​MICROORGANISM WITH ENHANCED L-THREONINE PRODUCTIVITY AND THREONINE PRODUCTION METHOD USING SAME
​State of the art
[1]
​The present application relates to a microorganism with enhanced L-threonine productivity, and a threonine production method using same.
[2]
​BACKGROUND ART
[3]
​Threonine is widely used as a feed, a food additive, and an animal growth promoter as a kind of essential amino acid, and is used as an infusion agent and a medicine synthetic raw material for medicine. The present invention relates to a method for producing threonine using microorganisms, which is a method for blocking a threonine decomposition pathway together with a threonine biosynthesis gene (Ex PPC, Aspain, Thrashing) enhancement in order to improve yield (Kwang Chemo-Ho, Lee, et al. Molecular Dynamics Specification 2007). The genes on the threonine decomposition pathway include TDH, TDCZT, glycoprotein, ILVET, and the like, among them, is known as the highest threonine decomposition gene.​Since the yield of threonine is greatly improved when the ILVSEG gene is deleted in the decomposition gene, it is generally known to apply a high sensitivity variation to ILVET activity weakening and isoleudine.
[4]
​On the other hand, with respect to the relationship between threonine and glycine, the main precursor of serine hydroxymethyltransferase involved in glycine synthesis is serine, and the enzyme activity is 24-fold higher when using threonine as a precursor when the threonine is used as a precursor. 2002). However, there is no known bar for glycine inflow and threonine production.
[5]
​DETAILED DESCRIPTION OF THE INVENTION
​Technical Problem
[6]
​According to the present invention, glycine transporter Cyca derived from Corynebacterium ammoniagenes is introduced to develop an available microorganism by introducing glycine discharged out of a cell, and a strain producing L-threonine in a high yield is completed.
[7]
​PROBLEM SOLVING MEANS
[8]
​The present application provides a Corynebacterium sp. Microorganism producing L-threonine with enhanced glycine transporter activity.
[9]
​The present application provides a composition for producing L-threonine, comprising a microorganism of the present application.
[10]
​The present application provides a method for preparing L-threonine, comprising a step of culturing a microorganism of the present application.
[11]
​The present application provides a use of L-threonine production of Corynebacterium sp. Microorganism with enhanced glycine transporter activity.
[12]
​EFFECT OF THE PRESENT INVENTION
[13]
​The microorganism for producing L-threonine of the present invention has excellent threonine productivity, and thus can be utilized for mass production of efficient L-threonine.
[14]
​Best Mode for Carrying out the Invention
[15]
​The method of the present invention will be described in detail. Meanwhile, each of the descriptions and embodiments disclosed in the present application can 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 is not considered that the scope of the present application is limited by the specific description described below.
[16]
​In addition, a person skilled in the art could recognize or identify a plurality of equivalents for a particular aspect of the present application described in the present application using only a normal experiment. Such equivalents are also intended to be included in the present application.
[17]
[18]
​One aspect of the present application provides a Corynebacterium sp. Microorganism producing L-threonine with enhanced glycine transporter activity.
[19]
​The term "glycine transporter" of the present application is a protein having a function of introducing glycine into cells and, more specifically, can be a D-serine/D-alanine/glycine transporter. The glycine transporter can be used in combination with a D-D-serine/D-alanine/glycine transporter or glycine inflow protein.
[20]
​The "D-serine/D-alanine/glycine transporter" is a protein capable of being involved in both serine, alanine, and glycine transport, and can obtain the information by searching for D-serine/D-alanine/glycine transporter sequences in NCBI_Genbank specification, which is a known database. The transporter can be, specifically, a YYCZT protein, and more specifically, can be a chatbot protein, but is not limited thereto.
[21]
​The term "CYCA protein" of the present application refers to a protein involved in serine, alanine, and glycine uptake. The CysCA protein is coded by a Cyca gene, and the Cyca gene is known to be present in microorganisms such as Escherichia coli, Klebsiola pneumoniae, Mycobacterium bovis, Salmonella enterica, Erwinia amylovora, and Corynebacterium ammoniagenes.
[22]
​For the purpose of the present application, the peptidyl protein of the present application can be included in any of which can enhance threonine productivity. Specifically, the peptidyl protein can be derived from a Corynebacterium sp. Microorganism and, more specifically, is derived from Corynebacterium ammoniagenes, but is not limited thereto. The Corynebacterium ammoniagenes have been classified into the same classification group as Corynebacterium ammoniagenes, and are classified into the same classification group as Corynebacterium sp.​In addition, the Brevibacterium ammoniagenes are designated by being changed to Corynebacterium stages.
[23]
​Therefore, in the present application, the terms Corynebacterium ammoniagenes, Brevibacterium ammoniagenes, Corynebacterium states, and Brevibacterium stats can be used and used.
[24]
[25]
​The amphiphilic protein of the present application may comprise an amino acid sequence having homology or identity of SEQ ID NO: 16 or 70% or more.
[26]
​Specifically, the chimeric protein May comprise an amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity with the amino acid sequence of SEQ ID NO: 16. In addition, if the homology or identity is the amino acid sequence indicating the efficacy corresponding to the protein, it is apparent that some sequences are included within the scope of the present application even though some sequences have deletion, modification, substitution or added amino acid sequences.
[27]
​In addition, a polypeptide which can be formulated from known gene sequences, for example, a polypeptide encoded by a polynucleotide hybridized under stringent conditions with a complementary sequence for the entire or part of a base sequence encoding the polypeptide, can be included without limitation as a polypeptide having serine, alanine, and glycine inflow activity.
[28]
​That is, although the present application describes a protein or polypeptide comprising an amino acid sequence as a specific sequence number, a protein or polypeptide consisting of an amino acid sequence as a specific sequence number, or a polypeptide or polypeptide having an amino acid sequence as a specific sequence number, it is obvious that some sequences can be used in the present application in the present application. For example, the amino acid sequence N ′-terminal and/or the C ′-terminal has a sequence that does not alter the function of the protein, a mutation that can be naturally occurring, a potential mutation or conservative substitution thereof.
[29]
​The term "conservative substitution" means that one amino acid is substituted with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may occur generally based on the polarity of the moiety, the charge, solubility, hydrophobicity, hydrophilicity, and/or the similarity in the amphiphilic nature. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.
[30]
[31]
​In the present application, the term "polynucleotide" has the meaning inclusive of a DNA molecule or RNA molecule molecule, and the nucleotide, which is a basic configuration unit in the polynucleotide, can include a natural nucleotide as well as a similar body in which a sugar or base site is modified (Schit et al. Nucleotide et al. John Wiley & Sons, New York VII (1980); Uhlman VII and Peyman et al. Chemical Engineers, 90: 543-584 (1990)).
[32]
​The polynucleotide may be a polynucleotide encoding the peptidyl protein of the present application, or a polynucleotide encoding a polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity of the polynucleotide. Specifically, a polynucleotide encoding a protein comprising an amino acid sequence having homology or identity of SEQ ID NO: 16 or SEQ ID NO: 16 or SEQ ID NO: 16 is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity.
