Dihydrodipicolinic acid reductase mutant polypeptide and L-threonine production method using same
technical field
[1]
The present application relates to a mutant polypeptide having weakened dihydrodipicolinate reductase activity and a method for producing L-threonine using the same.
[2]
background
[3]
Corynebacterium genus (the genus Corynebacterium ) Microorganisms, for example, Corynebacterium glutamicum ( Corynebacterium glutamicum ) is a gram-positive microorganism that is widely used for the production of L- amino acids and other useful substances. In order to produce the L-amino acid and other useful substances, various studies are being conducted for the development of high-efficiency production microorganisms and fermentation process technology. For example, a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in L-lysine biosynthesis or removing a gene unnecessary for L-lysine biosynthesis is mainly used (US Patent Registration) 8048650).
[4]
On the other hand, among L-amino acids, L-lysine, L-threonine, L-methionine, L-isoleucine, and L-glycine are aspartate-derived amino acids, and biosynthetic branches from aspartate to L-lysine and other aspartate-derived amino acids. Effective conversion of aspartyl semialdehyde, a common precursor acting on ), into homoserine may affect the synthesis level of L-threonine.
[5]
Dihydrodipicolinate reductase is an enzyme that acts on the lysine biosynthesis pathway of microorganisms. It is used for biosynthesis of lysine from aspartyl semialdehyde, a common precursor of microbial threonine biosynthesis and lysine biosynthesis. It is an important enzyme that acts immediately after dihydrodipicolinate synthase in transporting it to the pathway.
[6]
Since diaminopimelate, a precursor of lysine biosynthesis, is used in the formation of peptidoglycan constituting the cell wall of microorganisms, dihydrodipicoline acting during the diaminopimelate production pathway When the dapB gene encoding an acid reductase is deleted, the cell wall synthesis of the microorganism is inhibited, and thus the growth of the strain is inhibited.
[7]
Therefore, in order to improve L-threonine production capacity, an approach for reducing the gene acting on the biosynthetic pathway of L-lysine to an appropriate level rather than deletion is still required.
[8]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[9]
Under this background, the present inventors studied dihydrodipicolinic acid reductase activity to a specific level as a result of intensive research to increase the production of L- threonine while decreasing the production of L-lysine without delaying the growth rate of the strain. This application was completed by confirming that the production of L-threonine as well as the growth of microorganisms is maintained when the novel mutant polypeptide is used.
[10]
means of solving the problem
[11]
One object of the present application is to provide a dihydrodipicolinate reductase (dihydrodipicolinate reductase) variant polypeptide derived from Corynebacterium glutamicum.
[12]
Another object of the present application is to provide a polynucleotide encoding the variant polypeptide.
[13]
Another object of the present application is to provide a microorganism of the genus Corynebacterium (Corynebacterium sp.) comprising the dihydrodipicolinate reductase (dihydrodipicolinate reductase) variant polypeptide.
[14]
Another object of the present application is to provide a method for producing L-threonine using the microorganism.
[15]
Another object of the present application is to provide a use of the microorganism for producing L-threonine.
[16]
Effects of the Invention
[17]
When a novel mutant polypeptide with weakened dihydrodipicolinic acid reductase activity of the present application is used, compared to the strain having wild-type dihydrodipicolinic acid reductase activity, the production of lysine is reduced and the production of threonine is reduced without delay in the growth rate. It can be used widely for mass production of threonine as it can increase production.
[18]
Best mode for carrying out the invention
[19]
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.
[20]
[21]
One aspect of the present application for achieving the above object provides a dihydrodipicolinate reductase (dihydrodipicolinate reductase) variant polypeptide derived from Corynebacterium glutamicum.
[22]
Specifically, the Corynebacterium glutamicum-derived dihydrodipicolinate reductase variant polypeptide has the same sequence as the Corynebacterium glutamicum-derived dihydrodipicolinic acid reductase sequence. It provides a polypeptide having dihydrodipicolinic acid reductase activity, which serves as a reference sequence to be mutated and contains one or more amino acid substitutions. More specifically, the substitution of the amino acid provides a dihydrodipicolinic acid reductase variant polypeptide comprising the thirteenth amino acid substituted with another amino acid.
[23]
The dihydrodipicolinic acid reductase (dihydrodipicolinate reductase) variant polypeptide may have the same sequence as the dihydrodipicolinic acid reductase sequence derived from Corynebacterium glutamicum. For example, the 13th amino acid in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 98% or more identity therewith is asparagine, Threonine, Cysteine, Tyrosine, Serine , Lysine (Lysine) or glutamine (Glutamine) will be substituted, it may be a variant polypeptide. For example, it provides a variant polypeptide having dihydrodipicolinic acid reductase activity in which the 13th amino acid is substituted with asparagine in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 98% or more identity or homology thereto.
[24]
In this case, the amino acid sequence having 98% or more identity may consist of SEQ ID NO: 51. In addition, the dihydrodipicolinate reductase (dihydrodipicolinate reductase) variant polypeptide may consist of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 53.
[25]
[26]
In the present application, "dihydrodipicolinate reductase (Ec 1.3.1.26)" refers to the common biosynthesis of aspartate-derived amino acids L-methionine, L-threonine, L-isoleucine, and L-lysine in microorganisms. Aspartyl semialdehyde, an intermediate material, is converted to 2,3-dihydeopicolinate through the enzyme dihydrodipicolinate synthase, and this is It refers to an enzyme that catalyzes lysine biosynthesis by converting it to piperidine 2,6-dicarboxylate, a precursor of lysine biosynthesis.
[27]
In the present application, dihydrodipicolinic acid reductase may be included regardless of origin as long as it is a polypeptide having the conversion activity, and an enzyme derived from any organism (plants and microorganisms, etc.) may be used. Specifically, the dihydrodipicolinic acid reductase is the same as the sequence derived from the microorganism of the genus Corynebacterium, and more specifically, it may be identical to the sequence derived from Corynebacterium glutamicum .
[28]
In the present application, Corynebacterium glutamicum-derived dihydrodipicolinic acid reductase means that the same sequence as the microorganism-derived dihydrodipicolinic acid reductase is also included.
[29]
For example, it may be SEQ ID NO: 1 or a sequence having 98% or more identity or 99% or more identity thereto, but is not limited thereto. Such a sequence may be, for example, a polypeptide comprising the amino acid sequence of SEQ ID NO: 51.
[30]
The polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 51 may be used in combination with a polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 51, or a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 51 .
[31]
In the present application, various methods well known in the art are applicable to the method of securing dihydrodipicolinic acid reductase. Examples of the method include gene synthesis technology including codon optimization to secure polypeptides with high efficiency in microorganisms of the genus Corynebacterium, which are commonly used for polypeptide expression, and bioinformatics methods based on large-scale genomic information of microorganisms. It can be secured through a screening method of useful enzyme resources by the present invention, but is not limited thereto.
[32]
[33]
The "polypeptide having the activity of dihydrodipicolinic acid reductase" in the present application refers to the amino acid sequence of a polypeptide having the activity of dihydrodipicolinic acid reductase, for example, the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 51, before and after It does not exclude the addition of meaningless sequence or a naturally occurring mutation, or a silent mutation thereof, and exhibits the same or corresponding activity to the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 51 If it has, it corresponds to a polypeptide having the activity of dihydrodipicolinic acid reductase of the present application.
