阅读说明：本技术 修饰的高丝氨酸脱氢酶和使用其产生高丝氨酸或高丝氨酸衍生的l-氨基酸的方法 (Modified homoserine dehydrogenase and method for producing homoserine or homoserine-derived L-amino acid using the same ) 是由 权秀渊 李光雨 许兰 金径林 M·白 孙承珠 李在旼 于 2019-04-10 设计创作，主要内容包括：本公开涉及修饰的高丝氨酸脱氢酶和使用其产生高丝氨酸或高丝氨酸衍生的L-氨基酸的方法。(The present disclosure relates to modified homoserine dehydrogenase and a method of producing homoserine or homoserine-derived L-amino acid using the same.)

1. A modified homoserine dehydrogenase in which the amino acid at position 407 in the amino acid sequence of SEQ ID NO:1 is substituted with histidine.

2. A polynucleotide encoding the modified homoserine dehydrogenase of claim 1.

3. A microorganism of the genus Corynebacterium (genus Corynebacterium) comprising the modified homoserine dehydrogenase of claim 1.

4. The microorganism according to claim 3, wherein the corynebacterium genus microorganism produces homoserine or homoserine-derived L-amino acid.

5. The microorganism according to claim 4, wherein the homoserine-derived L-amino acid is at least one selected from the group consisting of: l-threonine, L-isoleucine, O-acetylhomoserine, and L-methionine.

6. The microorganism according to claim 3, wherein the microorganism of the Corynebacterium genus is Corynebacterium glutamicum (Corynebacterium glutamicum).

7. A method for producing homoserine or homoserine-derived L-amino acid, which comprises culturing a microorganism of Corynebacterium genus comprising the modified homoserine dehydrogenase of claim 1 in a culture medium.

8. The method according to claim 7, further comprising recovering homoserine or homoserine-derived L-amino acid from the cultured microorganism or culture medium.

9. The method according to claim 7, wherein the homoserine-derived L-amino acid is at least one selected from the group consisting of: l-threonine, L-isoleucine, O-acetylhomoserine, and L-methionine.

10. The method according to claim 7, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

11. A method for increasing homoserine or homoserine-derived L-amino acid production in a microorganism, comprising enhancing the activity of the modified homoserine dehydrogenase of claim 1.

12. Use of the modified homoserine dehydrogenase of claim 1 for increasing the production of homoserine or homoserine-derived L-amino acids.

Technical Field

The present disclosure relates to modified homoserine dehydrogenase, and in particular, to a modified homoserine dehydrogenase having a polypeptide comprising one or more amino acid substitutions in the amino acid sequence of a protein having homoserine dehydrogenase activity, wherein the amino acid substitution includes substitution of the amino acid at position 407 of the amino acid sequence with histidine, and a method of producing homoserine or homoserine-derived L-amino acid using the modified homoserine dehydrogenase.

Background

Among L-amino acids, L-threonine, L-isoleucine, and L-methionine generally use homoserine produced from aspartate-semialdehyde (hereinafter, "ASA") by homoserine dehydrogenase (hereinafter, "Hom"; EC: 1.1.1.3). Therefore, in order to produce amino acids by fermentation methods, it is necessary to maintain the activity of enzymes used in biosynthetic pathways at a certain level or higher, and intensive research has been conducted thereon.

Specifically, it is known that the activity of homoserine dehydrogenase acting at branch point (branch point) of L-lysine and L-threonine biosynthetic pathway is regulated by L-threonine and L-isoleucine. Recently, there have been several reports on desensitization of feedback inhibition by Hom to L-threonine and methods of producing L-threonine using the same. In 1991 Eikmann et al reported in Germany desensitization of Hom by substitution of glycine, which is the amino acid residue at position 378 of Hom, with glutamic acid (Eikmanns BJ et al, appl.Microbial Biotechnol.34: 617-622, 1991); and in 1991, Archer et al reported desensitization when the C-terminus of Hom was damaged by frameshift mutations (Archer JA et al, Gene 107: 53-59,1991).

