters include CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but is not limited thereto.

3) Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a nucleotide sequence encoding another start codon having a higher expression rate of the polypeptide compared to the intrinsic start codon. It may be a substitution, but is not limited thereto.

The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may include deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide; A combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity, but is not limited thereto. The replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. In this case, the vector used may further include a selection marker for confirming whether or not the chromosome is inserted. The selection marker is the same as described above.

6) The introduction of the foreign polynucleotide exhibiting the activity of the polypeptide may be the introduction of the foreign polynucleotide encoding the polypeptide exhibiting the same/similar activity as the polypeptide into a host cell. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same/similar activity as the polypeptide. The method used for the introduction can be performed by appropriately selecting a known transformation method by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to generate a polypeptide and increase its activity.

7) Codon optimization of the polynucleotide encoding the polypeptide is codon-optimized so that the transcription or translation of the endogenous polynucleotide is increased in the host cell, or the transcription and translation of the foreign polynucleotide is optimized in the host cell. It may be that its codons are optimized so that the

8) Selecting an exposed site by analyzing the tertiary structure of the polypeptide and modifying or chemically modifying it is, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored to determine the degree of sequence similarity. Accordingly, it may be to determine a template protein candidate, check the structure based on this, and select an exposed site to be modified or chemically modified and modified or modified.

Such enhancement of polypeptide activity is to increase the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-modified microbial strain, or increase the amount of product produced from the polypeptide. However, the present invention is not limited thereto.

More specifically, the Corynebacterium glutamicum strain of the present application is a microorganism in which a polypeptide comprising SEQ ID NO: 117, a nucleotide encoding a polypeptide comprising SEQ ID NO: 117 or a nucleotide comprising SEQ ID NO: 118 is further deleted can

Part or all of the polynucleotide in the microorganism of the present application is modified by (a) homologous recombination using a vector for chromosome insertion in the microorganism or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and/or (b) It may be induced by light and/or chemical treatments such as, but not limited to, ultraviolet and radiation. The method for modifying part or all of the gene may include a method by DNA recombination technology. For example, by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to a target gene into the microorganism to cause homologous recombination, a part or all of the gene may be deleted. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

Another aspect of the present application is (i) one or more variants of the present application (ii) one or more polynucleotides encoding the variant or (iii) a Corynebacterium glutamicum strain comprising a combination thereof in a medium It provides a method for producing L-glutamic acid, comprising the step of culturing in the.

The L-glutamic acid production method of the present application may include culturing a Corynebacterium glutamicum strain comprising the mutant of the present application or the polynucleotide of the present application or the vector of the present application in a medium.

In the present application, the term "cultivation" means growing the Corynebacterium glutamicum strain of the present application under moderately controlled environmental conditions. The culture process of the present application may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culture may be a batch, continuous and/or fed-batch, but is not limited thereto.

As used herein, the term "medium" refers to a material in which nutrients required for culturing the Corynebacterium glutamicum strain of the present application are mixed as a main component, and nutrients including water essential for survival and growth and growth factors. Specifically, any medium and other culture conditions used for culturing the Corynebacterium glutamicum strain of the present application may be used without any particular limitation as long as it is a medium used for culturing conventional microorganisms, but the Corynebacterium glutamicum of the present application Lium glutamicum strain can be cultured while controlling the temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid and / or vitamin and the like.

Specifically, the culture medium for the Corynebacterium sp. strain can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981)].

In the present application, the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, and the like; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; amino acids such as glutamic acid, methionine, lysine, and the like may be included. In addition, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugar cane offal and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e., converted to reducing sugar). molasses) may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more, but is not limited thereto.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.

The phosphorus may include potassium first potassium phosphate, second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and in addition, amino acids, vitamins and/or suitable precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, the present invention is not limited thereto.

In addition, during the culture of the Corynebacterium glutamicum strain of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without or without gas to maintain anaerobic and microaerobic conditions, it is not

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

L-glutamic acid produced by the culture of the present application may be secreted into the medium or remain in the cells.

The L- glutamic acid production method of the present application includes the steps of preparing the Corynebacterium glutamicum strain of the present application, preparing a medium for culturing the strain, or a combination thereof (regardless of the order, in any order) ), for example, prior to the culturing step, may further include.

The method for producing L- glutamic acid of the present application may further include recovering L- glutamic acid from the culture medium (the culture medium) or the Corynebacterium glutamicum strain. The recovering step may be further included after the culturing step.

The recovery may be to collect the desired L-glutamic acid using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method. . For example, centrifugation, filtration, treatment with a crystallized protein precipitating agent (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity Various chromatography such as island chromatography, HPLC, or a combination thereof may be used, and a desired L-glutamic acid may be recovered from a medium or a microorganism using a suitable method known in the art.

In addition, the L-glutamic acid production method of the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, when the method for producing L-glutamic acid of the present application includes both the recovery step and the purification step, the recovery step and the purification step are performed continuously or discontinuously, regardless of the order, or integrated into one step. may be performed, but is not limited thereto.

In the method of the present application, variants, polynucleotides, vectors, strains, and the like are as described in the other aspects above.

Another aspect of the present application is Corynebacterium comprising one or more variants of the present application, one or more polynucleotides encoding the variants, one or more vectors including the polynucleotides, or one or more polynucleotides of the present application glutamicum strain; the culture medium; Or to provide a composition for producing L- glutamic acid comprising a combination of two or more of them.

The composition of the present application may further include any suitable excipients commonly used in compositions for the production of amino acids, and these excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents or isotonic agents, etc. However, the present invention is not limited thereto.

In the composition of the present application, variants, polynucleotides, vectors, strains and L-glutamic acid are the same as those described in the other aspects above.

Hereinafter, the present application will be described in more detail by way of Examples. However, the following examples are merely preferred embodiments for illustrating the present application, and therefore, are not intended to limit the scope of the present application thereto. On the other hand, technical matters not described in this specification can be sufficiently understood and easily implemented by those skilled in the art or similar technical fields of the present application.

