Microorganism comprising variant LysE and method for producing l-amino acid using same

Abstract

The present application provides a microorganism comprising a variant LysE, and a method for producing an L-amino acid using the same. Compared with a wild type, the variant LysE can improve the secretion and / or production capacity of L-amino acid.

Description

Technical Field

[0001] Cross-references to related applications

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0063271, filed on May 14, 2024, and the entire contents disclosed in the relevant Korean patent application are incorporated herein by reference.

[0003] Throughout this disclosure, numerous papers and patent documents are cited, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety, thereby providing a clearer understanding of the technical level to which this invention pertains and the content of this invention.

[0004] This application relates to microorganisms containing the variant LysE, and methods for producing L-amino acids using them. The variant LysE can improve L-amino acid export capacity and / or L-amino acid production capacity compared to the wild type. Background Technology

[0005] Corynebacterium are Gram-positive microorganisms widely used in the production of L-amino acids. L-amino acids, especially L-lysine, are used in animal feed, human pharmaceuticals, and cosmetics industries, and are primarily produced through fermentation using Corynebacterium strains.

[0006] Numerous attempts have been made to improve methods for producing L-amino acids using Corynebacterium strains. Among these efforts, some studies have focused on improving L-amino acid production by using recombinant DNA technology to disrupt or attenuate specific genes. Furthermore, studies have been conducted to improve L-amino acid production by amplifying genes involved in the biosynthesis of various L-amino acids and analyzing their impact on L-amino acid production. Additionally, the introduction of heterologous genes from other bacteria has also been explored.

[0007] Despite these efforts, there is still a need to develop technologies to improve the production capacity of useful substances such as L-amino acids. Summary of the Invention

[0008] Technical issues

[0009] One implementation provides a variant lysine export protein.

[0010] Another implementation provides a polynucleotide that encodes the polypeptide.

[0011] Another implementation provides a recombinant vector containing the polynucleotide.

[0012] Another embodiment provides a recombinant microorganism comprising the polypeptide, a polynucleotide encoding the polypeptide, or a recombinant vector comprising the polynucleotide.

[0013] Another embodiment provides a method for producing L-amino acids, which includes culturing the recombinant microorganism or a microorganism that produces L-amino acids.

[0014] Another embodiment provides the use of the recombinant microorganism for the production of L-amino acids.

[0015] Technical solution

[0016] This specification provides a technique relating to the search for novel L-amino acid export proteins, confirmation of the effects of gene mutations in said novel export proteins, and strain improvement using said technique, with the aim of increasing the L-amino acid production capacity of Corynebacterium microorganisms. Taking L-lysine as a representative example, there are typically methods to increase the L-lysine yield of a strain or to increase the L-lysine production per unit time (productivity) in order to increase lysine production capacity. L-lysine export proteins are membrane proteins that export lysine produced through biosynthesis, and improvements to these proteins are important for increasing lysine yield and productivity. However, increasing the expression level of the L-lysine export protein (LysE, Ncgl1214) in Corynebacterium microorganisms has limitations in improving L-lysine production capacity.

[0017] Therefore, this specification provides recombinant strains with improved L-amino acid export and / or L-amino acid production capabilities by searching for novel heterologous L-amino acid exporting proteins with high L-amino acid exporting activity, improving heterologous L-amino acid exporting proteins, and introducing them into lysine-producing strains.

[0018] In this specification, as a representative example of novel heterologous L-amino acid export proteins, a novel L-amino acid export protein derived from *Shewanella atlantica* and the gene encoding it were discovered, and they were improved through mutation. Results from expressing mutants of the novel L-amino acid export protein from *Shewanella atlantica* into L-amino acid-producing microorganisms demonstrated significantly enhanced L-amino acid export and production capabilities compared to microorganisms that did not express the gene.

[0019] One embodiment provides a polypeptide in which the amino acid at position 8 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 46 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 1, or a combination thereof, is replaced with an amino acid different from the original amino acid. The polypeptide may function as an L-amino acid export protein. The L-amino acid may be L-lysine, L-arginine, or a combination thereof; specifically, it may be L-lysine.

[0020] Another implementation provides a polynucleotide that encodes the polypeptide.

[0021] Another implementation provides a recombinant vector containing the polynucleotide.

[0022] Another embodiment provides a recombinant microorganism comprising the polypeptide, a polynucleotide encoding the polypeptide, or a recombinant vector comprising the polynucleotide. The recombinant microorganism may have L-amino acid export and / or L-amino acid production capabilities compared to unmodified microorganisms, or may have increased L-amino acid export and / or L-amino acid production capabilities.

[0023] Another embodiment provides a method for producing L-amino acids, which includes culturing the recombinant microorganism or a microorganism that produces L-amino acids.

[0024] It will be described in more detail below.

[0025] One embodiment of this application provides a polypeptide in which the amino acid at the 8th amino acid position from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at the 46th amino acid position from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 1, or a combination thereof, is replaced with an amino acid different from the original amino acid.

[0026] In one embodiment, the polypeptide may function as an L-amino acid exporting protein. The L-amino acid may be L-lysine, L-arginine, or a combination thereof; specifically, it may be L-lysine.

[0027] In this specification, lysine exporting protein (LysE) can refer to a protein that is a transmembrane protein and functions to export products of intracellular biosynthesis (e.g., L-amino acids such as L-lysine). In one embodiment, the lysine exporting protein may be a heterologous lysine exporting protein derived from a microorganism belonging to a genus different from the parent strain (the pre-mutant microorganism) or a different species of microorganism. For example, it may be a protein derived from microorganisms other than those belonging to the genus Corynebacterium and has the ability to export L-amino acids (e.g., L-lysine). In one embodiment, the heterologous lysine exporting protein may be a membrane protein derived from a microorganism of the genus Shewanella (e.g., Shewanella Atlantica). In one specific embodiment, the lysine-exporting protein may be a protein represented by the amino acid sequence of SEQ ID NO: 1, and may be a protein having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more sequence identity or homology with said protein. In this specification, the polypeptide of the amino acid sequence of SEQ ID NO: 1 may be derived from *Shewanella Atlantica*.

[0028] In one embodiment, the amino acid at the 8th amino acid position from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 1 may be valine (Val, V), but is not limited thereto.

[0029] In one embodiment, the amino acid at the 46th amino acid position from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 1 may be aspartic acid (Asp, D), but is not limited thereto.

