L-citrulline-producing microorganisms and method for producing L-citrulline using the same

Abstract

This application relates to a method for producing L-citrulline, comprising the step of culturing a Corynebacterium microorganism having L-citrulline-producing ability in which the activity of a protein containing the amino acid sequence of Sequence ID No. 1 is weakened compared to endogenous activity, in a culture medium, and further relates to a composition for L-citrulline production comprising the microorganism, a culture of the microorganism, a ferment of the microorganism, or a combination of at least two thereof, and further relates to the use of the microorganism for L-citrulline production.

Description

Technical Field

[0001] The present application relates to a method for producing L-citrulline, which includes a step of culturing a microorganism belonging to the genus Corynebacterium having an L-citrulline-producing ability in which the activity of a protein containing the amino acid sequence of SEQ ID NO: 1 is weakened compared to the intrinsic activity, in a medium, and relates to a composition for producing L-citrulline containing the microorganism, a culture of the microorganism, a fermented product of the microorganism, or a combination of at least two of them, and relates to the use of the microorganism for producing L-citrulline.

Background Art

[0002] L-citrulline, an L-amino acid derived from glutamic acid, is industrially useful because it has a function of dilating blood vessels in the body and promoting ammonia excretion (K Takeda et al. 2013).

[0003] Microorganisms belonging to the genus Corynebacterium, particularly Corynebacterium glutamicum, are Gram-positive microorganisms that are widely used for L-amino acid production. In order to produce L-amino acids, in Corynebacterium genus strains, an approach specific to a target substance that mainly increases the expression level of genes encoding enzymes involved in the biosynthesis of L-amino acids or removes genes unnecessary for the biosynthesis of L-amino acids has been mainly used (Patent Document 1). However, there is still a need for research on methods for efficiently producing L-amino acids.

[0004] Therefore, research on effectively improving the L-citrulline-producing ability is still required.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-licensed literature

[0006] [Non-licensed document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-licensed document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

[0007] This application aims to provide an L-citrulline-producing microorganism and an L-citrulline production method using the same. [Means for Solving the Problems]

[0008] One aspect of this application provides an L-citrulline production method including the step of culturing a Corynebacterium microorganism having an L-citrulline production ability in which the activity of the protein containing the amino acid sequence of SEQ ID NO: 1 is weakened compared to the endogenous activity in a medium.

[0009] In one specific example, the protein containing the amino acid sequence of SEQ ID NO: 1 may be encoded by a gene containing the nucleotide sequence of SEQ ID NO: 2.

[0010] In the microorganism according to any of the above-described specific examples, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0011] In another specific example, the method may further include the step of recovering L-citrulline from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium.

[0012] Another aspect of the present application provides a Corynebacterium microorganism having an L-citrulline-producing ability in which the activity of the protein containing the amino acid sequence of SEQ ID NO: 1 is weakened compared to the endogenous activity, a culture of the microorganism, a fermented product of the microorganism, or a composition for producing L-citrulline containing at least two combinations thereof.

Advantages of the Invention

[0013] The Corynebacterium microorganism having an L-citrulline-producing ability in which the activity of the protein containing the amino acid sequence of SEQ ID NO: 1 of the present application is weakened compared to the endogenous activity can produce L-citrulline in a high yield, and thus is useful for the industrial production of L-citrulline.

Modes for Carrying Out the Invention

[0014] Hereinafter, these will be specifically described. Each explanation and embodiment disclosed in the present application is also applicable to other explanations and embodiments. That is, any combination of various elements disclosed in the present application is included in the present application. Further, the present application is not limited to the following specific descriptions.

[0015] In addition, those having ordinary knowledge in the technical field can recognize and confirm many equivalents of specific aspects of the present application described in the present application by using only ordinary experiments. Furthermore, it is intended that such equivalents are also included in the present application.

[0016] As used in the specification and claims of the present application, the singular forms of the articles ( "a", "an", and "the") include plural objects unless otherwise specified. Unless otherwise specified, singular terms include plural forms, and plural terms include singular forms. In the specification and claims of the present application, unless otherwise specified, "or" is used in the sense of including "and / or".

[0017] In this application, "about" is used before a specific number. In this application, "about" includes not only the exact number that follows the term "about," but also a range that is approximately that number or close to it. Considering the context in which the number is used, it is possible to determine whether it is close to or approximately that specific number. For example, "about" indicates a range of -10% to +10% of a given number. Another example is that "about" indicates a range of -5% to +5% of a given number. However, it is not limited to these examples.

[0018] In this application, terms such as "first, second, third," "i), ii), iii)," and "(a), (b), (c), (d)," are used to distinguish similar configurations and do not imply that they are continuous or performed in order. For example, when the above terms are used in relation to steps of a method, use, or analysis, there may be no time interval between those steps, they may be performed simultaneously, or they may be performed with intervals of a few seconds, a few minutes, a few hours, a few days, or a few months.

[0019] In this application, "consisting essentially of" means that the presence of the unspecified component is not substantially affected by the presence of the unspecified component in which the features claimed by this application are subject to this application.

[0020] In this application, "consisting of" means that the proportion of a particular component totals 100%. The component or feature referred to as "consisting of" is either essential or mandatory. In a specific example, any other optional or non-essential component is excluded from the component or feature referred to as "consisting of".

[0021] In this application, “comprising” means that the features, steps, or components referred to by the above terms are present, and does not exclude the presence or addition of one or more features, steps, or components. While the components or features referred to as “comprising” in this application are essential or mandatory, in a specific example, other optional or non-essential components or features may be further included.