Disclosure of Invention

Technical problem

The present inventors have conducted studies on desensitization to feedback inhibition by threonine, and as a result, they have isolated a novel gene encoding modified Hom and confirmed that L-amino acid-producing ability is improved in a microorganism into which the novel gene is transduced, thereby completing the present disclosure.

Technical scheme

It is an object of the present invention to provide a modified homoserine dehydrogenase in which the amino acid at position 407 of the amino acid sequence is substituted with histidine in the amino acid sequence of a protein having homoserine dehydrogenase activity.

Another object of the present invention is to provide a polynucleotide encoding a modified dehydrogenase.

It is still another object of the present invention to provide a microorganism of the genus Corynebacterium comprising the modified homoserine dehydrogenase.

It is still another object of the present invention to provide a method for producing homoserine or homoserine-derived L-amino acid, which comprises culturing a microorganism in a culture medium; and recovering homoserine or homoserine-derived L-amino acid from the cultured microorganism or culture medium (cultivated medium).

It is a further object of the present invention to provide a method for increasing homoserine or homoserine-derived L-amino acid production in a microorganism, the method comprising enhancing the activity of a modified homoserine dehydrogenase.

It is a further object of the present invention to provide the use of a modified homoserine dehydrogenase for increasing the production of homoserine or homoserine-derived L-amino acids.

Advantageous effects

The modified homoserine dehydrogenase of the present disclosure can be widely used for efficient mass production of homoserine or homoserine-derived L-amino acid (effective mass production) because the feedback inhibition of the final product is desensitized compared to the native or wild type.

Detailed Description

Hereinafter, the present disclosure will be described in detail. Meanwhile, each of the explanation and exemplary embodiments disclosed herein may be applied to other explanations and exemplary embodiments. That is, all combinations of the various factors disclosed herein fall within the scope of the present disclosure. Further, the scope of the present disclosure should not be limited by the specific description provided below.

To achieve the above objects, aspects of the present disclosure provide modified homoserine dehydrogenase having a polypeptide comprising one or more amino acid substitutions in the amino acid sequence of a protein having homoserine dehydrogenase activity, wherein the amino acid substitution comprises substitution of the amino acid at position 407 of the amino acid sequence with another amino acid.

Specifically, the present disclosure provides modified homoserine dehydrogenase having a polypeptide comprising one or more amino acid substitutions in the amino acid sequence of a protein having homoserine dehydrogenase activity, wherein the amino acid substitution comprises a substitution of the amino acid at position 407 of the amino acid sequence with histidine. More specifically, the present disclosure provides a modified homoserine dehydrogenase in which the amino acid at position 407 of the amino acid sequence of SEQ ID NO:1 is substituted with histidine.

In the present disclosure, homoserine dehydrogenase (EC:1.1.1.3) refers to an enzyme that catalyzes the synthesis of homoserine, a common intermediate in the biosynthesis of methionine, threonine, and isoleucine in plants and microorganisms. In the present disclosure, homoserine dehydrogenase (regardless of its source) may be included as long as it has the above transformation activity, and an enzyme derived from any organism (plant, microorganism, etc.) may be used as the homoserine dehydrogenase. Specifically, the homoserine dehydrogenase may be derived from a microorganism of the genus Corynebacterium, and more specifically, may be derived from Corynebacterium glutamicum (Corynebacterium glutamicum). For example, homoserine dehydrogenase may be a protein comprising the amino acid sequence of SEQ ID NO: 1. A protein comprising the amino acid sequence of SEQ ID NO. 1 can be used interchangeably with the term "protein having the amino acid sequence of SEQ ID NO. 1" or "protein consisting of the amino acid sequence of SEQ ID NO. 1".

In the present disclosure, various methods known in the art may be used as a method for obtaining homoserine dehydrogenase. Examples of such methods may include: including a gene synthesis technique for optimizing codons to efficiently obtain proteins in microorganisms of corynebacterium genus, which are commonly used for protein expression, and a method for screening useful enzyme resources using a bioinformatics method based on metagenomic (metagenomic) information of microorganisms, but the method is not limited thereto.

