L-histidine export protein and method for producing L-histidine using same

Abstract

Disclosed is a histidine export protein or a variant thereof having an L-histidine export ability, which can significantly increase the yield of L-histidine when expressed in a microorganism having an L-histidine production ability.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority based on Korean Patent Application No. 10-2022-0076773 filed on June 23, 2022, and all contents described in the file of the corresponding Korean Patent Application are incorporated as a part of this specification. Technical Field

[0003] The present application relates to a novel protein having histidine exporting activity, an L-histidine producing microorganism modified to express the protein, and a method for producing L-histidine using the microorganism. Background Art

[0004] L-histidine is one of the 20 standard amino acids. From a nutritional point of view, adults do not need a lot of L-histidine, but it is listed as an essential amino acid for growing children. In addition, L-histidine is involved in important physiological processes such as anti-oxidation and immunomodulation, and is used in the pharmaceutical industry, such as raw materials for gastrointestinal therapeutic agents and circulatory therapeutic agents and amino acid infusion preparations.

[0005] L-histidine is mainly produced by protein hydrolysis extraction using blood meal as raw material because it is contained in hemoglobin in large quantities. However, this method has disadvantages such as low efficiency and environmental pollution. On the other hand, it is possible to produce L-histidine by microbial fermentation, but large-scale industrialization has not yet been achieved. This is because the biosynthesis of L-histidine competes with the nucleotide synthesis precursor phosphoribosyl pyrophosphate (PRPP) and has a complex and high-energy biosynthesis process and regulatory mechanism.

[0006] Examples are known in which the production of the corresponding amino acids is increased when the expression and / or function of proteins having the ability to export different kinds of amino acids is enhanced, but there have been few previous studies on proteins having the ability to export L-histidine specifically.

[0007] In this context, it is necessary to discover proteins with histidine-specific export capabilities and develop technologies for producing histidine using them. Summary of the invention

[0008] Technical issues

[0009] One object of the present application is to provide a mutant L-histidine exporter protein, wherein: (1) the amino acid corresponding to the 72nd amino acid residue of the amino acid sequence of SEQ ID NO: 43 is substituted by another amino acid, or

[0010] (2) the amino acid at residue 124 of the amino acid sequence corresponding to SEQ ID NO: 43 is substituted by another amino acid, or

[0011] (3) (1) and (2) are both replaced.

[0012] Another object of the present application is to provide a microorganism comprising the protein or a polynucleotide encoding the protein.

[0013] Another object of the present application is to provide a composition for producing L-histidine, which comprises the protein, a polynucleotide encoding the protein or the microorganism.

[0014] Another object of the present application is to provide use of the protein, the polynucleotide encoding the protein or the microorganism for producing L-histidine.

[0015] Another object of the present application is to provide use of the protein, the polynucleotide encoding the protein or the microorganism in preparing a composition for producing L-histidine.

[0016] Another object of the present application is to provide a method for producing L-histidine, comprising culturing the microorganism in a culture medium.

[0017] Technical Solution

[0018] The present application shows that the production of L-histidine can be significantly improved by discovering a variant of a histidine export protein having L-histidine export ability and expressing the variant in a microorganism having L-histidine production ability.

[0019] In the present specification, it has been confirmed that a microorganism expressing an AzlC family ABC transporter permease derived from Helcobacillus massiliensis has excellent L-histidine production ability, and it has been confirmed that when an amino acid substitution mutation is introduced at a specific position of the AzlC family ABC transporter permease, the L-histidine production ability is further increased.

[0020] Proteins, polynucleotides and recombinant vectors

[0021] On the one hand, a mutant protein (or polypeptide) having L-histidine export activity is provided. The protein may be a protein having L-histidine specific export ability. In this specification, the mutant protein may be represented by a mutant histidine export protein. The mutant protein may have AzlC family ABC transporter permeation activity.

[0022] In one embodiment, the mutant protein may have an equivalent or enhanced L-histidine export activity compared to a wild-type L-histidine export protein (e.g., a protein containing an AzlD domain, or an AlzC family ABC transporter permease, etc.). The AzlD domain-containing protein or the AzlC family ABC transporter permease protein may be derived from Bacillus vulnificus. In the present specification, the AzlD domain-containing protein derived from Bacillus vulnificus may be described as an HmaE protein (or HmaE), and the AzlC family ABC transporter permease protein derived from Bacillus vulnificus may be described as an HmaF protein (or HmaF).

[0023] In one embodiment, the mutant protein (e.g., a mutant protein of a protein containing an AzlD domain, specifically, a mutant protein of a protein containing an AzlD domain derived from Bacillus massiliense) can be expressed together with an AzlC family ABC transporter permease protein in the same operon gene. In one embodiment, the mutant protein can have L-histidine export activity by binding to an AzlC family ABC transporter protein.

[0024] In one embodiment, the mutant protein may be a mutant protein of the AzlC family ABC transporter permease protein derived from Bacillus vulnificus.

[0025] The mutant protein may be a mutant protein in which one or more amino acid residues of the wild-type AzlC family ABC transporter permease protein derived from Bacillus vulnificus are substituted, deleted or inserted.

[0026] The wild-type AzlC family ABC transporter permease protein derived from Bacillus vulnificus may comprise the amino acid sequence of SEQ ID NO: 43 (WP_055090792.1), or consist of the amino acid sequence of SEQ ID NO: 43.

[0027] In one embodiment, the mutant protein may be a protein in which, starting from the N-terminus of the amino acid sequence of SEQ ID NO: 43, (1) the amino acid corresponding to residue 72 is substituted with another amino acid, or

[0028] (2) the amino acid corresponding to residue 124 is substituted by another amino acid, or

[0029] (3) (1) and (2) are both replaced.

[0030] As described above, counting amino acids from the N-terminus of an amino acid sequence may mean counting methionine (Met, M) translated from the start codon as the first amino acid.

[0031] In one embodiment, the mutant protein, starting from the N-terminus of the amino acid sequence of SEQ ID NO: 43, (1) may comprise a sequence in which the amino acid corresponding to residue 72 is substituted by another amino acid, wherein the other amino acid is leucine (Leu, L), arginine (Arg, R), histidine (His, H), lysine (Lys, K), aspartic acid (Asp, D), glutamic acid (Glu, E), serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Gln, Q), cysteine ​​(Cys, C), glycine (Gly, G), proline (Pro, P), alanine (Ala, A), valine (Val, V), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y) or tryptophan (Trp, W), i.e., an amino acid different from the original amino acid, or

[0032] (2) comprising a sequence in which the amino acid corresponding to residue 124 is substituted by another amino acid, wherein the other amino acid is valine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tyrosine or tryptophan, i.e., an amino acid different from the original amino acid, or

[0033] (3) includes sequences in which both (1) and (2) are substituted.

[0034] In one embodiment, the mutant protein may comprise a sequence wherein, starting from the N-terminus of the amino acid sequence of SEQ ID NO:43, (1) the amino acid corresponding to residue 72 is substituted with leucine, glycine, proline, alanine, valine or methionine;

[0035] (2) the amino acid corresponding to residue 124 is substituted with valine, glycine, proline, alanine, leucine or methionine; or

[0036] (3) (1) and (2) are both replaced.

[0037] In a specific embodiment, the mutant protein may be a protein in which, starting from the N-terminus of the amino acid sequence of SEQ ID NO: 43, the amino acid corresponding to the 72nd residue is substituted with leucine, and the amino acid corresponding to the 124th residue is substituted with valine. Obviously, even if some amino acid sequences other than the amino acids corresponding to the 72nd and / or 124th amino acid residues from the N-terminus of SEQ ID NO: 43 in the mutant protein are deleted, modified, substituted or added, they may also be included in the mutant proteins of the present application as long as they exhibit AzlC family ABC transporter permease activity.

[0038] In addition, in one embodiment, the mutant protein may comprise a polypeptide wherein, in an amino acid sequence having at least 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, or 99.5% or more sequence homology or sequence identity to the amino acid sequence described as SEQ ID NO:43, starting from the N-terminus of the amino acid sequence of SEQ ID NO:43, (1) the amino acid corresponding to residue position 72 is substituted with another amino acid, or (2) the amino acid corresponding to residue position 124 is substituted with another amino acid, or (3) both (1) and (2) are substituted. In other words, a polypeptide which comprises a substitution with another amino acid at the position corresponding to the 72nd and / or 124th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 43, and has at least 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 or 99.5% or more sequence homology or sequence identity with the amino acid sequence of SEQ ID NO: 43, and has AzlC family ABC transporter permease activity, can be included in the mutant protein of the present application.

