L-Histidine excretion protein and method for producing L-histidine using the same

Abstract

This application can dramatically improve the L-histidine production amount as a result of discovering a histidine excretion protein having an L-histidine excretion ability or a variant thereof and expressing the same in a microorganism having an L-histidine production ability.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims the benefit of priority based on Korean Patent Application No. 10 - 2022 - 0076773 filed on Jun. 23, 2022, and all contents disclosed in the literature of the Korean Patent Application are incorporated herein by reference in their entirety.

[0002] This application relates to a novel protein having histidine excretion activity, an L - histidine - producing microorganism modified to express the protein, and a method for producing L - histidine using the microorganism.

Background Art

[0003] L - histidine is one of the 20 standard amino acids. From a nutritional perspective, it is not required in large amounts for adults, but is classified as an essential amino acid required for growing children. Also, L - histidine is involved in important physiological processes such as antioxidation and immune regulation, and is used in the medical industry such as gastric ulcer therapeutic agents, raw materials for cardiovascular therapeutic agents, and amino acid infusion preparations.

[0004] L - histidine is particularly abundant in hemoglobin and is mainly produced by the protein hydrolysis extraction method using blood as a raw material. However, such a method has disadvantages such as low efficiency and environmental pollution. On the other hand, it is possible to produce L - histidine by the microbial fermentation method, but large - scale industrialization has not yet been carried out. This is because the biosynthesis of L - histidine is in a competitive relationship with phosphoribosyl pyrophosphate (PRPP), which is a nucleotide synthesis precursor, and has a complex biosynthesis process and regulatory mechanism that require high energy.

[0005] Examples are known in which the production of an amino acid increases when the expression and / or function of a protein having the ability to excrete other types of amino acids is enhanced, but little prior research has been conducted on proteins having L - histidine - specific excretion ability.

[0006] Under such a background, there is a demand for the discovery of proteins having histidine-specific excretion ability and the development of histidine production technology using the same.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The object of the present application is (1) whether the amino acid corresponding to the 72nd amino acid residue of the amino acid sequence of SEQ ID NO: 43 is substituted with another amino acid, (2) whether the amino acid corresponding to the 124th residue of the amino acid sequence of SEQ ID NO: 43 is substituted with another amino acid, (3) to provide a mutant L-histidine excretion protein in which both of the above (1) and (2) are substituted.

[0008] Another object of the present application is to provide a microorganism containing the protein or a polynucleotide encoding the protein.

[0009] Still another object of the present application is to provide a composition for producing L-histidine containing the protein, a polynucleotide encoding the protein, or the microorganism.

[0010] Still another object of the present application is to provide a use of the protein, a polynucleotide encoding the protein, or the microorganism for producing L-histidine.

[0011] Still another object of the present application is to provide a use of the protein, a polynucleotide encoding the protein, or the microorganism for manufacturing a composition for producing L-histidine.

[0012] Still another object of the present application is to provide a method for producing L-histidine, which includes a step of culturing the microorganism in a medium.

Means for Solving the Problems

[0013] This application proposes that by discovering mutants of histidine excretion proteins having L-histidine excretion ability and expressing them in microorganisms having the ability to produce L-histidine, it is possible to dramatically improve the L-histidine production amount.

[0014] In this specification, it was confirmed that microorganisms expressing the AzlC family ABC transporter permease derived from Helcobacillus massiliensis are excellent in the ability to produce L-histidine, and when an amino acid substitution mutation is introduced at a specific position of the AzlC family ABC transporter permease, it was confirmed that the ability to produce L-histidine further increases.

[0015] Protein, polynucleotide, and recombinant vector

[0016] One aspect provides a mutant protein (or polypeptide) having L-histidine excretion activity. The protein may be a protein having L-histidine specific excretion ability. In this specification, the mutant protein can be expressed as a mutant L-histidine excretion protein. The mutant protein may have AzlC family ABC transporter permease activity.

[0017] In one example, the variant protein may have L-histidine excretion activity equivalent to or stronger than that of a wild-type L-histidine excretion protein (for example, an AzlD domain-containing protein, or an AzlC family ABC transporter permease, etc.). The AzlD domain-containing protein or the AzlC family ABC transporter permease may be derived from Helcobacillus massiliensis. In the present specification, the AzlD domain-containing protein derived from Helcobacillus massiliensis can be described as the HmaE protein (or HmaE), and the AzlC family ABC transporter permease derived from Helcobacillus massiliensis can be described as the HmaF protein (or HmaF).

[0018] In one example, the variant protein (for example, a variant protein of an AzlD domain-containing protein, specifically a variant protein of an AzlD domain-containing protein derived from Helcobacillus massiliensis) may be expressed together with the AzlC family ABC transporter permease in the same operon gene. In one example, the variant protein may bind to the AzlC family ABC transporter permease and have L-histidine excretion activity.

[0019] In one example, the variant protein may be a variant protein of an AzlC family ABC transporter permease derived from Helcobacillus massiliensis.

[0020] The variant protein may be a variant protein into which a mutation has been introduced in which one or more amino acid residues of the wild-type AzlC family ABC transporter permease derived from Helcobacillus massiliensis have been substituted, deleted, or inserted.

[0021] The wild-type AzlC family ABC transporter permease protein derived from Helicobacter pylori may contain the amino acid sequence of SEQ ID NO: 43 (WP_055090792.1) or may consist of said sequence.

[0022] In one example, the mutant protein has the amino acid sequence of SEQ ID NO: 43, starting from the N-terminus (1) the amino acid corresponding to the 72nd residue is substituted with another amino acid, (2) the amino acid corresponding to the 124th residue is substituted with another amino acid, or (3) may contain an amino acid sequence in which both (1) and (2) are substituted.

[0023] Counting amino acids from the N-terminus in the amino acid sequence as described above can mean counting with the methionine (Met, M) translated from the start codon as the first amino acid.

[0024] In one example, the mutant protein has the amino acid sequence of SEQ ID NO: 43, starting from the N-terminus (1) the amino acid corresponding to the 72nd residue is another amino acid, i.e., an amino acid different from the original amino acid, and is substituted with 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), or contains a sequence substituted therewith, (2) The amino acid corresponding to the 124th residue is an amino acid different from the original amino acid, i.e., a different amino acid, and is substituted with valine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tyrosine, or tryptophan, or (3) It may include a sequence in which all of the above (1) and (2) are substituted.

[0025] In one example, the mutant protein has the amino acid sequence of SEQ ID NO: 43, starting from the N-terminus (1) The amino acid corresponding to the 72nd residue is substituted with leucine, glycine, proline, alanine, valine, or methionine; (2) The amino acid corresponding to the 124th residue is substituted with valine, glycine, proline, alanine, leucine, or methionine; or (3) It may include a sequence in which all of the above (1) and (2) are substituted.

[0026] In one specific example, the mutant protein may have the amino acid sequence of SEQ ID NO: 43, in which the amino acid corresponding to the 72nd residue from the N-terminus is substituted with leucine, and the amino acid corresponding to the 124th residue is substituted with valine. Even if a part of the amino acid sequence excluding the amino acids corresponding to the 72nd and / or 124th amino acid residues from the N-terminus of SEQ ID NO: 43 of the mutant protein is deleted, modified, substituted, or added, as long as it exhibits the AzlC family ABC transporter permease activity, it is obvious that it can be included in the mutant protein of the present application.

[0027] Also, in one example, the variant protein has 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 with the amino acid sequence set forth in SEQ ID NO: 43, and from the N-terminus with the amino acid sequence of SEQ ID NO: 43 (1) the amino acid corresponding to the 72nd residue is substituted with another amino acid, (2) the amino acid corresponding to the 124th residue is substituted with another amino acid, or (3) it can include a polypeptide in which both of the above (1) and (2) are substituted. That is, it includes substitutions with other amino acids at positions corresponding to the 72nd and / or 124th residues 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 a polypeptide having AzlC system ABC transporter permease activity can be included in the variant protein of the present application.