[34]
As a specific example, the polypeptide having the activity of dihydrodipicolinic acid reductase of the present disclosure is the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 51 or at least 60%, 70%, 80%, 83%, 84%, 85% thereof. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% homology or identity comprising an amino acid sequence comprising It may be a polypeptide. In addition, if an amino acid sequence having such homology or identity and exhibiting biological activity corresponding to the polypeptide, a polypeptide having an amino acid sequence in which some sequences are deleted, modified, substituted or added in addition to the amino acid sequence at the 13th position is also disclosed in the present application. It is obvious that it is included in the scope.
[35]
For example, the polypeptide having the activity of dihydrodipicolinic acid reductase in the present application may be a dihydrodipicolinic acid reductase derived from Corynebacterium glutamicum . More specifically, the amino acid sequence of the dihydrodipicolinic acid reductase derived from Corynebacterium glutamicum ATCC13032 (SEQ ID NO: 1) or the amino acid of the dihydrodipicolinic acid reductase derived from Corynebacterium glutamicum ATCC13869 sequence (SEQ ID NO: 51). The dihydrodipicolinic acid reductase having the above sequence shows homology or identity to each other and exhibits corresponding efficacy as a dihydrodipicolinic acid reductase. The inclusion is obvious.
[36]
The "homology" or "identity" refers to the percent identity between two given 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. The terms homology and identity can often be used interchangeably. 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).
[37]
[38]
As used herein, the term “variant”, “variant” 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 in the amino acid sequence corresponding to the polypeptide having the activity of dihydrodipicolinic acid reductase may refer to a mutant whose activity is weakened compared to that of the wild type, native type or non-mutated type.
[39]
In the present application, the dihydrodipicolinic acid reductase variant polypeptide is, "mutant dihydrodipicolinic acid reductase", "mutated dihydrodipicolinic acid reductase", "dihydrodipicolinic acid reductase variant", It may be used interchangeably as "mutant dihydrodipicolinic acid reductase" or "dihydrodipicolinic acid reductase variant". Meanwhile, such a mutant may be non-naturally occurring.
[40]
Mutation in the present application is a method for improving enzymes in general, and known methods known in the art can be used without limitation, and there are strategies such as rational design and directed evolution. For example, a rational design strategy includes a method of site-directed mutagenesis or site-specific mutagenesis of an amino acid at a specific position, and a directed evolution strategy includes a method of causing random mutagenesis, etc. There is this. Also, it may be mutated without external manipulation due to natural mutation. Specifically, the dihydrodipicolinic acid reductase mutant polypeptide may be an isolated one, a recombinant polypeptide, or may be non-naturally occurring. However, the present invention is not limited thereto.
[41]
[42]
The "dihydrodipicolinic acid reductase variant polypeptide" of the present application is a polypeptide having dihydrodipicolinic acid reductase activity comprising one or more amino acid substitutions, wherein the amino acid substitution is a thirteenth amino acid substituted with another amino acid. It provides a dihydrodipicolinic acid reductase variant polypeptide comprising that.
[43]
'Substitution with another amino acid' is not limited as long as it is substituted with an amino acid different from the amino acid before the substitution. Specifically, lysine, histidine, glutamic acid, arpartic acid, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine are substituted with any one amino acid it may have been More specifically, the dihydrodipicolinic acid reductase (dihydrodipicolinate reductase) variant polypeptide has a polar amino acid or It may be substituted with a basic amino acid.
[44]
More specifically, in the dihydrodipicolinate reductase variant polypeptide, the amino acid at the position corresponding to the 13th amino acid in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 98% or more identity therewith is asparagine (Asparagine). ), threonine, cysteine, tyrosine, serine, lysine, or glutamine substituted with, may be a variant polypeptide.
[45]
In addition, the substituted amino acid residue may include a natural amino acid as well as a non-natural amino acid. The unnatural amino acid may be, for example, a D-amino acid, a homo-amino acid, a beta-homo-amino acid, an N-methyl amino acid, an alpha-methyl amino acid, an unconventional amino acid (eg, citrulline or naph thylalanine, etc.), but is not limited thereto. On the other hand, in the present application, when it is expressed that 'a specific amino acid has been substituted', it is obvious that the amino acid is substituted with an amino acid different from the amino acid before the substitution, even if it is not separately indicated that it is substituted with another amino acid.
[46]
In one embodiment, the amino acid at the position corresponding to the 13th amino acid in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence having 98% or more identity therewith is substituted with asparagine, a variant having dihydrodipicolinic acid reductase activity polypeptides can be provided. In this case, the amino acid sequence having 98% or more identity may consist of SEQ ID NO: 51.
[47]
[48]
As used herein, the term “corresponding to” refers to an amino acid residue at a position listed in a protein or peptide, or an amino acid residue similar to, identical to, or homologous to a residue listed in a protein or peptide. As used herein, "corresponding region" generally refers to a similar position in a related protein or reference protein.
[49]
In the present application, specific numbering may be used for amino acid residue positions within the polypeptides used in this application. For example, by aligning the polypeptide sequence of the present application with the target polypeptide to be compared, it is possible to renumber the position corresponding to the amino acid residue position of the polypeptide of the present application.
[50]
In the variant of dihydrodipicolinic acid reductase provided in the present application, amino acids at specific positions in the above-described dihydrodipicolinic acid reductase are substituted, so that the production capacity of L-threonine is increased compared to the polypeptide before the mutation. The variant polypeptide comprises a substitution with asparagine at the position corresponding to the 13th amino acid in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 98% or more identity therewith, and the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 51, having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more, but less than 100% sequence homology to the amino acid sequence, and having an activity of dihydrodipicolinic acid reductase. it could be
[51]
The activity of dihydrodipicolinic acid reductase of the variant polypeptide may be weaker than the activity of dihydrodipicolinic acid reductase having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 98% or more identity thereto. .
[52]
As a specific example, the variant dihydrodipicolinic acid reductase of the present application may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 53, or an amino acid sequence having homology or identity therewith. In addition, if the amino acid sequence has such homology or identity and exhibits biological activity corresponding to the polypeptide, a polypeptide having an amino acid sequence in which some amino acids are deleted, modified, substituted or added in addition to the amino acid at the position corresponding to the 13th amino acid It is obvious that it is included in the scope of the present application.
[53]
[54]
In addition, unlike the dihydrodipicolinic acid reductase mutant polypeptide of the present application or a wild-type or natural polypeptide having the activity of dihydrodipicolinic acid reductase in a microorganism containing the same, or an unmodified polypeptide, the final product lysine It may have a characteristic that the activity to be generated is weakened.
[55]
In the present application, "weakened" means that the function of a protein or polypeptide is reduced, and due to the weakening of the activity of the dihydrodipicolinic acid reductase, aspartyl semialdehyde, a common precursor of threonine biosynthesis and lysine biosynthesis, is The function of sending lysine to the pathway for biosynthesis is lowered, and as a result, the production capacity of the final product, lysine, is reduced, while the production capacity of threonine is increased, thereby increasing the productivity of threonine and threonine-derived amino acids. can be raised The threonine-derived amino acid means an amino acid that can be biosynthesized using threonine as a precursor, and is not limited as long as it is a material that can be biosynthesized from threonine.
