Microorganisms capable of producing L-histidine efficiently, and methods for producing histidine using these microorganisms.

Abstract

The present application relates to a microorganism with enhanced L-histidine production capacity and a method for producing histidine by using same.

Description

Microorganisms with enhanced L-histidine production capacity and histidine production method using the same

[0001] The present application relates to a microorganism with enhanced L-histidine production ability and a histidine production method using the same.

[0002]

[0003] L-histidine is one of the 20 standard amino acids. While adults do not require large amounts from a nutritional standpoint, it is classified as an essential amino acid for growing children. Furthermore, L-histidine is involved in important physiological processes, including antioxidant and immune regulation, and is used in the medical industry as a raw material for gastric ulcer treatments, circulatory system treatments, and amino acid rehydration solutions.

[0004] Histidine is particularly abundant in hemoglobin, and is primarily produced through protein hydrolysis extraction using blood meal. However, this process has drawbacks such as low efficiency and environmental pollution. While L-histidine can be produced through microbial fermentation, large-scale industrialization has not yet been achieved. This is because L-histidine biosynthesis competes with PRPP, a nucleotide synthesis precursor, and requires a complex, energy-intensive biosynthetic process and regulatory mechanisms.

[0005] The L-histidine production capacity of microorganisms used in fermentation methods has been improved in the past by mutagenesis and mutant selection methods, and by controlling the metabolism of strains through genetic modification. Recently, it has been known that histidine production using microorganisms is biosynthesized through several steps from PRPP. However, among the enzymes involved in histidine biosynthesis, the first enzyme, ATP phosphoribosyl transferase, suffers from feedback inhibition by the final product, L-histidine or its derivatives, which poses a problem for industrial mass production of L-histidine (International Patent Publication No. WO2014-029376). Due to this complex biosynthetic process and control mechanism, an approach from various perspectives related to microbial metabolism was required to produce L-histidine through microbial culture.

[0006]

[0007] The present inventors introduced the glycine transporter cycA from Corynebacterium ammoniagenes to develop a microorganism capable of utilizing glycine released from the cell, and as a result, completed a microorganism that produces L-histidine in high yield.

[0008]

[0009] The present application provides a microorganism of the genus Corynebacterium that produces L-histidine and has enhanced glycine transporter activity.

[0010] The present application provides a composition for producing L-histidine comprising the microorganism of the present application.

[0011] The present application provides a method for producing L-histidine, which comprises a step of culturing the microorganism of the present application.

[0012] The present application provides a use of a Corynebacterium genus microorganism with enhanced glycine transporter activity for producing L-histidine.

[0013]

[0014] The microorganism for producing L-histidine of the present application has excellent histidine production ability and can be utilized for efficient mass production of L-histidine.

[0015]

[0016] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0017] Furthermore, those skilled in the art will recognize or be able to ascertain, using only routine experimentation, numerous equivalents to the specific embodiments of the present application described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0018]

[0019] One aspect of the present application provides a Corynebacterium genus microorganism having enhanced glycine transporter activity for producing L-histidine.

[0020] The term "glycine transporter" in the present application includes, without limitation, any protein having the function of importing glycine into cells, and specifically may be a D-serine / D-alanine / glycine transporter. The glycine transporter may be used interchangeably with a D-serine / D-alanine / glycine transporter or a glycine import protein.

[0021] The above "D-serine / D-alanine / glycine transporter" is a protein that can be involved in the transport of serine, alanine, and glycine, and its information can be obtained by searching for the D-Serine / D-Alanine / glycine transporter sequence in a known database such as NCBI Genbank. The transporter may be specifically CycA or AapA, and more specifically, may be a CycA protein, but is not limited thereto.

[0022] The "CycA protein" of the present application refers to a protein involved in the uptake of serine, alanine, and glycine. The CycA protein is encoded by the cycA gene, and the cycA gene is known to exist in microorganisms such as Escherichia coli, Klebsialla pneumoniae, Mycobacterium bovis, Salmonella enterica, Erwinia amylovora, and Corynebacterium ammoniagenes.

[0023] For the purpose of the present application, the CycA protein of the present application may include any protein that can enhance histidine production ability. Specifically, the CycA protein may be derived from a microorganism of the genus Corynebacterium or the genus Escherichia, and more specifically, may be derived from Corynebacterium ammoniagenes, but is not limited thereto. The Corynebacterium ammoniagenes is homologous to Brevibacterium ammoniagenes and is classified into the same taxon as Corynebacterium stationis and Brevibacterium stationis (International Journal of Systematic and Evolutionary Microbiology 60: 874-879). Additionally, the above Brevibacterium ammoniagenes was renamed Corynebacterium stationaris.

[0024] Therefore, in the present application, the terms Corynebacterium ammoniagenes, Brevibacterium ammoniagenes, Corynebacterium stationarity, and Brevibacterium stationarity may be used interchangeably.

[0025] The CycA protein of the present application may comprise an amino acid sequence having SEQ ID NO: 1 or at least 70% homology or identity therewith.

[0026] Specifically, the CycA protein may comprise an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity to the amino acid sequence of SEQ ID NO: 1. In addition, it is obvious that an amino acid sequence having the homology or identity and exhibiting an effect corresponding to the protein is included within the scope of the present application even if the amino acid sequence has an amino acid sequence in which some of the sequences are deleted, modified, substituted, or added.

[0027] Additionally, probes that can be prepared from known genetic sequences, for example, polypeptides encoded by polynucleotides that hybridize under stringent conditions with a complementary sequence to all or part of the base sequence encoding the polypeptide, may also be included without limitation, polypeptides having serine, alanine and glycine uptake activity.

[0028] That is, even if the present application describes a "protein or polypeptide comprising an amino acid sequence described by a specific sequence number," a "protein or polypeptide consisting of an amino acid sequence described by a specific sequence number," or a "protein or polypeptide having an amino acid sequence described by a specific sequence number," it is obvious that a protein having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted, or added can also be used in the present application, as long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number. For example, this includes cases in which the protein has an addition of a sequence that does not alter the function of the protein at the N-terminus and / or C-terminus, a mutation that may occur naturally, a silent mutation thereof, or a conservative substitution.

[0029] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0030]

[0031] In this application, the term "polynucleotide" has a meaning that comprehensively includes DNA or RNA molecules, and nucleotides, which are the basic structural units in polynucleotides, may include not only natural nucleotides but also analogs in which sugar or base moieties are modified (see Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0032] The above polynucleotide may be a polynucleotide encoding the CycA protein of the present application or a polynucleotide encoding a polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the CycA protein of the present application. Specifically, for example, a polynucleotide encoding a protein comprising SEQ ID NO: 1 or an amino acid sequence having at least 70% homology or identity with SEQ ID NO: 1 may be a polynucleotide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity with SEQ ID NO: 2 or a polynucleotide sequence of SEQ ID NO: 2.

[0033] In addition, it is obvious that a polynucleotide that can be translated into a protein comprising SEQ ID NO: 1 or an amino acid sequence having 70% or more identity with SEQ ID NO: 1 due to codon degeneracy, or a protein having homology or identity therewith, may also be included. Alternatively, a probe that can be prepared from a known gene sequence, for example, a polynucleotide sequence that encodes a protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 1 by hybridizing under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence, may be included without limitation. The "stringent conditions" above mean conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). For example, a condition in which genes having a high degree of homology or identity, 70% or more, 80% or more, specifically 85% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and particularly specifically 99% or more, hybridize with each other, and genes having a lower degree of homology or identity than that do not hybridize, or a washing condition of a typical Southern hybridization, 60°C, 1ХSSC, 0.1% SDS, specifically 60°C, 0.1ХSSC, 0.1% SDS, more specifically 68°C, 0.1ХSSC, 0.Conditions for washing once, or more specifically, twice or three times, at a salt concentration and temperature equivalent to 1% SDS can be enumerated. Hybridization requires that two polynucleotides have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the present application may also include isolated polynucleotide fragments that are complementary to the entire sequence, as well as substantially similar polynucleotide sequences.

