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

Abstract

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 L-histidine production amount can be dramatically improved.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims the benefit of priority based on Korean Patent Application No. 10 - 2022 - 0076772, filed on June 23, 2022, and all of the 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 it 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 treatment agents, raw materials for cardiovascular system treatment 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] Although it is known that 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, little prior research has been conducted on proteins having L - histidine - specific excretion ability.

[0006] Under such a background, there is a need to discover proteins having histidine-specific excretion ability and develop histidine production technology using the same.

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present application is to provide a mutant L-histidine excretion protein in which the amino acid corresponding to the 92nd residue of the amino acid sequence of SEQ ID NO: 13 is substituted with another amino acid.

[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 L-histidine-excreting proteins having L-histidine excretion ability and expressing them in microorganisms having L-histidine production ability, it is possible to dramatically improve the L-histidine production amount.

[0014] In this specification, it was confirmed that a microorganism expressing an AzlD domain-containing protein derived from Dermabacter vaginalis has excellent L-histidine production ability, and it was confirmed that when an amino acid substitution mutation is introduced at a specific position of the AzlD domain-containing protein, the L-histidine production ability 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 may be expressed as a mutant L-histidine-excreting protein. The mutant protein may have an activity equivalent to that of the AzlD domain-containing protein.

[0017] In one example, the mutant protein may have L-histidine efflux activity equivalent to or enhanced from that of a wild-type L-histidine efflux protein (such as an AzlD domain-containing protein or an AzlC family ABC transporter permease). The AzlD domain-containing protein or the AzlC family ABC transporter permease may be derived from Dermabacter vaginalis. In the present specification, the AzlD domain-containing protein derived from Dermabacter vaginalis may be described as the DvaE protein (or DvaE), and the AzlC family ABC transporter permease derived from Dermabacter vaginalis may be described as the DvaF protein (or DvaF).

[0018] In one example, the mutant protein (such as a mutant protein of an AzlD domain-containing protein, specifically a mutant protein of an AzlD domain-containing protein derived from Dermabacter vaginalis) may be expressed together with the AzlC family ABC transporter permease in the same operon gene. In one example, the mutant protein may bind to the AzlC family ABC transporter permease and have L-histidine efflux activity.

[0019] In one example, the mutant protein may be a mutant protein of an AzlD domain-containing protein derived from Dermabacter vaginalis.

[0020] The mutant protein may be a mutant protein into which a mutation has been introduced in which one or more amino acid residues of the wild-type AzlD domain-containing protein derived from Dermabacter vaginalis have been substituted, deleted, or inserted.

[0021] The wild-type AzlD domain-containing protein derived from Delftibacter vaginalis may contain the amino acid sequence of SEQ ID NO: 13 (WP_065248527.1) or may consist of said sequence.

[0022] In one example, the mutant protein may contain an amino acid sequence in which the amino acid corresponding to the 92nd residue from the N-terminus in the amino acid sequence of SEQ ID NO: 13 is substituted with another amino acid. Counting amino acids from the N-terminus in the amino acid sequence as described above can mean counting the methionine (Met, M) translated from the start codon as the first amino acid.

[0023] In one example, the mutant protein has an amino acid sequence in which the amino acid corresponding to the 92nd residue from the N-terminus in the amino acid sequence of SEQ ID NO: 13 is another amino acid, i.e., an amino acid different from the original amino acid, and is substituted with cysteine (Cys, C), 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), glycine (Gly, G), proline (Pro, P), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), or tryptophan (Trp, W). In one example, the other amino acid may be cysteine, glutamic acid, serine, threonine, asparagine, or glutamine. In one specific example, the other amino acid may be cysteine. Among the mutant proteins, as long as a partial amino acid sequence excluding the amino acid corresponding to the 92nd amino acid residue from the N-terminus in the amino acid sequence of SEQ ID NO: 13 shows activity equivalent to that of the AzlD domain-containing protein, it is obvious that it can be included in the mutant protein of the present application.