[0022] One aspect of this application provides a method for producing L-citrulline, comprising the step of culturing a Corynebacterium microorganism having L-citrulline-producing ability in which the activity of a protein containing the amino acid sequence of SEQ ID NO: 1 is weaker than that of endogenous activity, in a culture medium.

[0023] In this application, "protein containing the amino acid sequence of Sequence ID No. 1" means a protein that is endogenously present in microorganisms of the genus Corynebacterium, or a hypothetical protein whose function is unknown.

[0024] Specifically, the protein containing the amino acid sequence of Sequence ID No. 1 of this application is a protein having the activity of the protein containing the amino acid sequence of Sequence ID No. 1 encoded by the gene containing the base sequence of Sequence ID No. 2. However, the type of protein is not particularly limited as long as it has activity equivalent to the protein containing the amino acid sequence of Sequence ID No. 1 and improves the production capacity of L-citrulline in Corynebacterium microorganisms by weakening its activity compared to endogenous activity. Proteins containing the amino acid sequence of Sequence ID No. 1 encoded by the gene containing the base sequence of Sequence ID No. 2 are publicly known in the art, and the amino acid and polynucleotide sequences of proteins containing the amino acid sequence of Sequence ID No. 1 can be obtained from known databases, such as NCBI's GenBank, but are not limited to these.

[0025] In this application, the protein containing the amino acid sequence of Sequence ID No. 1 may be, but is not limited to, a protein containing the amino acid sequence of Sequence ID No. 1 derived from a microorganism of the genus Corynebacterium, specifically Corynebacterium glutamicum.

[0026] For example, a protein containing the amino acid sequence of SEQ ID NO: 1 contains the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having 60% or more homology or identity with it, but is not limited to these, as long as it has the activity of a protein containing the amino acid sequence of SEQ ID NO: 1. Specifically, a polypeptide having the activity of a protein containing the amino acid sequence of SEQ ID NO: 1 may have an amino acid sequence that has at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 1, may contain the aforementioned amino acid sequence, may consist of the aforementioned amino acid sequence, or may be substantially composed of the aforementioned amino acid sequence. For example, a protein containing the amino acid sequence of SEQ ID NO: 1 is a protein endogenously present in microorganisms of the genus Corynebacterium or Corynebacterium glutamicum, but is not limited to these.

[0027] In this application, even if a polypeptide is described as "containing" an amino acid sequence represented by a specific sequence number, a polypeptide "consisting of" an amino acid sequence represented by a specific sequence number, or a polypeptide or protein "having" an amino acid sequence represented by a specific sequence number, it goes without saying that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, conserved substituted, or added are also included in this application, as long as they have the same or equivalent activity as a protein consisting of the amino acid sequence of said sequence number. For example, if a protein has the same or equivalent activity as the aforementioned modified protein, this does not exclude the addition of sequences before or after the amino acid sequence that do not change the function of the protein, naturally occurring mutations, silent mutations, or conserved substitutions, and it goes without saying that proteins having such sequence additions or mutations are also included in this application.

[0028] For example, the mutant polypeptide may have an addition of a sequence that does not alter the function of the mutant polypeptide, a spontaneous mutation, a silent mutation, or a conserved substitution at its N-terminus, C-terminus, and / or within the amino acid sequence. For example, the polypeptide may be bound to the N-terminal signal (or leader) sequence of a protein involved in protein transfer co-translationally or post-translationally. Alternatively, the polypeptide may be bound to other sequences or linkers so that the polypeptide can be identified, purified, or synthesized.

[0029] In this application, "conservative substitution" means that one amino acid is replaced by 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. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Other examples include amino acids with electrically charged side chains (arginine, lysine, histidine, glutamic acid, and aspartic acid) and amino acids with uncharged side chains (also called neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Further examples include phenylalanine, tryptophan, and tyrosine, which are classified as aromatic amino acids. Still further examples include valine, leucine, and isoleucine, which are classified as branched amino acids.As another example, the 20 amino acids are classified by size and, in order of increasing relative volume, into five groups: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, and arginine; and phenylalanine, tryptophan, and tyrosine. However, this is not necessarily the only classification. Typically, conservative substitutions have little to no effect on polypeptide activity.

[0030] Furthermore, the base sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 1 may also be a base sequence encoding a protein that has the activity of a protein containing the amino acid sequence of SEQ ID NO: 1, and whose activity is weakened compared to endogenous activity in Corynebacterium microorganisms, thereby improving L-citrulline production. For example, this could be the NCgl2383 gene, which is an endogenously present gene in Corynebacterium microorganisms, or a gene encoding a hypothetical protein whose function is unknown, but is not limited to these.

[0031] For example, a protein containing the amino acid sequence of SEQ ID NO: 1 is encoded by a polynucleotide having, or containing, or consisting of, or substantially composed of, the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 2, or by a polynucleotide substantially composed of the nucleotide sequence of said nucleotide. Furthermore, the nucleotide sequence of SEQ ID NO: 2 can be obtained from a known database, such as NCBI's GenBank, but is not limited to these.

[0032] In this application, the gene containing the nucleotide sequence of SEQ ID NO: 2 is used interchangeably with a polynucleotide containing the nucleotide sequence of SEQ ID NO: 2, a gene or polynucleotide having the nucleotide sequence of SEQ ID NO: 2, a gene or polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, or the NCgl2383 gene.

[0033] For example, the NCgl2383 gene, which contains the nucleotide sequence of SEQ ID NO: 2, has two types of ORFs (open reading frames), such as ORF1 and ORF2, but is not limited to these. For example, ORF1 of the NCgl2383 gene codes for a protein containing the amino acid sequence of SEQ ID NO: 17, and ORF2 of the NCgl2383 gene codes for a protein containing the amino acid sequence of SEQ ID NO: 18, but is not limited to these.

