Microorganisms capable of producing L-ornithine and a method for producing L-ornithine using the same

Abstract

The present application relates to a microorganism having the ability to produce L-ornithine and a method for producing L-ornithine using the same.

Description

Technical Field

[0001] This application relates to a microorganism having the ability to produce L-ornithine and a method for producing L-ornithine using the same.

Background Art

[0002] L-amino acids are used in the animal feed, human pharmaceuticals, and cosmetics industries, and in some cases of L-amino acids, they are produced by fermentation using microorganisms. In order to improve the method for producing L-amino acids using microorganisms, research using recombinant DNA technology has been advanced. For example, by deleting and attenuating the expression of some genes or amplifying genes related to L-amino acid biosynthesis, the ability of microorganisms to produce L-amino acids could be increased. In particular, improvement of the ability of microorganisms to excrete L-amino acids has been considered as a major technique for increasing the production ability of L-amino acids (US 10995378 B2). Therefore, by introducing and amplifying genes whose ability to excrete L-amino acids has been clarified, it is possible to excrete high-concentration L-amino acids accumulated inside microorganisms and greatly increase the production ability.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0004]

Non-Patent Document 1

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-Patent Document 19

Summary of the Invention

Problems to be Solved by the Invention

[0005] The inventors completed this application by confirming that when a foreign LysE / ArgO family amino acid transporter protein is introduced into a microorganism, the ability to produce L-ornithine is increased compared to a non-transformed microorganism.

Means for Solving the Problems

[0006] One object of this application is to provide a recombinant microorganism of the genus Corynebacterium having the ability to produce L-ornithine, which contains a LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or a polynucleotide encoding a LysE / ArgO family amino acid transporter protein.

[0007] Another object of this application is to provide a method for producing L-ornithine, which includes culturing a recombinant microorganism of the genus Corynebacterium having the ability to produce L-ornithine, which contains a LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or a polynucleotide encoding a LysE / ArgO family amino acid transporter protein, in a medium.

Effects of the Invention

[0008] The microorganism of the present application has an increased ability to produce L-ornithine compared to the existing non-modified microorganism by introducing a foreign LysE / ArgO family amino acid transporter protein.

Mode for Carrying Out the Invention

[0009] Specifically, it is as follows. On the other hand, each explanation and embodiment disclosed in the present application is also applicable to each other explanation and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. Also, it is not considered that the category of the present application is limited by the specific descriptions described below. Also, throughout this specification, a number of papers and patent documents are referenced and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present application belongs and the content of the present invention are more clearly explained.

[0010] One aspect of the present application provides a recombinant microorganism of the genus Corynebacterium having an ability to produce L-ornithine, which comprises a LysE / ArgO family amino acid transporter protein derived from a Shewanella genus strain or a polynucleotide encoding the LysE / ArgO family amino acid transporter protein.

[0011] In the present application, the term "LysE / ArgO family amino acid transporter" is a protein having LysE (lysine exporter) and / or ArgO (arginine exporter) functions belonging to the amino acid exporter family in bacteria. That is, the LysE / ArgO family amino acid transporter means a protein having lysine and / or arginine excretion activity.

[0012] The amino acid sequences of the aforementioned LysE / ArgO family amino acid transporter proteins can be obtained from known databases such as NCBI's Genebank.

[0013] For example, the LysE / ArgO family amino acid transporter proteins of this application may be derived from microorganisms.

[0014] As another example, the LysE / ArgO family amino acid transporter proteins of this application may be exogenous proteins that are not endogenously present in microorganisms of the genus Corynebacterium.

[0015] As another example, the LysE / ArgO family amino acid transporter protein of this application may be derived from a microorganism. The aforementioned microorganisms may specifically be derived from microorganisms of the genus Shewanella, more specifically from Shewanella corallii, Shewanella oneidensis, Shewanella putrefaciens, Shewanella putrefaciens, Shewanella sp. MR-4, Shewanella sp. BC20, Shewanella xiamenensis, Shewanella decolorationis, Shewanella sp. HN-41, Shewanella baltica, Shewanella sp. ISTPL2, etc., and more specifically from Shewanella corallii or Shewanella oneidensis, but are not limited thereto.

[0016] As another example, the amino acid sequence of the LysE / ArgO family amino acid transporter of this application may be WP_115137742.1 derived from Shewanella kollarii or WP_011072781.1 derived from Shewanella oneidensis MR-1, but is not necessarily limited to these, and it is obvious that it includes proteins with LysE / ArgO family amino acid transporter activity derived from a variety of Shewanella microorganisms.

[0017] In this application, the term "Shewanella microorganisms" refers to a type of marine bacteria that can inhabit both oxygen-rich and oxygen-deprived environments and is known for its ability to induce the reduction and precipitation of toxic metals such as chromium and uranium.

[0018] In this application, the LysE / ArgO family amino acid transporter protein derived from a Shewanella strain may have, contain, or be composed of the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 3.

[0019] In this application, LysE / ArgO family amino acid transporter proteins derived from Shewanella strains may include amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 3, or amino acid sequences having at least 80%, 80.3%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity thereto. Furthermore, it is obvious that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, conservedly substituted, or added are also included within the scope of this application, as long as they have such homology or identity and exhibit efficacy corresponding to proteins containing the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 3.

