Modified LysE Polypeptide, microorganisms containing this polypeptide, and methods for producing L-amino acids using these microorganisms.

Abstract

The invention proposes a LysE variant polypeptide, a polynucleotide encoding this polypeptide, a recombinant vector containing this polynucleotide, and a microorganism containing the polynucleotide, and a method for producing L-amino acids using them. The LysE variant is capable of improving the secretion and / or production of L-amino acids compared to the wild-type.

Description

Microorganism containing mutant LysE, and method for producing L-amino acid using the same

[0001] Cross-citation with related application(s)

[0002] This disclosure claims the benefit of priority to Republic of Korea Patent Application No. 10-2024-0063271, filed May 14, 2024, the entire contents of which are incorporated herein by reference.

[0003] Numerous papers and patents are referenced and cited throughout this disclosure. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0004] The present application relates to a microorganism comprising a mutant LysE, and a method for producing L-amino acids using the same. The mutant LysE may have improved L-amino acid excretion and / or production capacity compared to the wild type.

[0005]

[0006] Microorganisms of the genus Corynebacterium are Gram-positive microorganisms widely used for the production of L-amino acids. L-amino acids, particularly L-lysine, are used in the animal feed, human pharmaceutical, and cosmetic industries, and are primarily produced through fermentation using Corynebacterium strains.

[0007] Many attempts have been made to improve the production of L-amino acids using strains of the genus Corynebacterium. Among these, research has been conducted to improve Corynebacterium strains that produce L-amino acids by disrupting or attenuating specific genes using recombinant DNA technology. Furthermore, research has been conducted to improve L-amino acid-producing Corynebacterium strains by amplifying individual genes involved in L-amino acid biosynthesis and analyzing their effects on L-amino acid production. Furthermore, attempts have been made to introduce exogenous genes from other bacteria.

[0008] Despite these efforts, the development of technologies to improve the production of useful substances such as L-amino acids is still required.

[0009]

[0010] An example provides a mutant lysine excretor.

[0011] Another example provides a polynucleotide encoding the above polypeptide.

[0012] Another example provides a recombinant vector comprising the above polynucleotide.

[0013] Another example provides a recombinant microorganism comprising the polypeptide, a polynucleotide encoding the polypeptide, or a recombinant vector comprising the polynucleotide.

[0014] Another example provides a method for producing L-amino acids, comprising a step of culturing the recombinant microorganism or L-amino acid producing microorganism.

[0015] Another example provides the use of the recombinant microorganism for the production of L-amino acids.

[0016]

[0017] In this specification, for the purpose of increasing the L-amino acid productivity of microorganisms of the genus Corynebacterium, a novel extruder of L-amino acids is searched for, the influence of genetic mutations of the novel extruder is confirmed, and a technology related to strain improvement using the same is provided. Taking L-lysine as a representative example, in general, to increase lysine productivity, there is a method of improving the L-lysine yield of the strain or increasing the L-lysine production per hour (productivity). The L-lysine extruder is a membrane protein that extrudes lysine produced through biosynthesis, and improvement of this protein is important for increasing lysine yield and productivity. However, increasing the expression level of the L-lysine extruder (LysE, Ncgl1214) possessed by microorganisms of the genus Corynebacterium has limitations in improving L-lysine productivity.

[0018] Accordingly, in this specification, a new foreign L-amino acid excretor having high L-amino acid excretion activity is searched for, and the foreign L-amino acid excretor is improved and introduced into a lysine producing strain, thereby providing a recombinant strain having improved L-amino acid excretion ability and / or production ability.

[0019] In this specification, a novel L-amino acid excretor derived from Shewanella atlantica and a gene encoding the same, as a representative example of a novel foreign L-amino acid excretor, were discovered and improved through mutation. When a mutant form of the novel L-amino acid excretor derived from Shewanella atlantica was expressed in a microorganism producing L-amino acids, it was confirmed that the L-amino acid excretion / production ability was significantly improved compared to a microorganism in which the gene was not expressed.

[0020] An example provides a polypeptide in which an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1, an amino acid corresponding to the 46th amino acid position from the N-terminus, or a combination thereof is replaced with an amino acid different from the original. The polypeptide may have an L-amino acid exporter function. The L-amino acid may be L-lysine, L-arginine, or a combination thereof, and specifically, may be L-lysine.

[0021] Another example provides a polynucleotide encoding the above polypeptide.

[0022] Another example provides a recombinant vector comprising the above polynucleotide.

[0023] Another example provides a recombinant microorganism comprising the polypeptide, a polynucleotide encoding the polypeptide, or a recombinant vector comprising the polynucleotide. The recombinant microorganism may have an L-amino acid excretion and / or production ability, or may have an increased L-amino acid excretion and / or production ability compared to an unmodified microorganism.

[0024] Another example provides a method for producing L-amino acids, comprising a step of culturing the recombinant microorganism or L-amino acid producing microorganism.

[0025] Below, it is explained in more detail.

[0026] An example of the present application provides a polypeptide in which an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1, an amino acid corresponding to the 46th amino acid position from the N-terminus, or a combination thereof is replaced with an amino acid different from the original.

[0027] In one embodiment, the polypeptide may have the function of an L-amino acid excretor. The L-amino acid may be L-lysine, L-arginine, or a combination thereof, and specifically may be L-lysine.

[0028] In the present specification, a lysine exporter protein (LysE) is a transmembrane protein that functions to export a product synthesized within a cell, such as an L-amino acid such as L-lysine, to the outside of the cell. In one example, the lysine exporter may be a foreign lysine exporter derived from a microorganism belonging to a genus different from the parent strain (microorganism before mutation) or a microorganism of a different species, for example, a protein derived from a microorganism other than a microorganism belonging to the genus Corynebacterium, and having the ability to export an L-amino acid, such as L-lysine. In one example, the foreign lysine exporter may be a membrane protein derived from a microorganism of the genus Shewanella, for example, Shewanella atlantica. In one specific example, the lysine exporter may be a protein represented by the amino acid sequence of SEQ ID NO: 1, and may be a protein having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity or homology with the protein. In the present specification, the polypeptide of the amino acid sequence of SEQ ID NO: 1 may be derived from Shewanella atlantica.

[0029] In one embodiment, the amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 may be, but is not limited to, valine (Val, V).

[0030] In one embodiment, the amino acid corresponding to the 46th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 may be, but is not limited to, aspartic acid (Asp, D).