[0039] In a specific embodiment, the mutant protein may comprise or consist of the amino acid sequence of SEQ ID NO: 56, but is not limited thereto. Obviously, even if some amino acid sequences other than the 72nd and / or 124th amino acid residues at the N-terminus of SEQ ID NO: 56 in the mutant protein are deleted, modified, substituted or added, they may also be included in the mutant proteins of the present application, as long as they exhibit AzlC family ABC transporter permease activity. In addition, in one embodiment, the mutant protein may comprise a polypeptide in which the amino acid corresponding to the 72nd and / or 124th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 56 is fixed, and has at least 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 or 99.5% or more sequence homology or sequence identity with the amino acid sequence of SEQ ID NO: 56. In other words, a polypeptide in which the amino acid corresponding to the 72nd and / or 124th residue from the N-terminus in the amino acid sequence of SEQ ID NO:56 is substituted with other amino acids, and has at least 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 or 99.5% or more homology or identity with the amino acid sequence of SEQ ID NO:56, and has AzlC family ABC transporter permease activity, can be included in the mutant protein of the present application.

[0040] In one embodiment, the mutant protein may have enhanced L-histidine export activity compared to the wild-type protein (e.g., a protein containing a wild-type AzlD domain). In one embodiment, when expressed with a wild-type AzlC family ABC transporter permease protein, the mutant protein may have further enhanced L-histidine export activity.

[0041] The wild-type L-histidine export protein can be a protein having a sequence homology of 60% or more to SEQ ID NO: 43 (a wild-type AzlC family ABC transporter permease protein derived from Bacillus massiliense), SEQ ID NO: 44 (a protein containing a wild-type AzlD domain derived from Bacillus massiliense), or a combination thereof. For example, in a specific embodiment, the wild-type L-histidine export protein can have a sequence homology 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, 99% or more, or 99.5% or more to SEQ ID NO: 43, SEQ ID NO: 44, or a combination thereof. The protein represented by SEQ ID NO:43 can be encoded by the nucleic acid sequence of SEQ ID NO:45, the protein represented by SEQ ID NO:44 can be encoded by the nucleic acid sequence of SEQ ID NO:46, or the protein represented by SEQ ID NO:43 and / or SEQ ID NO:44 can be encoded by the nucleic acid sequence of SEQ ID NO:47 (an operator sequence fused to the overlapping region at the 3' end of SEQ ID NO:45 and the 5' end of SEQ ID NO:46).

[0042] In another aspect, a polynucleotide encoding the mutant protein is provided.

[0043] In the present application, the term "polynucleotide" refers to a polymer of nucleotides, in which nucleotide monomers are linked by covalent bonds to form a long chain, i.e., a DNA or RNA chain of a certain length or longer, and more specifically, refers to a polynucleotide fragment encoding a mutant polypeptide.

[0044] The polynucleotide encoding the mutant protein of the present application may comprise a base sequence encoding the amino acid sequence of SEQ ID NO:56.

[0045] In the present specification, a polynucleotide (which can be used interchangeably with "gene") or a polypeptide (which can be used interchangeably with "protein") "comprising, consisting of, or represented by a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide substantially comprises a specific nucleic acid sequence or amino acid sequence, and may be interpreted as including "substantially equivalent sequences", wherein mutations (deletions, substitutions, modifications and / or additions) are applied to the specific nucleic acid sequence or amino acid sequence (or mutations are not excluded) within the scope of maintaining the original function and / or desired function of the polynucleotide or polypeptide.

[0046] In one embodiment, the nucleic acid sequence or amino acid sequence provided in the present specification may include a sequence modified by ordinary mutagenesis, such as direct evolution and / or site-directed mutagenesis, etc., within the scope of maintaining its original function and / or desired function. In one embodiment, a polynucleotide or polypeptide "comprising a specific nucleic acid sequence or amino acid sequence, or consisting of a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide (i) substantially comprises a specific nucleic acid sequence or amino acid sequence, or (ii) consists of a specific nucleic acid sequence or amino acid sequence having 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 The present invention relates to an amino acid sequence having 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher or 99.5% or higher homology to a microorganism, or basically comprises an amino acid sequence having 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher or 99.5% or higher homology to a specific nucleotide sequence or amino acid sequence, and keeps original function and / or desired function. In this manual, desired function can refer to the L-Histidine export activity and / or the L-Histidine production capacity that gives or increases microorganism.

[0047] In the nucleic acid sequence described in the present specification, various modifications can be made to the coding region within the range that the amino acid sequence and / or function of the protein expressed by the coding region is not changed, taking into account codons preferred in the microorganism to express the protein due to codon degeneracy.

[0048] In this specification, the term "homology (identity)" can refer to the degree of consistency with a given nucleic acid sequence or amino acid sequence, and can be expressed as a percentage (%). For example, in the case of identity with a nucleic acid sequence, the BLAST algorithm (see: Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) or Pearson's FASTA algorithm (see: Methods Enzymol., 183, 63, 1990) can be used to determine. Based on this algorithm BLAST, a program named BLASTN or BLASTX has been developed (see: http: / / www.ncbi.nlm.nih.gov).

[0049] In one embodiment, the polynucleotide comprising the specific nucleic acid sequence provided in this specification 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, which can be adjusted by those skilled in the art according to the purpose, for example, a Tm value of 55°C, 60°C, 63°C or 65°C, and analyzed under the following conditions: These conditions are specifically described in the known literature. For example, conditions that allow 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, 99% or more, or 99.5% or more to hybridize, and conditions that do not hybridize genes with complementarity lower than that, or washing conditions for ordinary southern hybridization, i.e., conditions for washing once, specifically, twice to three times, at a salt concentration and temperature corresponding to 60°C, 1x SSC (saline-sodium citrate buffer) and 0.1% (w / v) SDS (sodium dodecyl sulfate); 60°C, 0.1x SSC and 0.1% SDS; or 68°C, 0.1x SSC and 0.1% SDS, can be listed, but are not limited thereto. Hybridization requires that the two nucleotides have complementary sequences, or, depending on the stringency of the hybridization, allow mismatches between bases. The term "complementary" can be used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in the case of DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. The appropriate stringency of polynucleotide hybridization depends on the length and degree of complementarity of the polynucleotide, which is well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0050] The introduction of polynucleotides or vectors can be carried out by appropriately selecting known transformation methods by those skilled in the art. In this specification, the term "transformation" refers to the process of introducing a specific polynucleotide or a vector comprising the polynucleotide into a host cell, and the transformed polynucleotide can be located in the chromosome of the host cell (i.e., inserted into the chromosome) or outside the chromosome. As an example, transformation can introduce a polynucleotide encoding a target protein (exogenous protein) or a vector comprising the polynucleotide into a host cell so that the protein encoded by the polynucleotide is expressed in the host cell. In addition, the polynucleotide can include DNA and / or RNA encoding the target protein. As long as the polynucleotide can be introduced into the host cell and expressed, there is no restriction on the form of its introduction. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, and the expression cassette is a gene construct comprising all elements that need to be expressed by itself. The expression cassette can generally include expression regulatory elements, such as promoters, transcription termination signals, ribosome binding sites and / or translation termination signals that are operably connected to the polynucleotide. The expression cassette can be in the form of an expression vector that can replicate itself. In addition, the polynucleotide can be introduced into the host cell in its own form and can be operably connected to the sequence necessary for expression in the host cell.

[0051] In the above, the term "operably linked" may refer to the functional connection of an expression control element (e.g., a promoter) and a polynucleotide, so that the expression control element performs transcriptional regulation (e.g., transcription initiation) on the polynucleotide encoding the target protein (exogenous protein). The operable connection can be performed using gene recombination techniques known in the art, for example, by common site-specific DNA cleavage and connection, but is not limited thereto.

[0052] The method of transforming a polynucleotide into a host cell can be carried out by any method for introducing a nucleic acid into a cell (microorganism), and can be carried out by appropriately selecting a transformation technique known in the art according to the host cell. As known transformation methods, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation (polyethylene glycol-mediated uptake), DEAE-dextran method, cationic liposome method, lipofection, lithium citrate-DMSO method, etc. can be exemplified, but are not limited thereto.

[0053] The introduction (insertion) of the polynucleotide into the host cell genome (chromosome) can be carried out by appropriately selecting a known method by a person skilled in the art, for example, an RNA-guided endonuclease system (or CRISPR system; for example, at least one selected from a mixture (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA, etc.), a complex (e.g., a ribonucleic acid fusion protein (RNP), a recombinant vector (e.g., a vector comprising an RNA-guided endonuclease encoding gene and a guide RNA encoding DNA together, etc.), which comprises (a) an RNA-guided endonuclease (e.g., Cas9 protein, etc.), a gene encoding it, or a vector comprising the gene; and (b) a guide RNA (e.g., a single guide RNA (sgRNA), etc.), a DNA encoding it, or a vector comprising the DNA)), but is not limited thereto.