[33]
​In addition, it is obvious that a polynucleotide containing an amino acid sequence having the identity of SEQ ID NO: 16 or SEQ ID NO: 16 and 70% or more can also be included by codon degeneration. For example, a polynucleotide sequence encoding a protein comprising an amino acid sequence having an amino acid sequence of SEQ ID NO: 16 and an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 16 can be included without limitation. The term "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions are described in, for example, J Sambrook et al. J Sambrook et al.​, Molecular Cloning VII, A Laboratory Manual Arch, 2 nd Edition, Cold Spring Spring Harbor Laboratory Press, Cold Spring Harbor Harbor, New York, 1989; F M Engausubel et al. Current Protocols in Molecular Biology VII, John Wiley & Amp; Sons Tar, Inc. New York, NY). For example, genes having high homology or identity are hybridized to genes having at least 70%, at least 80%, specifically at least 85%, specifically at least 90%, more specifically at least 95%, and more specifically at least 97%, particularly at least 99% homology or identity, or a condition in which genes having homology or identity of 99% or more are hybridized, or a condition in which genes having low homology or identity are not hybridized, or 60° C, 1 × SSC experience, 0. 1 ​% SDS, specifically 60° C. 0.1 × SSC, 0.1% SDS, more specifically 68° C. 0.1 × SSC, 0.1% SDS, and more specifically a condition for cleaning one or more times, specifically 2-3 times.
[34]
[35]
​Hybridization requires that two polynucleotides have complementary sequences, although a mismatch between bases is possible depending on the degree of hybridization of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can be hybridized with each other. For example, with respect to DNA, adenosine is complementary to thymine and is complementary to guanine. Accordingly, the present application can also include substantially similar polynucleotide sequences as well as isolated polynucleotide fragments complementary to the entire sequence.
[36]
​Specifically, a polynucleotide having homology or identity can be detected by using a hybridization condition comprising a hybridization step at a Tm value of 55°C and using the above conditions. In addition, the Tm value may be 60° C, 63° C, or 65° C. but is not limited thereto, and can be appropriately controlled by a person skilled in the art according to the purpose.
[37]
[38]
​The term "homology" or "identity" in the present application refers to a degree associated with two given amino acid sequences or base sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably. The sequence identity or identity of a conserved polynucleotide or polypeptide is determined by a standard arrangement algorithm and a default gap penalty established by the program being used can be used together. Substantially, homologous or identical sequences can hybridize to at least about 50%, 60%, 70%, 80%, or 90% or more of the entire sequence or total-length in intermediate or high stringent conditions. The hybridization is also considered to be a polynucleotide containing livestock codons instead of codon in a polynucleotide.
[39]
​The homology or identity to the polypeptide or polynucleotide sequence is, for example, the algorithm BLAST specification by the literature (Karlin et al. Altschul et al. Pro. Natl. Acad. Sci USA Arch, 90, 5873 (1993)) or FASTA Specification by Pearson (see Methods, Enzymol). 183, 63, 1990). Based on this algorithm BLAST specification, a program called BLAST or BLSTX is developed (see http://www.ncbi.nlm.nih.gov). In addition, whether any amino acid or polynucleotide sequence has homology, similarity or identity can be confirmed by comparing the sequence with Southern hybridization experiments under defined stringent conditions, and the appropriate hybridization conditions defined therein are within the corresponding technical range and are well known to those skilled in the art (eg, J Sambrook et al.)​, Molecular Cloning Algorithm, A Laboratory Manual Arch, 2 nd Edition, Cold Spring Spring Harbor Laboratory Press, Cold Spring Harbor Harbor, New York, 1989; F M Engausubel et al. Current Protocols in Molecular Biology Concept, John Wiley & Amp; Sons Tar, Inc. New York, NY).
[40]
[41]
​The term "activity enhancement" of the term "protein" of the present application means that activity is improved compared to the intrinsic activity of a protein having microorganisms or the activity before deformation. The activity enhancement may include introducing a foreign protein and both the active enhancement of the intrinsic protein. That is, a foreign protein is introduced into a microorganism having an intrinsic activity of a specific protein, and the protein is introduced into a microorganism having no intrinsic activity. The "protein introduction" means that the activity of a particular protein is modified to be introduced into the microorganism. This can be expressed as the activity enhancement of the corresponding protein.
[42]
​The term "intrinsic" of the present application refers to a state in which a strain before a change in trait is originally present when a microorganism is changed into genetic variation due to natural or artificial factors.
[43]
[44]
​ACTIVE REINFORCEMENT IN THE PRESENT APPLICATION
[45]
1 ​METHOD FOR INCREASING COPY NUMBER OF POLYNUCLEOTIDE ENCODING PROTEIN, AND METHOD FOR INCREASING COPY NUMBER OF POLYNUCLEOTIDE ENCODING THE PROTEIN
[46]
2 ​of an expression regulatory sequence to increase the expression of the polynucleotide; and a method for modifying the expression regulatory sequence to increase the expression of the polynucleotide,
[47]
3 ​The present invention relates to a method for modifying a polynucleotide sequence on a chromosome to enhance the activity of the protein,
[48]
4 ​METHOD FOR INTRODUCING FOREIGN POLYNUCLEOTIDE SHOWING ACTIVITY OF PROTEIN OR MUTANT POLYNUCLEOTIDE OPTIMIZED FOR CODON OF POLYNUCLEOTIDE
[49]
5 ​or a combination thereof, but is not limited thereto.
[50]
[51]
​The increase in copy number of the polynucleotide is not particularly limited thereto, but can be performed in a form operably linked to a vector, or inserted into a chromosome in a host cell. Specifically, a polynucleotide encoding a protein of the present application can be operably linked to a vector capable of being replicated and functioning independently of a host to be introduced into a host cell, or the polynucleotide can be operably linked to a vector capable of inserting the polynucleotide into a chromosome in a host cell to be introduced into a host cell, thereby increasing the copy number of the polynucleotide in the chromosome of the host cell.
[52]
​The modification of the expression regulatory sequence to increase the expression of the polynucleotide is not particularly limited, but the nucleic acid sequence is deleted, inserted, non-complementary, or complementary substituted or a combination thereof so as to further enhance the activity of the expression regulatory sequence, or by replacing a nucleic acid sequence having stronger activity. The expression regulatory sequence may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosomal binding site, a sequence for regulating the termination of transcription and decryption, and the like.
[53]
​A strong heterologous promoter can be connected to an upper portion of the polynucleotide expression unit instead of an original promoter, and an example of the strong promoter is a CJ7 promoter (Korean Patent No. 0620092 and WO2006/065095), a LYSDIO -1 promoter (WO2009/096689), an EF VIII-Tu promoter, a grove promoter, an acetyl or acetyl promoter, and the like. In addition, the modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but the nucleic acid sequence is deleted, inserted, non-complementary, or complementary substituted or a combination thereof so as to further enhance the activity of the polynucleotide sequence, or by replacing a polynucleotide sequence improved to have a stronger activity.
[54]
​In addition, the introduction of a foreign polynucleotide sequence can be carried out by introducing a foreign polynucleotide encoding a protein exhibiting the same/similar activity to the protein, or a codon-optimized variant polynucleotide thereof into a host cell. The foreign polynucleotide can be used without limitation on the origin or sequence which exhibits the same/similar activity as that of the protein. In addition, the introduced foreign polynucleotide can be introduced into a host cell by optimizing the codon thereof so as to perform optimized transcription and translation within a host cell. The introduction can be carried out by selecting a known transformation method by a person skilled in the art, and a protein can be generated by expressing the introduced polynucleotide in a host cell, thereby increasing its activity.
[55]
​Finally, the method for transforming the protein to be reinforced by the combination of 1) to 4) can be carried out by applying one or more methods of modification of the expression regulatory sequence, deformation of the polynucleotide sequence on chromosome, deformation of the polynucleotide sequence on the chromosome, or deformation of the codon-optimized variant polynucleotide thereof so as to increase the copy number of the polynucleotide encoding the protein, the expression of the polynucleotide sequence on the chromosome, or the modification of the codon-optimized variant polynucleotide thereof.