[0034] The polynucleotides of this application can be modified in various ways in the coding region, either through codon degeneracy or by considering preferred codons in organisms intending to express a protein containing the amino acid sequence of SEQ ID NO: 1 of this application, as long as the amino acid sequence of the protein containing the amino acid sequence of SEQ ID NO: 1 of this application remains unchanged. Therefore, it goes without saying that polynucleotides that are translated by codon degeneracy into polypeptides consisting of the amino acid sequence of a protein containing the amino acid sequence of SEQ ID NO: 1 of this application, or polypeptides homologous or identical thereto, are also included. For example, the polynucleotides of this application may be SEQ ID NO: 2 or its degenerated sequence.

[0035] Furthermore, the polynucleotide of this 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 this application, and encodes a protein containing the amino acid sequence of SEQ ID NO: 1 of this application.

[0036] In this application, "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are related, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0037] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, and a default gap penalty established by the program used may also be applied. Substantially, homologous or identical sequences generally hybridize with at least 50%, 60%, 70%, 80%, or 90% of the entire sequence or its total length under moderate to high stringent conditions. Hybridization also includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy in the polynucleotide.

[0038] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined, for example, using default parameters as described in Non-Patent Document 1 and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as performed in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, homology, similarity, or identity can be determined using BLAST or Clustal W from the National Center for Biotechnology Information.

[0039] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 3, as disclosed in Non-Patent Document 8, for example. In summary, the GAP program is defined as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of two sequences. Default parameters for the GAP program include (1) unitary matrices (where identity is 1 and non-identity is 0), a PAM Matrix (see disclosure in Non-Patent Document 9), and a weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) from 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 extended penalty of 0.5); and (3) no penalty for terminal gaps.

[0040] Furthermore, whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions. The defined appropriate hybridization conditions are within the scope of the art and are determined by methods well known to those skilled in the art (e.g., Non-Patent Documents 11 and 12), but are not limited to these.

[0041] In this application, "stringent condition" refers to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Document 13). For example, this could involve hybridizing polynucleotides with high homology or identity, such as polynucleotides with 60% or more, 70% or more, 75% or more, 80% 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, while not hybridizing polynucleotides with lower homology or identity. Alternatively, it could involve washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a typical Southern hybridization: 60°C, 1×SSC, 0.1%SDS, more specifically 60°C, 0.1×SSC, 0.1%SDS, or more specifically 68°C, 0.1×SSC, 0.1%SDS.

[0042] The aforementioned hybridization requires that the two nucleotides have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this application may include not only substantially similar base sequences, but also isolated nucleic acid fragments whose entire sequences are complementary.

[0043] For example, polynucleotides homologous or identical to the polynucleotides of this 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. The Tm value may be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0044] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (e.g., Non-Patent Document 11).

[0045] In this application, "L-citrulline" refers to the L-amino acid having the chemical formula H2NC(O)NH(CH2)3CH(NH2)CO2H.

[0046] In this application, "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially. These are microorganisms in which a specific mechanism has been weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of endogenous gene activity, and which have been genetically modified for the production of a target polypeptide, protein, or product. In this application, "microorganism" and "strain" are used interchangeably and are used together.

[0047] In this application, "microorganisms having L-citrulline production ability" means prokaryotic or eukaryotic microbial strains that produce L-citrulline within their bodies, and includes all microorganisms in which L-citrulline production ability has been conferred from a parent strain that lacked L-citrulline production ability, as well as microorganisms that inherently possess L-citrulline production ability. L-citrulline production ability can be conferred or improved through selective breeding.

[0048] For example, the microorganisms having L-citrulline production ability in this application are microorganisms (e.g., recombinant strains) in which the activity of the protein containing the amino acid sequence of SEQ ID NO: 1 is attenuated compared to its endogenous activity.

[0049] In this application, "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but rather refers to wild-type strains or natural strains themselves, or strains before genetic mutation and changes in phenotype due to natural or artificial factors. The aforementioned "unmodified microorganism" is used interchangeably with "pre-modification strain," "pre-modification microorganism," "non-mutant strain," "unmodified strain," "non-mutant microorganism," "pre-mutation parent strain," "wild-type microorganism," "reference microorganism," or "standard microorganism." In this application, "unmodified microorganism" refers to strains in which the activity of the protein containing the amino acid sequence of SEQ ID NO: 1 has not been weakened compared to its endogenous activity, or strains in which the activity of the protein containing the amino acid sequence of SEQ ID NO: 1 has not been weakened compared to its endogenous activity, but is not limited to these. Furthermore, in this application, "unmodified microorganism" refers to microorganisms containing the amino acid sequence consisting of SEQ ID NO: 1, or the polynucleotide consisting of SEQ ID NO: 2, but is not limited to these.

[0050] For the purposes of this application, the unmodified microorganisms of this application may also be microorganisms that intrinsically contain i) a protein consisting of the amino acid sequence of SEQ ID NO: 1, or a protein consisting of an amino acid sequence having at least 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 of SEQ ID NO: 1, and / or ii) a polynucleotide sequence encoding a protein containing an amino acid sequence having at least 80% homology to SEQ ID NO: 1, the base sequence of SEQ ID NO: 2, or a base sequence having 80% 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 base sequence of SEQ ID NO: 2, and less than 100%.