[0034] Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0035]

[0036] As used herein, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences or base sequences are related, and may be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably. Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can hybridize, typically at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or its full length, under moderate or high stringency conditions. Hybridization also contemplates polynucleotides that contain degenerate codons in place of codons in the polynucleotide.

[0037] Homology or identity of the above polypeptide or polynucleotide sequences can be determined, for example, using the algorithm BLAST according to the literature [see: Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] or FASTA by Pearson (see: Methods Enzymol., 183, 63, 1990). Based on the algorithm BLAST, programs called BLASTN and BLASTX have been developed (see: http: / / www.ncbi.nlm.nih.gov). Additionally, whether any amino acid or polynucleotide sequence has homology, similarity or identity can be determined by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and appropriate hybridization conditions are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0038] The term "enhancing protein activity" in this application refers to enhancing the activity of a protein in a microorganism compared to its intrinsic activity or activity before modification. This activity enhancement may encompass both introducing a foreign protein and enhancing the activity of an endogenous protein. In other words, it includes introducing a foreign protein into a microorganism that possesses the intrinsic activity of a specific protein, as well as introducing the protein into a microorganism that lacks the intrinsic activity. The term "protein introduction" refers to the modification of a specific protein so that its activity is introduced into the microorganism and expressed. This can also be expressed as enhancing the activity of the protein.

[0039] The term "intrinsic" in this application means the state that the parent strain originally had before the change in traits, when the traits of a microorganism change due to genetic mutation caused by natural or artificial factors.

[0040]

[0041] In this application, active enhancement is

[0042] 1) Increase in the copy number of the polynucleotide encoding the above protein,

[0043] 2) Modification of the expression control sequence to increase the expression of the above polynucleotide,

[0044] 3) Modification of the polynucleotide sequence on the chromosome to enhance the activity of the above protein;

[0045] 4) Introduction of a foreign polynucleotide exhibiting the activity of the above protein or a codon-optimized variant polynucleotide of the above polynucleotide, or

[0046] 5) It can be performed by a method of transforming it to be strengthened by a combination of these, but is not limited thereto.

[0047]

[0048] The above 1) increase in the copy number of the polynucleotide may be performed in a form operably linked to a vector, or by insertion into a chromosome in a host cell, but is not particularly limited thereto. Specifically, the increase may be performed by introducing a polynucleotide encoding the protein of the present invention into a host cell by being operably linked to a vector capable of replicating and functioning independently of the host, or by introducing the polynucleotide into a host cell by being operably linked to a vector capable of inserting the polynucleotide into a chromosome in the host cell, thereby increasing the copy number of the polynucleotide in the chromosome of the host cell.

[0049] Next, 2) Modification of the expression control sequence to increase the expression of the polynucleotide may be performed by, but is not particularly limited to, inducing a sequence mutation in the nucleic acid sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control sequence, or by replacing the nucleic acid sequence with a nucleic acid sequence having stronger activity. The expression control sequence may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, etc.

[0050] A strong heterologous promoter may be linked upstream of the above polynucleotide expression unit instead of the original promoter. Examples of the strong promoter include, but are not limited to, the CJ7 promoter (Korean Patent No. 0620092 and WO2006 / 065095), the lysCP1 promoter (WO2009 / 096689), the EF-Tu promoter, the groEL promoter, the aceA or aceB promoter, etc. In addition, 3) the modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but may be performed by inducing a mutation in the expression control sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof of the nucleic acid sequence to further enhance the activity of the polynucleotide sequence, or by replacing it with a polynucleotide sequence that has been improved to have stronger activity.

[0051] In addition, 4) introduction of a foreign polynucleotide sequence can be performed by introducing a foreign polynucleotide encoding a protein exhibiting the same / similar activity as the protein, or a codon-optimized mutant polynucleotide thereof, into a host cell. The foreign polynucleotide can be used without limitation in its origin or sequence as long as it exhibits the same / similar activity as the protein. In addition, the introduced foreign polynucleotide can be introduced into a host cell by optimizing its codons so that optimized transcription and translation can occur within the host cell. The introduction can be performed by a person skilled in the art appropriately selecting a known transformation method, and the introduced polynucleotide can be expressed within the host cell, thereby producing a protein and increasing its activity.

[0052] Finally, 5) the method of modifying to be strengthened by a combination of the above 1) to 4) can be performed by applying at least one of the following methods together: increasing the copy number of a polynucleotide encoding the protein, modifying an expression regulatory sequence to increase its expression, modifying the polynucleotide sequence on a chromosome, and modifying a foreign polynucleotide exhibiting the activity of the protein or a codon-optimized mutant polynucleotide thereof.

[0053]

[0054] The term "vector" in this application refers to a DNA construct containing a polynucleotide sequence encoding a target protein operably linked to suitable regulatory sequences so as to enable expression of the target protein in a suitable host. The regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. The vector, after being transformed into a suitable host cell, may replicate or function independently of the host genome, and may be integrated into the genome itself. For example, a vector for intracellular chromosomal integration may be used to replace a polynucleotide encoding a target protein in a chromosome with a mutated polynucleotide. The insertion of the polynucleotide into the chromosome may be accomplished by any method known in the art, such as, but not limited to, homologous recombination.

[0055] The vector of the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. 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, pCC1BAC vectors, etc. can be used.

[0056] The term "transformation" in this application refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell, thereby enabling expression of the protein encoded by the polynucleotide within the host cell. The transformed polynucleotide may be located within the chromosome of the host cell or outside the chromosome, as long as it can be expressed within the host cell. Furthermore, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form, as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0057] Additionally, the term "operably linked" in the present application means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target protein of the present application.

[0058] The method for transforming the vector of the present application includes any method for introducing nucleic acids into cells, and can be performed by selecting an appropriate standard technique known in the art depending on the host cell. Examples thereof include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0059]

[0060] The term "microorganism producing L-histidine" in the present application includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may refer to microorganisms that naturally have L-histidine production capabilities or microorganisms in which L-histidine production capabilities have been conferred on a parent strain that does not have L-histidine production capabilities. It may be a microorganism in which a specific mechanism has been weakened or strengthened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and may be a microorganism in which genetic mutation has occurred or activity has been enhanced for the purpose of L-histidine production.

[0061] For example, the microorganism producing L-histidine may be a microorganism with enhanced glycine transporter activity. Alternatively, the microorganism may further produce histidine by suppressing feedback restriction of histidine biosynthetic enzymes, enhancing or inhibiting enzymes involved in the histidine biosynthetic pathway, or inactivating the activity of enzymes or proteins that do not affect histidine biosynthesis, thereby facilitating metabolism in the histidine biosynthetic pathway.

[0062] Specifically, it may be a microorganism in which the activity of the CycA protein is enhanced, or in which the HisG polypeptide is additionally mutated to suppress feedback limitation of the histidine biosynthetic pathway, or in which the expression of one or more genes encoding enzymes of the histidine biosynthetic pathway including hisE, hisG, hisA, hisF, hisI, hisD, hisC, hisB, and hisN is enhanced. In addition, it may be a microorganism in which an enzyme of the histidine degradation pathway is inactivated, or the activity of a protein or enzyme in a pathway that consumes an intermediate, cofactor, or energy source in the histidine biosynthetic pathway is inactivated, or a protein that introduces histidine, which is a target product, is inactivated. For example, it may be a microorganism in which gamma-aminobutyrate permease (NCgl1108) is inactivated.

[0063] Additionally, it may be a microorganism in which the activity of a protein or enzyme not associated with microbial growth or histidine biosynthesis is inactivated. More specifically, it may be a microorganism in which the activity of formyltetrahydrofolate deformylase (PurU) or transposase (NCgl2131), which do not affect microbial growth or L-histidine biosynthesis, is weakened.