[0020] For example, the amino acid sequence may have additions or deletions of sequences that do not alter the function of the protein of this application, spontaneous mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within it.

[0021] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0022] In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, and may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0023] The homology or identity of sequences of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, and a default gap penalty established by the program used is available. Substantially homologous or identical sequences are generally hybridizable in whole or in part with other sequences under moderate to high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing codons in general or codon degeneracy in polynucleotides.

[0024] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.] Academic Press, San (Including Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0025] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include: (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (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 opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0026] The gene encoding the LysE / ArgO family amino acid transporter protein in this application may be a gene named lysE and / or argO.

[0027] For example, the lysE gene and / or argO gene may be a polynucleotide encoding WP_115137742.1 from Shewanella corallii or WP_011072781.1 from Shewanella oneidensis MR-1, but is not necessarily limited to this, and it is obvious that it includes lysE genes and / or argO genes from a variety of Shewanella microorganisms encoding proteins having LysE / ArgO family amino acid transporter activity.

[0028] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are linked together in a long chain by covalent bonds, and is a DNA or RNA chain of a certain length or longer. More specifically, it means a polynucleotide fragment that codes for a protein.

[0029] The polynucleotide encoding the LysE / ArgO family amino acid transporter protein of this application may include a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 3. As an example of this application, the polynucleotide of this application may have or include the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Furthermore, the polynucleotide of this application may consist of or be essentially composed of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Specifically, the LysE / ArgO family amino acid transporter protein may be encoded by a polynucleotide described in the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0030] The polynucleotides of this application may undergo various modifications to the coding region, taking into consideration the degeneracy of the codons or the preferred codons in organisms that intend to express the LysE / ArgO family amino acid transporter proteins of this application, as long as these modifications do not alter the amino acid sequence of the LysE / ArgO family amino acid transporter proteins. Specifically, the polynucleotides of this application may have, contain, or consist of a nucleotide sequence that has 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more homology or identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or may consist of, or be essentially composed of, a nucleotide sequence that has 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more homology or identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0031] Furthermore, the polynucleotides of this application may include, without limitation, any probes produced from known gene sequences, such as sequences that can hybridize under stringent conditions with complementary sequences to all or part of the polynucleotide sequences of this application. “Stringent conditions” means conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, we can list conditions under which polynucleotides with high homology or identity hybridize with each other, with homology or identity levels of 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, but do not hybridize with polynucleotides with lower homology or identity levels. Alternatively, we can list conditions under which the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to that of normal southern hybridization, which is 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.

[0032] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationships between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the overall sequence.

[0033] Specifically, polynucleotides homologous or identical to the polynucleotides of this application can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may also be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0034] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0035] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and is a microorganism in which a specific mechanism has been weakened or strengthened due to the insertion of an external gene or the strengthening or weakening of the activity of an endogenous gene, and may be a microorganism that has undergone genetic modification for the production of a target polypeptide, protein, or product.

[0036] The strains of this application may be strains that naturally possess L-ornithine production ability or microorganisms to which L-ornithine production ability has been conferred from strains that do not possess L-ornithine production ability. For example, the microorganisms may have weakened activity of ornithine carbamoyltransferase subunit F (ArgF) and / or arginine repressor (ArgR); and / or lack endogenous LysE protein. As another example, the microorganisms may have improved L-ornithine efflux ability by introducing LysE / ArgO family amino acid transporter proteins or polynucleotides encoding them derived from the Shewanella strains of this application.

[0037] The strains of this application may be microorganisms having increased L-ornithine production capacity compared to Corynebacterium strains or wild-type Corynebacterium strains that do not contain the LysE / ArgO family amino acid transporter protein derived from the Shewanella strain of this application. Specifically, the strains of this application are recombinant microorganisms into which a LysE / ArgO family amino acid transporter protein derived from Shewanella corallii or Shewanella oneidensis has been introduced, having increased activity compared to its endogenous activity. The strains may have improved L-ornithine production capacity because the activity of the LysE / ArgO family amino acid transporter protein is increased compared to its endogenous activity, thereby improving L-ornithine efflux. In other words, the strains of this application may have increased activity of the LysE / ArgO family amino acid transporter protein compared to its endogenous activity.

[0038] As an example, the target strains used to compare the presence or absence of increased L-ornithine production capacity, namely "non-mutated microorganisms that have not been introduced with LysE / ArgO family amino acid transporter proteins derived from Shewanella strains," may be, but are not limited to, a Corynebacterium glutamicum strain having L-ornithine production capacity in which the serine at position 55 from the N-terminus of the amino acid sequence of endogenous ArgF is replaced with a stop codon, and the glutamate at position 47 from the N-terminus of the amino acid sequence of endogenous ArgR is replaced with a stop codon; or a Corynebacterium glutamicum strain lacking endogenous LysE.