[0051] For the purposes of this application, the microorganisms described herein include all microorganisms that produce the target L-citrulline, in which the activity of a protein containing the amino acid sequence of SEQ ID NO: 1 is weakened compared to its endogenous activity. For example, the microorganisms described herein are genetically modified or recombinant microorganisms characterized by improved L-citrulline production capacity due to weakened protein activity of the amino acid sequence of SEQ ID NO: 1 compared to its endogenous activity, but are not limited to these. Specifically, the recombinant strains with improved L-citrulline production capacity are microorganisms that have improved L-citrulline production capacity compared to natural wild-type microorganisms or unmodified microorganisms with endogenous activity of a protein containing the amino acid sequence of SEQ ID NO: 1, but are not limited to these.

[0052] For example, microorganisms capable of producing L-citrulline are prokaryotic or eukaryotic microbial strains that produce L-citrulline within their bodies, and include all microorganisms that inherently possess L-citrulline production ability, as well as microorganisms in which L-citrulline production ability has been conferred to a parent strain that lacks L-citrulline production ability by the activity of a protein containing the amino acid sequence of Sequence ID No. 1, which is weaker than the endogenous activity described in this application. L-citrulline production ability can be conferred or improved through selective breeding.

[0053] The microorganisms described in this application include all microorganisms in which the activity of a protein containing the amino acid sequence of Sequence ID No. 1 has been weakened compared to its endogenous activity by various known methods.

[0054] The aforementioned "endogenous activity" refers to the activity of a specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change, when a trait is altered due to genetic mutation caused by natural or artificial factors. This term is often used interchangeably with "activity before deformation."

[0055] In this application, "weakening" of polypeptide activity is a concept that includes all cases where the activity is reduced compared to endogenous activity or where the activity is eliminated. The term "weakening" is used interchangeably with terms such as deficiency, inactivation, deletion, disruption, down-regulation, decline, attenuation, repression, and reduction.

[0056] For example, the weakening of polypeptide activity includes a state in which polypeptide activity is exhibited within the host cell (microorganism) but is not completely inactivated due to deletion, where polypeptide activity is reduced compared to endogenous activity or activity before modification, polypeptide expression is not expressed at all compared to the host cell (microorganism) before the trait change or the unmodified microorganism, or even if expressed, its activity is absent or reduced (inactivated). The weakening includes the weakening or removal of the polypeptide activity itself compared to the original polypeptide activity of the microorganism due to mutations in the polynucleotide encoding the polypeptide, a decrease in the overall level of polypeptide activity within the cell compared to the natural strain due to inhibition of gene expression of the polynucleotide encoding it or inhibition of translation into polypeptide, complete absence of gene expression, and absence of polypeptide activity even if the gene is expressed.

[0057] The weakening of polypeptide activity compared to endogenous activity means that the activity of the specific polypeptide is reduced compared to the activity originally possessed by the parent strain or unmodified microorganism before the trait change. Whether or not the polypeptide activity has been weakened can be confirmed by a decrease in the degree of polypeptide activity, expression level, or the amount of product produced from the polypeptide.

[0058] For example, the weakening described above means that the activity of the protein containing the amino acid sequence of SEQ ID NO: 1 becomes less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 0% of the activity of the protein containing the amino acid sequence of SEQ ID NO: 1 in the parental strain or unmodified microorganism before the trait change.

[0059] For example, inactivation means that the protein containing the amino acid sequence of Sequence ID No. 1 is not expressed at all compared to unmodified microorganisms, or if it is expressed, its activity is absent or weakened.

[0060] Such weakening of polypeptide activity is not limited to these methods and can be achieved by applying various methods well known in the field (e.g., Non-Patent Documents 14, 15, etc.).

[0061] Specifically, the weakening of polypeptide activity in this application includes: 1) deleting all or part of the gene encoding the polypeptide; 2) modifying the expression regulatory region (or expression regulatory sequence) so as to weaken the expression of the gene encoding the polypeptide; 3) modifying the amino acid sequence constituting the polypeptide so as to delete or weaken the polypeptide's activity (for example, deleting / substituting / adding one or more amino acids in the amino acid sequence); and 4) modifying the gene sequence encoding the polypeptide so as to delete or weaken the polypeptide's activity (for example, modifying the gene sequence to encode a polypeptide that has been modified so as to delete or weaken the polypeptide's activity). 1) Deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of a peptide gene; 2) Modifying the base sequence of the start codon or 5'UTR region of the polypeptide-encoding gene transcript; 3) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the polypeptide-encoding gene transcript; 4) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the polypeptide-encoding gene so that a secondary structure is formed that prevents ribosome attachment; 5) Adding a promoter to the 3' end of the open reading frame (ORF) of the polypeptide-encoding gene sequence to reverse transcription (Reverse transcription engineering, RTE); 6) Regulating the cellular localization of the protein (polypeptide); or 7) Combining two or more of the above 1) to 9), but not being particularly limited thereto.

[0062] For example, the deletion of part or all of the gene encoding the polypeptide described in 1) above may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, or by substituting it with a polynucleotide or marker gene in which part of the nucleotide sequence is deleted.

[0063] Methods for deleting some or all of such polynucleotides include, but are not limited to, deleting polynucleotides by homologous recombination using a chromosome introduction vector in a microorganism, or inducing mutations using light such as ultraviolet light or chemical substances, and selecting a bacterial strain lacking the target gene from the resulting mutants. Methods for deleting some or all of the aforementioned genes include methods using DNA recombination technology. For example, some or all of the gene can be deleted by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism and inducing homologous recombination. The introduced nucleotide sequence or vector contains a dominant selection marker, but is not limited to that.

