Strain for producing high-concentration L-glutamic acid and method for producing L-glutamic acid using the same

Abstract

The present application relates to a bacterial strain for producing high-concentration L-glutamic acid and a method for producing L-glutamic acid using the same.

Description

Technical Field

[0001] The present application relates to a strain for producing high-concentration L-glutamic acid and an L-glutamic acid production method using the same.

Background Art

[0002] L-glutamic acid is a typical amino acid produced by fermentation, has a unique and characteristic taste, and is one of the important amino acids widely used in the food field as well as in the pharmaceutical field and other animal feed fields. L-glutamic acid is produced using microorganisms such as the genus Corynebacterium, Escherichia coli, Bacillus subtilis, Streptomyces, the genus Penicillum, the genus Klebsiella, Erwinia, and the genus Pantoea (Patent Documents 1, 2).

[0003] Currently, various studies are being conducted for the development of microorganisms for highly efficient production of L-glutamic acid and fermentation process technologies. For example, approaches specific to target substances such as increasing the expression of genes encoding enzymes involved in amino acid biosynthesis in Corynebacterium genus microorganisms or removing genes unnecessary for amino acid biosynthesis are mainly used for improving the production yield of L-glutamic acid.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

[0005] [Non-Patent Document 1] Vitamins & Hormones Volume 78, 2008, Pages 103 - 130 [Non-Patent Document 2] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-Patent Document 3] Rice et al., 2000, Trends Genet. 16: 276 - 277 [Non-Patent Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443 - 453 [Non-Patent Document 5] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-Patent Document 6] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) [Non-Patent Document 7] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 [Non-Patent Document 8] [CARILLO ET AL / .](1988) SIAM J Applied Math 48: 1073 [Non-Patent Document 9] Smith and Waterman, Adv. Appl. Math (1981) 2:482 [Non-Patent Document 10] Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) [Non-Patent Document 11] Gribskov et al(1986) Nucl. Acids Res. 14: 6745 [Non-Patent Document 12] J. Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 [Non-Patent Document 13] F.M. Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8 [Non-Patent Document 14] Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793 [Non-Patent Document 15] Sambrook et al. Molecular Cloning 2012 [Non-Patent Document 16] Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986 [Non-Patent Document 17] Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16 [Non-Patent Document 18] "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981) [Non-Patent Document 19] Appl Environ Microbiol. 2007 Feb;73(4):1308-19. Epub 2006 Dec 8. [Non-Patent Document 20] DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix [Non-Patent Document 21] Appl. Microbiol. Biothcenol.(1999) 52:541-545 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] As a result of intensive efforts to produce L-glutamic acid in high yields, the present inventors confirmed that the ability to produce L-glutamic acid is improved by an inactivated VKOR protein, and thus completed the present application. [Means for Solving the Problems]

[0007] This application aims to provide a Corynebacterium microorganism in which the VKOR (vitamin K epoxide reductase family protein) protein is inactivated.

[0008] Furthermore, this application aims to provide a method for producing L-glutamic acid, which includes the step of culturing a Corynebacterium microorganism in which the VKOR protein is inactivated in a medium.

[0009] Furthermore, this application aims to provide a composition for producing L-glutamic acid, which includes a Corynebacterium microorganism in which the VKOR protein is inactivated, a medium in which the microorganism has been cultured, or a combination thereof.

[0010] Furthermore, this application aims to provide a method for producing a Corynebacterium microorganism, which includes the step of inactivating the VKOR protein.

[0011] Furthermore, this application aims to provide a composition for producing L-glutamic acid, which includes a Corynebacterium microorganism in which the VKOR protein is inactivated, a medium in which the microorganism has been cultured, or a combination thereof.

[0012] Furthermore, this application aims to provide a method for producing a Corynebacterium microorganism, which includes the step of inactivating the VKOR protein.

[0013] Furthermore, this application aims to provide the use of a Corynebacterium microorganism in which the VKOR protein is inactivated for producing L-glutamic acid.

Effects of the Invention

[0014] The L-glutamic acid-producing Corynebacterium microorganism in which the VKOR protein of this application is inactivated can produce L-glutamic acid in a high yield, and thus is useful for the industrial production of L-glutamic acid.

Modes for Carrying Out the Invention

[0015] Hereinafter, these will be specifically described. Note that each description and embodiment disclosed in the present application is applicable to other descriptions and embodiments respectively. That is, any combination of various elements disclosed in the present application is included in the present application. Also, the present application is not limited to the following specific descriptions. Furthermore, those with ordinary knowledge in the relevant technical field will be able to recognize and confirm many equivalents of the specific aspects of the present application described in the present application using only ordinary experiments. Furthermore, it is intended that such equivalents are also included in the present application.

[0016] One aspect of the present application provides a Corynebacterium microorganism in which a VKOR (vitamin K epoxide reductase family protein) protein is inactivated.

[0017] In the present application, "VKOR (vitamin K epoxide reductase family protein)" means an enzyme having the activity of reducing vitamin K 2,3-epoxide and vitamin K to vitamin K hydroquinone (Non-Patent Document 1).

[0018] In one example, the VKOR protein of the present application may be derived from a microorganism. Specifically, the microorganism is derived from a Corynebacterium microorganism, and more specifically, it is derived from Corynebacterium glutamicum, Corynebacterium deserti, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium callunae, etc., but is not limited thereto.

[0019] The amino acid sequence of the VKOR protein may be encoded by the VKOR gene. For example, the VKOR gene may be NCgl0775 derived from Corynebacterium glutamicum ATCC13032, or BBD29_04485 derived from Corynebacterium glutamicum ATCC13869, but is not limited thereto.

[0020] The VKOR protein of the present application may include a polypeptide represented by an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, or may be composed of the polypeptide. Further, the VKOR protein of the present application may have an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, or may be essentially consisting of the amino acid sequence. Specifically, the protein may be composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 1.

[0021] The amino acid sequence of SEQ ID NO: 1 is obtained from the GenBank of the National Institutes of Health (NIH GenBank), a well-known database. In the present application, the amino acid sequence of SEQ ID NO: 1 may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity with the amino acid sequence represented by SEQ ID NO: 1. Also, as long as it has such homology or identity and shows an efficacy corresponding to the protein containing the amino acid sequence of SEQ ID NO: 1, it goes without saying that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added is also included in the present application.