[0024] In one example, the mutant protein can include 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: 13, and the amino acid corresponding to the 92nd residue of the amino acid sequence of SEQ ID NO: 13 is replaced by another amino acid. That is, it includes substitution with another amino acid at the position corresponding to the 92nd residue of the amino acid sequence of SEQ ID NO: 13, 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: 13, and a polypeptide having an activity equivalent to that of the AzlD domain-containing protein can be included in the mutant protein of the present application.

[0025] In one specific example, the mutant protein may include, but is not limited to, the amino acid sequence of SEQ ID NO: 48 or may consist of the said sequence. As long as a mutant protein consisting of the amino acid sequence of SEQ ID NO: 48 shows equivalent activity to the AzlD domain-containing protein even when a partial amino acid sequence excluding the amino acid corresponding to the 92nd residue is deleted, modified, substituted or added, 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 acid corresponding to the 92nd residue of the amino acid sequence of SEQ ID NO: 48 is 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: 48. That is, a polypeptide having equivalent activity to the AzlD domain-containing protein, in which the amino acid corresponding to the 92nd residue of the amino acid sequence of SEQ ID NO: 48 is substituted with another amino acid 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: 48, can be included in the mutant protein of the present application.

[0026] 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.

[0027] The wild-type L-histidine efflux protein may be a protein having 60% or more sequence homology with SEQ ID NO: 12 (wild-type AzlD domain-containing protein derived from Dermabacter vaginalis), SEQ ID NO: 13 (AzlC system ABC transporter permease protein derived from Dermabacter vaginalis), 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: 12, 13, or a combination thereof.

[0028] The protein represented by SEQ ID NO: 12 is encoded by the nucleic acid sequence of SEQ ID NO: 14, the protein represented by SEQ ID NO: 13 is encoded by the nucleic acid sequence of SEQ ID NO: 15, or the protein represented by SEQ ID NO: 12 and / or SEQ ID NO: 13 may be encoded by the nucleic acid sequence of SEQ ID NO: 16 (an operon sequence fused at the overlapping site of the 3'-end of SEQ ID NO: 14 and the 5'-end of SEQ ID NO: 15).

[0029] Another aspect provides a polynucleotide that encodes (codes for) the mutant protein. In the present application, the term "polynucleotide" is a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds and is a DNA or RNA strand having a length of a certain length or more. More specifically, it means a polynucleotide fragment that encodes the mutant polypeptide.

[0030] 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: 48.

[0031] As used herein, a polynucleotide (which may be used interchangeably with "gene") or a polypeptide (which may be used interchangeably with "protein") being "comprising, consisting of, or represented by a specific nucleic acid sequence or amino acid sequence" can mean that the polynucleotide or polypeptide necessarily contains the specific nucleic acid sequence or amino acid sequence, and includes (or does not exclude) "substantially equivalent sequences" in which mutations (deletions, substitutions, modifications, and / or additions) are made to the specific nucleic acid sequence or amino acid sequence within the scope of maintaining the original function and / or the intended function of the polynucleotide or polypeptide.

[0032] In one example, the nucleic acid sequences or amino acid sequences provided herein can include those modified by conventional mutagenesis methods, such as directed evolution and / or site-directed mutagenesis, within the scope of maintaining their original or intended functions. In one example, a polynucleotide or polypeptide being "comprising or consisting of a specific nucleic acid sequence or amino acid sequence" or the polynucleotide or polypeptide can mean that (i) it necessarily contains the specific nucleic acid sequence or amino acid sequence, or (ii) it consists of or necessarily contains 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 its original function and / or intended function. As used herein, the intended function can mean a function of conferring or increasing the L-histidine excretion activity and / or L-histidine production ability of a microorganism.

[0033] The nucleic acid sequences described in this specification can be modified in various ways in the coding region without changing the amino acid sequence and / or function of the protein expressed from the coding region, taking into account the codons preferred by the microorganism in which the protein is to be expressed due to the degeneracy of the codons.

[0034] As used herein, the term "homology (identity)" refers to 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) and 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).