[0064] Furthermore, modifying the regulatory expression sequence described in 2) above may be done by causing a mutation in the regulatory expression region (or regulatory expression sequence) through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting it with a sequence having weaker activity. The regulatory expression region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and translation.

[0065] Modifying the amino acid sequence or polynucleotide sequence described in 3) and 4) above is carried out by introducing a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, so as to weaken the activity of the polypeptide, or by substituting it with an amino acid sequence or polynucleotide sequence modified to have lower activity, or an amino acid sequence or polynucleotide sequence modified to eliminate activity, but is not limited to these methods. For example, gene expression is inhibited or weakened by introducing a mutation into the polynucleotide sequence to form a stop codon, but is not limited to this method.

[0066] Furthermore, modifying the base sequence of the start codon or 5'UTR region of the gene transcript encoding the polypeptide (as described in 5) above can be done, for example, by substituting it with another start codon that has a lower polypeptide expression rate compared to the endogenous start codon, but is not limited to this.

[0067] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide (6) above may be carried out, for example, as described in Non-Patent Document 16.

[0068] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the polypeptide-coding gene so that a secondary structure is formed that makes ribosome attachment impossible may be done by making mRNA translation impossible or slowing down the rate of mRNA translation.

[0069] The process of adding a promoter to the 3' end of the open reading frame (ORF) of the polynucleotide 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 its activity.

[0070] 9) The regulation of the intracellular location of a protein (polypeptide) may be carried out by targeting the protein (polypeptide) to a specific intracellular organelle or specific intracellular space. For example, this can be done by adding or removing a leader sequence that functions to target the protein (polypeptide), thereby targeting the periplasm or cytoplasm, but is not limited to these.

[0071] Such weakening of polypeptide activity is achieved by reducing the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or by reducing the amount of product produced from the polypeptide, but is not limited to these methods.

[0072] In the microorganisms of this application, modification of part or all of the polynucleotides can be induced by (a) homologous recombination using a chromosome introduction vector in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment such as ultraviolet light or radiation. The methods for modifying part or all of the genes include methods using DNA recombination technology. For example, deletion of part or all of the gene can be achieved by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The introduced nucleotide sequence or vector contains, but is not limited to, a dominant selection marker.

[0073] For example, the recombinant microorganisms having L-citrulline production ability of this application include any microorganism that is transformed by a vector and produces L-citrulline by having the activity of a protein containing the amino acid sequence of SEQ ID NO: 1 of this application weakened.

[0074] The vector of this application comprises a DNA product comprising a polynucleotide sequence encoding a target polypeptide operably linked to a suitable regulatory region (or regulatory sequence) so as to enable expression of the target polypeptide in a suitable host. The regulatory region comprises a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0075] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. 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, pSK series, pSKH series, pET series, etc. can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0076] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using an intracellular chromosome introduction vector. 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. The vector may further include a selection marker to confirm whether or not the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed by the vector, that is, to confirm whether or not the target nucleic acid molecule has been inserted, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, and expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit different phenotypes, thus allowing for the selection of transformed cells.

[0077] In this application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism to express the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any form that is expressed in the host cell, regardless of whether it is inserted into or outside the host cell's chromosome. The polynucleotide also contains DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form that is expressed therein. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. Typically, the expression cassette includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequence necessary for expression in the host cell, but is not limited to this.

[0078] In this application, "operably linked" means a configuration in which a regulatory sequence is positioned appropriately so that the regulatory sequence indicates the expression of a coding sequence. Therefore, "operably linked" includes a regulatory region of a well-known or desired functional domain, such as a promoter, terminator, signal sequence, or enhancer region, that is attached to or linked to a target (gene or polypeptide) so that its expression, secretion, or function can be regulated according to its well-known or desired activity. For example, this means a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a target variant polypeptide of this application, and the polynucleotide sequence is functionally linked to the said polynucleotide sequence.

[0079] In this application, "expression" includes, but is not limited to, any steps involved in the production of polypeptides, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0080] In this application, "expression vector" means a linear or cyclic nucleic acid molecule comprising a coding sequence and a regulatory sequence operably linked for its expression.

[0081] In this application, "regulatory sequence" refers to a polynucleotide sequence necessary for the expression of a coding sequence. Each regulatory sequence may be a sequence native to (of the same origin as) the coding sequence, or a foreign sequence (derived from another gene). Examples of regulatory sequences include leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and translation termination. The smallest unit of a regulatory sequence includes a promoter and transcription and translation termination sequences.

[0082] With respect to cells, polynucleotides, polypeptides, or vectors, “recombinant” in this application means that cells, polynucleotides, polypeptides, or vectors are modified by the introduction of heterologous nucleic acids or polypeptides, or by alteration of native polynucleotides or polypeptides, or that cells are derived from such modified cells. For example, recombinant cells express genes that are not found in the cell’s native (non-recombinant) form, or they express genes that are not expressed at all, or they express native genes that are abnormally expressed.

[0083] The microorganisms described in this application include all microorganisms in which the activity of a protein containing the amino acid sequence of Sequence ID No. 1 has been weakened compared to its endogenous activity by various known methods.

[0084] As a specific example, a microorganism in which the activity of a protein containing the amino acid sequence of SEQ ID NO: 1 of this application is weakened compared to its endogenous activity is a microorganism in which the polynucleotide encoding the protein containing the amino acid sequence of SEQ ID NO: 1 is deleted or modified, but is not limited to this. Specifically, it is a microorganism in which the base sequence of SEQ ID NO: 2 is deleted and the protein containing the amino acid sequence of SEQ ID NO: 1 is inactive, but is not limited to this.