[0028] In one specific example, the mutant protein includes, but is not limited to, the amino acid sequence of SEQ ID NO: 56 or consists of the said sequence. For a mutant protein consisting of the amino acid sequence of SEQ ID NO: 56, as long as a partial amino acid sequence excluding the amino acids corresponding to the 72nd and / or 124th residues from the N-terminus in the amino acid sequence of SEQ ID NO: 56 shows AzlC family ABC transporter permease activity after deletion, modification, substitution or addition, it is obvious that it can be included in the mutant protein of the present application. In another example, in the mutant protein, the amino acids corresponding to the 72nd and / or 124th residues from the N-terminus in the amino acid sequence of SEQ ID NO: 56 are fixed, and it can include a polypeptide 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 with the amino acid sequence of SEQ ID NO: 56. That is, a polypeptide having AzlC family ABC transporter permease activity, in which the amino acids corresponding to the 72nd and / or 124th residues from the N-terminus in the amino acid sequence of SEQ ID NO: 56 are substituted with other amino acids and 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 homology or identity with the amino acid sequence of SEQ ID NO: 56 can be included in the mutant protein of the present application.

[0029] In one example, the mutant protein may have enhanced L-histidine excretion activity compared to the wild-type protein (e.g., wild-type AzlD domain-containing protein). In one example, when the mutant protein is expressed together with the wild-type AzlC family ABC transporter permease protein, the L-histidine excretion activity can be further enhanced.

[0030] The wild-type L-histidine efflux protein may be a protein having 60% or more sequence homology with SEQ ID NO: 43 (wild-type AzlC-based ABC transporter permease protein derived from Helicobacter marismortui), SEQ ID NO: 44 (wild-type AzlD domain-containing protein derived from Helicobacter marismortui), or a combination thereof. For example, in one specific example, the wild-type L-histidine efflux protein may have 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 with SEQ ID NO: 43, 44, or a combination thereof. The protein represented by SEQ ID NO: 43 is encoded by the nucleic acid sequence of SEQ ID NO: 45, the protein represented by SEQ ID NO: 44 is 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 may be encoded by the nucleic acid sequence of SEQ ID NO: 47 (an operon sequence fused at the overlapping site of the 3' end of SEQ ID NO: 45 and the 5' end of SEQ ID NO: 46).

[0031] Another aspect provides a polynucleotide that encodes (codes for) the mutant protein.

[0032] In the present application, the term "polynucleotide" is a polymer of nucleotides in which nucleotide monomers are covalently linked in a long chain and is a DNA or RNA strand having a certain length or more. More specifically, it means a polynucleotide fragment that encodes the mutant polypeptide.

[0033] The polynucleotide encoding the mutant protein of the present application may include a base sequence that encodes the amino acid sequence of SEQ ID NO: 56.

[0034] As used herein, a polynucleotide (which may be used interchangeably with "gene") or a polypeptide (which may be used interchangeably with "protein") is said to "comprise, consist of, or be represented by a specific nucleic acid sequence or amino acid sequence" means that the polynucleotide or polypeptide must contain the specific nucleic acid sequence or amino acid sequence, and within the range that maintains the original function and / or the intended function of the polynucleotide or polypeptide, it can be interpreted to include (or not exclude) a "substantially equivalent sequence" in which mutations (deletions, substitutions, modifications, and / or additions) are made to the specific nucleic acid sequence or amino acid sequence.

[0035] In one example, the nucleic acid sequences or amino acid sequences provided herein can include those modified by ordinary mutagenesis methods, such as directed evolution and / or site-directed mutagenesis, within the range that maintains their original or intended functions. In one example, a polynucleotide or polypeptide "comprises or consists of a specific nucleic acid sequence or amino acid sequence" or the polynucleotide or polypeptide (i) must contain the specific nucleic acid sequence or amino acid sequence, or (ii) consists of or must contain an 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, 98% or more, 99% or more, or 99.5% or more homology with the specific nucleic acid sequence or amino acid sequence, and maintains the original function and / or the intended function. As used herein, the intended function can mean a function that confers or increases the L-histidine excretion activity and / or L-histidine production ability of a microorganism.

[0036] The nucleic acid sequences described in this specification can be variously modified in the coding region within the range that does not change the amino acid sequence and / or function of the protein expressed from the coding region, taking into account the codons preferred in the microorganism in which the protein is to be expressed due to the degeneracy of the codons.

[0037] As used herein, the term "homology (identity)" means the degree of identity with a given nucleic acid sequence or amino acid sequence and is expressed as a percentage (%). In the case of identity to a nucleic acid sequence, for example, it can be determined using algorithms such as BLAST (see Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (see Methods Enzymol., 183, 63, 1990). Based on such an algorithm as BLAST, programs called BLASTN and BLASTX have been developed (see http: / / www.ncbi.nlm.nih.gov).

[0038] In one example, a polynucleotide containing the specific nucleic acid sequence provided herein can be interpreted to include not only the specific nucleic acid sequence or a nucleic acid sequence substantially equivalent thereto, but also a polynucleotide fragment containing a nucleic acid sequence complementary to the specific nucleic acid sequence. Specifically, the polynucleotide having such complementarity hybridizes at a Tm value that can be appropriately 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 can be analyzed under the conditions described below: such conditions are specifically described in known literature. For example, genes having 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 hybridize, and genes having lower complementarity do not hybridize, or conditions for washing in normal Southern hybridization, which are 60°C, 1×SSC (saline-sodium citrate buffer), and 0.1% (w / v) SDS (Sodium Dodecyl Sulfate); 60°C, 0.1×SSC, and 0.1% SDS; or 68°C, 0.1×SSC, and 0.1% SDS, including washing once, specifically 2 to 3 times, at the corresponding salt concentration and temperature, but not limited thereto. For hybridization, it is required that two nucleotides have complementary sequences, or base mismatches are allowed 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 the case of DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, which is well known in the relevant technical field (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0039] The introduction of the polynucleotide or vector can be appropriately selected and carried out by those skilled in the art using known transformation methods. In this specification, the term "transformation" means that the polynucleotide transformed in the process of introducing a specific polynucleotide or a vector containing the same into a host cell can be inserted into or located outside the chromosome within the host cell. In one example, transformation may be to introduce a polynucleotide encoding a target protein (foreign protein) or a vector containing the same into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. Further, the polynucleotide can contain DNA and / or RNA encoding the target protein. The form of the polynucleotide introduced into the host cell is not limited 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 of a gene construct containing all the elements necessary for its own expression. The expression cassette can usually contain expression regulatory elements such as a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and / or a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. Further, the polynucleotide may be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell. In the above, the term "operably linked" means that the expression regulatory element (e.g., promoter) and the polynucleotide are functionally linked so that the expression regulatory element can perform transcriptional regulation (e.g., transcription initiation) of the polynucleotide encoding the target protein (foreign protein). Operable linkage can be carried out using genetic recombination techniques known in the art, for example, by ordinary site-specific DNA cleavage and ligation, but is not limited thereto.

[0040] The method of transforming the polynucleotide into a host cell can be carried out by any method of introducing nucleic acid into a cell (microorganism), and the transformation technique known in the art can be appropriately selected according to the host cell and carried out. As the known transformation methods, electroporation, calcium phosphate (CaPO4) precipitation method, calcium chloride (CaCl2) precipitation method, microinjection, polyethylene glycol (PEG) precipitation method (polyethylene glycol-mediated uptake), DEAE-dextran method, cationic liposome method, lipofection, lithium acetate-DMSO method, etc. are exemplified, but not limited thereto.

[0041] The introduction (insertion) of the polynucleotide into the host cell genome (chromosome) can be carried out by a person skilled in the art appropriately selecting a known method. For example, an RNA-guided endonuclease system or CRISPR system; for example, (a) an RNA-guided endonuclease (e.g., Cas9 protein, etc.), its coding gene, or a vector containing the gene; and (b) a guide RNA (e.g., single guide RNA (sgRNA), etc.), its coding DNA, or a mixture containing the DNA (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA, etc.), a complex (e.g., a ribonucleic acid fusion protein (RNP), a recombinant vector (e.g., a vector containing an RNA-guided endonuclease coding gene and a guide RNA coding DNA, etc.), etc.) selected from the group consisting of one or more, but not limited thereto.

