The present application relates to a variant homoserine dehydrogenase, and specifically, a variant homoserine dehydrogenase having a polypeptide comprising one or more amino acid substitutions in the amino acid sequence of a protein having homoserine dehydrogenase activity. As a first step, the amino acid substitution relates to a mutant homoserine dehydrogenase comprising the 407 amino acid substitution with histidine, and a method for producing homoserine or homoserine-derived L-amino acid using the same.
[2]
background
[3]
Among L-amino acids, L-threonine, L-isoleucine, and L-methionine are converted from aspartate-semialdehyde (ASA) to homoserine dehydrogenase (Hom, EC: 1.1.1.3). ), which is produced by homoserine, is commonly used. Therefore, in order to produce the amino acids by the fermentation method, it is essential to maintain the activity of the enzymes used in the biosynthetic pathway at a certain level or more, and intensive research has been made on this.
[4]
In particular, it is known that homoserine dehydrogenase, which acts at a branch point of L-lysine and L-threonine biosynthetic pathways, is regulated by L-threonine and L-isoleucine. There have been several reports on Hom desensitized to feedback inhibition by L-threonine and a method for producing L-threonine using the same. In 1991, Germany's Eikmann et al . reported that Hom was desensitized by conversion of glycine, the 378th amino acid residue of Hom, to glutamate (Eikmanns BJ et al ., Appl. Microbial Biotechnol. 34: 617-622, 1991), 1991 Archer et al . of the United States reported that when the C-terminus of Hom is damaged due to a frameshift mutation, it exhibits desensitization properties (Archer JA et al ., Gene 107: 53-59, 1991).
[5]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[6]
The present inventors separated the novel gene encoding the variant Hom while conducting the feedback inhibition desensitization study by threonine, and confirmed that the L-amino acid production ability is improved in the microorganism transduced with the novel gene. completed.
[7]
means of solving the problem
[8]
One object of the present application is to provide a variant homoserine dehydrogenase in which the 407th amino acid is substituted with histidine in the amino acid sequence of a protein having homoserine dehydrogenase activity.
[9]
Another object of the present application is to provide a polynucleotide encoding the variant dehydrogenase.
[10]
Another object of the present application is to provide a microorganism of the genus Corynebacterium, including the variant homoserine dehydrogenase.
[11]
Another object of the present application, the step of culturing the microorganism in a medium; To provide a method for producing homoserine or homoserine-derived L-amino acids, comprising the step of recovering homoserine or homoserine-derived L-amino acids from the cultured microorganism or medium.
[12]
Another object of the present application is to provide a method for increasing the production of homoserine or homoserine-derived L-amino acids, comprising enhancing the activity of the mutant homoserine dehydrogenase in a microorganism.
[13]
Another object of the present application is to provide a use for increasing the production of homoserine or homoserine-derived L-amino acids of the variant homoserine dehydrogenase.
[14]
Effects of the Invention
[15]
The mutant homoserine dehydrogenase of the present application can be widely used for mass production of homoserine or homoserine-derived L-amino acids more efficiently because feedback inhibition by the final product is desensitized compared to the native or wild type.
[16]
Best mode for carrying out the invention
[17]
This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.
[18]
[19]
One aspect of the present application for achieving the above object is a variant homoserine dehydrogenase having a polypeptide comprising one or more amino acid substitutions in the amino acid sequence of a protein having the activity of homoserine dehydrogenase, the amino acid The substitution provides a variant homoserine dehydrogenase, comprising the substitution of amino acid at position 407 with another amino acid.
[20]
Specifically, as a variant homoserine dehydrogenase having a polypeptide comprising one or more amino acid substitutions in the amino acid sequence of a protein having homoserine dehydrogenase activity of SEQ ID NO: 1, wherein the amino acid substitution is at position 407 Provided is a variant homoserine dehydrogenase comprising one substituted with histidine. More specifically, the 407th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine, it provides a variant homoserine dehydrogenase.
[21]
[22]
In the present application, homoserine dehydrogenase (EC: 1.1.1.3) refers to an enzyme that catalyzes the synthesis of homoserine, a common intermediate of methionine, threonine, and isoleucine biosynthesis in plants and microorganisms, Also called homoserine dehydrogenase. In the present application, homoserine dehydrogenase may be included regardless of origin as long as it is a protein having the conversion activity, and an enzyme derived from any organism (plants and microorganisms, etc.) may be used. Specifically, the homoserine dehydrogenase may be derived from a microorganism of the genus Corynebacterium, and more specifically , may be derived from Corynebacterium glutamicum . For example, it may be a protein comprising the amino acid sequence of SEQ ID NO: 1. The protein comprising the amino acid sequence of SEQ ID NO: 1 may be used in combination with the protein having the amino acid sequence of SEQ ID NO: 1 and the protein consisting of the amino acid sequence of SEQ ID NO: 1.
[23]
In the present application, various methods well known in the art are applicable as a method of securing homoserine dehydrogenase. Examples of the method include gene synthesis technology that includes codon optimization to secure proteins with high efficiency in microorganisms of the genus Corynebacterium, which are commonly used for protein expression, and bioinformatics methods based on large-scale genome information of microorganisms. It can be secured through a screening method of useful enzyme resources by the present invention, but is not limited thereto.
[24]
In the present application, the protein having the activity of homoserine dehydrogenase is the amino acid sequence of the protein having the activity of homoserine dehydrogenase, for example, the amino acid sequence of SEQ ID NO: 1, the addition of meaningless sequences before and after or naturally occurring Possible mutations, or latent mutations thereof, are not excluded, and homoserine dehydrogenase of the present application is not excluded if it has the same or corresponding activity as the protein comprising the amino acid sequence of SEQ ID NO: 1 It corresponds to an active protein. For example, the protein having the activity of homoserine dehydrogenase of the present application is a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80%, 90%, 95%, or 97% or more homology thereto. can be
[25]
In addition, even if it is described as 'a protein or a polypeptide comprising the amino acid sequence described in a specific SEQ ID NO:' in the present application, if it is an amino acid sequence that has such homology and exhibits efficacy corresponding to the protein, some sequences may be deleted, modified, It is apparent that proteins having substituted or added amino acid sequences are also included within the scope of the present application. For example, a protein having the activity of homoserine dehydrogenase in the present application is Corynebacterium glutamicum ( corynebacterium glutamicum ) can homoserine dehydrogenase derived from the best. More specifically, 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 (SEQ ID NO: 41) derived from Corynebacterium glutamicum ATCC13869. Homoserine dehydrogenase having the above sequence shows 80%, 90%, 95%, or 97% or more homology with each other, and shows the corresponding efficacy as homoserine dehydrogenase, so the homoserine dehydrogenase of the present application It is self-evident that it is included in the protein having the activity of the genase.
[26]
[27]
The term "homology" refers to the percent identity between two polynucleotide or polypeptide moieties. It refers to the degree of correspondence with a given amino acid sequence or base sequence, and may be expressed as a percentage. In the present specification, a homologous sequence having the same or similar activity to a given amino acid sequence or base sequence is expressed as "% homology". Homology between sequences from one moiety to another can be determined by known art. For example, by using standard software, specifically BLAST 2.0, which calculates parameters such as score, identity, and similarity, or by Southern hybridization experiments under defined stringent conditions. Appropriate hybridization conditions defined, which can be confirmed by comparing Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).