[0031] The polypeptide of the present application may be a polypeptide in which an amino acid substitution mutation is induced in the lysine exporter described above (a mutant lysine exporter). In one example, the polypeptide may be an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., valine (Val, V)), an amino acid corresponding to the 46th amino acid position from the N-terminus (e.g., aspartic acid (Asp, D)), or a combination thereof, in which an amino acid different from the original is substituted (e.g., an amino acid sequence in which an amino acid valine (Val, V) at the 8th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1, an amino acid aspartic acid (Asp, D) at the 46th amino acid from the N-terminus, or a combination thereof is substituted with another amino acid). More specifically, the polypeptide may be one in which the amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., valine (Val, V)) is replaced with an amino acid different from the original (e.g., glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), or asparagine (N); specifically, alanine (A)), and the amino acid corresponding to the 46th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., aspartic acid (Asp, D)) is replaced with an amino acid different from the original (e.g., glutamic acid (E), Glycine (G), Alanine (A), Serine (S), Threonine (T), Cysteine ​​(C), Valine (V), Leucine (L), Isoleucine (I), Methionine (M), Proline (P), Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Glutamine (Q), Histidine (H), Lysine (K), Arginine (R), or Asparagine (N);Specifically, it may be substituted with glycine (G)), or the amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., valine (Val, V)) is substituted with an amino acid different from the original (e.g., glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), or asparagine (N); specifically, alanine (A)), and the 46th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., aspartic acid (Asp, D)) is substituted with an amino acid different from the original (e.g., glutamic acid (E), It may be substituted with glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), glutamine (Q), histidine (H), lysine (K), arginine (R), or asparagine (N); specifically, glycine (G). In one specific example, the polypeptide may include a polypeptide represented by the amino acid sequence of SEQ ID NO: 3 (wherein the 8th amino acid valine (V) is replaced with alanine (A)), a polypeptide represented by the amino acid sequence of SEQ ID NO: 4 (wherein the 46th amino acid aspartic acid (D) is replaced with glycine (G)), or a polypeptide represented by the amino acid sequence of SEQ ID NO: 5 (wherein the 8th amino acid valine (V) is replaced with alanine (A) and the 46th amino acid aspartic acid (D) is replaced with glycine (G)), but is not limited thereto. A polypeptide in which a mutation is induced in this manner may have an L-amino acid (e.g., L-lysine) exporter function.;

[0032] In one embodiment, the polypeptide of the present application may have an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., valine (Val, V)), an amino acid corresponding to the 46th amino acid position from the N-terminus (e.g., aspartic acid (Asp, D)), or a combination thereof, substituted with a different amino acid, and may have a homology or identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%; and less than 100% with the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0033] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that references a particular sequence. As used herein, the term "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0034] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position at which a modification such as a substitution, insertion, or deletion occurs, by comparing it to a query sequence (also referred to as a “reference sequence”).

[0035] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) can be used, but is not limited thereto, and any sequence alignment program known in the art, pairwise sequence comparison algorithm, etc. can be appropriately used.

[0036] Another example provides a polynucleotide encoding a polypeptide of the present invention as described above.

[0037] In this application, "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, a DNA or RNA strand of a certain length or longer, and more specifically, a polynucleotide fragment encoding the variant.

[0038] In one example, a polynucleotide encoding a polypeptide of the present application may be represented by one nucleic acid sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 8.

[0039]

[0040] In this specification, the phrase "a polynucleotide (which may be used interchangeably with a "gene") or a polypeptide (which may be used interchangeably with a "protein") "contains a specific nucleic acid sequence or amino acid sequence or is expressed by a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide consists of or essentially includes the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including (or not excluding) a "substantially equivalent sequence" in which a mutation (deletion, substitution, modification, and / or addition) is added to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or the desired function of the polynucleotide or polypeptide is maintained.

[0041] In one example, the nucleic acid sequence or amino acid sequence provided herein may include a modification by conventional mutagenesis, such as directed evolution and / or site-directed mutagenesis, to the extent that it maintains its original function or desired function. In one example, a polynucleotide or polypeptide “comprises a particular nucleic acid sequence or amino acid sequence” can mean that the polynucleotide or polypeptide (i) consists of or essentially comprises the particular nucleic acid sequence or amino acid sequence, or (ii) consists of or essentially comprises an amino acid sequence that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology with the particular nucleic acid sequence or amino acid sequence and retains its original function and / or desired function. In the present specification, the original function may be an L-amino acid (e.g., L-lysine) exporter function (in the case of an amino acid sequence), or a function (in the case of a nucleic acid sequence) of encoding a protein having an L-amino acid (e.g., L-lysine) exporter function, and the desired function may mean a function of increasing or imparting an L-amino acid (e.g., L-lysine) export ability and / or production ability of a microorganism.

[0042] The nucleic acid sequence described herein can be modified in various ways in the coding region within a range that does not change the amino acid sequence and / or function of the protein expressed from the coding region, taking into account the codon preferred in the microorganism that is to express the protein (lysine exporter) due to the degeneracy of the codon.

[0043] In this application, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0044] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences are generally capable of hybridizing under moderate or high stringency conditions, typically along at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or its full length. It should be appreciated that hybridization also encompasses polynucleotides containing common codons or codons considered codon degeneracy in polynucleotides.

[0045] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented 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) can be determined using the 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, homology, similarity, or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information Database.

[0046] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program, such as that disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or, for example, Needleman et al. (1970), J Mol Biol. 48:443. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0047] Additionally, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and the appropriate hybridization conditions defined are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 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).

[0048]

[0049] Another example provides a recombinant vector comprising the polynucleotide of the present invention described above. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0050] As used herein, the term "vector" refers to a DNA construct containing a base sequence of a polynucleotide encoding a target protein operably linked to suitable regulatory sequences so as to enable expression of the target protein in a suitable host. The regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence regulating the termination of transcription and / or translation. After being transformed into a suitable host microorganism, the vector may be expressed independently of the genome (genome) of the host microorganism, or may be integrated into the genome of the host microorganism.

[0051] The vector usable in this specification is not particularly limited as long as it is replicable in a host cell, and may be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, cosmids, viruses, bacteriophages, etc. in a natural or recombinant state. For example, as the vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. can be used as a phage vector or a cosmid vector, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series, etc. can be used as a plasmid vector. Specifically, examples include, but are not limited to, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors.

[0052] The vector usable herein may be a known expression vector and / or a vector for insertion of a polynucleotide into a host cell chromosome. The insertion of the polynucleotide into the host cell chromosome may be accomplished by any method known in the art, for example, but is not limited to, homologous recombination. The vector may further include a selection marker for confirming whether the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the polynucleotide has been inserted, and may be selected from genes that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface protein. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, thereby allowing the selection of transformed cells.

[0053] The introduction of the polynucleotide or vector can be performed by a person skilled in the art by appropriately selecting a known transformation method. As used herein, the term "transformation" refers to introducing a vector containing a polynucleotide encoding a target protein (lysine exporter) into a host microorganism so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can include both an integrated location within the chromosome of the host microorganism or an extrachromosomal location, as long as it can be expressed within the host microorganism. In addition, the polynucleotide includes DNA and / or RNA encoding the target protein. As long as the polynucleotide can be introduced into the host microorganism and expressed, there is no limitation on the form in which it is introduced. For example, the polynucleotide can be introduced into the host microorganism in the form of an expression cassette, which is a genetic construct containing all the elements necessary for its own expression. The above expression cassette may typically include expression control elements such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal that are operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell. The term "operably linked" as described above may mean that an expression control element (e.g., a promoter) and a polynucleotide are functionally linked so that the expression control element can perform transcriptional control (e.g., transcription initiation) of a polynucleotide encoding a target protein (lysine exporter).Operable linking can be accomplished using genetic recombination techniques known in the art, including, but not limited to, conventional site-specific DNA cleavage and ligation.

[0054] The method for transforming the above polynucleotide into a host microorganism can be performed by any method for introducing a nucleic acid into a cell (microorganism), and can be performed by appropriately selecting a transformation technique known in the art depending on the host microorganism. Examples of the known transformation methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation, DEAE-dextran, cationic liposome, lipofection, and lithium acetate-DMSO.