[0064] The term "inactivation of protein activity" in the present application means that the expression of an enzyme or protein is not expressed at all, or even if it is expressed, the activity is absent or reduced compared to a natural wild-type strain, a parent strain, or a strain in which the protein is not modified. In this case, the reduction is a concept that includes cases in which the activity of the protein is reduced compared to the activity of the protein originally possessed by the microorganism due to mutation of the gene encoding the protein, modification of the expression regulatory sequence, deletion of part or all of the gene, etc., and cases in which the overall activity of the protein in the cell is lower than that of the natural strain or the strain before modification due to inhibition of expression or translation of the gene encoding the protein, etc., and combinations thereof. In the present application, the inactivation can be achieved by applying various methods well known in the art. Examples of the methods include: 1) a method of deleting all or part of the gene encoding the protein; 2) Modification of an expression regulatory sequence to reduce the expression of the gene encoding the protein, 3) Modification of the gene sequence encoding the protein to eliminate or weaken the activity of the protein, 4) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to a transcript of the gene encoding the protein; 5) A method of forming a secondary structure by adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the protein to make ribosome attachment impossible; 6) A method of adding a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polynucleotide sequence of the gene encoding the protein (Reverse transcription engineering, RTE), etc., and a combination thereof can also be achieved, but the present invention is not particularly limited thereto.

[0065] However, this is only one example, and the present invention is not limited thereto, and may be a microorganism that promotes the expression of a gene encoding an enzyme of various known L-histidine biosynthetic pathways, inactivates an enzyme of a degradation pathway, or inactivates an enzyme of a pathway that consumes an intermediate, cofactor, or energy source in the histidine biosynthetic pathway. The above L-histidine-producing microorganism can be produced by applying various known methods.

[0066] For the purpose of this application, the microorganism of this application may be any microorganism that contains the above glycine transporter and can produce L-histidine.

[0067] In the present application, the above “microorganism capable of producing L-histidine” may be used interchangeably with “microorganism producing L-histidine,” “microorganism having L-histidine production ability,” and “microorganism for producing L-histidine.”

[0068]

[0069] The histidine-producing microorganism of the present invention may further enhance the activity of a glycine-decomposing protein. The "histidine-producing microorganism" and "enhanced protein activity" are as described above.

[0070] The term "glycine cleavage protein" in the present application may be used to mean a protein that is directly or indirectly involved in the glycine cleavage pathway, and may mean each protein that constitutes the "glycine cleavage system (GCV)", or a complex of the proteins, or the glycine cleavage system itself.

[0071] Specifically, the glycine degradation protein may be at least one protein selected from the group consisting of T-protein (GcvT), P-protein (GcvP), L-protein (GcvL), H-protein (GcvH) constituting the glycine degradation system, and LipB and LipA, which are coenzymes of the glycine degradation system, but is not limited thereto (John E. Cronan, Microbiology and Molecular Biology Reviews., 13 April 2016). The glycine degradation protein may be derived from a microorganism of the genus Corynebacterium, specifically, from Corynebacterium ammoniagenes, but is not limited thereto. For example, the GcvP protein may have SEQ ID NO: 26, the GcvT protein may have SEQ ID NO: 27, the GcvH may have SEQ ID NO: 28, the LipA protein may have SEQ ID NO: 29, the LipB protein may have SEQ ID NO: 30, or may have at least 70% homology or identity with each of the above sequences, but is not limited thereto. Specifically, the GcvP protein may include the amino acid sequence of SEQ ID NO: 26, or may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with the amino acid sequence of SEQ ID NO: 26. The above description of homology or identity applies equally to GcvT, GcvH, LipA, and LipB. Furthermore, it is apparent that any amino acid sequence that has the above homology or identity and exhibits an effect corresponding to the protein is within the scope of the present application, even if it has an amino acid sequence with some of the sequence deleted, modified, substituted, or added.

[0072] In addition, a polypeptide having glycine decomposition activity may also be included without limitation as a probe that can be prepared from a known genetic sequence, for example, a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary sequence to all or part of the base sequence encoding the polypeptide.

[0073] The above homology or identity is as described above.

[0074]

[0075] In the present application, the term "microorganism of the genus Corynebacterium that produces L-histidine" may refer to a microorganism that produces L-histidine and belongs to the genus Corynebacterium. The microorganism that produces L-histidine is as described above. Specifically, the microorganism of the genus Corynebacterium that has L-histidine production ability in the present application may refer to a microorganism of the genus Corynebacterium that has enhanced L-histidine production ability by having the activity of the glycine transporter of the present application enhanced or by being transformed with a vector containing a gene encoding the glycine transporter. In addition, the term "microorganism of the genus Corynebacterium" may refer to a microorganism of the genus Corynebacterium that has enhanced L-histidine production ability by having the activity of the glycine degradation protein enhanced or by being transformed with a vector containing a gene encoding the glycine degradation protein. The above "Corynebacterium genus microorganism having improved L-histidine production ability" refers to a microorganism having improved L-histidine production ability compared to a parent strain or an unmodified microorganism before transformation. The above "unmodified microorganism" refers to a naturally occurring Corynebacterium genus strain itself, a microorganism that does not contain a gene encoding the glycine transporter, or a microorganism that has not been transformed with a vector containing a gene encoding the glycine transporter.

[0076] In this application, “microorganism of the genus Corynebacterium” may include all microorganisms of the genus Corynebacterium. Specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli pollutisoli), Corynebacterium imitans), Corynebacterium testudinoris or Corynebacterium flavescens, and more specifically Corynebacterium glutamicum.

[0077] Another aspect of the present application provides a composition for producing L-histidine, comprising the microorganism for producing L-histidine of the present application.

[0078] The composition for producing L-histidine may refer to a composition capable of producing L-histidine by the L-histidine-producing microorganism of the present application. The composition comprises the L-histidine-producing microorganism, and may include, without limitation, an additional component capable of producing histidine using the strain. The additional component capable of producing histidine may further include, for example, any suitable excipient commonly used in fermentation compositions, or a component of a medium. Such excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0079] Another aspect of the present application provides a use of a Corynebacterium genus microorganism having enhanced glycine transporter activity for producing L-histidine.

[0080] “Glycine transporter”, “enhancer” or “microorganism of the genus Corynebacterium” are as described above.

[0081]

[0082] Another aspect of the present application provides a method for producing L-histidine, comprising the step of culturing the microorganism.

[0083] The medium and other culture conditions used for culturing the microorganism of the present application may be any medium used for culturing microorganisms of the genus Corynebacterium without any particular limitation. Specifically, the microorganism of the present application may be cultured in a general medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins, etc., under aerobic or anaerobic conditions while controlling temperature, pH, etc.

[0084] In the present application, the carbon source may include, but is not limited to, carbohydrates such as glucose, fructose, sucrose, maltose, etc.; alcohols such as sugar alcohols, glycerol, etc.; fatty acids such as palmitic acid, stearic acid, linoleic acid; organic acids such as pyruvic acid, lactic acid, acetic acid, citric acid; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, carbohydrates such as sterilized pretreated molasses (i.e., molasses converted to reducing sugar), etc. may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more.

[0085] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0086] The above-mentioned personnel may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc.

[0087] In addition, the medium may contain vitamins and / or appropriate precursors. The medium or precursor may be added to the culture in a batch or continuous manner, but is not limited thereto.

[0088] In the present application, the pH of the culture can be adjusted by appropriately adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture during the cultivation of microorganisms. Furthermore, foaming can be suppressed during the cultivation by using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, to maintain an aerobic state of the culture, oxygen or an oxygen-containing gas can be injected into the culture. To maintain an anaerobic or microaerobic state, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide can be injected.

[0089] The temperature of the culture may be between 25°C and 40°C, more specifically between 28°C and 37°C, but is not limited thereto. The culture period may continue until the desired amount of useful substance is produced, and may be between 1 hour and 100 hours, but is not limited thereto.

[0090]

[0091] The above L-histidine production method may include a step of recovering L-histidine from at least one material selected from the microorganism, the medium, the culture thereof, the supernatant of the culture, the extract of the culture, and the lysate of the microorganism after the culturing step.

[0092] The above recovery step can recover the target substance, L-histidine, from the culture solution using a suitable method known in the art, depending on the culturing method of the microorganism of the present application, such as a batch, continuous, or fed-batch cultivation method. For example, the recovery of the L-histidine can be accomplished using methods such as precipitation, centrifugation, filtration, chromatography, and crystallization. For example, the culture solution can be centrifuged at low speed to remove biomass, and the obtained supernatant can be separated through ion exchange chromatography, but is not limited thereto.