[0030] The polypeptide of this application may be a polypeptide (variant lysine export protein) that has undergone an amino acid substitution mutation induced in the above-mentioned lysine export protein. In one embodiment, the polypeptide may be a polypeptide in which the amino acid corresponding to the 8th amino acid position from the N-terminus of 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 of the amino acid sequence of SEQ ID NO: 1 (e.g., aspartic acid (Asp, D)), or a combination thereof, is replaced with an amino acid different from the original amino acid (e.g., a polypeptide with an amino acid sequence in which the 8th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1, valine (Val, V), the 46th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1, aspartic acid (Asp, D), or a combination thereof, is replaced with a different amino acid).More specifically, the polypeptide can be: a polypeptide in which the amino acid corresponding to the 8th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 (e.g., valine (Val, V)) is replaced with an amino acid different from the original amino acid (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)); such a polypeptide, in which the amino acid corresponding to the 8th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid different from the original amino acid (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)); a polypeptide in which the amino acid corresponding to the 8th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid different from the original amino acid (e.g., glutamic acid (E), glycine (G), alanine (A)); The amino acid at position 46 from the N-terminus of amino acid 1 (e.g., aspartic acid (Asp, D)) is replaced with an amino acid different from the original amino acid (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, glycine (G)); or such a polypeptide, wherein the amino acid corresponding to SEQ ID NO: The amino acid at the 8th amino acid position from the N-terminus of the amino acid sequence SEQ ID NO: 1 (e.g., valine (Val, V)) is replaced with an amino acid different from the original amino acid (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 at the 46th amino acid position from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 1 (e.g., aspartic acid (Asp, V)) is replaced with an amino acid different from the original amino acid (e.g., glutamic acid (E), glycine (G), alanine (A), glutamine (Q), histidine (H), lysine (K), arginine (R), or asparagine (N); specifically, alanine (A)). D)) is replaced with an amino acid different from the original amino acid (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, glycine (G)).In one specific embodiment, the polypeptide may comprise, but is not limited to, the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 (where the 8th amino acid valine (V) is replaced with alanine (A)), the polypeptide represented by the amino acid sequence of SEQ ID NO: 4 (where the 46th amino acid aspartic acid (D) is replaced with glycine (G)), or the polypeptide represented by the amino acid sequence of SEQ ID NO: 5 (where the 8th amino acid valine (V) is replaced with alanine (A) and the 46th amino acid aspartic acid (D) is replaced with glycine (G)). The polypeptide inducing this mutation may function as an L-amino acid exporting protein (e.g., an L-lysine exporting protein).

[0031] In one embodiment, the polypeptide of this application may be a polypeptide in which the amino acid corresponding to the 8th amino acid position from the N-terminus of 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 of the amino acid sequence of SEQ ID NO: 1 (e.g., aspartic acid (Asp, D)), or a combination thereof, is replaced with an amino acid different from the original amino acid, and may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more and less than 100% homology or identity with the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0032] In this application, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar to, identical to, or homologous to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position may be done by referring to a specific sequence to determine a specific amino acid in the sequence. As used in this application, the term "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0033] For example, any amino acid sequence can be compared with SEQ ID NO: 1. Based on this, each amino acid residue in 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, the sequence alignment algorithm described in this application can identify locations where variations (such as amino acid positions, or substitutions, insertions, or deletions) have occurred compared to the query sequence (also called the "reference sequence").

[0034] Such alignments can be performed using, for example, the Needleman–Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), but are not limited to these. Sequence alignment programs known in the art, such as pairwise sequence comparison algorithms, can also be used as appropriate.

[0035] Another embodiment provides a polynucleotide that encodes the polypeptide described above in this application.

[0036] In this application, "polynucleotide" refers to a polymer of nucleotides, wherein nucleotide monomers are covalently linked together in a long chain form, and is a DNA or RNA chain of a predetermined length or longer, and more specifically, refers to a polynucleotide fragment encoding the aforementioned variants.

[0037] In one embodiment, the polynucleotide encoding the polypeptide of this application may be represented by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 8.

[0038] In this specification, when a polynucleotide (which may be used interchangeably with "gene") or polypeptide (which may be used interchangeably with "protein") is described as "comprising a specific nucleic acid sequence or amino acid sequence" or "represented by a specific nucleic acid sequence or amino acid sequence," this may mean that the polynucleotide or polypeptide is composed of, or substantially comprises, that specific nucleic acid sequence or amino acid sequence. Furthermore, it can be interpreted as including (or not excluding) a "substantially equivalent sequence" of that specific nucleic acid sequence or amino acid sequence in which mutations (deletions, substitutions, modifications, and / or additions) are applied to, within the scope of maintaining the original and / or intended function of the polynucleotide or polypeptide.

[0039] In one embodiment, the nucleic acid or amino acid sequences provided in this specification may comprise those nucleic acid or amino acid sequences that have been modified by conventional mutagenesis methods, such as direct evolution and / or site-directed mutagenesis, to the extent that their original or intended function is maintained. In one embodiment, when a polynucleotide or polypeptide is described as “comprising a specific nucleic acid or amino acid sequence,” it may mean that the polynucleotide or polypeptide (i) consists of, or substantially comprises, the specific nucleic acid or amino acid sequence, or (ii) is composed of, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more of the specific nucleic acid or amino acid sequence. The sequence comprises, or substantially contains, an amino acid sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with the specific nucleic acid sequence or amino acid sequence, and retains the original function and / or intended function. In this specification, the original function may be the function of an L-amino acid exporting protein (e.g., an L-lysine exporting protein) (in the case of the amino acid sequence), or the function of a protein encoding an L-amino acid exporting protein (e.g., an L-lysine exporting protein) (in the case of the nucleic acid sequence). The intended function may refer to the function of increasing or conferring the L-amino acid (e.g., L-lysine) exporting capacity and / or L-amino acid (e.g., L-lysine) production capacity of microorganisms.

[0040] Due to the degeneracy of codons, and considering the preferred codons in the microorganisms from which the protein (lysine export protein) is to be expressed, the nucleic acid sequences described in this specification can be modified in various ways in the coding region without altering the amino acid sequence and / or the function of the protein expressed from the coding region.

[0041] In this application, the terms "homology" or "identity" refer to the degree of correlation between two given amino acid sequences or base sequences, and may be expressed as a percentage. The terms homology and identity are generally used interchangeably.

[0042] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms and can be achieved using default gap penalties established by the procedure employed. Essentially, homologous or identical sequences can typically be hybridized along at least approximately 50%, 60%, 70%, 80%, or 90% of the entire sequence or the full length of the sequence under moderate to high stringency conditions. Clearly, hybridization also includes polynucleotides containing universal codons or codons that account for codon degeneracy in the polynucleotide.

[0043] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined, for example, using known computer algorithms, such as the “FASTA” program with default parameters as described in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, this determination can be made using the Needleman–Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), such as in the Needle program (version 5.0.0 or later) within the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, TrendsGenet. 16: 276-277) (including the GCG package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Altschul, SF et al., J. Mol. Biol. 215: 403 (1990); Guide to Huge Computers, Martin J. Bishop, Ed., Academic Press, San Diego, 1994 and Carillo et al. (1988), SIAM J. Applied Math. 48: As performed in 1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0044] Homology, similarity, or identity of polynucleotides or polypeptides can be determined, for example, by comparing sequence information using the GAP computer program, as described in Smith and Waterman, Adv. Appl. Math. (1981) 2:482, and, for example, Needleman et al. (1970), J. Mol. Biol. 48:443. In summary, the GAP program can be defined as a value obtained by dividing the number of similarly arranged symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP procedure may include (1) a binary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix, such as those published by Schwartz and Dayhoff, Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353–358 (1979), Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol in each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy.