[0055] The insertion of the polynucleotide into the host cell genome (chromosome) or the introduction of a mutation so that a host cell endogenous gene (e.g., LysE gene) encodes the polypeptide of the present application can be performed by a person skilled in the art by appropriately selecting a known method, and can be performed using, for example, an RNA-guided endonuclease system (e.g., one or more selected from the group consisting of (a) an RNA-guided endonuclease (e.g., Cas9 protein, etc.), an encoding gene thereof, or a vector containing the gene; and (b) a guide RNA (e.g., single guide RNA (sgRNA), etc.), an encoding DNA thereof, or a vector containing the DNA (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA, etc.), a complex (e.g., a ribonucleic acid fusion protein (RNP), a recombinant vector (e.g., a vector containing together an RNA-guided endonuclease encoding gene and a guide RNA encoding DNA, etc.), etc.), but is not limited thereto.

[0056]

[0057] Another example provides an L-amino acid producing microorganism comprising the polypeptide of the present invention described above, a polynucleotide encoding the same, or a recombinant vector comprising the polynucleotide.

[0058] In this specification, the term "L-amino acid producing microorganism" may be used to mean a case where a lysine excretor mutation as described above is introduced into a microorganism having L-amino acid excretion and / or production ability, thereby causing the microorganism to have increased L-amino acid excretion and / or production ability, and / or a case where a lysine excretor mutation as described above is introduced into a microorganism having no L-amino acid excretion and / or production ability, thereby causing the microorganism to have L-amino acid excretion and / or production ability. As used herein, "microorganism" encompasses unicellular bacteria and may be used interchangeably with "cell".

[0059] The above L-amino acid may be L-lysine, L-arginine, or a combination thereof, and may be specifically, but is not limited to, L-lysine.

[0060] In one example, the microorganism may be selected from all microorganisms having the ability to excrete and / or produce L-amino acids (e.g., L-lysine). In one example, the host microorganism may be a microorganism that naturally has the ability to excrete and / or produce L-amino acids, or a microorganism that has the ability to excrete and / or produce L-amino acids by introducing a mutation into a parent strain that has no or significantly low ability to excrete and / or produce L-amino acids.

[0061] In one specific example, the microorganism may be at least one selected from among all microorganisms of the genus Corynebacterium and microorganisms of the genus Escherichia, which naturally have L-amino acid excretion and / or production ability, or microorganisms that have L-amino acid excretion and / or production ability by introducing a mutation into a parent strain that has no or significantly low L-amino acid excretion and / or production ability. The above Corynebacterium genus microorganism may include, but is not necessarily limited to, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes, Corynebacterium efficiens, etc. More specifically, the Corynebacterium genus microorganism may be Corynebacterium glutamicum. The Escherichia genus strain may be Escherichia coli.

[0062] In one example, an L-amino acid producing microorganism into which a lysine exporter mutation has been introduced may have increased L-amino acid excretion and / or production ability compared to an identical, unmodified microorganism. The term “unmodified microorganism” does not exclude a microorganism containing a mutation that may occur naturally, and may refer to a natural (wild-type) strain itself, a microorganism of the same species into which a lysine exporter mutation has not been introduced, or a microorganism before introduction, and may refer to a microorganism that does not include a polypeptide, and / or a polynucleotide encoding the polypeptide, in which an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 of the present application described above, an amino acid corresponding to the 46th amino acid position from the N-terminus, or a combination thereof is substituted with an amino acid different from the original. The term “unmodified” may be used interchangeably with “before modification,” “unmutated,” and “before mutation.”

[0063] In one example, the microorganism of the present application having increased L-amino acid excretion ability and / or productivity (production amount) may have an L-amino acid excretion ability and / or productivity (production amount) increased by about 1% or more, about 3.5% or more, about 5% or more, about 8% or more, about 9% or more, about 10% or more, about 12% or more, about 15% or more, or about 19% or more (the upper limit is not particularly limited and may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, or about 20% or less) compared to a pre-mutation microorganism or an unmodified microorganism (e.g., a microorganism without lysE introduced or a microorganism with wild-type lysE introduced), but is not limited thereto. In another example, the microorganism of the present application having increased L-amino acid excretion ability and / or productivity (production amount) may have an L-amino acid excretion ability and / or productivity (production amount) increased by about 1.01 times or more, about 1.035 times or more, about 1.05 times or more, about 1.08 times or more, about 1.09 times or more, about 1.10 times or more, about 1.12 times or more, about 1.15 times or more, or about 1.19 times or more (the upper limit is not particularly limited and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, or about 1.5 times or less), compared to a pre-mutation microorganism or an unmodified microorganism (e.g., a microorganism without lysE introduced or a microorganism with wild-type lysE introduced), but is not limited thereto.

[0064] The term “about” above includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all ranges of values ​​equal to or similar to the value following the term “about,” but is not limited thereto.

[0065] In one example, an L-amino acid producing microorganism into which the lysine efflux mutation has been introduced may have an increased strain survival rate due to the introduction of the lysine efflux mutation. More specifically, an L-amino acid producing microorganism into which the lysine efflux mutation has been introduced may have an increased strain survival rate due to the introduction of the lysine efflux mutation compared to a microorganism before the mutation or an unmodified microorganism (e.g., a microorganism into which lysE has not been introduced or a microorganism into which wild-type lysE has been introduced).

[0066] In one example, the L-amino acid producing microorganism into which the lysine efflux mutation has been introduced may have an increased L-amino acid excretion ability and / or production ability due to the introduction of the lysine efflux mutation. More specifically, the L-amino acid producing microorganism into which the lysine efflux mutation has been introduced may have an increased L-amino acid excretion ability and / or production ability due to the introduction of the lysine efflux mutation compared to a microorganism before the mutation or an unmodified microorganism (e.g., a microorganism into which lysE has not been introduced or a microorganism into which wild-type lysE has been introduced).

[0067] The above “strain survival rate” may refer to the extent to which the strain survives under specific conditions, and for the purpose of the present application, the strain survival rate may be used interchangeably with terms such as “strain growth rate,” “strain growth rate,” “strain activity,” “cell survival rate,” “cell growth rate,” “cell growth rate,” and “cell activity.” In one example, the strain survival rate can be determined by measuring the OD (optical density) value of the strain (bacterial body) culture at a specific point in time during cultivation.

[0068] In the present specification, in order to distinguish a microorganism before the introduction of a lysine efflux mutation from an "L-amino acid producing microorganism" in which the L-amino acid excretion ability and / or production ability is increased or the L-amino acid excretion ability and / or production ability is granted by the introduction of the lysine efflux mutation, the microorganism before the introduction of the lysine efflux mutation may be expressed as a host microorganism.

[0069] In this specification, “introduction of a lysine exporter mutant” may mean any operation in which the polypeptide of the present application (a mutant lysine exporter) described above is introduced into a host microorganism.

[0070] The above "L-amino acid producing microorganism having a lysine exporter mutation introduced" may be a microorganism having increased L-amino acid export and / or production ability or endowed with L-amino acid export and / or production ability, including (1) a polypeptide in which an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 as described above (e.g., valine (Val, V)), an amino acid corresponding to the 46th amino acid position from the N-terminus (e.g., aspartic acid (Asp, D)) or a combination thereof is replaced with an amino acid different from the original, (2) a polynucleotide encoding the same, and / or (3) a recombinant vector including the polynucleotide.