실시예 1: ABC 트랜스포터 ATP-결합 단백질 변이체를 발현하는 미생물의 L-글루탐산 생산능 평가Example 1: Evaluation of L-glutamic acid production ability of microorganisms expressing the ABC transporter ATP-binding protein variant

실시예 1-1: 미생물내 ABC 트랜스포터 ATP-결합 단백질 변이체 발현을 위한 벡터 제작Example 1-1: Construction of vector for expression of ABC transporter ATP-binding protein mutants in microorganisms

ABC transporter ATP-binding protein amino acid sequence (SEQ ID NO: 3) to determine the effect of the mutant (G32D; SEQ ID NO: 1) in which the glycine at position 32 of the amino acid sequence (SEQ ID NO: 3) is substituted for L-glutamic acid production. The vector was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

PCR was performed using the primer pair of the sequences of SEQ ID NOs: 45 and 46 and the primer pair of the sequences of SEQ ID NOs: 47 and 48 using gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 45 and SEQ ID NO: 48 to obtain a fragment. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-BBD29_01215 (G32D).

실시예 1-2: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산주 제작 및 ABC 트랜스포터 ATP-결합 단백질 변이체 도입 균주 제작Example 1-2: wild-type Corynebacterium glutamicum-derived L-glutamic acid production strain production and ABC transporter ATP-binding protein mutant introduction strain production

실시예 1-2-1: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산능을 갖는 코리네박테리움 글루타미쿰 균주 제작Example 1-2-1: Preparation of Corynebacterium glutamicum strain having L-glutamic acid-producing ability derived from wild-type Corynebacterium glutamicum

Corynebacterium glutamicum ATCC13869-derived odhA based on the prior literature (Appl Environ Microbiol. 2007 Feb; 73(4): 1308-19. Epub 2006 Dec 8.) to produce a strain having L-glutamic acid-producing ability derived from ATCC13869. A strain of Corynebacterium glutamicum ATCC13869 ΔodhA in which the gene was deleted was prepared.

Specifically, PCR was performed using the primer pairs of SEQ ID NO: 111 and SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114 using Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template for odhA deletion. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 111 and SEQ ID NO: 114 to obtain a fragment. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-ΔodhA.

The prepared pDCM2-ΔodhA vector was transformed into the Corynebacterium glutamicum ATCC13869 strain by electroporation, and then a strain in which the odhA gene was deleted was obtained through a secondary crossover process. The gene deletion was confirmed through PCR and genome sequencing using SEQ ID NO: 115 and SEQ ID NO: 116, and the prepared strain was named ATCC13869ΔodhA.

The primer sequences used herein are shown in Table 1 below.

서열번호SEQ ID NO:	명칭designation	서열order
111111	odhA_up_FodhA_up_F	TGAATTCGAGCTCGGTACCCTTGAACGGAATTGGGTGGTGAATTCGAGCTCGGTACCCTTGAACGGAATTGGGTGG
112112	odhA_up_RodhA_up_R	CCCAGGTGGCATCGGTACCTTCACCCAGCGCCACGCAGCCCAGGTGGCATCGGTACCTTCACCCAGCCGCCACGCAG
113113	odhA_down_FodhA_down_F	CGCTGGGTGAAGGTACCGATGCCACCTGGGTTGGTCAAGCGCTGGGTGAAGGTACCGATGCCACCTGGGTTGGTCAAG
114114	odhA_down_RodhA_down_R	GTCGACTCTAGAGGATCCCCGGACAAGGAATGGAGAGAGTCGACTCTAGAGGATCCCCGGACAAGGAATGGAGAGA
115115	odhA_del_FodhA_del_F	CTTACCGTTGTTGCCCTTCTTACCGTTGTTGCCCTT
116116	odhA_del_RodhA_del_R	CTCCTTCACCCACATCATTCTCCTTCACCCACATCATT

실시예 1-2-2: ABC 트랜스포터 ATP-결합 단백질 변이체 발현 균주 제작Example 1-2-2: ABC transporter ATP-binding protein mutant expression strain construction

The vector prepared in Example 2 was transformed into ATCC13869ΔodhA prepared in Example 1-2-1.

In the strain in which the homologous recombination occurred, the strain into which the mutant was introduced was selected using SEQ ID NOs: 49 and 50. Each selected strain was named ATCC13869 ΔodhA_BBD29_01215_G32D.

실시예 1-2-3: ABC 트랜스포터 ATP-결합 단백질 변이체 발현 균주의 L-글루탐산 생산능 비교Example 1-2-3: Comparison of L-glutamic acid production capacity of ABC transporter ATP-binding protein mutant expression strains

In order to check the L-glutamic acid production ability of the strain prepared in Example 1-2-1, the strain ATCC13869ΔodhA was used as a control and cultured in the following manner.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours with shaking at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and incubated at 30° C. for 40 hours at 200 rpm. After completion of the culture, L-glutamic acid production capacity was measured using high performance liquid chromatography (HPLC), and the measurement results are shown in Table 2 below. The glutamic acid concentration and the concentration increase rate in the culture medium for each strain tested are shown in Table 2 below.

Glucose 1%, broth 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, agar 2%, urea 0.2%, pH 7.2

Raw sugar 6%, calcium carbonate 5%, ammonium sulfate 2.25%, potassium monophosphate 0.1%, magnesium sulfate 0.04%, iron sulfate 10 mg/L, thiamine hydrochloride 0.2 mg/L, biotin 50㎍/L

균주명strain name	L-글루탐산 농도(g/L)L-glutamic acid concentration (g/L)	L-글루탐산 농도 증가율(%)L-glutamic acid concentration increase rate (%)
ATCC13869 △odhAATCC13869 △odhA	1.91.9	--
ATCC13869 △odhA_ BBD29_01215_G32DATCC13869 △odhA_ BBD29_01215_G32D	2.32.3	21.121.1

As shown in Table 2, it was confirmed that the concentration of L-glutamic acid was significantly increased in ATCC13869 ΔodhA_BBD29_01215_G32D into which the BBD29_01215 (G32D) gene was introduced compared to the ATCC13869 ΔodhA strain.

The ATCC13869 △odhA_BBD29_01215_G32D was named CA02-1605, and it was deposited with the Korea Microorganism Conservation Center, a trustee under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12846P.