[0045] Furthermore, whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be confirmed by comparing the sequences under specific, stringent conditions using Southern hybridization experiments. These appropriate hybridization conditions are within the scope of the relevant field 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).

[0046] Another embodiment provides a recombinant vector comprising the polynucleotides described above in this application. The vector may be an expression vector for expressing the polynucleotides in a host cell, but is not limited thereto.

[0047] In this specification, the term "vector" refers to a DNA construct containing a polynucleotide base sequence encoding a target protein, operatively linked to a suitable regulatory sequence to allow expression of the target protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any operon sequence regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence regulating transcription and / or translation termination. After transformation into a suitable host microorganism, the vector may be expressed independently of the host microorganism's genome or may be integrated into the host microorganism's genome.

[0048] There are no particular limitations on the vectors that can be used in this specification, as long as they can replicate in the host cell and can be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, entrapments, viruses, and bacteriophages in their native or recombinant states. For example, as vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or entrapment vectors, while pBR-based vectors, pUC-based vectors, pBluescriptII-based vectors, pGEM-based vectors, pTZ-based vectors, pCL-based vectors, and pET-based vectors can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be listed, but are not limited to these.

[0049] The vectors available in this specification may be known expression vectors and / or vectors for inserting polynucleotides into the chromosomes of host cells. Insertion of polynucleotides into the chromosomes of host cells can be performed by any method known to those skilled in the art, such as homologous recombination, but not limited thereto. The vector may also contain a selection marker to confirm whether a selection marker has been inserted into the chromosome. The selection marker is used to select cells transformed by the vector, i.e., to confirm the insertion of the polynucleotide, and can select and use genes conferring selectable phenotypes (such as drug resistance, auxotrophic phenotypes, cytotoxic agent resistance, or surface protein expression). In an environment treated with a selection agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, thereby allowing for the selection of transformed cells.

[0050] The introduction of polynucleotides or vectors can be performed using known transformation methods appropriately selected by those skilled in the art. In this specification, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target protein (lysine export protein) into a host microorganism, such that the protein encoded by the polynucleotide can be expressed in the host cells. All cases are covered as long as the transformed polynucleotide can be expressed in the host microorganism, whether it is inserted into the host microorganism's chromosome or located extrachromosomally. Furthermore, the polynucleotide contains DNA and / or RNA encoding the target protein. The form of introduction is not limited as long as the polynucleotide can be introduced into the host microorganism and expressed. For example, the polynucleotide can be introduced into the host microorganism in the form of an expression cassette, which is a gene construct containing all the elements required for autonomous expression. The expression cassette may contain expression regulatory elements, such as promoters, transcription termination signals, ribosome binding sites, and / or translation termination signals operatively linked to the polynucleotide. The expression cassette can be in the form of an expression vector capable of autonomous replication. Furthermore, the polynucleotide can be introduced into the host cell in its own form and operatively linked to the sequence required for expression in the host cell. As used herein, the term "operationally ligated" can refer to the functional ligation of an expression regulatory element to a multinucleotide encoding a target protein (lysine export protein) such that the expression regulatory element can be regulated transcriptionally (e.g., transcription initiation). Operable ligation can be performed using gene recombination techniques known in the art, such as, but not limited to, conventional site-specific DNA cutting and ligation.

[0051] The conversion of polynucleotides into host microorganisms can be carried out by any method of introducing nucleic acids into cells (microorganisms), and can be carried out by appropriately selecting known conversion techniques in the art according to the host microorganism. Examples of known conversion methods may include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation (PEG-mediated uptake), DEAE-dextran method, cationic liposome method, lipid transfection, and lithium acetate-DMSO method.

[0052] The insertion of mutations can be performed by those skilled in the art using known methods of appropriate selection, thereby inserting a polynucleotide into the host cell genome (chromosome) or a host cell endogenous gene (e.g., the LysE gene) encoding the polypeptide of this application, and can be performed, for example, using 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.), a gene encoding it, or a vector containing the gene; and (b) a guide RNA (e.g., a single guide RNA (sgRNA), etc.), DNA encoding it, or a vector containing the DNA), said system including, but not limited to, mixtures (e.g., a mixture of RNA-guided endonuclease protein and guide RNA), complexes (e.g., ribonucleoprotein (RNP)) and recombinant vectors (e.g., a vector containing an RNA-guided endonuclease-encoding gene and a guide RNA-encoding DNA).

[0053] Another embodiment provides a microorganism for producing L-amino acids, which contains the polypeptide, a polynucleotide encoding the polypeptide, or a recombinant vector containing the polynucleotide of this application.

[0054] In this specification, the term "microorganism producing L-amino acids" can be used to refer to a situation where a microorganism with L-amino acid exporting and / or L-amino acid production capacity has increased L-amino acid exporting and / or L-amino acid production capacity by introducing the lysine exporting protein mutation as described above, and / or a situation where a microorganism without L-amino acid exporting and / or L-amino acid production capacity becomes L-amino acid exporting and / or L-amino acid production capacity by introducing the lysine exporting protein mutation as described above. In this specification, the term "microorganism" includes single-celled bacteria and may be used interchangeably with "cell".

[0055] The L-amino acid can be L-lysine, L-arginine, or a combination thereof. Specifically, it can be L-lysine, but is not limited thereto.

[0056] In one embodiment, the microorganism may be selected from any microorganism having L-amino acid (e.g., L-lysine) export and / or L-amino acid production capabilities. In one embodiment, the host microorganism may be a microorganism naturally possessing L-amino acid export and / or L-amino acid production capabilities, or a microorganism possessing L-amino acid export and / or L-amino acid production capabilities by introducing mutations into a parent strain that does not have significantly low L-amino acid export and / or L-amino acid production capabilities.

[0057] In one specific embodiment, the microorganism may be at least one selected from the genus *Corynebacterium* or the genus *Escherichia*, including microorganisms naturally possessing L-amino acid export and / or L-amino acid production capabilities, and microorganisms possessing L-amino acid export and / or L-amino acid production capabilities by introducing mutations into parental strains that do not possess L-amino acid export and / or L-amino acid production capabilities or have significantly low L-amino acid export and / or L-amino acid production capabilities. The *Corynebacterium* genus microorganisms may include, but are not limited to, *Corynebacterium glutamicum*, *Corynebacterium ammoniagenes*, *Brevibacterium lactofermentum*, *Brevibacterium flavum*, *Corynebacterium thermoaminogenes*, and *Corynebacterium efficiens*. More specifically, the *Corynebacterium* genus microorganism may be *Corynebacterium glutamicum*. The Escherichia coli strain mentioned can be Escherichia coli.