[0035] 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 with each other, and conditions where genes having lower complementarity do not hybridize with each other, or the washing conditions of normal Southern hybridization, such as 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 washing once, specifically 2 to 3 times, at a salt concentration and temperature corresponding to 68°C, 0.1×SSC, and 0.1% SDS can be mentioned, but are 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 with 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 polynucleotide, which is well known in the relevant technical field (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0036] 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 in 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.

[0037] The method of transforming the host cell with the polynucleotide 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.

[0038] The introduction (insertion) of the polynucleotide into the host cell genome (chromosome) can be carried out by those skilled in the art appropriately selecting known methods. For example, an RNA-guided endonuclease system or a 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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. The insertion of the polynucleotide into the host cell chromosome is performed by any method known in the art, such as homologous recombination or the CRISPR system, but is not limited thereto. 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 for use. 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.

[0043] Microorganism

[0044] 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.

[0045] The mutant protein may be exogenous. As used herein, "exogenous" means not inherently present in the microorganism but derived from a species different from the microorganism.

[0046] As used herein, the "microorganism with enhanced L-histidine excretion activity and / or L-histidine production ability" refers to a microorganism 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 an L-histidine excretion activity and / or L-histidine production ability that is higher than its original L-histidine excretion activity and / or L-histidine production ability.

[0047] As used herein, the term "microorganism" includes single-celled bacteria and can be used interchangeably with the term "cell".

[0048] In the present application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modifications, and microorganisms in which specific mechanisms have been weakened or strengthened due to causes such as the insertion of foreign genes, the enhancement of the activity of endogenous genes, or the inactivation of endogenous genes, and may include microorganisms that have been genetically modified (modified) for the production of a target polypeptide, protein, or product (e.g., L-histidine).

[0049] The microorganism of the present application is, but not limited to, a microorganism (e.g., a recombinant microorganism) that has been genetically modified 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.

[0050] 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 the "parent microorganism or parent strain" or "host cell".

[0051] 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.

[0052] The microorganism of the present application is a microorganism containing 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 containing 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 activity of the mutant protein of the present application (for example, a recombinant strain), but not limited thereto.

[0053] 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.

[0054] 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 for expressing the mutant protein may be carried out by introducing a 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 or additionally contain the AzlD domain-containing protein-encoding gene inherent in the parent strain.

[0055] 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. In one example, the microorganism with enhanced L-histidine excretion activity and / or L-histidine production ability has an L-histidine excretion activity and / or L-histidine production ability that is increased by about 5% or more, 10% or more, 11% or more, 12% or more, 17% or more, 20% or more, 50% or more, 80% or more, or 100% or more compared to the pre-mutation parental strain, unmodified microorganism, and microorganism containing the wild-type L-histidine excretion protein, but is not limited thereto.

[0056] In another example, the microorganism with enhanced L-histidine excretion activity and / or L-histidine production ability has an L-histidine excretion activity and / or L-histidine production ability that is increased by 0.5 g / L or more, 0.7 g / L or more, 0.8 g / L or more, 2 g / L or more, 3 g / L or more, 3.2 g / L or more, 4 g / L or more, 5 g / L or more, 5.2 g / L or more, 6 g / L or more, or 10 g / L or more compared to the pre-mutation parental strain, unmodified microorganism, and microorganism containing the wild-type L-histidine excretion protein, but is not limited thereto.

[0057] 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.

[0058] Another aspect provides a composition for L-histidine production containing the mutant protein, the polynucleotide, the recombinant vector, or the microorganism.

[0059] Another aspect provides the use of the mutant protein, the polynucleotide, the recombinant vector, or the microorganism for L-histidine production.

[0060] 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.

[0061] Another aspect provides a method for producing (manufacturing) L-histidine, which includes the step of culturing the microorganism in a medium. The production method may further include a step of recovering L-histidine from the cultured microorganism, the medium, or all of them after the culturing step.

[0062] 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 imparting 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.

[0063] The step of introducing the mutation may include the step of introducing (transforming) a polynucleotide encoding a mutant protein or a recombinant vector containing the polynucleotide into a microorganism, or may include a step of artificially generating a mutation (for example, Error-prone PCR, etc.).