[0022] For example, those having an addition or deletion of a sequence that does not change the function of the protein of the present application, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus, C-terminus, and / or inside of the amino acid sequence are included.

[0023] The "conservative substitution" means that one amino acid is substituted with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Usually, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0024] The "homology" or "identity" in the present application means the degree to which two given amino acid sequences or nucleotide sequences are similar, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0025] The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard sequence algorithms, and the default gap penalty established by the program used may be used together. Substantially, homologous or identical sequences generally hybridize with all or part of the sequence under moderately or highly stringent conditions. It goes without saying that hybridization also includes hybridization with a polynucleotide having a codon that takes into account general codons or codon degeneracy in the polynucleotide.

[0026] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as default parameters like those in Non-Patent Document 2 and the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 4) as performed by the needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later versions) (including the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, FASTA (Non-Patent Documents 6, 7 and 8)). For example, homology, similarity or identity can be determined using BLAST or Clustal W of the National Center for Biotechnology Information.

[0027] The homology, similarity or identity of a polynucleotide or polypeptide can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 4, as disclosed in Non-Patent Document 9 for example. In summary, the GAP program defines it as the value obtained by dividing the number of similar sequence symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program include (1) a binary comparison matrix (with a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Non-Patent Document 11 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 10, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0028] In the present application, the polynucleotide encoding the VKOR protein may be a VKOR gene. Examples of the VKOR gene include NCgl0775, BBD29_04485 gene, etc., as described above.

[0029] The "polynucleotide" in the present application refers to a polymer of nucleotides in which nucleotide monomers are covalently linked in a long chain, meaning a DNA or RNA strand longer than a predetermined length, and more specifically refers to a polynucleotide fragment encoding the protein.

[0030] The polynucleotide encoding the VKOR protein of the present application may include a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. As an example of the present application, the polynucleotide of the present application may have the sequence of SEQ ID NO: 2, or may include the sequence of SEQ ID NO: 2. Further, the polynucleotide of the present application may consist of the sequence of SEQ ID NO: 2, or may be substantially composed of the sequence of SEQ ID NO: 2. Specifically, the VKOR protein may be encoded by a polynucleotide represented by the base sequence of SEQ ID NO: 2.

[0031] The polynucleotide of the present application can be variously modified in the coding region within the range where the amino acid sequence of the VKOR protein does not change, due to the degeneracy of codons or considering the codons preferred in the organism in which the VKOR protein of the present application is to be expressed. Specifically, the polynucleotide of the present application has a base sequence having 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more homology or identity with the sequence of SEQ ID NO: 2, or includes the said base sequence, or consists of a base sequence having 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more homology or identity with the sequence of SEQ ID NO: 2, or is substantially composed of the said base sequence, but is not limited thereto.

[0032] In addition, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or part of the polynucleotide sequence of the present application. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 12 and 13). For example, conditions under which polynucleotides having high homology or identity, 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or conditions for washing in ordinary Southern hybridization, specifically, washing once, specifically 2 to 3 times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, more specifically 60°C, 0.1×SSC, 0.1% SDS, and even more specifically 68°C, 0.1×SSC, 0.1% SDS, can be mentioned.

[0033] Hybridization requires that two nucleic acids have complementary sequences, even if base mismatches are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include not only nucleic acid sequences that are substantially similar, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0034] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. Further, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to the purpose.

[0035] The appropriate stringency for hybridizing the polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the variables are known in the art (for example, Non-Patent Document 12).

[0036] The "microorganism (or strain)" in the present application includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or strengthened due to reasons such as insertion of an exogenous gene or enhancement or inactivation of the activity of an endogenous gene, and is a microorganism that has been genetically modified for the production of a target polypeptide, protein, or product.

[0037] The "inactivation" of polypeptide activity in this application encompasses all concepts where the activity decreases compared to the intrinsic activity or the activity disappears. The said inactivation is interchangeably used with terms such as weakening (inactivation), deficiency, down-regulation, decrease, reduction, attenuation, etc.

[0038] The said inactivation includes at least one of the following: the activity of the polypeptide itself is decreased or removed compared to the activity of the polypeptide originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide; the overall degree and / or concentration (expression level) of polypeptide activity in the cell decreases compared to the natural strain due to inhibition of gene expression of the polynucleotide encoding it or inhibition of translation into the polypeptide; the expression of the polynucleotide is completely absent; and even if the polynucleotide is expressed, the polypeptide has no activity. The "intrinsic activity" means the activity of a specific polypeptide originally possessed by the parental strain, wild type, or unmodified microorganism before the trait change when the trait changes genetically due to natural or artificial factors. This is interchangeably used with the "activity before modification". That the activity of a polypeptide is "inactivated, weakened, deficient, decreased, down-regulated, reduced, attenuated" compared to the intrinsic activity means that it decreases compared to the activity of the specific polypeptide originally possessed by the parental strain or unmodified microorganism before the trait change.

[0039] The inactivation of the activity of such polypeptides can be achieved by applying various methods well-known in the art, not limited to these (for example, Non-Patent Documents 14, 15, etc.).

[0040] Specifically, the inactivation of the polypeptide activity of the present application is achieved by: 1) deleting all or part of the gene encoding the polypeptide; 2) modifying the expression regulatory region (or expression regulatory sequence) so that the expression of the gene encoding the polypeptide is decreased; 3) modifying the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is weakened or decreased (for example, deleting / substituting / adding one or more amino acids in the amino acid sequence); 4) modifying the gene sequence encoding the polypeptide so that the activity of the polypeptide is deleted or decreased (for example, deleting / substituting / adding one or more nucleobases in the nucleobase sequence of the polypeptide gene so as to encode a polypeptide modified so that the activity of the polypeptide is deleted or weakened); 5) modifying the start codon, Shine-Dalgarno sequence or the base sequence encoding the 5'UTR region of the gene transcript encoding the polypeptide; 6) introducing an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide so that a secondary structure that makes it impossible for ribosomes to attach is formed; 8) adding a promoter so as to reverse transcribe to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE), or 9) by a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0041] For example, the above 1) deleting all or part of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, substituting with a polynucleotide with some nucleotides deleted or a marker gene.