In the present disclosure, the protein having homoserine dehydrogenase activity does not exclude mutations that may occur due to the addition of a nonsense sequence upstream or downstream of the amino acid sequence of the protein having homoserine dehydrogenase activity (e.g., the amino acid sequence of SEQ ID NO:1), or naturally occurring mutations, or silent mutations therein. In addition, the protein also corresponds to the protein having the homoserine dehydrogenase activity of the present disclosure as long as the protein has the same or corresponding activity as the protein including the amino acid sequence of SEQ ID NO: 1. As a specific example, the protein having homoserine dehydrogenase activity of the present disclosure may be a protein consisting of the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% homology thereto.

In addition, although it is described as "a protein or polypeptide including an amino acid sequence of a specific SEQ ID NO" in the present disclosure, it is apparent that any protein having an amino acid sequence deleted, modified, substituted, or added in a partial sequence may also fall within the scope of the present disclosure as long as the protein has an amino acid sequence of any one of the above homologies and exhibits the action corresponding to the above protein. For example, in the present disclosure, the protein having homoserine dehydrogenase activity may be homoserine dehydrogenase derived from corynebacterium glutamicum. More specifically, the protein having homoserine dehydrogenase activity may be the amino acid sequence of homoserine dehydrogenase derived from Corynebacterium glutamicum ATCC13032 (SEQ ID NO:1), the amino acid sequence of homoserine dehydrogenase derived from Corynebacterium glutamicum ATCC14067 (SEQ ID NO:40), or the amino acid sequence of homoserine dehydrogenase derived from Corynebacterium glutamicum ATCC13869 (SEQ ID NO: 41). Since homoserine dehydrogenases having the above sequences show homology of at least 80%, at least 90%, at least 95%, or at least 97% or each other, and since these homoserine dehydrogenases exhibit actions corresponding to those of homoserine dehydrogenases, it is apparent that they are contained in proteins having homoserine dehydrogenase activity of the present disclosure.

As used herein, the term "homology" refers to the percentage of identity between two polynucleotide or polypeptide portions. Homology refers to the degree of match with a given amino acid sequence or nucleotide sequence, and can be expressed as a percentage. In the present disclosure, homologous sequences having the same or similar activity as a given amino acid sequence or nucleotide sequence are denoted as "% homology". Homology between sequences from one part to another can be determined by techniques known in the art. For example, homology can be confirmed using standard software (i.e., BLAST 2.0) for calculating parameters (e.g., score, identity, and similarity) or by comparing sequences by Southern hybridization experiments. The appropriate hybridization conditions to be defined can be determined by methods known to those skilled in the art (e.g., J.Sambrook et al, Molecular Cloning, A Laboratory Manual, 2)^ndEdition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; m. Ausubel et al, Current Protocols in Molecular Biology, John Wiley&Sons,Inc.,New York)。

As used herein, the term "modified", or "variant" refers to a culture or an individual that exhibits heritability or non-heritability alternations in one stable phenotype. Specifically, these terms may refer to variants in which one or more amino acids in the amino acid sequence corresponding to the protein having homoserine dehydrogenase activity are modified to effectively increase its activity, as compared to the wild type, the natural type, or the non-modified type; a variant wherein feedback inhibition of isoleucine, threonine, or an analog or derivative thereof is released; or a variant in which both increased activity and feedback-inhibited release are achieved.

In the present disclosure, the term "modified homoserine dehydrogenase" is used interchangeably with "homoserine dehydrogenase variant". Also, such variants may be non-naturally occurring.