[0064] In the method, the step of culturing the microorganism can be carried out by, but is 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 (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., 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 culture can be secreted into the medium or remain intracellularly.

[0065] The medium that can be used for the culture 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.

[0066] 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, or after that.

Advantages of the Invention

[0067] This application can discover a histidine efflux protein having the ability to efflux 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

[0068] 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.

[0069] Example 1. Search for foreign histidine efflux genes and candidate selection In order to select protein candidates having the ability to specifically efflux L-histidine, using the amino acid sequences of efflux 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)) as query sequences, based on the PSI-BLAST search results from NCBI and Kegg database, candidate genes predicted as membrane proteins that may efflux L-histidine and the microorganisms carrying them were selected.

[0070] Among these, considering the biosafety level and the possibility of ensuring a level applicable to production strains, 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 as shown in Table 1 below:

[0071]

Table 1

[0072] (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).)

[0073] 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 produced.

[0074] Example 2-1. Production of the vector pDZΔN2131 for inserting a target gene In order 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 (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).

[0075] To replace the NCgl2131 gene with an efflux 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. 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, 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 QIAGEN's PCR Purification kit, cloning was performed using the pDZ vector (Korean Registered Patent No. 10-0924065) and TaKaRa's Infusion Cloning Kit to construct a vector pDZΔN2131 for NCgl2131 gene deletion and target gene insertion.

[0076] Example 2-2. Preparation of 6 vectors for introducing foreign L-histidine efflux 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 a 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 the polymerization reaction was carried out 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 a 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 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 haq fragment, PgapA fragment, and the pDZΔN2131 vector digested 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.

[0077] 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, and then the polymerization reaction was carried out 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, 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 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.

[0078] The nucleotide sequence information of the gene encoding the Kluyvera cryocrescens-derived protein (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, 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 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, 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 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.

[0079] The nucleotide sequence information of the gene encoding the protein derived from Corynebacterium stationis (hereinafter referred to as Cst, SEQ ID NO: 7) (hereinafter referred to as cst, SEQ ID NO: 8) was obtained from the GeneBank of the National Institutes of Health (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.

[0080] The nucleotide sequence information of the operon (hereinafter referred to as lsa, SEQ ID NO: 11) encoding the protein derived from Leucobacter salsicius (hereinafter referred to as 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 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 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.

[0081] 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 Gene Bank (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 a 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 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 a 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 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.

[0082] Example 2-3. Production of Recombinant Corynebacterium Strains To confirm the L-histidine excretion ability of the foreign L-histidine excretion gene candidates, the prepared NCgl2131 deletion vector (pDZΔN2131) and six types of 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 crossover process, seven types of recombinant strains in which the NCgl2131 gene on the chromosome was deleted or replaced with a foreign L-histidine excretion gene candidate were produced, and 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.

[0083] 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, 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 spot-cultured on minimal solid medium supplemented with L-histidine. The composition of the minimal solid medium used was as follows:

[0084] 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 1 liter of distilled water), Agar 20 g

[0085] *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 1 liter of distilled water)

[0086] 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:

[0087]

Table 2

[0088] (In Table 2, the number of + indicates the relative growth degree of the strain, and each indicates the following: +: 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)

[0089] 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 the medium containing L - histidine above the minimum inhibitory concentration.

[0090] From this, the protein Dva derived from Dermabacter vaginalis was selected as a protein having the ability to confer resistance to L - histidine above the minimum inhibitory concentration to Corynebacterium strains and having the ability to specifically efflux L - histidine.

[0091] Example 4. Production of a strain into which a gene (dva) derived from Dermabacter vaginalis is introduced based on a 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).

[0092] For this purpose, the vectors pDZΔN2131 and pDZΔN2131-PgapA-Dva prepared in Example 2 were each transformed into the KCCM80179 strain by electroporation. After undergoing 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.