[0085] For example, the microorganisms with improved L-citrulline production ability described in this application are microorganisms that have improved L-citrulline production ability compared to unmodified microorganisms, but are not limited to these. For example, the unmodified microorganism used as a comparison for whether or not the L-citrulline production ability has improved is the C.gl::argR*_argG* strain, but is not limited to this.

[0086] As an example, the microorganism with improved L-citrulline production ability showed an improvement of approximately 1% or more compared to the L-citrulline production ability of the parent strain before mutation or the unmodified microorganism. Specifically, this improvement was approximately 1% or more, approximately 2.5% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 11% or more, approximately 12% or more, approximately 13% or more, approximately 14% or more, approximately 15% or more, approximately 16% or more, approximately 17% or more, and approximately 18% or more. The above represents an improvement of approximately 19% or more, approximately 20% or more, or approximately 21% or more (there are no particular restrictions on the upper limit; for example, it can be approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 50% or less, approximately 45% or less, approximately 40% or less, approximately 35% or less, approximately 30% or less, approximately 25% or less, or approximately 22% or less), but any product that shows a positive increase compared to the productivity of the parent strain or unmodified microorganism before mutation is acceptable. As another example, recombinant strains with improved L-citrulline production ability are those in which L-citrulline production ability has improved by approximately 1.1 times or more, approximately 1.11 times or more, approximately 1.12 times or more, approximately 1.13 times or more, approximately 1.14 times or more, approximately 1.15 times or more, approximately 1.16 times or more, approximately 1.17 times or more, approximately 1.18 times or more, approximately 1.19 times or more, approximately 1.2 times or more, or approximately 1.21 times or more (there is no particular upper limit, for example, approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, approximately 1.5 times or less, approximately 1.4 times or less, approximately 1.3 times or less, or approximately 1.25 times or less), but are not limited to these.

[0087] For example, the microorganism having the ability to produce L-citrulline may be either a prokaryotic cell or a eukaryotic cell, and specifically a prokaryotic cell. Examples of such prokaryotic cells include microbial strains belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, Pseudomonas, Leptospira, Salmonella, Brevibacterium, Hyphomonas, Chromobacterium, and Norcardia, or fungi or yeast. Specifically, these are microbial strains and yeasts belonging to the genera Escherichia, Corynebacterium, and Leptospira. More specifically, they are microbial strains of the genus Corynebacterium.

[0088] In any of the above-mentioned specific examples of microorganisms, the microorganism of this application may be a microorganism of the genus Corynebacterium.

[0089] As an example of this application, the microorganisms of this application are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. The microorganisms of this application may be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganisms of this application are microorganisms of the genus Corynebacterium, and more specifically, Corynebacterium glutamicum or Corynebacterium stationis, but are not limited to these.

[0090] On the other hand, the microorganisms having L-citrulline production ability described in this application include all of the following: naturally occurring wild-type microorganisms themselves, microorganisms whose L-citrulline production ability has been improved by enhancing or weakening the activity of genes related to the L-citrulline production mechanism, and microorganisms whose L-citrulline production ability has been improved by introducing or enhancing the activity of external genes.

[0091] In this application, "cultivation" means growing the bacterial strain of this application under appropriately adjusted environmental conditions. The cultivation process of this application can be carried out using suitable culture media and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used by those skilled in the art depending on the selected bacterial strain. Specifically, the cultivation is batch, continuous, and / or fed-batch culture, but is not limited to these.

[0092] In this application, "culture medium" refers to a substance that is a mixture mainly composed of nutrients necessary for culturing the microorganisms of this application, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the strains of this application may be any that are used for culturing ordinary microorganisms, and the microorganisms of this application can be cultured in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions, with the temperature, pH, etc. adjusted. For example, a culture medium for Corynebacterium strains is disclosed in Non-Patent Document 17.

[0093] In this application, the carbon source can be carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and 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 maceration liquid can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and any other carbon source in an appropriate amount may be used. These carbon sources can be used individually or in combination of two or more, but are not limited to these uses.

[0094] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, as well as organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration liquid, casein hydrolysates, fish or their decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources can be used individually or in combination of two or more, but are not limited to these uses.

[0095] As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or equivalent sodium-containing salts can be used. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., can be used, and in addition, amino acids, vitamins, and / or suitable precursors can be used. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these.

[0096] Furthermore, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in a suitable manner during the cultivation of the microorganisms of this application. In addition, during cultivation, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, oxygen or oxygen-containing gas may be injected into the culture medium to maintain an aerobic state, and to maintain an anaerobic and microaerobic state, it is not necessary to inject gas, but nitrogen, hydrogen, or carbon dioxide gas may be injected, but the invention is not limited to these.

[0097] In the culture described in this application, the culture temperature is maintained at 27-37°C, specifically 30-33°C, and the culture is performed for approximately 20-120 hours, but is not limited to these values.

[0098] In this application, "culture" means a culture solution, concentrated culture solution, dried culture solution, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing a specific microorganism in a culture medium, wherein the culture solution means one containing the specific microorganism, and the culture filtrate means one that substantially does not contain the specific microorganism (here, this means substantially removing the specific microorganism separated by filtration, etc., and does not mean completely removing microorganisms from the filtrate). The culture may be in any dosage form, for example, a liquid, emulsion, or solid. Specifically, for the purposes of this application, the culture may contain L-citrulline.

[0099] In this application, "fermentation" refers to the process by which microorganisms use their enzymes to decompose organic matter, excluding putrefaction reactions. Fermentation and putrefaction reactions proceed through similar processes, but if useful substances are produced as a result of the decomposition, it is called fermentation, while if foul odors are produced or harmful substances are created, it is called putrefaction.