실시예 2: ABC 트랜스포터 ATP-결합 단백질 변이체를 발현하는 미생물의 L-글루탐산 생산능 평가Example 2: Evaluation of L-glutamic acid production ability of microorganisms expressing the ABC transporter ATP-binding protein variant

실시예 2-1: 미생물내 ABC 트랜스포터 ATP-결합 단백질Example 2-1: ABC transporter ATP-binding protein in microorganisms 변이체 발현을 위한 벡터 제작Vector construction for mutant expression

To determine the effect of the mutant (A282T; SEQ ID NO: 5) in which the alanine at position 282 of the ABC transporter ATP-binding protein amino acid sequence (SEQ ID NO: 7) is substituted with threonine (A282T; SEQ ID NO: 5) on L-glutamic acid production A vector for constructing an expression strain thereof was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 51 and 52 and a pair of primers of sequences of SEQ ID NOs: 53 and 54, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 51 and SEQ ID NO: 54 to obtain a fragment. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-BBD29_01305 (A282T).

실시예 2-2: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산주 제작 및 ABC 트랜스포터 ATP-결합 단백질 변이체 도입 균주 제작Example 2-2: Production of wild-type Corynebacterium glutamicum-derived L-glutamic acid production strain and ABC transporter ATP-binding protein mutant introduction strain production

실시예 2-2-1: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산능을 갖는 코리네박테리움 글루타미쿰 균주 제작Example 2-2-1: Preparation of Corynebacterium glutamicum strain having L-glutamic acid-producing ability derived from wild-type Corynebacterium glutamicum

The primer sequences used herein are shown in Table 1 above.

실시예 2-2-2: ABC 트랜스포터 ATP-결합 단백질Example 2-2-2: ABC transporter ATP-binding protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 2-1 was transformed into ATCC13869 ΔodhA prepared in Example 2-2-1.

In the strain in which the homologous recombination occurred, the strain into which the mutant was introduced was selected using SEQ ID NOs: 55 and 56. Each selected strain was named ATCC13869 ΔodhA_BBD29_01305_A282T.

실시예 2-2-3: ABC 트랜스포터 ATP-결합 단백질 변이체 발현 균주의 L-글루탐산 생산능 비교Example 2-2-3: Comparison of L-glutamic acid production ability of ABC transporter ATP-binding protein mutant expression strains

In order to confirm the L-glutamic acid production ability of the strain prepared in Example 2-2-1, the strain ATCC13869ΔodhA was used as a control and cultured in the following manner.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and cultured with shaking at 30° C. for 40 hours at 200 rpm. After completion of the culture, L-glutamic acid production capacity was measured using high performance liquid chromatography (HPLC), and the measurement results are shown in Table 3 below. The glutamic acid concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 3 below.

균주명strain name	L-글루탐산 농도(g/L)L-glutamic acid concentration (g/L)	L-글루탐산 농도 증가율(%)L-glutamic acid concentration increase rate (%)
ATCC13869 △odhAATCC13869 △odhA	1.91.9	--
ATCC13869 △odhA_BBD29_01305_A282TATCC13869 △odhA_BBD29_01305_A282T	2.652.65	39.539.5

As shown in Table 3, it was confirmed that the concentration of L-glutamic acid was significantly increased in ATCC13869 ΔodhA_BBD29_01305_A282T into which the BBD29_01305 (A282T) gene was introduced compared to the ATCC13869ΔodhA strain.

The ATCC13869 △odhA_BBD29_01305_A282T was named CA02-1614, and it was deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty on November 30, 2020, and was given an accession number KCCM12855P.

실시예 3: D-알라닌-D-알라닌 리가아제 AExample 3: D-alanine-D-alanine ligase A 변이체를 발현하는 미생물의 L-글루탐산 생산능 평가Evaluation of L-glutamic acid production ability of microorganisms expressing variants

실시예 3-1: 미생물내 D-알라닌-D-알라닌 리가아제 AExample 3-1: D-alanine-D-alanine ligase A in microorganisms 변이체 발현을 위한 벡터 제작Vector construction for mutant expression

D-alanine-D-alanine ligase A variant (G256S; SEQ ID NO: 9) in which glycine at position 256 of the amino acid sequence (SEQ ID NO: 11) is substituted with serine (G256S; SEQ ID NO: 9) to determine the effect on L-glutamic acid production, the expression strain production A vector for was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 57 and 58 and a pair of primers of sequences of SEQ ID NOs: 59 and 60, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a pair of primers of SEQ ID NO: 57 and SEQ ID NO: 60 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-ddlA (G256S).

실시예 3-2: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산주 제작 및 D-알라닌-D-알라닌 리가아제 A 변이체 도입 균주 제작Example 3-2: Production of wild-type Corynebacterium glutamicum-derived L-glutamic acid production strain and D-alanine-D-alanine ligase A mutant introduction strain production

실시예 3-2-1: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산능을 갖는 코리네박테리움 글루타미쿰 균주 제작Example 3-2-1: Preparation of Corynebacterium glutamicum strain having L-glutamic acid-producing ability derived from wild-type Corynebacterium glutamicum

Specifically, PCR was performed using the primer pairs of SEQ ID NO: 111 and SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114 using Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template for odhA deletion. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 111 and SEQ ID NO: 114 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-ΔodhA.

The primer sequences used herein are shown in Table 1 above.

실시예 3-2-2: D-알라닌-D-알라닌 리가아제 AExample 3-2-2: D-alanine-D-alanine ligase A 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 3-1 was transformed into ATCC13869ΔodhA prepared in Example 3-2-1.

In the strain in which the homologous recombination occurred, the strain into which the mutant was introduced was selected using SEQ ID NOs: 61 and 62. Each selected strain was named ATCC13869 ΔodhA_ddlA_G256S.

실시예 3-2-3: D-알라닌-D-알라닌 리가아제 A 변이체 발현 균주의 L-글루탐산 생산능 비교Example 3-2-3: Comparison of L-glutamic acid production capacity of D-alanine-D-alanine ligase A mutant expression strain

In order to check the L-glutamic acid production ability of the strain prepared in Example 3-2-1, the strain ATCC13869ΔodhA was used as a control and cultured in the following manner.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and cultured with shaking at 30° C. for 40 hours at 200 rpm. After completion of the culture, L-glutamic acid production capacity was measured using high performance liquid chromatography (HPLC), and the measurement results are shown in Table 4 below. The glutamic acid concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 4 below.