[0093] The above recovery step may include a purification process.

[0094]

[0095] Hereinafter, this application will be described in more detail through examples and experimental examples. However, these examples and experimental examples are intended to exemplify this application and the scope of this application is not limited to these examples and experimental examples.

[0096]

[0097] Example 1. Production of a high-performance histidine-producing artificial mutant strain.

[0098] To obtain an artificial mutant strain with high L-histidine production capacity, mutation of a microorganism was induced using the following method.

[0099] Specifically, a mutant strain was obtained using the histidine-producing strain KCCM11795P (Korean Patent Application No. 10-2016-0030092) derived from Corynebacterium glutamicum ATCC13032 and produced through NTG treatment. The KCCM11795P strain was cultured in an activation medium for 16 hours, and the activated strain was inoculated into a seed medium and cultured for 14 hours, after which 5 ml of the culture was harvested. The harvested culture was washed with 100 mM citric buffer, and NTG (N-Methyl-N'-nitro-N-nitrosoguanidine) was added to a final concentration of 200 mg / L, treated for 20 minutes, and washed with 100 mM phosphate buffer. The mortality rate was calculated by spreading the strain treated with NTG on minimal medium, and the mortality rate was 85%.

[0100] In order to obtain a resistant mutant strain to 1, 2, 4-triazole-3-alanine (TRA), which is a derivative of L-histidine, the strain treated with NTG was plated on minimal medium supplemented with 1, 2, 4-triazole-3-alanine at concentrations of 0.2 g / L, 0.5 g / L, and 1 g / L, respectively, and cultured at 30°C for 5 days. Among the mutant strains found at the three concentrations, the 1, 2, 4-triazole-3-alanine-resistant artificial mutant strain with the highest histidine production ability was obtained, and this strain was named CA14-0682.

[0101] Activation Badge

[0102] 1% meat juice, 1% polypeptone, 0.5% sodium chloride, 1% yeast extract, 2% agar, pH 7.2

[0103] <Jongbaeji>

[0104] Glucose 5%, Bactopeptone 1%, Sodium Chloride 0.25%, Yeast Extract 1%, Urea 0.4%, pH 7.2

[0105] Minimum badge

[0106] Glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium phosphate monobasic 0.1%, urea 0.1%, thiamine 0.001%, biotin 200 ㎍ / L, agar 2%, pH 7.0

[0107]

[0108] To confirm the L-histidine production ability and L-glycine production amount of the selected CA14-0682 strain, the culture was performed using the following method. Each strain was inoculated into a 250-mL corner-baffle flask containing 25 mL of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm. Then, 1 mL of the seed culture was inoculated into a 250-mL corner-baffle flask containing 25 mL of production medium and cultured at 30°C for 24 hours with shaking at 200 rpm. After completion of the culture, the L-histidine and L-glycine production amounts were measured by HPLC.

[0109]

[0110] <Production medium>

[0111] Glucose 5%, ammonium sulfate 2%, monobasic potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, CSL (corn steep liquor) 2.0%, biotin 200 ㎍ / L, calcium carbonate, pH 7.2,

[0112]

[0113] L-histidine and L-glycine production by CA14-0682 strain OD Glucose used (g / L) Histidine production (g / L) Glycine production (g / L) KCCM11795P110.21002.991.41 CA14-068250.110014.256.99

[0114] The culture results confirmed that the artificial mutant strain CA14-0682, which has resistance to high concentrations of TRA, has the ability to produce L-histidine at a yield of 15%.

[0115] The above CA14-0682 strain was safely deposited in the Korea Center for Microbiological Conservation (KCCM) and assigned the accession number KCCM 80179.

[0116]

[0117] Example 2. Construction of a vector introducing a glycine transporter (CycA(Cam)) derived from Corynebacterium ammoniagenes.

[0118] In order to insert the gene cycA (hereinafter referred to as cycA(cam), SEQ ID NO: 2) encoding the CycA protein derived from Corynebacterium ammoniagenes (hereinafter referred to as CycA(Cam), SEQ ID NO: 1) into the Corynebacterium glutamicum chromosome, purU was used as the insertion site in Corynebacterium glutamicum (Journal of Biotechnology 104, 5-25 Jorn Kalinowski et al, 2003). To construct the purU deletion and target gene insertion vectors, PCR was performed using the chromosome of ATCC13032 as a template and the primer pairs of SEQ ID NO: 3 and SEQ ID NO: 4, and SEQ ID NO: 5 and SEQ ID NO: 6, respectively. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were as follows: denaturation 95°C, 30 sec; annealing 55°C, 30 sec; And after repeating the polymerization reaction at 72℃ for 2 minutes 28 times, the polymerization reaction was performed at 72℃ for 5 minutes. As a result, DNA fragments of 1606 bp del-purU (SEQ ID NO: 7) and 1625 bp del-purU (SEQ ID NO: 8) were obtained, respectively. The obtained DNA products were purified using QIAGEN's PCR Purification kit and then cloned using pDZ (Korean Patent No. 10-0924065) vector and TaKaRa's Infusion Cloning Kit, thereby constructing the vector pDZΔpurU for purU deletion and target gene insertion.

[0119] In order to obtain a promoter-linked cycA(Cam) DNA fragment (hereinafter referred to as Pn-cycA(Cam)), PCR was performed using the primers of SEQ ID NO: 9 and SEQ ID NO: 10 using the chromosome of Corynebacterium ammoniagenes ATCC 6872 as a template. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were as follows: denaturation at 95°C for 30 sec; annealing at 55°C for 30 sec; and polymerization at 72°C for 90 sec, repeated 28 times, followed by polymerization at 72°C for 5 min. As a result, a 1970-bp Pn-cycA(cam) DNA fragment was obtained, and this amplified product was purified using a QIAGEN PCR Purification kit and used as an insert DNA fragment for vector construction (SEQ ID NO: 11). The obtained DNA product was purified using QIAGEN's PCR Purification kit and then cloned using the above-mentioned pDZΔpurU vector and TaKaRa's Infusion Cloning Kit to produce the cycA(Cam) introduction vector pDZΔpurU::Pn-cycA(Cam).

[0120]

[0121] Example 3. Production of a strain introducing a glycine transporter derived from CA14-0682 strain and evaluation of histidine production ability.

[0122] The vector pDZΔpurU::Pn-cycA(cam) produced in the above Example 2 was transformed into the CA14-0682 strain, and through a second crossing process, a strain in which the purU gene on the chromosome was substituted in the form of Pn-cycA(cam) was produced, and this was named CA14-0682ΔpurU::Pn-cycA(cam).

[0123] To confirm the L-histidine production ability and L-glycine production amount of the produced CA14-0682ΔpurU strain and CA14-0682ΔpurU::Pn-cycA(Cam) strain, they were cultured using the method performed in Example 1.

[0124] L-histidine and L-glycine production of cycA(cam)-introduced strains derived from CA14-0682 OD Glucose used (g / L) Histidine production (g / L) Glycine production (g / L) CA14-0682 50.2100 14.857.41 CA14-0682ΔpurU 50.1100 14.887.42 CA14-0682ΔpurU::Pn-cycA(Cam) 49.7100 15.496.51

[0125] The evaluation results showed that the parent strain CA14-0682 had the ability to produce 14.85 g / L of L-histidine and 7.41 g / L of L-glycine, and the purU deletion strain had the same level of L-histidine production as the parent strain, whereas the CA14-0682ΔpurU::Pn-cycA(Cam) strain had a 4.3% increase in L-histidine production and a 13.8% decrease in L-glycine production. This confirmed that introducing a glycine import gene into cells by introducing extracellular L-glycine increases L-histidine production.

[0126]

[0127] Example 4. Construction of a CycA (Cam) overexpression recombinant vector

[0128] To more strongly express intracellular cycA(Cam), a cycA(Cam) overexpression recombinant vector was constructed. The known promoter pcj7 derived from a Corynebacterium spp. (Korean Patent No. 10-0620092) and the promoter of the gene glyA encoding the known serine hydroxymethyltransferase (hereinafter referred to as PglyA) were used.