[0071] The above "L-amino acid producing microorganism having a lysine exporter mutation introduced" may be a microorganism having increased strain survival rate, including (1) a polypeptide in which an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 described above (e.g., valine (Val, V)), an amino acid corresponding to the 46th amino acid position from the N-terminus (e.g., aspartic acid (Asp, D)) or a combination thereof is replaced with an amino acid different from the original, (2) a polynucleotide encoding the same, and / or (3) a recombinant vector including the polynucleotide.

[0072] A microorganism including a polynucleotide encoding a polypeptide, or a recombinant vector including the same, in which the amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., valine (Val, V)), the amino acid corresponding to the 46th amino acid position from the N-terminus (e.g., aspartic acid (Asp, D)), or a combination thereof is replaced with an amino acid different from the original, may mean that the microorganism further includes the polynucleotide or the recombinant vector in addition to the genome.

[0073] In one specific example, the L-amino acid producing microorganism may be a microorganism including a polynucleotide encoding an amino acid sequence in which an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., valine (V)) is substituted with a different amino acid (e.g., an amino acid selected from the group consisting of glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), aspartic acid (D), glutamine (Q), histidine (H), lysine (K), arginine (R), and asparagine (N); specifically, alanine (A)) and / or a recombinant vector including the same. More specifically, the L-amino acid producing microorganism may be a polynucleotide encoding an amino acid sequence in which an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., valine (V)) is substituted with an amino acid different from the original, alanine (A), and / or a recombinant vector including the same, and more specifically, may be a microorganism of the genus Corynebacterium, such as Corynebacterium glutamicum, including a polynucleotide encoding an amino acid sequence of SEQ ID NO: 3 (more specifically, a polynucleotide represented by a nucleic acid sequence of SEQ ID NO: 6) and / or a recombinant vector including the same.

[0074] In one specific example, the L-amino acid producing microorganism may be a microorganism including a polynucleotide encoding an amino acid sequence in which an amino acid corresponding to the 46th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., aspartic acid (D)) is substituted with a different amino acid (an amino acid selected from the group consisting of glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), glutamine (Q), histidine (H), lysine (K), arginine (R), and asparagine (N); specifically, glycine (G)) and / or a recombinant vector including the same. More specifically, the L-amino acid producing microorganism may include a polynucleotide encoding an amino acid sequence in which an amino acid corresponding to the 46th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., aspartic acid (D)) is substituted with a different amino acid, glycine (G), and / or a recombinant vector including the same, and more specifically, may be a microorganism of the genus Corynebacterium, such as Corynebacterium glutamicum, including a polynucleotide encoding an amino acid sequence of SEQ ID NO: 4 (more specifically, a polynucleotide represented by a nucleic acid sequence of SEQ ID NO: 7) and / or a recombinant vector including the same.

[0075] In one specific example, the L-amino acid producing microorganism is a microorganism in which the amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., valine (Val, V)) is replaced with an amino acid different from the original (e.g., an amino acid selected from the group consisting of glutamic acid (E), glycine (G), alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), glutamine (Q), histidine (H), lysine (K), arginine (R), and asparagine (N); specifically, alanine (A)), and the amino acid corresponding to the 46th amino acid position from the N-terminus (e.g., aspartic acid (D)) is replaced with an amino acid different from the original (e.g., glutamic acid (E), glycine (G), It may be a microorganism comprising a polynucleotide encoding an amino acid sequence substituted with an amino acid selected from the group consisting of alanine (A), serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F), tyrosine (Y), tryptophan (W), glutamine (Q), histidine (H), lysine (K), arginine (R), and asparagine (N); specifically, glycine (G)) and / or a recombinant vector comprising the same.More specifically, the L-amino acid producing microorganism may include a polynucleotide encoding an amino acid sequence in which an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 (e.g., valine (V)) is substituted with an amino acid different from the original (e.g., aspartic acid (D)) and an amino acid corresponding to the 46th amino acid position from the N-terminus (e.g., aspartic acid (D)) is substituted with an amino acid different from the original (e.g., glycine (G)) and / or a recombinant vector including the same, and more specifically, may be a microorganism of the genus Corynebacterium, such as Corynebacterium glutamicum, including a polynucleotide encoding an amino acid sequence of SEQ ID NO: 5 (more specifically, a polynucleotide represented by a nucleic acid sequence of SEQ ID NO: 8) and / or a recombinant vector including the same.

[0076] Another example provides a method for increasing the L-amino acid excretion and / or production ability of a microorganism or a method for imparting the L-amino acid excretion and / or production ability to a microorganism, comprising the step of introducing into a microorganism (transformation) a polypeptide in which an amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of SEQ ID NO: 1, an amino acid corresponding to the 46th amino acid position from the N-terminus, or a combination thereof is replaced with an amino acid different from the original; a polynucleotide encoding the same; or a recombinant vector comprising the polynucleotide.

[0077] The above modified lysine excretor, polynucleotide, recombinant vector, introduction (transformation) and microorganism are as described above.

[0078] Another example provides a method for producing L-amino acids, comprising a step of culturing the above-described L-amino acid-producing microorganism in a medium. The method may further comprise, after the culturing step, a step of recovering L-amino acids from the cultured microorganism, the medium, or both.

[0079] In one example, the L-amino acid may be L-lysine, L-arginine, or a combination thereof, and specifically may be L-lysine.

[0080] In this application, the term "cultivation" refers to growing the L-amino acid-producing microorganism of this application (e.g., a microorganism of the genus Corynebacterium) under appropriately controlled environmental conditions. The culturing process of this application can be performed using appropriate media and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0081] In this application, the term "medium" means a material mixed as a main component with nutrients required for culturing the L-amino acid-producing microorganism of the present application (e.g., microorganism of the genus Corynebacterium), and supplies nutrients and growth factors, as well as water essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of the present application may be any medium used for culturing general microorganisms without particular limitation, but the L-amino acid-producing microorganism of the present application (e.g., microorganism of the genus Corynebacterium) may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid, and / or vitamin, etc.

[0082] In the above method, the step of culturing the microorganism is not particularly limited thereto, but may be performed by a known batch culture method, continuous culture method, fed-batch culture method, etc. At this time, the culture conditions are not particularly limited thereto, but may be adjusted to an appropriate pH (e.g., pH 5 to 9, specifically pH 6 to 8, most specifically pH 7) using a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid), and an aerobic condition may be maintained by introducing oxygen or an oxygen-containing gas mixture into the culture. The culture temperature may be maintained at 20 to 45°C, or 25 to 40°C, and the culture may be performed for about 10 to 160 hours, but is not limited thereto. The L-amino acid (e.g., L-lysine) produced by the culture may be secreted into the medium or may remain within the cells.

[0083] The medium usable for the above culture may be selected from among media commonly used for microbial culture. In one example, the medium may be selected from the group consisting of sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oils and fats (e.g., soybean oil, sunflower seed oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), organic acids (e.g., acetic acid), etc., as a carbon source, and may be used individually or in combination of two or more, but is not limited thereto. As a nitrogen source, one or more selected from the group consisting of nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea), inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate) may be used individually or in combination of two or more, but is not limited thereto. As a phosphorus source, one or more selected from the group consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium-containing salts, but is not limited thereto, may be used individually or in combination of two or more. In addition, the medium may contain essential growth-promoting substances such as other metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, and / or vitamins.