[0039] For example, the recombinant strain with increased production capacity may have an increase of approximately 1% or more compared to the L-ornithine production capacity of the parent strain or non-myxoid before mutation. Specifically, this could be approximately 1% or more, approximately 2% or more, approximately 5% or more, approximately 10% or more, approximately 15% or more, approximately 18% or more, approximately 18.9% or more, approximately 19% or more, approximately 19.1% or more, approximately 19.7% or more, approximately 20% or more, or approximately 20.3% or more (there are no special restrictions on the upper limit; for example, it may be approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 50% or less, approximately 40% or less, approximately 30% or less, approximately 20% or less, or approximately 15% or less). However, it is not limited to this, as long as it has a positive increase compared to the production capacity of a microorganism in which LysE / ArgO family amino acid transporter proteins or polynucleotides encoding them derived from the parent strain, non-myxoid, or Shewanella strain before mutation have not been introduced. In other examples, the recombinant strain with increased L-ornithine production capacity may be, but is not limited to, a strain in which L-ornithine production capacity has increased by approximately 1.01 times or more, approximately 1.02 times or more, approximately 1.05 times or more, approximately 1.10 times or more, approximately 1.15 times or more, approximately 1.18 times or more, approximately 1.189 times or more, approximately 1.19 times or more, approximately 1.191 times or more, approximately 1.197 times or more, approximately 1.20 times or more, or approximately 1.203 times or more (there is no special limit on the upper limit; for example, it may be approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, or approximately 2 times or less).

[0040] In this application, the term "non-mutant microorganism" means a strain that is either wild-type or naturally occurring, or a strain before its characteristics are altered by genetic mutations due to natural or artificial factors, and does not exclude strains containing naturally occurring mutations in microorganisms. Furthermore, the microorganism refers to a strain that has not been introduced, or is in the process of being introduced, the LysE / ArgO family amino acid transporter protein derived from the Shewanella strains described herein. The non-mutant microorganisms of this application do not exclude strains that include the introduction or deformation of other proteins or genes other than the LysE / ArgO family amino acid transporter protein derived from the Shewanella strains or the polynucleotide encoding it.

[0041] In this application, the term "non-mutant microorganism" may be used interchangeably with "pre-deformation strain," "pre-deformation microorganism," "non-mutant strain," "non-mutant myxomycete strain," "non-mutant microorganism," or "reference microorganism."

[0042] The microorganisms of this application may be, but are not limited to, microorganisms into which LysE / ArgO family amino acid transporter proteins or polynucleotides encoding them derived from Shewanella strains have been introduced; or microorganisms that have been genetically modified (e.g., recombinant microorganisms) to which LysE / ArgO family amino acid transporter proteins or polynucleotides encoding them derived from Shewanella strains have been introduced. The term "intrinsic activity" refers to the activity of a specific polypeptide that was originally present in the parental strain, wild type, or non-mutant microorganism before the trait change due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification."

[0043] As another example of this application, the microorganisms of this application are Corynebacterium stationis, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. It may also be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and more specifically, it may be Corynebacterium glutamicum.

[0044] In addition to introducing LysE / ArgO family amino acid transporter proteins derived from the Shewanella strain of this application, the microorganisms of this application can have their LysE / ArgO family amino acid transporter activity enhanced by known methods.

[0045] In this application, the term “enhancement” of polypeptide activity (including, for example, proteins identified by the names of each enzyme) means that the polypeptide activity is increased compared to its intrinsic activity. Such enhancement may be used interchangeably with terms such as activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase all include exhibiting activity that was not originally present, or exhibiting activity that is improved compared to the intrinsic activity or activity before the mutation. “Intrinsic activity” means the activity of a specific polypeptide that was originally present in the parent strain or non-mutant microorganism before the mutation occurred, in cases where the trait changes due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before the mutation.” “Enhancement,” “upregulation,” “overexpression,” or “increase” of polypeptide activity compared to its intrinsic activity means that the activity and / or concentration (expression level) of the specific polypeptide that was originally present in the parent strain or non-mutant microorganism before the mutation occurred is improved.

[0046] The aforementioned enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether or not the polypeptide's activity has been enhanced can be confirmed by an increase in the polypeptide's activity level, expression level, or the amount of product excreted from the polypeptide.

[0047] The enhancement of the activity of the polypeptide can be achieved by applying a variety of methods well known in the field, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before deformation. Specifically, this may involve, but is not limited to, the use of genetic engineering and / or protein engineering, which are routine methods in molecular biology and are well known to ordinary technicians in this field (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0048] Specifically, the enhancement of the polypeptide activity of this application is 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides; 2) Replace gene expression regulatory regions on chromosomes encoding polypeptides with highly active sequences; 3) Modification of the nucleotide sequence encoding the start codon or 5'UTR region of a polypeptide-encoding gene transcript; 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity; 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified to enhance polypeptide activity); 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding such activity; 7) Codon optimization of polynucleotides encoding polypeptides; 8) Analyze the tertiary structure of the polypeptide, select exposed sites, and deform or chemically modify them; or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0049] More specifically, The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described in 1) above may be achieved by introducing into the host cell a vector that is operablely linked to the polynucleotide encoding the polypeptide, replicates independently of the host, and functions. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosomes within the host cell. The introduction into the chromosome is performed by introducing into the host cell a vector that causes the polynucleotide to be inserted into the chromosomes within the host cell, but is not limited to this. The vector is as described above.

