Microorganism expressing a protein derived from Shewanella oneidensis and a method for producing L-amino acids using the same

Abstract

A microorganism capable of expressing a foreign protein and a method for producing an L-amino acid using the same are provided. The microorganism capable of expressing the foreign protein may have improved L-amino acid secretion and / or production capabilities compared to a wild-type microorganism.

Description

[Technical Field]

[0001] The present invention relates to a microorganism capable of expressing a foreign protein and a method for producing an L-amino acid using the same. The microorganism capable of expressing the foreign protein may have improved L-amino acid secretion and / or production capabilities compared to the wild-type microorganism. [Background technology]

[0002] The genus Corynebacterium is a Gram-positive microorganism that is widely used for the production of L-amino acids, which are used in animal feed, human pharmaceuticals, and cosmetics industries, and are produced by fermentation using Corynebacterium strains.

[0003] Many attempts have been made to improve methods for producing L-amino acids using Corynebacterium strains. Among these, research has been conducted to improve Corynebacterium strains that produce L-amino acids by disrupting or attenuating the expression of specific genes using recombinant DNA technology. Furthermore, many studies have been conducted to improve L-amino acid-producing Corynebacterium strains by amplifying genes involved in the biosynthesis of each L-amino acid and studying the effect on L-amino acid production. Despite these efforts, there is still a need for the development of strains with improved L-amino acid production capabilities. Summary of the Invention [Problem to be solved by the invention]

[0004] One embodiment of the present invention provides a microorganism expressing a foreign protein. The foreign protein may be a protein derived from a microorganism of a different species from the microorganism, and may be a protein newly identified in the present invention for its lysine excretion function. The microorganism expressing the foreign protein may have improved L-amino acid excretion and / or production capabilities compared to the same microorganism not expressing the foreign protein.

[0005] Another embodiment provides a composition for producing an L-amino acid, comprising a microorganism expressing the exogenous protein.

[0006] Another embodiment provides a method for producing an L-amino acid, comprising culturing a microorganism that expresses the exogenous protein.

[0007] The microorganism may be a Corynebacterium microorganism. [Means for solving the problem]

[0008] In this specification, we have sought to improve the L-amino acid production ability of Corynebacterium microorganisms by searching for and introducing novel L-amino acid export proteins. Taking L-lysine as a representative example, methods for increasing lysine production generally involve improving the L-lysine yield of the strain or increasing the amount of L-lysine produced per hour (productivity). L-lysine export proteins are membrane proteins that export lysine produced through biosynthesis, and improving these proteins is important for increasing lysine yield and productivity. However, there is a limit to how much the expression of L-lysine export proteins (lysE, Ncgl1214) possessed by Corynebacterium microorganisms can improve L-lysine production ability.

[0009] Therefore, in this specification, we have explored new foreign L-amino acid export proteins with high L-amino acid export activity and introduced them into lysine-producing strains to provide recombinant strains with improved L-amino acid export and / or production capabilities.

[0010] In this specification, we have discovered a novel L-amino acid export protein derived from Shewanella oneidensis and the gene encoding it as a representative example of a new foreign L-amino acid export protein. When this protein was expressed in a microorganism that produces L-amino acids, we confirmed that the L-amino acid export / production ability was significantly improved compared to a microorganism that does not express the gene.

[0011] One embodiment provides a microorganism expressing a foreign protein. The foreign protein may be a protein derived from a microorganism of a different species from the microorganism, and may be a protein newly identified herein as having a lysine excretion function. The microorganism expressing the foreign protein may have improved ability to excrete and / or produce one or more amino acids selected from the group consisting of L-lysine, L-arginine, and L-histidine, compared to the same microorganism not expressing the foreign protein.

[0012] Another embodiment provides a composition for producing an L-amino acid, comprising a microorganism expressing the exogenous protein.

[0013] Another embodiment provides a method for producing an L-amino acid, comprising culturing a microorganism that expresses the exogenous protein.

[0014] The microorganism may be a Corynebacterium microorganism.

[0015] The L-amino acid can include one or more amino acids selected from the group consisting of L-lysine, L-arginine, and L-histidine.

[0016] This will be explained in more detail below.