[0093] 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 culture were as follows:

[0094] <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

[0095] <Seed medium> 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

[0096] <Production Medium> Glucose 10% (w / v), Ammonium Sulfate 2% (w / v), Potassium Phosphate Monobasic 0.1% (w / v), Magnesium Sulfate Heptahydrate 0.05% (w / v), CSL (Corn Steep Liquor) 2.0% (w / v), Biotin 200 μg / L, Calcium Carbonate, pH 7.2

[0097] 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:

[0098]

Table 3

[0099] As shown in Table 3 above, the NCgl2131-deficient strain has an L-histidine production ability equivalent to that of the parental strain, the KCCM80179 strain. On the other hand, it was confirmed that the KCCM80179ΔN2131-PgapA-Dva strain into which the gene derived from Dermabacter vaginalis was introduced had a 23% and 21% or more increase in L-histidine production ability compared to the NCgl2131-deficient strain and the parental strain, the KCCM80179 strain, respectively.

[0100] 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 efflux L-histidine.

[0101] Example 5. Preparation of a Dva mutant library using the artificial mutagenesis method To obtain a mutant Dva with increased L-histidine excretion ability, a mutant protein expression vector library for primary crossover was constructed. For this purpose, using the chromosomal DNA of the Dermabacter vaginalis strain (KCTC39585) as a template, a dva operon with randomly introduced base substitution mutations was obtained by the Error-prone PCR method using the primer pair of SEQ ID NO: 40 and SEQ ID NO: 41. Error-prone PCR was performed using the GenemorphII Random Mutagenesis Kit (Stratagene) under the condition that 0 to 3.5 mutations per kb were introduced into the amplified gene fragment. 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 dva, PCR was performed using the chromosomal DNA 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 to obtain a PgapA fragment. The obtained mutant dva operon, 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, transformed into DH5α, and spread on an LB solid medium containing kanamycin (25 mg / L). After screening 20 transformed colonies and obtaining plasmids for nucleotide sequence analysis, it was confirmed that mutations were introduced at different positions with an average frequency of 1.5 mutations / kb. Approximately 10,000 transformed Escherichia coli colonies were taken to extract plasmids, which were named the pDZΔN2131-PgapA-Dva(mt) library.

[0102] Example 6: Introduction of Two Artificial Mutation Libraries and Selection of Strains with Increased L-Histidine Production Ability Using the prepared ATCC13032ΔN2131 strain as the parental strain, the prepared pDZΔN2131-PgapA-Dva(mt) library was transformed by homologous chromosomal recombination and spread on a complex plate medium containing kanamycin (25 mg / L) to obtain approximately 3,300 colonies, and each colony was named ATCC13032ΔN2131-PgapA-Dva(mt)-1 to ATCC13032ΔN2131-PgapA-Dva(mt)-3300.

[0103] <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)

[0104] The 3,300 colonies obtained were subjected to the minimum inhibitory concentration (MIC) experiment for L-histidine as performed in Example 3. For efficient screening, it was performed 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 at 32°C and 1,000 rpm for about 18 hours in a 96-well plate, and then the OD600 value was measured to select colonies with excellent growth. As the control group, the prepared ATCC13032ΔN2131 strain and ATCC13032ΔN2131::Dva strain were used. Through the primary experiment, 413 were selected, and through the repeated experiment, 78 colonies were selected. The medium components used were as follows.

[0105] 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)

[0106] *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)

[0107] For the minimum inhibitory concentration experiment, 3 g / L of L-histidine was added to the medium and cultured for 18 hours.

[0108] The 78 selected colonies were subjected to solid substrate screening by the method performed in Example 3, and 3 colonies were finally screened, and the results are shown in Table 4 below.

[0109]

Table 4

[0110] (In Table 4, the number of + indicates the relative growth degree of the strain, and each indicates the following: +: no single colony is 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 so that it cannot be distinguished from single colonies)

[0111] As shown in Table 4 above, the NCgl2131 deletion strain (ATCC13032ΔN2131) could not grow smoothly in the minimal medium containing 1 g / L of L-histidine, but the ATCC13032ΔN2131::Dva strain into which the gene derived from Dermabacter vaginalis was introduced grew smoothly, and three strains (ATCC13032ΔN2131-PgapA-Dva(mt)-517, ATCC13032ΔN2131-PgapA-Dva(mt)-1236, and ATCC13032ΔN2131-PgapA-Dva(mt)-2543) selected by solid medium screening showed a higher growth level in the minimal medium containing 1 g / L of histidine.