[56]
In addition, in the present application, since the dihydrodipicolinic acid reductase function is not inhibited but weakened, there is no problem in producing diaminopimelic acid, a precursor of lysine biosynthesis, so the synthesis of the cell wall of the microorganism is not inhibited. It is characterized in that there is no problem in growth. That is, the activity can be attenuated to an appropriate level by mutation in which the 13th amino acid is substituted with another amino acid, rather than the deletion of the gene (dapB) encoding dihydrodipicolinic acid reductase.
[57]
[58]
Another aspect of the present application provides a polynucleotide encoding the dihydrodipicolinic acid reductase variant polypeptide.
[59]
The dihydrodipicolinic acid reductase and variant polypeptides are as described above.
[60]
As used herein, the term "polynucleotide" is a polymer of nucleotides in which nucleotide monomers are connected in a long chain by covalent bonds, and has a meaning including DNA or RNA strands of a certain length or more, and its basic constituent units Phosphorus nucleotides include not only natural nucleotides, but also analogs in which sugar or base sites are modified. In the present application, the polynucleotide may be a polynucleotide isolated from a cell or an artificially synthesized polynucleotide, but is not limited thereto.
[61]
Specifically, it may be a polynucleotide encoding the dihydrodipicolinic acid reductase variant polypeptide. The polynucleotide encoding the variant polypeptide of the present application may be included without limitation as long as it is a polynucleotide sequence encoding the variant polypeptide having dihydrodipicolinic acid reductase activity of the present application. In one embodiment, the polynucleotide encoding the variant polypeptide of the present application is a nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 54 or at least 60%, 70%, 80%, 83%, 84%, 85%, 86 thereof A polynucleotide comprising a nucleotide sequence having %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% homology or identity can be In addition, if a nucleotide sequence encoding an amino acid sequence having such homology or identity and exhibiting biological activity corresponding to the dihydrodipicolinic acid reductase polypeptide, some sequences other than the nucleotide sequence encoding the 13th amino acid are deleted, modified, It is apparent that polynucleotides having a substituted or added base sequence are also included in the scope of the present application.
[62]
[63]
In the present application, the polynucleotide encoding the amino acid sequence of the dihydrodipicolinic acid reductase 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.
[64]
Due to the genetic code degeneracy, a nucleotide sequence encoding the same amino acid sequence and variants thereof may also be included in the present application. Specifically, the polynucleotide encoding the polypeptide having dihydrodipicolinic acid reductase activity is expressed from the coding region in consideration of codons preferred in the organism in which the polypeptide is to be expressed due to codon degeneracy. Various modifications may be made to the coding region within a range that does not change the amino acid sequence of the polypeptide, and may be included without limitation as long as it has the activity of dihydrodipicolinic acid reductase.
[65]
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 a polypeptide consisting of the amino acid sequence of SEQ ID NO: Any sequence encoding a polypeptide having a may be included without limitation.
[66]
The "stringent conditions" means conditions that allow specific hybridization between polynucleotides. Such conditions are described in methods well known to those skilled in the art (eg, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). For example, genes with high homology are hybridized between genes having homology of 80%, 90% or more, more specifically 95% or more, more specifically 97% or more, particularly specifically 99% or more, , conditions under which genes with lower homology do not hybridize, or washing conditions of conventional Southern hybridization, such as 60°C, 1SSC, 0.1% SDS, specifically 60°C, 0.1SSC, 0.1% SDS, more specifically The conditions of washing once, specifically two to three times, at 68° C., at a salt concentration and temperature corresponding to 0.1 SSC, 0.1% SDS, can be enumerated. Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization.
[67]
In the present application, the term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing with each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also include substantially similar nucleotide sequences as well as isolated nucleotide fragments 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).
[68]
[69]
Another aspect of the present application is a vector comprising a polynucleotide encoding the variant polypeptide.
[70]
As used herein, the term "vector" refers to a DNA preparation containing the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable regulatory sequence so that the target polypeptide 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.
[71]
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, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used, but is not limited thereto.
[72]
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 polypeptide of interest may 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 the vector, that is, to confirm whether a target nucleic acid molecule is inserted, and confer a selectable phenotype such as drug resistance, auxotrophicity, resistance to cytotoxic agents, or surface protein expression. markers may 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.
[73]
[74]
Another aspect of the present application is a transformant into which the vector is introduced.
[75]
In the present application, the term "transformation" means introducing a vector including a polynucleotide encoding a target polypeptide into a host cell so that the polypeptide 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 outside the chromosome, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA or RNA encoding a target polypeptide. 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, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are usually operably linked to the polynucleotide. 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 nucleic acid 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.
[76]
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 polypeptide of the present application and the polynucleotide sequence are functionally linked. An 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.
[77]
[78]
Another aspect of the present application provides a microorganism comprising a variant dihydrodipicolinic acid reductase. Specifically, it provides a microorganism of the genus of Corynebacterium that produces L-threonine, including the dihydrodipicolinic acid reductase mutant polypeptide .
[79]
The dihydrodipicolinic acid reductase, variant polypeptide is as described above.
[80]
In the present application, the term "microorganism" includes wild-type microorganisms or microorganisms that have been genetically modified either naturally or artificially, and a specific mechanism is It is a concept that includes both weakened and enhanced microorganisms.
[81]
In the present application, the terms "threonine" or "L-threonine" may be used interchangeably, and "lysine" or "L-lysine" may also be used interchangeably.
[82]
Specifically, the microorganism containing the dihydrodipicolinic acid reductase mutant polypeptide of the present application is endowed with the ability to produce L-amino acids to the microorganisms having the ability to naturally produce L-amino acids or the parent strains without the ability to produce L-amino acids means microorganisms. Specifically, in the microorganism containing the dihydrodipicolinic acid reductase, the 13th amino acid in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 51 is asparagine, threonine, cysteine, tyrosine ), may be a microorganism expressing a mutant dihydrodipicolinic acid reductase substituted with serine, lysine, or glutamine, and in one embodiment, arginine, amino acid 13 in the amino acid sequence It may be a microorganism expressing a mutant dihydrodipicolinic acid reductase substituted with asparagine, but is not limited thereto.
[83]
The microorganism is characterized in that it does not interfere with growth by including the dihydrodipicolinic acid reductase mutant polypeptide in which the 13th amino acid is substituted, and the production of lysine can be decreased while the production of threonine can be increased.
[84]
The enzyme activity of dihydrodipicolinic acid reductase is weakened by the position of the mutation, thereby reducing the production of lysine and increasing the production of threonine without delaying the growth rate of the strain.
[85]
[86]
The microorganism containing the mutant dihydrodipicolinic acid reductase of the present application has a reduced ability to produce lysine compared to the microorganism containing a polypeptide having the activity of a wild-type or unmodified dihydrodipicolinic acid reductase, and threonine Since the productivity is improved, threonine can be obtained in high yield from these microorganisms.