[0042] Modifying the expression regulatory region (or expression regulatory sequence) in 2) above may be performed by introducing mutations in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting it with a sequence having lower activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0043] Modifying the amino acid sequence or polynucleotide sequence in 3) and 4) above may be performed by deleting, inserting, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so that the activity of the polypeptide is weakened, or by substituting it with an amino acid sequence or polynucleotide sequence improved to have lower activity, or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, by introducing a mutation into the polynucleotide sequence to form a stop codon, the expression of the gene can be inhibited or reduced, but is not limited thereto.

[0044] Modifying the start codon of the gene transcript encoding the polypeptide in 5) above or the base sequence encoding the 5'UTR region may be performed, for example, by substituting it with a base sequence encoding another start codon having a lower polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0045] Introducing an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide in 6) above can be referred to, for example, Non-Patent Document 16.

[0046] Adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide so that a secondary structure that prevents the attachment of the ribosome is formed may be carried out by making mRNA translation impossible or by reducing the rate.

[0047] In addition, adding a promoter so as to reverse-transcribe at the 3'-end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide and weakening the activity.

[0048] The "enhancement" of polypeptide activity in the present application means improving the activity of the polypeptide compared to the intrinsic activity. The enhancement is used interchangeably with activation, up-regulation, overexpression, increase, etc. Here, activation, enhancement, up-regulation, overexpression, and increase all include the appearance of an activity that was not originally present and the improvement of the activity compared to the intrinsic activity or the activity before modification. The "intrinsic activity" means the activity of a specific polypeptide originally possessed by the parental strain or unmodified microorganism before the trait change when the trait changes due to genetic mutation by natural or artificial factors. This is used interchangeably with the "activity before modification". That the activity of a polypeptide is "enhanced", "up-regulated", "overexpressed", or "increased" compared to the intrinsic activity means an improvement compared to the activity and / or concentration (expression level) of the specific polypeptide originally possessed by the parental strain or unmodified microorganism before the trait change.

[0049] The enhancement may be achieved by introducing a foreign polypeptide, or may also be achieved by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by an increase in the degree of activity of the polypeptide, the expression level, or the amount of the product produced from the polypeptide.

[0050] For enhancing the activity of the polypeptide, various methods well-known in the art can be applied, and any method can be used as long as it can enhance the activity of the target polypeptide more than that of the microorganism before modification. Specifically, it is a usual method in molecular biology and uses genetic engineering and / or protein engineering well-known to those having ordinary knowledge in the technical field, but is not limited thereto (for example, Non-Patent Documents 15, 17, etc.).

[0051] Specifically, the enhancement of the polypeptide activity in the present application includes: 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of the gene on the chromosome encoding the polypeptide with a sequence having strong activity; 3) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying the polynucleotide sequence encoding the polypeptide so that the polypeptide activity is enhanced (for example, modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so that the polypeptide activity is enhanced); 6) introducing a foreign polypeptide showing the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) optimizing the codons of the polynucleotide encoding the polypeptide; 8) analyzing the three-dimensional structure of the polypeptide, selecting and modifying the exposed portion, or chemically modifying it; or 9) performing by a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0052] More specifically, increasing the intracellular copy number of the polynucleotide encoding the polypeptide 1) is achieved by introducing into the host cell a vector that replicates and functions regardless of the host and in which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be achieved by introducing one copy or two or more copies of the polynucleotide encoding the polypeptide into the chromosome in the host cell. The introduction into the chromosome is carried out by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome in the host cell, but is not limited thereto.

[0053] Substituting the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide 2) with a sequence having strong activity is carried out, for example, by causing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof so that the activity of the expression regulatory region is further enhanced, or by substituting it with a sequence having higher activity. The expression regulatory region includes, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, etc. For example, it is carried out by substituting the original promoter with a strong promoter, but is not limited thereto.

[0054] Examples of known strong promoters include, but are not limited to, CJ1 to CJ7 promoters (Patent Document 3), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (Patent Document 4), O2 promoter (Patent Document 5), tkt promoter, yccA promoter, etc.

[0055] ​Modifying the start codon of the gene transcript encoding the polypeptide in 3) above or the nucleotide sequence encoding the 5'UTR region, for example, is done by substituting the nucleotide sequence encoding another start codon with a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0056] Modifying the amino acid sequence or polynucleotide sequence in 4) and 5) above is to generate sequence mutations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof so that the activity of the polypeptide is enhanced, or substituting with an amino acid sequence or polynucleotide sequence improved to have higher activity, or an amino acid sequence or polynucleotide sequence improved to have improved activity, but is not limited thereto. Specifically, the substitution is done by inserting a polynucleotide into the chromosome by homologous recombination, but is not limited thereto. Here, the vector used may further contain a selection marker for confirming whether it has been inserted into the chromosome. The selection marker is as described above.

[0057] Introducing a foreign polynucleotide showing the activity of the polypeptide in 6) above is done by introducing a foreign polynucleotide encoding a polypeptide showing the same / similar activity as the polypeptide into the host cell. The origin and sequence of the foreign polynucleotide may be any as long as it shows the same / similar activity as the polypeptide. The introduction can be appropriately selected by those skilled in the art using known transformation methods. When the polynucleotide introduced as described above is expressed in the host cell, a polypeptide is generated and its activity is improved.

[0058] Optimizing the codons of the polynucleotide encoding the polypeptide (7) is achieved by optimizing the codons of the endogenous polynucleotide so that transcription or translation increases in the host cell, or by optimizing the codons of the foreign polynucleotide so that optimized transcription and translation occur in the host cell.

[0059] In addition, analyzing the tertiary structure of the polypeptide (8), selecting and modifying the exposed portion, or chemically modifying it is, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which the sequence information of known proteins is stored, determining candidates for the template protein according to the degree of sequence similarity, confirming the structure based on this, and selecting and modifying or chemically modifying the exposed portion to be modified.

[0060] Enhancement of such polypeptide activity is achieved by improving the activity or concentration, expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in the wild-type or unmodified microorganism, or by increasing the amount of the product produced from the polypeptide, but is not limited thereto.