[0054] On the other hand, a recombinant vector comprising a polynucleotide is provided. The recombinant vector can be used as an expression vector for a polypeptide. The recombinant vector can be used to insert the polynucleotide into the genome of a host cell, or to replace the corresponding gene of the host cell genome.

[0055] In this specification, the term "vector" refers to a DNA product containing a base sequence of a polynucleotide encoding a target protein, which is operably linked to a suitable regulatory sequence so as to express 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 cell, the vector may be expressed independently of the host cell's genome, or may be integrated into the host cell's genome.

[0056] The vector that can be used in this specification is not particularly limited, as long as it can replicate in the host cell, and it can be selected from all commonly used vectors. Examples of commonly used vectors can include plasmids, cosmids, viruses, phages, etc. in a natural state or a recombinant state. For example, as vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A can be used as phage vectors or cosmid vectors, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series and pET series can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be cited, but are not limited to this.

[0057] The vector that can be used for this specification sheet can be a known expression vector and / or a vector for inserting a polynucleotide into a host cell chromosome. The polynucleotide can be inserted into a host cell chromosome by any method known in the art, such as homologous recombination or CRISPR system, but is not limited thereto. The vector may further include a selection marker for confirming whether it is inserted into a chromosome. The selection marker is used to select cells transformed with the vector, that is, to confirm the insertion of the polynucleotide, and can be selected and used from genes that give selectable phenotypes such as drug resistance, auxotrophy, cytotoxic agent resistance or surface protein expression. Because only cells expressing the selection marker survive or show different phenotypes, the cells that can be transformed can be selected.

[0058] microorganism

[0059] On the other hand, provide and comprise at least one (1 kind, 2 kinds or all 3 kinds) microorganism that comprises the group that is composed of being selected from by mutant protein, polynucleotide of encoding this mutant protein and the recombinant vector that comprises this polynucleotide.This microorganism can have L-Histidine export activity and / or L-Histidine production capacity.Compare with not comprising at least one microorganism that is selected from by mutant protein, polynucleotide of encoding this mutant protein and the recombinant vector that comprises this polynucleotide, this microorganism can have enhanced (or increase, improvement) L-Histidine export activity and / or L-Histidine production capacity.

[0060] The mutant protein may be exogenous. In the present specification, "exogenous" may mean that it is not endogenous to the microorganism, but is derived from a species other than the microorganism.

[0061] In the present specification, "a microorganism having enhanced L-histidine export activity and / or L-histidine production capacity" may be a microorganism that originally did not have L-histidine export activity and / or L-histidine production capacity, but after being engineered (mutated) to express the above-mentioned mutant protein, it has acquired L-histidine export activity and / or L-histidine production capacity; or, a microorganism whose original L-histidine export activity and / or L-histidine production capacity has been improved after engineering (mutation).

[0062] In the present specification, "microorganism" includes unicellular bacteria and can be used interchangeably with "cell".

[0063] In the present application, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and may be microorganisms in which a specific mechanism is weakened or enhanced due to reasons such as the insertion of exogenous genes or the enhancement or inactivation of the activity of endogenous genes, and is a genetically modified microorganism comprising a gene for producing a desired polypeptide, protein or product (e.g., L-histidine).

[0064] The microorganism of the present application may be a genetically modified microorganism (eg, a recombinant microorganism) to enhance the activity of the L-histidine export protein or the polynucleotide encoding the same, but is not limited thereto. The vector is as described above.

[0065] In the present specification, in order to distinguish from the microorganism after mutation, the microorganism used to express the mutant protein before mutation may be referred to as "parent strain (parent microorganism) or host cell".

[0066] A microorganism (or strain, recombinant cell) has L-histidine export activity and / or L-histidine production capacity, or has enhanced L-histidine export activity and / or L-histidine production capacity, which may mean that the L-histidine export activity and / or L-histidine production capacity is different from that of an unmodified microorganism, a cell before recombining, a parent strain and / or a wild-type strain, wherein the unmodified microorganism, a cell before recombining, a parent strain and / or a wild-type strain does not have L-histidine export activity and / or L-histidine production capacity; or that the L-histidine export activity and / or L-histidine production capacity is enhanced compared to an unmodified microorganism, a cell before recombining, a parent strain and / or a wild-type strain.

[0067] The microorganism of the present application can be any one or more microorganisms comprising the mutant protein of the present application, the polynucleotide encoding the mutant protein of the present application, and the vector comprising the polynucleotide of the present application; a microorganism modified to express the mutant protein of the present application or the polynucleotide of the present application; a microorganism (e.g., a recombinant strain) expressing the mutant protein of the present application or the polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) having the activity of the mutant protein of the present application, but is not limited thereto.

[0068] In one embodiment, microorganism can be at least one selected from the group consisting of microorganisms of Corynebacterium, microorganisms of Escherichia etc. The microorganism of Corynebacterium can include Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes, Corynebacterium efficiens etc., but is not limited thereto. More specifically, the microorganism of Corynebacterium can be Corynebacterium glutamicum. The bacterial strain of Escherichia can be Escherichia coli.

[0069] The microorganism may contain at least one (1, 2 or all 3) selected from the group consisting of a mutant protein, a polynucleotide encoding the mutant protein and a recombinant vector comprising the polynucleotide. In one embodiment, the mutation for expressing the mutant protein may be performed by introducing a polynucleotide encoding the mutant protein or a recombinant vector comprising the polynucleotide, or by artificial mutation (e.g., error-prone PCR, etc.), or the like. The polynucleotide encoding the mutant protein to be introduced into the parent strain may thus replace the gene encoding the AzlC family ABC transporter permease in the present strain, or further be included therein.

[0070] In one embodiment, when the microorganism comprises a mutant protein and a wild-type AzlC family ABC transporter permease protein, it may have further enhanced L-histidine export activity and / or L-histidine production ability.

[0071] In one embodiment, the microorganism with enhanced L-histidine export activity and / or L-histidine production capacity may have an L-histidine production capacity increased by about 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more compared to the parent strain before mutation, an unmodified microorganism, and a microorganism comprising a wild-type L-histidine export protein, but is not limited thereto.

[0072] In other embodiments, the microorganism with enhanced L-histidine export activity and / or L-histidine production capacity may have an increased L-histidine production capacity of about 1 g / L or more, 1.5 g / L or more, 2 g / L or more, 2.5 g / L or more, 3 g / L or more, 3.5 g / L or more, or 4 g / L or more, compared to the parent strain before mutation, an unmodified microorganism, and a microorganism comprising a wild-type L-histidine export protein, but is not limited thereto.

[0073] The term "about" means a range including all values ​​such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and also includes all values ​​equal to or similar to the value following the term "about", but is not limited thereto.

[0074] In another aspect, a composition for producing L-histidine is provided, which comprises a mutant protein, a polynucleotide, a recombinant vector or a microorganism.

[0075] In another aspect, provided is a use of a mutant protein, a polynucleotide, a recombinant vector or a microorganism for producing L-histidine.

[0076] In another aspect, provided is a use of a mutant protein, a polynucleotide, a recombinant vector or a microorganism in preparing a composition for producing L-histidine.

[0077] In another aspect, a method for producing (preparing) L-histidine is provided, comprising culturing a microorganism in a culture medium. The preparation method may further comprise recovering L-histidine from the cultured microorganism, the culture medium, or both after the culturing.

[0078] On the other hand, a method for increasing the L-histidine export activity and / or the L-histidine production capacity of a microorganism is provided, or a method for conferring L-histidine export activity and / or L-histidine production capacity to a microorganism is provided, comprising enhancing the L-histidine export activity and / or L-histidine production capacity of a microorganism.

[0079] Introducing mutations may include introducing (transforming) a polynucleotide encoding a mutant protein or a recombinant vector comprising the polynucleotide into a microorganism, or may include artificially generating mutations (eg, error-prone PCR, etc.).

[0080] Other embodiments provide a method for producing L-histidine, comprising culturing a microorganism with enhanced L-histidine export activity and / or L-histidine production capacity in a culture medium. The method may further comprise recovering L-histidine from the cultured microorganism, culture medium, or both after culturing.