Specifically, the modified homoserine dehydrogenase of the present disclosure may be a modified protein having a polypeptide comprising one or more amino acid substitutions in the amino acid sequence of the protein having homoserine dehydrogenase activity, wherein the amino acid substitution comprises a substitution of histidine for the amino acid at position 407 of the amino acid sequence. The amino acid sequence of the protein having homoserine dehydrogenase activity is as described above, and may be, for example, the amino acid sequence of SEQ ID NO: 1. In addition, "amino acid 407" may refer to an amino acid at a position corresponding to the amino acid 407 from the N-terminus of the amino acid sequence of SEQ ID NO:1, and specifically, may refer to an amino acid 407 from the N-terminus of the amino acid sequence of SEQ ID NO: 1. The amino acid at position 407 may be an amino acid in which arginine is substituted with histidine. More specifically, the modified homoserine dehydrogenase of the present disclosure may be a protein comprising the amino acid sequence of SEQ ID NO 8. In addition, the protein does not exclude mutations that may occur due to addition of a nonsense sequence upstream or downstream of the amino acid sequence, naturally occurring mutations, or silent mutations therein, and any protein having the same or corresponding activity as the modified homoserine dehydrogenase activity corresponds to a protein having the modified homoserine dehydrogenase activity of the present disclosure. As a specific example, the modified homoserine dehydrogenase of the present disclosure may be a protein consisting of the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% homology with the above amino acid sequence, while the amino acid at position 407 from the N-terminus of the amino acid sequence of SEQ ID NO:1 is a fixed (fixed) protein.

In addition, unlike the wild-type or natural protein, or the unmodified protein having homoserine dehydrogenase activity, the modified homoserine dehydrogenase of the present disclosure may be a modified homoserine dehydrogenase in which feedback inhibition of the final product (i.e., isoleucine, threonine, methionine, homoserine, or derivatives or analogs thereof) is released or desensitized. As used herein, the term "feedback inhibition" refers to the prevention of early responses by the end products of metabolism. Therefore, when the feedback inhibition by homoserine dehydrogenase is released or desensitized, the homoserine and homoserine-derived L-amino acid production capacity can be improved as compared to when the feedback inhibition is not released or desensitized.

Homoserine-derived L-amino acid refers to an L-amino acid that can be biosynthesized using L-homoserine as a precursor, and is not limited as long as it is a substance biosynthesized from L-homoserine. The homoserine-derived L-amino acid may include not only homoserine-derived L-amino acids but also derivatives thereof. For example, the homoserine-derived L-amino acid may be L-threonine, L-isoleucine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, O-phospho-L-homoserine, L-methionine, and/or glycine, but the homoserine-derived L-amino acid is not limited thereto. More specifically, the homoserine-derived L-amino acid may be L-threonine, L-isoleucine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, and/or L-methionine, but the homoserine-derived L-amino acid is not limited thereto.

Another aspect of the present disclosure provides a polynucleotide encoding a modified homoserine dehydrogenase.

Homoserine dehydrogenase and variants (modified homoserine dehydrogenase) are described above.

As used herein, the term "polynucleotide" refers to a nucleotide polymer composed of nucleotide monomers covalently bonded in a long chain (e.g., a DNA or RNA strand having a predetermined or longer length), and more specifically, it refers to a polynucleotide fragment encoding a modified homoserine dehydrogenase. The polynucleotide encoding the modified protein of the present disclosure may be included without limitation as long as it has a polynucleotide sequence encoding the modified protein having the homoserine dehydrogenase activity of the present disclosure.

In the present disclosure, the polynucleotide encoding the amino acid sequence of the homoserine dehydrogenase variant can be derived in particular from a microorganism of the genus corynebacterium, and more particularly from corynebacterium glutamicum. However, the microorganism is not limited thereto.

In addition, in a polynucleotide encoding a protein, various modifications may be made in the coding region without changing the amino acid sequence of the protein due to codon degeneracy or considering codons preferred in an organism in which the protein is expressed. In particular, the polynucleotide may be a polynucleotide comprising a polynucleotide sequence encoding a protein or a polynucleotide sequence having at least 80%, at least 90%, at least 95%, or at least 97% homology to the above polynucleotide sequences. In addition, it is apparent that a polynucleotide sequence having a deletion, modification, substitution, or addition in a partial sequence may also fall within the scope of the present disclosure as long as it is a polynucleotide sequence encoding a protein having the above homology and exhibiting substantially the same or corresponding action as the above protein. The polynucleotide encoding a protein having homoserine dehydrogenase activity of the present disclosure may have a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO. 1. For example, the polynucleotide may have the polynucleotide sequence of SEQ ID NO. 2, but is not limited thereto. In addition, the polynucleotide encoding the modified homoserine dehydrogenase of the present disclosure may have a polynucleotide sequence encoding a polypeptide comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO. 1, and specifically, may have a polynucleotide sequence encoding SEQ ID NO. 8. For example, the polynucleotide may have the polynucleotide sequence of SEQ ID NO. 7, but is not limited thereto.