[56]
[57]
​The term "vector" of the present application refers to a DNA jam preparation containing a polynucleotide sequence encoding the target protein operably linked to a regulatory sequence suitable for expressing a target protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for adjusting such transcription, a sequence encoding a suitable MDIO coronavirus binding site, and a sequence for regulating the termination of transcription and translation. The vector may be transformed into a suitable host cell, replicated or functioning independently of the host genome, and incorporated into the genome itself. For example, a polynucleotide encoding a target protein in a chromosome can be replaced with a mutated polynucleotide through a vector for inserting chromosome in a cell. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, for example, by homologous recombination.
[58]
​The vector of the present application is not specifically limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmid, cosmid, virus, and bacteriophage in a natural state or in a recombined state. For example, a phage vector or a cosmid vector can be used as a phage vector or a cosmid vector, and can be used as a plasmid vector, PQI_15, AISHII_2, APII_2, T10, T11, HAHRON4QI, and HAHRON21L, and the like, and can be used as a plasmid vector, and can be used as a PQI-based, a PQI-based, a pBluescript-based, a pGEM-based, a PTZ-based, a PQI-based, and a PET-based, and the like. In particular, PQI, PQI, PQI, PQI, PQI, PQI, PQI, PQI, PQI, PQI, PQI, PQI, and PQI may be used.
[59]
​The term "transformation" of the present application means that a vector comprising a polynucleotide encoding a target protein is introduced into a host cell so that a protein encoding the polynucleotide can be expressed in a host cell. A transformed polynucleotide can be expressed in a host cell, and can include all of these without being inserted into the chromosome of the host cell or being located in addition to the chromosome. In addition, the polynucleotide comprises a DNA molecule encoding a target protein and an RNA. The polynucleotide can be introduced into a host cell and can be expressed in any form. For example, the polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a gene structure including all elements required to be expressed by itself.​The expression cassette may typically 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 is introduced into a host cell in the form of itself to be operably connected to a sequence required for expression in a host cell, and is not limited thereto.
[60]
​In addition, the term "operably linked" refers to a promoter sequence for initiating and mediating transcription of a polynucleotide encoding a target protein of the present application, and the gene sequence being functionally connected.
[61]
​Methods for transforming a vector of the present application include any method of introducing nucleic acids into cells, and can be performed by selecting a suitable standard technique as is known in the art according to host cells. For example, electroporation, calcium phosphate (potassium phosphate) precipitation, calcium chloride (potassium chloride) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE alpha-dextran method, cationic liposome method, and acetic acid lithium-DMSO method.
[62]
[63]
​The term "microorganism producing L-threonine," according to the present application, can mean a microorganism in which the productivity of L-threonine is applied to a parent strain without the productivity of microorganism or L-threonine having L-threonine productivity, by including all microorganisms in which a wild-type microorganism or a natural or artificially genetic modification has occurred. The present invention can be a microorganism in which a specific mechanism is weakened or enhanced due to the cause of the insertion of an external gene or the activity of an intrinsic gene, or the like, and thus a genetic mutation occurs for the production of the desired L-threonine, or a microorganism having enhanced activity can be provided.
[64]
​For example, the microorganism producing L-threonine may be a microorganism with enhanced glycine transporter activity. Alternatively, the feedback restriction of the enzyme on the threonine biosynthesis pathway can be suppressed, or an enzyme involved in the threonine biosynthesis pathway can be reinforced or suppressed to produce threonine. The present invention can be a microorganism producing threonine by inactivating the activity of an enzyme or protein which does not have an effect on threonine biosynthesis and smooth metabolism to a threonine biosynthesis pathway. Alternatively, the present invention can be a microorganism in which the activity of a protein or enzyme on a route that consumes an intermediate, an auxiliary factor, or an energy source on a threonine biosynthesis pathway can be inactivated.
[65]
​More specifically, the present invention can be a microorganism in which a lysozyme kinase (LYSDOM), a homoloserine dehydrogenase (HOM) polypeptide is mutated to suppress feedback restriction of a threonine biosynthesis pathway, or enhance the expression of a protein involved in threonine production increase, or inactivated an enzyme of a threonine decomposition pathway.
[66]
​However, the present invention is merely an example, and is not limited thereto, and can be a microorganism which enhances the expression of a gene encoding an enzyme of various known L-threonine biosynthesis pathway or inactivated an enzyme of a decomposition pathway. The microorganism producing L-threonine can be manufactured by applying various methods known in the art.
[67]
​The term "inactivation of protein activity" in the present application means that the expression of an enzyme or protein is not expressed or decreased even if the expression of an enzyme or protein is not expressed or expressed at all, compared to a wild-type strain, a parent strain, or a strain that is not modified. The reduction of the gene encoding the protein, the modification of the expression regulatory sequence, a part of the gene, or the deletion of the whole protein is reduced compared to the activity of the protein having the original microorganism, and when the activity of the entire protein in the cell is lower than that of the natural or modified strain in the cell by inhibiting the expression or translation of the gene encoding the protein, a combination thereof is also included. In the present application, the deactivation can be achieved by application of various methods well known in the art.​METHOD FOR THE DELETION OF THE WHOLE OR PART OF THE GENE ENCODING THE PROTEIN;; 2) the modification of the expression regulatory sequence so as to reduce the expression of the gene encoding the protein; 3) the modification of the gene sequence encoding the protein such that the activity of the protein is removed or attenuated; 4) the introduction of an antisense oligonucleotide (eg, an antisense RNA molecule) which complementarily binds to the transcript of the gene encoding the protein; 5) forming a secondary structure by adding a sequence complementary to a sign-egg sequence to the front end of a sine-Dalgarno sequence of the gene encoding the protein; 6) a method for adding a promoter which is transferred in the opposite direction to the 3'end of the ORF (open reading frame) of the polynucleotide sequence of the gene encoding the protein.​, RTE_2), and the like, or a combination thereof, but is not particularly limited thereto.
[68]
​According to the present application, the microorganism of the present application comprises the glycine transporter and can all be a microorganism capable of producing L-threonine.
[69]
​The microorganism capable of producing L-threonine in the present application can be used in combination with a microorganism producing L-threonine, a microorganism having L-threonine productivity, and a microorganism for producing L-threonine.
[70]
[71]
​The microorganism producing threonine of the present application can further enhance the activity of the glycine decomposition protein. The "threonine-producing microorganism" and "active reinforcing" of the protein are as described above.
[72]
​The term "glycine decomposition protein" of the present application can be used as a protein directly or indirectly related to a glycine decomposition pathway, or a complex of the protein or a complex of the protein or the glycine decomposition system itself.
[73]
​Specifically, the glycine decomposition protein can be any one or more proteins selected from the group consisting of T-protein, P-protein, L-protein, H-protein, and LIPA which are the coenzyme of the glycine decomposition system, but is not limited thereto.. 13 April 2016). The glycine decomposition protein can be derived from Corynebacterium sp. Microorganism, specifically, Corynebacterium ammoniagenes, but is not limited thereto.​The CVCV protein has a sequence number of SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 40, which is a sequence of SEQ ID NO: 40, SEQ ID NO: 40, and SEQ ID NO: 42, but is not limited to a protein comprising an amino acid sequence of SEQ ID NO: 42 or a protein having 70% or more homology or identity with the protein. Specifically, the CVCV protein May comprise an amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity with the amino acid sequence of SEQ ID NO: 38. The description of the homology or identity is the same for CVIV, CVIV, SVipStar, and Sipipain.​In addition, if the homology or identity is an amino acid sequence showing efficacy corresponding to the protein, it is obvious that some sequences are included within the scope of the present application even though some sequences have deletion, modification, substitution or added amino acid sequences.