균주명strain name	L-글루탐산 농도(g/L)L-glutamic acid concentration (g/L)	L-글루탐산 농도 증가율(%)L-glutamic acid concentration increase rate (%)
ATCC13869 △odhAATCC13869 △odhA	1.91.9	--
ATCC13869 △odhA_ddlA_G256SATCC13869 △odhA_ddlA_G256S	2.312.31	21.621.6

As shown in Table 4, it was confirmed that the concentration of L-glutamic acid was significantly increased in ATCC13869 ΔodhA_ddlA_G256S into which the ddlA (G256S) gene was introduced compared to the ATCC13869 ΔodhA strain.

The ATCC13869 △odhA_ddlA_G256S was named CA02-1613, and it was deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12854P.

실시예 4: 글루코사민-6-포스페이트 디아미나제Example 4: Glucosamine-6-phosphate deaminase 변이체를 발현하는 미생물의 L-글루탐산 생산능 평가Evaluation of L-glutamic acid production ability of microorganisms expressing variants

실시예 4-1: 미생물내 글루코사민-6-포스페이트 디아미나제Example 4-1: Glucosamine-6-phosphate deaminase in microorganisms 변이체 발현을 위한 벡터 제작Vector construction for mutant expression

Glucosamine-6-phosphate deaminase amino acid sequence (SEQ ID NO: 15) of the variant (A137V; SEQ ID NO: 13) in which alanine at position 137 of the amino acid sequence (SEQ ID NO: 15) is substituted with valine (A137V; SEQ ID NO: 13) to determine the effect on L-glutamic acid production for its expression strain production The vector was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

PCR was performed using a primer pair of sequences of SEQ ID NOs: 63 and 64 and a pair of primers of sequences of SEQ ID NOs: 65 and 66 using wild-type Corynebacterium glutamicum ATCC (gDNA (genomic DNA) of 13869) as a template, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 63 and SEQ ID NO: 66 to obtain a fragment.PCR was denatured at 94°C for 5 minutes, 30 seconds at 94° C., 30 seconds at 55° C., and 1 minute and 30 seconds at 72° C. were repeated 30 times, followed by 5 minutes at 72° C. The pDCM2 vector was treated with smaI, and the PCR product obtained above was fusion cloned. Fusion cloning was performed using In-Fusion® HD cloning kit (Clontech) The resulting plasmid was named pDCM2-nagB (A137V).

실시예 4-2: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산주 제작 및 글루코사민-6-포스페이트 디아미나제 변이체 도입 균주 제작Example 4-2: Preparation of wild-type Corynebacterium glutamicum-derived L-glutamic acid production strain and glucosamine-6-phosphate deaminase mutant introduction strain

실시예 4-2-1: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산능을 갖는 코리네박테리움 글루타미쿰 균주 제작Example 4-2-1: Preparation of Corynebacterium glutamicum strain having L-glutamic acid-producing ability derived from wild-type Corynebacterium glutamicum

The prepared pDCM2-ΔodhA vector was transformed into the Corynebacterium glutamicum ATCC13869 strain by electroporation, and then a strain in which the odhA gene was deleted was obtained through a secondary crossover process. The gene deletion was confirmed by PCR and genome sequencing using SEQ ID NO: 115 and SEQ ID NO: 116, and the prepared strain was named ATCC13869ΔodhA.

The primer sequences used herein are shown in Table 1 above.

실시예 4-2-2: 글루코사민-6-포스페이트 디아미나제Example 4-2-2: Glucosamine-6-phosphate deaminase 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 4-1 was transformed into ATCC13869ΔodhA prepared in Example 4-2-1.

In the strain in which the homologous recombination occurred, the strain into which the mutant was introduced was selected using SEQ ID NOs: 67 and 68. Each selected strain was named ATCC13869 ΔodhA_nagB_A137V.

실시예 4-2-3: 글루코사민-6-포스페이트 디아미나제 변이체 발현 균주의 L-글루탐산 생산능 비교Example 4-2-3: Comparison of L-glutamic acid production capacity of glucosamine-6-phosphate deaminase mutant expression strains

In order to check the L-glutamic acid production ability of the strain prepared in Example 4-2-1, the strain ATCC13869 ΔodhA was used as a control and cultured in the following manner.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and cultured with shaking at 30° C. for 40 hours at 200 rpm. After completion of the culture, L-glutamic acid production capacity was measured using high performance liquid chromatography (HPLC), and the measurement results are shown in Table 5 below. The glutamic acid concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 5 below.

균주명strain name	L-글루탐산 농도(g/L)L-glutamic acid concentration (g/L)	L-글루탐산 농도 증가율(%)L-glutamic acid concentration increase rate (%)
ATCC13869 △odhAATCC13869 △odhA	1.91.9	--
ATCC13869 △odhA_nagB_A137VATCC13869 △odhA_nagB_A137V	2.62.6	36.836.8

As shown in Table 5, it was confirmed that the concentration of L-glutamic acid was significantly increased in ATCC13869 ΔodhA_nagB_A137V into which the nagB (A137V) gene was introduced compared to the ATCC13869 ΔodhA strain.

The ATCC13869 △odhA_nagB_A137V was named CA02-1611, and it was deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12852P.

실시예 5: 엑시뉴클레아제 ABC 서브유닛 AExample 5: Exinuclease ABC Subunit A 변이체를 발현하는 미생물의 L-글루탐산 생산능 평가Evaluation of L-glutamic acid production ability of microorganisms expressing variants

실시예 5-1: 미생물내 엑시뉴클레아제 ABC 서브유닛 AExample 5-1: Exinuclease ABC subunit A in microorganisms 변이체 발현을 위한 벡터 제작Vector construction for mutant expression

In order to determine the effect of the glycine at position 575 of the exinuclease ABC subunit A amino acid sequence (SEQ ID NO: 19) on the production of L-glutamic acid, the mutant (G575D; SEQ ID NO: 17) in which the glycine at position 575 is substituted with aspartic acid was produced. A vector was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 69 and 70 and a pair of primers of sequences of SEQ ID NOs: 71 and 72, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 69 and SEQ ID NO: 72 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-uvrA (G575D).