[0058] In one embodiment, a microorganism that has been introduced with a lysine export protein mutation to produce L-amino acids may have increased L-amino acid export capacity and / or L-amino acid production capacity compared to a homologous, unmodified microorganism. The term "unmodified microorganism" does not exclude naturally occurring mutant microorganisms and may refer to the natural (wild-type) strain itself, homologous microorganisms without the lysine export protein mutation, or microorganisms prior to the mutation. The term may also refer to microorganisms that do not contain such polypeptides and / or polynucleotides encoding said polypeptides, wherein the amino acid corresponding to the 8th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1, the amino acid corresponding to the 46th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1, or a combination thereof, is replaced with an amino acid different from the original amino acid. The term "unmodified" may be used interchangeably with "before modification," "unmutated," or "before mutation."

[0059] In one embodiment, the microorganism of this application having increased L-amino acid export capacity and / or L-amino acid production capacity (production amount) may have an increase of 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 (no particular upper limit is imposed, 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) of L-amino acid export capacity and / or L-amino acid production capacity (production amount) compared to the unmutated microorganism or the unmodified microorganism (e.g., the microorganism without lysE or the microorganism with wild-type lysE), but is not limited thereto. In another embodiment, the microorganism of this application having increased L-amino acid export capacity and / or L-amino acid production capacity (production amount) may have an increase of 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 (no particular upper limit is imposed, 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) of L-amino acid export capacity and / or L-amino acid production capacity (production amount), but is not limited thereto.

[0060] As used herein, the term “about” refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all values ​​equal to or similar to the value following the term “about”.

[0061] In one embodiment, when a lysine export protein mutation is introduced, the L-amino acid-producing microorganism with the lysine export protein mutation can have increased strain viability. More specifically, when a lysine export protein mutation is introduced, the L-amino acid-producing microorganism with the lysine export protein mutation can have increased strain viability compared to the unmutated microorganism or the unmodified microorganism (e.g., microorganisms without lysE or with wild-type lysE).

[0062] In one embodiment, when a lysine export protein mutation is introduced, the L-amino acid-producing microorganism with the lysine export protein mutation may have increased L-amino acid export capacity and / or L-amino acid production capacity. More specifically, when a lysine export protein mutation is introduced, the L-amino acid-producing microorganism with the lysine export protein mutation may have increased L-amino acid export capacity and / or L-amino acid production capacity compared to the unmutated microorganism or the unmodified microorganism (e.g., microorganisms without lysE or with wild-type lysE).

[0063] The term "strain viability" can refer to the degree to which a strain survives under specific conditions, and for the purposes of this application, strain viability can be used interchangeably with terms such as "strain growth rate," "strain survival rate," "strain activity," "cell viability," "cell growth rate," "cell survival rate," and "cell activity." In one embodiment, strain viability can be determined by measuring the OD (optical density) value of the strain (cell) culture at specific time points during culture.

[0064] In this specification, in order to distinguish the microorganism prior to the introduction of the lysine export protein mutation from the "L-amino acid producing microorganism" in which the lysine export protein mutation is introduced to increase L-amino acid export capacity and / or L-amino acid production capacity or to endow L-amino acid export capacity and / or L-amino acid production capacity, the microorganism prior to the introduction of the lysine export protein mutation may be referred to as the host microorganism.

[0065] In this specification, "introducing a lysine export protein mutation" can refer to all operations that introduce the polypeptide (mutated lysine export protein) of this application as described above into a host microorganism.

[0066] "Microorganisms that produce L-amino acids by introducing lysine export protein mutations" can be microorganisms in which L-amino acid export capacity and / or L-amino acid production capacity are increased or endowed by including: (1) the polypeptide of the present application as described above, for example, a polypeptide in which the amino acid corresponding to the 8th amino acid position from the N-terminus of 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 of the amino acid sequence of SEQ ID NO: 1 (e.g., aspartic acid (Asp, D)), or a combination thereof, is replaced with an amino acid different from the original amino acid; (2) a polynucleotide encoding the polypeptide; and / or (3) a recombinant vector containing the polynucleotide.

[0067] “Microorganisms that produce L-amino acids by introducing lysine export protein mutations” can be microorganisms that have increased strain activity by including the following: (1) the polypeptide of this application as described above, for example, a polypeptide in which the amino acid corresponding to the 8th amino acid position from the N-terminus of 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 of the amino acid sequence of SEQ ID NO: 1 (e.g., aspartic acid (Asp, D)) or a combination thereof is replaced with an amino acid different from the original amino acid, (2) a polynucleotide encoding the polypeptide, and / or (3) a recombinant vector containing the polynucleotide.

[0068] Microorganisms can refer to microorganisms that, in addition to their genome, also contain the polynucleotide or recombinant vector: the polypeptide, for example, in which the amino acid corresponding to the 8th amino acid position from the N-terminus of 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 of the amino acid sequence of SEQ ID NO: 1 (e.g., aspartic acid (Asp, D)), or a combination thereof, is replaced with an amino acid different from the original amino acid; the polynucleotide encoding the polypeptide; and / or the recombinant vector containing the polynucleotide.

[0069] In one specific embodiment, the microorganism producing L-amino acids may be a microorganism containing a polynucleotide encoding an amino acid sequence and / or a recombinant vector containing the polynucleotide, wherein the amino acid sequence corresponding to the 8th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 (e.g., valine (V)) is replaced with an amino acid different from the original 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)). More specifically, the microorganism producing L-amino acids may contain a polynucleotide encoding an amino acid sequence and / or a recombinant vector containing said polynucleotide, wherein the amino acid sequence corresponding to the 8th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 (e.g., valine (V)) is replaced with an amino acid alanine (A) that is different from the original amino acid. More specifically, it may be a Corynebacterium genus microorganism, such as Corynebacterium glutamicum, which contains a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 (more specifically, the polynucleotide shown by the nucleic acid sequence of SEQ ID NO: 6) and / or a recombinant vector containing said polynucleotide.

[0070] In one specific embodiment, the microorganism producing L-amino acids may be a microorganism containing a polynucleotide encoding an amino acid sequence and / or a recombinant vector containing the polynucleotide, wherein the amino acid sequence corresponding to the 46th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 (e.g., aspartic acid (D)) is replaced with an amino acid different from the original 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), 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)). More specifically, the microorganisms producing L-amino acids may contain a polynucleotide encoding an amino acid sequence and / or a recombinant vector containing said polynucleotide, wherein the amino acid sequence corresponding to the 46th amino acid position from the N-terminus of the amino acid sequence in SEQ ID NO: 1 (e.g., aspartic acid (D)) is replaced with an amino acid glycine (G) that is different from the original amino acid. More specifically, it may be a Corynebacterium genus microorganism, such as Corynebacterium glutamicum, which contains a polynucleotide encoding the amino acid sequence in SEQ ID NO: 4 (more specifically, the polynucleotide shown by the nucleic acid sequence in SEQ ID NO: 7) and / or a recombinant vector containing said polynucleotide.