[28]
[29]
As used herein, the term "variant", "mutant" or "variant" refers to a culture or individual that exhibits a stable phenotypic change, either genetically or non-genetically. Specifically, one or more amino acids are mutated in the amino acid sequence corresponding to the protein having the activity of homoserine dehydrogenase so that the activity is efficiently increased compared to the wild type, native type or unmodified type, or isoleucine, threonine, It may refer to a mutant in which feedback inhibition to their analogs or derivatives is canceled or both activity increase and feedback inhibition are canceled.
[30]
In the present application, mutant homoserine dehydrogenase may be used interchangeably as "mutated homoserine dehydrogenase" or "homoserine dehydrogenase mutant". Meanwhile, such a mutant may be non-naturally occurring.
[31]
The variant homoserine dehydrogenase of the present application is specifically a variant protein 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 is 407 It may be a mutant homoserine dehydrogenase comprising the first amino acid being substituted with histidine. The amino acid sequence of the protein having the activity of homoserine dehydrogenase is the same as described above, and may be, for example, the amino acid sequence of SEQ ID NO: 1. In addition, the '407th amino acid' may mean an amino acid at a position corresponding to the 407 amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1, specifically, 407th from the N-terminus of the amino acid sequence of SEQ ID NO: 1 may mean amino acids. The 407th amino acid may be one in which arginine is substituted with histidine. More specifically, the variant homoserine dehydrogenase of the present application may be a protein comprising the amino acid sequence of SEQ ID NO: 8. In addition, it does not exclude the addition of meaningless sequences before and after the sequence, or a mutation that may occur naturally, or a silent mutation thereof, and has the same or corresponding activity to the mutant homoserine dehydrogenase. If it is, it corresponds to a protein having the activity of the mutant homoserine dehydrogenase of the present application. For a specific example, the variant homoserine dehydrogenase of the present application has the amino acid sequence of SEQ ID NO: 8 or amino acid 407 from the N-terminus in the amino acid sequence of SEQ ID NO: 8 is fixed, and 80%, 90%, 95 %,
[32]
[33]
In addition, the mutant homoserine dehydrogenase of the present application is different from the wild-type or native protein having the activity of homoserine dehydrogenase, or the unmodified protein, the final product isoleucine, threonine, methionine, homoserine Alternatively, feedback inhibition by derivatives or analogs thereof may be canceled or may have desensitization characteristics. As used herein, the term "feedback inhibition" refers to inhibition of a reaction in which the final product of metabolism is higher than that. Therefore, when the feedback inhibition of homoserine dehydrogenase is canceled or desensitized, the productivity of homoserine and homoserine-derived L-amino acids of microorganisms can be increased compared to the case where it is not.
[34]
The homoserine-derived L-amino acid means an L-amino acid that can be biosynthesized using L-homoserine as a precursor, and is not limited as long as it is a material that can be biosynthesized from L-homoserine. The homoserine-derived L-amino acid may include not only homoserine-derived L-amino acid, but also derivatives thereof. For example L-threonine, L-isoleucine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, O-phospho-L-homoserine, L-methionine and/or L- It may be glycine, but is not limited thereto. More specifically, it may be L-threonine, L-isoleucine, O-acetyl-L-homoserine, O-succinyl-L-homoserine and/or L-methionine, but is not limited thereto.
[35]
[36]
Another aspect of the present application provides a polynucleotide encoding the variant homoserine dehydrogenase.
[37]
The homoserine dehydrogenase and variants are as described above.
[38]
As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, the mutant homoserine. It refers to a polynucleotide fragment encoding a dehydrogenase. The polynucleotide encoding the variant protein of the present application may be included without limitation as long as it is a polynucleotide sequence encoding the variant protein having homoserine dehydrogenase activity of the present application.
[39]
In the present application, the polynucleotide encoding the amino acid sequence of the homoserine dehydrogenase variant may be specifically derived from a microorganism of the genus Corynebacterium, and more specifically, may be derived from Corynebacterium glutamicum. However, the present invention is not limited thereto.
[40]
In addition, the polynucleotide encoding the protein is located in the coding region within a range that does not change the amino acid sequence of the protein due to codon degeneracy or in consideration of codons preferred in the organism to express the protein. Various modifications may be made. Specifically, it may be a polynucleotide comprising a polynucleotide sequence encoding the protein or a polynucleotide sequence having 80%, 90%, 95%, or 97% or more homology thereto. In addition, as long as it is a polynucleotide sequence encoding a protein having such homology and exhibiting substantially the same or corresponding efficacy as the protein, a polynucleotide sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application. self-evident The polynucleotide encoding the protein having homoserine dehydrogenase activity of the present application may be a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. For example, it may be the polynucleotide sequence of SEQ ID NO: 2, but is not limited thereto. In addition, the polynucleotide encoding the variant homoserine dehydrogenase of the present application may be a polynucleotide sequence encoding a polypeptide comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1, specifically, SEQ ID NO: 8 It may be a polynucleotide sequence encoding For example, it may be the polynucleotide sequence of SEQ ID NO: 7, but is not limited thereto.
[41]
Or by hybridizing under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence, the activity of the variant homoserine dehydrogenase of the present application Any sequence encoding a protein having a may be included without limitation. The "stringent conditions" means conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., supra). For example, genes with high homology between genes having 80% or more, specifically 90% or more, more specifically 95% or more, still more specifically 97% or more, particularly specifically 99% or more homology between genes Conditions that hybridize with each other and do not hybridize with genes with lower homology, or wash conditions for general Southern hybridization at 60° C., 1×SSC, 0.1% SDS, specifically 60° C., 0.1×SSC, 0.1 % SDS, more specifically, at a salt concentration and temperature corresponding to 68° C., 0.1×SSC, 0.1% SDS, washing conditions once, specifically 2 to 3 times, can be enumerated. Hybridization requires that two polynucleotides have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between bases of nucleotides that are capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. thus, The present application may also cover substantially similar nucleotide sequences as well as isolated nucleotide fragments that are complementary to the overall sequence. Specifically, polynucleotides having homology 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 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose. The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).
[42]
[43]
Another aspect of the present application provides a microorganism comprising a mutant homoserine dehydrogenase. Specifically, it provides a microorganism of the genus of Corynebacterium that produces homoserine or homoserine-derived L-amino acids, including the variant homoserine dehydrogenase .
[44]
The homoserine dehydrogenase and variant are the same as described above.
[45]
Specifically, the microorganism containing the mutant homoserine dehydrogenase of the present application is a microorganism having the ability to naturally produce homoserine or homoserine-derived L-amino acid, or a parent without the ability to produce homoserine or homoserine-derived L-amino acid. It refers to a microorganism to which the production ability of homoserine or homoserine-derived L-amino acids is imparted to the strain. Specifically, the microorganism containing the homoserine dehydrogenase may be a microorganism expressing a mutant homoserine dehydrogenase in which the 407th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine, but is not limited thereto. The microorganism is transformed with a vector containing a polynucleotide encoding the variant homoserine dehydrogenase or a polynucleotide encoding a variant homoserine dehydrogenase to express the variant polypeptide. As a capable cell or microorganism, for the purpose of the present application, the host cell or microorganism may be any microorganism capable of producing homoserine or homoserine-derived L-amino acids, including the variant polypeptide.
