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

Abstract

The present application finds a histidine exporting protein having an ability to export L-histidine or a variant thereof, and expresses the protein in a microorganism having an ability to produce L-histidine, thereby significantly improving the yield of L-histidine.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority based on Korean Patent Application No. 10-2022-0076772 filed on June 23, 2022, and all contents described in the file of the corresponding Korean patent application are incorporated as a part of this specification. Technical Field

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

[0004] L-histidine is one of the 20 standard amino acids. From a nutritional perspective, adults do not require large amounts of L-histidine, but it is listed as an essential amino acid for growing children. Furthermore, L-histidine participates in important physiological processes such as antioxidant and immunomodulatory functions, and is used in the pharmaceutical industry as a raw material for gastrointestinal and circulatory therapeutics and in amino acid infusion preparations.

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

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

[0007] In this context, it is necessary to discover proteins with histidine-specific export capabilities and develop technologies to utilize them to produce histidine. Summary of the Invention

[0008] Technical issues

[0009] One object of the present application is to provide a mutant L-histidine exporter, wherein the amino acid corresponding to the 92nd residue of the amino acid sequence of SEQ ID NO: 13 is substituted by another amino acid.

[0010] Another object of the present application is to provide a microorganism comprising the protein or a polynucleotide encoding the protein.

[0011] Another object of the present application is to provide a composition for producing L-histidine, which comprises the protein, a polynucleotide encoding the protein, or the microorganism.

[0012] Another object of the present application is to provide use of the protein, the polynucleotide encoding the protein, or the microorganism for producing L-histidine.

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

[0014] Another object of the present application is to provide a method for producing L-histidine, comprising culturing the microorganism in a culture medium.

[0015] Technical Solution

[0016] The present application shows that the production of L-histidine can be significantly improved by discovering a variant of a histidine export protein having L-histidine export ability and expressing the variant in a microorganism having L-histidine production ability.

[0017] In the present specification, it has been demonstrated that a microorganism expressing a protein containing an AzID domain derived from Dermabacter vaginalis has excellent L-histidine production ability, and it has been demonstrated that when an amino acid substitution mutation is introduced at a specific position of the AzID domain-containing protein, the L-histidine production ability is further increased.

[0018] Proteins, polynucleotides and recombinant vectors

[0019] In one aspect, a mutant protein (or polypeptide) having L-histidine export activity is provided. The protein may be a protein having L-histidine-specific export capability. In this specification, the mutant protein may be represented by a mutant histidine export protein. The mutant protein may have AzlC family ABC transporter permeation activity.

[0020] In one embodiment, the mutant protein may have equivalent or enhanced L-histidine export activity compared to a wild-type L-histidine export protein (e.g., an AzlD domain-containing protein, or an AlzC family ABC transporter permease, etc.). The AzlD domain-containing protein or the AzlC family ABC transporter permease protein may be derived from Dermatobacterium vaginalis. In this specification, the AzlD domain-containing protein derived from Dermatobacterium vaginalis may be described as the DvaE protein (or DvaE), and the AzlC family ABC transporter permease protein derived from Dermatobacterium vaginalis may be described as the DvaF protein (or DvaF).

[0021] In one embodiment, the mutant protein (e.g., a mutant protein of an AzlD domain-containing protein, specifically, a mutant protein of an AzlD domain-containing protein derived from Dermatobacterium vaginalis) can be expressed together with an AzlC family ABC transporter permease protein in the same operon gene. In one embodiment, the mutant protein can have L-histidine export activity by binding to an AzlC family ABC transporter protein.

[0022] In one embodiment, the mutant protein may be a mutant protein of an AzID domain-containing protein derived from Dermatobacterium vaginalis.

[0023] The mutant protein may be a mutant protein in which one or more amino acid residues of the wild-type AzID domain-containing protein derived from Dermatobacterium vaginalis are substituted, deleted or inserted.

[0024] The wild-type AzlD domain-containing protein derived from Dermatobacterium vaginalis may comprise the amino acid sequence of SEQ ID NO: 13 (WP_065248527.1), or consist of the amino acid sequence of SEQ ID NO: 13.

[0025] In one embodiment, the mutant protein may comprise an amino acid sequence in which the amino acid at position 92 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 13 is substituted with another amino acid. As described above, counting amino acids from the N-terminus of the amino acid sequence may mean counting methionine (Met, M) translated from the start codon as the first amino acid.

[0026] In one embodiment, the mutant protein may comprise a sequence in which the amino acid corresponding to residue 92 is substituted with another amino acid, wherein the other amino acid is 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), i.e., an amino acid different from the original amino acid. In one embodiment, the other amino acid may be cysteine, glutamic acid, serine, threonine, asparagine, or glutamine. In a specific embodiment, the other amino acid may be cysteine. Obviously, even if some amino acid sequences other than the amino acid corresponding to the 92nd amino acid residue from the N-terminus of SEQ ID NO: 13 in the mutant protein are deleted, modified, substituted or added, they may be included in the mutant proteins of the present application as long as they exhibit activity comparable to that of the AzlD domain-containing protein.

[0027] In addition, in one embodiment, the mutant protein may comprise a polypeptide wherein the amino acid corresponding to residue 92 of the amino acid sequence of SEQ ID NO: 13 is substituted by another amino acid in an amino acid 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 depicted as SEQ ID NO: 13. In other words, a polypeptide which comprises a substitution with another amino acid at a position corresponding to residue 92 from the N-terminus in 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 to the amino acid sequence of SEQ ID NO: 13, and has an activity comparable to that of a protein containing an AzID domain, can be included in the mutant proteins of the present application.

[0028] In a specific embodiment, the mutant protein may comprise or consist of the amino acid sequence of SEQ ID NO: 48, but is not limited thereto. Obviously, even if some amino acid sequences other than the amino acid corresponding to residue 92 from the N-terminus of SEQ ID NO: 48 in the mutant protein are deleted, modified, substituted, or added, they may also be included in the mutant proteins of the present application, as long as they exhibit activity comparable to that of the AzID domain-containing protein. Furthermore, in one embodiment, the mutant protein may comprise a polypeptide in which the amino acid corresponding to the 92nd residue from the N-terminus in the amino acid sequence of SEQ ID NO: 48 is fixed and has a sequence homology or sequence identity of 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 to the amino acid sequence of SEQ ID NO: 48. In other words, a polypeptide having an amino acid corresponding to the 92nd residue from the N-terminus in the amino acid sequence of SEQ ID NO: 48 being substituted with another amino acid 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: 48, and having an activity comparable to that of a protein containing an AzID domain, can be included in the mutant proteins of the present application.

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

[0030] The wild-type L-histidine export protein can be a protein having 60% or greater sequence homology to SEQ ID NO: 12 (a wild-type AzlD domain-containing protein derived from Dermatobacterium vaginalis), SEQ ID NO: 13 (an AzlC family ABC transporter permease protein derived from Dermatobacterium vaginalis), or a combination thereof. For example, in a specific embodiment, the wild-type L-histidine export protein can have 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 99.5% or greater homology to SEQ ID NO: 12, 13, or a combination thereof.

[0031] The protein represented by SEQ ID NO:12 can be encoded by the nucleic acid sequence of SEQ ID NO:14, the protein represented by SEQ ID NO:13 can be 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 can be encoded by the nucleic acid sequence of SEQ ID NO:16 (an operator sequence fused to the overlapping region at the 3' end of SEQ ID NO:14 and the 5' end of SEQ ID NO:15).

[0032] In another aspect, a polynucleotide encoding the mutant protein is provided. In this application, the term "polynucleotide" refers to a polymer of nucleotides, wherein nucleotide monomers are linked by covalent bonds to form a long chain, i.e., a DNA or RNA chain of a certain length or longer, and more specifically, refers to a polynucleotide fragment encoding a mutant polypeptide.