[0050] The replacement of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may, for example, involve sequence mutation by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with a sequence having stronger activity, in order to further enhance the activity of the expression regulatory region. The expression regulatory region may include, 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 decoding. For example, the original promoter may be replaced with a potent promoter.

[0051] Examples of well-known powerful promoters include, but are not limited to, the CJ1-CJ7 promoters (US Patent No. 7662943 B2), the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent No. 10584338 B2), O2 promoter (US Patent No. 10273491 B2), tkt promoter, and yccA promoter.

[0052] The sequence modification encoding the start codon or 5'UTR region of the polypeptide-encoding gene transcript described in 3) above may, but is not limited to, substitution with a sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon.

[0053] The modifications of the amino acid sequence or polynucleotide sequence described in 4) and 5) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or replacement with an improved amino acid sequence or polynucleotide sequence that has stronger activity or an improved amino acid sequence or polynucleotide sequence that has increased activity. Specifically, the replacement may be, but is not limited to, insertion of a polynucleotide into the chromosome by homologous recombination. The vector used in this case may further include a selection marker to confirm the presence or absence of chromosomal insertion.

[0054] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The foreign polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction is carried out by a person skilled in the art appropriately selecting a known transformation method, and the introduction of the polynucleotide in the host cell generates the polypeptide, thereby increasing its activity.

[0055] The codon optimization of the polynucleotide encoding the polypeptide described in 7) above may be codon optimization of the endogenous polynucleotide so that transcription or translation increases in the host cell, or optimization of the codon of the exogenous polynucleotide so that optimized transcription or translation occurs in the host cell.

[0056] 8) Analyzing the tertiary structure of a polypeptide and selecting exposed sites to deform or chemically modify may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on these candidates, and selecting exposed sites to deform or chemically modify.

[0057] Such enhancement of polypeptide activity may be achieved by increasing the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-deformation microbial strain, or by increasing the amount of product produced from the polypeptide, but is not limited to these methods.

[0058] Modification of some or all of the polynucleotides in the microorganisms of this application may be induced by (a) homologous recombination using a chromosome insertion vector within 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 and radiation. The method for modifying some or all of the genes may include methods using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene is injected into the microorganism to induce homologous recombination, thereby deleting some or all of the genes. The injected nucleotide sequence or vector may, but is not limited to, contain a dominant selection marker.

[0059] The vector of this application may include a DNA product comprising a polynucleotide sequence encoding a target polypeptide operably linked to a suitable regulatory region (or regulatory sequence) for expressing the target polypeptide in a suitable host. The regulatory region may include a promoter on which transcription can be initiated, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and decoding. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome and integrate into the genome itself.

[0060] 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, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0061] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosome insertion vector within a cell. The insertion of the polynucleotide into the chromosome may be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker may further be included to confirm the presence or absence of the chromosome insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the target nucleic acid molecule insertion, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, thus allowing for the selection of transformed cells.

[0062] In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them, regardless of whether they are inserted into or outside the chromosomes of the host cell, as long as they can be expressed in the host cell. The polynucleotide also includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form that allows it to be introduced and expressed in the host cell. 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 autonomous expression. The expression cassette may typically include 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 sequences necessary for expression in the host cell, but is not limited to this.

[0063] Furthermore, the term "operably linked" in the foregoing means that a 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.

[0064] As yet another example of this application, the microorganism of this application may be a microorganism in which the activity of some proteins in the L-ornithine biosynthesis pathway is further enhanced, or the activity of some proteins in the L-ornithine degradation pathway is further weakened, thereby enhancing the L-ornithine production capacity.

[0065] Specifically, the microorganisms of this application may be microorganisms in which the activity of endogenous ArgF is further weakened or the argF gene encoding it is further deficient, microorganisms in which the activity of endogenous ArgR is further weakened or the argR gene encoding it is further deficient, and / or microorganisms in which the activity of LysE is further weakened or the lysE gene encoding it is further deficient.

[0066] The amino acid sequences of ArgF, ArgR, or LysE can be obtained from known databases such as NCBI's Genebank. For example, the amino acid sequence of ArgF in this application may include ANU33618.1 (SEQ ID NO: 5) derived from Corynebacterium glutamicum ATCC 13869 or an amino acid sequence having 80% or more sequence identity thereto; the amino acid sequence of ArgR may include ANU33619.1 (SEQ ID NO: 7) derived from Corynebacterium glutamicum ATCC 13869 or an amino acid sequence having 80% or more sequence identity thereto; and the amino acid sequence of LysE may include ANU33473.1 (SEQ ID NO: 9) derived from Corynebacterium glutamicum ATCC 13869 or an amino acid sequence having 80% or more sequence identity thereto. However, the application is not limited thereto, and it is obvious that it includes proteins with ArgF, ArgR, or LysE activity from a variety of origins.

[0067] However, the weakening of ArgF, ArgR, and / or LysE protein activity or the deletion of argF, argR, and / or lysE genes are not limited to these examples, and the microorganisms of this application may also be microorganisms in which the protein activity of various known L-ornithine biosynthesis pathways is enhanced or the protein activity of degradation pathways is weakened.