[0061] In the microorganism of the present application, some or all of the modifications of the polynucleotide can be induced by (a) homologous recombination using a chromosomal integration vector in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) treatment with light such as ultraviolet rays or radiation and / or chemical substances, but is not limited thereto. The method for modifying some or all of the gene includes methods by DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene is introduced into the microorganism to cause homologous recombination, resulting in deletion of some or all of the gene. The introduced nucleotide sequence or vector may contain a dominant selection marker, but is not limited thereto.

[0062] As used herein, the vector of the present application means a DNA product containing the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression regulatory region (or expression regulatory sequence) so as to be able to express the target polypeptide in a suitable host. The expression regulatory region includes a promoter that initiates transcription, any operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate or function regardless of the host genome and is integrated into the genome itself.

[0063] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant states. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0064] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker for confirming whether the polynucleotide has been introduced into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, that is, for confirming whether the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show different phenotypes, so that transformed cells can be selected.

[0065] As used in this application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide as long as it is expressed in the host cell, regardless of whether it is inserted into the host cell chromosome or located extrachromosomally. Further, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its own expression. Usually, the expression cassette includes 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 a self-replicating expression vector. Further, the polynucleotide may be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited thereto.

[0066] Further, the "operably linked" means that the promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target polypeptide of this application is functionally linked to the polynucleotide sequence.

[0067] The microorganism of this application may be a microorganism in which the VKOR protein or the polynucleotide encoding the same is inactivated, or a microorganism (for example, a recombinant microorganism) genetically modified by a vector so that the VKOR protein or the polynucleotide encoding the same is inactivated, but is not limited thereto. The vector is as described above.

[0068] The microorganism of the present application may be a microorganism having the ability to produce L-glutamic acid.

[0069] The microorganism of the present application may be a microorganism that naturally has the ability to produce L-glutamic acid, or may be a microorganism in which the VKOR protein or the polynucleotide encoding the same is inactivated in a parent strain lacking the ability to produce L-glutamic acid and the ability to produce L-glutamic acid is imparted, but is not limited thereto.

[0070] For example, the recombinant microorganism of the present application is transformed with a vector so that the VKOR protein or the polynucleotide encoding the same is inactivated, and is a microorganism in which the VKOR protein or the polynucleotide encoding the same is inactivated, and any microorganism may be used as long as the VKOR protein or the polynucleotide encoding the same is inactivated and the microorganism produces L-glutamic acid.

[0071] For the purpose of the present application, the recombinant microorganism of the present application is a natural wild-type microorganism, or a microorganism that produces L-glutamic acid including the VKOR protein or the polynucleotide encoding the same, in which the VKOR protein or the polynucleotide encoding the same is inactivated, and compared with the natural wild-type microorganism, or a microorganism that produces L-glutamic acid including the VKOR protein or the polynucleotide encoding the same, it is a microorganism with improved L-glutamic acid production ability, but is not limited thereto. For example, the target strain for comparing whether the L-glutamic acid production ability is improved, and the unmodified microorganism in which the VKOR protein is not inactivated is the Corynebacterium glutamicum ATCC13869 strain lacking the odhA gene known as an L-glutamic acid producing strain, and the Corynebacterium glutamicum BL2 strain (KFCC11074, Patent Document 6) known as an L-glutamic acid producing NTG mutant strain, but is not limited thereto.

[0072] One example As such, the recombinant strain with improved production ability has an improvement of about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 14.3% or more, about 20% or more, about 28.6% or more, about 30% or more, about 31.9% or more, about 33.3% or more, about 37.7% or more, about 40% or more, about 42.9% or more, about 46.3% or more, about 50% or more, or about 52.4% or more (the upper limit is not particularly limited, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 15% or less) compared to the L-glutamic acid production ability of the parental strain before mutation or the non-modified microorganism. However, any strain that shows an increase in the + value compared to the production ability of the parental strain before mutation or the non-modified microorganism may be used. As another example, the microorganism with improved production ability has an L-glutamic acid production ability that is about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.05 times or more, about 1.06 times or more, about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.1 times or more, about 1.14 times or more, about 1.28 times or more, about 1.32 times or more, about 1.33 times or more, about 1.37 times or more, about 1.43 times or more, about 1.46 times or more, or about 1.52 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) compared to the parental strain before mutation or the non-modified microorganism, but is not limited thereto.

[0073] In the present application, the "non-modified microorganism" does not exclude strains containing mutations that can occur naturally in microorganisms, but means the wild-type strain or the natural strain itself, or the strain before genetic mutation and phenotypic change due to natural or artificial factors. For example, the non-modified microorganism means a strain in which the VKOR protein or the polynucleotide encoding the same described in the present specification has not been inactivated or before inactivation. The "non-modified microorganism" is used interchangeably with the "strain before modification", "microorganism before modification", "non-mutant strain", "non-modified strain", "non-mutant microorganism", or "reference microorganism".

[0074] As another example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, specifically Corynebacterium glutamicum, but is not limited thereto.

[0075] As yet another example, the recombinant microorganism of the present application may be a microorganism in which the activity of some of the proteins in the L-glutamic acid biosynthesis pathway is further enhanced, or the activity of some of the proteins in the L-glutamic acid degradation pathway is further inactivated, thereby enhancing the L-glutamic acid production ability.

[0076] Specifically, the microorganism of the present application may be a microorganism in which the OdhA protein is further inactivated or the odhA gene is further deleted. More specifically, the microorganism of the present application may be Corynebacterium glutamicum in which the OdhA protein is inactivated in Corynebacterium glutamicum ATCC 13869, or a microorganism in which the odhA gene is deleted in Corynebacterium glutamicum ATCC 13869. The OdhA protein contains the amino acid sequence of NCBI Sequence ID WP_060564343.1 (SEQ ID NO: 32), and the odhA gene contains the nucleotide sequence of NCBI GenBank BBD29_06050, but is not limited thereto.

[0077] However, the inactivation of the OdhA protein or the deletion of the odhA gene is only an example and is not limited thereto. The microorganism of the present application may be a microorganism in which the protein activity of various known L-glutamic acid biosynthesis pathways is enhanced, or a microorganism in which the protein activity of the degradation pathway is inactivated.

[0078] Another aspect of the present application provides a method for producing L-glutamic acid, which includes the step of culturing a Corynebacterium microorganism in which the VKOR protein is inactivated in a medium.