[0033] The polynucleotide encoding the mutant protein of the present application may comprise a base sequence encoding the amino acid sequence of SEQ ID NO: 48.

[0034] In this specification, a polynucleotide (which can be used interchangeably with "gene") or a polypeptide (which can be used interchangeably with "protein") "comprising a specific nucleic acid sequence or amino acid sequence, consisting of a specific nucleic acid sequence or amino acid sequence, or represented by a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide basically contains a specific nucleic acid sequence or amino acid sequence, and can be interpreted as including "substantially equivalent sequences", wherein mutations (deletions, substitutions, modifications and / or additions) are applied to the specific nucleic acid sequence or amino acid sequence (or mutations are not excluded) within the scope of maintaining the original function and / or desired function of the polynucleotide or polypeptide.

[0035] In one embodiment, the nucleic acid sequence or amino acid sequence provided in the present specification may include sequences modified by ordinary mutagenesis, such as direct evolution and / or site-directed mutagenesis, etc., within the scope of maintaining its original function and / or desired function. In one embodiment, a polynucleotide or polypeptide "comprising a specific nucleic acid sequence or amino acid sequence, or consisting of a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide (i) substantially comprises a specific nucleic acid sequence or amino acid sequence, or (ii) consists of a specific nucleic acid sequence or amino acid sequence that has 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or more. In some embodiments, the present invention relates to an amino acid sequence having 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher or 99.5% or higher homology to the specific nucleotide sequence or amino acid sequence, and keeps original function and / or desired function. In this manual, desired function can refer to giving or increasing the L-Histidine export activity and / or the L-Histidine productivity of microorganisms.

[0036] In the nucleic acid sequences described in this specification, various modifications can be made to the coding region within the range that does not change the amino acid sequence and / or function of the protein expressed in the coding region, taking into account codons preferred in the microorganism to express the protein due to codon degeneracy.

[0037] In this specification, the term "homology (identity)" can refer to the degree of identity with a given nucleic acid sequence or amino acid sequence and can be expressed as a percentage (%). For example, in the case of identity with a nucleic acid sequence, it can be determined using the BLAST algorithm (see: Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90, 5873, 1993) or Pearson's FASTA algorithm (see: Methods Enzymol., 183, 63, 1990). Based on this BLAST algorithm, programs named BLASTN or BLASTX have been developed (see: http: / / www.ncbi.nlm.nih.gov).

[0038] In one embodiment, a polynucleotide comprising a specific nucleic acid sequence provided herein can be interpreted as including not only the specific nucleic acid sequence or a nucleic acid sequence substantially equivalent thereto, but also a polynucleotide fragment comprising a nucleic acid sequence complementary to the specific nucleic acid sequence. Specifically, the complementary polynucleotides can hybridize at a Tm value, which can be adjusted by those skilled in the art according to the purpose, for example, a Tm value of 55°C, 60°C, 63°C, or 65°C, and analyzed under the following conditions: these conditions are specifically described in known literature. For example, conditions that allow genes with high complementarity of 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more to hybridize, and conditions that do not hybridize genes with less than this complementarity, or washing conditions for ordinary southern hybridization, i.e., conditions of washing once, specifically, two to three times, at a salt concentration and temperature corresponding to 60°C, 1x SSC (saline-sodium citrate buffer) and 0.1% (w / v) SDS (sodium dodecyl sulfate); 60°C, 0.1x SSC and 0.1% SDS; or 68°C, 0.1x SSC and 0.1% SDS, can be listed, but are not limited thereto. Hybridization requires that the two nucleotides have complementary sequences, or, depending on the stringency of the hybridization, allow mismatches between bases. The term "complementary" can be used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in the case of DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. The appropriate stringency of polynucleotide hybridization depends on the length and complementary degree of the polynucleotide, which is well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0039] The introduction of polynucleotides or vectors can be carried out by appropriately selecting known transformation methods by those skilled in the art. In this specification, the term "transformation" refers to the process of importing a specific polynucleotide or a vector comprising the polynucleotide into a host cell, and the transformed polynucleotide can be located in the chromosome of the host cell (i.e., inserted into the chromosome) or outside the chromosome. As an example, transformation can import a polynucleotide encoding a target protein (exogenous protein) or a vector comprising the polynucleotide into a host cell so that the protein encoded by the polynucleotide is expressed in the host cell. In addition, the polynucleotide can comprise DNA and / or RNA encoding the target protein. As long as the polynucleotide can be imported into the host cell and expressed, there is no restriction on the form of its introduction. For example, the polynucleotide can be imported into the host cell in the form of an expression cassette, and the expression cassette is a genetic construct comprising all elements that need to be expressed. The expression cassette can generally comprise expression regulatory elements, such as promoters, transcription termination signals, ribosome binding sites, and / or translation termination signals that are operably connected to the polynucleotide. The expression cassette can be in the form of an expression vector that can self-replicate. In addition, the polynucleotide can be imported into the host cell in its own form and be operably connected to the sequence necessary for expression in the host cell. In the above, the term "operably linked" may refer to the functional connection between an expression control element (e.g., a promoter) and a polynucleotide, so that the expression control element performs transcriptional regulation (e.g., transcription initiation) on the polynucleotide encoding the target protein (exogenous protein). Operable connection can be performed using gene recombination techniques known in the art, for example, by common site-specific DNA cleavage and ligation, but is not limited thereto.

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

[0041] The introduction (insertion) of the polynucleotide into the host cell genome (chromosome) can be performed by appropriately selecting a known method by those skilled in the art, for example, an RNA-guided endonuclease system (or CRISPR system; for example, at least one selected from the group consisting of a mixture (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA, etc.), a complex (e.g., a ribonucleic acid fusion protein (RNP), a recombinant vector (e.g., a vector comprising an RNA-guided endonuclease encoding gene and a guide RNA encoding DNA, etc.), which comprises (a) an RNA-guided endonuclease (e.g., Cas9 protein, etc.), a gene encoding the same, or a vector comprising the same; and (b) a guide RNA (e.g., a single guide RNA (sgRNA), etc.), a DNA encoding the same, or a vector comprising the same)), but is not limited thereto.

[0042] On the other hand, a recombinant vector comprising a polynucleotide is provided. The recombinant vector can be used as an expression vector for a polypeptide. The recombinant vector can be used to insert the polynucleotide into the genome of a host cell, or to replace the corresponding gene in the genome of a host cell.

[0043] In this specification, the term "vector" refers to a DNA product containing a base sequence of a polynucleotide encoding a target protein, which is operably linked to an appropriate regulatory sequence to express the target protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence that regulates transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence that regulates transcription and / or translation termination. After transformation into a suitable host cell, the vector can be expressed independently of the host cell's genome or integrated into the host cell's genome.

[0044] There are no particular limitations on the vectors that can be used in this specification, as long as they can replicate in host cells, and they can be selected from all commonly used vectors. Examples of commonly used vectors can include plasmids, cosmids, viruses, phages, and the like in their natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be cited, but are not limited thereto.

[0045] The vector that can be used for this specification sheet can be a known expression vector and / or a vector for inserting a polynucleotide into a host cell chromosome. The polynucleotide can be inserted into the host cell chromosome by any method known in the art, such as homologous recombination or CRISPR system, but is not limited thereto. The vector may further include a selection marker for confirming whether it is inserted into the chromosome. The selection marker is used to select cells transformed with the vector, that is, to confirm the insertion of the polynucleotide, and can be selected and used from genes that give selectable phenotypes such as drug resistance, auxotrophy, cytotoxic agent resistance or surface protein expression. Because only cells expressing the selection marker survive or show different phenotypes, the transformed cells can be selected.