In addition, probes prepared from known gene sequences, for example, any sequences that hybridize under stringent conditions to a sequence complementary to all or part of a polynucleotide sequence to encode a protein having homoserine dehydrogenase activity of the present disclosure, may also be included without limitation. "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (e.g., j.sambrook et al, supra). Stringent conditions may include, for example, conditions under which genes having high homology, 80% or more homology, specifically 90% or more homology, more specifically 95% or more homology, more specifically 97% or more homology, still more specifically 99% or more homology hybridize to each other, while genes having homology lower than the above do not hybridize to each other; or general washing conditions for Southern hybridization (i.e., in salt)The concentration and temperature correspond to 60 ℃,1 × SSC, 0.1% SDS, specifically, 60 ℃, 0.1 × SSC, 0.1% SDS, and more specifically 68 ℃, 0.1 × SSC, 0.1% SDS washing once, specifically, two or three times)_mHybridization step at value. Further, T_mThe value may be 60 ℃, 63 ℃, or 65 ℃, but is not limited thereto, and may be appropriately regulated by those skilled in the art according to the purpose thereof. The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (see Sambrook et al, supra, 9.50-9.51, 11.7-11.8).

Yet another aspect of the present disclosure provides a microorganism comprising a modified homoserine dehydrogenase. Specifically, the present disclosure provides a homoserine or homoserine-derived L-amino acid-producing microorganism of Corynebacterium genus comprising modified homoserine dehydrogenase.

Homoserine dehydrogenase and variants are described above.

Specifically, a microorganism comprising the modified homoserine dehydrogenase of the present disclosure refers to a microorganism which naturally has the ability to produce homoserine or homoserine-derived L-amino acid, or a microorganism in which the ability to produce homoserine or homoserine-derived L-amino acid is imparted to a parent strain thereof which lacks the ability to produce homoserine or homoserine-derived L-amino acid. Specifically, the microorganism containing homoserine dehydrogenase can be a microorganism capable of expressing modified homoserine dehydrogenase in which the amino acid at position 407 of the amino acid sequence of SEQ ID NO:1 is substituted with histidine, but the microorganism is not limited thereto. The microorganism can be a cell or a microorganism comprising a polynucleotide encoding a modified homoserine dehydrogenase or a vector capable of being transformed with a polynucleotide comprising a polynucleotide encoding a modified homoserine dehydrogenase for expression of a modified polypeptide. For the purposes of the present disclosure, a host cell or microorganism may be any microorganism which is capable of producing homoserine or homoserine-derived L-amino acid comprising a modified polypeptide.

The microorganism comprising the modified homoserine dehydrogenase of the present disclosure has improved homoserine and homoserine-derived L-amino acid production ability compared to wild-type or microorganisms comprising a protein having unmodified homoserine dehydrogenase activity. Therefore, homoserine and homoserine-derived L-amino acids can be obtained in high yield from microorganisms comprising the modified homoserine dehydrogenase of the present disclosure.

In the present disclosure, the type of microorganism containing the modified homoserine dehydrogenase is not particularly limited, but may be an Enterobacter (genus Enterobacter) microorganism, an Escherichia (genus Escherichia) microorganism, an Erwinia (genus Erwinia) microorganism, a Serratia (genus Serratia) microorganism, a pseudomonas (genus pseudomonas) microorganism, a Providencia (genus Providencia) microorganism, a corynebacterium (genus corynebacterium) microorganism, or a Brevibacterium (genus Brevibacterium) microorganism. More specifically, the microorganism may be a microorganism of Corynebacterium genus.