[46]
The microorganism containing the mutant homoserine dehydrogenase of the present application has improved production ability of homoserine and homoserine-derived L-amino acids compared to the microorganism containing a protein having the activity of wild-type or unmodified homoserine dehydrogenase Therefore, homoserine and homoserine-derived L-amino acids can be obtained in high yield from these microorganisms.
[47]
In the present application, the type of microorganism containing the mutant homoserine dehydrogenase is not particularly limited, but Enterobacter genus, Escherichia genus, Erwinia genus, Serratia ( Serratia ) genus Pseudomonas ( Pseudomonas ) genus Providencia ( Providencia ) genus Corynebacterium ( of Corynebacterium ) genus and Brevibacterium ( Brevibacterium may be a microorganism belonging to the genus). More specifically , it may be a microorganism belonging to the genus Corynebacterium .
[48]
In the present application, "Corynebacterium genus microorganism" is specifically Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum ( Brevibacterium lactofermentum ), Brevibacterium flavum ( Brevibacterium flavu m), Corynebacterium thermo amino to Ness ( Corynebacterium thermoaminogenes ), Corynebacterium epi syeonseu ( Corynebacterium efficiens or the like), it is not limited thereto. More specifically, in the present application, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum .
[49]
Meanwhile, the microorganism containing the mutant homoserine dehydrogenase may be a microorganism into which a vector containing a polynucleotide encoding a mutant homoserine dehydrogenase is introduced. Specifically, the introduction may be made by transformation, but is not limited thereto.
[50]
As used herein, the term "vector" refers to a DNA preparation containing the base sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed 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 a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
[51]
The vector used in the present application 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 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 may be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors may be used. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used. Specifically, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. may be used, but is not limited thereto.
[52]
The vector usable in the present application is not particularly limited, and a known expression vector may be used. In addition, a polynucleotide encoding a target protein can be inserted into a chromosome through a vector for intracellular chromosome insertion. Insertion of the polynucleotide into a chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted. The selection marker is used to select cells transformed with a vector, that is, to determine whether a target polynucleotide molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface protein expression. Markers to be given can be used. In an environment treated with a selective agent, only cells expressing a selectable marker survive or exhibit other expression traits, and thus transformed cells can be selected.
[53]
As used herein, the term "transformation" refers to introducing a vector including a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located extrachromosomally, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding a target protein. The polynucleotide may be introduced into a host cell and expressed in any form, as long as it can be expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto. The transformation method includes any method of introducing a polynucleotide into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (Ca(H 2PO 4 ) 2 , CaHPO 4 , or Ca 3 (PO 4 ) 2 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method and the like, but is not limited thereto.
[54]
In addition, as used herein, the term “operably linked” means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein of the present application and the polynucleotide sequence are functionally linked. The operable linkage may be prepared using a genetic recombination technique known in the art, and site-specific DNA cleavage and ligation may be made using a cleavage and ligation enzyme in the art, but is not limited thereto.
[55]
The microorganism containing the mutant homoserine dehydrogenase may be one transformed to include the mutant homoserine dehydrogenase in a microorganism of the genus Corynebacterium. The microorganism of the genus Corynebacterium is illustratively a strain having resistance to 2-amino-3-hydroxy-valerate (AHV); In order to solve the feedback inhibition of aspartate kinase (lysC), which acts as the first important enzyme in the threonine biosynthesis pathway, amino acid 377 of lysC, Leucine, is substituted with Lysine, and L-threo strains that produce nin; L-threonine dehydratase (the first enzyme of the isoleucine biosynthetic pathway) in the L-threonine-producing strain was substituted with alanine at the 323th amino acid of the gene ilvA to L- strains that produce isoleucine ( Appl. Enviro. Microbiol. , Dec. 1996, p.4345-4351); O-acetyl-homoserine (thiol)-lyase and cystathionine gamma-synthase proteins of the O-acetyl-homoserine degradation pathway are inactivated, resulting in O-acetyl homo strains that produce serine; Alternatively, a strain in which methionine cysteine ​​transcription regulator protein is inactivated to produce methionine may be included, but is not limited thereto.
[56]
[57]
Another aspect of the present application provides a method for producing homoserine or homoserine-derived L-amino acids, comprising culturing the described microorganism in a medium.
[58]
The method for producing L-amino acids may further include recovering homoserine or homoserine-derived L-amino acids from the cultured microorganism or cultured medium.
[59]
The microorganism may be a microorganism of the genus Corynebacterium including the homoserine dehydrogenase variant of the present application as described above, and more specifically, may be Corynebacterium glutamicum. In addition, the microorganism of the genus Corynebacterium or Corynebacterium glutamicum may be homoserine or a microorganism producing homoserine-derived L-amino acids. The homoserine-derived L-amino acid may include not only homoserine-derived L-amino acid, but also derivatives thereof. For example L-threonine, L-isoleucine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, O-phospho-L-homoserine, L-methionine and/or L- It may be glycine, but is not limited thereto. More specifically, it may be L-threonine, L-isoleucine, O-acetyl-L-homoserine, O-succinyl-L-homoserine and/or L-methionine, but is not limited thereto.
[60]
The homoserine or homoserine-derived L-amino acid may be a culture solution of homoserine or homoserine-derived L-amino acid produced by the microorganism described in the present application, or may be in a purified form. In addition, it is apparent to those skilled in the art that it includes both its own form as well as its salt form.
[61]
The method for producing homoserine or homoserine-derived L-amino acid can be easily determined by those skilled in the art under optimized culture conditions and enzyme activity conditions known in the art.
[62]
In the method, the step of culturing the microorganism is not particularly limited, but may be performed by a known batch culture method, a continuous culture method, a fed-batch culture method, and the like. At this time, the culture conditions are not particularly limited thereto, but use a basic compound (eg, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg, phosphoric acid or sulfuric acid) to an appropriate pH (eg, pH 5 to 9, specifically can control pH 6 to 8, most specifically pH 6.8) and maintain aerobic conditions by introducing oxygen or an oxygen-containing gas mixture into the culture. The culture temperature may be maintained at 20°C to 45°C, specifically 25°C to 40°C, and may be cultured for about 10 to 160 hours, but is not limited thereto. Threonine, isoleucine, or acetylhomoserine produced by the above culture may be secreted into the medium or remain in cells.
[63]
In addition, the culture medium used is a carbon source that includes sugars and carbohydrates (eg, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oils and fats (eg, soybean oil, sunflower seed). Oil, peanut oil and coconut oil), fatty acids (eg palmitic acid, stearic acid and linoleic acid), alcohols (eg glycerol and ethanol) and organic acids (eg acetic acid) may be used individually or in combination. , but not limited thereto. Nitrogen sources include nitrogen-containing organic compounds (such as peptone, yeast extract, broth, malt extract, corn steep liquor, soy meal and urea), or inorganic compounds (such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) may be used individually or in combination, but is not limited thereto. As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium-containing salt corresponding thereto, etc. may be used individually or in combination, but is not limited thereto. In addition, the medium may contain essential growth-promoting substances such as other metal salts (eg, magnesium sulfate or iron sulfate), amino acids and vitamins.