[0042] Another aspect provides a recombinant vector containing the polynucleotide. The recombinant vector can be used as an expression vector for the polypeptide. The recombinant vector may be for inserting the polynucleotide into the genome of the host cell or replacing the corresponding gene in the host cell genome.

[0043] As used herein, the term "vector" means a DNA construct containing the nucleotide sequence of a polynucleotide encoding a target protein operably linked to a suitable regulatory sequence so as to be capable of expressing the target protein in a suitable host. The regulatory sequence can include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence for regulating the termination of transcription and / or translation. After being transformed into a suitable host cell, the vector can be expressed regardless of the host cell genome or integrated into the host cell genome.

[0044] As used herein, the usable vector is not particularly limited as long as it can replicate in a host cell, and can be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, cosmids, viruses, bacteriophages, etc. in their natural or recombinant states. For example, as the vector, phage vectors or cosmid vectors such as pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used, and as plasmid vectors, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be exemplified, but are not limited thereto.

[0045] In this specification, the usable vector may be a known expression vector and / or a vector for inserting a polynucleotide into the host cell chromosome. Insertion of the polynucleotide into the host cell chromosome is carried out by any method known in the art, such as, but not limited to, homologous recombination or the CRISPR system. The vector can further include a selection marker for confirming whether it has been inserted into the chromosome. The selection marker is for selecting cells transformed with the vector, that is, for confirming the necessity of insertion of the polynucleotide, and can be selected from 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, transformed cells can be selected because only cells expressing the selection marker survive or show another phenotypic trait.

[0046] microorganism

[0047] Another aspect provides a microorganism comprising one or more (1, 2, or all 3) selected from the group consisting of the mutant protein, the polynucleotide encoding the mutant protein, and the recombinant vector containing the polynucleotide. The microorganism may have L-histidine excretion activity and / or L-histidine production ability. The L-histidine excretion activity and / or L-histidine production ability of the microorganism is enhanced (or increased, improved) compared to a microorganism that does not contain one or more selected from the group consisting of the mutant protein, the polynucleotide encoding the mutant protein, and the recombinant vector containing the polynucleotide.

[0048] The mutant protein may be exogenous. As used herein, "exogenous" means not inherently present in the microorganism and derived from a species different from the microorganism.

[0049] As used herein, the "microorganism with enhanced L-histidine excretion activity and / or L-histidine production ability" is an organism that has been engineered (mutated) to express the aforementioned mutant protein, such that a microorganism that originally lacked L-histidine excretion activity and / or L-histidine production ability has acquired such activity and / or ability, or has acquired L-histidine excretion activity and / or L-histidine production ability that is higher than its original level.

[0050] As used herein, the term "microorganism" includes single-celled bacteria and can be used interchangeably with the term "cell".

[0051] In the present application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modifications. Due to causes such as the insertion of foreign genes, enhancement or inactivation of the activity of endogenous genes, etc., the specific mechanism of the microorganism may be weakened or strengthened. The microorganism may include genetic modifications for the production of a target polypeptide, protein or product (e.g., L-histidine).

[0052] The microorganism of the present application is, but not limited to, a genetically modified microorganism (e.g., a recombinant microorganism) via a vector such that the activity of an L-histidine excretion protein or a polynucleotide encoding the same is enhanced. The vector is as described above.

[0053] As used herein, in order to distinguish the microorganism before being mutated to express the mutant protein from the mutated microorganism, it can be expressed as "parent microorganism or parent strain" or "host cell".

[0054] When the microorganism (or strain, recombinant cell) has L-histidine excretion activity and / or L-histidine production ability, or the L-histidine excretion activity and / or L-histidine production ability is enhanced, it means that, different from an unmodified microorganism, a cell before recombination, a parental strain, and / or a wild-type strain that has no L-histidine excretion activity and / or L-histidine production ability, it is provided with L-histidine excretion activity and / or L-histidine production ability, or the L-histidine excretion activity and / or L-histidine production ability is improved as compared with an unmodified microorganism, a cell before recombination, a parental strain, and / or a wild-type strain.

[0055] The microorganism of the present application is a microorganism including any one or more of the mutant protein of the present application, a polynucleotide encoding the mutant protein of the present application, and a vector including 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 expressing the mutant protein of the present application or the polynucleotide of the present application (for example, a recombinant strain); or a microorganism having the mutant protein activity of the present application (for example, a recombinant strain), but is not limited thereto.

[0056] In one example, the microorganism may be one or more selected from the group consisting of microorganisms of the genus Corynebacterium, microorganisms of the genus Escherichia, etc. The microorganisms of the genus Corynebacterium may include, but are not necessarily limited to, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes, Corynebacterium efficiens, etc. More specifically, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum. The strain of the genus Escherichia may be Escherichia coli.

[0057] The microorganism may include one or more (one kind, two kinds, or all three kinds) selected from the group consisting of a mutant protein, a polynucleotide encoding the mutant protein, and a recombinant vector containing the polynucleotide. In one example, the mutation to express the mutant protein may be carried out by introducing the polynucleotide encoding the aforementioned mutant protein or a recombinant vector containing the same, or may be carried out by artificial mutagenesis (for example, error-prone PCR, etc.). The polynucleotide encoding the mutant protein thus introduced into the parent strain may replace the AzlC-type ABC transporter permease-encoding gene endogenous to the parent strain, or may additionally be included in addition thereto.

[0058] In one example, when the microorganism contains the mutant protein together with the wild-type AzlC family ABC transporter permease protein, the L-histidine excretion activity and / or the L-histidine production ability can be further enhanced.

[0059] In one example, the microorganism with enhanced L-histidine excretion activity and / or L-histidine production ability has an increased L-histidine production ability of 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, the unmodified microorganism, and the microorganism containing the wild-type L-histidine excretion protein, but is not limited thereto.

[0060] In another example, the microorganism with enhanced L-histidine excretion activity and / or L-histidine production ability has an increased L-histidine production ability 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, the unmodified microorganism, and the microorganism containing the wild-type L-histidine excretion protein, but is not limited thereto.

[0061] The term "about" includes all ranges such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values in a range equivalent or similar to the numerical value following the term "about", but is not limited thereto.

[0062] Another aspect provides a composition for L-histidine production containing the mutant protein, the polynucleotide, the recombinant vector, or the microorganism.

[0063] Another aspect provides the use of the mutant protein, the polynucleotide, the recombinant vector, or the microorganism for L-histidine production.

[0064] Another aspect provides a use of the mutant protein, the polynucleotide, the recombinant vector, or the microorganism for the production of a composition for L-histidine production.

[0065] Another aspect provides a method for L-histidine production, which includes the step of culturing the microorganism in a medium. The production method may further include the step of recovering L-histidine from the cultured microorganism, the medium, or all of them after the culturing step.

[0066] Another aspect provides a method for increasing the L-histidine excretion activity and / or L-histidine production ability of the microorganism, or a method for conferring L-histidine excretion activity and / or L-histidine production ability to the microorganism, which includes the step of enhancing the L-histidine excretion activity and / or L-histidine production ability of the microorganism.

[0067] The step of introducing the mutation may include the step of introducing (transforming) a polynucleotide encoding the mutant protein or a recombinant vector containing the polynucleotide into the microorganism, or may include the step of artificially generating a mutation (for example, Error-prone PCR, etc.).

[0068] Another example provides a method for L-histidine production, which includes the step of culturing a microorganism with enhanced L-histidine excretion activity and / or L-histidine production ability in a medium. The method may further include the step of recovering L-histidine from the cultured microorganism, the medium, or all of them after the culturing step.

[0069] In the above method, the step of culturing the microorganism can be carried out by, but not particularly limited to, known batch culture methods, continuous culture methods, fed-batch culture methods, etc. At this time, the culture conditions are not particularly limited thereto, but an appropriate pH (for example, pH 5 to 9, specifically pH 6 to 8) can be adjusted using a basic compound (for example, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (for example, phosphoric acid or sulfuric acid), 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 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 above culture can be secreted into the medium or remain intracellularly.