실시예 5-2: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산주 제작 및 엑시뉴클레아제 ABC 서브유닛 A 변이체 도입 균주 제작Example 5-2: Production of wild-type Corynebacterium glutamicum-derived L-glutamic acid producing strain and exinuclease ABC subunit A mutant introduction strain

실시예 5-2-1: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산능을 갖는 코리네박테리움 글루타미쿰 균주 제작Example 5-2-1: Preparation of Corynebacterium glutamicum strain having L-glutamic acid-producing ability derived from wild-type Corynebacterium glutamicum

The prepared pDCM2-ΔodhA vector was transformed into the Corynebacterium glutamicum ATCC13869 strain by electroporation, and then a strain in which the odhA gene was deleted was obtained through a secondary crossover process. The gene deletion was confirmed by PCR and genome sequencing using SEQ ID NO: 115 and SEQ ID NO: 116, and the prepared strain was named ATCC13869 ΔodhA.

The primer sequences used herein are shown in Table 1 above.

실시예 5-2-2: 엑시뉴클레아제 ABC 서브유닛 AExample 5-2-2: Exinuclease ABC Subunit A 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 5-1 was transformed into ATCC13869ΔodhA prepared in Example 5-2-1.

In the strain in which the homologous recombination occurred, the strain into which the mutant was introduced was selected using SEQ ID NOs: 73 and 74. Each selected strain was named ATCC13869 ΔodhA_uvrA_G575D.

실시예 5-2-3: 엑시뉴클레아제 ABC 서브유닛 A 변이체 발현 균주의 L-글루탐산 생산능 비교Example 5-2-3: Comparison of L-glutamic acid production capacity of exinuclease ABC subunit A mutant expression strains

In order to confirm the L-glutamic acid production ability of the strain prepared in Example 5-2-1, the strain ATCC13869 ΔodhA was used as a control and cultured in the following manner.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and cultured with shaking at 30° C. for 40 hours at 200 rpm. After completion of the culture, L-glutamic acid production capacity was measured using high performance liquid chromatography (HPLC), and the measurement results are shown in Table 6 below. The glutamic acid concentration and the concentration increase rate in the culture medium for each strain tested are shown in Table 6 below.

균주명strain name	L-글루탐산 농도(g/L)L-glutamic acid concentration (g/L)	L-글루탐산 농도 증가율(%)L-glutamic acid concentration increase rate (%)
ATCC13869 △odhAATCC13869 △odhA	1.91.9	--
ATCC13869 △odhA_uvrA_G575DATCC13869 △odhA_uvrA_G575D	2.32.3	21.121.1

As shown in Table 6, it was confirmed that the concentration of L-glutamic acid was significantly increased in ATCC13869 ΔodhA_uvrA_G575D into which the uvrA_G575D gene was introduced compared to the ATCC13869 ΔodhA strain.

The ATCC13869 △odhA_uvrA_G575D was named CA02-1612, and it was deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12853P.

실시예 6: 리보뉴클레아제 P 변이체를 발현하는 미생물의 L-글루탐산 생산능 평가Example 6: Evaluation of L-glutamic acid production ability of microorganisms expressing ribonuclease P variants

실시예 6-1: 미생물내 리보뉴클레아제 P 변이체 발현을 위한 벡터 제작Example 6-1: Construction of a vector for the expression of ribonuclease P variants in microorganisms

In order to determine the effect of the ribonuclease P amino acid sequence (SEQ ID NO: 23) in which histidine at position 32 is substituted with tyrosine (H32Y; SEQ ID NO: 21) on L-glutamic acid production, a vector for constructing an expression strain thereof was developed. The plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Nebacterium chromosome was used as follows.

PCR was performed using the primer pair of sequences of SEQ ID NOs: 75 and 76 and the primer pair of sequences of SEQ ID NOs: 77 and 78, respectively, using gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 75 and SEQ ID NO: 78 to obtain a fragment. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-rnpA(H32Y).

실시예 6-2: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산주 제작 및 리보뉴클레아제 P 변이체 도입 균주 제작Example 6-2: Production of wild-type Corynebacterium glutamicum-derived L-glutamic acid production strain and ribonuclease P mutant introduction strain

실시예 6-2-1: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산능을 갖는 코리네박테리움 글루타미쿰 균주 제작Example 6-2-1: Preparation of Corynebacterium glutamicum strain having L-glutamic acid-producing ability derived from wild-type Corynebacterium glutamicum

After transforming the prepared pDCM2-ΔodhA vector into the Corynebacterium glutamicum ATCC13869 strain by electroporation, a secondary crossover process was performed to obtain a strain lacking the odhA gene on the chromosome. The gene deletion was confirmed through PCR and genome sequencing using SEQ ID NO: 115 and SEQ ID NO: 116, and the prepared strain was named ATCC13869ΔodhA.

The primer sequences used herein are shown in Table 1 above.

실시예 6-2-2: 리보뉴클레아제 P 변이체 발현 균주 제작Example 6-2-2: Ribonuclease P variant expression strain production

The vector prepared in Example 6-1 was transformed into ATCC13869ΔodhA prepared in Example 6-2-1.

In the strain in which the homologous recombination occurred, the strain into which the mutant was introduced was selected using SEQ ID NOs: 79 and 80. Each selected strain was named ATCC13869 ΔodhA_rnpA_H32Y.

실시예 6-2-3: 리보뉴클레아제 P 변이체 발현 균주의 L-글루탐산 생산능 비교Example 6-2-3: Comparison of L-glutamic acid production capacity of ribonuclease P variant expression strain

In order to check the L-glutamic acid production ability of the strain prepared in Example 6-2-1, the strain ATCC13869ΔodhA was used as a control and cultured in the following manner.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and cultured with shaking at 30° C. for 40 hours at 200 rpm. After completion of the culture, L-glutamic acid production capacity was measured using high performance liquid chromatography (HPLC), and the measurement results are shown in Table 7 below. The glutamic acid concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 7 below.

균주명strain name	L-글루탐산 농도(g/L)L-glutamic acid concentration (g/L)	L-글루탐산 농도 증가율(%)L-glutamic acid concentration increase rate (%)
ATCC13869 △odhAATCC13869 △odhA	1.91.9	--
ATCC13869 △odhA_rnpA_H32YATCC13869 △odhA_rnpA_H32Y	2.182.18	14.714.7

As shown in Table 7, it was confirmed that the concentration of L-glutamic acid was significantly increased in ATCC13869 ΔodhA_rnpA_H32Y into which the rnpA (H32Y) gene was introduced compared to the ATCC13869 ΔodhA strain.

The ATCC13869 △odhA_rnpA_H32Y was named CA02-1607, and it was deposited with the Korea Microorganism Conservation Center, a trustee under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12848P.