[0017] In this specification, the foreign protein may be a protein derived from a microorganism belonging to a different genus or species from that of the parent strain (pre-mutation microorganism), for example, a membrane protein derived from a microorganism other than a microorganism belonging to the genus Corynebacterium, and may be a protein having the ability to secrete one or more amino acids selected from the group consisting of L-amino acids, such as L-lysine, L-arginine, and L-histidine, which have been newly identified herein. In one example, the foreign protein may be a membrane protein (e.g., represented by the amino acid sequence of SEQ ID NO: 1) derived from a Shewanella microorganism, such as Shewanella oneidensis; or a protein (e.g., a membrane 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 to the protein.

[0018] The microorganism may contain or express one or more of the foreign proteins. The microorganism expressing the foreign protein may be a recombinant microorganism into which a polynucleotide encoding the foreign protein has been introduced. In one example, the polynucleotide encoding the protein of SEQ ID NO: 1 may be expressed as the nucleic acid sequence of SEQ ID NO: 2 or a sequence 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 to the nucleic acid sequence of SEQ ID NO: 2.

[0019] The microorganism expressing the foreign protein may be an L-amino acid-producing microorganism having the ability to secrete and / or produce L-amino acids.

[0020] As used herein, the term "L-amino acid-producing microorganism" may refer to a microorganism having the ability to secrete and / or produce L-amino acids, which has been mutated to express a foreign protein as described above, thereby increasing the ability to secrete and / or produce L-amino acids, and / or a microorganism not having the ability to secrete and / or produce L-amino acids, which has been mutated to express the foreign protein, thereby acquiring the ability to secrete and / or produce L-amino acids. As used herein, the term "microorganism" encompasses unicellular bacteria and may be used interchangeably with "cell." In order to distinguish a microorganism before being mutated to express the foreign protein from a microorganism after being mutated, the microorganism may be referred to as a "parent microorganism or parent strain" or a "host cell."

[0021] The L-amino acid may be one or more amino acids selected from the group consisting of L-lysine, L-arginine, and L-histidine, for example, L-lysine.

[0022] In one example, the microorganism can be selected from all microorganisms having the ability to secrete and / or produce L-amino acids. In one example, the microorganism, for example, the parent strain before mutation, can be (1) a microorganism that naturally has the ability to secrete and / or produce L-amino acids, or (2) a microorganism that has the ability to secrete and / or produce L-amino acids or has an improved ability to secrete and / or produce L-amino acids by introducing a mutation into the microorganism naturally having the ability to secrete and / or produce L-amino acids or a strain that has no or significantly reduced ability to secrete and / or produce L-amino acids.

[0023] In one embodiment, the microorganism may be one or more selected from the group consisting of (1) microorganisms that naturally have the ability to excrete and / or produce L-amino acids, or (2) all microorganisms of the genus Corynebacterium that have been given L-amino acid excretion and / or production ability or have improved L-amino acid excretion and / or production ability by introducing a mutation into a microorganism that naturally has the ability to excrete and / or produce L-amino acids or a parent strain that has no or significantly reduced L-amino acid excretion and / or production ability. The Corynebacterium microorganism may include, but is not limited to, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes, Corynebacterium efficiens, etc. More specifically, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0024] In one example, the microorganism expressing the foreign protein may be a microorganism into which a mutation that allows the expression of the foreign protein has been introduced. Thus, a microorganism that has been mutated to express a foreign protein may have increased L-amino acid secretion and / or production capabilities compared to an unmodified microorganism of the same species. The unmodified microorganism is a microorganism that does not express the foreign protein, and may refer to a microorganism of the same species that does not have a mutation that allows the expression of the foreign protein introduced therein, or a microorganism before the mutation was introduced.

[0025] As used herein, the term "mutation that allows expression of a foreign protein" refers to any manipulation that allows a parent strain to express the foreign protein. In one example, the mutation that allows expression of a foreign protein may involve introducing a polynucleotide encoding the foreign protein or a recombinant vector containing the same into the parent strain.

[0026] The "microorganism into which a mutation that allows it to express a foreign protein has been introduced" or "a microorganism mutated to express a foreign protein" may be a microorganism into which a polynucleotide encoding the foreign protein or a recombinant vector containing the same has been introduced, and may be conferred or enhanced with L-amino acid secretion and / or production capabilities compared to unmodified microorganisms.

[0027] In one example, the parent strain may be a wild-type strain or may be mutated to increase its ability to excrete and / or produce L-amino acids, for example, a strain in which the activity of proteins involved in the biosynthesis or metabolism of L-amino acids is modulated (increased (promoted) or decreased (inhibited)) compared to the wild-type strain, but is not limited thereto.

[0028] In one embodiment, the L-amino acid-producing microorganism in which the foreign protein is expressed may be the microorganism with accession number KCCM12827P.