[0081] 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. At this time, the culture conditions are not particularly limited to this, but the use of alkaline compounds (e.g., sodium hydroxide, potassium hydroxide or ammonia) or acidic compounds (e.g., phosphoric acid or sulfuric acid) can adjust a suitable pH (e.g., pH 5 to 9, specifically, pH 6 to 8, most specifically, pH 7.2), and aerobic conditions can be maintained by introducing oxygen or oxygen-containing gas mixtures into the culture. The culture temperature can be maintained at 20 to 45°C, or 25 to 40°C, and can be cultured for about 10 to 160 hours, but is not limited thereto. The L-histidine produced by the culture can be secreted into the culture medium or retained in the cell.

[0082] The culture medium that can be used for the culture can use at least one substance 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 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) as a carbon source, but not limited thereto. As a nitrogen source, at least one substance selected from the group consisting of nitrogen-containing organic compounds (e.g., peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal, and urea), inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate) can be used alone or in combination, but not limited thereto. As a phosphorus source, at least one substance selected from the group consisting of potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding nitrogen-containing salts can be used alone or in combination, but not limited thereto. In addition, the culture medium may contain necessary growth-promoting substances, such as other metal salts (eg, magnesium sulfate, iron sulfate, manganese sulfate, etc.), amino acids and / or vitamins, etc.

[0083] Reclaiming L-Histidine can be to use suitable method known in the art to collect required amino acid from culture medium, culture fluid or microorganism, and this depends on culture method.For example, can be reclaimed by one or more methods selected from centrifugal, filtration, anion exchange chromatography, crystallization, HPLC etc. The method of reclaiming L-Histidine can be included in addition before, simultaneously or afterwards and purify.

[0084] Beneficial Effects

[0085] The present application discloses a histidine export protein or a variant thereof having the ability to export L-histidine, which can significantly increase the yield of L-histidine when expressed in a microorganism having the ability to produce L-histidine. DETAILED DESCRIPTION

[0086] Hereinafter, the present invention will be described in more detail by the following examples. However, they are only used to illustrate the present invention, but the scope of the present invention is not limited by these examples.

[0087] Example 1. Search for exogenous histidine export genes and candidate selection

[0088] In order to select protein candidates having L-histidine-specific export ability, as a result of PSI-BLAST search based on NCBI and Kegg databases, amino acid sequences of amino acids classified by category (exported proteins are as follows: for basic amino acids: L-lysine (L-lys), for aromatic amino acids: tryptophan (Trp), for branched-chain amino acids: isoleucine (Ile) (LysE (Arch Microbiol 180: 155-160), Wex (Korean registered patent No. 10-1968317), BrnFE (Arch Microbiol 180: 155-160)) were selected as query sequences, and candidate genes predicted as membrane proteins that may export L-histidine and microorganisms having them were selected.

[0089] Among them, taking into account the biosafety level and the possibility of ensuring a level suitable for the production strains therein, one protein based on LysE, three proteins based on Wex, and two proteins based on BrnFE, as well as the genes encoding these proteins and the microorganisms containing these proteins were selected as shown in Table 1 below:

[0090] [Table 1]

[0091] List of L-histidine export candidates

[0092]

[0093]

[0094]

[0095] (In Table 1 above, the biosafety level is based on the microbial pathogenicity index (level 1 to 4) defined by the Centers for Disease Control and Prevention (the lower the level, the safer)

[0096] Example 2. Production of vectors introducing exogenous L-histidine export gene candidates and recombinant Corynebacterium strains introducing the same gene candidates

[0097] Six vectors were produced for introducing the six exogenous L-histidine export gene candidates selected in Example 1 into Corynebacterium strains.

[0098] Example 2-1. Production of vector pDZΔN2131 for inserting target gene

[0099] In order to import the exogenous L-histidine export gene candidate, the NCgl2131 gene in the gene encoding the Corynebacterium glutamicum transposon is used as an insertion site (Journal of Biotechnology 104, 5-25 Jorn Kalinowski et al, 2003). In addition, it is designed to express the exogenous L-histidine export gene candidate under the promoter of the gapA gene (hereinafter referred to as PgapA, SEQ ID NO: 17) in the Corynebacterium source.

[0100] In order to replace the NCgl2131 gene with the export gene, a vector of NCgl2131 deletion and target gene insertion was produced. In order to produce the vector, the chromosome of the Corynebacterium glutamicum strain ATCC13032 was used as a template, and the primers of SEQ ID NO:18 and SEQ ID NO:19 and SEQ ID NO:20 and SEQ ID NO:21 were used to perform PCR. As the polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, 531bp of del-N2131L (SEQ ID NO:22) and 555bp of del-N2131R (SEQ ID NO:23) DNA fragments were obtained respectively. The obtained DNA product was purified using a PCR purification kit from QIAGEN and then cloned using the pDZ vector (Korean registered patent number 10-0924065) and TaKaRa's Infusion cloning kit to produce the vector pDZΔN2131 for NCgl2131 gene deletion and target gene insertion.

[0101] Example 2-2. Production of 6 vectors introducing L-histidine export gene candidates

[0102] The base sequence information of the gene (hereinafter referred to as haq, SEQ ID NO: 2) encoding the protein derived from the water grass spirilla (hereinafter referred to as Haq, SEQ ID NO: 1) was obtained from the GenBank (NIH GenBank) of the National Institutes of Health of the United States. In order to amplify haq, a primer pair of SEQ ID NO: 24 and SEQ ID NO: 25 was used to perform PCR with the chromosomal DNA of the water grass spirilla strain (KCTC42001) as a template. As a polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 977 bp haq fragment containing 945 bp of haq (SEQ ID NO: 2) was obtained. In order to obtain a PgapA fragment that can be connected to haq, PCR was performed using a primer pair of SEQ ID NO: 26 and SEQ ID NO: 27, with the chromosome of ATCC13032 as a template. As a polymerase for the PCR reaction, PfuUltraTM High Reliability DNA Polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 441 bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO: 17) was obtained. The obtained haq fragment and PgapA fragment and the pDZΔN2131 vector cut with ScaI restriction enzyme were cloned using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Haq.

[0103] The base sequence information of the gene (hereinafter referred to as cpi, SEQ ID NO: 4) encoding the protein derived from the copper-grasping bacteria (hereinafter referred to as Cpi, SEQ ID NO: 3) was obtained from the GenBank (NIH GenBank) of the National Institutes of Health. In order to amplify the cpi derived from the copper-grasping bacteria, the primer pair of SEQ ID NO: 28 and SEQ ID NO: 29 was used, and the chromosomal DNA of the copper-grasping bacteria strain (KCTC22125) was used as a template for PCR. As a polymerase for the PCR reaction, PfuUltraTM high-reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 977bp cpi fragment containing 945bp of cpi (SEQ ID NO: 4) was obtained. In order to obtain a PgapA fragment that can be connected to cpi, PCR was performed using a primer pair of SEQ ID NO: 26 and SEQ ID NO: 30, with the chromosome of ATCC13032 as a template. As a polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 441 bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO: 17) was obtained. The obtained cpi fragment and PgapA fragment and the pDZΔN2131 vector cut with the ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Cpi.

[0104] The base sequence information of the gene (hereinafter referred to as kcr, SEQ ID NO: 6) encoding the protein derived from Kluyvera refracta (hereinafter referred to as Kcr, SEQ ID NO: 5) was obtained from the GenBank (NIH GenBank) of the National Institutes of Health. In order to amplify the kcr derived from Kluyvera refracta, PCR was performed using the primer pair of SEQ ID NO: 31 and SEQ ID NO: 32, with the chromosomal DNA of the Kluyvera refracta strain (KCTC2580) as a template. As a polymerase for the PCR reaction, PfuUltraTM High Reliability DNA Polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 914bp kcr fragment containing 882bp of kcr (SEQ ID NO: 6) was obtained. In order to obtain a PgapA fragment that can be connected to kcr, PCR was performed using a primer pair of SEQ ID NO: 26 and SEQ ID NO: 33, with the chromosome of ATCC13032 as a template. As a polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 441bp PgapA fragment containing 409bp of PgapA (SEQ ID NO: 17) was obtained. The obtained kcr fragment and PgapA fragment and the pDZΔN2131 vector cut with the ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Kcr.

[0105] The base sequence information of the gene (hereinafter referred to as cst, SEQ ID NO: 8) encoding the protein derived from Corynebacterium stagnation (hereinafter referred to as Cst, SEQ ID NO: 7) was obtained from GenBank (NIH GenBank) of the National Institutes of Health. In order to amplify the cst derived from Corynebacterium stagnation, the primer pair of SEQ ID NO: 34 and SEQ ID NO: 35 was used, and the chromosomal DNA of Corynebacterium stagnation strain (ATCC6872) was used as a template to perform PCR. As the polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, a cst fragment of 749bp containing 717bp of cst (SEQ ID NO: 8) was obtained. In order to obtain a PgapA fragment that can be connected to cst, PCR was performed using a primer pair of SEQ ID NO: 26 and SEQ ID NO: 36, with the chromosome of ATCC13032 as a template. As a polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 441 bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO: 17) was obtained. The obtained cst fragment and PgapA fragment and the pDZΔN2131 vector cut with the ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Cst.