[87]
In the present application, the microorganism containing the dihydrodipicolinic acid reductase mutant polypeptide is not particularly limited in its type, but Enterobacter genus , Escherichia genus, Erwinia genus, sera It may be a microorganism belonging to the genus Serratia , Pseudomonas , Providencia, Corynebacterium , and Brevibacterium . More specifically , it may be a microorganism belonging to the genus Corynebacterium .
[88]
In the present application, "Corynebacterium genus microorganism" is specifically Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Brevibacterium lactofermentum ( Brevibacterium lactofermentum ) , Brevibacterium flavum ( Brevibacterium flavu m ), Corynebacterium thermoaminogenes , Corynebacterium efficiens ), etc., but are not necessarily limited thereto. In one embodiment, the microorganism of the genus Corynebacterium in the present application may be Corynebacterium glutamicum .
[89]
The microorganism may be a microorganism into which a vector containing a polynucleotide encoding the dihydrodipicolinic acid reductase mutant polypeptide or a polynucleotide encoding a mutant dihydrodipicolinic acid reductase is introduced. . Specifically, the introduction may be made by transformation, but is not limited thereto, and for the purpose of the present application, the host cell or microorganism may include lysine or threonine or the amino acid lysine or threonine including the variant polypeptide. Any microorganism capable of producing the derived amino acid is possible.
[90]
[91]
The microorganism of the genus Corynebacterium is specifically, in order to enhance the biosynthetic pathway of L-threonine, for example, thrC encoding threonine synthetase, phosphoenol pyruvate carboxylase ( ppc gene encoding phosphoenolpyruvate carboxylase, galP gene involved in glucose uptake, lysC gene encoding lysine-sensitive aspartokinase 3, hom encoding homoserine dehydrogenase Expression of a gene or pyc gene that induces an increase in the oxaloacetate pool can be enhanced or increased in microorganisms.
[92]
In order to release the feedback inhibition for the L-threonine, for example, lysC gene, hom gene, or thrA having a dual function of aspartokinase and homoserine dehydrogenase 1 (Bifunctional aspartokinase / homoserine dehydrogenase 1) Genetic mutations can be introduced into genes and the like.
[93]
In order to inactivate a gene that weakens the biosynthetic pathway of L-threonine, for example, oxaloacetate (OAA), which is an L-threonine biosynthesis intermediate, is converted to phosphoenol pyruvate (PEP) Expression of the pckA gene involved in lysC gene, the tyrR gene that suppresses the lysC gene, the galR gene that suppresses the expression of the galP gene involved in glucose uptake, or the mcbR gene that is a DNA-binding transcriptional dual regulator can be attenuated or inactivated within the microorganism.
[94]
In order to increase the activity of the L-threonine operon, aspartokinase (aspartokinase), homoserine dehydrogenase (homoserine dehydrogenase), homoserine kinase (homoserine kinase) and threonine synthase (threonine synthase) By introducing a plasmid containing a threonine operon (Japanese Patent Laid-Open No. 2005-227977) composed of a gene encoding a threonine operon derived from E. coli or the like into a microorganism (TURBA E, et al, Agric. Biol. Chem) 53:2269~2271, 1989), may increase the expression of the threonine operon in microorganisms.
[95]
In addition, resistance to L-threonine analogs such as α-amino-β-hydroxy valeric acid or D,L-threonine hydroxamate can be conferred.
[96]
In addition, dapA (dihydrodipicolinate synthase), lysA (diaminopimelate decarboxylase), which are genes acting on the biosynthetic pathway of L-lysine, which has a precursor in common with L-threonine, , or the ddh (diaminopimelate dehydrogenase) gene.
[97]
However, the present invention is not limited thereto, and L-threonine production ability can be enhanced by a method for regulating gene expression known in the art.
[98]
In the present application, the term “enhancement/increase” is a concept that includes all those in which activity is increased compared to intrinsic activity.
[99]
This enhancement or increase in gene activity can be achieved by application of various methods well known in the art. Examples of the method include increasing the intracellular copy number of a gene; a method of introducing a mutation into an expression control sequence of a gene; a method of replacing a gene expression control sequence with a sequence with strong activity; a method of additionally introducing a mutation into the gene to enhance the activity of the gene; And it may consist of any one or more methods selected from the group consisting of a method of introducing a foreign gene into a microorganism, and may be achieved by a combination thereof, but is not particularly limited by the above example.
[100]
In the present application, the term "inactivation" is a concept that includes all of the activity is weakened or no activity compared to the intrinsic activity.
[101]
Inactivation or attenuation of such gene activity can be achieved by application of various methods well known in the art. Examples of the method include a method of deleting all or part of a gene on a chromosome, including when the activity of the gene is removed; a method of replacing a gene encoding the polypeptide on a chromosome with a gene mutated to reduce the activity of the polypeptide; a method of introducing a mutation into an expression control sequence of a gene on a chromosome encoding the polypeptide; a method of replacing the expression control sequence of the gene encoding the polypeptide with a sequence with weak or no activity (eg, a method of replacing the promoter of the gene with a promoter weaker than the endogenous promoter); a method of deleting all or part of a gene on a chromosome encoding the polypeptide; a method of introducing an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of the gene on the chromosome and inhibits translation from the mRNA to the polypeptide; A method of making the attachment of ribosomes impossible by artificially adding a sequence complementary to the SD sequence to the front end of the SD sequence of the gene encoding the polypeptide to form a secondary structure and ORF (open reading frame) of the sequence There is a reverse transcription engineering (RTE) method of adding a promoter so as to be reverse transcribed at the 3' end, and the like, which can also be achieved by a combination thereof, but is not particularly limited by the above example.
[102]
For example, increasing the intracellular copy number of a gene to enhance the activity of lysC, hom, or pyc; a method of introducing a mutation into an expression control sequence of a gene; a method of replacing a gene expression control sequence with a sequence with strong activity; a method of additionally introducing a mutation into the gene to enhance the activity of the gene; and a method of introducing a foreign gene into a microorganism, but is not limited thereto, and a known method for enhancing or increasing activity may be used without limitation.
[103]
For example, a method of deleting all or part of a gene on a chromosome, including a case in which the activity of the gene is removed to attenuate the activity of dapA, ddh, or lysA; a method of replacing a gene encoding the polypeptide on a chromosome with a gene mutated to reduce the activity of the polypeptide; a method of introducing a mutation into an expression control sequence of a gene on a chromosome encoding the polypeptide; a method of replacing the expression control sequence of the gene encoding the polypeptide with a sequence with weak or no activity (eg, a method of replacing the promoter of the gene with a promoter weaker than the endogenous promoter); It may consist of a method of deleting all or part of a gene on a chromosome encoding the polypeptide, but is not limited thereto, and a known method for weakening activity may be used without limitation.
[104]
[105]
In addition, the microorganism containing the dihydrodipicolinic acid reductase mutant polypeptide may further include any one or more of the following mutant polypeptides.