[0079] The method for producing L-glutamic acid of the present application may include the step of culturing a microorganism in which the VKOR protein or the polynucleotide encoding the same is inactivated, or a Corynebacterium microorganism genetically modified by a vector so that the VKOR protein or the polynucleotide encoding the same is inactivated in a medium.

[0080] In the present application, "cultivation" means growing the microorganisms of the present application under appropriately adjusted environmental conditions. The cultivation process of the present application can be carried out using a suitable medium and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used according to the microorganisms selected by those skilled in the art. Specifically, the cultivation is batch, continuous and / or fed-batch cultivation, but is not limited thereto.

[0081] In the present application, "medium" means a substance mixed mainly with the nutrients necessary for culturing the microorganisms of the present application, and supplies nutrients such as water, which is indispensable for survival and growth, and growth factors. Specifically, the medium and other cultivation conditions used for culturing the microorganisms of the present application can be any as long as they are those used for culturing ordinary microorganisms, and the microorganisms of the present application can be cultured by adjusting the temperature, pH, etc. under aerobic conditions in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin.

[0082] Specifically, the culture medium for culturing microorganisms of the genus Corynebacterium is disclosed in Non-Patent Document 18.

[0083] Examples of the carbon source in the present application include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, sugar alcohols such as mannitol and sorbitol, organic acids such as pyruvic acid, lactic acid and citric acid, and amino acids such as glutamic acid, methionine and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) can be used, and any other appropriate amount of carbon source can be used. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0084] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used as organic nitrogen sources. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0085] Examples of the phosphorus source include potassium dihydrogen phosphate, dipotassium hydrogen phosphate or sodium-containing salts corresponding thereto. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. can be used. In addition, amino acids, vitamins and / or suitable precursors, etc. can be used. These components or precursors can be added to the medium in a batch or continuous manner. However, it is not limited thereto.

[0086] Also, during the cultivation of the microorganism of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in a suitable manner. Furthermore, during the cultivation, an antifoaming agent such as a fatty acid polyglycol ester can be used to suppress the generation of bubbles. Furthermore, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium. In order to maintain the anaerobic and microaerobic states, it is not necessary to inject gas, and nitrogen, hydrogen or carbon dioxide gas may be injected, but it is not limited thereto.

[0087] In the cultivation of the present application, the cultivation temperature is maintained at 20 to 45 °C, specifically 25 to 40 °C, and cultivated for about 10 to 160 hours, but is not limited thereto.

[0088] The L-glutamic acid produced by culturing in this application is secreted into the culture medium or remains intracellular.

[0089] The method for producing L-glutamic acid in this application may further include the step of preparing the microorganism of this application, the step of preparing a culture medium for culturing the microorganism, or a combination thereof (in any order), for example, even before the step of culturing.

[0090] The method for producing L-glutamic acid in this application may further include the step of recovering L-glutamic acid from the culture medium (the medium in which the culture was performed) or the cultured microorganism used in the culture. The step of recovering may further be included after the step of culturing.

[0091] The recovery may be to recover the target L-glutamic acid using a suitable method known in the art according to the method for culturing the microorganism of this application, for example, batch, continuous, fed-batch culture methods, etc. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and other various chromatographies, HPLC, or a combination thereof may be used, and the target L-glutamic acid can be recovered from the culture medium or the microorganism using a suitable method known in the art.

[0092] Also, the method for producing L-glutamic acid in this application may further include a purification step. The purification can be performed by a suitable method known in the art. For example, when the method for producing L-glutamic acid in this application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, simultaneously, or integrated as one step, but are not limited thereto.

[0093] The VKOR protein, polynucleotide, vector, microorganism, etc. in the method of the present application are as described above.

[0094] Still another aspect of the present application provides a composition for producing L-glutamic acid, which comprises a corynebacterium microorganism in which the VKOR protein is inactivated, a medium in which the microorganism is cultured, or a combination thereof.

[0095] The composition of the present application may further contain any suitable excipient commonly used in the composition for producing L-glutamic acid. Such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffering agents, stabilizing agents, isotonic agents, and the like.

[0096] Still another aspect of the present application provides a method for producing a corynebacterium microorganism, which comprises a step of inactivating the VKOR protein. of the present application.

[0097] Still another aspect of the present application provides the use of a corynebacterium microorganism in which the VKOR protein is inactivated for producing L-glutamic acid.

[0098] The VKOR protein, inactivation, corynebacterium microorganism, etc. are as described above.

Examples

[0099] Hereinafter, the present application will be described in more detail with reference to examples. However, these examples are only preferred embodiments for exemplifying the present application, and the present application is not limited thereto. Technical matters not described in this specification can be fully understood and easily implemented by those skilled in the technical field of the present application or similar technical fields.

Examples

[0100] Generation of a Random Mutation Library Using Transposons Example 1-1: Generation of a Corynebacterium glutamicum Strain Having the Ability to Produce L-Glutamic Acid Derived from Wild-Type Corynebacterium glutamicum To generate a strain having the ability to produce L-glutamic acid derived from wild-type Corynebacterium glutamicum ATCC13869, a Corynebacterium glutamicum ATCC13869△odhA strain with the odhA gene deleted was generated based on the prior art literature (Non-Patent Document 19).

[0101] Specifically, for the deletion of the odhA gene, using the chromosomal DNA of wild-type Corynebacterium glutamicum ATCC13869 as a template and primer sets of SEQ ID NO: 3 and SEQ ID NO: 4, and SEQ ID NO: 5 and SEQ ID NO: 6, the upstream and downstream regions of the odhA gene were obtained by PCR. As the polymerase, Solg TM Pfu-X DNA polymerase was used, and the PCR amplification conditions were: after denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds, followed by a polymerization reaction at 72°C for 5 minutes.

[0102] The amplified upstream and downstream regions of odhA, and the chromosomal transformation vector pDCM2 (Patent Document 7) digested with SmaI restriction enzyme were cloned by the Gibson assembly (Non-Patent Document 20) method to obtain a recombinant vector, which was named pDCM2-△odhA. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts and then storing at 50°C for 1 hour.