[0106] The base sequence information of the operon (hereinafter referred to as lsa, SEQ ID NO: 11) encoding the protein derived from Bacillus salcisius (hereinafter referred to as LsaFE, SEQ ID NO: 9, 10) was obtained from the GenBank (NIH GenBank) of the National Institutes of Health. In order to amplify lsa derived from Bacillus salcisius, PCR was performed using the primer pair of SEQ ID NO: 37 and SEQ ID NO: 38, using the chromosomal DNA of Bacillus salcisius strain (KCTC19904) as a template. As a polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 1080bp lsa fragment containing 1048bp of lsa (SEQ ID NO: 11) was obtained. In order to obtain a PgapA fragment that can be connected to lsa, PCR was performed using a primer pair of SEQ ID NO: 26 and SEQ ID NO: 39, with the chromosome of ATCC13032 as a template. As a polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 441 bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO: 17) was obtained. The obtained lsa fragment and PgapA fragment and the pDZΔN2131 vector cut with the ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Lsa.

[0107] The base sequence information of the operon (hereinafter referred to as dva, SEQ ID NO: 16) encoding the protein derived from vaginal bacillus (hereinafter referred to as DvaFE, SEQ ID NO: 12, 13) was obtained from the GenBank of the National Institutes of Health (NIH GenBank). In order to amplify vaginal bacillus derived dva, PCR was performed using the primer pair of SEQ ID NO: 40 and SEQ ID NO: 41 and the chromosomal DNA of vaginal bacillus strain (KCTC39585) as a template. As a polymerase for the PCR reaction, PfuUltraTM High Reliability DNA Polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 1113 bp dva fragment containing 1081 bp of dva (SEQ ID NO: 16) was obtained. In order to obtain a PgapA fragment that can be connected to dva, PCR was performed using a primer pair of SEQ ID NO: 26 and SEQ ID NO: 42, with the chromosome of ATCC13032 as a template. As a polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 441 bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO: 17) was obtained. The obtained dva fragment and PgapA fragment and the pDZΔN2131 vector cut with the ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Dva.

[0108] Example 2-3. Production of recombinant strains of Corynebacterium

[0109] In order to confirm the L-histidine export ability of the exogenous L-histidine export gene candidates, the produced NCgl2131 deletion vector (pDZΔN2131) and 6 vectors for introducing exogenous L-histidine export gene candidates (pDZΔN2131-PgapA-Haq, pDZΔN2131-PgapA-Cpi, pDZΔN2131-PgapA-Kcr, pDZΔN2131-PgapA-Cst, pDZΔN2131-PgapA-Lsa, pDZΔN2131-PgapA-Dva) were introduced into the Corynebacterium glutamicum ATCC13032 strain respectively. More specifically, the vectors were transformed into ATCC13032 strains by electroporation, and seven recombinant strains (in which the NCgl2131 gene on the chromosome was deleted or replaced by a candidate L-histidine export gene) were produced through a secondary hybridization process. They were named ATCC13032ΔN2131 (N2131 gene deleted), ATCC13032ΔN2131::Haq (N2131 gene replaced by haq), ATCC13032Δ N2131::Cpi (the N2131 gene was replaced by cpi), ATCC13032ΔN2131::Kcr (the N2131 gene was replaced by kcr), ATCC13032ΔN2131::Cst (the N2131 gene was replaced by cst), ATCC13032ΔN2131::Lsa (the N2131 gene was replaced by lsa) and ATCC13032ΔN2131::Dva (the N2131 gene was replaced by dva).

[0110] Example 3. MIC determination of Corynebacterium strains into which exogenous L-histidine export gene candidates are introduced

[0111] In order to confirm the retention of the activity of L-histidine export ability of 7 recombinant Corynebacterium glutamicum strains (ATCC13032ΔN2131, ATCC13032ΔN2131::Haq, ATCC13032ΔN2131::Cpi, ATCC13032ΔN2131::Kcr, ATCC13032ΔN2131::Cst, ATCC13032ΔN2131::Lsa and ATCC13032ΔN2131::Dva) produced in Example 2 above, a minimum inhibitory concentration (MIC) experiment was performed using L-histidine. The 7 strains were cultured in a minimal liquid medium at 30°C for 24 hours and then treated with 1×10 3 and 1×10 4 The cells were diluted and spotted in a basic solid medium supplemented with L-histidine. The composition of the basic solid medium used was as follows:

[0112] Basic medium (pH 7.2)

[0113] Glucose 10g, KH2PO4 1g, K2HPO4 2g, MgSO4 7H2O 0.4g, urea 2g, (NH4)2SO4 5g, NaCl 0.5g, nicotinamide 5μg, calcium pantothenate 0.1μg, biotin 0.2μg, thiamine hydrochloride 3μg, trace element solution * 1ml (based on 1L of distilled water), agar 20g

[0114] *Trace element solution

[0115] Na2B4O7 10H2O 0.09g, (NH4)6Mo7O 27 4H2O 0.04g, ZnSO4 7H2O 0.01g, CuSO4 5H2O0.27g, MnCl2 4H2O 0.01g, FeCl3 6H2O 1g, CaCl2 0.01g (based on 1L of distilled water)

[0116] For the minimum inhibitory concentration experiment, 1 g / L of L-histidine was added to the basic solid culture medium. After 48 hours, the growth of cells was observed. The results are shown in Table 2 below:

[0117] [Table 2]

[0118] Growth level of Corynebacterium strains introduced with exogenous L-histidine export gene candidates in a minimal medium containing L-histidine

[0119]

[0120] (In Table 2, the number of + indicates the relative growth level of the strain, and each indicates the following: +: does not form a single colony, but forms overlaps (does not grow into a single colony, but grows in a cluster);

[0121] ++: overlap and less than 5 single colonies were formed;

[0122] +++: overlap and formation of less than 50 single colonies;

[0123] ++++: Overlapping and indistinguishable from single colonies)

[0124] As shown in Table 2 above, except for the ATCC13032ΔN2131::Dva strain, all strains grew steadily in a minimal medium without L-histidine. However, in a minimal medium containing 1 g / L of L-histidine, the growth of the strain into which the candidate gene for L-histidine export was introduced was not obvious, and only the ATCC13032ΔN2131::Dva strain into which the gene derived from vaginal bacillus was introduced showed growth superior to that of ATCC13032ΔN2131. This suggests that even in a medium containing L-histidine above the minimum inhibitory concentration, the protein derived from vaginal bacillus introduced can have the ability to export L-histidine.

[0125] Thus, the Dva protein derived from Dermatobacterium vaginalis was selected as a protein capable of conferring resistance to L-histidine exceeding the minimum inhibitory concentration in the coryneform bacteria strain and having the ability to specifically export L-histidine.

[0126] Example 4. Production of a strain into which a gene (dva) derived from Dermatobacterium vaginalis was introduced based on an L-histidine-producing strain derived from Corynebacterium (KCCM 80179) and evaluation of its L-histidine-producing ability

[0127] In order to confirm the L-histidine export ability of the Dva protein derived from vaginal bacillus, the Dva gene derived from vaginal bacillus was introduced into the L-histidine producing strain KCCM 80179 (Korean Patent Publication No. 10-2019-0065984).

[0128] To this end, the vectors pDZΔN2131 and pDZΔN2131-PgapA-Dva produced in Example 2 were respectively transformed into the KCCM80179 strain, and through a secondary hybridization process, two strains in which the NCgl2131 gene on the chromosome was deleted or replaced by the L-histidine export gene candidate (dva) were obtained, and they were named KCCM 80179ΔN2131 (NCgl2131 gene deleted) and KCCM 80179ΔN2131-PgapA-Dva (NCgl2131 gene replaced by dva), respectively.