[0084] The step of recovering the L-amino acid (e.g., L-lysine, L-arginine, or a combination thereof) may be to collect the desired amino acid from a medium, culture medium, or microorganism using a suitable method known in the art, depending on the culturing method. For example, the step of recovering may be performed by one or more methods selected from centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. The method may additionally include a purification step before, simultaneously with, or after the step of recovering the L-amino acid (e.g., L-lysine, L-arginine, or a combination thereof).

[0085] Another example of the present application provides a composition for producing L-amino acids comprising the L-amino acid producing microorganism, a medium in which the microorganism is cultured, or a combination thereof.

[0086] The composition may further comprise any suitable excipient commonly used in compositions for producing L-amino acids, including but not limited to preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents.

[0087] Another example provides a use for imparting and / or increasing L-amino acid production, L-amino acid excretion ability and / or production ability of the microorganism.

[0088] Another example provides the use of the microorganism for the preparation of a composition for producing L-amino acids.

[0089] In the above L-amino acid production, L-amino acid excretion ability and / or production ability imparting and / or increasing use and / or use for the preparation of a composition for L-amino acid production, the modified lysine excretor, polynucleotide, recombinant vector, introduction (transformation) and microorganism, etc. are as described above.

[0090]

[0091] Other examples of the present application provide compositions, methods, products, processes, or uses featuring one or more elements disclosed herein.

[0092]

[0093] The present specification provides a technique for increasing the ability of a microorganism to excrete and / or produce L-amino acids, and for this purpose, a technique is provided for improving the productivity of L-amino acids compared to a parent strain by inducing a mutation in a foreign protein for which an ability to excrete L-amino acids has been newly discovered, and introducing the mutant of the foreign protein into a microorganism.

[0094]

[0095]

[0096] Hereinafter, the present application will be described in more detail through examples. These examples are intended solely to illustrate the present application more specifically, and it will be apparent to those skilled in the art that the scope of the present application is not limited by these examples, in accordance with the gist of the present application.

[0097]

[0098] Example

[0099]

[0100] (Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise stated.)

[0101]

[0102] Example 1: Construction of a library of mutant foreign lysE gene vectors and an introduced strain.

[0103] To search for enzymes with improved excretion ability of L-lysine excretor, a library was constructed using the following method to obtain a foreign lysE mutant gene.

[0104] The amino acid sequence (SEQ ID NO: 1) of lysE (membrane protein) derived from Shewanella atlantica (S.at), which was selected as a foreign lysE, is shown in Table 1 below. Information on the gene encoding the membrane protein (Table 1 below, SEQ ID NO: 2) and the surrounding nucleic acid sequence (accession number NZ_RXNV01000002.1) was obtained from the National Institutes of Health (NIH GenBank) in the United States.

[0105] Sequence name Sequence (5'→3') Sequence number S. at lysE protein MQAFIQGVGIGGSLIMAVGAQNAFVLKQGLKRSHSLPIAGLCSLIDALMITAGVAGLGHLILAFPLIKDIASIGGAIFLLVYGAKALRSSFNEQTISQGATKGTETLKAAAVLTTLGISLLNPHLYLDTVVLLGSISAQFEGVDRPLFGAGAVLASFVWFFSLSFGARYLSPLFQNPKAWSYLDRFICMTMWTIAAVLLWPYL1 S. at lysE gene ATGCAGGCATTTATTCAGGGTGTTAGGAATTGGCGGAAGCCTTATTATGGCGGTGGGCGCACAAAATGCGTTGTATTAAAGCAGGGGCTGAAGCGTTCGACTCTTTACCCATTGCCGGACTGTGTTCATTGATCGATGCCTTTAATGATAACGGCTGGTGTGCAGGCCTGGGTCATTTGATTTTGGCCTTTCATTGATTAAGGATATTGCCAGTATTGGGGGCGATCTTCTTGCTGTTTATGGTGTGCAAAGGCTTTAAGGTCTTCGTTTAATGGAGCAAACCATATCTCAAGGTGGCGACG AAAGGGACTGAAACCCTTAAAGCTGCGGTGTTGACAACTTTGGGTATTAGTTTGCTCAACCCGCATCTGTATCTGGACACGGTTGTCTTGCTTGGTAGCATCAGTGCTCAGTTTGAAGGTGTTGACCGTCCTCTGTTTGGCGAGGTGCCGTACTGGCATCGTTTGTGTGGTTTTTTAGTTTAAGTTTTGGTGCGAGATACTTAAGTCCATATTTCAAAACCCTAAAGCCTGAGCTATTTAGACAGGTTTATCTGCATGACTATGTGGACATTGCGGCGGTCTTGTTATGGCCTTACTTGTAA2

[0106] Using the GenemorphII Random Mutagenesis Kit (Stratagene), 0-4.5 mutations per kb were introduced into a DNA fragment containing the lysE gene (609 bp; SEQ ID NO: 2) of Shewanella atlantica. Error-prone PCR was performed using the chromosome of the wild type (WT) of Shewanella atlantica as a template and the primers of SEQ ID NO: 9 and SEQ ID NO: 10 in Table 3 below. A reaction mixture containing WT strain (Shewanella atlantica) chromosome (500 ng), primers (125 ng each), Mutazyme II reaction buffer (1x), dNTP mix (40 mM), and Mutazyme II DNA polymerase (2.5 U) was denatured at 94°C for 2 minutes, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 10 minutes. As a result, a 664 bp gene fragment including a 609 bp gene (SEQ ID NO: 2) was obtained.

[0107] To obtain the gapA promoter derived from Corynebacterium glutamicum, PCR (SolgTM Pfu-X DNA polymerase) was performed using the genomic DNA of Corynebacterium glutamicum ATCC 13032 as a template and the primers of SEQ ID NO: 11 and SEQ ID NO: 12 in Table 3 below. The PCR conditions were as follows.

[0108] Step Temperature Time Initialization 95 ℃ 2 minutes Denaturation 95 ℃ 20 seconds Annealing 62 ℃ 40 seconds Elongation 72 ℃ 0.5~1 minutes Post Elongation 72 ℃ 5 minutes

[0109] The gene fragment obtained through this process was ligated to the pECCG117 vector (Korean Patent No. 10-0057684) obtained after 1 hour reaction with restriction enzyme BamHI-HF (NEB) at 37°C and 30 minutes treatment with CIP (NEB) enzyme at 37°C using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix), and then transformed into E. coli DH5α, and plated on LB solid medium containing kanamycin (25 mg / L).

[0110] After screening 30 transformed colonies, plasmids were obtained and sequenced. As a result, it was confirmed that mutations were introduced at different positions at a frequency of 0.8 mutations / kb. Finally, approximately 10,000 transformed E. coli colonies were collected, and plasmids were extracted using a plasmid prep kit (QIAGEN) and named the p117-PgapA_lysE(sat.mt) library. In addition, a vector in which the Shewanella atlantica wild-type (WT) lysE was introduced into the pECCG117 vector was constructed to be used as a control in the screening. A wild-type lysE gene fragment was prepared by PCR using the sequences of SEQ ID NOs: 9 and 10, and the p117-PgapA_lysE(sat.WT) vector was constructed in the same manner as above. PCR conditions included denaturation at 94°C for 2 minutes, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 10 minutes.