[0070] The medium that can be used for the cultivation can individually use one or more selected from the group consisting of sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oils and fats (e.g., soybean oil, sunflower seed oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), organic acids (e.g., acetic acid), etc. as a carbon source, or can use two or more in combination, but is not limited thereto. As a nitrogen source, one or more selected from the group consisting of nitrogen-containing organic compounds (e.g., peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal, and urea), inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), etc. can be individually used or two or more can be used in combination, but is not limited thereto. As a phosphorus source, one or more selected from the group consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium-containing salts corresponding thereto can be individually used or two or more can be used in combination, but is not limited thereto. Further, the medium can contain essential growth-promoting substances such as other metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, and / or vitamins.

[0071] The step of recovering the L-histidine may collect the target amino acid from the medium, the culture solution, or the microorganism using a suitable method known in the art according to the culture method. For example, the step of recovering can be performed by one or more methods selected from centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. The method of recovering the L-histidine can further include a purification step before, simultaneously with, or after that.

Advantages of the Invention

[0072] This application can discover a histidine-excreting protein having the ability to excrete L-histidine or a variant thereof, and as a result of expressing this in a microorganism having the ability to produce L-histidine, the production amount of L-histidine can be dramatically improved.

Mode for Carrying Out the Invention

[0073] Hereinafter, the present invention will be described more specifically by the following examples. However, these are for exemplifying the present invention, and the scope of the present invention is not limited by these examples.

[0074] Example 1. Search for foreign histidine excretion gene and candidate selection In order to select protein candidates having the ability to specifically excrete L-histidine, the amino acid sequences of excretion proteins (LysE (Arch Microbiol 180: 155-160), Wex (Korean Registered Patent No. 10-1968317), BrnFE (Arch Microbiol 180: 155-160)) for classified amino acids (basic amino acid: L-lysine (L-lys), aromatic amino acid: tryptophan (Trp), side chain amino acid: isoleucine (Ile)) were used as query sequences, and based on the PSI-BLAST search results from the NCBI and Kegg database, candidate genes predicted to be membrane proteins that may excrete L-histidine and the microorganisms carrying them were selected.

[0075] Among these, considering the biosafety level and the possibility of ensuring a level applicable to production strains, as shown in Table 1 below, one protein based on LysE, three proteins based on Wex, and two proteins based on BrnFE, the genes encoding them, and the microorganisms containing them were selected:

[0076]

Table 1

[0077] (In Table 1 above, the biosafety level follows the microbial pathogenicity index (levels 1 to 4) defined by the Centers for Disease Control and Prevention in the United States (the lower the level, the safer it is).)

[0078] Example 2. Production of a vector for introducing a foreign L-histidine excretion gene candidate and a recombinant Corynebacterium strain into which the vector has been introduced Six vectors for introducing six foreign L-histidine excretion gene candidates selected in Example 1 into Corynebacterium strains were prepared.

[0079] Example 2-1. Production of the vector pDZΔN2131 for inserting a target gene To introduce a foreign L-histidine excretion gene candidate, among the genes encoding the transposon of Corynebacterium glutamicum, the NCgl2131 gene was used as the insertion site (Journal of Biotechnology 104, 5-25 Jorn Kalinowski et al, 2003). Also, the foreign L-histidine excretion gene candidate was designed to be expressed under the promoter of the Corynebacterium-derived gapA gene (hereinafter, PgapA, SEQ ID NO: 17).

[0080] To replace the NCgl2131 gene with an excretion gene, an NCgl2131 deletion and target gene insertion vector was constructed. To construct the vector, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum strain ATCC13032 as a template and primer pairs of SEQ ID NO: 18 and SEQ ID NO: 19, and SEQ ID NO: 20 and SEQ ID NO: 21, respectively. PfuUltraTM High-Fidelity DNA Polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were as follows: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization reaction at 72°C for 2 minutes, repeated 28 times, followed by a polymerization reaction at 72°C for 5 minutes. As a result, DNA fragments of 531 bp del-N2131L (SEQ ID NO: 22) and 555 bp del-N2131R (SEQ ID NO: 23) were obtained, respectively. After purifying the obtained DNA products using the QIAGEN PCR Purification kit, cloning was performed using the pDZ vector (Korean Registered Patent No. 10-0924065) and the TaKaRa Infusion Cloning Kit to construct a vector pDZΔN2131 for NCgl2131 gene deletion and target gene insertion.

[0081] Example 2-2. Preparation of Six Kinds of Vectors for Introducing Foreign L-Histidine Excretion Gene Candidates The nucleotide sequence information of the gene encoding the protein derived from Herbaspirillum aquaticum (hereinafter, Haq, SEQ ID NO: 1) (hereinafter, haq, SEQ ID NO: 2) was obtained from the GeneBank of the National Institutes of Health (NIH GenBank). To amplify haq, PCR was performed using the chromosomal DNA of Herbaspirillum aquaticum strain (KCTC42001) as a template and the primer pair of SEQ ID NO: 24 and SEQ ID NO: 25. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 2 minutes, repeated 28 times, and then polymerization reaction at 72°C for 5 minutes. As a result, a 977-bp haq fragment containing 945-bp haq (SEQ ID NO: 2) was obtained. To obtain a PgapA fragment that can be ligated to haq, PCR was performed using the chromosome of ATCC13032 as a template and the primer pair of SEQ ID NO: 26 and SEQ ID NO: 27. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 1 minute, repeated 28 times, and then polymerization reaction at 72°C for 5 minutes. As a result, a 441-bp PgapA fragment containing 409-bp PgapA (SEQ ID NO: 17) was obtained. The obtained haq fragment, PgapA fragment, and the pDZΔN2131 vector cleaved 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.

[0082] The nucleotide sequence information of the gene encoding the protein derived from Cupriavidus pinatubonensis (hereinafter, Cpi, SEQ ID NO: 3) (hereinafter, cpi, SEQ ID NO: 4) was obtained from the National Institutes of Health GeneBank (NIH GenBank). To amplify cpi derived from Cupriavidus pinatubonensis, PCR was performed using the chromosomal DNA of Cupriavidus pinatubonensis strain (KCTC22125) as a template and a pair of primers of SEQ ID NO: 28 and SEQ ID NO: 29. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 2 minutes, repeated 28 times, followed by a polymerization reaction at 72°C for 5 minutes. As a result, a 977-bp cpi fragment containing 945-bp cpi (SEQ ID NO: 4) was obtained. To obtain a PgapA fragment that can be ligated to cpi, PCR was performed using the chromosome of ATCC13032 as a template and a pair of primers of SEQ ID NO: 26 and SEQ ID NO: 30. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 1 minute, repeated 28 times, followed by a polymerization reaction at 72°C for 5 minutes. As a result, a 441-bp PgapA fragment containing 409-bp PgapA (SEQ ID NO: 17) was obtained. The obtained cpi fragment, PgapA fragment, and the pDZΔN2131 vector digested with ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Cpi.

[0083] The nucleotide sequence information of the gene encoding the protein derived from Kluyvera cryocrescens (hereinafter, Kcr, SEQ ID NO: 5) (hereinafter, kcr, SEQ ID NO: 6) was obtained from the National Institutes of Health GeneBank (NIH GenBank). To amplify kcr derived from Kluyvera cryocrescens, PCR was performed using the chromosomal DNA of Kluyvera cryocrescens strain (KCTC2580) as a template and a primer pair of SEQ ID NO: 31 and SEQ ID NO: 32. As the polymerase for the PCR reaction, PfuUltra™ High-Fidelity 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 reaction at 72°C for 2 minutes, repeated 28 times, and then the polymerization reaction was carried out at 72°C for 5 minutes. As a result, a 914-bp kcr fragment containing 882-bp kcr (SEQ ID NO: 6) was obtained. To obtain a PgapA fragment that can be ligated to kcr, PCR was performed using the chromosome of ATCC13032 as a template and a primer pair of SEQ ID NO: 26 and SEQ ID NO: 33. As the polymerase for the PCR reaction, PfuUltra™ High-Fidelity 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 reaction at 72°C for 1 minute, repeated 28 times, and then the polymerization reaction was carried out at 72°C for 5 minutes. As a result, a 441-bp PgapA fragment containing 409-bp PgapA (SEQ ID NO: 17) was obtained. The obtained kcr fragment, PgapA fragment, and the pDZΔN2131 vector digested with ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Kcr.