[0112] Example 7. Preparation of a strain 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 dva containing three types of colonies selected in Example 6 into the L-histidine-producing strain. For this purpose, using the chromosomal DNAs of ATCC13032ΔN2131-PgapA-Dva(mt)-517, ATCC13032ΔN2131-PgapA-Dva(mt)-1236, and ATCC13032ΔN2131-PgapA-Dva(mt)-2543 as templates, PCR was performed using the primer pair of SEQ ID NO: 40 and SEQ ID NO: 41 to obtain three types of mutant dva. 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 the mutant dva, PCR was performed using the chromosomal DNA 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, followed by a polymerization reaction at 72°C for 5 minutes to obtain the PgapA fragment. The three obtained mutant dva fragments, the 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(mt)-517, pDZΔN2131-PgapA-Dva(mt)-1236, and pDZΔN2131-PgapA-Dva(mt)-2543 according to the derived library colony.

[0113] Subsequently, the constructed vector was transformed into the KCCM80179 strain by electroporation, and three types of strains into which three types of mutant dva were introduced through a secondary crossover process were constructed, which were named KCCM80179ΔN2131-PgapA-Dva(mt)-517, KCCM80179ΔN2131-PgapA-Dva(mt)-1236, and KCCM80179ΔN2131-PgapA-Dva(mt)-2543, respectively.

[0114] To confirm the L-histidine production ability of the prepared KCCM80179ΔN2131-PgapA-Dva(mt)-517, KCCM80179ΔN2131-PgapA-Dva(mt)-1236, and KCCM80179ΔN2131-PgapA-Dva(mt)-2543 strains, the prepared KCCM80179ΔN2131-PgapA-Dva strain was used as a control group and measured in the same manner as in Example 4, and the results are shown in Table 5.

[0115]

Table 5

[0116] As shown in Table 5 above, the NCgl2131-deficient strain (KCCM80179ΔN2131) has an L-histidine production ability comparable to that of the parental strain, KCCM80179 strain. On the other hand, the KCCM80179ΔN2131-PgapA-Dva strain into which the Dermabacter vaginalis-derived gene 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 parental strain, KCCM80179 strain, respectively.

[0117] The three strains into which the three types of dva mutations were introduced each had an increased L-histidine production ability at the same or higher level than the KCCM80179ΔN2131-PgapA-Dva strain compared to the NCgl2131-deficient strain and the parental strain, KCCM80179 strain. In particular, the KCCM80179ΔN2131-PgapA-Dva(mt)-2543 strain was confirmed to have an approximately 12% increase in L-histidine production ability compared to the KCCM80179ΔN2131-PgapA-Dva strain into which the wild-type dva was introduced.

[0118] Example 8. Confirmation of Mutations in the L-Histidine Production Ability-Increasing Mutant Dva Gene To confirm the mutations introduced into Dva of the KCCM80179ΔN2131-PgapA-Dva(mt)-2543 strain in which the L-histidine production ability-increasing effect was confirmed in Example 7, the nucleotide sequence of the Dva 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-Dva(mt)-2543 strain as a template. By nucleotide sequence analysis, the nucleotide sequence of the mutant dva operon and the protein sequences of DvaF or DvaE were confirmed and compared with the amino acid sequences of SEQ ID NO: 12 or SEQ ID NO: 13, and the mutation information of the amino acid sequence of the mutant DvaFE thus confirmed is shown in Table 6.

[0119]

Table 6

[0120] As a result of sequence confirmation, it was confirmed that the mutant DvaFE introduced into the KCCM80179ΔN2131-PgapA-Dva(mt)-2543 strain is a mutant exporter in which DvaF has a R92C mutation (the 92nd arginine (Arg, R) in SEQ ID NO: 13 is mutated to cysteine (Cys, C)) introduced into the wild type and DvaE, and the L-histidine excretion ability is increased.