[0068] In this application, the term "weakening" of polypeptide activity encompasses all concepts of reduced or absent activity compared to endogenous activity. This weakening may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decline, reduce, and attenuation.

[0069] The aforementioned weakening may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, cases where the overall polypeptide activity and / or concentration (expression level) in the cell is lower than that of the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or inhibition of translation into the polypeptide, cases where the polynucleotide is not expressed at all, and / or cases where there is no polypeptide activity despite the expression of the polynucleotide. "Inactivation, deficiency, reduction, downregulation, decrease, and attenuation" of polypeptide activity compared to its endogenous activity means that it has decreased compared to the activity of the specific polypeptide originally possessed by the parent strain or non-mutant microorganism before the trait change.

[0070] The weakening of the activity of such polypeptides can be achieved by any method known in the art, but is not limited to these methods, and can be achieved by applying a variety of well-known methods in the field (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0071] Specifically, the weakening of the polypeptide activity of this application is 1) Deletion of all or part of the gene encoding the polypeptide; 2) Modification of the gene expression regulatory region (or gene expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced; 3) Modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so as to remove or weaken the activity of the polypeptide; 4) Modification of the gene sequence encoding the polypeptide so that the polypeptide activity is removed or weakened (for example, deletion / substitution / addition of one or more nucleic acid bases in the nucleic acid sequence of the polypeptide gene so that it encodes a polypeptide that has been modified so that the polypeptide activity is removed or weakened); 5) Modifications of the nucleotide sequence encoding the start codon or 5'UTR region of a polypeptide-encoding gene transcript; 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in a polypeptide-encoding gene to form a secondary structure that cannot be attached to a ribosome; 8) Addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polypeptide-coding gene sequence (reverse transcription engineering, RTE); or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0072] for example, The deletion of part or all of the gene encoding the polypeptide described in 1) above may be the removal of the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

[0073] Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) described in 2) above may involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, resulting in a mutation on the expression regulatory region (or expression regulatory sequence), or replacement with a sequence having weaker 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 that regulates the termination of transcription and decoding.

[0074] The modifications of the amino acid sequence or polynucleotide sequence described in 3) and 4) above may include, but are not limited to, deletion, insertion, non-conservative or conservative substitution, or combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby causing sequence mutations, or replacement with an amino acid sequence or polynucleotide sequence modified to have weaker activity or an amino acid sequence or polynucleotide sequence modified to have no activity, in order to weaken the activity of the polypeptide. For example, gene expression can be inhibited or weakened by introducing mutations within a polynucleotide sequence to form a stop codon, but is not limited to this.

[0075] The sequence modification encoding the start codon or 5'UTR region of the polypeptide-encoding gene transcript described in 5) above may, but is not limited to, substitution with a sequence encoding another start codon that has a lower polypeptide expression rate compared to the endogenous start codon.

[0076] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide (6) can be done by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0077] 7) In order to form a secondary structure that ribosomes cannot attach to, the addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the polypeptide-encoding gene may make mRNA translation impossible or reduce its rate.

[0078] Furthermore, the addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) may weaken the activity by creating complementary antisense nucleotides in the transcript of the gene encoding the polypeptide.

[0079] Another aspect of this application provides a method for producing L-ornithine, comprising the step of culturing a recombinant Corynebacterium microorganism capable of producing L-ornithine in a culture medium, which contains a LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or a polynucleotide encoding a LysE / ArgO family amino acid transporter protein.

[0080] The method for producing L-ornithine according to this application may include the step of culturing a genetically modified microorganism in a culture medium to contain the LysE / ArgO family amino acid transporter protein or the polynucleotide encoding it derived from the Shewanella strain of this application.

[0081] In this application, the term "culture" means growing the microorganisms of this application under appropriately controlled environmental conditions. The culture process of this application is carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adapted and used by those skilled in the art depending on the selected microorganisms. Specifically, the culture may be batch, continuous, and / or fed-batch.

[0082] In this application, the term "culture medium" means a substance mixed primarily with nutrients necessary for culturing the microorganisms of this application, supplying water, which is essential for survival and growth, as well as nutrients and growth factors. Specifically, the culture medium and other culture conditions used for culturing the microorganisms of this application can be any culture medium used for ordinary microbial cultivation without any special restrictions, but the microorganisms of this application can be cultured under aerobic conditions in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while adjusting the temperature, pH, etc.

[0083] Specifically, culture media for microorganisms of the genus Corynebacterium can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0084] In this application, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can also be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited to these uses.

[0085] The nitrogen sources used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, and are not limited to these uses.

[0086] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used, and other components such as amino acids, vitamins, and / or appropriate precursors may also be included. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these methods.

[0087] Furthermore, during the cultivation of the microorganisms of this application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress the formation of bubbles. Furthermore, in order to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium, or in order to maintain an anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, or by injection.

[0088] In the culture described in this application, the culture temperature can be maintained at 20-45°C, specifically 25-40°C, and the culture can be performed for approximately 10-160 hours, but is not limited to this.