[0111] The nucleic acid sequences of the primers used above are summarized in Table 3 below:

[0112] Primer sequence (5'→3') SEQ ID NO: 9CTGCACTTCACGTTTTGAcgcgtATGCAGGCATTTATTCAGGG SEQ ID NO: 10GGGTGAGCAGCGTTTTCGTCGGcccACGCGTTACAAGTAAGGCCATAACA SEQ ID NO: 11GGATCCATGGTGATCAACGACCGAGCCTATTGGG SEQ ID NO: 12CTgCACCCTGAATAAATGCCTGCATGTTGTGTCTCCTCTAAAGAT

[0113]

[0114] Example 2: Construction and screening of vector library-introduced strains

[0115] In order to construct a strain in which the lysE gene is deleted from the wild-type Corynebacterium glutamicum ATCC13032, a vector for deleting the lysE gene was constructed. Specifically, a recombinant vector was constructed in which DNA fragments (each 600 bp) located at the 5' and 3' ends of the lysE gene were linked to the pDZ vector (Korean Patent No. 2009-0094433). Based on the base sequence of the lysE gene (SEQ ID NO: 2), primers SEQ ID NO: 13 and SEQ ID NO: 14 and primers SEQ ID NO: 15 and SEQ ID NO: 16 were synthesized at positions 600 bp apart from them, respectively (see Table 4 below).

[0116] Using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and the primers of SEQ ID NO: 13 and SEQ ID NO: 15, PCR was performed to prepare a 5'-terminal gene fragment of the lysE gene. In the same way, PCR was performed using the primers of SEQ ID NO: 14 and SEQ ID NO: 16 to prepare a gene fragment located at the 3'-terminal of the lysE gene. The PCR was performed by denaturing at 94°C for 2 minutes, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 10 minutes. The amplified DNA fragment was purified using a PCR Purification kit from Quiagen and then used as an insert DNA fragment for vector construction.

[0117] Meanwhile, the pDZ vector (Korean Patent No. 2009-0094433) treated with restriction enzyme XbaI and heat-treated at 65°C for 20 minutes and the prepared insert DNA fragment amplified through the PCR were ligated using an Infusion Cloning Kit, transformed into E. coli DH5α, and plated on LB solid medium containing kanamycin (25 mg / L). Colonies transformed with the vector in which the desired gene was inserted were selected through PCR using primers SEQ ID NO: 13 and SEQ ID NO: 14, and then the plasmid was obtained using a commonly known plasmid extraction method, which was named pDZ-ΔlysE.

[0118] The nucleic acid sequences of the primers used above are summarized in Table 4 below:

[0119] Primer sequence (5'→3') Sequence number 13 GTACCCGGGGATCCTCTAGAGTCTGGAAAGGCTCTTTACG Sequence number 14 GCCTGCAGGTCGACTCTAGATCTAGTTTCCCATCAACCATGT Sequence number 15 AAGTACTTCCATAGGTCACGTTTTCGCGGGTTTTGGAATC Sequence number 16 GATTCCAAAACCCGCGAAAACGTGACCTATGGAAGTACTT

[0120] The vector pDZ-ΔlysE constructed above was transformed into Corynebacterium glutamicum ATCC13032 by the electric pulse method (Van der Rest et al., Appl. Microbiol. Biotecnol. 52:541-545, 1999), and a strain with a deletion of the lysE gene was constructed by homologous chromosomal recombination. The strain with a deletion of the lysE gene was named Corynebacterium glutamicum 13032::ΔlysE. The p117-PgapA_lysE(sat.mt) library constructed in Example 1 was transformed into the 13032::ΔlysE by the electric pulse method, and about 5,000 colonies were obtained by spreading on a complex plate medium containing kanamycin (25 mg / L). To produce a control group, 13032::ΔlysE was transformed with the p117-PgapA_lysE(sat.WT) vector in the same manner to obtain colonies.

[0121] <Composite plate (pH 7.0)>

[0122] Glucose 10 g, Peptone 10 g, Beef extract 5 g, Yeast extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, Urea 2 g, Sorbitiol 91 g, Agar 20 g (per 1 liter of distilled water)

[0123] The above-mentioned secured control libraries 13032::ΔlysE_p117-PgapA_lysE(sat.WT) and 13032::ΔlysE_p117-PgapA_lysE(sat.mt) were each inoculated into a 96-Deep Well Plate-Dome (Bioneer) containing 400 μl of seed medium and cultured in a plate shaking incubator (TAITEC) at 32°C and 12,000 rpm for approximately 12 hours.

[0124] <Seed medium (pH 7.0)>

[0125] Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4·7H2O 0.5 g, biotin 0.1 mg, thiamine HCl 1 mg, calcium-pantothenic acid 22 mg, nicotinamide 2 mg (per 1 liter of distilled water)

[0126] When the cultured colonies of about 5,000 were serially diluted in a complex plate medium containing 100 g / L of L-lysine hydrochloride and subjected to an MIC (minimum inhibitory concentration) test, 12 colonies with significantly higher MIC values ​​were obtained compared to the control strain. Each colony was subjected to a secondary screening. Each colony was inoculated into a 96-Deep Well Plate-Dome (Bioneer) containing 400 μl of seed medium and cultured in a plate shaking incubator (TAITEC) at 32°C and 12,000 rpm for about 12 hours. In addition, in the secondary screening step, the initial OD (optical density) values ​​of the final cultured colonies were adjusted to the same value, and the MIC test was performed by serially diluting them in a complex plate medium containing 100 g / L of L-lysine hydrochloride. Through this, three strains with significantly higher MIC values ​​compared to the control strain into which the wild-type foreign lysE library gene was introduced were named 13032::lysE(sat.mt) and further experiments were conducted.

[0127]

[0128] Example 3: Confirmation of the mutant lysE gene sequence

[0129] In order to confirm the sequence of the gene inserted into strain 13032::lysE(mt) selected in Example 2, a gene fragment was amplified by PCR using primers of SEQ ID NO: 17 and SEQ ID NO: 18 in Table 5. The PCR conditions were the same as in Example 1, and the amplified DNA fragment was obtained using GeneAll Expin GEL SV kit (Seoul, KOREA) and subjected to base sequence analysis.

[0130] The nucleic acid sequences of the primers used above are summarized in Table 5 below:

[0131] Primer sequence (5' → 3') SEQ ID NO: 17TACATCGGTGTGTCAATGGG SEQ ID NO: 18GATCGATGAGATTTCCCACG

[0132] Analysis of the base sequence of the amplified gene revealed that the first 13032::lysE(sat.mt) strain contained a mutant foreign lysE gene encoding an L-lysine exporter mutant in which the 22nd to 24th base sequence downstream from the start codon of the Shewanella atlantica lysE gene ORF (open reading frame) was changed from the existing GTA to GCA, and the 8th amino acid from the N-terminus of the wild-type lysE amino acid sequence of Shewanella atlantica (SEQ ID NO: 1) was substituted for alanine (V8A). The second 13032::lysE(sat.mt) strain was confirmed to contain a mutant foreign lysE gene encoding an L-lysine exporter mutant in which the 136th to 138th base sequence downstream from the start codon of the Shewanella atlantica lysE gene ORF was changed from the existing GAT to GGT, and aspartic acid, the 46th amino acid from the N-terminus of the wild-type lysE amino acid sequence of Shewanella atlantica (SEQ ID NO: 1), was substituted with glycine (D46G). Finally, the third 13032::lysE(sat.mt) strain was confirmed to contain a mutant foreign lysE gene encoding an L-lysine exporter mutant in which the 22nd to 24th base sequence of the Shewanella atlantica lysE gene ORF was changed from the existing GTA to GCA, and the 136th to 138th base sequence was changed from the existing GAT to GGT, so that valine, the 8th amino acid from the N-terminus, was substituted with alanine (A) (V8A) and aspartic acid (D), the 46th amino acid from the N-terminus, was substituted with glycine (G) (D46G) of the wild-type lysE amino acid sequence of Shewanella atlantica (SEQ ID NO: 1).