[106]
The variant polypeptide further included is a dihydrodipicolinate synthase variant polypeptide in which the 119th amino acid is substituted from tyrosine to phenylalanine in the amino acid sequence of SEQ ID NO: 80, 302 in the amino acid sequence of SEQ ID NO: 81 Diaminopimelate decarboxylase (Diaminopimelate decarboxylase) variant polypeptide in which the amino acid is substituted from arginine to alanine; And in the amino acid sequence of SEQ ID NO: 82, the 169th amino acid may be any one or more selected from a diaminopimelate dehydrogenase variant polypeptide in which threonine is substituted with leucine.
[107]
The amino acid sequence of the dihydrodipicolinate synthase variant polypeptide in which the 119th amino acid in the amino acid sequence of SEQ ID NO: 80 is substituted from tyrosine to phenylalanine may be SEQ ID NO: 65, but is not limited thereto. The dihydrodipicolinate synthase (dapA) is an enzyme for biosynthesis of lysine from aspartyl semialdehyde, a common precursor of lysine and threonine, wherein the 119th amino acid sequence is phenylalanine from tyrosine As the function of dihydropicolinic acid synthase is weakened as it is substituted, the production capacity of lysine may be reduced. In the examples of the present application, it was confirmed that the production of lysine was decreased and the production of threonine was increased due to the introduction of the variant polypeptide.
[108]
[109]
The amino acid sequence of the diaminopimelate decarboxylase variant polypeptide in which the 302th amino acid is substituted from arginine to alanine in the amino acid sequence of SEQ ID NO: 81 may be SEQ ID NO: 70, but is not limited thereto. The diaminopimelate decarboxylase (lysA) is the last enzyme acting on lysine biosynthesis. As the 302th amino acid sequence is substituted from arginine to alanine, the function of diaminopimelate decarboxylase is weakened. The production capacity of lysine may be reduced. In the examples of the present application, it was confirmed that the production of lysine was decreased and the production of threonine was increased due to the introduction of the variant polypeptide.
[110]
[111]
The amino acid sequence of the diaminopimelate dehydrogenase variant polypeptide in which the 169th amino acid is substituted from threonine to leucine in the amino acid sequence of SEQ ID NO: 82 may be SEQ ID NO: 75, but is not limited thereto . The diaminopimelate dehydrogenase (diaminopimelate dehydrogenase, ddh) is an enzyme that acts on the biosynthesis of lysine. As the 169th amino acid is substituted from threonine to leucine, the function of diaminopimelate dehydrogenase is reduced. As it is weakened, the production capacity of lysine may be reduced. In the examples of the present application, it was confirmed that the production of lysine was decreased and the production of threonine was increased due to the introduction of the variant polypeptide.
[112]
Another aspect of the present application provides a method for producing threonine or threonine-derived L-amino acids, comprising the step of culturing the described microorganism in a medium.
[113]
In addition, the above production method further comprises the step of recovering threonine or threonine-derived L-amino acid from the cultured microorganism or cultured medium, production of threonine or threonine-derived L-amino acid provide a way
[114]
The microorganism may be a microorganism of the genus Corynebacterium comprising the dihydrodipicolinic acid reductase variant polypeptide 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 a microorganism producing threonine or L-amino acid derived from threonine. The threonine-derived L-amino acid may include not only threonine-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.
[115]
The threonine or threonine-derived L-amino acid may be a culture solution of threonine or threonine-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.
[116]
The method for producing the threonine or L-amino acid derived from threonine can be easily determined by those skilled in the art under optimized culture conditions and enzyme activity conditions known in the art.
[117]
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 may control pH 6 to 8, most specifically pH 6.8), but is not limited thereto. In addition, oxygen or oxygen-containing gas mixtures may be introduced into the culture to maintain aerobic conditions. The culture temperature may be maintained at 20°C to 45°C, specifically, 25°C to 40°C, but is not limited thereto. Incubation time may be cultured for about 10 to 160 hours, but is not limited thereto. The amino acids produced by the culture, in one embodiment, threonine or lysine may be secreted into the medium or remain in the cells.
[118]
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.
[119]
In the method for recovering threonine or L-amino acid derived from threonine 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 threonine or threonine-derived L- that is a target substance from a medium or microorganism using a suitable method known in the art amino acids 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.
[120]
[121]
Another aspect of the present application is a dihydrodipicolinic acid reductase variant polypeptide of the present application; a polynucleotide encoding the variant; and a vector comprising the polynucleotide; It provides a composition for producing L-threonine, including a microorganism or a culture solution thereof, including any one or more.
[122]
The dihydrodipicolinic acid reductase, variant polypeptides, polynucleotides, vectors and microorganisms thereof are as described above.
[123]
The microorganism may be of the genus Corynebacterium, specifically Corynebacterium glutamicum, but is not limited thereto. This is the same as described above.
[124]
[125]
The composition for producing L-threonine may refer to a composition capable of producing L-threonine by the dihydrodipicolinic acid reductase mutant polypeptide of the present application. The composition may include, without limitation, the composition capable of activating the dihydrodipicolinic acid reductase variant polypeptide or the dihydrodipicolinic acid reductase variant polypeptide. The dihydrodipicolinic acid reductase mutant polypeptide may be in a form included in a vector so that the operably linked gene can be expressed in the introduced host cell.
[126]
The composition may further comprise a cryoprotectant or an excipient. The cryoprotectant or excipient may be a non-naturally occurring material or a naturally occurring material, but is not limited thereto. In another embodiment, the cryoprotectant or excipient may be a material that the microorganism does not naturally contact, or a material that is not naturally included with the microorganism at the same time, but is not limited thereto.
[127]
[128]
Another aspect of the present application is the dihydrodipicolinic acid reductase variant polypeptide of the present application; a polynucleotide encoding the variant; a vector comprising the polynucleotide; Or it provides a use for the production of L- threonine or threonine-derived L-amino acid of a microorganism comprising at least one of them.
[129]
Modes for carrying out the invention
[130]
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.
[131]
[132]
Example 1: Preparation of vector library for mutation in dapB gene ORF
[133]
[134]
Corynebacterium glutamicum (Corynebacterium glutamicum) of the dapB gene expression level or a library was prepared in the following way for the purpose of excavating a weakened variant of its activity.
[135]
First , the GenemorphII Random Mutagenesis Kit (Stratagene) was used for the purpose of introducing 0-4.5 mutations per kb of a DNA fragment (747 bp) containing the dapB (747 bp) gene. Error-prone PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 (WT) as a template and primers SEQ ID NOs: 5 and 6. Specifically, a reaction solution containing the chromosomes of the WT strain (500 ng), primers 5 and 6 (125 ng each), Mutazyme II reaction buffer (1Х), dNTP mix (40 mM), Mutazyme II DNA polymerase (2.5U) After denaturing at 94˚C for 2 minutes, denaturation at 94˚C for 1 minute, annealing at 56˚C for 1 minute, and polymerization for 2 minutes at 72˚C were repeated 25 times, followed by polymerization at 72˚C for 10 minutes. did
[136]
The amplified gene fragment was ligated to the pCRII vector using the TOPO TA Cloning Kit (Invitrogen), transformed into E. coli DH5α, and plated on LB solid medium containing kanamycin (25 mg/l). After selecting 20 transformed colonies, a plasmid was obtained, and as a result of analyzing the nucleotide sequence, it was confirmed that mutations were introduced at different positions with a frequency of 0.5 mutations/kb. Finally, about 10,000 transformed E. coli colonies were taken and plasmids were extracted, and this was named pTOPO- dapB (mt) library.