In the present disclosure, the "microorganism of Corynebacterium genus" may be specifically Corynebacterium glutamicum, Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), Brevibacterium lactofermentum (Brevibacterium lactofermentum), Brevibacterium flavum (Brevibacterium flavum), Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes), Corynebacterium effectum (Corynebacterium efficiens), etc., but the microorganism of Corynebacterium genus is not limited thereto. More specifically, in the present disclosure, the microorganism of Corynebacterium genus may be Corynebacterium glutamicum.

Meanwhile, the microorganism containing the modified homoserine dehydrogenase may be a microorganism into which a vector including a polynucleotide encoding a homoserine dehydrogenase variant is introduced. Specifically, the introduction may be performed by transformation, but the method of introduction is not limited thereto.

As used herein, the term "vector" refers to a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a target protein, wherein the target protein is operably linked to suitable control sequences such that the target protein can be expressed in a suitable host. The control sequences may include a promoter capable of initiating transcription, any operator sequence used to control transcription, sequences encoding the appropriate mRNA ribosome binding domain, and sequences which control termination of transcription and translation. Upon transformation into a suitable host cell, the vector may replicate or function independently of the host genome, or may be integrated into the host genome itself.

The vector used in the present disclosure is not particularly limited as long as it can replicate in a host cell, and any vector known in the art may be used. Examples of conventional vectors may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, xii, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or cosmid vector; and pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL type, pET type, etc. can be used as plasmid vectors. Specifically, vectors pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC and the like can be used, but the vectors are not limited thereto.

The vector that can be used in the present disclosure is not particularly limited, and any known expression vector can be used. In addition, a polynucleotide encoding a target protein can be inserted into a chromosome by a vector for chromosomal insertion. The polynucleotide may be inserted into the chromosome by any method known in the art (e.g., homologous recombination), but the method is not limited thereto. The vector may further comprise a selection marker (selection marker) to confirm insertion of the polynucleotide into the chromosome. The selectable marker is used to screen cells transformed with the vector, i.e., to determine whether a polynucleotide molecule is inserted. Markers that provide alternative phenotypes (e.g., drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins) can be used. In the context of treatment with a selection agent, only cells expressing the selection marker can survive, or the cells can exhibit a different phenotype, and transformed cells can therefore be selected by this method.

As used herein, the term "transformation" refers to the introduction of a vector comprising a polynucleotide encoding a target protein into a host cell in such a way that the protein encoded by the polynucleotide is expressed in the host cell. It does not matter whether the transformed polynucleotide is integrated into the chromosome of the host cell and placed therein or is extrachromosomally located, so long as the transformed polynucleotide can be expressed in the host cell. Further, polynucleotides include DNA and RNA encoding target proteins. The polynucleotide may be introduced in any form as long as it can be introduced into and expressed in a host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct that includes all of the elements required for its autonomous expression. The expression cassette can include a promoter, transcription terminator, ribosome binding site, or translation termination signal operably linked to a polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into the host cell as it is and operably linked to a sequence required for expression in the host cell, but the method of introducing the polynucleotide is not limited thereto. The transformation method includes any method of introducing a polynucleotide into a cell, and it can be performed by selecting an appropriate standard technique known in the art according to the host cell. Examples of methods include electroporation, calcium phosphate (Ca (H)₂PO₄)₂、CaHPO₄Or Ca₃(PO₄)₂) Precipitate, calcium chloride (CaCl)₂) Precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, etc., but the transformation method is not limited thereto.

In addition, the term "operably linked" refers to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein of the disclosure is functionally linked to a polynucleotide sequence. The operable linkage may be prepared using a gene recombination technique known in the art, and site-specific DNA cleavage and linkage may be prepared using known restriction enzymes and ligases, but the method of operable linkage is not limited thereto.