[0103] The prepared pDCM2-△odhA vector was transformed into the wild-type Corynebacterium glutamicum ATCC13869 strain by electroporation, and after undergoing a secondary crossover process, a strain with the odhA gene deleted on the chromosome was obtained. The presence or absence of gene deletion was confirmed by PCR using SEQ ID NO: 7 and SEQ ID NO: 8 and genome sequencing, and the prepared strain was named ATCC13869△odhA.

[0104] The primer sequences used here are shown in Table 1.

[0105] [Table 1]

[0106] Example 1-2: Preparation of a Random Mutation Library Using a Transposon To obtain a strain with improved L-glutamic acid-producing ability, a vector library was prepared by the following method.

[0107] Using Corynebacterium glutamicum ATCC13869△odhA as the parental strain, the plasmid obtained using the EZ-Tn5 TM <R6Kγori / KAN-2>Tnp Transposome TM Kit (Epicentre) was transformed by electroporation (Non-Patent Document 21), and spread on a complex plate medium containing kanamycin (25 mg / l) to secure approximately 5,000 colonies. <Complex plate medium (pH 7.0)> Glucose 10 g, peptone 10 g, beef extract 5 g, yeast extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, urea 2 g, sorbitol 91 g, agar 20 g (in 1 liter of distilled water)

Example

[0108] Screening of Random Mutation Library Using Transposon Approximately 5,000 colonies obtained in Examples 1-2 were each inoculated into 300 μL of the following selection medium and cultured at 37°C and 1,000 rpm for approximately 48 hours in a 96-deep well plate.<Selection Medium (pH 7.0)>Raw sugar 5%, 1M phosphate buffer (pH 8.0) 100 ml, hydrolysed soybean meal (HSM) 0.096%, ammonium sulfate 2.25%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.04%, iron sulfate 10 mg / L, thiamine hydrochloride 0.2 mg / L, biotin 0.3 mg / L (in 1 L of distilled water)

[0109] After the cultivation, L-glutamic acid was measured using a YSI (YSI 2900 Biochemistry Analyzer). Ten colonies were selected as mutant strains showing higher L-glutamic acid values compared to the parent strain Corynebacterium glutamicum ATCC13869ΔodhA. The other colonies showed L-glutamic acid values equivalent to or lower than those of the Corynebacterium glutamicum ATCC13869ΔodhA strain used as the parent strain.

[0110] The 10 selected strains were cultured again in the same manner as above, and finally, the top mutant strain ATCC13869ΔodhA / mt-8 with improved L-glutamic acid productivity was selected compared to the parent strain Corynebacterium glutamicum ATCC13869ΔodhA strain.

Example

[0111] Elucidation of the cause of improved L-glutamic acid production ability in the selected mutant strain. For ATCC13869△odhA / mt-8 selected in Example 2, the gene disrupted by random insertion of a transposon was analyzed using the manufacturer's manual and Primers 1 (SEQ ID NO: 9) and 2 (SEQ ID NO: 10) of the Kit. As a result, based on the nucleotide sequence reported in the National Institutes of Health's gene bank (NIH Genbank), it was confirmed that the gene (BBD29_04485) containing the polynucleotide sequence of SEQ ID NO: 2 was inactivated.

[0112] The primer sequences used here are shown in Table 2.

[0113]

Table 2

Example

[0114] Preparation of a recombinant vector for inactivating the VKOR protein. Example 4-1: Preparation of a recombinant vector for deleting the BBD29_04485 gene encoding the VKOR protein. As confirmed in Example 3, to confirm whether deleting the BBD29_04485 gene (SEQ ID NO: 2) encoding the VKOR protein on the chromosome of Corynebacterium sp. strain improves the production ability of L-glutamic acid, a gene deletion recombinant vector was prepared.

[0115] Therefore, first, primers of SEQ ID NOs: 11 to 14 were synthesized to prepare a fragment for deleting the gene.

[0116] Specifically, using the chromosomal DNA of wild-type Corynebacterium glutamicum ATCC13869 as a template and primer sets of SEQ ID NO: 11 and SEQ ID NO: 12, and SEQ ID NO: 13 and SEQ ID NO: 14, a BBD29_04485 gene fragment was obtained by PCR. As the polymerase, Solg TMUsing Pfu-X DNA polymerase, the PCR amplification conditions were as follows: after denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds were performed, and then a polymerization reaction at 72°C for 5 minutes was carried out.

[0117] The amplified gene fragment and the chromosomal transformation vector pDCM2 digested with SmaI restriction enzyme were cloned by the Gibson assembly method to obtain a recombinant vector, which was named pDCM2-△BBD29_04485. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts and then storing at 50°C for 1 hour.

[0118] The primer sequences used here are shown in Table 3.

[0119]

Table 3

[0120] Example 4-2: Preparation of a recombinant vector for changing the start codon of BBD29_04485 encoding VKOR protein To confirm whether changing the start codons of the BBD29_04485 gene to TTG and CTG respectively on the chromosome of Corynebacterium strains can improve the production ability of L-glutamic acid, first, a start codon-changing recombinant vector was prepared.

[0121] For this purpose, first, primers of SEQ ID NOs: 15 to 20 were synthesized to prepare a fragment for changing the start codon of the gene.

[0122] Specifically, using the chromosomal DNA of wild-type Corynebacterium glutamicum ATCC13869 as a template and primer sets of SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 15 and SEQ ID NO: 19, and SEQ ID NO: 18 and SEQ ID NO: 20, gene fragments were obtained by PCR. As the polymerase, Solg TMUsing Pfu-X DNA polymerase, the PCR amplification conditions were as follows: after denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds were performed, followed by a polymerization reaction at 72°C for 5 minutes.

[0123] The amplified gene fragment and the chromosomal transformation vector pDCM2 digested with SmaI restriction enzyme were cloned by the Gibson assembly method to obtain a recombinant vector, which was named pDCM2-△BBD29_04485::BBD29_04485(g1t) vector and pDCM2-△BBD29_04485::BBD29_04485(g1c), respectively. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts and then storing at 50°C for 1 hour.

[0124] The primer sequences used here are shown in Table 4.

[0125]

Table 4

[0126] Example 4-3: Preparation of a recombinant vector for changing the ribosome binding site (RBS) of BBD29_04485 encoding VKOR protein To weaken the BBD29_04485 gene on the chromosome of Corynebacterium sp., the Shine-Dalgarno sequence (SD) was predicted from the nucleotide sequence containing 35 base pairs upstream of the gene and 35 base pairs from the N-terminus of the open reading frame (ORF).