[0129] In order to confirm the L-histidine production ability of the produced KCCM 80179ΔN2131 and KCCM 80179ΔN2131-PgapA-Dva strains, they were cultured by the following method: KCCM 80179ΔN2131 and KCCM 80179ΔN2131-PgapA-Dva strains were cultured in an activation medium for 16 hours, and then each strain was inoculated into a 250 mL baffled flask containing 25 mL of a seed 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 baffled flask containing 25 mL of a production medium and cultured at 30°C with shaking at 200 rpm for 48 hours. The composition of the culture medium used for the culture is as follows:

[0130] <Activation medium>

[0131] Meat broth 1% (w / v), polypeptone 1% (w / v), sodium chloride 0.5% (w / v), yeast extract 1% (w / v), agar 2% (w / v), pH 7.2

[0132] <Seed culture medium>

[0133] Glucose 5% (w / v), Bacto-peptone 1% (w / v), Sodium chloride 0.25% (w / v), Yeast extract 1% (w / v), Urea 0.4% (w / v), pH 7.2

[0134] <Production medium>

[0135] Glucose 10% (w / v), ammonium sulfate 2% (w / v), potassium dihydrogen phosphate 0.1% (w / v), magnesium sulfate heptahydrate 0.05% (w / v), CSL (corn syrup) 2.0% (w / v), biotin 200 μg μg / L, calcium carbonate, pH 7.2,

[0136] After the cultivation was completed, the L-histidine yield (histidine content in the culture medium) was measured by HPLC, and the results are shown in the following Table 3:

[0137] [Table 3]

[0138] L-histidine production by strains into which genes derived from KCCM 80179-derived Dermatobacterium vaginalis were introduced

[0139]

[0140] As shown in Table 3 above, it was confirmed that the NCgl2131 deletion strain had a level of L-histidine production capacity comparable to that of the parent strain KCCM 80179 strain, and the L-histidine production capacity of the KCCM 80179ΔN2131-PgapA-Dva strain into which the gene derived from vaginal Clostridium difficile was introduced was increased by 23% and 21% or more, respectively, compared with the NCgl2131 deletion strain and the parent strain.

[0141] The results of Examples 3 and 4 confirmed that by introducing the gene derived from vaginal bacillus, not only the resistance to L-histidine concentrations higher than the minimum inhibitory concentration was increased, but also the L-histidine production capacity was significantly increased. This result shows that the protein derived from vaginal bacillus is an L-histidine exporting protein capable of specifically exporting L-histidine.

[0142] Example 5. Obtaining L-histidine export-like proteins from other vaginal skin bacilli

[0143] Since the L-histidine export ability of the protein derived from vaginal dermatobacterium was confirmed in the above Examples 3 and 4, in order to further obtain similar proteins with high homology to the protein and amino acid sequence, a BLAST search was performed using the DvaF sequence (SEQ ID NO: 12) in DvaFE as the query sequence (see Table 4).

[0144] [Table 4]

[0145]

[0146] Based on the BLAST search results, 1 additional L-histidine export candidate that showed 60% or higher sequence homology and did not belong to the genus Dermatobacterium was selected and is shown in Table 5 below:

[0147] [Table 5]

[0148] List of other L-histidine export candidates

[0149]

[0150]

[0151] Example 6. Production of vectors for introducing other exogenous L-histidine export gene candidates

[0152] Vectors were produced for introducing into the Corynebacterium strain the two L-histidine export gene candidates additionally selected in Example 5. As in Example 2, the NCgl2131 gene was used as a defective site and PgapA was used as a promoter.

[0153] The base sequence information of the operon (hereinafter referred to as hma, SEQ ID NO: 47) encoding the protein derived from Bacillus vulnificus (hereinafter referred to as HmaFE, SEQ ID NO: 43, 44) was obtained from the GenBank (NIH GenBank) of the National Institutes of Health. In order to obtain haq DNA, DNA was synthesized using the gene synthesis service of Bionics. In order to amplify the synthesized DNA, PCR was performed using the primer pair of SEQ ID NO: 48 and SEQ ID NO: 49. As a polymerase for the PCR reaction, PfuUltraTM High Reliability DNA Polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 1113 bp hma fragment containing 1081 bp of hma (SEQ ID NO: 47) was obtained. In order to obtain a PgapA fragment that can be connected to hma, PCR was performed using a primer pair of SEQ ID NO: 26 and SEQ ID NO: 50, with the chromosome of ATCC13032 as a template. As a polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 441 bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO: 17) was obtained. The obtained hma fragment and PgapA fragment and the pDZΔN2131 vector cut with the ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Hma.

[0154] Example 7. Production of a strain into which a gene (hma) derived from Bacillus vulnificus was introduced based on an L-histidine-producing strain (KCCM 80179) and evaluation of its L-histidine-producing ability

[0155] In order to confirm the L-histidine export ability of the protein Hma derived from Bacillus vulnificus massiliense, it was introduced into the L-histidine-producing strain KCCM 80179.

[0156] To this end, the vector pDZΔN2131-PgapA-Hma produced in Example 6 was transformed into the KCCM80179 strain by electroporation, and through a secondary hybridization process, a strain in which the NCgl2131 gene on the chromosome was replaced by an L-histidine export gene candidate was produced and named KCCM 80179ΔN2131-PgapA-Hma (NCgl2131 gene replaced by hma).

[0157] In order to confirm the L-histidine production ability of the produced KCCM 80179ΔN2131-PgapA-Hma strain, the culture was carried out by the method performed in Example 4, and the L-histidine production was measured. As a control group, the KCCM 80179ΔN2131 strain and the KCCM 80179ΔN2131-PgapA-Dva strain produced in Example 4 were cultured and the L-histidine production (histidine content in the culture medium) was measured by the same method. The results obtained are shown in Table 6.

[0158] [Table 6]

[0159] L-histidine production of strains into which genes derived from KCCM 80179-derived Bacillus massiliense vulnificus were introduced

[0160]

[0161]

[0162] As shown in Table 6, the L-histidine production of the KCCM 80179ΔN2131-PgapA-Hma strain increased by 19% and 19% respectively compared with the NCgl2131 deletion strain (KCCM 80179ΔN2131) and the parent strain KCCM 80179. This result indicates that proteins derived from Bacillus massiliense can also be selected as L-histidine exporters capable of specifically exporting L-histidine.

[0163] Example 8: Production of Hma mutant library using artificial mutation method

[0164] In order to obtain mutant Hma with increased L-histidine export ability, a mutant protein expression vector library for primary crossover was produced. To this end, the hma operon with randomly introduced base substitution mutations was obtained by error-prone PCR method using the primer pair of SEQ ID NO:48 and SEQ ID NO:49, with the generated pDZΔN2131-PgapA-Hma as a template. For error-prone PCR, PCR was performed using GenemorphII random mutagenesis kit (Stratagene) under the condition of introducing 0 to 3.5 mutations per 1 kb into the amplified gene fragment, and the PCR conditions were denaturation at 96°C for 30 seconds; annealing at 53°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 30 times. In order to obtain a PgapA fragment that can be connected to hma, PCR was performed using the primer pair of SEQ ID NO:26 and SEQ ID NO:50, with the chromosome of ATCC13032 as a template. As the polymerase for PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: by denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes, the PgapA fragment was obtained. The mutant hma operon and PgapA fragment obtained by Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method were cloned and the pDZΔN2131 vector cut with ScaI restriction enzyme was transformed into DH5α and applied to LB solid medium containing kanamycin (25 mg / L). As a result of selecting 20 transformed colonies and then obtaining plasmids and analyzing base sequences, it was confirmed that mutations were introduced at different positions at an average frequency of 2 mutations / kb. The plasmid was extracted by extracting about 10,000 transformed E. coli colonies and named as pDZΔN2131-PgapA-Hma(mt) library.

[0165] Example 9: Introduction of Hma artificial mutant library and selection of strains with increased L-histidine production capacity

[0166] The produced ATCC13032ΔN2131 strain was used as the parent strain, and the produced pDZΔN2131-PgapA-Hma(mt) library was transformed by homologous chromosome recombination and plated on a composite plate medium containing kanamycin (25 mg / L) to obtain approximately 7,000 colonies, each of which was named TCC13032ΔN2131-PgapA-Hma(mt)-1 to ATCC13032ΔN2131-PgapA-Hma(mt)-7000.

[0167] <Compound plate medium (pH 7.0)>

[0168] Glucose 10g, peptone 10g, beef extract 5g, yeast extract 5g, brain heart infusion 18.5g, NaCl 2.5g, urea 2g, sorbitol 91g, agar 20g (based on 1L of distilled water)

[0169] For the 7000 colonies obtained, the minimum inhibitory concentration (MIC) experiment of L-histidine carried out in Example 3 was carried out. For effective screening, it was carried out based on a liquid minimal culture medium containing 3g L-histidine. The colonies were immediately inoculated into 300ul of liquid minimal culture medium and cultured in a 96-deep well plate at 32°C and 1000rpm for 18 hours, and then the OD600 value was measured to select well-grown colonies. As a control group, the produced ATCC13032ΔN2131 strain and ATCC13032ΔN2131::Hma strain were used. By primary experiments, 251 colonies were selected, and by repeated experiments, 36 colonies were selected. The culture medium components used are as follows.