[0029] As used herein, the phrase "a polynucleotide (which may be used interchangeably with "gene") or a polypeptide (which may be used interchangeably with "protein") that "comprises, consists of, or is expressed by a specific nucleic acid sequence or amino acid sequence" is an expression that has an equivalent meaning and is interchangeable, and may mean that the polynucleotide or polypeptide essentially contains the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including a "substantially equivalent sequence" in which mutations (deletions, substitutions, modifications, and / or additions) have been made to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or intended function of the polynucleotide or polypeptide is maintained (or as not excluding the mutations).

[0030] In one example, the nucleic acid sequences or amino acid sequences provided herein may be modified by conventional mutagenesis methods, such as directed evolution and / or site-directed mutagenesis, to the extent that their original or intended functions are maintained. Unless otherwise specified herein, a polynucleotide or polypeptide "comprising or consisting of a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide (i) essentially contains the specific nucleic acid sequence or amino acid sequence, or (ii) consists of or essentially contains an amino acid sequence that has 60% or more, 65% 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 homology to the specific nucleic acid sequence or amino acid sequence, and maintains its original and / or intended functions. As used herein, the original function may be a lysine export protein function (in the case of an amino acid sequence) or a function encoding a protein having the lysine export protein function (in the case of a nucleic acid sequence), and the target function may refer to a function of increasing or imparting the ability of a microorganism to export and / or produce an L-amino acid (e.g., L-lysine, L-arginine, L-histidine, or a combination thereof) to the microorganism.

[0031] As used herein, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid or nucleotide sequences, and may be expressed as a percentage. The terms homology and identity may sometimes be used interchangeably. Sequence homology or identity of conserved polynucleotides or polypeptides can be determined using standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences generally hybridize over at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or entire length under moderately or highly stringent conditions. Hybridization obviously also includes polynucleotides containing common codons or codons that take codon degeneracy into account.

[0032] 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, it can be determined using 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), (GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387(1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,][ET AL, J MOLEC BIOL 215]:403(1990); Guide to Huge Computers, Martin J. Bishop, [Ed.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48:1073. For example, BLAST from the National Database Center for Biotechnology Information, or ClustalW, can be used to determine homology, similarity, or identity.

[0033] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that of Needleman et al. (1970), J Mol Biol. 48:443, as known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program can be defined as the number of similar aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0034] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions. Defining appropriate hybridization conditions is 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).

[0035] The nucleic acid sequences described herein may be modified in various ways in the coding region, taking into account the codons preferred by the microorganism in which the protein (lysine export protein) is to be expressed due to codon degeneracy, as long as the amino acid sequence and / or function of the protein expressed from the coding region is not altered.

[0036] In one example, a polynucleotide comprising a specific nucleic acid sequence provided herein can be interpreted as including not only the specific nucleic acid sequence or a nucleic acid sequence substantially equivalent thereto, but also a polynucleotide fragment comprising a nucleic acid sequence complementary to the specific nucleic acid sequence. Specifically, the complementary polynucleotide can hybridize at a Tm value that can be appropriately adjusted by those skilled in the art depending on the purpose, for example, a Tm value of 55°C, 60°C, 63°C, or 65°C, and can be analyzed under the conditions described below; such conditions are specifically described in known literature. For example, the hybridization conditions may be such that genes with high complementarity of 60% or more, 65% 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, 98% or more, 99.5% or more, or 99.9% or more hybridize with each other, but genes with lower complementarity do not hybridize with each other, or the washing conditions for ordinary Southern hybridization, which are 60°C, 1×SSC (saline-sodium citrate buffer), and 0.1% (w / v) SDS (sodium dodecyl sulfate). Sulfate; 60°C, 0.1×SSC, and 0.1% SDS; or 68°C, 0.1×SSC, and 0.1% SDS, followed by washing once, specifically two to three times. Hybridization requires that two nucleotides have complementary sequences, although mismatches between bases may be tolerated depending on the stringency of hybridization. The term "complementary" can be used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and is well known in the relevant art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0037] Introduction of the polynucleotide or vector can be performed by those skilled in the art using a known transformation method. As used herein, the term "transformation" refers to the introduction of a polynucleotide encoding a target protein (foreign protein) or a vector containing the polynucleotide into a host cell, thereby enabling the expression of the protein encoded by the polynucleotide in the host cell. A transformed polynucleotide can include any polynucleotide, whether it is located intrachromosomally or extrachromosomally, as long as it can be expressed in the host cell. The polynucleotide can also include DNA and / or RNA encoding the target protein. The polynucleotide may be introduced into a host cell in any form, as long as it can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette typically includes expression regulatory elements, such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal, operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Alternatively, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell. The term "operably linked" as used herein may mean that the polynucleotide is functionally linked to an expression regulatory element (e.g., a promoter) so that the expression regulatory element can regulate transcription (e.g., transcription initiation) of the polynucleotide encoding the target protein (foreign protein). Operable linkage can be achieved using recombinant DNA techniques known in the art, such as, but not limited to, conventional site-specific DNA cleavage and ligation.