[0046] microorganism

[0047] On the other hand, provide and comprise at least a (1 kind, 2 kinds or whole 3 kinds) microorganism that is selected from the group that the polynucleotide of free mutant protein, encoding this mutant protein and the recombinant vector that comprises these polynucleotide form.This microorganism can have L-Histidine export activity and / or L-Histidine productivity.Compare with at least a microorganism that does not comprise and is selected from the group that the polynucleotide of free mutant protein, encoding this mutant protein and the recombinant vector that comprises these polynucleotide form, this microorganism can have enhanced (or increase, improvement) L-Histidine export activity and / or L-Histidine productivity.Mutant protein may be exogenous.In this manual, " exogenous " can refer to not this microorganism endogenous existence, but derives from the species beyond this microorganism.

[0048] In this specification, "a microorganism having enhanced L-histidine export activity and / or L-histidine production capacity" may be a microorganism that originally did not have L-histidine export activity and / or L-histidine production capacity, but after being engineered (mutated) to express the above-mentioned mutant protein, it has acquired L-histidine export activity and / or L-histidine production capacity; or, a microorganism whose original L-histidine export activity and / or L-histidine production capacity has been improved after engineering (mutation).

[0049] In this specification, "microorganism" includes unicellular bacteria and can be used interchangeably with "cell".

[0050] In the present application, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and may be a microorganism in which a specific mechanism is weakened or enhanced due to reasons such as the insertion of exogenous genes or the enhancement or inactivation of the activity of endogenous genes, and is a microorganism comprising a genetic modification for producing a desired polypeptide, protein or product (e.g., L-histidine).

[0051] The microorganism of the present application may be a genetically modified microorganism (eg, a recombinant microorganism) to enhance the activity of the L-histidine export protein or the polynucleotide encoding the same, but is not limited thereto.

[0052] In this specification, in order to distinguish it from the microorganism after mutation, the microorganism used to express the mutant protein before mutation may be referred to as the "parent strain (parent microorganism) or host cell".

[0053] A microorganism (or bacterial strain, recombinant cell) has L-histidine export activity and / or L-histidine productivity, or has enhanced L-histidine export activity and / or L-histidine productivity, which may mean that the L-histidine export activity and / or L-histidine productivity is different from that of an unmodified microorganism, cell before the recombinant, parental strain and / or wild-type strain, which does not have L-histidine export activity and / or L-histidine productivity, or that its L-histidine export activity and / or L-histidine productivity is enhanced compared to an unmodified microorganism, cell before the recombinant, parental strain and / or wild-type strain. The microorganism of the present application may be any one or more microorganisms comprising the mutant protein of the present application, a polynucleotide encoding the mutant protein of the present application, and a vector comprising the polynucleotide of the present application; a microorganism modified to express the mutant protein of the present application or the polynucleotide of the present application; a microorganism expressing the mutant protein of the present application or the polynucleotide of the present application (e.g., a recombinant strain); or a microorganism having the activity of the mutant protein of the present application (e.g., a recombinant strain), but is not limited thereto.

[0054] In one embodiment, microorganism can be at least one in the group being formed of the microorganism of free Corynebacterium, the microorganism of Escherichia etc.The microorganism of Corynebacterium can comprise Corynebacterium glutamicum, ammonia-producing Corynebacterium (Corynebacterium ammoniagenes), lactose fermentation Brevibacterium (Brevibacteriumlactofermentum), yellow Brevibacterium (Brevibacterium flavum), heat ammonia-producing Corynebacterium (Corynebacterium thermoaminogenes), efficient Corynebacterium (Corynebacterium efficiens) etc., but is not limited to this.More specifically, the microorganism of Corynebacterium can be Corynebacterium glutamicum.The bacterial strain of Escherichia can be intestinal bacteria.

[0055] The microorganism may contain at least one (one, two, or all three) selected from the group consisting of a mutant protein, a polynucleotide encoding the mutant protein, and a recombinant vector comprising the polynucleotide. In one embodiment, the mutation to express the mutant protein can be performed by introducing a polynucleotide encoding the mutant protein or a recombinant vector comprising the polynucleotide, or by artificial mutagenesis (e.g., error-prone PCR, etc.), or the like. The polynucleotide encoding the mutant protein to be introduced into the parent strain can thus replace the gene encoding the protein containing the AzlD domain in the strain, or be further included therein.

[0056] In one embodiment, when a microorganism comprises a mutant protein and a wild-type AzlC family ABC transporter permease protein, it may have further enhanced L-histidine export activity and / or L-histidine production capacity. In one embodiment, the microorganism having enhanced L-histidine export activity and / or L-histidine production capacity may have an increase of 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 in L-histidine export activity and / or L-histidine production capacity compared to the parent strain before mutation, an unmodified microorganism, and a microorganism comprising a wild-type L-histidine export protein, but is not limited thereto.

[0057] In other embodiments, the microorganism with enhanced L-histidine export activity and / or L-histidine productivity can have an increase of 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 of L-histidine export activity and / or L-histidine productivity compared to the parent strain before mutation, an unmodified microorganism, and a microorganism comprising a wild-type L-histidine export protein, but is not limited thereto.

[0058] The term "about" represents a range including all values ​​such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and also includes all values ​​equal to or similar to the value following the term "about", but is not limited thereto.

[0059] In another aspect, provided is a composition for producing L-histidine, comprising a mutant protein, a polynucleotide, a recombinant vector or a microorganism.

[0060] In another aspect, provided is a use of a mutant protein, a polynucleotide, a recombinant vector or a microorganism for producing L-histidine.

[0061] In another aspect, provided is a use of a mutant protein, a polynucleotide, a recombinant vector or a microorganism in preparing a composition for producing L-histidine.

[0062] In another aspect, a method for producing (preparing) L-histidine is provided, comprising culturing a microorganism in a culture medium. The preparation method may further comprise recovering L-histidine from the cultured microorganism, the culture medium, or both after the culturing.

[0063] On the other hand, provided is a method for increasing the L-histidine export activity and / or L-histidine production capacity of a microorganism, or a method for conferring L-histidine export activity and / or L-histidine production capacity on a microorganism, comprising enhancing the L-histidine export activity and / or L-histidine production capacity of a microorganism.

[0064] Introducing mutations may include introducing (transforming) a polynucleotide encoding a mutant protein or a recombinant vector comprising the polynucleotide into a microorganism, or may include artificially generating mutations (eg, error-prone PCR, etc.).

[0065] In this method, cultivating microorganism is not particularly limited to this, but can be carried out by known batch culture method, continuous culture method, fed-batch culture method etc.At this moment, culture condition is not particularly limited to this, but uses alkaline compound (for example: sodium hydroxide, potassium hydroxide or ammonia) or acidic compound (for example: phosphoric acid or sulfuric acid) to regulate suitable pH (for example, pH 5 to 9, specifically, pH 6 to 8, most specifically, pH 7.2), and can by importing oxygen or oxygen-containing gas mixture into culture and maintain aerobic condition.Cultivation temperature can be maintained at 20 to 45 ℃, or 25 to 40 ℃, and can cultivate about 10 to 160 hours, but is not limited to this.The L-histidine produced by cultivating can be secreted into culture medium or retained in cell.

[0066] The culture medium that can be used for culture can use at least one substance selected from the group consisting of sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oils and fats (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), and organic acids (e.g., acetic acid) as a carbon source, but is not limited thereto. As a nitrogen source, at least one substance selected from the group consisting of nitrogen-containing organic compounds (e.g., peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal, and urea), inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate) can be used alone or in combination, but is not limited thereto. As a phosphorus source, at least one substance selected from the group consisting of potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding nitrogen-containing salts can be used alone or in combination, but is not limited thereto. In addition, the culture medium may contain necessary growth-promoting substances, such as other metal salts (eg, magnesium sulfate, iron sulfate, manganese sulfate, etc.), amino acids and / or vitamins.