[0127] Based on the above base sequence, three types of ribosome binding site candidate groups, namely RBS1, RBS2, and RBS3, were predicted using RBS Calculator (github). As a result, the expression levels of the three types of ribosome binding site candidate groups were predicted to decrease by 50%, 20%, and 10% respectively compared to the conventional ribosome binding site.

[0128] The predicted ribosome binding sites are shown in Table 5.

[0129]

Table 5

[0130] In order to confirm whether the production ability of L-glutamic acid is improved by changing the ribosome binding site of BBD29_04485 on the chromosome of Corynebacterium strains, ribosome binding site modification recombinant vectors were prepared for each of the three predicted ribosome binding sites as described above.

[0131] For this purpose, first, primers of SEQ ID NOs: 21 to 28 were synthesized to prepare a fragment for modifying the ribosome binding site of the gene.

[0132] Specifically, using the chromosomal DNA of wild-type Corynebacterium glutamicum ATCC13869 as a template, gene fragments were obtained by PCR using primer sets of SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 21 and SEQ ID NO: 25, SEQ ID NO: 24 and SEQ ID NO: 26, SEQ ID NO: 21 and SEQ ID NO: 27, and SEQ ID NO: 24 and SEQ ID NO: 28. As the polymerase, Solg TM Pfu-X DNA polymerase was used, and the PCR amplification conditions were as follows: after denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds were performed, and then a polymerization reaction at 72°C for 5 minutes was carried out.

[0133] The amplified gene fragment and the chromosomal transformation vector pDCM2 digested with SmaI restriction enzyme were cloned by the Gibson assembly method to obtain a recombinant vector, which was named pDCM2-△RBS(wt)::RBS1 vector, pDCM2-△RBS(wt)::RBS2 vector, and pDCM2-△RBS(wt)::RBS3 vector, respectively. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amount, and then storing at 50 °C for 1 hour.

[0134] The primer sequences used here are shown in Table 6.

[0135]

Table 6

Example

[0136] Preparation of a strain with L-glutamic acid production ability derived from wild-type Corynebacterium glutamicum in which BBD29_04485 encoding VKOR protein is attenuated. The ATCC13869△odhA strain prepared in Example 1 was used as the target, and the pDCM2-△BBD29_04485 vector, pDCM2-△BBD29_04485::BBD29_04485(g1t) vector, pDCM2-△BBD29_04485::BBD29_04485(g1c) vector, pDCM2-△RBS(wt)::RBS1 vector, pDCM2-△RBS(wt)::RBS2 vector, and pDCM2-△RBS(wt)::RBS3 vector prepared in Example 4 were introduced to confirm the effect on L-glutamic acid production ability.

[0137] First, the pDCM2-△BBD29_04485 vector was transformed into the Corynebacterium glutamicum ATCC13869△odhA strain by electroporation, and then through a secondary crossing process, a strain with the BBD29_04485 gene deleted on the chromosome was obtained.

[0138] The genetic manipulation was confirmed by PCR and genomic sequencing using SEQ ID NO: 29 and SEQ ID NO: 30, which amplify the upstream and downstream regions of the same recombination, respectively, and it was named CA02-1624.

[0139] Next, the pDCM2-△BBD29_04485::BBD29_04485(g1t) vector and the pDCM2-△BBD29_04485::BBD29_04485(g1c) vector were transformed into the Corynebacterium glutamicum ATCC13869△odhA strain by electroporation. Then, through the secondary crossing process, strains were obtained in which the start codon of the BBD29_04485 gene on the chromosome was changed from GTG to TTG and CTG, respectively.

[0140] The genetic manipulation was confirmed by PCR and genomic sequencing using SEQ ID NO: 31 and SEQ ID NO: 30, which amplify the upstream and downstream regions of the same recombination, respectively, and they were named CA02-1625 and CA02-1630, respectively.

[0141] Next, the pDCM2-△RBS(wt)::RBS1 vector, the pDCM2-△RBS(wt)::RBS2 vector, and the pDCM2-△RBS(wt)::RBS3 vector were transformed into the Corynebacterium glutamicum ATCC13869△odhA strain by electroporation. Then, through the secondary crossing process, strains were obtained in which the ribosome binding site of the BBD29_04485 gene on the chromosome was changed to RBS1, RBS2, and RBS3, respectively.

[0142] The genetic manipulation was confirmed by PCR and genomic sequencing using the primers of SEQ ID NO: 31 and SEQ ID NO: 30, which amplify the upstream and downstream regions of the same recombination, respectively, and they were named CA02-1631, CA02-1632, and CA02-1633, respectively.

[0143] The primer sequences used here are shown in Table 7.

[0144]

Table 7

[0145] Using the ATCC13869△odhA strain as the control group, the following method was used to culture six strains, namely CA02-1624, CA02-1625, CA02-1630, CA02-1631, CA02-1632, and CA02-1633, prepared as described above, to confirm their L-glutamic acid production ability.

[0146] The strain was inoculated onto a plate medium as the seed medium and cultured at 30 °C for 20 hours. Then, one platinum loop of the strain was inoculated into a 250 ml corner baffle flask containing 25 ml of the following production medium and cultured with shaking at 30 °C and 200 rpm for 40 hours. After the culture was completed, the L-glutamic acid production amount was measured using high performance liquid chromatography (HPLC). The measurement results are shown in Table 8. <Seed medium> Glucose 1%, gravy 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, agar 2%, urea 0.2%, pH 7.2 <Production medium> Raw sugar 6%, calcium carbonate 5%, ammonium sulfate 2.25%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.04%, iron sulfate 10 mg / L, thiamine hydrochloride 0.2 mg / L, biotin 50 μg / L

[0147]

Table 8

[0148] As shown in Table 8, compared with the wild-type-derived ATCC13869△odhA strain, in all of CA02-1624 with the BBD29_04485 gene deleted, CA02-1625, CA02-1630 with the start codon of the BBD29_04485 gene weakened to TTG and CTG respectively, CA02-1631, CA02-1632, and CA02-1633 with the ribosome binding site of the BBD29_04485 gene weakened to RBS1, RBS2, and RBS3 respectively, an increase in the concentration of L-glutamic acid was confirmed.