[0084] The nucleotide sequence information of the gene encoding the protein derived from Corynebacterium stationis (hereinafter, Cst, SEQ ID NO: 7) (hereinafter, cst, SEQ ID NO: 8) was obtained from the National Institutes of Health GeneBank (NIH GenBank). To amplify cst derived from Corynebacterium stationis, PCR was performed using the chromosomal DNA of Corynebacterium stationis strain (ATCC6872) as a template and the primer pair of SEQ ID NO: 34 and SEQ ID NO: 35. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 2 minutes, repeated 28 times, and then the polymerization reaction was carried out at 72°C for 5 minutes. As a result, a 749-bp cst fragment containing 717-bp cst (SEQ ID NO: 8) was obtained. To obtain a PgapA fragment that can be ligated to cst, PCR was performed using the chromosome of ATCC13032 as a template and the primer pair of SEQ ID NO: 26 and SEQ ID NO: 36. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 1 minute, repeated 28 times, and then the polymerization reaction was carried out at 72°C for 5 minutes. As a result, a 441-bp PgapA fragment containing 409-bp PgapA (SEQ ID NO: 17) was obtained. The obtained cst fragment, PgapA fragment, and the pDZΔN2131 vector digested with ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Cst.

[0085] The nucleotide sequence information of the operon (hereinafter, lsa, SEQ ID NO: 11) encoding the protein derived from Leucobacter salsicius (hereinafter, LsaFE, SEQ ID NO: 9, 10) was obtained from the National Institutes of Health GeneBank (NIH GenBank). To amplify lsa derived from Leucobacter salsicius, PCR was performed using the chromosomal DNA of Leucobacter salsicius strain (KCTC19904) as a template and the primer pair of SEQ ID NO: 37 and SEQ ID NO: 38. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 2 minutes, repeated 28 times, and then the polymerization reaction was carried out at 72°C for 5 minutes. As a result, a 1080 bp lsa fragment containing 1048 bp of lsa (SEQ ID NO: 11) was obtained. To obtain a PgapA fragment that can be ligated to lsa, PCR was performed using the chromosome of ATCC13032 as a template and the primer pair of SEQ ID NO: 26 and SEQ ID NO: 39. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 1 minute, repeated 28 times, and then the polymerization reaction was carried out at 72°C for 5 minutes. As a result, a 441 bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO: 17) was obtained. The obtained lsa fragment, PgapA fragment, and the pDZΔN2131 vector digested with ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Lsa.

[0086] The nucleotide sequence information of the operon (hereinafter, dva, SEQ ID NO: 16) encoding the protein derived from Dermabacter vaginalis (hereinafter, DvaFE, SEQ ID NO: 12, 13) was obtained from the National Institutes of Health GeneBank (NIH GenBank). To amplify dva derived from Dermabacter vaginalis, PCR was performed using the chromosomal DNA of the Dermabacter vaginalis strain (KCTC39585) as a template and the primer pair of SEQ ID NO: 40 and SEQ ID NO: 41. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 2 minutes, repeated 28 times, and then a polymerization reaction was performed at 72°C for 5 minutes. As a result, an 1113 bp dva fragment containing 1081 bp of dva (SEQ ID NO: 16) was obtained. To obtain a PgapA fragment that can be ligated to dva, PCR was performed using the chromosome of ATCC13032 as a template and the primer pair of SEQ ID NO: 26 and SEQ ID NO: 42. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 1 minute, repeated 28 times, and then a polymerization reaction was performed at 72°C for 5 minutes. As a result, a 441 bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO: 17) was obtained. The obtained dva fragment, PgapA fragment, and the pDZΔN2131 vector cleaved with ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Dva.

[0087] Example 2-3. Production of Recombinant Corynebacterium Strains To confirm the L-histidine excretion ability of the foreign L-histidine excretion gene candidate, the prepared NCgl2131 deletion vector (pDZΔN2131) and six foreign L-histidine excretion gene candidate-introduced vectors (pDZΔN2131-PgapA-Haq, pDZΔN2131-PgapA-Cpi, pDZΔN2131-PgapA-Kcr, pDZΔN2131-PgapA-Cst, pDZΔN2131-PgapA-Lsa, pDZΔN2131-PgapA-Dva) were each introduced into Corynebacterium glutamicum ATCC13032 strain. More specifically, each of the vectors was transformed into the ATCC13032 strain by electroporation, and through a secondary crossing process, seven recombinant strains in which the NCgl2131 gene on the chromosome was deleted or replaced with a foreign L-histidine excretion gene candidate were produced. These were named ATCC13032ΔN2131 (N2131 gene deletion), ATCC13032ΔN2131::Haq (N2131 gene replaced with haq), ATCC13032ΔN2131::Cpi (N2131 gene replaced with cpi), ATCC13032ΔN2131::Kcr (N2131 gene replaced with kcr), ATCC13032ΔN2131::Cst (N2131 gene replaced with cst), ATCC13032ΔN2131::Lsa (N2131 gene replaced with lsa), and ATCC13032ΔN2131::Dva (N2131 gene replaced with dva), respectively.

[0088] Example 3. MIC Measurement of Corynebacterium Strains Introduced with Foreign L-Histidine Excretion Gene Candidates To confirm the presence or absence of L-histidine excretion ability in the seven 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 using L-histidine was conducted. After culturing the seven strains in minimal liquid medium at 30 °C for 24 hours, they were diluted with 1×10 3 and 1×10 4 cells and spotted on minimal solid medium supplemented with L-histidine. The composition of the minimal solid medium used was as follows:

[0089] Minimum medium (pH 7.2) Glucose 10 g, KH2PO4 1 g, K2HPO4 2 g, MgSO4·7H2O 0.4 g, Urea 2 g, (NH4)2SO4 5 g, NaCl 0.5 g, Nicotinamide 5 μg, Calcium pantothenate 0.1 μg, Biotin 0.2 μg, Thiamine HCl 3 μg, Trace elements solution* 1 ml (per liter of distilled water), Agar 20 g

[0090] *Trace elements solution Na2B4O7·10H2O 0.09 g, (NH4)6Mo7O 27 4·4H2O 0.04 g, ZnSO4·7H2O 0.01 g, CuSO4·5H2O 0.27 g, MnCl2·4H2O 0.01 g, FeCl3·6H2O 1 g, CaCl2 0.01 g (per liter of distilled water)

[0091] For the minimum inhibitory concentration experiment, 1 g / L of L-histidine was added to the minimal solid medium, and cell growth was observed after 48 hours. The results are shown in Table 2 below:

[0092]

Table 2

[0093] (In Table 2, the number of + indicates the relative growth degree of the strain, and the following are shown respectively: +: A single colony is not formed, but heavy (a form that cannot grow into a single colony and grows in a clump) is formed; ++: Heavy is formed, and less than 5 single colonies are formed; +++: Heavy is formed, and less than 50 single colonies are formed; ++++: Heavy is formed so that it is not classified into single colonies)

[0094] As shown in Table 2 above, all strains except the ATCC13032ΔN2131::Dva strain grew smoothly in the minimal medium without L - histidine. However, in the minimal medium containing 1 g / L of L - histidine, the growth of most strains into which L - histidine efflux candidate genes were introduced was slight, and only the ATCC13032ΔN2131::Dva strain into which the gene derived from Dermabacter vaginalis was introduced showed better growth compared to ATCC13032ΔN2131. This indicates that the introduced protein derived from Dermabacter vaginalis can have the ability to efflux L - histidine even in a medium containing L - histidine above the minimum inhibitory concentration.

[0095] From this, the protein Dva derived from Dermabacter vaginalis was selected as a protein having the ability to specifically efflux L - histidine by conferring resistance to L - histidine at a concentration above the minimum inhibitory concentration to Corynebacterium strains.

[0096] Example 4. Production of a strain into which a gene (dva) derived from Dermabacter vaginalis is introduced based on the Corynebacterium - derived L - histidine - producing strain (KCCM80179) and evaluation of L - histidine - producing ability To confirm the L-histidine excretion ability of the protein Dva derived from Dermabacter vaginalis, the gene dva derived from Dermabacter vaginalis was introduced into the L-histidine-producing strain KCCM80179 (Korean Patent Publication No. 10-2019-0065984).