[64]
In the method for recovering homoserine or homoserine-derived L-amino acids produced in the culturing step of the present application, a desired product can be obtained from the culture medium using a suitable method known in the art according to the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. may be used, and homoserine or homoserine-derived L-amino acid, which is a target substance, is obtained from a medium or microorganism using a suitable method known in the art. can be recovered In addition, the recovery step may include an additional purification process, and may be performed using a suitable method known in the art.
[65]
[66]
Another aspect of the present application provides a use for increasing the production of homoserine or homoserine-derived L-amino acids of the variant homoserine dehydrogenase.
[67]
Another aspect of the present application provides a method for increasing homoserine or homoserine-derived L-amino acid production, comprising enhancing the activity of the mutant homoserine dehydrogenase in a microorganism.
[68]
In the present application, the term, "to be expressed / to be" a protein means a state in which the activity of the target protein is enhanced compared to before endogenous or modified when the target protein is introduced into a microorganism or exists in a microorganism.
[69]
Specifically, "introduction of a protein" means that the activity of a specific protein that the microorganism did not originally have or exhibited an improved activity compared to the intrinsic activity or activity before modification of the protein. For example, a polynucleotide encoding a specific protein may be introduced into a chromosome in a microorganism, or a vector including a polynucleotide encoding a specific protein may be introduced into the microorganism to exhibit its activity. In addition, "enhancement of activity" means that the activity is improved compared to the intrinsic activity or activity before modification of a specific protein of the microorganism. "Intrinsic activity" refers to the activity of a specific protein originally possessed by the parent strain before the transformation when the trait of a microorganism is changed due to genetic variation caused by natural or artificial factors.
[70]
Specifically, the enhancement of the activity of the present application includes increasing the intracellular copy number of the gene encoding the protein variant, a method of introducing a mutation into the expression control sequence of the gene encoding the protein variant, and gene expression regulation encoding the protein variant A method of replacing the sequence with a sequence with strong activity, a method of replacing a gene encoding a native protein having homoserine dehydrogenase activity on a chromosome with a gene encoding the protein variant, so that the activity of the protein variant is enhanced It may be carried out by any one or more methods selected from the group consisting of a method of further introducing a mutation into a gene encoding a protein having the homoserine dehydrogenase activity, and a method of introducing the protein mutant to a microorganism. not limited
[71]
In the above, the copy number increase of the gene is not particularly limited, but may be performed in a form operably linked to a vector or inserted into a chromosome in a host cell. Specifically, a vector capable of replicating and functioning independently of a host to which the polynucleotide encoding the protein of the present application is operably linked may be introduced into a host cell. Alternatively, a vector capable of inserting the polynucleotide into a chromosome in the host cell, to which the polynucleotide is operably linked, may be introduced into the chromosome of the host cell. Insertion of the polynucleotide into a chromosome may be accomplished by any method known in the art, for example, by homologous recombination.
[72]
Next, modifying the expression control sequence to increase the expression of the polynucleotide is not particularly limited thereto, but deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence to further enhance the activity of the expression control sequence, or their It can be carried out by inducing a mutation in the sequence in combination, or by replacing it with a nucleic acid sequence having a stronger activity. The expression control sequence is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence for regulating the termination of transcription and translation, and the like.
[73]
A strong promoter may be linked to the upper portion of the polynucleotide expression unit instead of the original promoter, but is not limited thereto. Examples of known strong promoters include cj1 to cj7 promoter (Republic of Korea Patent No. 10-0620092 call), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, P L promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (Republic of Korea Patent No. 10-1783170), O2 promoter (Korean Patent No. 10-1632642), tkt promoter, and yccA promoter, but are not limited thereto.
[74]
In addition, the modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but a mutation in the expression control sequence by deletion, insertion, non-conservative or conservative substitution of a nucleic acid sequence or a combination thereof to further enhance the activity of the polynucleotide sequence. It can be carried out by inducing or replacing the polynucleotide sequence improved to have stronger activity.
[75]
Incorporation and enhancement of such protein activity is generally performed such that the activity or concentration of the corresponding protein is at least 1%, 10%, 25%, 50%, based on the activity or concentration of the protein in the wild-type or unmodified microbial strain; It may be increased by 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000%, but is not limited thereto.
[76]
The amino acid sequence of the protein having the activity of homoserine dehydrogenase, the 407th amino acid, and the microorganism are the same as described above.
[77]
Modes for carrying out the invention
[78]
Hereinafter, the present application will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present application is not limited to these examples.
[79]
[80]
Example 1: Screening of AHV-resistant microorganisms through artificial mutagenesis
[81]
[82]
In this embodiment, L-threo of homoserine dehydrogenase (hereinafter, Hom, EC: 1.1.1.3) using Corynebacterium glutamicum KFCC10881 (Korean Patent No. 0159812) as the parent strain. In order to release the feedback inhibition by nin, an experiment was conducted to impart resistance to the L-threonine analog 2-amino-3-hydroxy-valerate (hereinafter, AHV).
[83]
Mutation was induced by artificial mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine: hereinafter, NTG). After the KFCC10881 strain cultured for 18 hours in the seed medium was inoculated into 4 ml of the seed medium, it was cultured until OD 660 became about 1.0. The culture medium was centrifuged to recover the cells, washed twice with 50 mM Tris-malate buffer (pH 6.5), and finally suspended in 4 ml of the same buffer. NTG solution (2 mg/ml in 0.05M Tris-malate buffer (pH6.5)) was added to the cell suspension to a final concentration of 150 mg/l, and then left at room temperature for 20 minutes, and then centrifuged to remove the cells. It was collected and washed twice with the same buffer to remove NTG. Finally, the washed cells were suspended in 4 ml of a 20% glycerol solution and stored at -70° C. until use. The NTG-treated strain was plated on a minimal medium containing 3 g/l of AHV, and through the above process, 126 strains of KFCC10881-derived AHV-resistant strain were obtained.
[84]
[85]
Seed medium (pH 7.0)
[86]
Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH 2 PO 4 4 g, K 2 HPO 4 8 g, MgSO 4 7H 2 O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium- Pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍ (based on 1 liter of distilled water)
[87]
[88]
Minimal medium (pH 7.2)
[89]
Glucose 5 g, KH 2 PO 4 1 g, (NH 4 ) 2 SO 4 5 g, MgSO 4 7H 2 O 0.4 g, NaCl 0.5 g Biotin 200 μg, Thiamine HCl 100 μg, Calcium-pantothenic acid 100 μg, Nicotinamide 0.03 g, urea 2 g, Na 2 B 4 O 7 10H 2 O 0.09 mg, (NH 4 ) 6 Mo 7 O 27 4H 2 O 0.04 mg, ZnSO 4 7H 2 O 0.01 mg, CuSO 4 5H 2O, MnCl 2 4H 2 O 0.01 mg, FeCl 3 6H 2 O 1 mg, CaCl 2 0.01 mg (based on 1 liter of distilled water)
[90]
[91]
Example 2: L-threonine production test against AHV resistant strain derived from KFCC10881
[92]
[93]
The L-threonine production ability test was performed on the 126 strains of AHV-resistant strains obtained in Example 1. The 126 strains obtained in Example 1 were inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium, and then cultured with shaking at 30° C. for 20 hours at 200 rpm. 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 24 ml of the following L-threonine production medium and cultured with shaking at 30° C. for 48 hours at 200 rpm.