[0067] Reclaiming L-Histidine can be to use suitable method known in the art to collect required amino acid from culture medium, culture fluid or microorganism, and this depends on culture method.For example, can reclaim by being selected from one or more methods of centrifugal, filtration, anion exchange chromatography, crystallization, HPLC etc. The method of reclaiming L-Histidine can be included in addition before, simultaneously or afterwards and purify.

[0068] Beneficial effects

[0069] The present application discloses a histidine export protein or a variant thereof having the ability to export L-histidine, which can significantly increase the yield of L-histidine when expressed in a microorganism having the ability to produce L-histidine. DETAILED DESCRIPTION

[0070] Hereinafter, the present invention will be described in more detail by the following examples. However, they are only used to illustrate the present invention, but the scope of the present invention is not limited by these examples.

[0071] Example 1. Search for exogenous histidine export genes and candidate selection

[0072] In order to select protein candidates having L-histidine-specific export ability, as a result of PSI-BLAST search based on NCBI and Kegg databases, amino acid sequences of amino acids classified by category (export proteins are as follows: for basic amino acids: L-lysine (L-lys), for aromatic amino acids: tryptophan (Trp), for branched-chain amino acids: isoleucine (Ile) (LysE (Arch Microbiol 180: 155-160), Wex (Korean registered patent number 10-1968317), BrnFE (Arch Microbiol 180: 155-160)) were used as query sequences, and candidate genes predicted to be membrane proteins that may export L-histidine and microorganisms having them were selected.

[0073] Among them, taking into account the biosafety level and the possibility of ensuring a level suitable for the production strains therein, as shown in Table 1 below, one LysE-based, three Wex-based, and two BrnFE-based proteins, as well as the genes encoding these proteins and the microorganisms containing these proteins were selected:

[0074] [Table 1]

[0075] List of L-histidine export candidates

[0076]

[0077]

[0078]

[0079] (In Table 1 above, the biosafety level is based on the microbial pathogenicity index (level 1 to 4) defined by the Centers for Disease Control and Prevention (CDC) of the United States (the lower the level, the safer it is).

[0080] Example 2. Production of Vectors Introducing Exogenous L-histidine Export Gene Candidates and Recombinant Corynebacterium Strains Introducing the Same Gene Candidates

[0081] Six vectors were produced for introducing the six exogenous L-histidine export gene candidates selected in Example 1 into Corynebacterium strains.

[0082] Example 2-1. Production of vector pDZΔN2131 for inserting target gene

[0083] In order to import the exogenous L-histidine export gene candidate, the NCgl2131 gene in the gene encoding the Corynebacterium glutamicum transposon was used as an insertion site (Journal of Biotechnology 104, 5-25 Jorn Kalinowski et al, 2003). In addition, it was designed to express the exogenous L-histidine export gene candidate under the promoter of the gapA gene (hereinafter referred to as PgapA, SEQ ID NO: 17) derived from Corynebacterium.

[0084] To replace the NCgl2131 gene with an export gene, a vector containing the NCgl2131 deletion and target gene insertion was produced. To produce the vector, PCR was performed using the chromosome of Corynebacterium glutamicum strain ATCC13032 as a template using primer pairs of SEQ ID NOs: 18 and 19, and SEQ ID NOs: 20 and 21, respectively. PfuUltra™ High Reliability 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 at 72°C for 2 minutes, repeated 28 times, followed by polymerization at 72°C for 5 minutes. As a result, 531 bp of del-N2131L (SEQ ID NO: 22) and 555 bp of del-N2131R (SEQ ID NO: 23) DNA fragments were obtained, respectively. The obtained DNA product was purified using a PCR purification kit from QIAGEN and then cloned using the pDZ vector (Korean registered patent number 10-0924065) and TaKaRa's Infusion cloning kit to produce the vector pDZΔN2131 for NCgl2131 gene deletion and target gene insertion.

[0085] Example 2-2. Production of 6 vectors introducing L-histidine export gene candidates

[0086] The base sequence information of the gene (hereinafter referred to as haq, SEQ ID NO: 2) encoding the protein derived from the water grass spirillum (hereinafter referred to as Haq, SEQ ID NO: 1) was obtained from the GenBank of the National Institutes of Health (NIH GenBank). In order to amplify haq, a primer pair of SEQ ID NO: 24 and SEQ ID NO: 25 was used, and PCR was performed using the chromosomal DNA of the water grass spirillum strain (KCTC42001) as a template. As the polymerase used for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 977 bp haq fragment containing 945 bp of haq (SEQ ID NO: 2) was obtained. To obtain a PgapA fragment capable of ligating to haq, PCR was performed using the primer pair of SEQ ID NO: 26 and SEQ ID NO: 27, using the chromosome of ATCC 13032 as a template. PfuUltra™ High Reliability 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 at 72°C for 1 minute, repeated 28 times, followed by polymerization at 72°C for 5 minutes. A 441 bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO: 17) was obtained. The obtained haq fragment and PgapA fragment and the pDZΔN2131 vector cut with ScaI restriction enzyme were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Haq.

[0087] The base sequence information of the gene (hereinafter referred to as cpi, SEQ ID NO: 4) encoding the protein derived from Copperobacterium (hereinafter referred to as Cpi, SEQ ID NO: 3) was obtained from the GenBank of the National Institutes of Health (NIH GenBank). In order to amplify the cpi derived from Copperobacterium, a primer pair of SEQ ID NO: 28 and SEQ ID NO: 29 was used, and PCR was performed using the chromosomal DNA of the Copperobacterium strain (KCTC22125) as a template. As a polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: as a result of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 977 bp cpi fragment containing 945 bp of cpi (SEQ ID NO: 4) was obtained. To obtain a PgapA fragment that can be linked to cpi, PCR was performed using the primer pair of SEQ ID NO:26 and SEQ ID NO:30, using the chromosome of ATCC13032 as a template. PfuUltra™ High Reliability 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 at 72°C for 1 minute, repeated 28 times, followed by polymerization at 72°C for 5 minutes. A 441-bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO:17) was obtained. The obtained cpi and PgapA fragments, along with the pDZΔN2131 vector cut with the ScaI restriction enzyme, were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was designated pDZΔN2131-PgapA-Cpi.

[0088] The base sequence information of the gene (hereinafter referred to as kcr, SEQ ID NO: 6) encoding the protein derived from Kluyvera refrigerata (hereinafter referred to as Kcr, SEQ ID NO: 5) was obtained from the GenBank of the National Institutes of Health (NIH GenBank). To amplify the kcr derived from Kluyvera refrigerata, PCR was performed using the primer pair of SEQ ID NO: 31 and SEQ ID NO: 32 and the chromosomal DNA of the Kluyvera refrigerata strain (KCTC2580) as a template. As the polymerase used for the PCR reaction, PfuUltraTM High Reliability DNA Polymerase (Stratagene) was used, and the PCR conditions were as follows: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, and then polymerization at 72°C for 5 minutes, a 914 bp kcr fragment containing 882 bp of kcr (SEQ ID NO: 6) was obtained. To obtain a PgapA fragment that can be linked to kcr, PCR was performed using the primer pair of SEQ ID NO: 26 and SEQ ID NO: 33, using the chromosome of ATCC13032 as a template. As the polymerase used for the PCR reaction, PfuUltra™ High Reliability DNA Polymerase (Stratagene) was used, and the PCR conditions were as follows: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, followed by polymerization at 72°C for 5 minutes. A 441bp PgapA fragment containing 409bp of PgapA (SEQ ID NO: 17) was obtained. The obtained kcr fragment and PgapA fragment, along with the pDZΔN2131 vector cut with the ScaI restriction enzyme, were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDZΔN2131-PgapA-Kcr.