[0149] Therefore, regardless of the specific means, it can be seen that as long as the activity of the VKOR protein is inactivated, the production ability of L-glutamic acid is enhanced.

[0150] Among the above strains, CA02-1624 was deposited with the Korean Collection for Type Cultures, an international depositary authority under the Budapest Treaty, on January 13, 2021 under the accession number KCCM12928P.

Example

[0151] Confirmation of the deletion effect of BBD29_04485 encoding VKOR protein in N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutant Corynebacterium glutamicum strains In addition to the wild-type Corynebacterium-derived strains, to confirm whether the gene also exhibits the same effect in NTG mutant Corynebacterium-derived strains with improved L-glutamic acid production ability, the attenuation effect of the gene confirmed in Example 5 was verified in Corynebacterium glutamicum BL2 strain (KFCC11074, Patent Document 6), a known L-glutamic acid-producing NTG mutant strain.

[0152] Specifically, three types of vectors, namely pDCM2-△BBD29_04485 vector, pDCM2-△BBD29_04485::BBD29_04485(g1c) vector, and pDCM2-△RBS(wt)::RBS3 vector, in which the L-glutamic acid concentration increased by 40% or more in Example 5, were introduced into the KFCC11074 strain, and the effects on L-glutamic acid production ability were confirmed.

[0153] First, the pDCM2-△BBD29_04485 vector was transformed into the KFCC11074 strain by electroporation, and then through a secondary crossing process, a strain with the BBD29_04485 gene deleted on the chromosome was obtained.

[0154] The genetic manipulation was confirmed by PCR and genomic sequencing using the primers of SEQ ID NO: 29 and SEQ ID NO: 30 that amplify the upstream and downstream regions of the same recombination, respectively, and it was named CA02-1634.

[0155] Next, the pDCM2-△BBD29_04485::BBD29_04485(g1c) vector was transformed into the KFCC11074 strain by electroporation, and then through a secondary crossover process, a strain was obtained in which the start codon of the BBD29_04485 gene on the chromosome was changed from GTG to CTG.

[0156] The genetic manipulation was confirmed by PCR and genomic sequencing using SEQ ID NO: 31 and SEQ ID NO: 30 that amplify the upstream and downstream regions of the same recombination, respectively, and it was named CA02-1635.

[0157] Next, the pDCM2-△RBS(wt)::RBS3 vector was transformed into the KFCC11074 strain by electroporation, and then through a secondary crossover process, a strain was obtained in which the ribosome binding site of the BBD29_04485 gene on the chromosome was changed to RBS3.

[0158] The genetic manipulation was confirmed by PCR and genomic sequencing using SEQ ID NO: 31 and SEQ ID NO: 30 that amplify the upstream and downstream regions of the same recombination, respectively, and it was named CA02-1636.

[0159] The KFCC11074 strain was used as the control group, and the following method was used to culture the three strains of CA02-1634, CA02-1635, and CA02-1636 prepared to confirm the L-glutamic acid production ability.

[0160] The strain was inoculated onto an agar plate as a seed medium and cultured at 30°C for 20 hours. Then, one platinum loopful of the strain was inoculated into a 250-ml conical baffle flask containing 25 ml of the following production medium and cultured with shaking at 30°C and 200 rpm for 40 hours. After the cultivation, the L-glutamic acid production was measured using high performance liquid chromatography (HPLC). The measurement results are shown in Table 9. <Seed medium> Glucose 1%, gravy 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, agar 2%, urea 0.2%, pH 7.2 <Production medium> Raw sugar 6%, calcium carbonate 5%, ammonium sulfate 2.25%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.04%, iron sulfate 10 mg / L, thiamine hydrochloride 0.2 mg / L, biotin 500 μg / L

[0161]

Table 9

[0162] As shown in the table, it was confirmed that in CA02-1634 with the BBD29_04485 gene deleted, the concentration of L-glutamic acid increased by about 46.4% compared to the KFCC11074 strain. Also, in CA02-1635 with the start codon of the BBD29_04485 gene weakened to CTG, it was confirmed that the concentration of L-glutamic acid increased by about 37.7%. Furthermore, in CA02-1636 with the ribosome binding site of the BBD29_04485 gene weakened to RBS3, it was confirmed that the concentration of L-glutamic acid increased by about 31.9%.

[0163] From the above description, those skilled in the technical field to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical idea and essential features. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all changes and modifications derived from the meaning and scope of the claims and their equivalent concepts, rather than the description of the specification.

[0164] TIFF0007690070000010.tif213150

Claims

1. A Corynebacterium microorganism in which the VKOR (vitamin K epoxide reductase family protein) protein is inactivated, wherein the VKOR protein consists of a polypeptide represented by an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO:

1.

2. The microorganism according to claim 1, wherein the VKOR protein is derived from Corynebacterium.

3. The microorganism according to claim 1, wherein the VKOR protein is encoded by a polynucleotide represented by the nucleotide sequence of SEQ ID NO:

2.

4. The microorganism according to claim 1, wherein the inactivation of the VKOR protein is a deletion or attenuation of the polynucleotide represented by the nucleotide sequence of SEQ ID NO:

2.

5. The microorganism according to claim 1, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

6. The microorganism according to claim 1, which has an improved L-glutamic acid-producing ability as compared with a parent strain or wild type in which the VKOR protein is not inactivated.

7. The microorganism according to claim 1, wherein the OdhA protein is further inactivated.

8. An L-glutamic acid production method comprising a step of culturing a Corynebacterium microorganism in which the VKOR protein is inactivated in a medium, wherein the VKOR protein consists of a polypeptide represented by an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO:

1.

9. The production method according to claim 8, wherein the VKOR protein is derived from Corynebacterium.

10. The production method according to claim 8, wherein the VKOR protein is encoded by a polynucleotide represented by the nucleotide sequence of SEQ ID NO:

2.

11. The production method according to claim 8, wherein the inactivation of the VKOR protein is a deletion or attenuation of the polynucleotide represented by the nucleotide sequence of SEQ ID NO:

2.

12. The production method according to claim 8, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

13. The production method according to claim 8, wherein the OdhA protein is further inactivated.

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