[0071] In one specific embodiment, the microorganism producing L-amino acids may be a microorganism comprising a polynucleotide encoding the following amino acid sequence and / or a recombinant vector comprising said polynucleotide, wherein the amino acid sequence 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 replaced with an amino acid different from the original 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), 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)). The amino acid at position 46 from the N-terminus of amino acid 1 (e.g., aspartic acid (D)) is replaced with an amino acid different from the original 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), 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)). More specifically, the microorganism producing L-amino acids may comprise a polynucleotide encoding the following amino acid sequence and / or a recombinant vector containing said polynucleotide, wherein in the amino acid sequence of SEQ ID NO: 1, the amino acid corresponding to the 8th amino acid position from the N-terminus (e.g., valine (V)) is replaced with an amino acid alanine (A) different from the original amino acid, 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 glycine (G) different from the original amino acid. More specifically, it may be a Corynebacterium genus microorganism, such as Corynebacterium glutamicum, which comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 (more specifically, the polynucleotide shown by the nucleic acid sequence of SEQ ID NO: 8) and / or a recombinant vector containing said polynucleotide.

[0072] Another embodiment provides a method for increasing the L-amino acid exporting capacity and / or L-amino acid production capacity of microorganisms, or a method for conferring L-amino acid exporting capacity and / or L-amino acid production capacity to microorganisms, comprising introducing (transforming) the microorganisms with: a polypeptide, wherein an amino acid corresponding to the 8th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1, an amino acid corresponding to the 46th amino acid position from the N-terminus of the amino acid sequence of SEQ ID NO: 1, or a combination thereof, is replaced with an amino acid different from the original amino acid; a polynucleotide encoding the polypeptide; or a recombinant vector comprising the polynucleotide.

[0073] The variant lysine export protein, polynucleotide, recombinant vector, introduction (transformation), and microorganisms are as described above.

[0074] Another embodiment provides a method for producing L-amino acids, comprising culturing the aforementioned L-amino acid-producing microorganisms in a culture medium. The method may further include recovering the L-amino acids from the cultured microorganisms, the culture medium, or both after culturing.

[0075] In one embodiment, the L-amino acid may be L-lysine, L-arginine, or a combination thereof; specifically, it may be L-lysine.

[0076] In this application, the term "culture" refers to the growth of microorganisms (e.g., Corynebacterium spp.) that produce L-amino acids under appropriately controlled environmental conditions. The culture process can be carried out according to suitable culture media and conditions known in the art. Such a culture process can be easily adapted and used by those skilled in the art based on the selected strain. Specifically, the culture can be batch, continuous, and / or fed-batch, but is not limited thereto.

[0077] In this application, the term "culture medium" refers to a substance that primarily contains the nutrients required for culturing the L-amino acid-producing microorganisms (e.g., Corynebacterium spp.) of this application, and provides the water necessary for their survival and growth, as well as nutrients and growth factors. Specifically, the culture medium and other culture conditions used to cultivate the microorganisms of this application can be any culture medium commonly used for culturing microorganisms without particular limitation. However, the amino acid-producing microorganisms of this application (e.g., Corynebacterium spp.) can be cultured under aerobic conditions in a conventional culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc.

[0078] In this method, the cultivation of microorganisms is not particularly limited to this, but can be carried out by known batch culture methods, continuous culture methods, fed-batch culture methods, etc. In this case, the culture conditions are not particularly limited to this, but can be controlled by adjusting the appropriate pH (e.g., pH 5 to 9, specifically pH 6 to 8, most specifically pH 7) using alkaline compounds (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or acidic compounds (e.g., phosphoric acid or sulfuric acid), and aerobic conditions can be maintained by introducing oxygen or a mixture of oxygen-containing gases into the culture. The culture temperature can be maintained at 20 to 45°C or 25 to 40°C, and the culture can be carried out for approximately 10 to 160 hours, but is not limited to this. L-amino acids (e.g., L-lysine) produced by the culture can be secreted into the culture medium or can be retained intracellularly.

[0079] The culture medium used for culturing can be selected from media commonly used for culturing microorganisms. In one embodiment, the culture medium may use one or more of the following groups as carbon sources, alone or in combination of two or more: sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oils and fats (e.g., soybean oil, sunflower 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., but not limited thereto. As nitrogen sources, one or more of the following groups may be used, alone or in combination of two or more: nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal, and urea), inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), etc., but not limited thereto. As phosphorus sources, one or more of the following groups may be used, alone or in combination of two or more: potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium-containing salts, but not limited thereto. In addition, the culture medium may contain essential growth promoters, such as other metal salts (e.g., magnesium sulfate or ferric sulfate), amino acids, and / or vitamins.

[0080] Recovery of L-amino acids (e.g., L-lysine, L-arginine, or combinations thereof) can be performed by collecting the target amino acids from a culture medium, culture, or microorganism using suitable methods known in the art, depending on the culturing method. For example, recovery can be carried out by one or more methods selected from centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. The method may also include purification before, during, or after the recovery of L-amino acids (e.g., L-lysine, L-arginine, or combinations thereof).

[0081] Another embodiment of this application provides a composition for producing L-amino acids, comprising the microorganisms for producing L-amino acids, a culture medium for culturing the microorganisms, or a combination thereof.

[0082] The composition may also contain any suitable excipients commonly used in compositions for the production of L-amino acids, including, but not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0083] Another embodiment provides the use of the microorganism for L-amino acid production, and for conferring and / or increasing L-amino acid export capacity and / or L-amino acid production capacity.

[0084] Another embodiment provides the use of the microorganism in the preparation of compositions for the production of L-amino acids.

[0085] In the use of L-amino acid production, conferring and / or increasing L-amino acid export capacity and / or L-amino acid production capacity, and / or preparing compositions for L-amino acid production, the mutant lysine export protein, polynucleotide, recombinant vector, introduction (transformation), and microorganisms, etc., are as described above.

[0086] Another embodiment of this application provides a composition, method, product, process, or use, characterized by one or more elements disclosed in this disclosure.

[0087] Beneficial effects

[0088] This specification provides techniques for increasing the L-amino acid export capacity and / or L-amino acid production capacity of microorganisms by inducing mutations in heterologous proteins whose L-amino acid export capacity has been recently identified and introducing variants of such heterologous proteins into microorganisms, as well as techniques for improving L-amino acid productivity compared to parental strains. Detailed Implementation

[0089] The present application will be described in more detail below by way of embodiments. It will be apparent to those skilled in the art that these embodiments are intended only to explain the present application in more detail, but the scope of the present application is not limited to these embodiments which have the essential points of the present application.

[0090] Example

[0091] (Throughout this instruction manual, unless otherwise stated, the "%" used to indicate the concentration of a particular substance refers to (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.)

[0092] Example 1: Preparation of a mutant heterologous lysE gene vector library and transformed strains

[0093] To obtain heterologous lysE variant genes for searching for enzymes with enhanced L-lysine exporting capabilities, a library was prepared using the following method.

[0094] Table 1 below shows the amino acid sequence (SEQ ID NO: 1) of lysE (a membrane protein) from *Shewanella at Atlantica* (S. at), which was selected as the heterologous lysE. Information on the gene encoding this membrane protein (Table 1, SEQ ID NO: 2) and its surrounding nucleic acid sequence (accession number NZ_RXNV01000002.1) was obtained from GenBank, National Institutes of Health (NIH).