[0121] From the above results, it was confirmed that the introduction of the mutant exporter derived from Dermabacter vaginalis not only increases the resistance to L-histidine concentrations equal to or higher than the minimum inhibitory concentration compared to the wild type, but also greatly increases the L-histidine production ability. Such results prove that the selected mutant protein derived from Dermabacter vaginalis is a mutant L-histidine excretion protein that can specifically excrete L-histidine.

[0122] Example 9. Production of a strain into which a mutant gene derived from Dermabacter vaginalis based on an L-histidine-producing strain (CA14-737) is introduced and evaluation of L-histidine production ability To reconfirm the ability of the Dva mutant of the protein derived from Dermabacter vaginalis to excrete L-histidine, it was introduced into the L-histidine-producing strain CA14-737 (KCCM12411P, Korean Patent Publication No. 10-2019-0065984) strain, which is derived from wild-type Corynebacterium glutamicum ATCC13032 and has a HisG polypeptide mutation that eliminates feedback inhibition by L-histidine and enhanced L-histidine biosynthetic genes.

[0123] For this purpose, three vectors (pDZΔN2131, pDZΔN2131-PgapA-Dva, pDZΔN2131-PgapA-Dva(mt)-2543) prepared in Examples 2 and 7 were each transformed into the CA14-737 strain by electroporation. Through a secondary crossover process, three strains were produced in which the NCgl2131 gene on the chromosome was deleted or replaced with the L-histidine excretion gene, and these were named CA14-737ΔN2131, CA14-737ΔN2131-PgapA-Dva, and CA14-737ΔN2131-PgapA-Dva(mt)-2543, respectively.

[0124] To confirm the L-histidine production ability of the prepared CA14-737ΔN2131, CA14-737ΔN2131-PgapA-Dva, and CA14-737ΔN2131-PgapA-Dva(mt)-2543 strains, they were cultured by the method performed in Example 4, and the L-histidine production amount (histidine content in the medium) was measured. The results are shown in Table 7 below.

[0125]

Table 7

[0126] As shown in Table 7, the CA14-737ΔN2131-PgapA-Dva strain into which the gene derived from Dermabacter vaginalis was introduced had a 60.0% increase in L-histidine production compared to the NCgl2131-deficient strain (CA14-737ΔN2131) and the parental strain CA14-737, and the CA14-737ΔN2131-PgapA-Dva(mt)-2543 strain into which the mutant dva was introduced had an 80.0% increase.

[0127] As a result, it was reconfirmed that both the wild-type and mutant proteins derived from Dermabacter vaginalis can specifically excrete L-histidine, and in the case of the selected mutant protein, it is an L-histidine exporter having a higher excretion ability than the wild-type protein.

[0128] Example 10. Preparation of vectors for expressing wild-type gene and mutant of Escherichia coli derived from Dermabacter vaginalis In order to confirm the L-histidine excretion ability of the protein Dva and mutants derived from Dermabacter vaginalis in various strains, vectors capable of expressing the wild-type Dva and Dva mutants in Escherichia coli were prepared.

[0129] Each gene was cloned into the Escherichia coli expression vector pCC1BAC (hereinafter, pBAC, Epicenter corp.), and the foreign L-histidine excretion gene candidate was designed to be expressed under the promoter of the yccA gene derived from the Escherichia coli strain MG1655 (hereinafter, PyccA, SEQ ID NO: 43).

[0130] To obtain the protein Dva derived from Dermabacter vaginalis and the mutant gene fragment, using the chromosomal DNA of ATCC13032ΔN2131-PgapA-Dva and ATCC13032ΔN2131-PgapA-Dva(mt)-2543 as templates, PCR was performed using the primer pairs of SEQ ID NO: 44 and SEQ ID NO: 45, respectively, to obtain wild-type and mutant dva DNA fragments. 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 the PyccA fragment, using the chromosome of MG1655 as a template, PCR was performed using the primer pair of SEQ ID NO: 46 and SEQ ID NO: 47. 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 the PyccA fragment. The obtained wild-type and mutant dva fragments, the PyccA fragment, and the pBAC vector digested with EcoRI were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pBAC-PyccA-Dva and pBAC-PyccA-Dva(mt)-2543.