[0100] In this application, the method for obtaining the fermented product from the strain is not particularly limited and can be obtained by methods commonly used in the art or similar fields.

[0101] In this application, "fermented product" includes not only the fermented substance itself, but also any kind of substance containing fermented products generated from the strain, such as a culture medium of the strain in which the strain and culture material coexist, a fermented product obtained by filtering the strain from the culture medium, a fermented product obtained by filtering a sterilized strain from the culture medium, an extract obtained by extracting the fermented product or a culture medium containing the same, a diluted solution or concentrate obtained by diluting the fermented product or its extract, a dried product obtained by drying the fermented product or its extract, and a lysate obtained by collecting and crushing the microbial cells of the strain.

[0102] In the method of this application, any culture conditions and methods known in the art are used for culturing microorganisms. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain.

[0103] L-citrulline produced by the culture described in this application is either secreted into the culture medium or remains within the cells.

[0104] In one specific example, the method for producing L-citrulline according to this application may further include, for example, the steps of preparing the microorganism of this application, preparing a culture medium for culturing the strain, or a combination thereof (in any order) before the culturing step.

[0105] The method for producing L-citrulline according to this application may further include a step of recovering a target substance, specifically L-citrulline, from the cultured microorganism, the culture product of the microorganism, the fermented product of the microorganism, or the culture medium. The recovery step may further include a step after the culture step.

[0106] The aforementioned recovery may involve collecting the target L-citrulline using a suitable method known in the art, depending on the microorganism culture method of this application, such as batch, continuous, or fed-batch culture. For example, various chromatography methods such as centrifugation, filtration, crystallization, treatment with protein precipitants (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used, and the target substance, specifically L-citrulline, can be recovered from the culture medium or microorganism using a suitable method known in the art.

[0107] Furthermore, the L-citrulline production method of this application may further include a purification step. The purification can be carried out by a preferred method known in the art. For example, if the L-citrulline production method of this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, regardless of order, simultaneously, or integrated as a single step, but are not limited thereto.

[0108] In the method of this application, the weakening of the activity of the protein containing the amino acid sequence of SEQ ID NO: 1, L-citrulline, etc., are as described above.

[0109] Another aspect of this application provides a composition for L-citrulline production comprising a Corynebacterium microorganism having L-citrulline production ability in which the activity of a protein containing the amino acid sequence of Sequence ID No. 1 of this application is attenuated compared to endogenous activity, a culture of the microorganism, a fermented product of the microorganism, or at least two combinations thereof.

[0110] The composition of this application may further contain any suitable excipients commonly used in compositions for L-citrulline production. Examples of such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0111] As a specific example, each component present in the composition of this application is included in a microbiologically effective amount or in an appropriate amount for production.

[0112] In the composition of this application, the weakening of the activity of the protein containing the amino acid sequence of SEQ ID NO: 1, L-citrulline, etc., are as described above.

[0113] A further aspect of this application provides the use of Corynebacterium microorganisms having L-citrulline-producing ability in which the activity of a protein comprising the amino acid sequence of Sequence ID No. 1 of this application is attenuated compared to endogenous activity, for L-citrulline production.

[0114] As previously mentioned, the use of this application involves the reduction in the activity of proteins containing the amino acid sequence of SEQ ID NO: 1, as well as L-citrulline, etc. [Examples]

[0115] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the application is not limited thereto. Technical matters not described herein can be fully understood and readily implemented by a skilled technician in the art of this application or a similar art. [Examples]

[0116] Creation of a recombinant vector that deletes the NCgl2383 gene To confirm the correlation between the inactivation of the protein containing the amino acid sequence of Sequence ID No. 1 and L-citrulline production ability, a recombinant plasmid was created in which the gene containing the nucleotide sequence of NCgl2383 was deleted on the chromosome of a Corynebacterium L-citrulline-producing strain.

[0117] First, in order to create a recombinant vector that deletes the aforementioned gene on the chromosome of a microorganism of the genus Corynebacterium, four primers corresponding to SEQ ID NOs. 3, 4, 5, and 6 were synthesized.

[0118] Specifically, in order to delete the ORF site (SEQ ID NO: 2) of the NCgl2383 gene, primers 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5), and 6 (SEQ ID NO: 6) were synthesized to have BamHI and XbaI restriction enzyme sites at the 5' and 3' ends, respectively. Using primers SEQ ID NOs. 3, 4, 5, and 6, PCR was performed using chromosomal DNA from Corynebacterium glutamicum ATCC13869 as a template. As a result, it was confirmed that DNA fragments corresponding to the anterior and posterior parts of the protein encoded by the gene were amplified by 1000 bp each. The PCR conditions were denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute, and 30 cycles, followed by polymerization at 72°C for 10 minutes. After that, the PCR fragments were extracted using a gel purification kit (QIAGEN). As described above, the recombinant arm gene fragment obtained and the vector pDCM2 (Patent Document 2), which was cleaved with BamHI and XbaI restriction enzymes, were linked using the Gibson assembly method (Non-Patent Document 18). Subsequently, the cells were transformed into Escherichia coli DH5α and spread on LB solid medium containing kanamycin (25 mg / l).

[0119] PCR was performed using primers SEQ ID NOs. 7 and 8 to select colonies transformed with a vector ligated with the target gene and pDCM2. Plasmids were obtained from the selected colonies using a well-known plasmid extraction method and named pDCM2-ΔNCgl2383. [Examples]

[0120] Production of L-citrulline-producing microorganisms To create an L-citrulline-producing microorganism, a vector was constructed in which the glutamic acid at position 47 of the argR (ANU33619.1) protein sequence was replaced with a stop codon. Using the genome of wild-type C. glutamicum ATCC 13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NOs. 9 and 10, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NOs. 11 and 12. Subsequently, a plasmid was obtained in the same manner as in Example 1. This plasmid was named pDCM2-argR(E47*).