[0170] Liquid basic medium (pH 7.2)

[0171] Glucose 10g, KH2PO4 1g, K2HPO4 2g, MgSO4 7H2O 0.4g, urea 2g, (NH4)2SO4 5g, NaCl 0.5g, nicotinamide 5μg, calcium pantothenate 0.1μg, biotin 0.2μg, thiamine hydrochloride 3μg, trace element solution * 1ml (based on 1L distilled water)

[0172] *Trace element solution

[0173] Na2B4O7 10H2O 0.09g, (NH4)6Mo7O 27 4H2O 0.04g, ZnSO4 7H2O 0.01g, CuSO4 5H2O0.27g, MnCl2 4H2O 0.01g, FeCl3 6H2O 1g, CaCl2 0.01g (based on 1L of distilled water)

[0174] For the minimum inhibitory concentration experiment, 3 g / L L-histidine was added to the culture medium and cultured for 18 hours.

[0175] The 36 selected colonies were screened on solid culture medium by the method in Example 3, and the 6 colonies with increased L-histidine export capacity were finally screened. The results are shown in Table 7 below:

[0176] [Table 7]

[0177] Growth of mutant strains in a minimal medium containing L-histidine

[0178]

[0179] (In Table 7, the number of + indicates the relative growth level of the strain, and each indicates the following: +: does not form a single colony, but forms overlaps (does not grow into a single colony, but grows in a cluster);

[0180] ++: overlap and less than 5 single colonies were formed;

[0181] +++: overlap and formation of less than 50 single colonies;

[0182] ++++: Overlapping and indistinguishable from single colonies)

[0183] As shown in Table 7 above, the strain lacking NCgl2131 (ATCC13032ΔN2131) could not grow stably in a minimal medium containing 1 g / L of L-histidine, but the ATCC13032ΔN2131::Hma strain into which a gene derived from Bacillus vulnificus was introduced grew stably, and six strains (ATCC13032ΔN2131-PgapA-Hma(mt)-1216, ATCC13032ΔN2131-PgapA-Hma(mt)-2305, ATCC13032ΔN2131-PgapA-Hma(mt)-3411, ATCC13032ΔN2131-PgapA-Hma(mt)-4426) were confirmed by screening based on solid culture medium. , ATCC13032ΔN2131-PgapA-Hma(mt)-5714, and ATCC13032ΔN2131-PgapA-Hma(mt)-6718 strains) were higher than the growth level in the minimal medium containing 1 g / L histidine.

[0184] Example 10. Production of strains introduced with a library selected from an L-histidine-producing strain derived from coryneform bacteria (KCCM 80179) and evaluation of L-histidine-producing ability

[0185] A vector for introducing the mutant hma contained in the six colonies selected in Example 9 into an L-histidine-producing strain was produced. To this end, PCR was performed using the primer pair of SEQ ID NO:48 and SEQ ID NO:49 and the chromosomal DNA of ATCC13032ΔN2131-PgapA-Hma(mt)-1216, ATCC13032ΔN2131-PgapA-Hma(mt)-2305, ATCC13032ΔN2131-PgapA-Hma(mt)-3411, ATCC13032ΔN2131-PgapA-Hma(mt)-4426, ATCC13032ΔN2131-PgapA-Hma(mt)-5714, and ATCC13032ΔN2131-PgapA-Hma(mt)-6718 as templates to obtain mutant hma. The PCR conditions were denaturation at 96°C for 30 seconds; annealing at 53°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 30 times. In order to obtain a PgapA fragment that can be connected to the mutant hma, a primer pair of SEQ ID NO: 26 and SEQ ID NO: 50 was used to perform PCR with the chromosome of ATCC13032 as a template. As a polymerase for the PCR reaction, PfuUltraTM High Reliability DNA Polymerase (Stratagene) was used, and the PCR conditions were as follows: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes to obtain the PgapA fragment. The six mutant hma fragments and PgapA fragments obtained were cloned using the Gibson assembly method and the pDZΔN2131 vector cut with ScaI restriction enzyme to obtain recombinant plasmids, which were named pDZΔN2131-PgapA-Hma(mt)-1216, pDZΔN2131-PgapA-Hma(mt)-2315, pDZΔN2131-PgapA-Hma(mt)-3411, pDZΔN2131-PgapA-Hma(mt)-4426, pDZΔN2131-PgapA-Hma(mt)-5714, and pDZΔN2131-PgapA-Hma(mt)-6718 according to the library colonies of their origin.

[0186] Afterwards, the produced vector was transformed into the KCCM 80179 strain by electroporation, and through a secondary hybridization process, six strains were produced, into which six mutant hma were introduced, and they were named KCCM80179ΔN2131-PgapA-Hma(mt)-1216, KCCM80179ΔN2131-PgapA-Hma(mt)-2315, KCCM80179ΔN2131-PgapA-Hma(mt)-3411, KCCM80179ΔN2131-PgapA-Hma(mt)-4426, and KCCM80179ΔN2131-PgapA-Hma(mt)-5714, respectively. , KCCM80179ΔN2131-PgapA-Hma(mt)-6718.

[0187] In order to confirm the L-histidine production capacity of the six strains produced, the KCCM80179ΔN2131-PgapA-Hma strain produced in Example 7 was used as a control group, and the measurement was performed by the same method as in Example 4. The results are shown in Table 8:

[0188] [Table 8]

[0189] L-histidine production of six strains derived from KCCM 80179 with hma mutation

[0190]

[0191] As shown in Table 8 above, the NCgl2131 deletion strain (KCCM 80179ΔN2131) has an L-histidine production capacity equivalent to the level of the parent strain KCCM80179 strain, but the ATCC13032ΔN2131::Hma strain into which the gene derived from Marseille vulnificus was introduced has an increased L-histidine production capacity compared to the NCgl2131 deletion strain and the parent strain KCCM 80179 strain.

[0192] It has been confirmed that compared with the NCgl2131 deletion strain and the parent strain KCCM 80179 strain, the six strains introduced with six mutant hma have L-histidine production capabilities equivalent to or higher than that of the KCCM 80179ΔN2131-PgapA-Hma strain, especially the KCCM80179ΔN2131-PgapA-Hma(mt)-5714 and KCCM80179ΔN2131-PgapA-Hma(mt)-6718 strains, whose L-histidine production capabilities were increased by 3.6% and 12.1%, respectively, compared with the KCCM 80179ΔN2131-PgapA-Hma introduced with the wild-type hma.

[0193] Example 11. Confirmation of mutant Hma gene mutation with increased L-histidine production ability

[0194] In order to confirm the mutation introduced into Hma of the KCCM80179ΔN2131-PgapA-Hma(mt)-6718 strain (the effect of increasing the L-histidine production capacity was confirmed in Example 10), the base sequence of the Hma variant was analyzed. In order to determine the base sequence, PCR was performed using the primer pair of SEQ ID NO: 18 and SEQ ID NO: 21 and the gDNA of the KCCM80179ΔN2131-PgapA-Hma(mt)-6718 strain as a template. The base sequence of the mutant hma operon and the protein sequence of HmaD or HmaE were confirmed by sequencing, and they were compared with the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 44, and thus, the mutation information of the amino acid sequence of the confirmed mutant HmaFE is shown in Table 9.

[0195] [Table 9]

[0196] KCCM80179ΔN2131-PgapA-Hma(mt)-6718 strain HmaF and HmaE mutations

[0197]

[0198] As a result of sequence confirmation, it was confirmed that the mutant HmaFE introduced into the KCCM80179ΔN2131-PgapA-Hma(mt)-6718 strain was a mutant exporter with increased L-histidine export ability due to the introduction of I72L (isoleucine (Ile, I) at position 72 of SEQ ID NO:43 was mutated to leucine (Leu, L)) and I124V (isoleucine (Ile, I) at position 124 of SEQ ID NO:43 was mutated to valine (Val, V)) mutations into HmaD.

[0199] This result not only confirmed that the resistance to L-histidine concentrations higher than the minimum inhibitory concentration was increased compared to the wild type, but also confirmed that the L-histidine production capacity was significantly increased by introducing the mutant exporter derived from Bacillus massiliense. This result shows that the selected mutant protein derived from Bacillus massiliense is a mutant L-histidine exporter protein capable of specifically exporting L-histidine.

[0200] Example 12. Production of strains into which genes derived from Bacillus vulnificus were introduced based on the L-histidine-producing strain (CA14-737) and evaluation of L-histidine production capacity

[0201] In order to confirm the L-histidine export ability of the protein Hma mutant derived from Bacillus massiliense, it was introduced into the L-histidine-producing strain CA14-737 (KCCM 12411P, Korean Patent Publication No. 10-2019-0065984) derived from wild-type Corynebacterium glutamicum ATCC13032, into which the HisG polypeptide mutation (in which feedback limitation is resolved by L-histidine) was introduced and the L-histidine biosynthesis gene was enhanced.