[0133]

[0134] Example 4: Construction of a vector introducing a mutant foreign lysE gene and construction of a strain.

[0135] 4-1. Construction of vectors introducing the V8A mutant foreign lysE gene and strain production

[0136] In order to introduce the V8A mutation confirmed in Example 3, a recombinant vector was constructed as follows. First, using the p117-PgapA_lysE (sat.WT) vector containing the gapA promoter as a template, primers of SEQ ID NO: 19 and SEQ ID NO: 20 (see Table 6) were synthesized, each having a 5' fragment and a 3' fragment with a restriction enzyme ScaI recognition site inserted at positions approximately 500 bp apart from the 22nd to 24th positions of the foreign lysE gene (SEQ ID NO: 2). In addition, primers of SEQ ID NO: 21 and SEQ ID NO: 22 were synthesized to introduce base substitution mutations at positions 600 bp apart from these (see Table 6).

[0137] Primer sequence (5' -> 3') SEQ ID NO: 19AACGACCGAGCCTATTGGG SEQ ID NO: 20AGGCTTCCGCCAATTCCTgC SEQ ID NO: 21GcAGGAATTGGCGGAAGCCT SEQ ID NO: 22TTACAAGTAAGGCCATAACA

[0138] The amplified gapA promoter region and the gene fragment derived from Shewanella atlantica containing the obtained mutation and the vector pDZTn (Korean Patent No. 10-1126041) digested with ScaI restriction enzyme were ligated using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix), and then transformed into Escherichia coli DH5α, which was plated on LB solid medium containing kanamycin (25 mg / L). To select colonies transformed with the vector linking the target gene and pDZTn, PCR was performed with primers of SEQ ID NO: 23 and SEQ ID NO: 24 (see Table 7). A plasmid was obtained from the selected colony using a commonly known plasmid extraction method, and this plasmid was named pDZTn-PgapA_lysE(sat.V8A). The constructed vector was transformed into the lysine-producing Corynebacterium glutamicum CJ3P strain (US Patent No. US 9556463 ​​B2) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and then a strain in which PgapA-Sat was inserted between transposon genes was obtained through a secondary crossing process. PCR and base sequence analysis were performed using primers of SEQ ID NOs: 23 and 24 (see Table 7) capable of amplifying the adjacent region including the position where the gene was inserted, and the genetic manipulation was confirmed. Thus, the Corynebacterium glutamicum strain into which the V8A mutant foreign lysE gene was introduced was named CJ3P::lysE(sat.V8A).

[0139] Primer sequence (5'→3') SEQ ID NO: 23ACGACGCTGGTATTTCTCCC SEQ ID NO: 24TGATTGTCGATATGACCGGG

[0140]

[0141] 4-2. Construction of vectors introducing the D46G mutant foreign lysE gene and strain production

[0142] In order to introduce the mutation D46G confirmed in Example 3, a recombinant vector was constructed as follows. First, using the p117-PgapA_lysE (sat.WT) vector containing the gapA promoter as a template, primers of SEQ ID NOs: 25 and 26 were synthesized, each having a 5' fragment and a 3' fragment having a ScaI recognition site inserted at positions 136 to 138 of the foreign lysE gene, which are approximately 500 bp apart in both directions. In addition, primers of SEQ ID NOs: 27 and 28 were synthesized to introduce base substitution mutations at positions 600 bp apart from these (see Table 8 below).

[0143] Primer sequence (5'→3') SEQ ID NO: 25AACGACCGAGCCTATTGGG SEQ ID NO: 26ACACCAGCCGTTATCATTAAGGCAcCGATC SEQ ID NO: 27GATCGgTGCCTTAATGATAACGGCTGGTGT SEQ ID NO: 28TTACAAGTAAGGCCATAACA

[0144] The amplified gapA promoter region and the gene fragment derived from Shewanella atlantica containing the obtained mutation and the vector pDZTn (Korean Patent No. 10-1126041) digested with ScaI restriction enzyme were ligated using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix), and then transformed into Escherichia coli DH5α, which was plated on LB solid medium containing kanamycin (25 mg / L). To select colonies transformed with the vector linking the target gene and pDZTn, PCR was performed with the primers of SEQ ID NO: 23 and SEQ ID NO: 24 (Table 7 above). A plasmid was obtained from the selected colony using a commonly known plasmid extraction method, and this plasmid was named pDZTn-PgapA_lysE(sat.D46G).

[0145] The constructed vector was transformed into the lysine-producing Corynebacterium glutamicum CJ3P strain (US Patent No. US 9556463 ​​B2) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and a strain in which PgapA-Sat was inserted between transposon genes was obtained through a second crossing-over process. PCR and base sequence analysis were performed using primers of SEQ ID NOs: 23 and 24 (Table 7 above) that can amplify adjacent regions including the position where the gene was inserted, and the genetic manipulation was confirmed. The strain into which the D46G mutant foreign lysE gene was introduced in this way was named CJ3P::lysE(sat.D46G).

[0146]

[0147] 4-3. Construction of vectors and strains introducing V8A and D46G mutant foreign lysE genes.

[0148] In order to introduce the mutations V8A and D46G identified in Example 3, a recombinant vector was constructed as follows. First, using the p117-PgapA_lysE (sat.WT) vector containing the gapA promoter as a template, primers of SEQ ID NOs: 29, 30, and 31, 32 (Table 9 below) were synthesized, each having a 5' fragment and a 3' fragment with a ScaI recognition site inserted at positions approximately 500 bp forward and backward from the 22nd to 24th bases and the 136th to 138th bases downstream of the foreign lysE gene. In addition, primers of SEQ ID NOs: 33 and 34 were synthesized to introduce base substitution mutations at positions 600 bp away from these (see Table 9 below).

[0149] Primer sequence (5' -> 3') SEQ ID NO: 29AACGACCGAGCCTATTGGG SEQ ID NO: 30AGGCTTCCGCCAATTCCTgC SEQ ID NO: 31GcAGGAATTGGCGGAAGCCT SEQ ID NO: 32ACACCAGCCGTTATCATTAAGGCAcCGATC SEQ ID NO: 33GATCGgTGCCTTAATGATAACGGCTGGTGT SEQ ID NO: 34TTACAAGTAAGGCCATAACA

[0150] The amplified gapA promoter region and the Shewanella atlantica gene fragment containing the obtained mutation and the vector pDZTn (Korean Patent No. 10-1126041) digested with ScaI restriction enzyme were ligated using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix), and then transformed into Escherichia coli DH5α, which was plated on LB solid medium containing kanamycin (25 mg / l). To select colonies transformed with the vector linking the target gene and pDZTn, PCR was performed with the primers of SEQ ID NO: 23 and SEQ ID NO: 24 (Table 7 above). A plasmid was obtained from the selected colony using a commonly known plasmid extraction method, and this plasmid was named pDZTn-PgapA_lysE(sat.V8A,D46G).