[0089] The base sequence information of the gene encoding the protein derived from Corynebacterium stagnantum (hereinafter referred to as Cst, SEQ ID NO: 7) (hereinafter referred to as cst, SEQ ID NO: 8) was obtained from the National Institutes of Health GenBank (NIH GenBank). To amplify cst derived from Corynebacterium stagnantum, PCR was performed using the primer pair of SEQ ID NO: 34 and SEQ ID NO: 35 and chromosomal DNA of Corynebacterium stagnantum strain (ATCC6872) as a template. PfuUltra™ High Reliability 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 at 72°C for 2 minutes, repeated 28 times, followed by polymerization at 72°C for 5 minutes. A 749 bp cst fragment containing 717 bp of cst (SEQ ID NO: 8) was obtained. To obtain a PgapA fragment that can be linked to cst, PCR was performed using the primer pair of SEQ ID NO:26 and SEQ ID NO:36, using the chromosome of ATCC13032 as a template. PfuUltra™ High Reliability 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 at 72°C for 1 minute, repeated 28 times, followed by polymerization at 72°C for 5 minutes. A 441-bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO:17) was obtained. The obtained cst and PgapA fragments were cloned using the Gibson assembly method, along with the pDZΔN2131 vector cut with the ScaI restriction enzyme, to obtain a recombinant plasmid, which was designated pDZΔN2131-PgapA-Cst.

[0090] The base sequence information of the operon (hereinafter referred to as lsa, SEQ ID NO: 11) encoding the protein derived from Leuconostoc salcidius (hereinafter referred to as LsaFE, SEQ ID NOs: 9, 10) was obtained from the National Institutes of Health GenBank (NIH GenBank). To amplify lsa derived from Leuconostoc salcidius, PCR was performed using the primer pair of SEQ ID NOs: 37 and 38 and chromosomal DNA of the Leuconostoc salcidius strain (KCTC19904) as a template. PfuUltra™ High Reliability 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 at 72°C for 2 minutes, repeated 28 times, followed by polymerization at 72°C for 5 minutes. A 1080 bp lsa fragment containing 1048 bp of lsa (SEQ ID NO: 11) was obtained. To obtain a PgapA fragment that can be linked to lsa, PCR was performed using the primer pair of SEQ ID NO:26 and SEQ ID NO:39, using the chromosome of ATCC13032 as a template. PfuUltra™ High Reliability 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 at 72°C for 1 minute, repeated 28 times, followed by polymerization at 72°C for 5 minutes. A 441-bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO:17) was obtained. The obtained lsa and PgapA fragments were cloned using the Gibson assembly method, along with the pDZΔN2131 vector cut with the ScaI restriction enzyme, to obtain a recombinant plasmid, which was designated pDZΔN2131-PgapA-Lsa.

[0091] The base sequence information for the operon (hereinafter referred to as dva, SEQ ID NO: 16) encoding a protein derived from Dermatobacterium vaginalis (hereinafter referred to as DvaFE, SEQ ID NOs: 12 and 13) was obtained from the National Institutes of Health GenBank (NIH GenBank). To amplify Dermatobacter vaginalis-derived dva, PCR was performed using the primer pair of SEQ ID NOs: 40 and 41, using chromosomal DNA from the Dermatobacter vaginalis strain (KCTC39585) as a template. PfuUltra™ High Reliability 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 at 72°C for 2 minutes, repeated 28 times, followed by polymerization at 72°C for 5 minutes. A 1113 bp dva fragment containing 1081 bp of dva (SEQ ID NO: 16) was obtained. To obtain a PgapA fragment capable of connecting to dva, PCR was performed using the primer pair of SEQ ID NO:26 and SEQ ID NO:42, using the chromosome of ATCC13032 as a template. PfuUltra™ High Reliability 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 at 72°C for 1 minute, repeated 28 times, followed by polymerization at 72°C for 5 minutes. This resulted in a 441-bp PgapA fragment containing 409 bp of PgapA (SEQ ID NO:17). The obtained dva and PgapA fragments were cloned using the Gibson assembly method, along with the pDZΔN2131 vector digested with the ScaI restriction enzyme, to obtain a recombinant plasmid, which was designated pDZΔN2131-PgapA-Dva.

[0092] Example 2-3. Production of recombinant strains of Corynebacterium

[0093] In order to confirm the L-histidine export ability of the exogenous L-histidine export gene candidate, the produced NCgl2131 deletion vector (pDZΔN2131) and 6 vectors for introducing exogenous L-histidine export gene candidate (pDZΔN2131-PgapA-Haq, pDZΔN2131-PgapA-Cpi, pDZΔN2131-PgapA-Kcr, pDZΔN2131-PgapA-Cst, pDZΔN2131-PgapA-Lsa, pDZΔN2131-PgapA-Dva) were respectively introduced into Corynebacterium glutamicum ATCC13032 strain. More specifically, the vectors were transformed into the ATCC13032 strain by electroporation, and through a secondary hybridization process, seven recombinant strains were produced (in which the NCgl2131 gene on the chromosome was deleted or replaced by a candidate L-histidine export gene), which were named ATCC13032ΔN2131 (N2131 gene deleted), ATCC13032ΔN2131::Haq (N2131 gene replaced by haq), ATCC13032ΔN2131::Haq (N2131 gene replaced by haq), and ATCC13032ΔN2131::Haq (N2131 gene replaced by haq). N2131::Cpi (the N2131 gene was replaced by cpi), ATCC13032ΔN2131::Kcr (the N2131 gene was replaced by kcr), ATCC13032ΔN2131::Cst (the N2131 gene was replaced by cst), ATCC13032ΔN2131::Lsa (the N2131 gene was replaced by lsa) and ATCC13032ΔN2131::Dva (the N2131 gene was replaced by dva).

[0094] Example 3. MIC determination of Corynebacterium strains into which exogenous L-histidine export gene candidates were introduced

[0095] In order to confirm the retention of the activity of the L-histidine export ability of 7 kinds of recombinant Corynebacterium glutamicum strains (ATCC13032ΔN2131, ATCC13032ΔN2131::Haq, ATCC13032ΔN2131::Cpi, ATCC13032ΔN2131::Kcr, ATCC13032ΔN2131::Cst, ATCC13032ΔN2131::Lsa and ATCC13032ΔN2131::Dva) produced in Example 2 above, a minimum inhibitory concentration (MIC) experiment was performed using L-histidine. The 7 strains were cultured in a minimal liquid medium at 30°C for 24 hours and then treated with 1×10 3 and 1×10 4 The cells were diluted and cultured in a minimal solid medium supplemented with L-histidine. The composition of the minimal solid medium used was as follows:

[0096] Minimal medium (pH 7.2)

[0097] Glucose 10g, KH2PO4 1g, K2HPO4 2g, MgSO4 7H2O 0.4g, urea 2g, (NH4)2SO4 5g, NaCl 0.5g, nicotinamide 5μg, calcium pantothenate 0.1μg, biotin 0.2μg, thiamine hydrochloride 3μg, trace element solution *1ml (based on 1L distilled water), agar 20g

[0098] *Trace element solution

[0099] Na2B4O7 10H2O 0.09g, (NH4)6Mo7O 27 4H2O 0.04g, ZnSO4 7H2O 0.01g, CuSO45H2O 0.27g, MnCl2 4H2O 0.01g, FeCl3 6H2O 1g, CaCl2 0.01g (based on 1 liter of distilled water)

[0100] For the minimum inhibitory concentration experiment, 1 g / L of L-histidine was added to the basic solid culture medium. After 48 hours, the cell growth was observed. The results are shown in Table 2 below.