[74]
​In addition, a polypeptide which can be formulated from known gene sequences, for example, a polypeptide encoded by a polynucleotide hybridized under stringent conditions with a complementary sequence for the entire or part of a base sequence encoding the polypeptide, can be included without limitation.
[75]
​The homology or identity is as described above.
[76]
[77]
​In the present application, the term " the genome of Corynebacterium sp. Microorganism for producing L-threonine is a microorganism producing L-threonine, and a microorganism belonging to the genus Corynebacterium can mean microorganisms belonging to the genus Corynebacterium. The microorganism producing L-threonine is as described above. Specifically, the Corynebacterium sp. Microorganism having L-threonine productivity in the present application can be transformed with a vector containing a gene encoding the glycine transporter, or a Corynebacterium sp. Microorganism having improved L-threonine productivity by being transformed with a vector containing a gene encoding the glycine transporter. In addition, the present invention can provide a Corynebacterium sp. Microorganism having improved L-threonine productivity by being transformed with a vector containing a gene encoding the glycine decomposition protein or enhancing the activity of the glycine decomposition protein.​According to the present invention, the Corynebacterium sp. Microorganism having improved L-threonine productivity means a microorganism having improved L-threonine productivity than a parent strain or a non-modified microorganism before change. The non-modified microorganism is a natural Corynebacterium sp. Strain itself or a microorganism which does not include a gene encoding the glycine transporter, or a microorganism which is not transformed with a vector containing a gene encoding the glycine transporter.
[78]
​In the present application, "Corynebacterium sp. Microorganism" can include all Corynebacterium sp. Microorganisms. Specifically, Corynebacterium glutamicum, Corynebacterium genus Corynebacterium, Corynebacterium sp. Corynebacterium sp. Corynebacterium halolinae, Corynebacterium sp. Corynebacterium ammoniagenes, Corynebacterium cholerae, Corynebacterium sp. Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium ammoniag@@​In addition, the present invention can be Corynebacterium glutamicum, Corynebacterium sp. Cxorn bacteria, Corynebacterium glutamicum, or Corynebacterium flavescens and, more specifically, can be Corynebacterium glutamicum.
[79]
[80]
​Another aspect of the present application provides a composition for producing L-threonine, comprising a microorganism for producing L-threonine of the present application.
[81]
​The L-threonine production composition can mean a composition capable of producing L-threonine by microorganisms producing L-threonine of the present application. The composition comprises a microorganism producing L-threonine, and can include, without limitation, an additional configuration capable of producing threonine by using the strain. Additional configurations capable of producing the threonine may further include, for example, any suitable excipient commonly used in the fermentation composition, or a component of the medium. Such excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers, or isotonic agents, and the like, but are not limited thereto.
[82]
[83]
​Another aspect of the present application provides a method for producing L-threonine, comprising a step of culturing the microorganism.
[84]
​The medium and other culture conditions used for culturing microorganisms of the present application can be used without any particular limitation if a medium used for culturing a microorganism of the present application is a medium used for culturing a conventional Corynebacterium sp. Microorganism. Specifically, the microorganism of the present application can be cultured while controlling temperature, PQI, etc. under aerobic or anaerobic conditions in a conventional medium containing a suitable carbon source, a nitrogen source, a personnel, an inorganic compound, an amino acid, and/or a vitamin.
[85]
​In the present application, the carbon source may include: carbohydrates such as glucose, fructose, sucrose, maltose, etc. alcohols such as sugar alcohols, glycerol, and the like; fatty acids such as palmitic acid, stearic acid, linoleic acid; organic acids such as pyruvic acid, lactic acid, acetic acid and citric acid; amino acids such as glutamic acid, methionine, lysine, and the like; and the like. In addition, natural organic nutrients such as starch hydrolysate, molasses, black strap molasses, rice bran, carnauba, sugar cane residue, and corn steep liquor can be used, and carbohydrates such as a sterilized pretreatment molasses (ie, molasses converted to reduced sugar) can be used, and other appropriate amounts of carbon sources can be variously used without limitation. These carbon sources may be used alone or in combination.
[86]
​The nitrogen source may be an organic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc. amino acids such as glutamic acid, methionine, glutamine, etc. peptone, NZ ′-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or decomposition product thereof, degreasing soybean cake or decomposition product thereof. These nitrogen sources may be used alone or in combination, but are not limited thereto.
[87]
​The phosphoric acid may include a phosphoric acid first potassium, a second potassium phosphate, or a sodium-containing salt corresponding thereto. The inorganic compound may be sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like.
[88]
​In addition, a vitamin and/or a suitable precursor may be included in the medium. The medium or precursor may be added to the culture in a batch or continuous manner, and is not limited thereto.
[89]
​In the present application, the pH of the culture can be adjusted by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid and the like in the culture of microorganisms in a suitable manner to the culture. In addition, during the culturing, bubble generation can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas can be injected into the culture or nitrogen, hydrogen, or carbon dioxide gas can be injected without the injection of gas in order to maintain anaerobic and unfavorable conditions.
[90]
​The temperature of the culture may be from 25°C to 40° C. more specifically 28°C to 37° C. but is not limited thereto. The culture period may continue until the amount of production of the desired useful material is obtained, specifically from 1 hour to 100 hours, but is not limited thereto.
[91]
[92]
​The method for preparing L-threonine can comprise the steps of: collecting L-threonine from at least one material selected from among the microorganisms, the culture medium, the culture thereof, the supernatant of the culture, the extract of the culture, and the lysate of the microorganism after the culturing step.
[93]
​In the recovery step, L L-threonine, which is a target material, can be recovered from a culture medium by using a suitable method known in the art according to a culture method of the microorganism of the present application, for example, batch, continuous or aerobic culture methods, and the like. For example, the number of L-threonine can be used as precipitation, centrifugation, filtration, chromatography, and crystallization. For example, the culture can be separated by low-speed centrifugation to remove biomass, and the obtained supernatant can be separated through ion exchange chromatography, but is not limited thereto.
[94]
​The recovery step may include a purification process.
[95]
[96]
​Another aspect of the present application provides the use of L-threonine production of Corynebacterium sp. Microorganisms with enhanced activity of glycine transporter.
[97]
​The "glycine transporter", "active strengthening" or "Corynebacterium sp. Microorganism" is as described above.
[98]
​FORM FOR THE PRACTICE OF THE INVENTION
[99]
​Hereinafter, the present disclosure will be described in more detail with examples and experimental examples. However, these examples and examples are for illustrative purposes only, and the scope of the present application is not limited to these examples and examples.
[100]
[101]
​PRODUCTION OF L-THREONINE PRODUCING STRAIN USING WILD-TYPE CORYNEBACTERIUM SP
[102]
[103]
​PRODUCTION OF CORYNEBACTERIUM SP. MICROORGANISM STRAIN HAVING L-THREONINE PRODUCTIVITY
[104]
[105]
​L-THREONINE PRODUCING STRAIN IS DEVELOPED FROM GENUS CORYNEBACTERIUM GLUTAMICUM ATCC13032. Specifically, a strain in which Leucine is substituted with Lysine (SEQ ID NO: 1) for feedback inhibition of AspARTate kinase (LYSAB) acting as a first important enzyme in a threonine biosynthesis pathway (SEQ ID NO: 1), Arginine (Arginine), which is a 398th amino acid of HOM, is substituted with Glutamine (SEQ ID NO: 6) for feedback inhibition of an important homoloserin dehydrogenase (HOM) in a threonine biosynthesis pathway (SEQ ID NO: 6).