[0202] To this end, the three vectors produced in Examples 2 and 10 (pDZΔN2131, pDZΔN2131-PgapA-Hma, and pDZΔN2131-PgapA-Hma(mt)-6718) were respectively transformed into the CA14-737 strain by electroporation, and through a secondary hybridization process, three strains in which the NCgl2131 gene on the chromosome was deleted or the L-histidine export gene was replaced were produced, and they were named CA14-737ΔN2131, CA14-737ΔN2131-PgapA-Hma, and CA14-737ΔN2131-PgapA-Hma(mt)-6718, respectively.

[0203] In order to confirm the L-histidine production ability of the produced CA14-737ΔN2131, CA14-737ΔN2131-PgapA-Hma, CA14-737ΔN2131-PgapA-Hma(mt)-6718 strains, they were cultured by the method performed in Example 4, and the L-histidine production (histidine content in the culture medium) was measured. The results are shown in the following Table 10:

[0204] [Table 10]

[0205] L-histidine production of strains into which wild-type genes and variants derived from Bacillus vulnificus from CA14-737 were introduced

[0206]

[0207] As shown in Table 10, compared with the NCgl2131 deletion strain (CA14-737ΔN2131) and the parent strain CA14-737 strain, the L-histidine production of the CA14-737ΔN2131-PgapA-Hma strain into which the gene derived from Massilia vulnificus was introduced increased by 54%, and the L-histidine production of the CA14-737ΔN2131-PgapA-Hma(mt)-6718 strain (in which the mutant hma was introduced) increased by 78%.

[0208] Thus, it was confirmed that all wild-type and mutant proteins derived from Bacillus vulnificus were able to specifically export L-histidine, and the selected mutant protein was an L-histidine export protein with an export capacity even higher than that of the wild-type protein.

[0209] Example 13. Production of E. coli vectors for expressing wild-type genes and variants from B. vulnificus

[0210] In order to confirm the L-histidine export ability of proteins Hma and variants derived from Bacillus massiliense for various strains, vectors capable of expressing wild-type Hma and Hma variants were produced. Each gene was cloned into the E. coli expression vector pCC1BAC (hereinafter, pBAC, Epicentre corp.) and designed to express exogenous L-histidine export gene candidates under the promoter of the yccA gene (hereinafter, PyccA, SEQ ID NO: 51) derived from the E. coli strain MG1655.

[0211] In order to obtain protein Hma and mutant gene fragments from Bacillus vulnificus, PCR was performed using primer pairs of SEQ ID NO: 52 and SEQ ID NO: 53, with chromosomal DNA of ATCC13032ΔN2131-PgapA-Hma and ATCC13032ΔN2131-PgapA-Hma (mt) -6718 as templates to obtain wild-type and mutant hma DNA fragments. The PCR conditions were denaturation at 96°C for 30 seconds; annealing at 53°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 30 times. In order to obtain the PyccA fragment, PCR was performed using primer pairs of SEQ ID NO: 54 and SEQ ID NO: 55, with the chromosome of MG1655 as a template. As a polymerase for PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes to obtain the PyccA fragment. The obtained wild-type and mutant hma fragments and PyccA fragments and the pBAC vector cut with EcoRI restriction enzyme were cloned using the Gibson assembly method to obtain recombinant plasmids, which were named pBAC-PyccA-Hma, pBAC-PyccA-Hma(mt)-6718.

[0212] Example 14. Production of strains into which the Hma wild-type gene and variants derived from Bacillus vulnificus were introduced based on an L-histidine-producing strain derived from Escherichia coli and evaluation of L-histidine production capacity

[0213] In order to confirm the L-histidine export ability of the protein Hma variant derived from Bacillus vulnificus based on the L-histidine producing strain derived from Escherichia coli, three strains were produced, in which the two vectors produced in Example 13 and the pBAC vector were introduced into the CA14-9003e strain (MG1655+hisGr hisL'_ΔΔpurR) having the previously reported genotype (purR deletion, hisL deletion, hisGr; The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants; Applied Biochemistry and Microbiology, 2013, Vol. 49, No. 2, pp. 130-135), and they were named CA14-9003e / pBAC, CA14-9003e / pBAC-PyccA-Hma, and CA14-9003e / pBAC-PyccA-Hma(mt)-6718, respectively.

[0214] In order to confirm the L-histidine production capacity of the CA14-9003e / pBAC, CA14-9003e / pBAC-PyccA-Hma, CA14-9003e / pBAC-PyccA-Hma(mt)-6718 strains produced, they are cultivated by the following method. The strain is cultured in LB solid medium (comprising 25 μg / mL chloramphenicol) for 16 hours, and then each strain is inoculated into a 250mL baffled flask containing 25mL LB liquid medium, and cultured at 37°C with 200rpm shaking for 20 hours. Then, 1mL of seed culture fluid is inoculated into a 250mL baffled flask containing 25mL of Escherichia coli production medium (Applied Biochemistry and Microbiology, 2013, Vol.49, No.2, pp.130-135), and cultured at 37°C with 200rpm shaking for 48 hours. The culture medium for cultivation is as follows:

[0215] <E. coli production medium>

[0216] Glucose 4% (w / v), yeast extract 0.2% (w / v), ammonium sulfate 1.6% (w / v), potassium phosphate dihydrate 0.06% (w / v), iron sulfate heptahydrate 0.0005% (w / v), magnesium sulfate pentahydrate 0.0005% (w / v), calcium carbonate, pH 7.2

[0217] After the cultivation was completed, the L-histidine production (histidine content in the medium) was measured by HPLC, and the results are shown in Table 11 below.

[0218] [Table 11]

[0219] L-histidine production of wild-type and variant strains derived from Bacillus vulnificus derived from CA14-9003e

[0220]

[0221]

[0222] As shown in Table 11, it was confirmed that the L-histidine production capacity of the CA14-9003e / pBAC-PyccA-Hma(mt)-6718 strain introduced with mutant Hma was 30% higher than that of the CA14-9003e / pBAC-PyccA-Hma strain introduced with wild-type Hma.

[0223] This result confirmed that even when the L-histidine export variant derived from Bacillus vulnificus was introduced into microorganisms other than Corynebacterium strains, the extracellular L-histidine export activity was significantly increased.

[0224] According to the above description, those skilled in the art to which the present disclosure belongs will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or basic features of the present invention. In this regard, the above embodiments should be understood to be illustrative and non-restrictive in all aspects. The scope of the present disclosure should be interpreted as including all changes or modifications derived from the meaning and scope of the claims to be described later and concepts equivalent thereto, without being limited to the above detailed description.

Claims

1. A mutant L-histidine exporter protein, wherein (1) the amino acid corresponding to the 72nd amino acid residue of the amino acid sequence of SEQ ID NO: 43 is substituted by another amino acid, (2) the amino acid at residue 124 of the amino acid sequence corresponding to SEQ ID NO: 43 is substituted by another amino acid, or (3) (1) and (2) are both replaced.

2. The protein according to claim 1, wherein (1) the amino acid at position 72 of the amino acid sequence of SEQ ID NO: 43 is substituted with leucine, glycine, proline, alanine, valine or methionine, or (2) the amino acid at residue 124 of the amino acid sequence corresponding to SEQ ID NO: 43 is substituted with valine, glycine, proline, alanine, leucine or methionine, or (3) (1) and (2) are both replaced. 3 . The protein according to claim 1 , wherein the protein has 99% or higher sequence homology with the amino acid sequence of SEQ ID NO:

56. A polynucleotide encoding the protein according to any one of claims 1 to 3. 5 . A microorganism comprising the protein according to claim 1 or a polynucleotide encoding the protein. The microorganism according to claim 5 , wherein the microorganism has the ability to produce L-histidine.

7. The microorganism according to claim 5, wherein the microorganism is of the genus Corynebacterium or the genus Escherichia.

8. The microorganism according to claim 7, wherein the microorganism is Corynebacterium glutamicum or Escherichia coli.

9. A composition for producing L-histidine, comprising The protein according to any one of claims 1 to 3, A polynucleotide encoding the protein, or A recombinant microorganism comprising the polynucleotide.

10. A method for producing L-histidine, comprising culturing a microorganism comprising the protein according to any one of claims 1 to 3 or a polynucleotide encoding the protein in a culture medium.

11. The method for producing L-histidine according to claim 10, further comprising recovering L-histidine from the cultured microorganism or the culture medium after the culture.

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