[0038] The method for transforming the polynucleotide into a host cell can be carried out by any method for introducing nucleic acids into cells (microorganisms), and can be carried out by appropriately selecting a transformation technique known in the art depending on the host cell. Known transformation methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation (polyethylene glycol-mediated uptake), DEAE-dextran, cationic liposome method, lipofection, and lithium acetate-DMSO method.

[0039] The polynucleotide can be introduced (inserted) into the host cell genome (chromosome) using a known method that can be appropriately selected by those skilled in the art. For example, the polynucleotide can be introduced (inserted) into the host cell genome (chromosome) using, but is not limited to, an RNA-guided endonuclease system (RNA-guided endonuclease system or CRISPR system; for example, a mixture containing (a) an RNA-guided endonuclease (e.g., Cas9 protein), a gene encoding the same, or a vector containing the gene; and (b) a guide RNA (e.g., single guide RNA (sgRNA)), a DNA encoding the same, or a vector containing the DNA (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA), a complex (e.g., a ribonucleic acid fusion protein (RNP)), a recombinant vector (e.g., a vector containing both a gene encoding an RNA-guided endonuclease and a DNA encoding a guide RNA), etc.).

[0040] As used herein, the term "vector" refers to a DNA construct containing a polynucleotide sequence encoding a target protein operably linked to a suitable regulatory sequence so as to enable the expression of the target protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal 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 host cell's genome (genetic body) or may be integrated into the host cell's genome.

[0041] The vectors usable herein are not particularly limited as long as they are replicable in host cells, and can be selected from any commonly used vector. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, bacteriophages, and the like. For example, phage or cosmid vectors such as pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used. Plasmid vectors such as pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used. Specific examples include, but are not limited to, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors.

[0042] The vectors usable herein may be known expression vectors and / or vectors for inserting polynucleotides into host cell chromosomes. Insertion of the polynucleotide into a host cell chromosome can be achieved by any method known in the art, including, but not limited to, homologous recombination or the CRISPR system. The vector may further include a selection marker for confirming the presence or absence of the chromosomal insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the polynucleotide insertion. It can be used to select among genes that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing for the selection of transformed cells.

[0043] Another example provides a method for increasing the L-amino acid excretion and / or production ability of a microorganism or a method for imparting L-amino acid excretion and / or production ability to a microorganism, the method comprising the step of introducing (transforming) the foreign protein, a polynucleotide encoding it, or a recombinant vector containing the polynucleotide into the microorganism.

[0044] The foreign protein, polynucleotide, and microorganism are as described above.

[0045] Another example provides a method for producing an L-amino acid, comprising culturing the L-amino acid-producing microorganism in a medium, which may further comprise recovering the L-amino acid from the cultured microorganism, the medium, or both.

[0046] In the above method, the step of culturing the microorganism is not particularly limited, and 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, but a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid) can be used to adjust the pH to an appropriate level (e.g., pH 5-9, specifically pH 6-8, most specifically pH 6.8), and oxygen or an oxygen-containing gas mixture can be introduced into the culture to maintain aerobic conditions. The culture temperature can be maintained at 20-45°C or 25-40°C, and the culture can be carried out for approximately 10-160 hours, but is not limited thereto. The L-amino acid produced by the culture can be secreted into the medium or remain intracellularly.

[0047] The media usable for the culture may include, but are not limited to, one or more carbon sources 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 linolenic acid), alcohols (e.g., glycerol and ethanol), and organic acids (e.g., acetic acid), either individually or in combination. The nitrogen sources may include, but are not limited to, one or more nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea), and inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), either individually or in combination. The phosphorus source may be one or more selected from the group consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium-containing salts, and may be used individually or in combination. The medium may also contain other essential growth-promoting substances such as metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, and / or vitamins.