[0129] To obtain a pcj7 promoter DNA fragment, PCR was performed using p117-cj7-gfp, which contains pcj7, as a template. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR reaction was performed using primers of SEQ ID NOs: 12 and 13. The reaction was performed under the following conditions: denaturation at 95°C for 30 s; annealing at 55°C for 30 s; and polymerization at 72°C for 30 s, repeated 28 times, followed by polymerization at 72°C for 1 min. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain a 350-bp pcj7 fragment.

[0130] To obtain a cycA(Cam) DNA fragment containing a portion of the pcj7 sequence at the 5' end, PCR was performed using the Corynebacterium ammoniagenes ATCC 6872 chromosome as a template. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR reaction was performed using primers of SEQ ID NO: 14 and SEQ ID NO: 10. The PCR reaction was performed at 95°C for 30 s, annealing at 55°C for 30 s, and polymerization at 72°C for 30 s for 28 cycles, followed by polymerization at 72°C for 1 min. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain a cycA(Cam) fragment containing a portion of the pcj7 sequence at the 5' end and measuring 1647 bp.

[0131] Using the pcj7 fragment and cycA (Cam) fragment obtained above as templates, a fusion (sewing) PCR was performed using primers of SEQ ID NO: 12 and SEQ ID NO: 10. The PCR reaction was performed with 28 cycles of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, followed by polymerization at 72°C for 5 minutes. As a result, a 1964-bp pcj7-cycA (Cam) gene fragment was obtained, and this amplified product was purified using a QIAGEN PCR Purification kit and used as an insert DNA fragment for vector construction (SEQ ID NO: 15). The obtained DNA product was purified using QIAGEN's PCR Purification kit, and then cloned using the above-mentioned pDZΔpurU vector and TaKaRa's Infusion Cloning Kit to produce a vector pDZΔpurU::pcj7-cycA(Cam) that replaces the purU gene with the pcj7-cycA(Cam) gene.

[0132] In addition, PCR was performed using the ATCC13032 chromosome as a template to obtain a PglyA DNA fragment. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR reaction was performed using primers of SEQ ID NO: 16 and SEQ ID NO: 17. The reaction was performed at 95°C for 30 s, annealing at 55°C for 30 s, and polymerization at 72°C for 30 s for 28 cycles, followed by polymerization at 72°C for 1 minute. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain a 340-bp PglyA fragment.

[0133] To obtain a cycA(Cam) DNA fragment containing a portion of the PglyA sequence at the 5' end, PCR was performed using the Corynebacterium ammoniagenes ATCC 6872 chromosome as a template. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR reaction was performed using primers of SEQ ID NO: 18 and SEQ ID NO: 10. The PCR reaction was performed at 95°C for 30 s, annealing at 55°C for 30 s, and polymerization at 72°C for 30 s for 28 cycles, followed by polymerization at 72°C for 1 min. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain a cycA(Cam) fragment containing a portion of the PglyA sequence at the 5' end and measuring 1647 bp.

[0134] Using the PglyA fragment and cycA(Cam) fragment obtained above as templates, fusion (sewing) PCR was performed using primers of SEQ ID NO: 16 and SEQ ID NO: 10. The PCR reaction was performed with 28 cycles of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, followed by polymerization at 72°C for 5 minutes. As a result, a 1963-bp PglyA-cycA(Cam) gene fragment was obtained, and this amplification product was purified using a QIAGEN PCR Purification kit and used as an insert DNA fragment for vector construction (SEQ ID NO: 19). The obtained DNA product was purified using QIAGEN's PCR Purification kit, and then cloned using the above-mentioned pDZΔpurU vector and TaKaRa's Infusion Cloning Kit to produce a vector pDZΔpurU::PglyA-cycA(Cam) that replaces the purU gene with the PglyA-cycA(Cam) gene.

[0135]

[0136] Example 5. Production of a vector introducing the E. coli-derived glycine transporter (CycA(Eco))

[0137] Meanwhile, in order to compare its activity with that of the CycA protein derived from Corynebacterium ammoniagenes, a vector was constructed to introduce the gene cycA (hereinafter, cycA(Eco), SEQ ID NO: 21) encoding the CycA protein derived from Escherichia coli K-12 (hereinafter, CycA(Eco), SEQ ID NO: 20) (Microbiology, 141(Pt 1); 133-40, 1995) by linking it to pcj7 so that it can be operably linked.

[0138] To obtain a pcj7 promoter DNA fragment, PCR was performed using p117-cj7-gfp, which contains pcj7, as a template. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR reaction was performed using primers of SEQ ID NO: 12 and SEQ ID NO: 22. The PCR reaction was performed at 95°C for 30 s, annealing at 55°C for 30 s, and polymerization at 72°C for 30 s for 28 cycles, followed by polymerization at 72°C for 1 min. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain a 350-bp pcj7 fragment.

[0139] To obtain a cycA (Eco) gene fragment containing a portion of the pcj7 sequence at 5', PCR was performed using the primers of SEQ ID NO: 23 and SEQ ID NO: 24 using the E. coli K-12 W3110 chromosome as a template. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were as follows: denaturation at 95°C for 30 sec; annealing at 55°C for 30 sec; and polymerization at 72°C for 1 min, repeated 28 times, followed by polymerization at 72°C for 5 min. As a result, a 1659 bp cycA (Eco) gene fragment was obtained, and this amplified product was purified using a QIAGEN PCR Purification kit and used as an insert DNA fragment for vector construction.

[0140] Using the pcj7 fragment and cycA(Eco) fragment as templates, fusion (sewing) PCR was performed using primers of SEQ ID NO: 12 and SEQ ID NO: 24. The PCR reaction was performed with 28 cycles of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 90 seconds, followed by polymerization at 72°C for 5 minutes. As a result, a 1985-bp pcj7-cycA(Eco) gene fragment was obtained (SEQ ID NO: 25). This amplified product was purified using a QIAGEN PCR Purification kit and then cloned into the pDZΔpurU vector using a TaKaRa Infusion Cloning Kit according to the provided manual, thereby constructing a vector pDZΔpurU::pcj7-cycA(Eco) that substitutes the purU gene with the pcj7-cycA(Eco) gene.

[0141]

[0142] Example 6. Production of cycA(Cam) or cycA(Eco) overexpressing strains derived from CA14-0682 strain and comparison of histidine production capacity.

[0143] To produce cycA(Cam) or cycA(Eco) overexpressing strains using CA14-0682 strain as a parent strain, four types of vectors (pDZΔpurU, pDZΔpurU::pcj7-cycA(Cam), pDZΔpurU::PglyA-cycA(Cam), pDZΔpurU::pcj7-cycA(Eco)) were each transformed into CA14-0682 strain by electroporation, and through a secondary crossing process, strains in which purU on the chromosome was deleted and substituted in the form of pcj7-cycA(Cam) or PglyA-cycA(Cam) or pcj7-cycA(Eco) were obtained. Through the above process, four strains (CA14-0682ΔpurU, CA14-0682ΔpurU::pcj7-cycA(Cam), CA14-0682ΔpurU::PglyA-cycA(Cam), CA14-0682ΔpurU::pcj7-cycA(Eco)) were produced.

[0144] To confirm the L-histidine production ability and L-glycine production amount of the four strains produced, they were cultured using the same method as Example 1.