[0095] [Table 1]

[0096]

[0097] 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 wild-type *Shewanella Atlantica* (WT) as a template and primers SEQ ID NO: 9 and SEQ ID NO: 10 from Table 3 below. The reaction mixture containing the WT strain (*Shewanella Atlantica*) chromosome (500 ng), primers (125 ng each), Mutazyme II reaction buffer (1×), dNTP mixture (40 mM), and Mutazyme II DNA polymerase (2.5 U) was denatured at 94 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, and extension at 72 °C for 30 sec, and then extended at 72 °C for 10 min. As a result, a 664 bp gene fragment containing a 609 bp gene (SEQ ID NO: 2) was obtained.

[0098] To obtain the gapA promoter from Corynebacterium glutamicum, genomic DNA from Corynebacterium glutamicum ATCC 13032 was used as a template, and PCR (Solg™ Pfu-X DNA polymerase) was performed using primers from SEQ ID NO: 11 and SEQ ID NO: 12 in Table 3 below. The PCR conditions are as follows.

[0099] [Table 2]

[0100]

[0101] The gene fragment obtained through the above process was ligated to the pECCG117 vector (Korean Patent No. 10-0057684) obtained by treating with restriction enzyme BamHI-HF (NEB) at 37°C for 1 hour and with CIP (NEB) enzyme at 37°C for 30 minutes using the Gibson assembly method (DG Gibson et al., NATURE METHODS, Vol. 6, No. 5, May 2009, NEBuilder HiFi DNA Assembly Master Mix) .... The ligation was then performed on the pECCG117 vector (Korean Patent No. 10-0057684) using restriction enzyme BamHI-HF (NEB) at 37°C for 1 hour and then on CIP (NEB) enzyme at 37°C for 30 minutes. The vector was then transformed into E. coli DH5α and then plated on LB solid medium containing kanamycin (25 mg / L).

[0102] After selecting 30 transformed colonies, plasmids were obtained, and analysis of the nucleotide sequences confirmed the introduction of mutations at different positions at a frequency of 0.8 mutations / kb. Finally, approximately 10,000 transformed *E. coli* colonies were collected, plasmids were extracted using a plasmid preparation kit (QIAGEN), and named the p117-PgapA_lysE(sat.mt) library. Furthermore, to serve as a control in the screening, a vector was prepared in which *Shewanella at Atlantica* wild-type (WT) lysE was introduced into the pECCG117 vector. The wild-type lysE gene fragment was prepared by PCR using the sequences of SEQ ID NO: 9 and SEQ ID NO: 10, and the p117-PgapA_lysE(sat.WT) vector was prepared using the same method described above. The PCR conditions were 30 cycles of denaturation at 94°C for 2 minutes, followed by denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, and extension at 72°C for 30 seconds, and then extension at 72°C for 10 minutes.

[0103] The nucleic acid sequences of the primers used are summarized in Table 3 below:

[0104] [Table 3]

[0105]

[0106] Example 2: Preparation and screening of strains for insertion into vector libraries

[0107] To prepare a strain with the lysE gene deleted from wild-type Corynebacterium glutamicum ATCC13032, a vector for deleting the lysE gene was prepared. Specifically, a recombinant vector was prepared in which DNA fragments (600 bp each) located at the 5′ and 3′ ends of the lysE gene were ligated into a pDZ vector (Korean Patent No. 2009-0094433). Based on the nucleotide 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, respectively, were synthesized at positions 600 bp apart (see Table 4 below).

[0108] PCR was performed using the chromosome of *Corynebacterium glutamicum* ATCC13032 as a template and primers SEQ ID NO: 13 and SEQ ID NO: 15 to prepare a gene fragment at the 5′ end of the lysE gene. Similarly, PCR was performed using primers SEQ ID NO: 14 and SEQ ID NO: 16 to prepare a gene fragment at the 3′ end of the lysE gene. PCR was performed using 30 cycles of denaturation at 94°C for 2 min, followed by 1 min of denaturation at 94°C, 1 min of annealing at 56°C, and 30 s of extension at 72°C, followed by a final extension at 72°C for 10 min. The amplified DNA fragments were purified using a Qiagen PCR purification kit and then used as insert DNA fragments for vector construction.

[0109] On the other hand, after treatment with restriction enzyme XbaI and heat treatment at 65°C for 20 minutes, the pDZ vector (Korean Patent No. 2009-0094433) and the inserted DNA fragment prepared by the above PCR amplification were ligated using the Infusion cloning kit, and then transformed into E. coli DH5α and plated on LB solid medium containing kanamycin (25 mg / L). Plasmids were obtained from colonies transformed with vectors containing the target gene inserted by PCR using primers of SEQ ID NO: 13 and SEQ ID NO: 14, using conventionally known plasmid extraction methods, and named pDZ-ΔlysE.

[0110] The nucleic acid sequences of the primers used are summarized in Table 4 below:

[0111] [Table 4]

[0112]

[0113] The prepared vector pDZ-ΔlysE was transformed into *Corynebacterium glutamicum* ATCC13032 using electroporation (Vander Rest et al., *Appl. Microbiol. Biotocol.* 52:541-545, 1999) to prepare a strain in which the lysE gene was deleted through homologous chromosome recombination. This strain with the lysE gene deleted in this manner was named *Corynebacterium glutamicum* 13032::ΔlysE. The p117-PgapA_lysE(sat.mt) library prepared in Example 1 was transformed into the above-mentioned 13032::ΔlysE using electroporation, and approximately 5,000 colonies were obtained by platenling on composite agar plates containing kanamycin (25 mg / L). To prepare a control group, the p117-PgapA_lysE(sat.WT) vector was transformed into 13032::ΔlysE in the same manner to obtain colonies.

[0114] <Compound Plate Culture Medium (pH 7.0)>

[0115] 10 g glucose, 10 g peptone, 5 g beef extract, 5 g yeast extract, 18.5 g brain and heart extract, 2.5 g NaCl, 2 g urea, 91 g sorbitol, 20 g agar (based on 1 liter of distilled water)

[0116] The obtained control group libraries 13032::ΔlysE_p117-PgapA_lysE(sat.WT) and 13032::ΔlysE_p117-PgapA_lysE(sat.mt) were inoculated into 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.

[0117] Seed culture medium (pH 7.0)

[0118] 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, HCl thiamine 1 mg, calcium pantothenate 22 mg, nicotinamide 2 mg (based on 1 liter of distilled water)

[0119] When approximately 5,000 cultured colonies were serially diluted on a composite plate medium containing 100 g / L L-lysine hydrochloride and subjected to MIC (minimum inhibitory concentration) testing, 12 colonies with significantly increased MIC values ​​compared to the control strain were obtained. Secondary screening was performed on each colony. Each colony was inoculated into a 96-well round-bottom plate (Bioneer) containing 400 μL of seed medium and incubated in a plate shaking incubator (TAITEC) at 32°C and 12,000 rpm for approximately 12 hours. Furthermore, in the secondary screening step, the initial OD (optical density) values ​​of the final cultured colonies were adjusted to be the same, and they were serially diluted on a composite plate medium containing 100 g / L L-lysine hydrochloride for MIC testing. Thus, three strains exhibiting significantly higher MIC values ​​compared to the control strain containing the wild-type heterologous lysE library were named 13032::lysE(sat.mt), and further experiments were conducted.