[0089] L-ornithine produced by the culture described in this application is either secreted into the culture medium or remains within the cells.

[0090] The method for producing L-ornithine according to this application may further include, for example, a step of preparing the microorganism of this application, a step of preparing a culture medium for culturing the microorganism, or a combination thereof (in any order), before the culturing step.

[0091] The method for producing L-ornithine according to this application may further include a step of recovering L-ornithine from the culture medium (the medium in which the culture was performed) or from the cultured microorganisms. The recovery step may further include a step after the culture step.

[0092] The aforementioned recovery may also involve collecting the target L-ornithine using appropriate methods known in the art, such as the microbial culture methods of this application, for example, batch, continuous, or fed-batch culture methods. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (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 to recover the target L-ornithine from the culture medium or microorganism using appropriate methods known in the art.

[0093] Furthermore, the L-ornithine production method of this application may further include a purification step. The purification can be carried out using a suitable method known in the art. For example, if the L-ornithine production method of this application includes both a recovery step and a purification step, the recovery step and the purification step can be carried out sequentially or discontinuously, regardless of the procedure, or simultaneously or integrated into a single step, but are not limited thereto.

[0094] The microorganisms of this application may be microorganisms in which the activity of some proteins in the L-ornithine biosynthesis pathway is further enhanced, or the activity of some proteins in the L-ornithine degradation pathway is further weakened, thereby enhancing L-ornithine production capacity.

[0095] As an example, the microorganisms of this application may be microorganisms in which the activity of endogenous ArgF is further weakened or the argF gene encoding it is further deficient, microorganisms in which the activity of endogenous ArgR is further weakened or the argR gene encoding it is further deficient, and / or microorganisms in which the activity of LysE is further weakened or the lysE gene encoding it is further deficient.

[0096] In the method of this application, the Shewanella strain, LysE / ArgO family amino acid transporter protein, polynucleotide, vector, and microorganism are as described in the other aspects above.

[0097] Another aspect of this application provides a composition for L-ornithine production comprising a LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or a recombinant Corynebacterium microorganism containing a polynucleotide encoding a LysE / ArgO family amino acid transporter protein; a culture medium in which the same is cultured; or a combination thereof.

[0098] The composition of this application may further contain any suitable excipients commonly used in compositions for L-ornithine production, such excipients may be, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0099] Another aspect of this application provides a method for producing recombinant Corynebacterium microorganisms for L-ornithine production, comprising the step of introducing a LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or a polynucleotide encoding a LysE / ArgO family amino acid transporter protein.

[0100] Another aspect of this application provides a use for L-ornithine production of recombinant Corynebacterium microorganisms into which LysE / ArgO family amino acid transporter proteins derived from Shewanella strains or polynucleotides encoding LysE / ArgO family amino acid transporter proteins have been introduced.

[0101] The aforementioned Shewanella strains, LysE / ArgO family amino acid transporter proteins, introductions, and Corynebacterium microorganisms are as described in the other sections above.

[0102] The present application will be described in more detail below with reference to experimental examples. However, the following embodiments are merely preferred embodiments for illustrating the present application and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be fully understood and easily performed by a person of ordinary skill who is skilled in the art of this application or a similar art.

[0103] Example 1: Selection of a protein with L-ornithine efflux activity and plasmid construction. As proteins with high L-ornithine efflux activity, we selected the LysE / ArgO family amino acid transporter (WP_115137742.1) (SEQ ID NO: 1), a membrane protein derived from Shewanella corallii A687, and the LysE / ArgO family amino acid transporter (WP_011072781.1) (SEQ ID NO: 3), a membrane protein derived from Shewanella oneidensis MR-1. To amplify the base sequence of the gene encoding these proteins, we obtained information (NC_004347.2) regarding the gene encoding the aforementioned membrane protein and its surrounding nucleic acid sequence from the NIH GenBank. Based on this sequence information, DNA synthesis was performed (Cosmo genetech, Korea).

[0104] Vectors were prepared to introduce the membrane proteins from the selected Shewanella coralis A687 and Shewanella oneidensis MR-1 into L-ornithine-producing strains. First, ANU34435.1, one of the transposases present in the genome of wild-type Corynebacterium glutamicum ATCC 13869 (NZ_CP016335.1), was used as the insertion site. Using the genome of wild-type Corynebacterium glutamicum ATCC 13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NOs. 11 and 12, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NOs. 13 and 14.

[0105] To secure the gene fragment encoding a membrane protein from Shewanella koralii A687 (SEQ ID NO: 2), synthesized DNA was used as a template, and primer pairs of SEQ ID NOs: 15 and 16 were used. To secure the gapA promoter, genomic DNA from wild-type Corynebacterium glutamicum ATCC 13032 (NC_006958.1) was used as a template, and primer pairs of SEQ ID NOs: 19 and 20 were used. PCR was performed in each case, denaturing at 95°C for 2 minutes, followed by denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, polymerization at 72°C for 1 minute, repeated 25 times, and then incubated at 72°C for 5 minutes. Solg was used in the PCR during this process. TM Pfu-X DNA polymerase was used.