[0101] [Table 2]

[0102] Growth level of Corynebacterium strains introduced with exogenous L-histidine export gene candidates in minimal medium containing L-histidine

[0103]

[0104]

[0105] (In Table 2 , the number of + indicates the relative growth level of the strain, and each indicates the following: +: does not form a single colony, but forms overlapping colonies (does not grow into a single colony, but grows in a cluster);

[0106] ++: overlap and less than 5 single colonies were formed;

[0107] +++: overlap and formation of less than 50 single colonies;

[0108] ++++: Overlapping and indistinguishable from single colonies)

[0109] As shown in Table 2 above, all strains, except the ATCC13032ΔN2131::Dva strain, grew steadily in minimal medium without L-histidine. However, in minimal medium containing 1 g / L of L-histidine, the strains introduced with the candidate L-histidine export gene showed insignificant growth, and only the ATCC13032ΔN2131::Dva strain, which had introduced genes derived from Dermatobacter vaginalis, showed superior growth to ATCC13032ΔN2131. This suggests that the introduced Dermatobacter vaginalis-derived protein can exhibit L-histidine export capability even in medium containing L-histidine above the minimum inhibitory concentration.

[0110] Thus, the Dva protein derived from Dermatobacterium vaginalis was selected as a protein capable of conferring resistance to L-histidine exceeding the minimum inhibitory concentration in coryneform bacteria strains and having the ability to specifically export L-histidine.

[0111] Example 4. Production of a strain into which a gene (dva) derived from Dermatobacterium vaginalis was introduced based on an L-histidine-producing strain derived from Corynebacterium (KCCM 80179) and evaluation of its L-histidine-producing ability

[0112] To confirm the L-histidine export ability of the Dva protein derived from Dermatobacterium vaginalis, the Dva gene derived from Dermatobacter vaginalis was introduced into the L-histidine-producing strain KCCM 80179 (Korean Patent Publication No. 10-2019-0065984).

[0113] To this end, the vectors pDZΔN2131 and pDZΔN2131-PgapA-Dva produced in Example 2 were respectively transformed into the KCCM80179 strain, and through a secondary hybridization process, two strains in which the NCgl2131 gene on the chromosome was deleted or replaced by the L-histidine export gene candidate (dva) were obtained, and they were named KCCM 80179ΔN2131 (NCgl2131 gene deleted) and KCCM 80179ΔN2131-PgapA-Dva (NCgl2131 gene replaced by dva), respectively.

[0114] To confirm the L-histidine-producing ability of the KCCM 80179ΔN2131 and KCCM 80179ΔN2131-PgapA-Dva strains, they were cultured as follows: KCCM 80179ΔN2131 and KCCM 80179ΔN2131-PgapA-Dva strains were cultured in activation medium for 16 hours. Each strain was then inoculated into a 250 mL baffled flask containing 25 mL of seed culture medium and cultured with shaking at 200 rpm at 30°C for 20 hours. Subsequently, 1 mL of the seed culture was inoculated into a 250 mL baffled flask containing 25 mL of production medium and cultured with shaking at 200 rpm at 30°C for 48 hours. The composition of the culture medium used was as follows:

[0115] <Activation Medium>

[0116] Meat broth 1% (w / v), polypeptone 1% (w / v), sodium chloride 0.5% (w / v), yeast extract 1% (w / v), agar 2% (w / v), pH 7.2

[0117] <Seed culture medium>

[0118] 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

[0119] <Production Medium>

[0120] Glucose 10% (w / v), ammonium sulfate 2% (w / v), potassium dihydrogen phosphate 0.1% (w / v), magnesium sulfate heptahydrate 0.05% (w / v), CSL (corn steep liquor) 2.0% (w / v), biotin 200 μg / L, calcium carbonate, pH 7.2,

[0121] After the cultivation was completed, the L-histidine production (histidine content in the culture medium) was measured by HPLC, and the results are shown in Table 3 below.

[0122] [Table 3]

[0123] L-histidine production by a strain introduced with a gene derived from KCCM 80179-derived Dermatobacterium vaginalis

[0124]

[0125] As shown in Table 3 above, it was confirmed that the NCgl2131 deletion strain had an L-histidine production capacity at a level comparable to that of the parent strain KCCM 80179 strain, and the L-histidine production capacity of the KCCM 80179ΔN2131-PgapA-Dva strain into which the gene derived from vaginal bacillus was introduced was increased by 23% and 21% or more, respectively, compared with the NCgl2131 deletion strain and the parent strain.

[0126] The results of Examples 3 and 4 demonstrate that the introduction of a gene derived from Dermatobacter vaginalis not only increases resistance to L-histidine concentrations exceeding the minimum inhibitory concentration, but also significantly enhances L-histidine production capacity. This result demonstrates that the protein derived from Dermatobacter vaginalis is an L-histidine-exporting protein capable of specifically exporting L-histidine.

[0127] Example 5. Production of a Dva mutant library using artificial mutagenesis

[0128] To obtain mutant Dva with increased L-histidine export capacity, a library of mutant protein expression vectors for primary crossover was generated. To this end, using the primer pair of SEQ ID NO:40 and SEQ ID NO:41, chromosomal DNA from the Dermatobacterium vaginalis strain (KCTC39585) was used as a template to generate the dva operon with randomly introduced base substitution mutations via error-prone PCR. For error-prone PCR, PCR was performed using the Genemorph II Random Mutagenesis Kit (Stratagene) to introduce 0 to 3.5 mutations per 1 kb of the amplified gene fragment. The PCR conditions were denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 2 minutes, repeated 30 times. To obtain a PgapA fragment that could be linked to dva, PCR was performed using the primer pair of SEQ ID NO:26 and SEQ ID NO:42, using the chromosome of ATCC13032 as a template. As the polymerase for the PCR reaction, PfuUltraTM high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: by denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, and then polymerization at 72°C for 5 minutes to obtain the PgapA fragment. The mutant dva operon and PgapA fragment obtained were cloned using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method, and the pDZΔN2131 vector cut with the ScaI restriction enzyme, and transformed into DH5α and plated on LB solid medium containing kanamycin (25 mg / L). As a result of selecting 20 transformed colonies and then obtaining plasmids and analyzing the base sequences, it was confirmed that mutations were introduced at different positions at an average frequency of 1.5 mutations / kb. The plasmid was extracted by extracting approximately 10,000 transformed E. coli colonies and named as pDZΔN2131-PgapA-Dva(mt) library.

[0129] Example 6: Introduction of Dva artificial mutant library and selection of strains with increased L-histidine production capacity

[0130] The produced ATCC13032ΔN2131 strain was used as the parent strain, and the produced pDZΔN2131-PgapA-Dva(mt) library was transformed by homologous chromosomal recombination and plated on a composite plate medium containing kanamycin (25 mg / L) to obtain approximately 3,300 colonies, each of which was designated TCC13032ΔN2131-PgapA-Dva(mt)-1 to ATCC13032ΔN2131-PgapA-Dva(mt)-3300.

[0131] <Compound Plate Medium (pH 7.0)>

[0132] Glucose 10g, peptone 10g, beef extract 5g, yeast extract 5g, brain heart infusion 18.5g, NaCl 2.5g, urea 2g, sorbitol 91g, agar 20g (based on 1L distilled water)

[0133] For the 3,300 colonies obtained, the minimum inhibitory concentration (MIC) experiment of L-histidine performed in Example 3 was carried out. For effective screening, it was carried out based on a liquid minimal culture medium containing 3g of L-histidine. The colony was immediately inoculated into 300ul of liquid minimal culture medium and cultured in a 96-deep well plate at 32°C and 1000rpm for 18 hours, and then the OD600 value was measured to select well-grown colonies. As a control group, the produced ATCC13032ΔN2131 strain and ATCC13032ΔN2131::Dva strain were used. By primary experiment, 413 colonies were selected, and by repeated experiment, 78 colonies were selected. The culture medium components used are as follows.