[0120] Example 3: Confirmation of the mutated lysE gene base sequence

[0121] To confirm the gene sequence inserted into strain 13032::lysE(mt) selected in Example 2, PCR was performed using primers of SEQ ID NO: 17 and SEQ ID NO: 18 in Table 5 to amplify the gene fragment. PCR was performed under the same conditions as in Example 1, and the amplified DNA fragment was obtained using a GeneAll Expin GEL SV kit (Seoul, South Korea) for base sequence analysis.

[0122] The nucleic acid sequences of the primers used are summarized in Table 5 below:

[0123] [Table 5]

[0124]

[0125] As a result of the analysis of the base sequence of the amplified gene, it was found that the first 13032::lysE (sat.mt) strain contained a mutated heterologous lysE gene encoding a variant of the L-lysine export protein. In this variant, the 22nd to 24th bases downstream of the start codon of the open reading frame (ORF) of the Shewanella Atlantica lysE gene were changed from GTA to GCA, thereby replacing the 8th amino acid (valine) from the N-terminus of the wild-type lysE amino acid sequence (SEQ ID NO: 1) of Shewanella Atlantica with alanine (V8A). It can be confirmed that the second strain 13032::lysE(sat.mt) contains a mutated heterologous lysE gene encoding a variant of the L-lysine export protein. In this variant, the 136th to 138th base sequence downstream of the start codon of the ORF of the Shewanella Atlantica lysE gene is changed from GAT to GGT, thereby replacing the 46th amino acid (D46G) from the N-terminus of the wild-type lysE amino acid sequence (SEQ ID NO:1) of Shewanella Atlantica with glycine. Finally, it can be confirmed that the third strain 13032::lysE(sat.mt) contains a mutated heterologous lysE gene encoding an L-lysine export protein, in which the 22nd to 24th base sequence downstream of the ORF of the Shewanella Atlantica lysE gene is changed from GTA to GCA, and the 136th to 138th base sequence is changed from GAT to GGT. This replaces the 8th amino acid from the N-terminus of the wild-type Shewanella Atlantica lysE amino acid sequence (SEQ ID NO: 1) with alanine (A) (V8A) and replaces the 46th amino acid from the N-terminus with aspartic acid (D) (G) (D46G).

[0126] Example 4: Construction of a mutant heterologous lysE gene transfer vector and preparation of bacterial strains

[0127] 4-1. Construction of a heterologous lysE gene insertion vector with V8A mutation and preparation of bacterial strains

[0128] To introduce the mutant V8A confirmed in Example 3, a recombinant vector was prepared using the following method. First, using the p117-PgapA_lysE(sat.WT) vector containing the gapA promoter as a template, primers SEQ ID NO: 19 and SEQ ID NO: 20, containing the restriction enzyme ScaI recognition site, were synthesized targeting the 5′ and 3′ fragments located approximately 500 bp upstream and downstream of positions 22 to 24 of the heterologous lysE gene (SEQ ID NO: 2), respectively (see Table 6). Furthermore, primers SEQ ID NO: 21 and SEQ ID NO: 22, for introducing base substitution mutations, were synthesized at positions 600 bp away from the aforementioned sites (see Table 6).

[0129] [Table 6]

[0130]

[0131] The amplified gapA promoter region and a gene fragment containing the aforementioned mutation from *Shewanella at Atlantica* 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). This ligation was then performed on *E. coli* DH5α and plated on LB agar containing kanamycin (25 mg / L). To select colonies transformed with the vector containing the target gene and pDZTn, PCR was performed using primers of SEQ ID NO: 23 and SEQ ID NO: 24 (see Table 7). Plasmids were obtained from the selected colonies using standard plasmid extraction methods and named pDZTn-PgapA_lysE(sat.V8A). The prepared vector was transformed into the lysine-producing Corynebacterium glutamicum CJ3P strain (US Patent 9556463 ​​B2) using electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541–545), and after a second exchange, a strain with PgapA-Sat inserted between the transposons was obtained. PCR and sequencing were performed using primers of SEQ ID NO: 23 and SEQ ID NO: 24 (see Table 7), which amplified the adjacent region including the gene insertion site, confirming the genetic manipulation. The Corynebacterium glutamicum strain with the heterologous lysE gene with the V8A mutation introduced in this manner was named CJ3P::lysE(sat.V8A).

[0132] [Table 7]

[0133]

[0134] 4-2. Construction of the D46G mutant heterologous lysE gene insertion vector and preparation of bacterial strains

[0135] To introduce the D46G mutation confirmed in Example 3, a recombinant vector was prepared using the following method. First, using the p117-PgapA_lysE(sat.WT) vector containing the gapA promoter as a template, primers SEQ ID NO: 25 and SEQ ID NO: 26, containing the restriction enzyme ScaI recognition site, were synthesized targeting the 5′ and 3′ fragments located approximately 500 bp upstream and downstream of positions 136 to 138 of the heterologous lysE gene, respectively. Furthermore, primers SEQ ID NO: 27 and SEQ ID NO: 28, for introducing base substitution mutations, were synthesized at positions 600 bp away from the aforementioned sites (see Table 8 below).

[0136] [Table 8]

[0137]

[0138] The amplified gapA promoter region and a gene fragment containing the aforementioned mutation from *Shewanella at Atlantica* 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). This ligation was then performed on *E. coli* DH5α and plated on LB agar containing kanamycin (25 mg / L). To select colonies transformed with the vector containing the target gene and pDZTn, PCR was performed using primers of SEQ ID NO: 23 and SEQ ID NO: 24 (Table 7 above). Plasmids were obtained from the selected colonies using standard plasmid extraction methods and named pDZTn-PgapA_lysE(sat.D46G).

[0139] The prepared vector was transformed into the lysine-producing Corynebacterium glutamicum CJ3P strain (US Patent 9556463 ​​B2) using electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541–545), and after a second exchange, a strain with PgapA-Sat inserted between the transposons was obtained. PCR and sequencing were performed using primers of SEQ ID NO: 23 and SEQ ID NO: 24 (Table 7 above), which amplified the adjacent region including the gene insertion site, confirming the genetic manipulation. The strain with the heterologous lysE gene containing the D46G mutation introduced in this manner was named CJ3P::lysE(sat.D46G).