[0097] For this purpose, the vectors pDZΔN2131 and pDZΔN2131-PgapA-Dva prepared in Example 2 were each transformed into the KCCM80179 strain by electroporation. Through a secondary crossover process, two strains in which the NCgl2131 gene on the chromosome was deleted or replaced with the L-histidine excretion gene candidate (dva) were prepared, and these were named KCCM80179ΔN2131 (NCgl2131 gene deleted) and KCCM80179ΔN2131-PgapA-Dva (NCgl2131 gene replaced with dva), respectively.

[0098] To confirm the L-histidine production ability of the prepared KCCM80179ΔN2131 and KCCM80179ΔN2131-PgapA-Dva strains, they were cultured in the following manner: The KCCM80179ΔN2131 and KCCM80179ΔN2131-PgapA-Dva strains were cultured in an activation medium for 16 hours, and then each strain was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of a seed medium and cultured with shaking at 30 °C for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of a production medium and cultured with shaking at 30 °C for 48 hours at 200 rpm. The medium compositions used for the above culture were as follows:

[0099] <Activation medium> Beef extract 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

[0100] <Seed medium> 5% (w / v) glucose, 1% (w / v) bactopeptone, 0.25% (w / v) sodium chloride, 1% (w / v) yeast extract, 0.4% (w / v) urea, pH 7.2

[0101] <Production medium> 10% (w / v) glucose, 2% (w / v) ammonium sulfate, 0.1% (w / v) potassium dihydrogen phosphate, 0.05% (w / v) magnesium sulfate heptahydrate, 2.0% (w / v) CSL (corn steep liquor), 200 μg / L biotin, calcium carbonate, pH 7.2

[0102] After the cultivation, the L - histidine production amount (histidine content in the medium) was measured by HPLC, and the results are shown in Table 3 below:

[0103]

Table 3

[0104] As shown in Table 3 above, the NCgl2131 - deficient strain has an L - histidine production ability comparable to that of the parent strain, KCCM80179 strain. On the other hand, the KCCM80179ΔN2131 - PgapA - Dva strain into which the gene derived from Dermabacter vaginalis was introduced was confirmed to have a 23% and 21% or more increase in L - histidine production ability compared to the NCgl2131 - deficient strain and the parent strain, KCCM80179 strain, respectively.

[0105] From the results of Examples 3 and 4 above, it was confirmed that the introduction of the gene derived from Dermabacter vaginalis not only increases the resistance to L - histidine concentration above the minimum inhibitory concentration but also greatly increases the L - histidine production ability. Such results prove that the protein derived from Dermabacter vaginalis is an L - histidine efflux protein that can specifically excrete L - histidine.

[0106] Example 5. Additional acquisition of L - histidine efflux - like protein derived from Dermabacter vaginalis Since the ability of the protein derived from Dermabacter vaginalis to excrete L-histidine was confirmed in Examples 3 and 4 above, in order to additionally secure similar proteins with high amino acid sequence homology to the said protein, a BLAST search was conducted using the sequence of DvaF in DvaFE (SEQ ID NO: 12) as a query (see Table 4).

[0107] [Table 4]

[0108] Based on the results of the BLAST search, one candidate for L-histidine exporter that shows a sequence homology of 60% or more and does not belong to the genus Dermabacter was additionally selected and shown in Table 5 below:

[0109] [Table 5]

[0110] Example 6. Preparation of vectors for introducing additional foreign L-histidine exporter gene candidates Vectors for introducing the two candidates for L-histidine exporter genes additionally selected in Example 5 above into corynebacterium strains were prepared. Similar to Example 2, the NCgl2131 gene was used as the deletion site, and PgapA was used as the promoter.

[0111] The nucleotide sequence information of the operon (hereinafter, hma, SEQ ID NO: 47) encoding the protein derived from Helcobacillus massiliensis (hereinafter, HmaFE, SEQ ID NO: 43, 44) was obtained from the National Institutes of Health GeneBank (NIH GenBank). DNA was synthesized using the gene synthesis service of Bionics to obtain haqDNA. To amplify the synthesized DNA, PCR was performed using the primer pair of SEQ ID NO: 48 and SEQ ID NO: 49. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 2 minutes, repeated 28 times, followed by a polymerization reaction at 72°C for 5 minutes. As a result, an 1113 bp hma fragment containing 1081 bp of hma (SEQ ID NO: 47) was obtained. To obtain a PgapA fragment that can be ligated 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 the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 1 minute, repeated 28 times, followed by a polymerization reaction at 72°C for 5 minutes. As a result, a 441 bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO: 17) was obtained. The obtained hma fragment, PgapA fragment, and the pDZΔN2131 vector digested with ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Hma.

[0112] Example 7. Production of a strain introduced with a gene (hma) derived from Helcobacillus massiliensis based on the L-histidine-producing strain (KCCM80179) and evaluation of L-histidine production ability To confirm the L-histidine excretion ability of the protein Hma derived from Helcobacillus massiliensis, it was introduced into the L-histidine-producing strain KCCM80179.

[0113] For this purpose, the vector pDZΔN2131-PgapA-Hma produced in Example 6 was transformed into the KCCM80179 strain by electroporation, and a strain in which the NCgl2131 gene on the chromosome was replaced with the L-histidine efflux gene candidate through a secondary crossover process was produced, and this was named KCCM80179ΔN2131-PgapA-Hma (the NCgl2131 gene was replaced with hma). To confirm the L-histidine production ability of the produced KCCM80179ΔN2131-PgapA-Hma strain, the strain was cultured by the method performed in Example 4, and the L-histidine production amount was measured. As a control group, the KCCM80179ΔN2131 strain and the KCCM80179ΔN2131-PgapA-Dva strain produced in Example 4 were cultured and the L-histidine production amount (histidine content in the medium) was measured in the same manner. The obtained results are shown in Table 6.

[0114]

Table 6

[0115] As shown in Table 6, the L-histidine production amount of the KCCM80179ΔN2131-PgapA-Hma strain increased by 19% and 19%, respectively, compared to the NCgl2131-deficient strain (KCCM80179ΔN2131) and the parent strain KCCM80179. Such results indicate that the protein derived from Helcobacillus massiliensis is also selected as an L-histidine effluxer that can specifically efflux L-histidine.

[0116] Example 8: Preparation of an Hma mutant library using the artificial mutagenesis method To obtain a mutant Hma with increased L-histidine excretion ability, a mutant protein expression vector library for primary crossover was constructed. For this purpose, using the prepared pDZΔN2131-PgapA-Hma as a template, a hma operon with randomly introduced base substitution mutations was obtained by error-prone PCR using the primer pair of SEQ ID NO: 48 and SEQ ID NO: 49. Error-prone PCR was performed under the condition that 0 to 3.5 mutations per kb were introduced into the amplified gene fragment using the GenemorphII Random Mutagenesis Kit (Stratagene). The PCR conditions were: denaturation at 96°C for 30 seconds; annealing at 53°C for 30 seconds; and polymerization reaction at 72°C for 2 minutes, repeated 30 times. To obtain a PgapA fragment that can be ligated to hma, PCR was performed using the chromosome of ATCC13032 as a template and the primer pair of SEQ ID NO: 26 and SEQ ID NO: 50. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 1 minute, repeated 28 times, and then a polymerization reaction was performed at 72°C for 5 minutes to obtain a PgapA fragment. The obtained mutant hma operon, PgapA fragment, and the pDZΔN2131 vector cleaved with ScaI restriction enzyme were cloned using the Gibson assembly (DGGibson et al., NATURE METHODS, VOL.6 NO.5, MAY2009, NEBuilder HiFi DNA Assembly Master Mix) method, transformed into DH5α, and spread on LB solid medium containing kanamycin (25 mg / L). After screening 20 transformed colonies, plasmids were obtained and the base sequences were analyzed. As a result, it was confirmed that mutations were introduced at different positions with an average frequency of 2 mutations / kb. Approximately 10,000 transformed Escherichia coli colonies were taken to extract plasmids, which were named pDZΔN2131-PgapA-Hma(mt) library.