[94]
[95]
L-threonine production medium (pH 7.2)
[96]
Glucose 30g, KH 2 PO 4 2g, 3g Urea, (NH 4 ) 2 SO 4 40g, Peptone 2.5g, CSL (Sigma) 5g (10 ml), MgSO 4 .7H 2 O 0.5g, Leucine 400mg, CaCO 3 20g (Based on 1 liter of distilled water)
[97]
[98]
After the end of the culture, the production of various amino acids produced was measured using HPLC. Table 1 shows the concentration of amino acids in the culture medium for the top 5 strains, which were shown to have excellent L-threonine-producing ability among the 126 strains tested. The five-week candidates identified through the above process were named KFCC10881-1 to KFCC10881-5, respectively.
[99]
[100]
[Table 1] Excellent AHV-resistant strain L-threonine production experiment
OD Thr Hse Gly Ile Lys Thr+Hse+Gly+Ile
KFCC10881 60.1 0.0 0.1 0.2 0.0 12.3 0.3
KFCC10881-1 53.6 4.1 1.3 1.4 1.2 2.0 8.0
KFCC10881-2 53.3 2.2 0.9 1.0 1.1 8.3 5.2
KFCC10881-3 68.5 1.5 1.2 1.1 0.2 10.8 4.0
KFCC10881-4 59.1 1.2 0.9 1.0 0.7 1.9 3.8
KFCC10881-5 49.6 2.4 1.1 1.2 0.9 5.4 5.6
[101]
[102]
As shown in Table 1, L-threonine, L-homoserine, L-glycine, and L-isoleucine produced by five strains with AHV resistance increased compared to the control, whereas L-lysine decreased. seemed
[103]
The biosynthetic pathway of L-threonine and L-lysine is split from aspartate-semialdehyde (hereinafter, ASA) as a starting point. That is, as the production of L-threonine increases, the production of L-lysine decreases. Accordingly, as the production of L-threonine increases, homoserine (Hse), L-glycine (Gly), and L-isoleucine (Ile), which can be by-products in the L-threonine biosynthesis pathway, may also increase. The total production (Thr + Hse + Gly + Ile) was also confirmed.
[104]
[105]
Therefore, among the AHV-resistant strains, a strain (KFCC10881-1) with a reduced production of L-lysine, a high production of L-threonine, and a high total production of Thr + Hse + Gly + Ile (KFCC10881-1) was selected as the most excellent AHV-resistant strain. was selected.
[106]
[107]
Example 3: Base sequence analysis of strains with excellent threonine-producing ability derived from KFCC10881
[108]
[109]
In order to analyze the nucleotide sequence of the L-threonine biosynthetic enzymes of the strains selected in Example 2, the following procedure was performed. Based on the genetic information provided by KEGG (Kyoto Encyclopedia of Genes and Genomes), the base sequence of hom encoding homoserine dehydrogenase of Corynebacterium glutamicum ATCC13032 (SEQ ID NO: 2, NCgl1136), homoserine The nucleotide sequence of thrB encoding the kinase (SEQ ID NO: 3, NCgl1137) was obtained, respectively. hom and thrB are known to form an operon structure (Peoples et al ., Mol. Biol. 2(1):63-72, 1988).
[110]
In order to secure a DNA fragment including the hom - thrB operon of the selected strains , PCR was performed with the primer combination of SEQ ID NO: 4 and SEQ ID NO: 5 using the strain cell as a template. As a polymerase for the PCR reaction, PfuUltraTM high-confidence DNA polymerase (Stratagene) was used, and PCR conditions were denaturation 96°C, 30 seconds; annealing 52° C., 30 seconds; And the polymerization reaction was repeated 30 times at 72° C. for 3 minutes. As a result, it was possible to amplify a gene fragment of 2778 bp including the base sequence of 300 bp including the promoter region at the top of the start codon of SEQ ID NO: 2 and the bottom 200 bp of the stop codon of SEQ ID NO: 3 (SEQ ID NO: 6).
[111]
[112]
The nucleotide sequence was determined using the prepared primers with ABI PRISM 3730XL Analyzer (96 capillary type, Applied Biosystems). The nucleotide sequence corresponding to hom among the hom - thrB operon in KFCC10881-1 is that guanine at base 1220 of SEQ ID NO: 2 is mutated to adenine, indicating that the CGT gene codon encoding the arginine residue has been mutated to the CAT gene codon encoding the histidine residue. (hereinafter referred to as R407H mutation; SEQ ID NO: 7). On the other hand , no mutation was found in thrB corresponding to SEQ ID NO: 3 .
[113]
[114]
When the results of the nucleotide sequence analysis are summarized, as a result, Hom (SEQ ID NO: 8) expressed in KFCC10881-1 is caused by L-threonine by mutation of arginine, the 407th amino acid residue, into histidine (hereinafter, R407H mutation). It was found that the feedback suppression was desensitized.
[115]
[116]
Example 4: Preparation of a new homoserine dehydrogenase-introduced strain
[117]
[118]
In order to prepare a strain in which the mutant (R407H) identified in Example 2 was introduced from the wild-type strain, primers of SEQ ID NO: 9 and SEQ ID NO: 10 were prepared.
[119]
[120]
To prepare a strain in which the R407H hom mutation was introduced, PCR was performed using the primers of SEQ ID NO: 9 and SEQ ID NO: 10 using the genomic DNA extracted from the KFCC10811-1 strain as a template. As a polymerase for the PCR reaction, PfuUltra TM high-confidence DNA polymerase (Stratagene) was used, and PCR conditions were denaturation 95°C, 30 seconds; annealing 55° C., 30 seconds; and the polymerization reaction at 72° C. for 2 minutes was repeated 28 times. As a result, each of the 1668 bp gene fragments including the 300 bp promoter region of the 1338 bp hom gene was obtained. The amplification product was purified using QIAGEN's PCR purification kit and used as an insert DNA fragment for vector construction. On the other hand , after treatment with restriction enzyme smaI , the molar concentration (M) ratio of the pDZ (Korean Patent No. 0924065) vector and the inserted DNA fragment amplified through PCR, which was heat-treated at 65° C. for 20 minutes, was 1:2. Thus, a vector pDZ-R407H for introducing the R407H mutation onto the chromosome was prepared by cloning using the Infusion Cloning Kit of TaKaRa according to the provided manual.
[121]
[122]
The prepared vector was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain in which each mutant base was substituted on the chromosome through a secondary crossover process was obtained. Whether or not the substitution is appropriate was determined by using the following primer combinations and using the MASA (Mutant Allele Specific Amplification) PCR technique (Takeda et al ., Hum. Mutation, 2, 112-117 (1993)) using a primer combination matching the mutant sequence. In (SEQ ID NO: 11 and SEQ ID NO: 12), it was determined primarily by selecting the amplified strain, and hom sequencing of the selected strain was mutated in the same manner as in Example 2 using the primer combination of SEQ ID NO: 11 and SEQ ID NO: 13 Secondary confirmation was carried out by type sequencing. The strain substituted with the variant base was named CA09-0900.
[123]
[124]
The strain CA09-0900 was internationally deposited with the Korea Microorganism Conservation Center (KCCM), an international depository under the Budapest Treaty, as of December 14, 2018, and was given a deposit number as KCCM12418P.