[0140] 4-3. Construction of heterologous lysE gene insertion vectors with V8A and D46G mutations and preparation of bacterial strains

[0141] To introduce the mutants V8A and D46G confirmed in Example 3, recombinant vectors were prepared using the following method. First, using the p117-PgapA_lysE(sat.WT) vector containing the gapA promoter as a template, primers SEQ ID NO: 29 and SEQ ID NO: 30, containing the restriction enzyme ScaI recognition site, were synthesized targeting the 5′ and 3′ fragments located approximately 500 bp upstream and downstream of the heterologous lysE gene at bases 22-24 and 136-138, respectively (see Table 9 below). Furthermore, primers SEQ ID NO: 33 and SEQ ID NO: 34, used to introduce base substitution mutations, were synthesized at positions 600 bp away from the aforementioned sites (see Table 9 below).

[0142] [Table 9]

[0143]

[0144] The amplified gapA promoter region and a gene fragment containing the aforementioned mutation from *Shewanella at Atlantica* 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). This ligation was then performed on *E. coli* DH5α and plated on LB agar containing kanamycin (25 mg / L). To select colonies transformed with the vector containing the target gene and pDZTn, PCR was performed using primers of SEQ ID NO: 23 and SEQ ID NO: 24 (Table 7 above). Plasmids were obtained from the selected colonies using standard plasmid extraction methods and named pDZTn-PgapA_lysE(sat.V8A,D46G).

[0145] The prepared vector was transformed into the lysine-producing Corynebacterium glutamicum CJ3P strain (US Patent 9556463 ​​B2) using electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541–545), and after a second exchange, a strain with PgapA-Sat inserted between the transposons was obtained. PCR and base sequence analysis were performed using primers of SEQ ID NO: 23 and SEQ ID NO: 34, which amplified the adjacent region including the gene insertion site, confirming the genetic manipulation. In this manner, the strain with the heterologous lysE gene containing the V8A and D46G mutations was named CJ3P::lysE(sat.V8A,D46G).

[0146] Example 5: Analysis of the growth and L-lysine production capacity of strains with introduced mutant heterologous lysE gene in production medium.

[0147] The three strains prepared 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 quality and lysine production capacity in neutral pH medium were compared.

[0148] First, each strain was inoculated into a 250 mL corner-baffle flask containing 25 mL of seed culture medium and cultured at 30°C with shaking at 200 rpm for 20 hours. Then, 1 mL of the seed culture was inoculated into a 250 mL corner-baffle flask containing 24 mL of production culture medium and cultured at 37°C with shaking at 200 rpm for 40 hours. After culturing, the yield of L-lysine was measured by HPLC. The above experiment was repeated three times.

[0149] Seed culture medium (pH 7.0)

[0150] Raw sugar 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4•7H2O 0.5 g, biotin 0.1 mg, HCl thiamine 1 mg, calcium pantothenate 2 mg, nicotinamide 2 mg (based on 1 liter of distilled water)

[0151] <Production medium (pH 7.0)>

[0152] Raw sugar 100 g, (NH4)2SO4 40 g, soybean protein 2.5 g, corn steep liquor solids 5 g, urea 3 g, KH2PO4 1 g, MgSO4•7H2O 0.5 g, biotin 100 ug, thiamine hydrochloride 1000 ug, calcium pantothenate 2000 ug, nicotinamide 3000 ug, CaCO3 30 g (based on 1 liter of distilled water).

[0153] 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) are compared and shown in Table 10 below.

[0154] [Table 10]

[0155] OD (600 nm) measurements and lysine productivity of CJ3P, CJ3P::lysE(sat.WT), CJ3P::lysE(sat.V8A), CJ3P::lysE(sat.D46G), and CJ3P::lysE(sat.V8A,D46G).

[0156]

[0157] As shown in Table 10 above, the mutant strain exhibited superior levels in cell density and lysine concentration compared to the parental strains CJ3P and CJ3P::lysE(sat.WT), thus confirming that its cell viability (survival rate) and lysine production capacity during culture were superior to those of the parental strains.

[0158] This confirms that the mutation in LysE from Shewanella Atlantica increases lysine export, leading to increased cell viability (survival rate) and lysine production capacity.

[0159] Based on the above description, those skilled in the art will understand that this application can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, the above embodiments should be understood in all respects as illustrative rather than restrictive. The scope of this application should be interpreted to include all changes or modifications (not the detailed description above) derived from the meaning and scope of the foregoing claims and their equivalents.

Claims

1. A polypeptide wherein the amino acid at the 8th amino acid position from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at the 46th amino acid position from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 1, or a combination thereof, is replaced with an amino acid different from the original amino acid.

2. The polypeptide according to claim 1, wherein the polypeptide is: The polypeptide is formed by replacing the amino acid corresponding to the 8th amino acid position 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 replaced with glutamic acid, glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, glutamine, histidine, lysine, arginine, or asparagine; or The polypeptide is formed by replacing the amino acid corresponding to the 8th amino acid position with glutamic acid, glycine, alanine, serine, threonine, cysteine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamine, histidine, lysine, arginine, or asparagine, and replacing the amino acid corresponding to the 46th amino acid position with glutamic acid, glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, glutamine, histidine, lysine, arginine, or asparagine.

3. The polypeptide according to claim 1 or 2, which has the function of an L-amino acid exporting protein.

4. The polypeptide according to claim 1 or 2, wherein the polypeptide of the amino acid sequence of SEQ ID NO: 1 is derived from Shewanella Atlantica.

5. A polynucleotide encoding a polypeptide according to claim 1 or 2.

6. The polynucleotide of claim 5, wherein the polynucleotide is represented by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6 to 8.

7. A recombinant vector comprising the polynucleotide according to claim 5.

8. A microorganism for producing L-amino acids, comprising the polypeptide according to claim 1, a polynucleotide encoding the polypeptide, or a recombinant vector comprising the polynucleotide.

9. The microorganism for producing L-amino acids according to claim 8, wherein the polypeptide functions as an L-amino acid exporting protein.

10. The microorganism for producing L-amino acids according to claim 8, wherein the microorganism for producing L-amino acids is a microorganism of the genus Corynebacterium or Escherichia.

11. The microorganism for producing L-amino acids according to claim 10, wherein the microorganism is Corynebacterium glutamicum or Escherichia coli.

12. The microorganism for producing L-amino acids according to any one of claims 8 to 11, wherein the microorganism has an increased L-amino acid export capacity or L-amino acid production capacity compared with unmodified microorganisms.

13. The microorganism for producing L-amino acids according to any one of claims 8 to 11, wherein the microorganism has increased strain activity compared to unmodified microorganisms.

14. The microorganism for producing L-amino acids according to claim 9, wherein the L-amino acid is L-lysine.

15. A method for producing L-amino acids, comprising culturing a microorganism for producing L-amino acids according to any one of claims 8 to 11 in a culture medium.

16. The method for producing L-amino acids according to claim 15, further comprising recovering the L-amino acids from the cultured microorganisms, culture medium, or both after culturing.

17. The method for producing L-amino acids according to claim 15, wherein the L-amino acid is L-lysine.

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

19. A composition, method, product, process, or use, characterized in that... One or more elements disclosed in this disclosure.

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