[137]
[138]
Example 2: dapB mutant screening based on the production and growth rate of the dapB-deficient strain
[139]
[140]
In order to construct a strain in which the dapB gene is deleted in the wild-type Corynebacterium glutamicum ATCC13032, a vector pDZ-Δ dapB in which the dapB gene is deleted was prepared as follows. Specifically, DNA fragments located at the 5' and 3' ends of the dapB gene (300 bp each) were prepared in a form linked to the pDZ vector (Korean Patent No. 2009-0094433). Based on the reported base sequence (SEQ ID NO: 2) of the dapB gene, primers SEQ ID NOs: 7 and 8 with restriction enzyme SalI recognition sites inserted into 5' fragments and 3' fragments, and primers SEQ ID NO: 9 at positions 300 bp away from them, respectively and 10 were synthesized. Using the chromosome of Corynebacterium glutamicum ATCC13032 as a template, the 5' end gene fragment was prepared by PCR using primers SEQ ID NOs: 7 and 9. In the same manner, a gene fragment located at the 3' end of the gltA gene was prepared through PCR using SEQ ID NOs: 8 and 10. PCR conditions include denaturation at 94˚C for 2 minutes, denaturation at 94˚C for 1 minute, annealing at 56˚C for 1 minute, and polymerization for 40 seconds at 72˚C, repeated 30 times, followed by polymerization at 72˚C for 10 minutes. was performed.
[141]
On the other hand, after treatment with restriction enzyme SalI , the pDZ vector heat-treated at 65° C. for 20 minutes and the insert DNA fragment amplified through the PCR were ligated using Infusion Cloning Kit, transformed into E. coli DH5α, and kanamycin (25 mg/l ) was plated on LB solid medium containing After selecting colonies transformed with the vector into which the desired gene was inserted through PCR using primers SEQ ID NOs: 7 and 8, a plasmid was obtained using a conventionally known plasmid extraction method, and this plasmid was named pDZ-Δ dapB .
[142]
The constructed vector pDZ-Δ dapB was transformed into Corynebacterium glutamicum ATCC13032 by an electric pulse method (Van der Rest et al ., Appl. Microbial . Biotechnol . 52:541-545, 1999) and homologous chromosomal recombination . A strain in which the dapB gene was deleted was prepared. As such , the strain in which the dapB gene is deleted was named Corynebacterium glutamicum 13032::Δ dapB .
[143]
In addition, the 13032::Δ dapB strain was transformed with the pTOPO- dapB (mt) library by the electric pulse method and spread on a complex plate medium containing kanamycin (25 mg/l) to secure about 100 colonies. The L-lysine production ability test was performed on the secured 100 strains. After inoculating the 100 strains obtained in a 250 ml corner-baffle flask containing 25 ml of the seed medium, it was 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-lysine production medium and cultured with shaking at 30° C. for 48 hours at 200 rpm.
[144]
[145]
Lysine production medium (pH 7.0)
[146]
Glucose 100 g, (NH 4 ) 2 SO 4 40 g, Soy Protein 2.5 g, Corn Steep Solids 5 g, Urea 3 g, KH 2 PO 4 1 g, MgSO 4 7H 2 O 0.5 g, Biotin 100 μg, thiamine hydrochloride 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 3000 μg, CaCO 3 30 g (based on 1 liter of distilled water).
[147]
[148]
13032 and 13032::Δ dapB strains were used as controls. After completion of the culture, the production of lysine was measured using HPLC. While the L-lysine concentration was decreased compared to the wild-type 13032 strain, 6 strains having a higher L-lysine concentration than the 13032::Δ dapB strain were selected, and the concentration of amino acids in the culture solution for 6 weeks is shown in Table 1. The six strains selected above were named 13032::dapB(mt)-1 to 6. The other 94 colonies showed a higher L-lysine concentration than 13032 used as a control or the same level as the 13032::Δ dapB strain. In addition, while the growth rate of the 13032::Δ dapB strain was significantly reduced compared to 13032, it was confirmed that the selected six strains maintained a higher growth rate than 13032::Δ dapB .
[149]
[150]
[Table 1]
[151]
[152]
As shown in Table 1, the selected 6 strains showed a result that the L-lysine concentration was lower than that of the WT strain and higher than the 13032::Δ dapB strain.
[153]
[154]
Example 3: Confirmation of nucleotide sequences of 6 dapB mutants
[155]
[156]
6 kinds of selection strain 13032:: To confirm the dapB gene sequence of dapB( mt)-1 to 6 , a DNA fragment containing the dapB gene in the chromosome using the primers specified in Example 1 (SEQ ID NOs: 5 and 6) PCR amplification. PCR conditions include denaturation at 94˚C for 2 minutes, denaturation at 94˚C for 1 minute, annealing at 56˚C for 1 minute, and polymerization for 40 seconds at 72˚C, repeated 30 times, followed by polymerization at 72˚C for 10 minutes. was performed.
[157]
As a result of analyzing the nucleotide sequence of the amplified gene, 13032::dapB(mt)-1 of the six strains sequentially changed the nucleotide sequence 37-39 of SEQ ID NO: 2 from CGT to AAC, the 13th amino acid from the N-terminus In the variant in which phosphorus arginine is substituted with asparagine, 13032::dapB(mt)-2, the nucleotide sequence at positions 106 to 108 of SEQ ID NO: 2 is changed from GTC to ATC, and valine, the 36th amino acid from the N-terminus, is replaced with methionine The substituted variant, 13032::dapB(mt)-3, is a mutant in which the nucleotide sequence at positions 175 to 177 of SEQ ID NO: 2 is changed from GCT to CTG, and alanine, the 59th amino acid from the N-terminus, is substituted with leucine. , 13032::dapB(mt)-4 is a mutant in which the 235-237 nucleotide sequence of SEQ ID NO: 2 is changed from ACG to GCA, threonine, the 79th amino acid from the N-terminus, is substituted with alanine, 13032::dapB (mt)-5 is a mutant in which the nucleotide sequence at positions 433-435 of SEQ ID NO: 2 is changed from ACG to GCG, threonine, the 145th amino acid from the N-terminus, is substituted with alanine, and finally 13032::dapB(mt)- 6 confirmed that the 414th nucleotide sequence of SEQ ID NO: 2 was changed from G to C, and lysine, the 138th amino acid from the N-terminus, was substituted with arginine.
[158]
Among the six strains, while the production of L-lysine decreased compared to 13032, the WT strain, 13032:: Δ dapB, the production rate of L-lysine was low and the growth rate was 13032:: dapB similar to the WT strain 13032 (mt)-1 strain was selected as the most excellent dihydrodipicolinic acid reductase activity attenuated strain.
[159]
[160]
Example 4: Preparation of various strains in which arginine, the 13th amino acid of the dapB gene, is substituted with another amino acid
[161]
[162]
At the position of the 13th amino acid in the amino acid sequence 1, substitution with other proteogenic amino acids except for arginine of the wild type was attempted.
[163]
In order to introduce 19 kinds of heterologous base substitution mutations including R13N, which is the mutation identified in Example 3, each recombinant vector was prepared in the following way.