[106]
[107]
​Examples 1-1-1: LYSDIO Mutation Introduction
[108]
[109]
​Specifically, PCR was performed by using the chromosome of ATCC13032 as a template in order to manufacture the strain into which the LYSC (L377K) mutation is introduced, by using the primer of SEQ ID NO: 2 and SEQ ID NO: 3 or SEQ ID NO: 4 and SEQ ID NO: 5. PCR conditions were used as a polymerase for PCR. The PCR conditions were 95° C. 30 seconds; annealing 55° C. 30 sec; and polymerization at 72°C for 1 minute. As a result, a 515 bp DNA fragment of the 5 ′ upper portion and a DNA fragment of 538 bp of the 3'lower portion were obtained on the basis of the mutation of the LYSC gene. The amplified two DNA fragments were subjected to PCR with primers of SEQ ID NO: 2 and SEQ ID NO: 5. The PCR conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and 72°C for 2 minutes, followed by polymerization at 72°C for 5 minutes.​As a result, a 1023 bp DNA fragment containing the mutation of the LYSDIO gene encoding the aspartic kinase mutant substituted with lysine was amplified. The amplified product was purified using PCR Amplification Kit of Qiagen, and used as an insertion DNA fragment for vector production. On the other hand, after treatment with restriction enzyme (I), a PQI (Korean Patent Registration Patent No. 10-09865) vector heat-treated at 65°C for 20 minutes and a molar concentration (M +) ratio of an insertion DNA fragment amplified through the PCR are 1: 2, thereby producing a vector PQI-D377QI for introducing the LYSVII (L377K +) mutation onto a chromosome.
[110]
​The produced vector is transformed into an ATCC13032 CC by electroporation, and a strain in which each base mutation is substituted with a mutant base is obtained through a second crossing process, and the strain is designated as CJP1.
[111]
[112]
​Examples 1-1-2: HOM Variation Introduction
[113]
[114]
​PCR was performed using a primer of SEQ ID NO: 7 and SEQ ID NO: 8 or SEQ ID NO: 9 and SEQ ID NO: 10. PCR conditions were used as a polymerase for PCR. The PCR conditions were 95° C. 30 seconds; annealing 55° C. 30 sec; and polymerization at 72°C for 1 minute. As a result, a 668 bp DNA fragment of the 5'top portion and a 659 bp DNA fragment of the 3'lower portion were obtained with respect to the mutation of the HOM gene. The amplified two DNA fragments were subjected to PCR with primers of SEQ ID NO: 7 and SEQ ID NO: 10.​The PCR conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and 72°C for 2 minutes, followed by polymerization at 72°C for 5 minutes. As a result, the 1327 bp DNA fragment containing the mutation of the HOM gene encoding the homoloserine dehydrogenase mutant in which the 398th arginine is substituted with glutamine was amplified. The amplified product was purified using PCR Purification kit of QIAGEN, and used as an insertion DNA fragment for vector production. Meanwhile, a vector pDZ-R398Q for introducing HOM (R398Q) mutation onto a chromosome by Cloning according to the manual provided using a pDZ vector heat-treated at 65°C for 20 minutes and a molar concentration (M) ratio of an insertion DNA fragment amplified through the PCR is 1: 2.
[115]
​The manufactured vector was transformed into Corynebacterium glutamicum CJP1 L obtained by electroporation, and the strain is substituted with a mutant base on chromosome through a second crossing process. The strain was designated as D1-S398Q, and deposited with accession number 12120P (Korean Patent Registration No. 10-1947959) by accession number 12120P (Korean Patent No. 10-1947959).
[116]
[117]
​The present invention relates to a method for producing L-threonine-producing strain in which a D-alpha-alanine/D-serine/glycine transporter gene is introduced
[118]
[119]
​Experiment for inserting D-alanine/D-serine/glycine transporter gene into Corynebacterium glutamicum chromosome was performed in the microorganism prepared in Example 1-1.
[120]
[121]
​EXAMPLES 1-2-1: PRODUCTION OF GLYCINE TRANSPORTER DERIVED FROM CORYNEBACTERIUM AMMONIAGENES
[122]
[123]
​In order to insert a gene CYPCA encoding a glycine transporter protein, a Ncgl2131 gene encoding Transposon is used as an insertion site (Korean Patent Registration No. 10-1126041, Journal of Biotechnology, 104, 5-25 Jorn, Kalinowski et al. 2003) (SEQ ID NO: 11). In order to manufacture the Transposon insertion vector, PCR was performed using a primer of SEQ ID NO: 12 and SEQ ID NO: 13 or SEQ ID NO: 14 and SEQ ID NO: 15. The PCR condition is 95° C. 30 seconds; annealing 55° C. 30 sec; and polymerization reaction of 72°C and 2 min. The polymerization reaction is carried out at 72°C for 5 minutes.​As a result, the 2041 bp DNA fragment of the 5 ′ top site and the 2040 bp DNA fragment of the 3 ′ bottom site were obtained from the NCGL2131 gene, respectively. The amplified two DNA fragments were subjected to PCR with primers of SEQ ID NO: 12 and SEQ ID NO: 15. The PCR conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and 72°C for 4 minutes, followed by polymerization at 72°C for 5 minutes. As a result, a 4066 bp DNA fragment containing a recognition site of SPI and XhoI and two restriction enzymes was amplified at the center site. The amplified product was purified using PCR purification kit of QIAGEN, and used as an insertion DNA fragment for vector production.​On the other hand, after treatment with restriction enzyme (I), a PZT vector heat-treated at 65°C for 20 minutes and a molar concentration (M) ratio of an insertion DNA fragment amplified through the PCR are 1: 2, so as to be cloned according to the manual provided by using the Expression Cloning Kit of Dapha, a vector PQI-S2131 for insertion into the Ncgl2131 wild-type gene position was manufactured.
[124]
​The manufactured vector was transformed to KCCM12120P strain obtained in Example 1-1 by electroporation, and a strain in which Ncgl2131 gene is deleted on chromosome is obtained through a second crossing process, and the strain was named KCCM12120P-N2131.
[125]
[126]
​Meanwhile, PCR was performed using a primer of SEQ ID NO: 18 and SEQ ID NO: 19 using a primer of SEQ ID NO: 18 and SEQ ID NO: 19 to obtain a gene fragment having D-alanine/D-serine/glycine transporter activity. The PCR was carried out at 72°C for 5 minutes after repeating the denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and a polymerization reaction of 72°C and 90 seconds at 72°C for 5 minutes. As a result, 1595 bp of cycA gene fragment was obtained, and the amplified product was purified by using PCR Purification kit of QIAGEN (SEQ ID NO: 17) to be used as an insertion DNA fragment for vector production (SEQ ID NO: 17).
[127]
​PCR was performed using p11.7-CJ7-GFP containing promoter CJ7 derived from Corynebacterium sp. Microorganism as a template (Korean Patent No. 10-0620092). Here, "P117" is an E coli-Corynebacterium shuttle vector (Biotechnology Letters: 13 (10): 721-726, 1991). The PCR reaction is carried out at a temperature of 95° C, 30 seconds, annealing 55° C, 30 seconds, and a polymerization reaction of 72°C and 30 seconds using a primer of SEQ ID NO: 21 and SEQ ID NO: 22, followed by polymerization at 72°C for 3 minutes. The amplified PCR product was purified using PCR Purification Kit of Qiagen, and a CJ7 fragment having a size of 350 bp was obtained (SEQ ID NO: 20).