The microorganism containing the modified homoserine dehydrogenase may be a microorganism that has been transformed to include the modified homoserine dehydrogenase in a microorganism of corynebacterium genus. For example, the microorganism of Corynebacterium genus may include a strain resistant to 2-amino-3-hydroxy-pentanoic Acid (AHV); a strain that produces L-threonine by substituting leucine (i.e., amino acid at position 377 of aspartokinase (lysC)) with lysine to solve feedback inhibition of lysC (i.e., the first important enzyme acting on the threonine biosynthetic pathway); a strain that produces L-isoleucine by substituting alanine for the amino acid at position 323 of the ilvA gene encoding L-threonine dehydratase (i.e., the first enzyme acting on the biosynthetic pathway of isoleucine) in an L-threonine-producing strain (appl. enviro. Microbiol., Dec.1996, p.4345-4351); a strain producing O-acetylhomoserine by inactivating O-acetylhomoserine (thiol) -lyase involved in the degradation pathway of O-acetylhomoserine and cystathionine γ -synthase; or a strain which produces methionine by inactivating the transcriptional regulators of methionine and cysteine, but the strain of the microorganism of Corynebacterium genus is not limited thereto.

Yet another aspect of the present disclosure provides a method for producing homoserine or homoserine-derived L-amino acid, comprising: culturing the above microorganism in a medium.

The method for producing an L-amino acid may comprise recovering homoserine or homoserine-derived L-amino acid from the cultured microorganism or culture medium.

As described above, the microorganism may be a microorganism of corynebacterium genus comprising the homoserine dehydrogenase variant of the present disclosure, and more specifically, may be corynebacterium glutamicum. In addition, the microorganism of Corynebacterium genus or Corynebacterium glutamicum may be a microorganism producing homoserine or homoserine-derived L-amino acid. The homoserine-derived L-amino acid may include not only homoserine-derived L-amino acids but also derivatives thereof. For example, the homoserine-derived L-amino acid may be L-threonine, L-isoleucine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, O-phospho-L-homoserine, L-methionine, and/or glycine, but the homoserine-derived L-amino acid is not limited thereto. More specifically, the homoserine-derived L-amino acid may be L-threonine, L-isoleucine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, and/or L-methionine, but the homoserine-derived L-amino acid is not limited thereto.

Homoserine or homoserine-derived L-amino acid can be the culture medium of homoserine or homoserine-derived L-amino acid produced by the microorganisms described in the present disclosure, or can be in a purified form. It will be apparent to those skilled in the art that homoserine or homoserine-derived L-amino acid includes not only itself but also salts thereof.

The methods for producing homoserine or homoserine-derived L-amino acid can be easily determined by those skilled in the art under the conditions of optimized culture conditions and enzyme activity known in the art.

Among the above methods, the microorganism may be cultured by a batch process (batch process), a continuous process, a fed-batch process (fed-batch process), and the like known in the art, but the culture method is not particularly limited thereto. Specifically, with respect to the culture conditions, the pH of the culture may be adjusted to a suitable pH (e.g., pH 5 to pH 9, specifically pH 6 to pH 8, and most specifically pH 6.8) with an appropriate basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or acidic compound (e.g., phosphoric acid or sulfuric acid), and aerobic conditions of the culture may be maintained by introducing oxygen or an oxygen-containing gas mixture into the culture. The culture temperature may be generally in the range of 20 ℃ to 45 ℃, and specifically 25 ℃ to 40 ℃, for about 10 to 160 hours, but the culture conditions are not limited thereto. Threonine, isoleucine, or acetylhomoserine produced by the culture process can be secreted into the culture or can be retained in the cell.

In addition, as a carbon source of the medium, sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose); oils and fats (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil); fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid); alcohols (e.g., glycerol and ethanol); organic acids (e.g., acetic acid) and the like may be used alone or in combination, but the carbon source is not limited thereto. As the nitrogen source of the medium, nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat extract (meal gravy), wort, corn steep liquor, soybean meal and urea), inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) and the like can be used alone or in combination, but the nitrogen source is not limited thereto. As the phosphorus source of the medium, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, corresponding sodium-containing salts and the like may be used alone or in combination, but the phosphorus source is not limited thereto. In addition, the medium may contain other metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, vitamins, etc., which are essential growth promoting substances.

In the present disclosure, the method for recovering homoserine or homoserine-derived L-amino acid produced during culture can be performed by collecting a target product from a culture broth (culture broth) using an appropriate method known in the art. For example, methods such as centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. may be used, and the target substance (which is homoserine or homoserine-derived L-amino acid) can be recovered from the culture medium or the cultured microorganism using an appropriate method known in the art. Further, recovery may include additional purification processes, and may be performed using appropriate methods known in the art.