[0134] Liquid minimal medium (pH 7.2)

[0135] Glucose 10g, KH2PO4 1g, K2HPO4 2g, MgSO4 7H2O 0.4g, urea 2g, (NH4)2SO4 5g, NaCl 0.5g, nicotinamide 5μg, calcium pantothenate 0.1μg, biotin 0.2μg, thiamine hydrochloride 3μg, trace element solution*1ml (based on 1L distilled water)

[0136] *Trace element solution

[0137] Na2B4O7 10H2O 0.09g, (NH4)6Mo7O 27 4H2O 0.04g, ZnSO4 7H2O 0.01g, CuSO4 5H2O 0.27g, MnCl2 4H2O 0.01g, FeCl3 6H2O 1g, CaCl2 0.01g (based on 1 liter of distilled water)

[0138] For the minimum inhibitory concentration experiment, 3 g / L L-histidine was added to the culture medium and cultured for 18 hours.

[0139] The 78 selected colonies were screened on a solid culture medium by the method performed in Example 3, and three colonies with increased L-histidine exporting capacity were finally screened. The results are shown in Table 4 below.

[0140] [Table 4]

[0141] Growth of the mutant strain in a minimal medium containing L-histidine

[0142]

[0143] (In Table 4, the number of + indicates the relative growth level of the strain, and each indicates the following: +: does not form a single colony, but forms overlapping (does not grow into a single colony, but grows in a cluster);

[0144] ++: overlap and less than 5 single colonies were formed;

[0145] +++: overlap and formation of less than 50 single colonies;

[0146] ++++: Overlapping and indistinguishable from single colonies)

[0147] As shown in Table 4 above, the strain lacking NCgl2131 (ATCC13032ΔN2131) could not grow steadily in a minimal medium containing 1 g / L of L-histidine, but the ATCC13032ΔN2131::Dva strain into which a gene derived from vaginal bacillus was introduced grew steadily, and by screening based on solid culture medium, it was confirmed that the growth levels of three strains (ATCC13032ΔN2131-PgapA-Dva(mt)-517, ATCC13032ΔN2131-PgapA-Dva(mt)-1236, and ATCC13032ΔN2131-PgapA-Dva(mt)-2543) were higher than the growth levels in a minimal medium containing 1 g / L histidine.

[0148] Example 7. Production of strains introduced with a library selected from an L-histidine-producing strain derived from Corynebacterium (KCCM 80179) and evaluation of L-histidine-producing ability

[0149] Vectors were produced for introducing the mutant dva contained in the three colonies selected in Example 6 into an L-histidine-producing strain. To this end, PCR was performed using the primer pair of SEQ ID NO:40 and SEQ ID NO:41, using chromosomal DNA from ATCC13032ΔN2131-PgapA-Dva(mt)-517, ATCC13032ΔN2131-PgapA-Dva(mt)-1236, and ATCC13032ΔN2131-PgapA-Dva(mt)-2543 as templates to obtain the three mutant dva. PCR conditions included denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 2 minutes, repeated 30 times. To obtain a PgapA fragment capable of ligating to the mutant dva, PCR was performed using the primer pair of SEQ ID NO:26 and SEQ ID NO:42, using the chromosome of ATCC13032 as a template. PfuUltra™ High Reliability 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 at 72°C for 1 minute, repeated 28 times, followed by polymerization at 72°C for 5 minutes to obtain the PgapA fragment. The three mutant dva fragments and PgapA fragment obtained, along with the pDZΔN2131 vector cut with the ScaI restriction enzyme, were cloned using the Gibson assembly method to obtain recombinant plasmids, which were named pDZΔN2131-PgapA-Dva(mt)-517, pDZΔN2131-PgapA-Dva(mt)-1236, and pDZΔN2131-PgapA-Dva(mt)-2543, based on the library colonies from which they originated.

[0150] Afterwards, the produced vector was transformed into the KCCM 80179 strain by electroporation, and through a secondary hybridization process, three strains were produced, into which the three mutant dva were introduced, and they were named KCCM 80179ΔN2131-PgapA-Dva(mt)-517, KCCM 80179ΔN2131-PgapA-Dva(mt)-1236, and KCCM 80179ΔN2131-PgapA-Dva(mt)-2543, respectively.

[0151] In order to confirm the L-histidine production ability of the produced KCCM 80179ΔN2131-PgapA-Dva(mt)-517, KCCM 80179ΔN2131-PgapA-Dva(mt)-1236, and KCCM 80179ΔN2131-PgapA-Dva(mt)-2543 strains, the produced KCCM 80179ΔN2131-PgapA-Dva strain was used as a control group and measured by the same method as in Example 4. The results are shown in Table 5.

[0152] [Table 5]

[0153] L-histidine production in three strains derived from KCCM 80179 with the dva mutation

[0154]

[0155] As shown in Table 5 above, the NCgl2131 deletion strain (KCCM 80179ΔN2131) has an L-histidine production ability equivalent to the level of the parent strain KCCM80179 strain, but the L-histidine production ability of the KCCM 80179ΔN2131-PgapA-Dva strain into which the gene derived from vaginal bacillus was introduced was increased by 23% and 21% or more, respectively, compared with the NCgl2131 deletion strain and the parent strain KCCM 80179 strain.

[0156] It has been confirmed that compared with the NCgl2131 deletion strain and the parent strain KCCM 80179 strain, the three strains introduced with the three mutant dva have L-histidine production capabilities equivalent to or higher than that of the KCCM 80179ΔN2131-PgapA-Dva strain, in particular, the KCCM 80179ΔN2131-PgapA-Dva(mt)-2543 strain, whose L-histidine production capacity increased by about 12% compared with the KCCM 80179ΔN2131-PgapA-Dva introduced with the wild-type dva.

[0157] Example 8. Confirmation of a mutant Dva gene mutation with increased L-histidine production capacity

[0158] To confirm the mutation introduced into Dva of the KCCM 80179ΔN2131-PgapA-Dva(mt)-2543 strain (whose effect on increasing L-histidine production was demonstrated in Example 7), the base sequence of the Dva variant was analyzed. To determine the base sequence, PCR was performed using the primer pair of SEQ ID NO:18 and SEQ ID NO:21, using gDNA from the KCCM 80179ΔN2131-PgapA-Dva(mt)-2543 strain as a template. Sequencing confirmed the base sequence of the mutant dva operon and the protein sequence of DvaF or DvaE. These sequences were then compared with the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:13. The mutation information for the amino acid sequence of the confirmed mutant DvaFE is shown in Table 6.

[0159] [Table 6]

[0160] DvaF and DvaE mutations in KCCM 80179ΔN2131-PgapA-Dva(mt)-2543 strain

[0161]

[0162] As a result of sequence confirmation, it was confirmed that since DvaF is wild type, but the R92C (arginine (Arg, R) at position 92 of SEQ ID NO: 13 is mutated to cysteine ​​(Cys, C)) mutation is introduced into DvaE, the mutant DvaFE introduced into the KCCM 80179ΔN2131-PgapA-Dva(mt)-2543 strain is a mutant exporter with increased L-histidine export ability.

[0163] These results not only demonstrated increased resistance to L-histidine concentrations above the minimum inhibitory concentration compared to the wild-type strain, but also confirmed a significant increase in L-histidine production capacity by introducing the Dermatobacterium vaginalis-derived mutant exporter. This result demonstrates that the selected Dermatobacterium vaginalis-derived mutant protein is a mutant L-histidine exporter capable of specifically exporting L-histidine.

[0164] Example 9. Production of a strain into which a gene derived from Dermatobacter vaginalis was introduced based on an L-histidine-producing strain (CA14-737) and evaluation of L-histidine production capacity

[0165] To confirm the L-histidine export ability of the Dva mutant of the protein derived from Dermatobacterium vaginalis, it was introduced into the L-histidine-producing strain CA14-737 (KCCM 12411P, Korean Patent Publication No. 10-2019-0065984) derived from the wild-type Corynebacterium glutamicum ATCC13032, into which the HisG polypeptide mutation (in which feedback limitation is resolved by L-histidine) was introduced and the L-histidine biosynthesis gene was enhanced.