[128]
​The CJ7 fragment and the CysCA fragment obtained above were prepared as a template, and a fusion PCR was performed using the primers of SEQ ID NO: 21 and SEQ ID NO: 19. The PCR reaction is carried out at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and a polymerization reaction at 72°C for 5 minutes. As a result, a CJ7-CYCA gene fragment of 1945 bp was obtained, and the amplified product was purified using a PCR purification kit of Qiagen to be used as an insertion DNA fragment for vector production. Meanwhile, the PDZ-N2131 vector produced above is treated with XhoI, heat-treated at 65°C for 20 minutes, and the molar concentration (M) ratio of the obtained CJ7-CYCA fragment is 1: 2, so that a vector PDZ-N2131/CJ7-CYCA (CAM) capable of inserting the CYPCA gene into the NCGL2131 position is manufactured.
[129]
​The manufactured vector was transformed to KCCM 12120P strain by electroporation, and a strain in which the position of Ncgl2131 is substituted in the form of CJ7-7 is obtained on the chromosome through a second crossing process, and the strain was designated as "Cam".
[130]
[131]
​EXAMPLE 1-2. Preparation of E coli-derived glycine transporter
[132]
[133]
​Meanwhile, in order to compare the CysCA gene derived from Corynebacterium ammoniagenes and its activity, the CysCA gene derived from Escherichia coli K -12 is introduced to the KCCM 12120P strain (Microbiology, 141 (Pt 1); 133-40, 1995). Specifically, PCR was performed by using a primer of SEQ ID NO: 25 and SEQ ID NO: 26 using a primer of SEQ ID NO: 25 and SEQ ID NO: 26. The PCR was carried out at 72°C for 5 minutes after repeating the denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and a polymerization reaction of 72°C for 5 minutes. As a result, a cycA gene fragment of 1544 bp was obtained, and the amplified product was purified using a PCR Purification kit of Qiagen to be used as an insertion DNA fragment for vector production (SEQ ID NO: 23).
[134]
​The CJ7 fragment and the CysCA fragment obtained above were prepared as a template, and a fusion PCR was performed using the primer of SEQ ID NO: 21 and SEQ ID NO: 26. The PCR reaction is carried out at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and a polymerization reaction of 72°C and 90 seconds, followed by polymerization at 72°C for 5 minutes. As a result, a CJ7-CYCA gene fragment of 1894 bp was obtained, and the amplified product was purified using a PCR purification kit of Qiagen to be used as an insertion DNA fragment for vector production. Meanwhile, the PDZ-N2131 vector produced above is treated with XhoI, heat-treated at 65°C for 20 minutes, and the molar concentration (M) ratio of the obtained CJ7-CYCA fragment is 1: 2, so that a vector PDZ-N2131/CJ7-CYCA (ECO) capable of inserting the CYPCA gene into the NCGL2131 position is manufactured.
[135]
​The manufactured vector was transformed to KCCM 12120P strain by electroporation, and a strain in which the position of Ncgl2131 is substituted in the form of CJ7-7 is obtained on the chromosome through a second crossing process, and the strain was named 12120V/cc (Eco).
[136]
[137]
​Example 1-3: Evaluation of L-threonine productivity of a strain into which CYCA is introduced
[138]
[139]
​The L-threonine production ability test of the strains manufactured in Examples 1 and 2 was performed. A 250 ml corner-basket flask containing 25 ml of paper medium was inoculated with each of the strains obtained above and incubated at 30°C for 20 hours at 200 rpm. A 250 ml corner-bottom flask containing 24 ml of L L-threonine production medium was inoculated with 1 ml of a culture medium and incubated at 30°C for 48 hours at 200 rpm
[140]
[141]
​PAPER MEDIUM (PETA 7.0)
[142]
​20 g of glucose, 10 g of peptone, 5 g of yeast extract, 1.5 g of urea, 4 g of KH2PO4, 8 g of K2HPO4, 7 H2O of KH2PO4, 100 μg of biotin, 1000 μg of thiamine, 2000 μg of calcium-pantothenic acid, 2000 μg of nicotinamide (based on 1 liter of distilled water)
[143]
[144]
​L-THREONINE PRODUCTION MEDIUM (P207.2)
[145]
​30 g of glucose, 2 g of KH2PO4, 3 g of Urea, 3 g of (NH4) 2SO4, 2.5 g of Peptone, 5 g (10 ml) of CSL_3 (Sigma_2O_3), 4.5 g of Peptone, 400 mg of Leucine, 3 g (of distilled water, 1 L of distilled water)
[146]
[147]
​The production of various amino acids produced using HPLC after completion of culture was measured. The results are shown in Table 1.
[148]
[149]
[표1]
​STRAIN ​Amino acid (g/L)
​TAR TAR ​Gly-ARCH ​Ser ​Lys TAR ​Ile ARCH ​Ala ​Val LANDING
​CCM12120 1.61 0.27 0.04 2.73 0.07 0.14 0.04
​International filing date (day/month/year) 1.60 0.28 0.04 2.74 0.08 0.15 0.05
​CCM12120/DCAM 1.81 0.22 0.03 2.61 0.07 0.00 0.03
​KCTCCM12120V/strontium (Eco) 1.37 0.30 0.05 2.92 0.07 0.02 0.03
[150]
​As the production amount of L-threonine is increased, the production of L-lysine decreases, while the L L-threonine production amount increases, L L-isoleucine, glycine (Gly), which can be a by-product of L L-threonine biosynthesis pathway, can be increased, and their production is confirmed together. Also, the productivity of serine (Ser), alanine (Ala), and valine (Val) is also confirmed.
[151]
​As with the results of Table 1, L L-threonine produced by the Corynebacterium ammoniagenes derived from Corynebacterium ammoniagenes is 12.5% increased while L L-lysine decreased by 4%. In addition, glycine is reduced by 18.5% compared to the parent strain, and serine and valine are reduced in small width compared to the parent strain, and alanine is remarkably reduced to confirm that it is not detected in the culture medium.
[152]
​On the other hand, the KCCM12120P/cycA (ECCO) strain in which the E coli-derived cycA gene has been introduced has a reduction of 14.9% of L-threonine production compared to the parent strain, whereas L-lysine has 6.9% of L-lysine, and 10% of glycine increases. As described above, the effect of introducing cycA gene derived from E coli and Corynebacterium ammoniagenes is very different, but the concentration of alanine in the culture medium is significantly reduced. In addition, in the case of introducing cycA derived from E coli-derived cycA or Corynebacterium ammoniagenes, the amount of isoleucine production was not changed.
[153]
​Based on the above results, it was confirmed that the introduction of Corynebacterium ammoniagenes from Corynebacterium ammoniagenes is effective in the production of L-threonine.
[154]
​According to the present invention, a deposit number of KCCM12484P is given by accession number KCCM12484P on Apr. 10, 2019, which is a Korean Institute of Microbial Preservation Center (KCCM12484P), which is a National Institute of Microbial Storage Center (KCCM12484P), which is a National Institute of Microbial Storage Center (KCCM12484P), which is a National Institute of Microbial Storage Center (KCCM12484P), which is a National Institute of Microorganisms under the Advenst.