[0117] Example 9: Introduction of Hma artificial mutation library and selection of L-histidine-producing strains with increased ability Using the prepared ATCC13032ΔN2131 strain as the parental strain, the prepared pDZΔN2131-PgapA-Hma(mt) library was transformed by homologous chromosomal recombination, and spread on a complex plate medium containing kanamycin (25 mg / L) to obtain about 7000 colonies. Each colony was named ATCC13032ΔN2131-PgapA-Hma(mt)-1 to ATCC13032ΔN2131-PgapA-Hma(mt)-7000.

[0118] <Complex plate medium (pH 7.0)> Glucose 10 g, Peptone 10 g, Beef extract 5 g, Yeast extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, Urea 2 g, Sorbitiol 91 g, Agar 20 g (based on 1 liter of distilled water)

[0119] The 7000 colonies obtained were subjected to the minimum inhibitory concentration (MIC) experiment for L-histidine as performed in Example 3 above. For efficient screening, it was carried out based on a liquid minimal medium containing 3 g of L-histidine. The colonies were immediately inoculated into 300 μl of the liquid minimal medium and cultured in a 96-well plate at 32°C and 1000 rpm for about 18 hours, and then the OD600 value was measured to select colonies with excellent growth. As the control group, the prepared ATCC13032ΔN2131 strain and ATCC13032ΔN2131::Hma strain were used. Through the first experiment, 251 were selected, and through the repeated experiment, 36 colonies were selected. The medium components used are as follows.

[0120] Liquid minimum medium (pH 7.2) 10 g of glucose, 1 g of KH2PO4, 2 g of K2HPO4, 0.4 g of MgSO4·7H2O, 2 g of urea, 5 g of (NH4)2SO4, 0.5 g of NaCl, 5 μg of nicotinamide, 0.1 μg of calcium pantothenate, 0.2 μg of biotin, 3 μg of thiamine HCl, 1 ml of Trace elements solution* (based on 1 liter of distilled water)

[0121] *Trace elements solution 0.09 g of Na2B4O7·10H2O, (NH4)6Mo7O 27 4H2O 0.04 g, 0.01 g of ZnSO4·7H2O, 0.27 g of CuSO4·5H2O, 0.01 g of MnCl2·4H2O, 1 g of FeCl3·6H2O, 0.01 g of CaCl2 (based on 1 liter of distilled water)

[0122] For the minimum inhibitory concentration experiment, 3 g / L of L-histidine was added to the medium and cultured for 18 hours.

[0123] The selected 36 colonies were subjected to solid medium screening by the method performed in Example 3, and 6 colonies with increased L-histidine excretion ability were finally screened, and the results are shown in Table 7 below:

[0124]

Table 7

[0125] (In Table 7, the number of + indicates the relative growth degree of the strain, and each indicates the following: +: single colony is not formed but heavy (a form that cannot grow into a single colony and grows in a lump) is formed; ++: heavy is formed and less than 5 single colonies are formed; +++: heavy is formed and less than 50 single colonies are formed; ++++: heavy is formed without being distinguished from single colonies)

[0126] As shown in Table 7 above, the NCgl2131-deficient strain (ATCC13032ΔN2131) could not grow smoothly in the minimal medium containing 1 g / L of L-histidine, but the ATCC13032ΔN2131::Hma strain into which the gene derived from Helcobacillus massiliensis was introduced grew smoothly, and six strains (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 strains) with increased L-histidine excretion ability were identified by solid medium screening and showed a higher growth level in the minimal medium containing 1 g / L of histidine.

[0127] Example 10. Production of strains introduced with a Corynebacterium-derived L-histidine-producing strain (KCCM80179) base selection library and evaluation of L-histidine production ability A vector was constructed for introducing the mutant hma contained in 6 types of colonies selected in Example 9 into an L-histidine-producing strain. For this purpose, using the chromosomal DNAs 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, PCR was performed using the primer pair of SEQ ID NO: 48 and SEQ ID NO: 49 to obtain mutant hma species. The PCR conditions were: denaturation at 96°C for 30 seconds; annealing at 53°C for 30 seconds; and polymerization reaction at 72°C for 2 minutes, repeated 30 times. To obtain a PgapA fragment that can be ligated with the mutant hma, PCR was performed using the chromosome of ATCC13032 as a template and the primer pair of SEQ ID NO: 26 and SEQ ID NO: 50. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 1 minute, repeated 28 times, and then a polymerization reaction was performed at 72°C for 5 minutes to obtain a PgapA fragment. The 6 types of mutant hma fragments, the PgapA fragment, and the pDZΔN2131 vector digested with ScaI restriction enzyme were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was 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 derived library colony.

[0128] The subsequently produced vector was transformed into the KCCM80179 strain by electroporation, and six strains into which six mutant hma were introduced through a secondary crossing process were produced, and these were named KCCM80179ΔN2131-PgapA-Hma(mt)-1216, KCCM80179ΔN2131-PgapA-Hma(mt)-2315, KCCM80179ΔN2131-PgapA-Hma(mt)-3411, KCCM80179ΔN2131-PgapA-Hma(mt)-4426, KCCM80179ΔN2131-PgapA-Hma(mt)-5714, and KCCM80179ΔN2131-PgapA-Hma(mt)-6718, respectively.

[0129] To confirm the L-histidine production ability of the six produced strains, the KCCM80179ΔN2131-PgapA-Hma strain produced in Example 7 was cultured as a control group and measured by the same method as in Example 4, and the results are shown in Table 8:

[0130]

Table 8

[0131] As shown in Table 8 above, the NCgl2131-deficient strain (KCCM80179ΔN2131) has an L-histidine production ability equivalent to that of the parental strain KCCM80179 strain, while the ATCC13032ΔN2131::Hma strain into which the gene derived from Helcobacillus massiliensis was introduced has an increased L-histidine production ability compared to the NCgl2131-deficient strain and the parental strain KCCM80179 strain.

[0132] The six strains into which six mutant hmas were introduced each had an increased L-histidine production ability at the same level as or higher than that of the KCCM80179ΔN2131-PgapA-Hma strain, which is an NCgl2131-deficient strain and the parental strain KCCM80179. In particular, the KCCM80179ΔN2131-PgapA-Hma(mt)-5714 and KCCM80179ΔN2131-PgapA-Hma(mt)-6718 strains were confirmed to have a 3.6% and 12.1% increase in L-histidine production ability, respectively, compared to the KCCM80179ΔN2131-PgapA-Hma strain into which the wild-type hma was introduced.

[0133] Example 11. Confirmation of Mutations in the Hma Gene Variant with Increased L-Histidine Production Ability To confirm the mutations introduced into Hma of the KCCM80179ΔN2131-PgapA-Hma(mt)-6718 strain, in which the L-histidine production ability was confirmed in Example 10, the nucleotide sequence of the Hma mutant was analyzed. To determine the nucleotide sequence, PCR was performed using the primer pair of SEQ ID NO: 18 and SEQ ID NO: 21 with the gDNA of the KCCM80179ΔN2131-PgapA-Hma(mt)-6718 strain as a template. By nucleotide sequence analysis, the nucleotide sequence of the mutant hma operon and the protein sequence of HmaF or HmaE were confirmed, compared with the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 44, and the mutation information of the amino acid sequence of the mutant HmaFE thus confirmed is shown in Table 9.

[0134]

Table 9

[0135] 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 excretor with increased L-histidine excretion ability due to the introduction of I72L (the 72nd isoleucine (Ile, I) in sequence number 43 is mutated to leucine (Leu, L)) and I124V (the 124th isoleucine (Ile, I) in sequence number 43 is mutated to valine (Val, V)) mutations into HmaF.

[0136] From the above results, it was confirmed that the introduction of the mutant excretor derived from Helcobacillus massiliensis not only increased the tolerance to L-histidine concentrations above the minimum inhibitory concentration compared to the wild type, but also significantly increased the L-histidine production ability. These results prove that the selected mutant protein derived from Helcobacillus massiliensis is a mutant L-histidine excretion protein that can specifically excrete L-histidine.