[0145]

[0146] L-histidine and L-glycine production of cycA-introduced strains derived from CA14-0682 OD Glucose used (g / L) Histidine production (g / L) Glycine production (g / L) CA14-0682 50.3100 15.117.46 CA14-0682ΔpurU 50.5100 15.057.42 CA14-0682ΔpurU::pcj7_cycA(Cam) 40.1100 16.185.99 CA14-0682ΔpurU::PglyA_cycA(Cam) 44.7100 16.145.89 CA14-0682ΔpurU::pcj7_cycA(Eco) 51.3100 15.017.25

[0147]

[0148] The evaluation results showed that the CA14-0682ΔpurU::pcj7-cycA(Eco) strain, into which cycA from E. coli was introduced, had almost no Gly uptake ability and histidine production ability that was lower than the parent strain. On the other hand, the CA14-0682ΔpurU::pcj7-cycA(Cam) and CA14-0682ΔpurU::PglyA_cycA(Cam) strains, into which cycA from Corynebacterium ammoniagenes was fortified, showed a decrease in Gly production ability and an increase in histidine production ability by 7.1% and 6.8%, respectively, compared to the parent strain. Through this, it was confirmed that cycA derived from Corynebacterium ammoniagenes introduced into Corynebacterium glutamicum had a higher glycine uptake ability than cycA derived from E. coli and showed a greater effect on increasing histidine production through the introduced glycine. In addition, it was confirmed that when cycA(Cam) was introduced and expressed through the glyA promoter, it was more advantageous in securing cell mass. The CA14-0682ΔpurU::PglyA-cycA(Cam) strain was named CA14-0682-cycA(Cam) strain.

[0149]

[0150] Example 7. Production of a vector for introducing a glycine degradation system derived from Corynebacterium ammoniagenes.

[0151] Since it was previously confirmed that the activity of the glycine transporter affects the increase in histidine productivity, we aimed to confirm the histidine productivity when the intracellular availability of glycine introduced into the cell is further increased. Specifically, the Glycine Cleavage System (GCV system) was introduced. Among the six proteins that constitute the GCV system, only the genes encoding L-protein, LipB, and LipA are known in the Corynebacterium glutamicum strain, while the genes encoding the remaining three proteins are unknown. Therefore, in order to introduce the GCV system derived from Corynebacterium ammoniagenes, a vector was constructed to introduce genes encoding P-protein (SEQ ID NO: 26), T-protein (SEQ ID NO: 27), H-protein (SEQ ID NO: 28), LipA (SEQ ID NO: 29), and LipB (SEQ ID NO: 30) (gcvP (SEQ ID NO: 31), gcvT (SEQ ID NO: 32), gcvH (SEQ ID NO: 33), lipA (SEQ ID NO: 34), and lipB (SEQ ID NO: 35)). The above genes form two pairs of operons (gcvP-gcvT, gcvH-lipB-lipA) within the Corynebacterium ammoniagenes chromosome (hereinafter, gcvPT, gcvH-lipBA). To introduce the GCV system, the NCgl2131 gene, which encodes the transposon of Corynebacterium glutamicum, was used as an insertion site (Journal of Biotechnology 104, 5-25 Jorn Kalinowski et al, 2003). To replace the NCgl2131 gene with gcv system genes, NCgl2131 deletion and target gene insertion vectors were constructed. To construct the vectors, PCR was performed using the ATCC13032 chromosome as a template and the primer pairs of SEQ ID NOs: 36 and 37, and 38 and 39, respectively.PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were as follows: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 2 minutes, repeated 28 times, followed by polymerization at 72°C for 5 minutes. As a result, DNA fragments of 531 bp del-N2131L (SEQ ID NO: 40) and 555 bp del-N2131R (SEQ ID NO: 41) were obtained, respectively. The obtained DNA products were purified using a QIAGEN PCR Purification kit and cloned using the pDZ vector and the TaKaRa Infusion Cloning Kit, thereby constructing the vector pDZΔN2131 for NCgl2131 gene deletion and target gene insertion.

[0152] To obtain a promoter-linked gcvPT gene fragment (hereinafter, Pn_gcvPT(cam)), PCR was performed using the primers of SEQ ID NO: 42 and SEQ ID NO: 43 using the Corynebacterium ammoniagenes ATCC 6872 chromosome as a template. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were 28 cycles of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 5 minutes, followed by polymerization at 72°C for 7 minutes. As a result, a 4499-bp Pn_gcvPT(Cam) gene fragment including the promoter was obtained, and this amplified product was purified using a QIAGEN PCR Purification kit and used as an insert DNA fragment for vector construction (SEQ ID NO: 44).

[0153] To obtain a promoter-linked gcvH-lipBA gene fragment (hereinafter referred to as Pn_gcvH-lipBA(Cam)), PCR was performed using the primers of SEQ ID NO: 45 and SEQ ID NO: 46 using the chromosome of Corynebacterium ammoniagenes ATCC 6872 as a template. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were 28 cycles of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, followed by polymerization at 72°C for 7 minutes. As a result, a 3053 bp Pn_gcvH-lipBA (Cam) gene fragment including the promoter was obtained, and this amplified product was purified using QIAGEN's PCR Purification kit and used as an insert DNA fragment for vector production (SEQ ID NO: 47).

[0154] Sewing PCR was performed using the above-obtained Pn_gcvPT(Cam) fragment and Pn_gcvH-lipBA(Cam) fragment as templates and primers of SEQ ID NO: 42 and SEQ ID NO: 46. The PCR reaction was performed with 28 cycles of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 10 minutes, followed by polymerization at 72°C for 12 minutes. As a result, a 8259 bp Pn_gcvPT(Cam)_Pn-gcvH-lipBA(Cam) gene fragment was obtained, and the amplified product was purified using a PCR Purification kit from QIAGEN, and cloned into the pDZΔN2131 vector using an Infusion Cloning Kit from TaKaRa, thereby constructing a vector pDZΔN2131::GCV(Cam) that substitutes the NCgl2131 gene with the Pn_gcvPT(Cam)-Pn_gcvH-lipBA(Cam) gene.

[0155]

[0156] Example 8. Production of strains introducing glycine degradation system and glycine transporter derived from CA14-0682 strain and evaluation of histidine production ability.

[0157] The vectors pDZΔN2131 and pDZΔN2131::GCV(Cam) constructed in Example 7 were transformed into the CA14-0682 strain and the CA14-0682-cycA(Cam) strain, and through a secondary crossing process, a NCgl2131 gene deletion strain (CA14-0682-cycA(Cam)ΔN2131), a strain introducing only the glycine degradation system, and two strains introducing both the glycine degradation system and the glycine transporter (CA14-0682ΔN2131::GCV(Cam), CA14-0682-cycA(Cam)ΔN2131::GCV(Cam)) were constructed. To confirm the L-histidine production ability and L-glycine production amount of the produced CA14-0682-cycA(Cam)ΔN2131, CA14-0682ΔN2131::GCV(Cam) strains, and CA14-0682-cycA(Cam)ΔN2131::GCV(Cam) strains, they were cultured using the method performed in Example 1.

[0158]

[0159] L-histidine and L-glycine production of strains introduced with cycA(cam) and glycine degradation system derived from CA14-0682 OD Glucose used (g / L) Histidine production (g / L) Glycine production (g / L) CA14-0682 53.6 100 15.0 5 7.47 CA14-0682- cycA(Cam) ΔN2131 45.1 100 16.1 9 6.68 CA14-0682 ΔN2131::GCV(Cam) 48.9 100 16.5 24.72 CA14-0682-cycA(Cam) ΔN2131::GCV(Cam) 42.3 100 17.1 12.31

[0160]

[0161] As a result of the evaluation, in the case of the CA14-0682-cycA(Cam)ΔN2131 strain, which only introduced the glycine importer cycA(Cam), histidine production increased by 7.6% and glycine production decreased by 10.6% compared to the CA14-0682 strain, which was confirmed to be at a level equivalent to the results of the CA14-0682ΔpurU::PglyA_cycA(Cam) (named CA14-0682-cycA(Cam)) strain in Table 3. In the case of the CA14-0682ΔN2131::GCV(Cam) strain, which introduced the glycine degradation system, histidine production increased by 9.8% and glycine production decreased by 36.8% compared to the CA14-0682 strain. On the other hand, in the CA14-0682-cycA(Cam)ΔN2131::GCV(Cam) strain, in which GCV was additionally introduced into the CA14-0682-cycA(Cam)ΔN2131 strain, histidine production increased by 13.7% and glycine production decreased by 69.1% compared to the parent strain. Therefore, although the introduction of only the glycine importer or glycine degradation gene is effective in increasing histidine productivity and decreasing glycine production, it was confirmed that when introduced together with the glycine degradation system, the histidine production ability increased even more as the intracellularly produced glycine was decomposed.