[0048] The step of recovering the L-amino acid may involve collecting the target amino acid from the culture medium, culture solution, or microorganism using a suitable method known in the art depending on the culture method. For example, the recovery step may be performed by one or more methods selected from centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. The method of recovering the L-amino acid may further include a purification step before, during, or after the recovery step. [Effects of the Invention]

[0049] The present invention provides a technique for increasing the L-amino acid secretion and / or production ability of microorganisms, and for this purpose, provides a foreign protein that has been newly identified as having the ability to secrete L-amino acids, and a technique for improving L-amino acid productivity compared to a parent strain by introducing the foreign protein into a microorganism. DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It will be obvious to those skilled in the art that the examples described below can be modified within the scope of the essential gist of the invention.

[0051] Example 1: Searching and screening for L-lysine efflux genes To search for an exporter with higher L-lysine export activity than the endogenous L-lysine exporter (lysE, Ncgl1214) of Corynebacterium spp., we performed an RPS-BLAST search using the NCBI CDD (Common Domain Database) and a BLAST search using the KEGG Protein Database. Candidate proteins that could be considered membrane proteins capable of exporting L-lysine were selected.

[0052] [Table 1]

[0053] Example 2: Construction of a foreign membrane protein gene transfer vector The membrane protein from Shewanella oneidensis (Son) selected in Example 1 has the amino acid sequence of SEQ ID NO: 1. Information on the gene encoding this membrane protein and its surrounding nucleic acid sequence (accession number NC_004347.2) was obtained from the National Institutes of Health GenBank. DNA was synthesized based on this gene sequence (Cosmo Genetech, Korea). To amplify the gene, PCR (Solg™ Pfu-X DNA polymerase) was performed using the synthesized DNA as a template and primers SEQ ID NOs: 3 and 4 (Table 2) (Table 3). As a result, a 651-bp gene fragment containing a 621-bp gene (SEQ ID NO: 2) was obtained.

[0054] [Table 2]

[0055] [Table 3]

[0056] To isolate the gapA promoter from Corynebacterium glutamicum, PCR (Solg™ Pfu-X DNA polymerase) was performed using the genomic DNA of Corynebacterium glutamicum ATCC 13032 as a template and primers SEQ ID NOs: 5 and 6 (Table 2) (Table 3). The amplified gapA promoter region was ligated with the obtained Shewanella oneidensis gene fragment and the vector pDZTn (Korean Patent No. 10-1126041) digested with NdeI restriction enzyme using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix). The ligated vector was then transformed into E. coli DH5α and plated on LB solid medium containing kanamycin (25 mg / L). To select colonies transformed with the vector containing the target gene and pDZTn, PCR was performed using primers shown in SEQ ID NOs: 7 and 8 (Table 2). A plasmid was isolated from the selected colonies using a conventional plasmid extraction method, and this plasmid was designated pDZTn-PgapA-Son.

[0057] Example 3: Construction of strains into which foreign membrane proteins have been introduced The constructed pDZTn-PgapA-Son vector was used to transform the L-lysine-producing Corynebacterium glutamicum KCCM11016P strain (Korean Patent No. 10-0159812) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). A secondary crossover process was then performed to obtain a strain with PgapA-Son inserted between the transposon genes. PCR and nucleotide sequence analysis were performed using primers SEQ ID NOs: 9 and 10 (Table 4), which can amplify the flanking region including the insertion site of the gene, to confirm the genetic manipulation. The resulting strain was designated Corynebacterium glutamicum KCCM11016P::PgapA-Son.

[0058] [Table 4]

[0059] Example 4: Comparison of L-amino acid production ability in KCCM11016P strains into which foreign membrane proteins have been introduced The KCCM11016P::PgapA-Son strain and the control KCCM11016P were cultured in the following manner, and the bacterial mass, sugar consumption ability, and amino acid production ability were compared.

[0060] First, each strain was inoculated into a 250-ml corner-baffled flask containing 25 ml of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm. Then, 1 ml of the seed culture was inoculated into a 250-ml corner-baffled flask containing 24 ml of production medium and cultured at 37°C for 42 hours with shaking at 200 rpm. After the culture was completed, the amount of L-amino acid produced was measured by HPLC.

[0061] <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium-pantothenic acid 22mg, nicotinamide 2mg (based on 1 liter of distilled water)

[0062] <Production medium (pH 7.0)> Glucose 45g, (NH4)2SO4 15g, soy protein 10g, molasses 10g, KH2PO4 0.55g, MgSO4·7H2O 0.6g, biotin 0.9mg, thiamine hydrochloride 4.5mg, calcium-pantothenate 4.5mg, nicotinamide 30mg, MnSO4 9mg, FeSO4 9mg, ZnSO4 0.45mg, CuSO4 0.45mg, CaCO3 30g (based on 1 liter of distilled water).