[125]
[126]
Example 5: Measurement of enzyme activity of homoserine dehydrogenase
[127]
[128]
Hom enzyme activity was measured for the prepared strain. CA09-0900 prepared in Example 4 and the wild-type strain (ATCC13032) as a control were inoculated into 25 ml of the following seed medium, and then cultured until the latter half of the log phase. Cells were recovered through centrifugation, washed twice with 0.1 M potassium phosphate (pH7.6) buffer, and finally suspended in 2 ml of the same buffer containing glycerol at a concentration of 30%. After physically crushing the cell suspension for 10 minutes by general glass bead vortexing, the supernatant is recovered through two centrifugations (13,000 rpm, 4°C, 30 minutes), and crude extract for measuring Hom enzyme activity was used as To measure Hom enzyme activity, 0.1 ml of crude enzyme solution was added to 0.9 ml of the reaction solution for enzyme activity measurement (potassium phosphate (pH7.0) buffer, 25 mM NADPH, 5 mM aspartate semi-aldehyde), and then reacted at 30°C. did Hom enzyme activity U was defined as the number of NADPH umol consumed per minute according to the presence or absence of L-threonine (0mM, 10mM), and the measurement results of enzyme activity are shown in Table 2 below.
[129]
[130]
[Table 2] Desensitization measurement by Hom enzyme activity (U) and L-threonine
strain Enzyme activity (U) according to the amount of L-threonine added (mM)
0 mM 10 mM
ATCC13032 0.91 0.02
CA09-0900 1.37 1.23
[131]
[132]
As a result of the experiment, it was found that in the case of Hom containing R407H mutation, the degree of inhibition of activity decreased in the condition that 10 mM L-threonine was included, unlike wild-type Hom, and thus it was desensitized to L-threonine. could
[133]
[134]
Example 6: Production and evaluation of microbial strains of the genus Corynebacterium having L-threonine-producing ability
[135]
[136]
A strain producing L-threonine was developed from the wild species Corynebacterium glutamicum ATCC13032. Specifically, in order to solve the feedback inhibition of aspartate kinase (lysC), which acts as the first important enzyme in the threonine biosynthesis pathway, Leucine, amino acid 377 of lysC, was substituted with Lysine (sequence number 14).
[137]
More specifically, PCR was performed using the primers of SEQ ID NO: 15 and SEQ ID NO: 16 or SEQ ID NO: 17 and SEQ ID NO: 18 using the chromosome of ATCC13032 as a template to prepare strains in which the lysC (L377K) mutation was introduced. As a polymerase for the PCR reaction, PfuUltra TM high-confidence DNA polymerase (Stratagene) was used, and PCR conditions were denaturation 95°C, 30 seconds; annealing 55° C., 30 seconds; And the polymerization reaction was repeated 28 times at 72° C. for 1 minute. As a result, a 515 bp DNA fragment at the 5' upper end and a 538 bp DNA fragment at the 3' lower end were obtained, respectively, centering on the mutation of the lysC gene. Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 15 and SEQ ID NO: 18. PCR conditions were repeated 28 times of denaturation at 95°C for 5 minutes, then 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 1023 bp DNA fragment containing a mutation in the lysC gene encoding an aspartokinase variant in which the leucine at position 377 was substituted with a lysine was amplified. The amplification product was purified using QIAGEN's PCR purification kit and used as an insert DNA fragment for vector construction. On the other hand, restriction enzyme smaIAfter treatment with the pDZ vector (Korean Patent No. 0924065), which was heat-treated at 65 ° C. for 20 minutes, the molar concentration (M) ratio of the inserted DNA fragment amplified through the PCR was 1:2 to obtain TaKaRa. A vector pDZ-L377K for introducing L377K mutations into the chromosome was constructed by cloning using the Infusion Cloning Kit of the provided manual.
[138]
The prepared vector was transformed into ATCC13032 by electroporation, and through a secondary crossover process, a strain in which each nucleotide mutation was substituted with a mutant nucleotide on the chromosome was obtained, which was named CJP1. The CJP1 was named CA01-2307 and was deposited with the Korea Microorganism Conservation Center (KCCM), an international depository under the Budapest Treaty, as of March 29, 2017, and was given an accession number KCCM12000P.
[139]
[140]
In order to clearly confirm the change in L-threonine production of the strain, the mutation identified in Example 4 was introduced into the gene encoding homoserine dehydrogenase. Specifically, in order to introduce the R407H mutation into the CJP1 strain, the pDZ-R407H vector prepared in Example 4 was transformed into CJP1 by electroporation, followed by a secondary crossover process in the same manner as in Example 4, and base mutation on the chromosome A strain in which the mutant base was substituted was obtained. The strain substituted with the variant base was named CJP1-R407H.
[141]
[142]
[Table 3] Confirmation of L-threonine production ability of production strains
strain Amino acid (g/l)
Thr Lys
CJP1 0.36 3.62
CJP1-R407H 1.50 2.47
[143]
[144]
As a result, the strain introduced with the mutation decreased the production of L-lysine and increased the production of L-threonine by 1.14 g/L compared to the control CJP1 strain, and thus it was confirmed that the desensitization effect of Hom was greatly improved. .
[145]
[146]
Example 7: Production and evaluation of microbial strains of the genus Corynebacterium having L-isoleucine-producing ability
[147]
[148]
In order to prepare an isoleucine-producing strain, a mutant gene ilvA ( V323A) ( Appl. Enviro. Microbiol. , Dec. 1996, p.4345-4351) was constructed to enhance the expression of the vector.
[149]
Specifically, ilvA around the mutation site to produce a mutagenic vector intended for gene 5 'primer for amplifying the top part one pairs (SEQ ID NO: 19 and 20) and 3' primer for amplifying a bottom portion one pairs (SEQ ID NO: Nos. 21 and 22) were devised. The primers of SEQ ID NOs: 19 and 22 inserted BamHI restriction enzyme sites at each end, and the primers of SEQ ID NOs: 20 and 21 had nucleotide substitution mutations located at sites designed to cross each other.
[150]
[151]
PCR was performed using the primers of SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 using the wild-type chromosome as a template. PCR conditions were denatured at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds repeated 30 times, followed by polymerization at 72°C for 7 minutes. As a result, a 627 bp DNA fragment at the 5' upper end and a 608 bp DNA fragment at the 3' lower end were obtained centering on the mutation of the ilvA gene.
[152]
Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 19 and SEQ ID NO: 22. After denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds were repeated 30 times, followed by polymerization at 72°C for 7 minutes. As a result, a 1217 bp DNA fragment containing a mutation in the ilvA gene encoding the IlvA variant in which valine at position 323 was substituted with alanine was amplified. pECCG117 (Republic of Korea Patent No. 10-0057684) vector and a DNA fragment of 1011 bp were treated with restriction enzyme BamHI, ligated using a DNA conjugation enzyme, and then cloned to obtain a plasmid, which was called pECCG117- ilvA (V323A). named.
[153]
The pECCG117- ilvA (V323A) vector was introduced into the CJP1-R407H strain prepared in Example 6 by electroporation, and then plated on a selective medium containing 25 mg/L of kanamycin to obtain a transformant. After culturing in the same manner as in the flask culture method shown in Example 2, the concentration of L-isoleucine in the culture medium was analyzed and shown in Table 4.