[0162]

[0163] Example 9. Production of an L-histidine-producing strain derived from wild-type Corynebacterium glutamicum

[0164] Next, to confirm the effect of introducing CycA and the GCV system in a wild-type Corynebacterium glutamicum strain, an L-histidine producing strain was first developed from the wild-type Corynebacterium glutamicum ATCC13032 strain.

[0165]

[0166] Example 9-1: Introduction of HisG polypeptide mutations

[0167] First, in order to resolve the feedback inhibition of HisG polypeptide, the first enzyme in the L-histidine biosynthetic pathway, the 233rd and 235th amino acids from the N-terminus of HisG were simultaneously substituted from glycine to histidine (hereinafter, G233H mutation) and from threonine to glutamine (hereinafter, T235Q), respectively (SEQ ID NO: 48) (ACS Synth. Biol., 2014, 3 (1), pp 21-29).

[0168] Specifically, in order to construct a vector for inserting hisG polypeptide mutation, the gene fragment of the upstream region of residues 233 and 235 of hisG polypeptide (hereinafter, G233H, T235Q-5') was obtained by PCR using the primers of SEQ ID NO: 49 and SEQ ID NO: 50 using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, and the gene fragment of the downstream region of residues 233 and 235 of hisG polypeptide (hereinafter, G233H, T235Q-3') was obtained by PCR using the primers of SEQ ID NO: 51 and SEQ ID NO: 52. The polymerase was Solg TM Pfu-X DNA polymerase was used, and the PCR amplification conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, followed by polymerization at 72°C for 5 minutes.

[0169] The amplified G233H, T235Q-5' fragment and G233H, T235Q-3' fragment were cloned using pDZ and Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method to construct the hisG polypeptide mutation introduction vector pDZ-hisG(G233H, T235Q).

[0170] The constructed pDZ-hisG (G233H, T235Q) vector was transformed into the wild-type Corynebacterium glutamicum ATCC13032 strain by electroporation, and then a second crossover process was performed to obtain a strain in which amino acids 233 and 235 of the HisG polypeptide on the chromosome were replaced from glycine to histidine and from threonine to glutamine, respectively. The genetic manipulation was confirmed through PCR and sequencing using SEQ ID NOs: 53 and 54, which can amplify the external regions of the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and was named CA14-0011.

[0171]

[0172] Example 9-2: Strengthening the histidine biosynthetic pathway

[0173] Next, to strengthen the L-histidine biosynthetic pathway, the biosynthetic genes separated into a total of four operons were introduced as clusters with substituted promoters. Specifically, the biosynthetic genes separated into a total of four operons (hisE-hisG, hisA-impA-hisF-hisI, hisD-hisC-hisB, cg0911-hisN) were operably linked to three previously known synthetic promoters (lysCP1 (Korean Patent No. 10-0930203), pcj7, or SPL13 (Korean Patent No. 10-1783170 B1)) or the gapA gene promoter, and each operon was clustered and introduced all at once. The insertion site used was the Ncgl1108 gene encoding gamma-aminobutyrate permease (Microb Biotechnol. 2014 Jan;7(1):5-25).

[0174] The specific experimental method is as follows. To construct the NCgl1108 gene deletion vector, the chromosomal DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and the NCgl1108 upstream region (hereinafter, N1108-5') was obtained by PCR using the primers of SEQ ID NO: 55 and SEQ ID NO: 56, and the Ncgl1108 downstream region (hereinafter, N1108-3') was obtained by PCR using the primers of SEQ ID NO: 57 and SEQ ID NO: 58. The polymerase was Solg. TM Pfu-X DNA polymerase was used, and the PCR amplification conditions were as follows: denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, followed by polymerization at 72°C for 5 minutes. The amplified N1108-5' fragment and N1108-3' fragment were cloned using pDZ and the Gibson assembly method to construct the NCgl1108 deletion vector pDZΔN1108 vector.

[0175] The constructed pDZ-ΔNCgl1108 vector was transformed into the CA14-0011 strain by electroporation, and a second crossover process was performed to obtain a strain in which the NCgl1108 gene was disrupted on the chromosome. The genetic manipulation was confirmed through PCR using SEQ ID NOs: 59 and 60, which can amplify the external regions upstream and downstream of the disrupted homologous recombination region, and sequencing, and this strain was named CA14-0736.

[0176] To strengthen the histidine biosynthetic cluster, we sought to secure four operon gene groups and promoter regions to be replaced. The lysCP1 promoter fragment and hisE-hisG fragment, the gapA promoter fragment and hisA-impA-hisF-hisI fragment, the SPL13 fragment and hisD-hisC-hisB fragment, and the pcj7 fragment and cg0911-hisN fragment were obtained.

[0177] To obtain the lysCP1 DNA fragment, PCR was performed using the chromosome of the KCCM10919P strain (Korean Patent No. 10-0930203) as a template. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR reaction was performed using primers of SEQ ID NO: 61 and SEQ ID NO: 62. The reaction was performed at 95°C for 30 s, annealing at 55°C for 30 s, and polymerization at 72°C for 30 s for 28 cycles, followed by polymerization at 72°C for 1 min. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain the lysCP1 fragment.

[0178] To obtain the hisE-hisG gene fragment, PCR was performed using the chromosome of the CA14-0011 strain as a template. The PCR reaction was performed using primers of SEQ ID NO: 63 and SEQ ID NO: 64. The reaction was performed 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. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain the hisE-hisG fragment.

[0179] To obtain a promoter DNA fragment of the gapA gene from Corynebacterium glutamicum (hereinafter referred to as PgapA), PCR was performed using the Corynebacterium glutamicum ATCC13032 chromosome as a template. The PCR reaction was performed using primers of SEQ ID NO: 65 and SEQ ID NO: 66. The reaction was performed under the following conditions: 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. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain the PgapA fragment.

[0180] To obtain the hisA-impA-hisF-hisI gene fragment, PCR was performed using the chromosome of the CA14-0011 strain as a template. The PCR reaction was performed using primers of SEQ ID NO: 67 and SEQ ID NO: 68. The PCR reaction was performed 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. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain the hisA-impA-hisF-hisI fragment.

[0181] To obtain the SPL13 DNA fragment, PCR was performed using SPL13 DNA as a template. The PCR reaction was performed using primers of SEQ ID NO: 69 and SEQ ID NO: 70. The reaction consisted of 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by a final extension at 72°C for 5 minutes. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain the SPL13 DNA fragment.

[0182] To obtain a pcj7 promoter DNA fragment, PCR was performed using p117-cj7-gfp, which contains pcj7, as a template. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR reaction was performed using primers of SEQ ID NO: 71 and SEQ ID NO: 72. The PCR reaction was performed at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s for 28 cycles, followed by polymerization at 72°C for 1 min. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain the pcj7 fragment.

[0183] To obtain the hisD-hisC-hisB gene fragment, PCR was performed using the chromosome of the CA14-0011 strain as a template. The PCR reaction was performed using primers of SEQ ID NO: 73 and SEQ ID NO: 74. The PCR reaction was performed at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 5 minutes, repeated 28 times, followed by polymerization at 72°C for 5 minutes. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain the hisD-hisC-hisB gene fragment.

[0184] To obtain the cg0911-hisN gene fragment, PCR was performed using the CA14-0011 strain chromosome as a template. The PCR reaction was performed using primers of SEQ ID NOs: 75 and 76. The reaction was performed at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 5 minutes, repeating 28 times, followed by polymerization at 72°C for 5 minutes. The amplified PCR product was purified using a QIAGEN PCR Purification kit to obtain the cg0911-hisN gene fragment.

[0185] The obtained lysCP1 DNA fragment, hisE-hisG DNA fragment, PgapA DNA fragment, hisA-impA-hisF-hisI DNA fragment, SPL13 DNA fragment, hisD-hisC-hisB DNA fragment, pcj7 DNA fragment, and cg0911-hisN DNA fragment were cloned into the pDZ-ΔNcgl1108 vector using the Gibson assembly method to construct the L-histidine biosynthesis enhanced cluster introduction vector pDZ-ΔNcgl1108::lysCP1_hisEG-PgapA_hisA-impA-hisFI-SPL13_HisDCB-pcj7_cg0911-hisN.