[0131] Example 11. Production of Escherichia coli-derived L-histidine-producing strain base, production of Dermabacter vaginalis-derived Dva wild-type gene and mutant-introduced strain, and evaluation of L-histidine-producing ability To confirm the L-histidine excretion ability of the Dva variant of the protein derived from Dermabacter vaginalis based on the Escherichia coli-derived L-histidine-producing strain, two vectors prepared in Example 10 and the pBAC vector were introduced into the CA14-9003e strain (MG1655+hisGrhisL’_Δ ΔpurR) with the previously reported genotype (purR deficiency, hisL deficiency, 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 produce three strains, which were named CA14-9003e / pBAC, CA14-9003e / pBAC-PyccA-Dva, and CA14-9003e / pBAC-PyccA-Dva(mt)-2543, respectively.

[0132] To confirm the L-histidine production ability of the prepared CA14-9003e / pBAC, CA14-9003e / pBAC-PyccA-Dva, and CA14-9003e / pBAC-PyccA-Dva(mt)-2543 strains, they were cultured in the following manner. The strains were cultured on an LB solid medium (containing 25 μg / ml of chloramphenicol) for 16 hours, and then each strain was inoculated into a 250-ml Erlenmeyer flask containing 25 ml of an LB liquid medium and cultured with shaking at 200 rpm at 37°C for 20 hours. Then, 1 ml of the seed culture solution was inoculated into a 250-ml Erlenmeyer flask containing 25 ml of an Escherichia coli production medium (Applied Biochemistry and Microbiology, 2013, Vol.49, No.2, pp.130-135) and cultured with shaking at 200 rpm at 37°C for 48 hours. The media used for the above culture are as follows:

[0133] <Escherichia coli production medium> 4% (w / v) glucose, 0.2% (w / v) yeast extract, 1.6% (w / v) ammonium sulfate, 0.06% (w / v) dipotassium phosphate trihydrate, 0.0005% (w / v) ferrous sulfate heptahydrate, 0.0005% (w / v) magnesium sulfate pentahydrate, calcium carbonate, pH 7.2

[0134] After the cultivation, the L-histidine production amount (histidine content in the medium) was measured by HPLC, and the results are shown in Table 8 below.

[0135]

Table 8

[0136] As shown in Table 8, it was confirmed that the CA14-9003e / pBAC-PyccA-Dva(mt)-2543 strain into which the mutant dva was introduced had about a 17.9% increase in L-histidine production ability compared to the wild-type dva-introduced strain CA14-9003e / pBAC-PyccA-Dva strain.

[0137] From the above results, it was confirmed that when the L-histidine exporter mutant derived from Dermabacter vaginalis was introduced into microorganisms other than Corynebacterium strains, the ability to excrete L-histidine extracellularly was greatly increased.

[0138] From the above description, those skilled in the technical field 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. A mutant L-histidine excretion protein in which the amino acid corresponding to the 92nd residue of the amino acid sequence of SEQ ID NO: 13 is substituted with another amino acid.

2. The protein according to claim 1, wherein the other amino acid is cysteine, glutamic acid, serine, threonine, asparagine or glutamine.

3. The protein according to claim 1, wherein the other amino acid is cysteine.

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

48.

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

6. A microorganism comprising the protein according to any one of claims 1 to 4, or the polynucleotide encoding the protein.

7. The microorganism according to claim 6, wherein the microorganism has the ability to produce L-histidine.

8. The microorganism according to claim 6, wherein the microorganism is a Corynebacterium or Escherichia.

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

10. The protein according to any one of claims 1 to 4, the polynucleotide encoding the protein, or a composition for producing L-histidine comprising the recombinant microorganism containing the polynucleotide.

11. A method for producing L-histidine, comprising culturing a microorganism comprising the protein according to any one of claims 1 to 4, or the polynucleotide encoding the protein, in a medium.

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

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