Yet another aspect of the present disclosure provides the use of a modified homoserine dehydrogenase for increasing the production of homoserine or homoserine-derived L-amino acids.

Yet another aspect of the present disclosure provides a method for increasing homoserine or homoserine-derived L-amino acid production in a microorganism, comprising enhancing the activity of a modified homoserine dehydrogenase.

As used herein, the term "to be expressed/expressed" refers to a state in which a target protein is introduced into a microorganism or, in the case where a protein is present in a microorganism, the activity of the protein is enhanced as compared with the activity of its endogenous protein or its activity before modification.

Specifically, the term "introduction of a protein" means that a microorganism exhibits an activity of a specific protein not originally present in the microorganism, or the microorganism exhibits an enhanced activity as compared with its endogenous activity or the activity of a protein before modification. For example, it may mean that a polynucleotide encoding a specific protein is introduced into the chromosome of a microorganism or a vector containing a polynucleotide encoding a specific protein is introduced into a microorganism, thereby exhibiting its activity. In addition, the term "activity enhancement" refers to an improvement in the activity of a particular protein as compared to its endogenous activity or activity prior to modification. The term "endogenous protein" refers to the condition in which a parent strain of a microorganism originally has the activity of a specific protein, wherein the trait of the microorganism is altered due to genetic modification by natural or artificial factors.

Specifically, in the present disclosure, enhancement of activity may be achieved by one or more of the following methods: a method of increasing the intracellular copy number of a gene encoding a protein variant; a method of introducing a modification into an expression control sequence of a gene encoding a protein variant; a method of substituting a sequence having a strong activity for an expression control sequence of a gene encoding a protein variant; a method of substituting a gene encoding a native protein on a chromosome having homoserine dehydrogenase activity with a gene encoding a protein variant; a method of introducing a further modification into a gene encoding a protein having homoserine dehydrogenase activity to enhance the activity of a protein variant; and a method of introducing the protein variant into a microorganism, but the method is not limited thereto.

In the above, the copy number of the gene may be increased in the form of operably linking the gene to a vector or by inserting the gene into the chromosome of the host cell, but the method is not particularly limited thereto. In particular, the copy number of a gene can be increased by introducing a vector into a host cell, wherein the vector is operably linked to a polynucleotide encoding a protein of the disclosure and is capable of replicating and functioning regardless of the host cell. Alternatively, the copy number of a gene can be increased by introducing a vector operably linked to a polynucleotide into the chromosome of a host cell. Insertion of the polynucleotide into the chromosome can be achieved by methods known in the art (e.g., homologous recombination).

Then, in order to increase the expression of the polynucleotide, modification may be induced therein by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control sequence; or by modifying the expression control sequence by replacing it with an accounting sequence having a stronger activity, but the method of modification is not particularly limited thereto. The expression control sequence may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling termination of transcription and translation, and the like, but the expression control sequence is not limited thereto.

The strong promoter may be linked to the upstream region of the expression unit of the polynucleotide instead of the original promoter, but the method is not limited thereto. Examples of strong promoters known in the art may include cj1 to cj7 promoter (KR patent No. 10-0620092), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (KR patent No. 10-1783170), O2 promoter (KR patent No. 10-1632642), tkt promoter, yccA promoter, etc., but the promoters are not limited thereto.

Further, modification of the polynucleotide sequence on the chromosome may induce modification on the expression control sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the polynucleotide sequence; or by replacing the polynucleotide sequence with a polynucleotide sequence modified to have stronger activity, but the method of modification is not particularly limited thereto.

Introduction and enhancement of the activity of a protein as described above can generally increase the activity or concentration of the corresponding protein by at least 1%, at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%, and at most 1,000% or 2,000%, based on the activity or concentration of the protein in the wild-type or unmodified microbial strain, although the scope is not limited thereto.

The amino acid sequence of the protein having homoserine dehydrogenase activity, the amino acid at position 407, and the microorganism are as described above.