[164]
First, using the genomic DNA extracted from the WT strain as a template, primers SEQ ID NOs: 11 and 12 with restriction enzyme SalI recognition sites inserted into the 5' fragment and 3' fragment at positions approximately 600 bp back and forth from positions 36 to 39 of the dapB gene. synthesized. In order to introduce 19 heterogeneous base substitution mutations, primers SEQ ID NOs: 13-50 for substituting the 36th to 39th nucleotide sequences of the dapB gene were synthesized.
[165]
Specifically, the pDZ- dapB (R13N) plasmid was constructed in a form in which DNA fragments located at the 5' and 3' ends of the dapB gene (600 bp each) were linked to the pDZ vector (Korean Patent No. 2009-0094433). Using the chromosome of the WT strain as a template, the 5' end gene fragment was prepared by PCR using primers SEQ ID NOs: 11 and 13. PCR conditions include denaturation at 94˚C for 2 minutes, denaturation at 94˚C for 1 minute, annealing at 56˚C for 1 minute, and polymerization for 40 seconds at 72˚C, repeated 30 times, followed by polymerization at 72˚C for 10 minutes. was performed. In the same manner, a gene fragment located at the 3' end of the dapB gene was prepared through PCR using SEQ ID NOs: 12 and 14. After the amplified DNA fragment was purified using Quiagen's PCR purification kit, it was used as an insert DNA fragment for vector construction.
[166]
Meanwhile, the pDZ vector, which was treated with restriction enzyme SalI and heat-treated at 65° C. for 20 minutes, and the inserted DNA fragment amplified through the PCR were ligated using the Infusion Cloning Kit, and then transformed into E. coli DH5α. The strain was plated on LB solid medium containing kanamycin (25 mg/l). After selecting colonies transformed with the vector into which the desired gene was inserted through PCR using primers SEQ ID NOs: 11 and 12, a plasmid was obtained using a commonly known plasmid extraction method. This plasmid was named pDZ- dapB (R13N).
[167]
In the same manner, pDZ-dapB (R13G) using primers SEQ ID NOs: 11 and 15, 12 and 16, pDZ-dapB (R13A) using primers SEQ ID NOs: 11 and 17, 12 and 18 , primers SEQ ID NOs: 11 and 19, pDZ- dapB (R13V) using 12 and 20, pDZ- dapB (R13L) using primers SEQ ID NOs: 11 and 21, 12 and 22, pDZ- dapB ( R13L ) using primers SEQ ID NOs: 11 and 23, 12 and 24 ( R13I), pDZ- dapB (R13M) using primers SEQ ID NOs: 11 and 25, 12 and 26, pDZ- dapB (R13F) using primers SEQ ID NOs: 11 and 27, 12 and 28 , primers SEQ ID NOs: 11 and 29, pDZ- dapB (R13W) using 12 and 30, pDZ- dapB (R13P) using primers SEQ ID NOs: 11 and 31, 12 and 32, pDZ- dapB using primers SEQ ID NOs: 11 and 33, 12 and 34 (R13S), pDZ- dapB (R13T) using primers SEQ ID NOs: 11 and 35, 12 and 36, pDZ- dapB (R13C) using primers SEQ ID NOs: 11 and 37, 12 and 38 , primers SEQ ID NOs: 11 and 39 , pDZ- dapB (R13Y) using , 12 and 40, pDZ- dapB (R13Q) using primers SEQ ID NOs: 11 and 41, 12 and 42, pDZ- dapB using primers SEQ ID NOs: 11 and 43, 12 and 44 (R13D), pDZ- dapB (R13E) using primers SEQ ID NOs: 11 and 45, 12 and 46, pDZ- dapB (R13K) using primers SEQ ID NOs: 11 and 47, 12 and 48 , primers SEQ ID NOs: 11 and 49 , 12 and 50 were used to construct pDZ- dapB (R13H).
[168]
[169]
In order to further clarify the lysine concentration and growth rate according to the introduction of dapB mutations, each prepared vector was transformed into a lysine-producing Corynebacterium glutamicum KCCM11016P strain (Korea Patent No. 10-0159812) by an electric pulse method. did. As such , 19 strains in which heterologous base substitution mutations were introduced into the dapB gene were KCCM11016P:: dapB (R13N), KCCM11016P:: dapB (R13G), KCCM11016P:: dapB (R13A), KCCM11016P:: dapB (R13V), KCCM11016P:: dapB ( R13V), KCCM11016P:: : dapB ( R13L), KCCM11016P:: dapB (R13I), KCCM11016P:: dapB (R13M), KCCM11016P:: dapB (R13F), KCCM11016P:: dapB (R13F), KCCM11016P:: dapB KC (R13W), KCCM11016P:: dapB (R13P), KCCM11016P:: dapB (R13S), KCCM11016P:: dapB (R13T), KCCM11016P:: dapB (R13C), KCCM11016P:: dapB (R13Y), KCCM11016P:: dapB (R13Q), KCCM11016P:: dapB (R13D), KCCM11016P:: dapB (R13E), KCCM11016P:: dapB (R13K)로 각각 명명하였다.
[170]
[171]
Example 5: Analysis of lysine production capacity for dapB mutants
[172]
[173]
Using the KCCM11016P strain as a control, 19 selected strains were cultured in the following manner to measure the rate of glucose consumption, lysine production yield, and threonine production yield.
[174]
First, each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of a seed medium, and cultured with shaking at 30 °C for 20 hours at 200 rpm. Then, a 250 ml corner-baffle flask containing 24 ml of the production medium was inoculated with 1 ml of the seed culture and cultured with shaking at 32 °C for 72 hours at 200 rpm. The composition of the species medium and the production medium is as follows, respectively. After completion of the culture, the concentrations of L-lysine and L-threonine were measured using HPLC (Waters 2478).
[175]
[176]

[177]
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 μg, nicotinamide 2000 μg (based on 1 liter of distilled water)
[178]
[179]

[180]
Glucose 100 g, (NH 4 ) 2 SO 4 40 g, Soy Protein 2.5 g, Corn Steep Solids 5 g, Urea 3 g, KH 2 PO 4 1 g, MgSO 4 7H 2 O 0.5 g, Biotin 100 μg, thiamine hydrochloride 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 3000 μg, CaCO 3 30 g (based on 1 liter of distilled water).
[181]
The measurement results of lysine and threonine production capacity, and sugar consumption rate are shown in Table 2 below.

[184]
In the case of a strain comprising a variant polypeptide in which the 13th amino acid of SEQ ID NO: 1 is substituted with another amino acid, when the substituted amino acid is polar (S, T, C, Y, N, Q, K) Growth of the strain It was confirmed that the production of lysine was weakened and the production of threonine was improved while maintaining the industrially usable level. Among them, in the case of the R13N mutant introduced strain, the growth of the most strain was similar to that of KCCM11016P, and the lysine production capacity was the lowest and the threonine production capacity increased the most. In addition, R13S, R13T, R13C, R13Y, R13Q, R13K As the growth of the mutant strain was maintained, the lysine production capacity was decreased while the threonine production capacity was improved.