[155]
[156]
​EXAMPLE 1-4. Production of L-threonine-producing strain in which the corresponding gene is introduced
[157]
[158]
​METHOD FOR PRODUCING VECTOR FOR INTRODUCING GLYCINE DEGRADING PROTEIN
[159]
[160]
​It has been confirmed that the activity of the glycine transporter affects the increase in threonine production capacity, and thus the ability to produce threonine in the case of further increasing the ability of intracellular utilization of glycine introduced into the cell is confirmed. Specifically, a Glycine cleavage system (GCV) system is introduced. In the Corynebacterium glutamicum strain, only a gene encoding L VII-protein, LIB, or LIPQI in six species constituting a GCV harvesting system is known, and a gene encoding the other three kinds of proteins is not known. Therefore, in order to introduce a GCV system derived from Corynebacterium ammoniagenes, GCV (SEQ ID NO: 27), GCVBS (SEQ ID NO: 28), GCVBS (SEQ ID NO: 29), LIPOP (SEQ ID NO: 30), and LIB (SEQ ID NO: 31) were produced. The following experiment was carried out.​The PCR was performed using a primer of SEQ ID NO: 32 and SEQ ID NO: 33 to obtain a gcvPT gene fragment from among the genes corresponding to the GCV system. The PCR was carried out at 72°C for 7 minutes after repeating 95° C. 30 seconds; annealing 55° C. 30 sec; and polymerization at 72°C for 7 minutes. As a result, a gcvPT gene fragment of 4936 bp including a promoter was obtained, and the amplified product was purified using a PCR Purification kit of QIAGEN to be used as an insertion DNA fragment for vector production (SEQ ID NO: 27, 28).
[161]
​In order to obtain a gcvH-lipBA gene fragment which is another GCV system, PCR was performed using a primer of SEQ ID NO: 34 and SEQ ID NO: 35 using a primer of SEQ ID NO: 34 and SEQ ID NO: 35. The PCR was carried out at 72°C for 5 minutes after repeating the denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and a polymerization reaction of 72°C for 5 minutes. As a result, a 3321 bp gcvH-lipBA gene fragment containing a promoter was obtained, and the amplified product was purified using a PCR Purification kit of QIAGEN to be used as an insertion DNA fragment for vector production (SEQ ID NO: 29, 30, 31).
[162]
​The GCVPT fragment and the GCCVH-LIPBA fragment obtained above were given as a template, and a fusion PCR was performed using the primers of SEQ ID NO: 32 and SEQ ID NO: 35. The PCR reaction is carried out at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and a polymerization reaction at 72°C for 12 minutes. As a result, 8257 bp of GCV-LIPBA gene fragment was obtained, and the amplified product was purified using PCR purification kit of QIAGEN to be used as an insertion DNA fragment for vector production.​On the other hand, the PQI-S2131 vector produced in Examples 1 and 2 was treated with SpeI and XhoI, and then, the GCV system gene can be inserted into the Ncgl2131 site by Cloning the GCV system gene according to the manual provided by using the Fusion Cloning Kit of Dapha.
[163]
[164]
​METHOD FOR PRODUCING A GLYCINE-DEGRADING PROTEIN-INTRODUCED STRAIN
[165]
[166]
​PCR was performed using a primer of SEQ ID NO: 36 and SEQ ID NO: 37 using the pDZ-N2131/CJ7-cycA (Cam) vector obtained in Example 1-2 as a template. The PCR was carried out at 72°C for 5 minutes after repeating the denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and a polymerization reaction of 72°C and 90 seconds at 72°C for 5 minutes. As a result, a CJ7-cycA (Cam) gene fragment of 1944 bp was obtained, and the amplified product was purified using a PCR Purification kit of Qiagen to be used as an insertion DNA fragment for vector production.​On the other hand, a vector PQI-Z2131/GCV-LIP-LIP (Cam) capable of inserting a GCV system gene and a D-alpha-alanine/D-serine/glycine transporter gene into an Ncgl2131 + position is manufactured by treating the PQI-Z2131/GCV-LIP-LIPQI vector produced above with restriction enzyme XhoI and heat-treating at 65°C for 20 minutes.
[167]
​The manufactured vector was transformed to KCCM 12120P strain by electroporation, followed by a second crossing process to obtain a strain in which the position of Ncgl2131 in chromosome is substituted in the form of GCV-VII-LIP-strontium (Cam +).
[168]
[169]
​METHOD FOR EVALUATING THREONINE PRODUCTION ABILITY OF GLYCINE TRANSPORTER AND GLYCINE DECOMPOSITION PROTEIN INTRODUCTION STRAIN
[170]
[171]
​L L-threonine productivity test of the strains manufactured in Examples 1-2 and 1-4-2 is performed. The strains obtained above were inoculated into a 250 ml corner-basket flask containing 25 ml of the above seed medium, and then cultured at 30°C for 20 hours at 200 rpm. The L-threonine production medium contains 24 ml of L-threonine production medium, and 1 ml of a culture medium is inoculated into a 250 ml corner-basket flask containing 24 ml of L-threonine production medium, and cultured at 30°C for 48 hours at 200 rpm.
[172]
​The production of various amino acids produced using HPLC after completion of culture was measured. The results are shown in Table 2.
[173]
[174]
[표2]
​STRAIN ​Amino acid (g/L)
​TAR TAR ​Gly-ARCH ​Ser ​Lys TAR ​Ile ARCH ​Ala ​Val LANDING
​CCM12120 1.61 0.27 0.04 2.73 0.07 0.14 0.04
​International filing date (day/month/year) 1.60 0.28 0.04 2.74 0.08 0.15 0.05
​CCM12120/DCAM 1.81 0.22 0.03 2.34 0.07 0.00 0.03
​CCM12120V/GCV-LIP-RATCHET (Cam ARCH) 2.04 0.13 0.02 2.36 0.10 0.00 0.01
[175]
​As the result of Table 2, the GCV system gene is introduced together with the D-alpha-alanine/D-serine/glycine transporter gene. The strain is 26.7% improved L L-threonine production compared to the strain of KCCM 12120P, which is a parent strain, which is a yield of 12.7% with respect to the strain of KCTC12120P/GCV. Based on the above, it was confirmed that the L L-threonine production amount of the strain introduced with the GCV system gene is further increased.
[176]
​The L L-lysine productivity of the KCC 12120P/GCV-LIP-SCF (CAM +) strain was 13.6% compared to KCCM12120P, and the strain was 14.3% compared to KCCM12120P. The L L-isoleucine production amount was increased compared to KCCM 12120P in the accession number of KCTC12120V/GCV-LIP-PTH (CAM +). However, when considering the results of Examples 1-3, it can be seen that L L-isoleucine, which is a by-product of a biosynthetic pathway, increases with an increase in L-threonine production.
[177]
​The glycine production was reduced 18.5% when only a wild-type gene was introduced from the KCCM 12120P strain and 51.9% compared to the KCCM 12120P strain when GCV system genes have been introduced together. Alanin was reduced to a large width only by the introduction of chimeric gene, and it was confirmed that there is no adverse effect due to the introduction of GCV system. According to the present invention, the production amount of serine and valine is very small, but it was confirmed that all of the strain KCCM 12120P/GCV-LIP-strontium (Cam) strain were reduced compared to the parent strain KCCM 12120P.
[178]
​The accession number 12120V/GCV-LIP-PTH (CAM +) strain of the present invention was designated as CA09-0905, and the deposit number is given as KCCM12485P in Korean Culture Center (KCCM) on Apr. 10, 2019, which is a Korean Institute of Microbial Storage Center (KCCM12485P), which is a National Institute of Microbial Storage Center (KCCM12485P).
[179]
[180]
​On the basis of the above results, it was confirmed that L-threonine production is increased when a wild-type gene, which is a Corynebacterium ammoniagenes-derived D-alanine/D-serine/glycine transporter gene, is introduced on the basis of a Corynebacterium sp. L-threonine production strain. In addition, when the GCV system corresponding to the GCV system is introduced together with the chatbot gene, the L-threonine production amount increases to a greater width.
[181]
[182]
​EXAMPLE 2. Confirmation of L-threonine production ability of L-threonine-producing strain
[183]
[184]
​As shown in Example 1, experiments were carried out to confirm the effect of introducing YCZT and GCV system into existing threonine production strains.