[0166] To this end, the three vectors produced in Examples 2 and 7 (pDZΔN2131, pDZΔN2131-PgapA-Dva, pDZΔN2131-PgapA-Dva(mt)-2543) were respectively transformed into the CA14-737 strain by electroporation, and through a secondary hybridization process, three strains in which the NCgl2131 gene on the chromosome was deleted or the L-histidine export gene was replaced were produced, and they were named CA14-737ΔN2131, CA14-737ΔN2131-PgapA-Dva, CA14-737ΔN2131-PgapA-Dva(mt)-2543, respectively.

[0167] In order to confirm the L-histidine production ability of the produced 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 (histidine content in the culture medium) was measured. The results are shown in Table 7 below.

[0168] [Table 7]

[0169] L-histidine production by strains into which wild-type genes and variants derived from CA14-737-derived Dermatobacterium vaginalis were introduced

[0170]

[0171] As shown in Table 7, compared with the NCgl2131 deletion strain (CA14-737ΔN2131) and the parent strain CA14-737 strain, the L-histidine production of the CA14-737ΔN2131-PgapA-Dva strain into which the gene derived from vaginal bacillus was introduced increased by 60.0%, and the L-histidine production of the CA14-737ΔN2131-PgapA-Dva(mt)-2543 strain (in which the mutant dva was introduced) increased by 80.0%.

[0172] Thus, it was confirmed that all the wild-type and mutant proteins derived from Dermatobacterium vaginalis could specifically export L-histidine, and the selected mutant proteins were L-histidine exporting proteins with an export capacity even higher than that of the wild-type protein.

[0173] Example 10. Production of E. coli vectors for expressing wild-type genes and variants derived from Dermatobacterium vaginalis

[0174] To confirm the L-histidine export ability of the Dva protein and variants derived from different strains of Dermatobacterium vaginalis, vectors capable of expressing wild-type Dva and Dva variants were produced, respectively.

[0175] Each gene was cloned into the E. coli expression vector pCC1BAC (hereinafter, pBAC, Epicentre corp.) and designed to express the exogenous L-histidine export gene candidate under the promoter of the yccA gene (hereinafter, PyccA, SEQ ID NO: 43) derived from the E. coli strain MG1655.

[0176] To obtain the Dva protein and mutant gene fragments from Dermatobacterium vaginalis, PCR was performed using the primer pair of SEQ ID NO:44 and SEQ ID NO:45 with the chromosomal DNA of TCC13032ΔN2131-PgapA-Dva and ATCC13032ΔN2131-PgapA-Dva(mt)-2543 as templates to generate wild-type and mutant dva DNA fragments. PCR conditions included denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 2 minutes, repeated 30 times. To obtain the PyccA fragment, PCR was performed using the primer pair of SEQ ID NO:46 and SEQ ID NO:47 with the chromosome of MG1655 as template. As the polymerase used for the PCR reaction, PfuUltra™ high reliability DNA polymerase (Stratagene) was used, and the PCR conditions were as follows: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 28 times, followed by polymerization at 72°C for 5 minutes to obtain the PyccA fragment. The obtained wild-type and mutant dva fragments and PyccA fragments and the pBAC vector cut with the EcoRI restriction enzyme were cloned using the Gibson assembly method to obtain recombinant plasmids, which were named pBAC-PyccA-Dva and pBAC-PyccA-Dva(mt)-2543.

[0177] Example 11. Production of L-histidine-producing strains derived from Escherichia coli into which the Dva wild-type gene and variants derived from Dermatobacter vaginalis were introduced and evaluation of L-histidine production capacity

[0178] In order to confirm the L-histidine export ability of the Dva protein variant derived from Dermatophagoides vaginalis based on the L-histidine-producing strain derived from Escherichia coli, three strains were produced, in which the two vectors produced in Example 10 and the pBAC vector were introduced into the CA14-9003e strain (MG1655+hisGr hisL'_ΔΔpurR) having a previously reported genotype (purR deletion, hisL deletion, hisGr; The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants; Applied Biochemistry and Microbiology, 2013, Vol. 49, No. 2, pp. 130-135), and they were named CA14-9003e / pBAC, CA14-9003e / pBAC-PyccA-Dva, and CA14-9003e / pBAC-PyccA-Dva(mt)-2543, respectively.

[0179] In order to confirm the L-histidine production capacity of the CA14-9003e / pBAC, CA14-9003e / pBAC-PyccA-Dva, CA14-9003e / pBAC-PyccA-Dva (mt) -2543 strains produced, they were cultivated by the following method. The strain was cultured in LB solid medium (comprising 25 μg / mL chloramphenicol) for 16 hours, and then each strain was inoculated into a 250mL baffled flask containing 25mL LB liquid medium, and cultured at 37°C with 200rpm shaking for 20 hours. Then, 1mL of seed culture fluid was inoculated into a 250mL baffled flask containing 25mL of Escherichia coli production medium (Applied Biochemistry and Microbiology, 2013, Vol.49, No.2, pp.130-135), and cultured at 37°C with 200rpm shaking for 48 hours. The culture medium for cultivation is as follows:

[0180] <E. coli production medium>

[0181] Glucose 4% (w / v), yeast extract 0.2% (w / v), ammonium sulfate 1.6% (w / v), potassium phosphate dihydrate 0.06% (w / v), ferrous sulfate heptahydrate 0.0005% (w / v), magnesium sulfate pentahydrate 0.0005% (w / v), calcium carbonate, pH 7.2

[0182] After the cultivation was completed, the L-histidine production (histidine content in the culture medium) was measured by HPLC, and the results are shown in Table 8 below.

[0183] [Table 8]

[0184] L-histidine production in strains introduced with wild-type dva and variant dva derived from Dermatobacterium vaginalis derived from CA14-9003e

[0185]

[0186]

[0187] As shown in Table 8, the L-histidine production capacity of the CA14-9003e / pBAC-PyccA-Dva(mt)-2543 strain into which the mutant dva was introduced was confirmed to be approximately 17.9% higher than that of the CA14-9003e / pBAC-PyccA-Dva strain into which the wild-type dva was introduced.

[0188] These results confirmed that even when the L-histidine exporting variant derived from Dermatobacterium vaginalis was introduced into microorganisms other than Corynebacterium strains, the extracellular L-histidine export activity was significantly increased.

[0189] Based on the above description, those skilled in the art to which the present disclosure belongs will be able to understand that, without changing the technical spirit or basic features of the present invention, the present invention can be implemented in other specific forms. In this regard, the above embodiments should be understood to be illustrative and not restrictive in all aspects. The scope of the present disclosure should be interpreted as including all variations or modifications derived from the meaning and scope of the claims to be described later and concepts equivalent thereto, without being limited to the above detailed description.

Claims

1. A mutant L-histidine exporter, wherein the amino acid corresponding to the 92nd residue of the amino acid sequence of SEQ ID NO: 13 is substituted by another amino acid.

2. The protein of claim 1, wherein the another amino acid is cysteine, glutamic acid, serine, threonine, asparagine or glutamine.

3. The protein of claim 1, wherein the another amino acid is cysteine. 4 . The protein according to claim 1 , wherein the protein has 99% or higher sequence identity with the amino acid sequence of SEQ ID NO:

48. A polynucleotide encoding the protein according to any one of claims 1 to 4. A microorganism comprising the protein according to any one of claims 1 to 4 or a polynucleotide encoding the protein. The microorganism according to claim 6 , wherein the microorganism has L-histidine-producing ability.

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

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

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

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

Images