실시예 7: MFS 트랜스포터 변이체를 발현하는 미생물의 L-글루탐산 생산능 평가Example 7: Evaluation of L-glutamic acid production ability of microorganisms expressing MFS transporter variants

실시예 7-1: 미생물내 MFS 트랜스포터 변이체 발현을 위한 벡터 제작Example 7-1: Construction of a vector for the expression of MFS transporter mutants in microorganisms

In order to determine the effect of the mutant (A382T; SEQ ID NO: 25) in which alanine at position 382 of the MFS transporter amino acid sequence (SEQ ID NO: 27) is substituted with threonine (A382T; SEQ ID NO: 25) on the production of L-glutamic acid, a vector for constructing an expression strain thereof was developed with Corynebacter Plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Rium chromosome was produced as follows.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 81 and 82 and a pair of primers of SEQ ID NOs: 83 and 84, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a primer pair of SEQ ID NO: 81 and SEQ ID NO: 84 to obtain a fragment. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-iolT2 (A382T).

실시예 7-2: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산주 제작 및 MFS 트랜스포터 변이체 도입 균주 제작Example 7-2: Wild-type Corynebacterium glutamicum-derived L-glutamic acid production strain production and MFS transporter mutant introduction strain production

실시예 7-2-1: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산능을 갖는 코리네박테리움 글루타미쿰 균주 제작Example 7-2-1: Preparation of Corynebacterium glutamicum strain having L-glutamic acid-producing ability derived from wild-type Corynebacterium glutamicum

The primer sequences used herein are shown in Table 1 above.

실시예 7-2-2: MFS 트랜스포터 변이체 발현 균주 제작Example 7-2-2: MFS transporter mutant expression strain construction

The vector prepared in Example 7-1 was transformed into ATCC13869ΔodhA prepared in Example 7-2-1.

In the strain in which the homologous recombination occurred, the strain into which the mutant was introduced was selected using SEQ ID NOs: 85 and 86. Each selected strain was named ATCC13869 ΔodhA_iolT2_A382T.

실시예 7-2-3: MFS 트랜스포터 변이체 발현 균주의 L-글루탐산 생산능 비교Example 7-2-3: Comparison of L-glutamic acid production capacity of MFS transporter mutant expression strains

In order to check the L-glutamic acid production ability of the strain prepared in Example 7-2-1, the strain ATCC13869ΔodhA was used as a control and cultured in the following manner.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and cultured with shaking at 30° C. for 40 hours at 200 rpm. After completion of the culture, L-glutamic acid production capacity was measured using high performance liquid chromatography (HPLC), and the measurement results are shown in Table 8 below. The glutamic acid concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 8 below.

균주명strain name	L-글루탐산 농도(g/L)L-glutamic acid concentration (g/L)	L-글루탐산 농도 증가율(%)L-glutamic acid concentration increase rate (%)
ATCC13869 △odhAATCC13869 △odhA	1.91.9	--
ATCC13869 △odhA_iolT2_A382TATCC13869 △odhA_iolT2_A382T	2.62.6	36.836.8

As shown in Table 8, it was confirmed that the concentration of L-glutamic acid was significantly increased in ATCC13869 ΔodhA_iolT2_A382T into which the iolT2(A382T) gene was introduced compared to the ATCC13869 ΔodhA strain.

The ATCC13869 △odhA_iolT2_A382T was named CA02-1608, and it was deposited with the Korea Microorganism Conservation Center, a trustee under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12849P.

실시예 8: 갈락토사이드 O-아세틸트랜스퍼라제 변이체를 발현하는 미생물의 L-글루탐산 생산능 평가Example 8: Evaluation of L-glutamic acid production ability of microorganisms expressing galactoside O-acetyltransferase variants

실시예 8-1: 미생물내 갈락토사이드 O-아세틸트랜스퍼라제 변이체 발현을 위한 벡터 제작Example 8-1: Construction of a vector for the expression of galactoside O-acetyltransferase mutants in microorganisms

To determine the effect of the leucine at position 106 of the galactoside O-acetyltransferase amino acid sequence (SEQ ID NO: 31) with phenylalanine substituted (L106F; SEQ ID NO: 29) on L-glutamic acid production, for the production of an expression strain thereof The vector was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 87 and 88 and a pair of primers of sequences of SEQ ID NOs: 89 and 90, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 87 and SEQ ID NO: 90 to obtain a fragment. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-maa (L106F).

실시예 8-2: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산주 제작 및 갈락토사이드 O-아세틸트랜스퍼라제 변이체 도입 균주 제작Example 8-2: Preparation of wild-type Corynebacterium glutamicum-derived L-glutamic acid production strain and production of galactoside O-acetyltransferase mutant introduction strain

실시예 8-2-1: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산능을 갖는 코리네박테리움 글루타미쿰 균주 제작Example 8-2-1: Preparation of Corynebacterium glutamicum strain having L-glutamic acid-producing ability derived from wild-type Corynebacterium glutamicum

The primer sequences used herein are shown in Table 1 above.

실시예 8-2-2: 갈락토사이드 O-아세틸트랜스퍼라제 변이체 발현 균주 제작Example 8-2-2: Galactoside O-acetyltransferase mutant expression strain production

The vector prepared in Example 8-1 was transformed into ATCC13869ΔodhA prepared in Example 8-2-1.

In the strain in which the homologous recombination occurred, the strain into which the mutant was introduced was selected using SEQ ID NOs: 91 and 92. Each selected strain was named ATCC13869 ΔodhA_maa_L106F.

실시예 8-2-3: 갈락토사이드 O-아세틸트랜스퍼라제 변이체 발현 균주의 L-글루탐산 생산능 비교Example 8-2-3: Comparison of L-glutamic acid production capacity of galactoside O-acetyltransferase mutant expression strains

In order to check the L-glutamic acid production ability of the strain prepared in Example 8-2-1, the strain ATCC13869ΔodhA was used as a control and cultured in the following manner.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and cultured with shaking at 30° C. for 40 hours at 200 rpm. After completion of the culture, L-glutamic acid production capacity was measured using high performance liquid chromatography (HPLC), and the measurement results are shown in Table 9 below. The glutamic acid concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 9 below.