[154]
[155]
[Table 4] Confirmation of L-isoleucine production ability of the production strain
strain L-isoleucine (g/L)
CJP1/pECCG117- ilvA (V323A) 0.7
CJP1-R407H/pECCG117- ilvA (V323A) 1.4
[156]
[157]
As a result, it was confirmed that 0.7 g/L of L-isoleucine production capacity was improved in the strain containing the hom(R407H) mutation compared to the control strain.
[158]
[159]
Example 8: Production and evaluation of O-acetyl-homoserine (OAH) producing strain substituted with mutant Hom
[160]
[161]
8-1. Production of ATCC13032 strain substituted with mutant Hom
[162]
[163]
In the same manner as in Example 4 , the R407H mutation was introduced into the hom gene of the ATCC13032 strain, and this strain was named ATCC13032::Hom FBR .
[164]
[165]
8-2. Deletion of metB gene
[166]
[167]
In this embodiment, through PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, the metB gene encoding O-acetyl-homoserine degradation pathway cystathionine gamma-synthase was obtained. secured. Based on the National Institutes of Health's GenBank (NIH GenBank), the nucleotide sequence information of the metB gene (NCBI registration number Ncgl2360, SEQ ID NO: 23) is secured, and based on this, the N-terminal part of the metB gene and the linker sequence containing the Primers (SEQ ID NOs: 24, 25) and primers (SEQ ID NOs: 26, 27) containing a C-terminal part and a linker part were synthesized. PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, and oligonucleotides of SEQ ID NOs: 24, 25, and 26 and 27 as primers. Polymerase PfuUltra TM high-confidence DNA polymerase (Stratagene) was used, PCR conditions denaturation 96 ℃, 30 seconds; Annealing 53° C., 30 seconds; And after repeating the polymerization reaction at 72°C for 1 minute 30 times, the polymerization reaction was performed at 72°C for 7 minutes. As a result, the 500bp amplified gene containing the N-terminal part and linker of the metB gene and A 500bp amplified gene containing the C-terminal part of the metB gene and a linker was obtained.
[168]
[169]
PCR was performed using the two amplified genes obtained above as a template and primers of SEQ ID NOs: 24 and 27, PCR conditions: denaturation 96°C, 60 seconds; Annealing 50° C., 60 seconds; And after repeating the polymerization reaction at 72°C for 1 minute 30 times, the polymerization reaction was performed at 72°C for 7 minutes. As a result, a 1000bps amplified ΔmetB gene, a metB inactivation cassette containing the N-terminal-linker-C-terminal of the metB gene, was obtained. The metB gene obtained through the PCR was treated with restriction enzymes XbaI and SalI contained at the end, and cloned through ligation into a pDZ vector treated with restriction enzymes XbaI and SalI, and finally the metB inactivation cassette was cloned. A pDZ- ΔmetB recombinant vector was constructed.
[170]
The prepared pDZ- ΔmetB vector was transformed into Corynebacterium glutamicum ATCC13032, ATCC13032::Hom FBR , and the metB gene was inactivated on the chromosome through a secondary crossover process Corynebacterium glutamicum ATCC13032 ΔmetB , ATCC13032::Hom FBR ΔmetB was obtained. The inactivated metB gene was finally confirmed by performing PCR using the primers of SEQ ID NOs: 24 and 27 and comparing it with ATCC13032 in which the metB gene was not inactivated.
[171]
[172]
8-3. Deletion of metY gene
[173]
[174]
In this example, through PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, O-acetyl-homoserine thiolase (O-acetylhomoserine (thiol)-lyase) of the O-acetyl-homoserine degradation pathway The metY gene encoding was obtained. Based on the National Institutes of Health's GenBank (NIH GenBank), the nucleotide sequence information of the metY gene (NCBI registration number Ncgl0625, SEQ ID NO: 28) is secured, and based on this, the N-terminal part and linker sequence of the metY gene are obtained. Primers (SEQ ID NOs: 29, 30) and primers (SEQ ID NOs: 31, 32) containing a C-terminal part and a linker part were synthesized.
[175]
[176]
PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, and oligonucleotides of SEQ ID NOs: 29 and 30 and SEQ ID NOs: 31 and 32 as primers. Polymerase PfuUltra TM high-confidence DNA polymerase (Stratagene) was used, PCR conditions denaturation 96 ℃, 30 seconds; Annealing 53° C., 30 seconds; And after repeating the polymerization reaction at 72°C for 1 minute 30 times, the polymerization reaction was performed at 72°C for 7 minutes. As a result, a 500bp amplified gene containing the N-terminal portion and linker of the metY gene and a 500bp amplified gene containing the C-terminal portion and linker of the metY gene were obtained. PCR was performed using the two amplified genes obtained above as a template and primers of SEQ ID NOs: 29 and 32, PCR conditions: denaturation 96° C., 60 seconds; Annealing 50° C., 60 seconds; And after repeating the polymerization reaction at 72° C. for 1 minute 10 times, the polymerization reaction was performed at 72° C. for 7 minutes. As a result, metY amplified in a metY inactivation of 1000bps cassette containing a N-terminal-linker-C- terminal of the gene ΔmetY to obtain the gene.
[177]
The metY gene obtained through the PCR was treated with restriction enzymes XbaI and SalI contained at the end, and cloned through ligation into a pDZ vector treated with restriction enzymes XbaI and SalI, and finally the metY inactivation cassette was cloned. A pDZ- ΔmetY recombinant vector was constructed.
[178]
The constructed pDZ- ΔmetY vector was transformed into Corynebacterium glutamicum ATCC13032, ATCC13032::Hom FBR , ATCC13032 ΔmetB , ATCC13032::Hom FBR ΔmetB strains, and the metY gene was inactivated on the chromosome through a secondary crossover process. Corynebacterium glutamicum ATCC13032 ΔmetY , ATCC13032::Hom FBR ΔmetY , ATCC13032 ΔmetB ΔmetY , ATCC13032::Hom FBR ΔmetB ΔmetY were obtained. The inactivated metY gene was finally confirmed by comparison with ATCC13032 in which the metY gene was not inactivated after PCR using the primers of SEQ ID NOs: 29 and 32 was performed .
[179]
[180]
8-4. O-acetyl-homoserine production strain production and evaluation
[181]
[182]
ATCC13032, ATCC13032 ΔmetB , ATCC13032 ΔmetY , ATCC13032 ΔmetBΔmetY , ATCC13032::Hom FBR , ATCC13032::Hom prepared in Examples 8-1 to 8-3, in which the metB, metY, and metBY genes are deleted and substituted with the mutant hom gene FBR ΔmetB, ATCC13032::Hom FBR ΔmetY, ATCC13032::Hom FBR ΔmetBΔmetY O-acetyl-homoserine production capacity was compared.
[183]
[184]
Specifically, a single colony cultured overnight in LB solid medium in an incubator at 32°C was inoculated by 1 platinum in 25 ml of O-acetyl-homoserine titer medium, and cultured at 32°C at 250 rpm for 42-64 hours. O-acetyl-homoserine from the culture was analyzed by HPLC. The analysis results are shown in Table 5 below.