[185]
On the other hand, when substituted with amino acids of the remaining properties, the threonine production ability is reduced, the sugar consumption rate of the strain is also equal to the control group, or otherwise it is significantly lowered, which is confirmed to be at a level that is not industrially applicable.
[186]
As a result of the above, it was confirmed that when the mutation of the present application was introduced, the growth of the strain was maintained at an appropriate level, while only the lysine yield was reduced and it was effective in improving the threonine productivity.
[187]
This result shows that, through the regulation of the activity of dihydrodipicolic acid reductase, the growth rate of the strain caused by the inhibition of cell wall synthesis due to the weakening of the lysine biosynthesis pathway is maintained while maintaining the lysine production capacity to an appropriate level, and at the same time, the resulting By sending the flow of carbon to threonine, it was confirmed that the production capacity of threonine could be improved.
[188]
[189]
Example 6: Preparation of a strain in which arginine, the 13th amino acid of the dapB gene based on ATCC 13032 of the genus Corynebacterium , is substituted with asparagine
[190]
[191]
In order to reconfirm the effect of the variant in which the 13th amino acid is substituted with asparagine in the wild-type strain, the amino acid sequence of dihydrodipicolinic acid reductase (SEQ ID NO: 1) inherently in Corynebacterium glutamicum ATCC 13032 (SEQ ID NO: 1) The amino acid arginine was substituted with asparagine.
[192]
Specifically, the pDZ-dapB(R13N) vector prepared in Example 4 was transformed into Corynebacterium glutamicum ATCC13032 by an electric pulse method. The strain in which the heterologous base substitution mutation was introduced into the dapB gene was named 13032:: dapB ( R13N ).
[193]
[194]
Example 7: Analysis of threonine and lysine production capacity for dapB mutants based on the microorganisms of the genus Corynebacterium ATCC13032
[195]
[196]
Using the 13032::Δ dapB strain and the Corynebacterium glutamicum ATCC13032 strain used in Example 1 as controls, the 13032::dapB(R13N) strain prepared in Example 6 was cultured in the following way to consume sugar The rate, threonine and lysine production yield were measured.
[197]
First, each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of a seed medium, and cultured with shaking at 30 °C for 20 hours at 200 rpm. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium and cultured with shaking at 32 °C for 24 hours at 200 rpm. The composition of the species medium and the production medium is as follows, respectively. After completion of the culture, the concentrations of L-lysine and L-threonine were measured using HPLC (Waters 2478).
[198]
[199]
Seed medium (pH 7.0)
[200]
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 μg, nicotinamide 2000 μg (based on 1 liter of distilled water)
[201]
[202]
L-threonine production medium (pH 7.2)
[203]
Glucose 30 g, 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, Leucine 400 mg, CaCO 3 20 g (based on 1 liter of distilled water).
[204]
[205]
The culture result, threonine and lysine production capacity, and the measurement result of sugar consumption rate are shown in Table 3 below.

Claims
[Claim 1]
The amino acid corresponding to the 13th position in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence having 98% or more identity is asparagine, Threonine, Cysteine, Tyrosine, Serine ), lysine (Lysine) or glutamine (Glutamine) is substituted, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 98% or more identity therewith and at least 80%, or more, less than 100% sequence homology, and dihydro A variant polypeptide having dipicolinic acid reductase (dihydrodipicolinate reductase) activity.
[Claim 2]
The activity of claim 1, wherein the dihydrodipicolinic acid reductase activity of the mutant polypeptide is weaker than the wild-type dihydrodipicolinic acid reductase activity having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 98% or more identity therewith. Having, a variant polypeptide.
[Claim 3]
The variant polypeptide according to claim 1, wherein the amino acid sequence having 98% or more identity to the amino acid sequence of SEQ ID NO: 1 is the amino acid sequence shown in SEQ ID NO: 51.
[Claim 4]
The variant polypeptide according to claim 1, wherein the variant polypeptide consists of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 53.
[Claim 5]
A polynucleotide encoding the variant polypeptide of claim 1 .
[Claim 6]
The polynucleotide of claim 5, wherein the polynucleotide consists of the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 54.
[Claim 7]
The amino acid corresponding to the 13th position in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence having 98% or more identity is asparagine, Threonine, Cysteine, Tyrosine, Serine ), lysine (Lysine) or glutamine (Glutamine) is substituted, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 98% or more identity therewith and at least 80%, or more, less than 100% sequence homology, and dihydro a variant polypeptide having dihydrodipicolinate reductase activity; a polynucleotide encoding the polypeptide; And Corynebacterium sp. microorganism comprising any one or more of the vector containing the polynucleotide .
[Claim 8]
The microorganism according to claim 7, wherein the amino acid sequence having 98% or more identity to the amino acid sequence of SEQ ID NO: 1 is SEQ ID NO: 51, Corynebacterium sp .
[Claim 9]
The method according to claim 7, wherein the Corynebacterium sp. microorganism additionally comprises any one or more selected from the following (1) to (3) variant polypeptides, Corynebacterium genus ( Corynebacterium sp.) microorganisms. (1) Dihydrodipicolinate synthase (dihydrodipicolinate synthase) activity is weakened mutant polypeptide. (2) mutant polypeptide with weakened diaminopimelate decarboxylase activity (3) mutant polypeptide with weakened diaminopimelate dehydrogenase activity.
[Claim 10]
The microorganism according to claim 9, wherein the variant polypeptide comprises any one or more selected from variant polypeptides of (1) to (3) . (1) In the amino acid sequence of SEQ ID NO: 65, the 119th amino acid is substituted from tyrosine to phenylalanine (dihydrodipicolinate synthase) variant polypeptide (2) in the amino acid sequence of SEQ ID NO: 70 Diaminopimelate decarboxylase (Diaminopimelate decarboxylase) variant polypeptide in which the 302th amino acid is substituted from arginine to alanine; and (3) a diaminopimelate dehydrogenase variant polypeptide in which the 169th amino acid in the amino acid sequence of SEQ ID NO: 75 is substituted with leucine from Threonine.
[Claim 11]
According to claim 7, wherein the microorganism is L- threonine (Threonine) production capacity is increased compared to the non-mutant strain, Corynebacterium sp. microorganisms.
[Claim 12]
The microorganism according to claim 7, wherein the microorganism is Corynebacterium glutamicum .
[Claim 13]
The amino acid corresponding to the 13th position in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence having 98% or more identity is asparagine, Threonine, Cysteine, Tyrosine, Serine ), lysine (Lysine) or glutamine (Glutamine) is substituted, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 98% or more identity therewith and at least 80%, or more, less than 100% sequence homology, and dihydro Dipicolinic acid reductase (dihydrodipicolinate reductase) comprising the step of culturing a microorganism of the genus Corynebacterium sp. containing a variant polypeptide having an activity in a medium, L- threonine producing (Threonine) Way.
[Claim 14]
The method of claim 13, wherein the amino acid sequence having 98% or more identity to the amino acid sequence of SEQ ID NO: 1 is SEQ ID NO: 51, L-threonine production method.
[Claim 15]
The method of claim 13, further comprising the step of recovering L-threonine from the cultured medium and the microorganism in the culturing of the microorganism.