[0121] To create a microorganism with further improved L-citrulline production capacity, a vector was constructed in which the phenylalanine at position 68 of the argG (ANU33620.1) protein sequence was replaced with a stop codon. Using the genome of C. glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using primers SEQ ID NOs. 13 and 14, and the homologous recombinant B arm was amplified using primers SEQ ID NOs. 15 and 16. Subsequently, a plasmid was obtained using the method described above. This plasmid was named pDCM2-argG(F68*).

[0122] Using the constructed pDCM2-argR(E47*) vector, wild-type C. glutamicum ATCC 13869 was transformed by electroporation (Non-Patent Literature 19). Subsequently, after a secondary cross-reaction, a strain was obtained in which the 139th base sequence of argR was replaced from guanine (g) to thymine (t), and the 47th protein sequence was replaced with a stop codon. Using primer pairs of Sequence ID No. 9 and 12, which amplify the adjacent region including the insertion site of the gene, PCR and nucleotide sequence analysis were performed to confirm the genetic manipulation. The microorganism thus obtained was named C. gl::argR*.

[0123] To create a microorganism with further enhanced citrulline production in C.gl::argR*, we obtained the microorganism using the pDCM2-argG(F68*) vector as described above. Using primer pairs SEQ ID NOs. 13 and 16, which amplify the adjacent region including the insertion site of the gene, PCR and sequencing analysis were performed to confirm the genetic manipulation. The microorganism thus obtained was named C.gl::argR*_argG*.

[0124] The primer sequences used here are shown in Table 1.

[0125] [Table 1] [Examples]

[0126] Creation of NCgl2383 gene knockout strains Based on the L-citrulline-producing Corynebacterium strain C.gl::argR*_argG*, a strain lacking the gene containing the nucleotide sequence of Sequence ID No. 2 was created.

[0127] Specifically, the recombinant plasmid pDCM2-ΔNCgl2383 prepared in Example 1 was transformed into Corynebacterium glutamicum C.gl::argR*_argG*, an L-citrulline-producing bacterial strain, by homologous recombination on the chromosome (Non-Patent Literature 20).

[0128] Subsequently, secondary recombination was performed in solid agar plates containing sucrose. Using PCR with primers 3 and 6, the recombinant Corynebacterium glutamicum strains that had undergone secondary recombination were identified as strains lacking the aforementioned gene. These recombinant strains were named Corynebacterium glutamicum C.gl::argR*_argG*_ΔNCgl2383. [Examples]

[0129] Evaluation of L-citrulline production capacity of NCgl2383 gene knockout strains. To analyze L-citrulline production capacity, the parent strain Corynebacterium glutamicum C.gl::argR*_argG* and the Corynebacterium glutamicum C.gl::argR*_argG*_ΔNCgl2383 strain, prepared as described above, were cultured using the following method.

[0130] Each bacterial strain was inoculated into a 250 ml corner baffle flask containing 25 ml of the following production medium, and cultured with shaking at 33°C and 200 rpm for 48 hours. After the culture period, the amount of bacterial cells and L-citrulline produced was measured using HPLC. <Production medium (pH 7.2)> Raw sugar 50g, (NH4)2SO4 30g, yeast extract 1g, KH2PO4 1.1g, MgSO4·7H2O 1.2g, L-arginine 0.2g, biotin 1mg, thiamine hydrochloride 5mg, calcium pantothenate 5mg, nicotinamide 15mg, MnSO4 10mg, FeSO4 10mg, ZnSO4 0.5mg, CuSO4 0.5mg, CaCO3 30g (in 1 liter of distilled water)

[0131] The above experiment was repeated twice. The culture results (average values) are shown in Table 2.

[0132] [Table 2]

[0133] As shown in Table 2, in C.gl::argR*_argG*_ΔNCgl2383, which was created by deleting the NCgl2383 gene from the L-citrulline-producing strain Corynebacterium glutamicum C.gl::argR*_argG*, it was confirmed that L-citrulline production capacity improved by an average of 21% compared to the parent strain, without affecting the bacterial cell.

[0134] Therefore, it was confirmed that L-citrulline production capacity is improved in microorganisms of the genus Corynebacterium by deleting the gene containing the nucleotide sequence of Sequence ID No. 2.

[0135] From the above explanation, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. This application should be interpreted as including all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A method for producing L-citrulline, comprising the step of culturing a Corynebacterium microorganism having L-citrulline-producing ability in which the activity of a protein containing the amino acid sequence of SEQ ID NO: 1 is weaker than that of endogenous activity, in a culture medium.

2. The method according to claim 1, wherein the protein containing the amino acid sequence of SEQ ID NO: 1 is encoded by a gene containing the base sequence of SEQ ID NO:

2.

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

4. The method according to claim 1, further comprising the step of recovering L-citrulline from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium.

5. A composition for L-citrulline production comprising a Corynebacterium microorganism having L-citrulline production ability in which the activity of a protein containing the amino acid sequence of Sequence ID No. 1 is weakened compared to its endogenous activity, a culture of the said microorganism, a fermented product of the said microorganism, or at least two combinations thereof.

6. Use of Corynebacterium microorganisms, which have L-citrulline production ability with protein activity weakened compared to endogenous activity, containing the amino acid sequence of SEQ ID NO: 1, for L-citrulline production.

Images