[0063] The experiment was repeated three times, and the culture results (average values) are shown in Table 5.

[0064] [Table 5]

[0065] As shown in Table 5, the KCCM11016P::PgapA-Son strain exhibited increased amino acid production ability compared to the control strain KCCM11016P. The KCCM11016P::PgapA-Son strain (Corynebacterium glutamicum CA03-1359) was internationally deposited with the Korea Center for Microorganisms (KCCM), an international depository under the Budapest Treaty, on October 29, 2020, and was assigned the deposit number KCCM12827P.

[0066] Example 5: Comparison of L-amino acid production ability in KCCM10770P strains into which foreign membrane proteins have been introduced A strain in which the PgapA-Son gene was inserted into the transposon site on the genome of Corynebacterium glutamicum KCCM10770P (Korean Patent No. 10-0924065) was obtained using the method described in Example 3. The resulting strain was named Corynebacterium glutamicum KCCM10770P::PgapA-Son.

[0067] The KCCM10770P::PgapA-Son strain and the control KCCM10770P were cultured to compare the bacterial mass, sugar consumption ability, and amino acid production ability (see Example 4). The experiment was repeated three times, and the culture results (average values) are shown in Table 6.

[0068] [Table 6]

[0069] As shown in Table 6, the KCCM10770P::PgapA-Son strain exhibited increased amino acid productivity compared to the control strain KCCM10770P.

[0070] Example 6: Comparison of L-amino acid production ability in CJ3P strains into which foreign membrane proteins have been introduced A strain in which the PgapA-Son gene was inserted into the transposon site on the genome of Corynebacterium glutamicum CJ3P (US 9556463 ​​B2), which had been made capable of producing L-lysine by introducing three mutations [pyc (P458S), hom (V59A), and lysC (T311I)] into a wild-type strain, was obtained using the method described in Example 3. The resulting strain was designated Corynebacterium glutamicum CJ3P::PgapA-Son.

[0071] The CJ3P::PgapA-Son strain and the control CJ3P were cultured to compare the bacterial mass, sugar consumption ability, and amino acid production ability (see Example 4). The experiment was repeated three times, and the culture results (average values) are shown in Table 7.

[0072] [Table 7]

[0073] As shown in Table 7, the CJ3P::PgapA-Son strain exhibited increased amino acid productivity compared to the control strain CJ3P.

[0074] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims below, rather than the above detailed description, and equivalent concepts thereof.

[0075] [Deposit number] Depository institution: Korea Microorganism Collection (International Deposit) Accession number: KCCM12827P Deposit date: October 29, 2020 JPEG0007774625000008.jpg240168

Claims

1. A Corynebacterium glutamicum microorganism expressing a membrane protein having the ability to excrete one or more L-amino acids selected from the group consisting of L-lysine, L-arginine, and L-histidine, which is derived from Shewanella oneidensis as shown in SEQ ID NO: 1, or a membrane protein having the ability to excrete one or more L-amino acids selected from the group consisting of L-lysine, L-arginine, and L-histidine, which has 90% or more sequence identity thereto and is derived from a microorganism of the genus Shewanella.

2. The Corynebacterium glutamicum microorganism according to claim 1 , wherein a polynucleotide encoding the membrane protein has been introduced into the Corynebacterium glutamicum microorganism.

3. The Corynebacterium glutamicum microorganism according to claim 2, wherein the polynucleotide is represented by SEQ ID NO: 2 or a nucleic acid sequence having 90% or more sequence identity thereto.

4. The Corynebacterium glutamicum microorganism according to any one of claims 1 to 3, which has an increased ability to produce one or more L-amino acids selected from the group consisting of L-lysine, L-arginine, and L-histidine, compared to a Corynebacterium glutamicum microorganism that does not express the membrane protein.

5. A composition for producing one or more L-amino acids selected from the group consisting of L-lysine, L-arginine, and L-histidine, comprising the Corynebacterium glutamicum microorganism according to any one of claims 1 to 3.

6. Cultivating the Corynebacterium glutamicum microorganism according to any one of claims 1 to 3 in a medium; and recovering the one or more L-amino acids selected from the group consisting of L-lysine, L-arginine, and L-histidine from the cultured microorganism, the medium, or both.

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