[185]
[186]
O-acetyl-L-homoserine production medium (pH 7.2)
[187]
G glucose 30, KH 2 PO 4 2 g, Urea 3 g, (NH 4 ) 2 SO 4 40 g, Peptone 2.5 g, CSL (Sigma) 5 g (10 ml), MgSO 4 .7H 2 O 0.5 g, Methionine 400 mg, Leucine 400 mg, CaCO 3 20 g (based on 1 liter of distilled water)
[188]
[189]
[Table 5] O-acetyl-homoserine production evaluation
strains O-AH production (g/L)
ATCC13032 - 0.0
metB 0.3
metY 0.3
metBY 0.5
ATCC13032::Hom FBR (R407H) - 0.0
metB 1.3
metY 1.5
metBY 3.7
[190]
[191]
As a result, as shown in Table 5 above, when culturing the control strain, Corynebacterium ATCC13032, O-acetyl-L-homoserine did not accumulate, while metB, metY, and metBY were inactivated ATCC13032 ΔmetB , ATCC13032 ΔmetY , ATCC13032 ΔmetBΔmetY It was confirmed that 0.3, 0.3, and 0.5 g/L of O-acetyl-L-homoserine were accumulated in the strain, respectively.
[192]
In addition, ATCC13032::Hom FBR strain in which the hom gene was substituted in the form of R407H, ATCC13032::Hom FBR ΔmetB, ATCC13032::Hom FBR ΔmetY, ATCC13032::Hom FBR ΔmetBΔmetY in which metB, metY, and metBY were inactivated, respectively In the case of the strain, it was confirmed that 1.3, 1.5, and 3.7 g/L of O-acetyl-L-homoserine were accumulated.
[193]
Therefore, it was confirmed from the above results that the production of target amino acids using homoserine as a precursor can be greatly improved by using the mutant hom of the present invention.
[194]
[195]
Example 9: Production and evaluation of L-methionine producing strains
[196]
[197]
Example 9-1: Recombinant vector construction for mcbR gene deletion
[198]
[199]
In this embodiment, in order to produce a methionine production strain, a strain in Example 6 group known cysteine methionine transcription control factor in the production in danbaekjilreul coding mcbR: (J. Biotechnol 103. 51-65 , 2003) the inactivation of the A vector was created for
[200]
Specifically, in order to delete the mcbR gene on the Corynebacterium ATCC13032 chromosome, a recombinant plasmid vector was prepared by the following method. Based on the nucleotide sequence reported to the National Institutes of Health's gene bank (NIH Genbank), the mcbR gene and peripheral sequence (SEQ ID NO: 33) of Corynebacterium glutamicum were obtained.
[201]
For the purpose of obtaining the deleted mcbR gene, using the primers of SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37 using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template, PCR was performed did PCR conditions were repeated 30 times of denaturation at 95 ° C. for 5 minutes, then denaturation at 95 ° C. for 30 seconds, annealing at 53 ° C. for 30 seconds, and polymerization at 72 ° C. for 30 seconds, followed by polymerization at 72 ° C. for 7 minutes. As a result, each 700 bp DNA fragment was obtained.
[202]
The pDZ vector and the amplified mcbR gene fragment, which cannot be replicated in Corynebacterium glutamicum, were treated with a restriction enzyme smaI for chromosome introduction, ligated using a DNA conjugation enzyme, and then transformed into E. coli DH5α and transformed into kanamycin. (25mg/ℓ) was plated on LB solid medium. After selecting colonies transformed with the vector into which the defective fragments of the desired genes were inserted through PCR, a plasmid was obtained using a plasmid extraction method, and it was named pDZ-Δ mcbR .
[203]
[204]
Example 9-2: Production and evaluation of microorganism strains of the genus Corynebacterium having L-methionine-producing ability
[205]
[206]
The pDZ-Δ mcbR vector prepared in Example 9-1 was transformed into CJP1-R407H and CJP1 strains prepared in Example 6 by homologous recombination on the chromosome by electroporation (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Thereafter, secondary recombination was performed in a solid medium containing X-gal. A strain lacking the mcbR gene was identified through PCR using primers (SEQ ID NOs: 38 and 39) for the Corynebacterium glutamicum transformant on which the secondary recombination was completed . The recombinant strains are, respectively, Corynebacterium glutamicum CJP1-R407H △ mcbR , CJP1 △ mcbR was named.
[207]
[208]
In order to analyze the L-methionine production ability of the CJP1-R407HΔ mcbR strain prepared above, it was cultured with the parent strain, Corynebacterium glutamicum CJP1Δ mcbR strain, in the following way.
[209]
Inoculated with Corynebacterium glutamicum CJP1Δ mcbR and the invention strain Corynebacterium glutamicum CJP1-R407HΔ mcbR in a 250 ml corner-baffle flask containing 25 ml of the following, and inoculated at 30 ° C. for 20 hours, Shaking culture was performed at 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium and cultured with shaking at 30° C. for 48 hours at 200 rpm. The composition of the species medium and the production medium is as follows, respectively.
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[211]

[212]
Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4 7H2O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 2000 μg ( based on 1 liter of distilled water)
[213]
[214]

[215]
Glucose 50 g, (NH4)2S2O3 12 g, Yeast extract 5 g, KH2PO4 1 g, MgSO4 7H2O 1.2 g, biotin 100 μg, thiamine hydrochloride 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 3000 μg, CaCO3 30 g ( based on 1 liter of distilled water).
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[217]
Table 6 shows the L-methionine concentration in the culture solution cultured by the above culture method.
[218]
[219]
[Table 6] Confirmation of L-methionine production ability of the production strain
strain L-Methionine (g/L)
CJP1△ mcbR 0.01
CJP1-R407H△ mcbR 0.19
[220]
[221]
As a result, it was confirmed that the L-methionine production capacity was improved by 0.18 g/L compared to the control strain in the strain containing the R407H hom mutation.
[222]
From the above results, it was confirmed that the production of L-methionine can be greatly improved by using the mutant hom of the present invention.
[223]
[224]
From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts thereof.
[225]

[226]

Claims
[Claim 1]
In the amino acid sequence of SEQ ID NO: 1, the 407th amino acid is substituted with histidine, a variant homoserine dehydrogenase.
[Claim 2]
A polynucleotide encoding the mutant homoserine dehydrogenase of claim 1.
[Claim 3]
A microorganism of the genus Corynebacterium, comprising the mutant homoserine dehydrogenase of claim 1.
[Claim 4]
The microorganism of the genus Corynebacterium according to claim 3, wherein the microorganism of the genus Corynebacterium produces homoserine or L-amino acids derived from homoserine.
[Claim 5]
The microorganism of the genus Corynebacterium 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.
[Claim 6]
The microorganism according to claim 3, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.
[Claim 7]
A method for producing homoserine or homoserine-derived L-amino acids, comprising culturing a microorganism of the genus Corynebacterium comprising the mutant homoserine dehydrogenase of claim 1 in a medium.
[Claim 8]
The method of claim 7, wherein the method further comprises recovering homoserine or homoserine-derived L-amino acids from the cultured microorganism or culture medium.
[Claim 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-acetyl-L-homoserine and L-methionine, homoserine or homoserine. A method for producing a derived L-amino acid.
[Claim 10]
The method of claim 7, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum, homoserine or homoserine-derived L-amino acid production method.
[Claim 11]
A method for increasing homoserine or homoserine-derived L-amino acid production, comprising enhancing the activity of the mutant homoserine dehydrogenase of claim 1 in a microorganism.
[Claim 12]
The use of the mutant homoserine dehydrogenase of claim 1 to increase the production of homoserine or homoserine-derived L-amino acids.