[0151] The constructed vector was transformed into the lysine-producing Corynebacterium glutamicum CJ3P strain (US Patent No. US 9556463 ​​B2) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and a strain in which PgapA-Sat was inserted between transposon genes was obtained through a second crossing process. PCR and base sequence analysis were performed using primers of SEQ ID NOs: 23 and 34 that can amplify adjacent regions including the position where the gene was inserted, and the genetic manipulation was confirmed. The strain into which the V8A and D46G mutant foreign lysE genes were introduced was named CJ3P::lysE(sat.V8A,D46G).

[0152]

[0153] Example 5: Analysis of growth and L-lysine production capacity of strains introducing a mutant foreign lysE gene in a titer medium.

[0154] The three strains produced in Example 4, the control strain CJ3, and the control strain CJ3P::lysE(sat.WT) containing lysE(sat, WT) were cultured using the following method, and the cell mass and lysine production ability in neutral medium were compared.

[0155] First, each strain was inoculated into a 250 mL corner-baffle flask containing 25 mL of seed medium and cultured at 30°C for 20 h with shaking at 200 rpm. 1 mL of the seed culture was inoculated into a 250 mL corner-baffle flask containing 24 mL of production medium and cultured at 37°C for 40 h with shaking at 200 rpm. After completion of cultivation, the amount of L-lysine produced was measured by HPLC. The above experiment was repeated three times.

[0156] <Seed medium (pH 7.0)>

[0157] 20 g of raw sugar, 10 g of peptone, 5 g of yeast extract, 1.5 g of urea, 4 g of KH2PO4, 8 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.1 mg of biotin, 1 mg of thiamine HCl, 2 mg of calcium-pantothenic acid, 2 mg of nicotinamide (per 1 liter of distilled water)

[0158]

[0159] <Production medium (pH 7.0)>

[0160] 100 g raw sugar, 40 g (NH4)2SO4, 2.5 g soy protein, 5 g corn steep solids, 3 g urea, 1 g KH2PO4, 0.5 g MgSO4·7H2O, 100 ug biotin, 1000 ug thiamine hydrochloride, 2000 ug calcium pantothenic acid, 3000 ug nicotinamide, 30 g CaCO3 (per 1 liter of distilled water).

[0161]

[0162] The culture results (average values) of CJ3P, CJ3P::lysE(sat.WT), CJ3P::lysE(sat.V8A), CJ3P::lysE(sat.D46G), and CJ3P::lysE(sat.V8A,D46G) were compared and shown in Table 10 below.

[0163] CJ3P, CJ3P:: lightE (sat.WT), CJ3P:: lightE (sat.V8A), CJ3P:: lightE (sat.D46G), CJ3P:: lightE (sat. V8A, D46G) OD (600 nm) and lysine production ability measurement Strain culture conditions Cells (OD 600 nm) Lysine concentration (g / L) Lysine concentration increase rate (%) 18h 40h 40h CJ3PpH 7.0 46.2 62.5 7.0 3 100 CJ3P::lysE (sat. WT) 46.8 65.2 7.4 6 106.1 CJ3P::lysE (sat. V8A) 48.6 73.3 8.4 119.4 (112.6 compared to CJ3P::lysE (sat. WT)) CJ3P::lysE (sat. D46G) 47.3 69.3 8.0 9 115.1 (112.6 compared to CJ3P::lysE (sat. WT)) 108.4)CJ3P::lysE(sat.V8A,D46G)46.967.87.72109.8(CJ3P::lysE(sat.WT) vs. 103.8)

[0164] As shown in Table 10 above, the strains in which mutations were introduced in both cell and lysine concentration showed superior levels than the parent strains CJ3P and CJ3P::lysE (sat.WT), and it was confirmed that the cell activity (survival rate) and lysine production ability according to the culture time were superior to those of the parent strain.

[0165] Through this, it was confirmed that mutations in LysE derived from Shewanella atlantica increased lysine excretion, thereby increasing cell activity (survival rate) and lysine production.

[0166]

[0167] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims

1. A polypeptide in which the amino acid corresponding to the 8th amino acid position from the N-terminus in the amino acid sequence of sequence number 1, the amino acid corresponding to the 46th amino acid position from the N-terminus, or a combination thereof is replaced with an amino acid different from the original.

2. In the first paragraph, the polypeptide A polypeptide in which the amino acid corresponding to the 8th amino acid position is substituted with glutamic acid, glycine, alanine, serine, threonine, cysteine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamine, histidine, lysine, arginine, or asparagine; A polypeptide in which the amino acid corresponding to the 46th amino acid position is substituted with glutamic acid, glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, glutamine, histidine, lysine, arginine, or asparagine; or A polypeptide wherein the amino acid corresponding to the 8th amino acid position is substituted with glutamic acid, glycine, alanine, serine, threonine, cysteine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamine, histidine, lysine, arginine, or asparagine, and the amino acid corresponding to the 46th amino acid position is substituted with glutamic acid, glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, glutamine, histidine, lysine, arginine, or asparagine.

3. A polypeptide having the function of an L-amino acid excretor according to claim 1 or 2.

4. A polypeptide according to claim 1 or 2, wherein the polypeptide having the amino acid sequence of sequence number 1 is derived from Shewanella atlantica.

5. A polynucleotide encoding the polypeptide of claim 1 or 2.

6. A polynucleotide expressed by one nucleic acid sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 8 in the fifth paragraph.

7. A recombinant vector comprising the polynucleotide of clause 5.

8. An L-amino acid producing microorganism comprising the polypeptide of paragraph 1, a polynucleotide encoding the same, or a recombinant vector comprising the polynucleotide.

9. An L-amino acid producing microorganism in claim 8, wherein the polypeptide has the function of an L-amino acid excretor.

10. In paragraph 8, the L-amino acid producing microorganism is a microorganism of the genus Corynebacterium or Escherichia.

11. In paragraph 10, the microorganism is an L-amino acid producing microorganism that is Corynebacterium glutamicum or Escherichia coli.

12. An L-amino acid producing microorganism having an increased L-amino acid excretion or production ability compared to a non-modified microorganism according to any one of claims 8 to 11.

13. An L-amino acid producing microorganism having an increased strain survival rate compared to a non-transformed microorganism according to any one of claims 8 to 11.

14. An L-amino acid producing microorganism in claim 9, wherein the L-amino acid is L-lysine.

15. A method for producing L-amino acid, comprising a step of culturing an L-amino acid producing microorganism of any one of claims 8 to 11 in a medium.

16. In paragraph 15, after the culturing step, A method for producing L-amino acids, further comprising a step of recovering L-amino acids from the cultured microorganism, medium, or both.

17. A method for producing L-amino acid in claim 15, wherein the L-amino acid is L-lysine.

18. Use of an L-amino acid producing microorganism according to any one of claims 8 to 11 for producing L-amino acids.

19. A composition, method, product, process, or use characterized by one or more elements disclosed herein.