[0106] Using DNA synthesized to secure the gene fragment encoding a membrane protein derived from Shewanella oneidensis MR-1 (SEQ ID NO: 4), PCR was performed under the same conditions as described above using primer pairs of SEQ ID NOs: 17 and 18.

[0107] The amplified gapA promoter region, a gene fragment encoding a membrane protein from Shewanella koralii A687 or Shewanella oneidensis MR-1, a homologous recombinant arm gene fragment, and the vector pDCM2 (WO2021-187781 A1) cleaved with SalI and BamHI restriction enzymes were ligated using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix). These ligated cells were then transformed into E. coli DH5α and streaked onto LB solid medium containing kanamycin (25 mg / l). To select colonies transformed with vectors containing the gene encoding the membrane protein from Shewanella koralii A687 or Shewanella oneidensis MR-1, PCR was performed using primer pairs of SEQ ID NOs. 21 and 22. Plasmids were extracted from the selected colonies using a plasmid prep kit (QIAGEN), and these plasmids were named pDCM2-PgapA-Sco and pDCM2-PgapA-Son, respectively.

[0108] The primer sequences used here are shown in Table 1 below.

[0109] [Table 1]

[0110] Example 2: Production of L-ornithine-producing and lysE-deficient bacterial strains To produce an L-ornithine-producing strain, a vector was created in which the serine at position 55 from the N-terminus of the amino acid sequence of wild-type Corynebacterium glutamicum-derived ArgF (ANU33618.1) (SEQ ID NO: 5) was replaced with a stop codon. Using the genome of wild-type Corynebacterium glutamicum ATCC 13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NOs: 23 and 24, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NOs: 25 and 26. Subsequently, a plasmid was obtained in the same manner as in Example 1, and the plasmid was named pDCM2-argF(S55*).

[0111] To produce a strain with improved L-ornithine production capacity, a vector was created in which the glutamate at position 47 from the N-terminus of the amino acid sequence of wild-type Corynebacterium glutamicum-derived ArgR (ANU33619.1) (SEQ ID NO: 7) was replaced with a stop codon. Using the genome of wild-type Corynebacterium glutamicum ATCC 13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NOs: 27 and 28, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NOs: 29 and 30. Subsequently, a plasmid was obtained using the method described above, and this plasmid was named pDCM2-argR(E47*).

[0112] Wild-type Corynebacterium glutamicum ATCC 13869 was transformed using the pDCM2-argF(S55*) vector via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). Following a secondary cross-reaction, a strain was obtained in which the serine at position 55 from the N-terminus of the ArgF amino acid sequence was replaced with a stop codon. PCR and nucleotide sequence analysis were performed using primer pairs SEQ ID NOs. 23 and 26, which can amplify the adjacent region including the insertion site of the gene, to confirm the genetic manipulation. The resulting strain was named C.gl::argF*.

[0113] C.gl::argF* was transformed with the pDCM2-argR(E47*) vector, and a strain was obtained in which the glutamate at position 47 from the N-terminus of the ArgR amino acid sequence was replaced with a stop codon using the same method as described above. PCR and nucleotide sequence analysis were performed using primer pairs of SEQ ID NOs. 27 and 30, which can amplify the adjacent region including the insertion site of the gene, to confirm the genetic manipulation. The strain thus obtained was named C.gl::argF*_argR*.

[0114] Next, in order to produce a LysE-deficient strain, a vector lacking the ORF (open reading frame) of wild-type Corynebacterium glutamicum LysE (ANU33473.1) (SEQ ID NO: 9) was created. Using the genome of wild-type Corynebacterium glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using primer pairs of SEQ ID NOs: 31 and 32, and the homologous recombinant B arm was amplified using primer pairs of SEQ ID NOs: 33 and 34. The homologous recombinant arm gene fragments were cloned into the vector pDCM2, which had been cleaved with SalI and BamHI restriction enzymes, in the same manner as in Example 1, and transformed into Escherichia coli DH5α. PCR was performed using primer pairs of SEQ ID NOs: 21 and 22 to select the transformed colonies. Plasmids were extracted from the selected colonies using a plasmid prep kit (QIAGEN), and the plasmid was named pDCM2-△lysE.

[0115] The L-ornithine-producing strain C.gl::argF*_argR* was transformed with the pDCM2-△lysE plasmid via electroporation. Following a secondary cross-reaction, a strain lacking the LysE-encoding gene (lysE) was obtained. PCR and sequencing analysis were performed using primer pairs (SEQ ID NOs. 31 and 34) capable of amplifying the adjacent region including the insertion site of the gene, confirming the genetic manipulation. The resulting strain was named C.gl::argF*_argR*_△lysE.

[0116] The primer sequences used here are shown in Table 2 below.