[185]
[186]
​EXAMPLE 2-1. Introduction to L L-Threonine Producing Strain KCCM11272P Strain
[187]
[188]
​According to the present invention, a strain is obtained by transforming a strain of Corynebacterium glutamicum KCCM112222P (Korean Registration Patent No. 10-1335853), which is a strain of L L-threonine production strain, which is a strain of L L-threonine production strain, and the positions of Ncgl2131 are respectively deleted on chromosome through a second crossing process.
[189]
[190]
​The L-threonine production ability test of the manufactured strains is performed. A 250 ml corner-basket flask containing 25 ml of paper medium was inoculated with each of the strains obtained above and incubated at 30°C for 20 hours at 200 rpm. A 250 ml corner-bottom flask containing 24 ml of L L-threonine production medium was inoculated with 1 ml of a culture medium and incubated at 32°C for 48 hours at 200 rpm
[191]
[192]
​PAPER MEDIUM (PETA 7.0)
[193]
​20 g of glucose, 10 g of peptone, 5 g of yeast extract, 1.5 g of urea, 4 g of KH2PO4, 8 g of K2HPO4, 7 H2O of KH2PO4, 100 μg of biotin, 1000 μg of thiamine, 2000 μg of calcium-pantothenic acid, 2000 μg of nicotinamide (based on 1 liter of distilled water)
[194]
[195]
​L-THREONINE PRODUCTION MEDIUM (PETA 7.0)
[196]
​100g of glucose, 2 g of KH2PO4, 3 g of Urea, 3 g of (NH4) 2SO4, 4.5 g of Peptone, 2.5 g of Peptone, 5 g of CSL (Sigma), 4.5 g of Peptone, 100 μg of biotin, 1000 μg of thiamine, 2000 μg of calcium-pantothenic acid, 3000 μg of nicotinamide, 30 g of thiamine (distilled water, 1 L of distilled water)
[197]
​The production of various amino acids produced using HPLC after completion of culture was measured. The results are shown in Table 3.
[198]
[199]
[표3]
​STRAIN ​Amino acid (g/L)
​TAR TAR ​Gly-ARCH ​Ser ​Lys TAR ​Ile ARCH ​Ala ​Val LANDING
​CCM11222T 7.64 1.30 0.86 3.68 1.46 0.60 0.46
​International filing date (day/month/year) 7.66 1.28 0.88 3.68 1.47 0.59 0.41
​ 8.80 1.02 0.85 3.13 1.42 0.01 0.42
​KCTCCM11222T/strontium (Eco) 6.67 1.35 0.90 4.42 1.49 0.05 0.40
[200]
​As shown in Table 3, an L-L-threonine production amount increased by 15.2% compared to KCCM112P, which is a parent strain, is shown in Table 3, while L L-lysine productivity was reduced by 14.9%. Glycine and alanine showed a result of 21.5% and 98.3%, respectively, compared to KCCM112P, and confirmed to be similar to the evaluation result on the basis of the KCCM12120P strain of Example 1. In the case of isoleucine, the strain is reduced by 3% compared to KCCM112P, and the accession number KCTC222T/strontium (ECO_2) strain is increased by 4% compared to KCCM112P, and thus it was confirmed that the introduction of protein protein is not significantly affected by the production of isoleucine. In particular, the present invention confirms that isoleucine production is reduced in the case of a strain in which a Corynebacterium ammoniagenes-derived chimeric gene is introduced.
[201]
​On the other hand, the Accession No. KCTC11222P/SCF (ECO) strain in which the E coli-derived chatbot gene has been introduced has an L L-threonine production rate of 12.7% and an L L-lysine productivity of 20.1%, respectively, compared to the KCCM 11272P strain. Therefore, it was confirmed that the E coli-derived mitotic gene has no effect on the production of L-threonine production.
[202]
​Accordingly, the present invention can increase the productivity of threonine by introducing Corynebacterium ammoniagenes derived from Corynebacterium ammoniagenes into a mutant having threonine productivity.
[203]
[204]
​The present invention also relates to a method for producing an L-threonine-producing strain (KCCM112P)
[205]
[206]
​In the same manner as in Example 1, a vector PQI-Z2131/GCV-LIP-motif (Cam SCF), which is manufactured in order to confirm a combination effect of a Cam receptor gene and a GCV system gene, is transformed to the KCCM112P strain strain, and a strain in which Ncgl2131 is substituted in the form of GCV-LIP-lactam is obtained through a second crossing process.
[207]
[208]
​The L-threonine production ability test of the manufactured strains is performed. The strains obtained above were inoculated into a 250 ml corner-basket flask containing 25 ml of the above seed medium, and then cultured at 30°C for 20 hours at 200 rpm. The L-threonine production medium contains 24 ml of L-threonine production medium, and 1 ml of a culture medium is inoculated into a 250 ml corner-basket flask containing 24 ml of L-threonine production medium, and cultured at 32°C for 48 hours at 200 rpm.
[209]
​The production of various amino acids produced using HPLC after completion of culture was measured. The results are shown in Table 4.
[210]
[211]
[표4]
​STRAIN ​Amino acid (g/L)
​TAR TAR ​Gly-ARCH ​Ser ​Lys TAR ​Ile ARCH ​Ala ​Val LANDING
​CCM11222T 7.63 1.32 0.87 3.70 1.45 0.60 0.45
​International filing date (day/month/year) 7.64 1.29 0.88 3.68 1.45 0.58 0.40
​ 8.81 1.04 0.85 3.11 1.42 0.01 0.41
​CCM11222T/GCV-LIP-RATCHET (Cam ARCH) 10.31 0.57 0.49 3.17 1.50 0.00 0.31
[212]
​As a result of Table 4, the L L-threonine production amount increased by 35.1% compared to KCCM112P, which is a parent strain, is exhibited, which is an L L-threonine production amount which is 17.0% increased compared to the accession number KCTC11222P/GCV-LIP (Cam +) strain. On the basis of the same, it was confirmed that the L L-threonine production amount of the strain introduced with the GCV system gene is further increased compared to the case in which the GCV system gene is introduced alone, as in Example 1, in the same manner as in Example 1.
[213]
​The L L-lysine productivity of the Bacillus subtilis 112222T/GCV-LIP-SCF (CAM +) strain was reduced 14.3% compared to KCCM112P, whereas the strain of KCTC11222P/GCV-LIP (CAM +) strain decreased 15.9% compared to KCCM112P. The L L-isoleucine production amount was 3.4% increased compared to KCCM 1122P in the accession number KCTC222DIO/GCV-LIP-IPAM strain. However, when considering the results of Example 2-1, it is possible to interpret the L L-threonine production as an incidental effect due to an increase in the production of L-threonine.
[214]
​The glycine production was reduced to 21.2% when only a wild-type gene was introduced from the KCCM112P strain strain, and 56.8% of the KCCM112P strain was reduced when GCV system genes have been introduced together. Alanin was reduced to a large width only by the introduction of wild-type gene, and was further reduced by GCV system introduction to not be measured in the GCV-Lip-Lip-Law strain. According to the present invention, the serine and valine productivity of the Bacillus subtilis 112222T/GCV-LIP-LSCF (Cam +) strain was 43.7% and 31.1%, respectively, compared to the parent strain KCCM 11272P.
[215]
[216]
​From the above description, those of ordinary skill in the art will appreciate that the present application may be practiced in other specific forms without altering the technical idea or essential features thereof. In this regard, it should be understood that the embodiments described above are exemplary and not restrictive in all respects. The scope of the present application is to be construed as being included in the scope of the present application in all changes or modifications derived from the meaning and range of the claims, and the equivalent concept of the appended claims rather than the detailed description.