[0186] The constructed pDZ-ΔNcgl1108::lysCP1_hisEG-PgapA_hisA-impA-hisFI-SPL13_hisDCB-pcj7_cg0911-hisN vector was transformed into the CA14-0011 strain by electroporation, and a strain with the biosynthetic gene inserted on the chromosome was obtained through a second crossing process. The genetic manipulation was confirmed through PCR using SEQ ID NOs: 59 and 60, which can amplify the external regions of the upstream and downstream regions of the homologous recombination region where the gene was inserted, and genome sequencing, and this was named CA14-0737.

[0187] The above CA14-0737 strain was deposited internationally with the Korea Center for Microbiological Cultures (KCCM), an international depository under the Budapest Treaty, on November 27, 2018, and was assigned the deposit number KCCM12411P.

[0188]

[0189] Example 10. Production of strains introducing glycine transporter and glycine degradation system derived from CA14-0737 strain.

[0190] The four types of vectors prepared above (pDZΔpurU, pDZΔpurU::PglyA-cycA(Cam), pDZΔpurU::pcj7-cycA(Cam), pDZΔpurU::pcj7-cycA(Eco)) were transformed into CA14-0737 strain, and through the second crossing process, purU gene deletion strain, cycA(Cam) introduction strain, and cycA(Eco) introduction strain were prepared, and CA14-0737ΔpurU, CA14-0737ΔpurU::PglyA-cycA(Cam), CA14-0737ΔpurU::pcj7-cycA(Eco) were prepared. The prepared CA14-0737ΔpurU, To confirm the L-histidine production ability and L-glycine production amount of CA14-0737ΔpurU::PglyA-cycA(Cam), CA14-0737ΔpurU::pcj7-cycA(Cam), and CA14-0737ΔpurU::pcj7-cycA(Eco) strains, they were cultured using the method performed in Example 1.

[0191]

[0192] L-histidine and L-glycine production of cycA-introduced strains derived from CA14-0737 OD Glucose used (g / L) Histidine production (g / L) Glycine production (g / L) CA14-0737 88.41004.112.21 CA14-0737ΔpurU 87.91004.20 2.24 CA14-0737ΔpurU::PglyA-cycA(Cam) 87.41004.93 1.90 CA14-0737ΔpurU::pcj7-cycA(Cam) 84.11004.97 1.95 CA14-0737ΔpurU::pcj7-cycA(Eco) 88.91004.29 2.20

[0193]

[0194] The evaluation results showed that the CA14-0737ΔpurU::pcj7-cycA(Eco) strain, into which cycA from E. coli was introduced, had almost no Gly influx and showed the same histidine production ability as the parent strain. On the other hand, in the CA14-0737ΔpurU::pcj7-cycA(Cam) strain, into which cycA from Corynebacterium ammoniagenes was introduced, histidine production ability increased by 20.9% and glycine production amount decreased by 11.8% compared to the parent strain. The strain expressing cycA(Cam) through the glyA promoter also showed a 20% increase in histidine production ability and a 14% decrease in glycine production. Through this, it was confirmed that cycA derived from Corynebacterium ammoniagenes introduced into Corynebacterium glutamicum had a higher glycine uptake ability than cycA derived from E. coli and showed a greater effect in increasing histidine production through the introduced glycine. Among these, the CA14-0737ΔpurU::PglyA-cycA(Cam) strain was named CA14-0737-cycA(Cam).

[0195] The vectors pDZΔN2131 and pDZΔN2131::GCV(Cam) constructed in Example 7 were transformed into the CA14-0737 strain and the CA14-0737-cycA(Cam) strain, and through a secondary crossing process, a NCgl2131 gene deletion strain (CA14-0737-cycA(Cam)ΔN2131), a strain introducing only the glycine degradation system, and two strains introducing both the glycine degradation system and the glycine transporter (CA14-0737ΔN2131::GCV(Cam), CA14-0737-cycA(Cam)ΔN2131::GCV(Cam)) were constructed. To confirm the L-histidine production ability and L-glycine production amount of the produced CA14-0737-cycA(Cam)ΔN2131 strain, CA14-0737ΔN2131::GCV(Cam) strain, and CA14-0737-cycA(Cam)ΔN2131::GCV(Cam) strain, they were cultured using the method performed in Example 1.

[0196]

[0197] L-histidine and L-glycine production by strains introduced with cycA(cam) and glycine degradation system derived from CA14-0737 OD Glucose used (g / L) Histidine production (g / L) Glycine production (g / L) CA14-0737 88.11004.152.17 CA14-0737-cycA(Cam)ΔN2131 75.11004.891.48 CA14-0737ΔN2131::GCV(Cam) 78.21005.420.94 CA14-0737-cycA(Cam)ΔN2131::GCV(Cam) 71.31005.970.46

[0198]

[0199] As a result of the evaluation, in the case of the CA14-0737-cycA(Cam)ΔN2131 strain, into which only the glycine transporter cycA(Cam) was introduced, histidine production increased by 17.8% and glycine production decreased by 13% compared to the parent strain, and in the CA14-0737-cycA(Cam)ΔN2131::GCV(Cam) strain, into which the glycine transporter cycA(cam) and GCV were introduced simultaneously, histidine production increased by 43.9% and glycine production decreased by 78.8% compared to the parent strain. In the case of the CA14-0737ΔN2131::GCV(Cam) strain with the glycine degradation system introduced, histidine production increased by 30.6% and glycine production decreased by 56.7% compared to the parent strain, but glycine still accumulated in the culture medium, and histidine productivity was also confirmed to be lower than that of the strain introduced simultaneously with cycA(Cam). Therefore, although the introduction of only the glycine transporter or the glycine degradation system is effective in increasing histidine productivity and decreasing glycine production, it was confirmed that when the glycine transporter and glycine degradation system were introduced together, the histidine production ability increased even more as the glycine produced within the cell was decomposed. The CA14-0737-cycA(Cam) strain was named CA14-0777, and the CA14-0737-cycA(Cam)ΔN2131::GCV(Cam) strain was named CA14-0809. The two strains were deposited internationally with the Korea Center for Microbiological Cultures (KCCM), an international depository under the Budapest Treaty, on April 15, 2019, and were assigned the deposit numbers KCCM12488P and KCCM12489P, respectively.

[0200]

[0201] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

[0202]

[0203]

[0204]

Claims

DEPCT651. Microorganisms of the genus Corynebacterium for the production of L-histidine; microorganisms with enhanced glycine transporter activity.

2. Claim 1 microorganisms, where the glycine transporter is derived from Corynebacteriumammoniagenes.

3. Claim 1 microorganisms, where the glycine transporter protein is CycA.

4. Claim 1 microorganisms, where the glycine transporter consists of the amino acid sequence SEQIDNO:1 or sequence...

5. Microorganism of claim 1, where the activity of the glycine decomposition protein is further enhanced.

6. Microorganism of claim 1, where the glycine decomposition protein is one or more selected proteins from a group consisting of GcvP, GcvT, GcvH, LipB, and LipA.

7. Microorganism of claim 6, where the glycine decomposition protein is derived from Corynebacteriumammoniagenes. 8.The microorganism of claim 6, where GcvP consists of the amino acid sequence SEQIDNO:26, GcvT consists of the amino acid sequence SEQIDNO:27, GcvH consists of the amino acid sequence SEQIDNO:28, LipA consists of the amino acid sequence SEQIDNO:29, and LipB consists of the amino acid sequence SEQIDNO:30, or each consists of an amino acid sequence with 90% or greater originality to each other amino acid sequence 9. The microorganism of claim 1, where the microorganism of the genus Corynebact for the production of L-histidine is Corynebact.

10. Components for the production of L-histidine, components comprising one of the microorganisms of claims 1 through 9.

11. Method of L-histidine production, a method comprising the steps of: culturing one of the microorganisms of claims 1 through 9 in a medium; and the recovery of L-histidine from the microorganisms and the medium.

12. Use of microorganisms of the genus Corynebacterium with improved glycine transporter activity to enhance L-histidine production.