[0117] [Table 2]

[0118] Example 3: Production of a bacterial strain with a foreign LysE / ArgO family amino acid transporter protein gene. To produce exogenous membrane protein gene-transformed bacterial strains, the pDCM2-PgapA-Sco vector and the pDCM2-PgapA-Son vector produced in Example 1 were used to transform the L-ornithine-producing strain C.gl::argF*_argR* and the lysE-deficient strain C.gl::argF*_argR*_△lysE, produced in Example 2, respectively, by electroporation. After a secondary crossover process, strains with PgapA-Sco inserted and strains with PgapA-Son inserted were obtained. PCR and nucleotide sequence analysis were performed using primer pairs of Sequence ID No. 26 and 29, which can amplify adjacent regions including the insertion site of the gene, to confirm the genetic manipulation. The strains obtained in this manner were named C.gl::argF*_argR*_PgapA-Sco, C.gl::argF*_argR*_△lysE_PgapA-Sco, C.gl::argF*_argR*_PgapA-Son, and C.gl::argF*_argR*_△lysE_PgapA-Son, respectively.

[0119] Example 4: Comparison of L-ornithine production capacity of exogenous LysE / ArgO family amino acid transporter protein-introduced bacterial strains. The wild-type Corynebacterium glutamicum ATCC 13869 and the strain produced in Example 3 were cultured with the strain produced in Example 2 using the following method, and their cell mass, sugar consumption capacity, and L-ornithine production capacity were compared.

[0120] First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium and cultured with shaking at 30°C for 20 hours at 200 rpm. Then, 1 ml of seed culture solution was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium and cultured with shaking at 33°C for 42 hours at 200 rpm. After the culture was complete, the amount of L-ornithine produced was measured by HPLC, and the results are shown in Tables 3 and 4 below.

[0121] <Seed medium (pH 7.0)> Glucose 20g, Peptone 10g, Yeast extract 5g, Urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, Biotin 0.1mg, Thiamine HCl 1mg, Calcium pantothenate 22mg, Nicotinamide 2mg (based on 1 liter of distilled water)

[0122] <Production medium (pH 7.0)> Raw sugar 50g, (NH4)2SO4 25g, yeast extract 1g, KH2PO4 0.55g, MgSO4·7H2O 0.6g, L-arginine 0.2g, biotin 0.9mg, thiamine hydrochloride 4.5mg, calcium pantothenate 4.5mg, nicotinamide 30mg, MnSO4 9mg, FeSO4 9mg, ZnSO4 0.45mg, CuSO4 0.45mg, CaCO3 30g (based on 1 liter of distilled water)

[0123] [Table 3]

[0124] [Table 4]

[0125] As shown in Table 3 above, the C.gl::argF*_argR*_△lysE_PgapA-Sco strain, into which a gene encoding a LysE / ArgO family amino acid transporter protein, a membrane protein derived from Shewanella koralii A687, was introduced as an exogenous membrane protein, showed a significant increase in L-ornithine production capacity and L-ornithine yield compared to the lysE-deficient C.gl::argF*_argR*_△lysE strain. Furthermore, the C.gl::argF*_argR*_PgapA-Sco strain into which the membrane protein derived from Shewanella koralii A687 was introduced showed increased L-ornithine production capacity and L-ornithine yield compared to C.gl::argF*_argR*.

[0126] Furthermore, as shown in Table 4 above, the C.gl::argF*_argR*_△lysE_PgapA-Son strain, in which a gene encoding a LysE / ArgO family amino acid transporter protein, a membrane protein derived from Shewanella oneidensis MR-1, was introduced as an exogenous membrane protein into a lysE-deficient strain, showed a significant increase in L-ornithine production capacity and L-ornithine yield compared to the lysE-deficient strain C.gl::argF*_argR*_△lysE. In addition, the C.gl::argF*_argR*_PgapA-Son strain, into which a membrane protein derived from Shewanella oneidensis MR-1 was introduced, showed increased L-ornithine production capacity and L-ornithine yield compared to C.gl::argF*_argR*.

[0127] From the above description, 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. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A recombinant microorganism of Corynebacterium glutamicum having L-ornithine production ability, comprising a LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or a polynucleotide encoding a LysE / ArgO family amino acid transporter protein, Recombinant microorganisms in which the activity of ornithine carbamoyltransferase subunit F is weakened.

2. The microorganism according to claim 1, wherein the protein contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO:

3.

3. The microorganism according to claim 1, wherein the strain of the genus Shewanella is a strain of Shewanella corali or Shewanella oneidensis.

4. The microorganism according to any one of claims 1 to 3, wherein the microorganism further has weakened arginine repressor activity.

5. A recombinant microorganism of Corynebacterium glutamicum having the ability to produce L-ornithine, comprising a LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or a polynucleotide encoding a LysE / ArgO family amino acid transporter protein, A recombinant microorganism in which the protein contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of Sequence ID No.

1.

6. A method for producing L-ornithine, comprising the step of culturing a recombinant microorganism of Corynebacterium glutamicum having L-ornithine-producing ability, which contains a LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or a polynucleotide encoding a LysE / ArgO family amino acid transporter protein, in a culture medium.

7. The method according to claim 6, wherein the protein contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO:

3.

8. The method according to claim 6, wherein the Shewanella strain is a Shewanella kollii or Shewanella oneidensis strain.

9. The method according to claim 6, wherein the microorganism further has weakened activity of ornithine carbamoyltransferase subunit F.

10. The method according to any one of claims 6 to 9, wherein the microorganism has further weakened arginine repressor activity.

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