[0137] Example 12. Construction of L-histidine producing strain (CA14-737) based on Helcobacillus massiliensis-derived mutant gene-introduced strain and evaluation of L-histidine production ability To reconfirm the L-histidine excretion ability of the protein Hma mutant derived from Helcobacillus massiliensis, a HisG polypeptide mutation was introduced to eliminate feedback restriction by L-histidine from wild-type Corynebacterium glutamicum ATCC13032, and the L-histidine biosynthesis gene was enhanced, and the mutant was introduced into the L-histidine-producing strain CA14-737 (KCCM12411P, Republic of Korea Patent Publication No. 10-2019-0065984).

[0138] For this purpose, three vectors (pDZΔN2131, pDZΔN2131-PgapA-Hma, pDZΔN2131-PgapA-Hma(mt)-6718) produced in Examples 2 and 10 were each transformed into the CA14-737 strain by electroporation. Through a secondary crossover process, three strains in which the NCgl2131 gene on the chromosome was deleted or replaced with the L-histidine excretion gene were produced, and these were named CA14-737ΔN2131, CA14-737ΔN2131-PgapA-Hma, and CA14-737ΔN2131-PgapA-Hma(mt)-6718, respectively.

[0139] To confirm the L-histidine production ability of the produced CA14-737ΔN2131, CA14-737ΔN2131-PgapA-Hma, and CA14-737ΔN2131-PgapA-Hma(mt)-6718 strains, they were cultured by the method performed in Example 4, the L-histidine production amount (histidine content in the medium) was measured, and the results are shown in Table 10 below:

[0140]

Table 10

[0141] As shown in Table 10, the CA14-737ΔN2131-PgapA-Hma strain into which the gene derived from Helcobacillus massiliensis was introduced had a 54% increase in L-histidine production compared to the NCgl2131-deficient strain (CA14-737ΔN2131) and the parental strain CA14-737, and the CA14-737ΔN2131-PgapA-Hma(mt)-6718 strain into which the mutant hma was introduced had a 78% increase.

[0142] This reconfirmed that both the wild-type and mutant proteins derived from Helcobacillus massiliensis can specifically excrete L-histidine, and that the selected mutant protein is an L-histidine exporter with a higher excretion ability than the wild-type protein.

[0143] Example 13. Construction of vectors for expressing wild-type genes and mutants from Helcobacillus massiliensis in Escherichia coli To confirm the L-histidine excretion ability of the protein Hma and mutants derived from Helcobacillus massiliensis in various strains, vectors capable of expressing wild-type Hma and Hma mutants in Escherichia coli were constructed. Each gene was cloned into the Escherichia coli expression vector pCC1BAC (hereinafter, pBAC, Epicenter corp.) and designed to express the foreign L-histidine excretion gene candidate under the promoter of the yccA gene (hereinafter, PyccA, SEQ ID NO: 51) derived from the Escherichia coli strain MG1655.

[0144] To obtain the protein Hma and mutant gene fragments derived from Helcobacillus massiliensis, chromosomal DNAs of ATCC13032ΔN2131-PgapA-Hma and ATCC13032ΔN2131-PgapA-Hma(mt)-6718 were used as templates, and PCR was performed using the primer pairs of SEQ ID NO: 52 and SEQ ID NO: 53 to obtain wild-type and mutant hma DNA fragments, respectively. The PCR conditions were as follows: denaturation at 96°C for 30 seconds; annealing at 53°C for 30 seconds; and polymerization reaction at 72°C for 2 minutes, repeated 30 times. To obtain the PyccA fragment, PCR was performed using the chromosomal DNA of MG1655 as a template and the primer pair of SEQ ID NO: 54 and SEQ ID NO: 55. As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity 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 reaction at 72°C for 1 minute, repeated 28 times, followed by a polymerization reaction at 72°C for 5 minutes to obtain the PyccA fragment. The obtained wild-type and mutant hma fragments, PyccA fragment, and the pBAC vector digested with the EcoRI restriction enzyme were cloned using the Gibson assembly method to obtain recombinant plasmids, which were named pBAC-PyccA-Hma and pBAC-PyccA-Hma(mt)-6718.

[0145] Example 14. Production of L-histidine-producing strains derived from Escherichia coli, construction of mutant-introduced strains using the wild-type gene of Hma derived from Helcobacillus massiliensis, and evaluation of L-histidine production ability To confirm the L-histidine excretion ability of the Hma mutant protein derived from Helcobacillus massiliensis based on the L-histidine-producing strain derived from Escherichia coli, two vectors prepared in Example 13 and the pBAC vector were introduced into the CA14-9003e strain (MG1655+hisGr hisL’_Δ ΔpurR) with the previously reported genotype (purR-deficient, hisL-deficient, 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) to prepare three strains, which were named CA14-9003e / pBAC, CA14-9003e / pBAC-PyccA-Hma, and CA14-9003e / pBAC-PyccA-Hma(mt)-6718, respectively.

[0146] To confirm the L-histidine production ability of the prepared CA14-9003e / pBAC, CA14-9003e / pBAC-PyccA-Hma, and CA14-9003e / pBAC-PyccA-Hma(mt)-6718 strains, they were cultured in the following manner. After culturing the strains on an LB solid medium (containing 25 μg / ml of chloramphenicol) for 16 hours, each strain was inoculated into a 250-ml Erlenmeyer flask containing 25 ml of LB liquid medium and cultured with shaking at 37 °C for 20 hours at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250-ml Erlenmeyer flask containing 25 ml of the Escherichia coli production medium (Applied Biochemistry and Microbiology, 2013, Vol.49, No.2, pp.130-135) and cultured with shaking at 37 °C for 48 hours at 200 rpm. The media used for the above culture were as follows:

[0147] <Escherichia coli production medium> Glucose 4% (w / v), yeast extract 0.2% (w / v), ammonium sulfate 1.6% (w / v), dipotassium phosphate trihydrate 0.06% (w / v), ferrous sulfate heptahydrate 0.0005% (w / v), magnesium sulfate pentahydrate 0.0005% (w / v), calcium carbonate, pH 7.2,

[0148] After the cultivation, the L-histidine production amount (histidine content in the medium) was measured by HPLC, and the results are shown in Table 11 below.

[0149]

Table 11

[0150] As shown in Table 11, it was confirmed that the CA14-9003e / pBAC-PyccA-Hma(mt)-6718 strain into which the mutant Hma was introduced had a 30% increase in L-histidine production ability compared to the wild-type Hma-introduced strain CA14-9003e / pBAC-PyccA-Hma strain.

[0151] From the above results, it was confirmed that when the L-histidine exporter mutant derived from Helcobacillus massiliensis was introduced into microorganisms other than Corynebacterium strains, the ability to excrete L-histidine to the outside of the cell was greatly increased.

[0152] From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including the meaning and scope of the claims described below, and all changes or modifications derived from the equivalent concepts thereof within the scope of the present invention.

Claims

1. (1) whether the amino acid corresponding to the 72nd amino acid residue of the amino acid sequence of SEQ ID NO: 43 is substituted with another amino acid, (2) whether the amino acid corresponding to the 124th residue of the amino acid sequence of SEQ ID NO: 43 is substituted with another amino acid, or (3) a mutant L-histidine excretion protein in which both (1) and (2) above are substituted.

2. (1) whether the amino acid corresponding to the 72nd amino acid residue of the amino acid sequence of SEQ ID NO: 43 is substituted with leucine, glycine, proline, alanine, valine or methionine, (2) whether the amino acid corresponding to the 124th residue of the amino acid sequence of SEQ ID NO: 43 is substituted with valine, glycine, proline, alanine, leucine or methionine, or (3) the protein according to claim 1, in which both (1) and (2) above are substituted.

3. The protein according to claim 1, wherein the protein has a sequence homology of 99% or more with the amino acid sequence of SEQ ID NO:

56.

4. A polynucleotide encoding the protein according to any one of claims 1 to 3.

5. A microorganism comprising the protein according to any one of claims 1 to 3, or the polynucleotide encoding the protein.

6. The microorganism according to claim 5, which has the ability to produce L-histidine.

7. The microorganism according to claim 5, which belongs to the genus Corynebacterium or the genus Escherichia.

8. The microorganism according to claim 7, which 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, the polynucleotide encoding the protein, or a recombinant microorganism containing the polynucleotide.

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

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

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