균주명strain name	L-글루탐산 농도(g/L)L-glutamic acid concentration (g/L)	L-글루탐산 농도 증가율(%)L-glutamic acid concentration increase rate (%)
ATCC13869 △odhAATCC13869 △odhA	1.91.9	--
ATCC13869 △odhA_maa_L106FATCC13869 △odhA_maa_L106F	2.22.2	15.815.8

As shown in Table 9, it was confirmed that the concentration of L-glutamic acid was significantly increased in ATCC13869 ΔodhA_maa_L106F into which the maa(L106F) gene was introduced compared to the ATCC13869 ΔodhA strain.

The ATCC13869 △odhA_maa_L106F was named CA02-1606, and it was deposited with the Korea Microorganism Conservation Center, an institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12847P.

실시예 9: 스퍼미딘 신타아제 변이체를 발현하는 미생물의 L-글루탐산 생산능 평가Example 9: Evaluation of L-glutamic acid production ability of microorganisms expressing spermidine synthase variants

실시예 9-1: 미생물내 스퍼미딘 신타아제 변이체 발현을 위한 벡터 제작Example 9-1: Construction of a vector for the expression of spermidine synthase mutants in microorganisms

Proline at position 453 of the spermidine synthase amino acid sequence (SEQ ID NO: 35) is substituted with serine (P453S; SEQ ID NO: 37), or alanine at position 396 is substituted with threonine (A396T; SEQ ID NO: 39), or proline at position 453 In order to confirm the effect of this serine-substituted mutant (P453S+A396T; SEQ ID NO: 33) in which alanine at position 396 is substituted with threonine on L-glutamic acid production, a vector for constructing an expression strain thereof was constructed as a gene in the Corynebacterium chromosome. Plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of was prepared as follows.

The following method was used to construct a plasmid in which the proline at position 453 was substituted with serine. PCR was performed using a primer pair of sequences of SEQ ID NOs: 93 and 94 and a pair of primers of sequences of SEQ ID NOs: 95 and 96 using gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 93 and SEQ ID NO: 96 to obtain a fragment. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-speE (P453S).

The following method was used to construct a plasmid in which alanine at position 396 was substituted with threonine. Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 99 and 100 and a pair of primers of sequences of SEQ ID NOs: 101 and 102, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 99 and SEQ ID NO: 102 to obtain a fragment. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-speE (A396T).

실시예 9-2: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산주 제작 및 스퍼미딘 신타아제 변이체 도입 균주 제작Example 9-2: Production of wild-type Corynebacterium glutamicum-derived L-glutamic acid production strain and spermidine synthase mutant introduction strain

실시예 9-2-1: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산능을 갖는 코리네박테리움 글루타미쿰 균주 제작Example 9-2-1: Preparation of Corynebacterium glutamicum strain having L-glutamic acid-producing ability derived from wild-type Corynebacterium glutamicum

The primer sequences used herein are shown in Table 1 above.

실시예 9-2-2: 스퍼미딘 신타아제 변이체 발현 균주 제작Example 9-2-2: Spermidine synthase mutant expression strain production

The vector prepared in Example 9-1 was transformed into ATCC13869ΔodhA prepared in Example 9-2-1.

In the strain in which the homologous recombination occurred, a strain into which the P453S mutant was introduced was selected using SEQ ID NOs: 97 and 98, and a strain into which the A396T mutant was introduced using SEQ ID NOs: 103 and 104 was selected. Each selected strain was named ATCC13869 ΔodhA_speE_P453S and ATCC13869 ΔodhA_speE_A396T. In addition, the strain introduced with both mutations was named ATCC13869 ΔodhA_speE_P453S+A396T.

실시예 9-2-3: 스퍼미딘 신타아제 변이체 발현 균주의 L-글루탐산 생산능 비교Example 9-2-3: Comparison of L-glutamic acid production ability of spermidine synthase mutant expression strains

In order to confirm the L-glutamic acid production ability of the strain prepared in Example 9-2-1, the strain ATCC13869ΔodhA was used as a control and cultured in the following manner.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and cultured with shaking at 30° C. for 40 hours at 200 rpm. After completion of the culture, L-glutamic acid production capacity was measured using high performance liquid chromatography (HPLC), and the measurement results are shown in Table 10 below. The glutamic acid concentration and the concentration increase rate in the culture medium for each strain tested are shown in Table 10 below.

균주명strain name	L-글루탐산 농도(g/L)L-glutamic acid concentration (g/L)	L-글루탐산 농도 증가율(%)L-glutamic acid concentration increase rate (%)
ATCC13869 △odhAATCC13869 △odhA	1.91.9	--
ATCC13869 △odhA_speE_P453SATCC13869 △odhA_speE_P453S	2.42.4	26.326.3
ATCC13869 △odhA_speE_A396TATCC13869 △odhA_speE_A396T	2.552.55	34.234.2
ATCC13869 △odhA_speE_P453S+A396TATCC13869 △odhA_speE_P453S+A396T	2.652.65	39.539.5

As shown in Table 10 above, the ATCC13869 △odhA_speE_P453S, ATCC13869 △odhA_speE_A396T and ATCC13869 △odhA_speE_A396T and ATCC13869 △odhA_speE_P45 concentrations of speE(P453S) and/or speE(A396T) genes were significantly increased compared to the ATCC13869 △odhA strain. was confirmed.

The ATCC13869 △odhA_speE_P453S was named CA02-1609, and was deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12850P. In addition, the ATCC13869 △odhA_speE_A396T was named CA02-1610, and it was deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty on November 30, 2020, and was given an accession number KCCM12851P.

실시예 10: 글루타메이트 합성 효소 서브 유니트 알파 변이체를 발현하는 미생물의 L-글루탐산 생산능 평가Example 10: Evaluation of L-glutamic acid production ability of microorganisms expressing glutamate synthase subunit alpha variant

실시예 10-1: 미생물내 글루타메이트 합성 효소 서브 유니트 알파 변이체 발현을 위한 벡터 제작Example 10-1: Construction of vector for expression of glutamate synthase subunit alpha variant in microorganisms

To determine the effect of the mutant (S1192F; SEQ ID NO: 41) in which the serine at position 1192 of the amino acid sequence (SEQ ID NO: 43) of the glutamate synthase subunit alpha protein is substituted with phenylalanine (S1192F; SEQ ID NO: 41) on the L-glutamic acid production The vector was constructed as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using a pair of primers of SEQ ID NOs: 105 and 106 and a pair of primers of SEQ ID NOs: 107 and 108, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 105 and SEQ ID NO: 108 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with SmaI and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-gltB (S1192F).

실시예 10-2: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산주 제작 및 글루타메이트 합성 효소 서브 유니트 알파 변이체 도입 균주 제작Example 10-2: Preparation of wild-type Corynebacterium glutamicum-derived L-glutamic acid production strain and production of glutamate synthase subunit alpha mutant introduction strain

실시예 10-2-1: 야생형 코리네박테리움 글루타미쿰 유래 L-글루탐산 생산능을 갖는 코리네박테리움 글루타미쿰 균주 제작Example 10-2-1: Preparation of Corynebacterium glutamicum strain having L-glutamic acid-producing ability derived from wild-type Corynebacterium glutamicum

Specifically, for odhA deletion, the upstream region and downstream region of the odhA gene using the primer sets of SEQ ID NO: 111 and SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114 using Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template was obtained through PCR. Solg ^TM Pfu-X DNA polymerase was used as the polymerase, and PCR amplification conditions were after denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and polymerization at 72°C for 60 seconds after repeating 30 times. , the polymerization was carried out at 72 °C for 5 minutes.

A recombinant plasmid was obtained by cloning the amplified odhA upstream and downstream regions, and the vector pDCM2 for chromosome transformation cut with SmaI restriction enzyme using the Gibson assembly method, and was named pDCM2-ΔodhA. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment with the calculated number of moles, and then storing at 50° C. for 1 hour.

The primer sequences used herein are shown in Table 1 above.

실시예 10-2-2: 글루타메이트 합성 효소 서브 유니트 알파 변이체 발현 균주 제작Example 10-2-2: Glutamate synthase subunit alpha mutant expression strain production

The vector prepared in Example 10-1 was transformed into ATCC13869ΔodhA prepared in Example 10-2-1.

In the strain in which the homologous recombination occurred, the strain into which the mutant was introduced was selected using SEQ ID NOs: 109 and 110. Each selected strain was named ATCC13869 ΔodhA_gltB_S1192F.

실시예 10-2-3: 글루타메이트 합성 효소 서브 유니트 알파 변이체 발현 균주의 L-글루탐산 생산능 비교Example 10-2-3: Comparison of L-glutamic acid production capacity of glutamate synthase subunit alpha variant expression strain

In order to check the L-glutamic acid production ability of the strain prepared in Example 10-2-1, the strain ATCC13869ΔodhA was used as a control and cultured in the following manner.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and incubated at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and cultured with shaking at 30° C. for 40 hours at 200 rpm. After completion of the culture, L-glutamic acid production capacity was measured using high performance liquid chromatography (HPLC), and the measurement results are shown in Table 11 below. The MSG concentration and the concentration increase rate in the culture medium for each strain tested are shown in Table 11 below.

균주명strain name	L-글루탐산 농도(g/L)L-glutamic acid concentration (g/L)	L-글루탐산 농도 증가율(%)L-glutamic acid concentration increase rate (%)
ATCC13869 △odhAATCC13869 △odhA	2.102.10	--
ATCC13869 △odhA_gltB_S1192FATCC13869 △odhA_gltB_S1192F	2.782.78	32.432.4

As shown in Table 11, it was confirmed that the concentration of L-glutamic acid increased by 32.4% in ATCC13869ΔodhA_gltB_S1192F into which the gltB (S1192F) gene was introduced compared to the ATCC13869ΔodhA strain.

From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims described below, rather than the above detailed description, and equivalent concepts thereof, to be included in the scope of the present application.

Claims

(i) any one or more variants selected from the group consisting of the following (a) to (l), (ii) one or more polynucleotides encoding the variant, or (iii) a combination thereof, Corynebacter Lium glutamicum strain:

(a) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which glycine, an amino acid corresponding to position 32 of SEQ ID NO: 3, is substituted with aspartic acid;

(b) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which alanine, an amino acid corresponding to position 282 of SEQ ID NO: 7, is substituted with threonine;

(c) a D-alanine-D-alanine ligase A variant comprising the amino acid sequence set forth in SEQ ID NO: 9 in which glycine, an amino acid corresponding to position 256 of SEQ ID NO: 11, is substituted with serine;

(d) a glucosamine-6-phosphate deaminase variant consisting of the amino acid sequence shown in SEQ ID NO: 13, in which alanine, an amino acid corresponding to position 137 of SEQ ID NO: 15, is substituted with valine;

(e) an exinuclease ABC subunit A variant comprising the amino acid sequence set forth in SEQ ID NO: 17 in which glycine, an amino acid corresponding to position 575 of SEQ ID NO: 19, is substituted with aspartic acid;

(f) a ribonuclease P variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which histidine, an amino acid corresponding to position 32 of SEQ ID NO: 23, is substituted with tyrosine;

(g) an MFS transporter variant consisting of the amino acid sequence set forth in SEQ ID NO: 25, in which alanine, which is an amino acid corresponding to position 382 of SEQ ID NO: 27, is substituted with threonine;

(h) a galactoside O-acetyltransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 29, wherein leucine, an amino acid corresponding to position 106 of SEQ ID NO: 31, is substituted with phenylalanine;

(i) spermidine synthase consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which proline, an amino acid corresponding to position 453 of SEQ ID NO: 35, is substituted with serine, and alanine, an amino acid corresponding to position 396, is substituted with threonine variant;

(j) a spermidine synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 37, wherein proline, which is the amino acid corresponding to position 453 of SEQ ID NO: 35, is substituted with serine;

(k) a spermidine synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 39 in which alanine, an amino acid corresponding to position 396 of SEQ ID NO: 35, is substituted with threonine; and

(l) a glutamate synthase subunit alpha variant comprising the amino acid sequence shown in SEQ ID NO: 41 in which serine, an amino acid corresponding to position 1192 of SEQ ID NO: 43, is substituted with phenylalanine.
The method of claim 1, wherein the strain comprises (i) two or more variants selected from the group consisting of (a) to (l), (ii) two or more polynucleotides encoding the variant, or (iii) their strains, including combinations.
A method for producing L-glutamic acid, comprising the step of culturing the Corynebacterium glutamicum strain of claim 1 or 2 in a medium.