Microorganisms having the ability to produce L-ornithine and 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 technology 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, high-concentration L-amino acids accumulated inside microorganisms can be excreted, and the production ability can be greatly increased.

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-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

[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 that of the non - modified microorganism. [[Means for Solving the Problems]]

[0006] One object of the present 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 the present application is to provide a method for producing L - ornithine, which includes the step of 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 description and embodiment disclosed in the present application is also applicable to each other description 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 description 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 sequence of the LysE / ArgO family amino acid transporter protein can be obtained from public databases such as Genebank of NCBI.

[0013] As an example, the LysE / ArgO family amino acid transporter protein of the present application may be derived from a microorganism.

[0014] As another example, the LysE / ArgO family amino acid transporter protein of the present application may be an exogenous protein that does not inherently exist in microorganisms of the genus Corynebacterium.

[0015] As still another example, the LysE / ArgO family amino acid transporter protein of the present application may be derived from a microorganism. Specifically, the microorganism may be derived from a microorganism of the genus Shewanella, and more specifically, it may be derived 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 even more specifically, it may be derived from Shewanella corallii or Shewanella oneidensis, but is not limited thereto.

[0016] As another example, the amino acid sequence of the LysE / ArgO family amino acid transporter of the present application may be WP_115137742.1 derived from Shewanella coralii or WP_011072781.1 derived from Shewanella oneidensis MR-1, but is not necessarily limited thereto, and it is obvious that it includes proteins having LysE / ArgO family amino acid transporter activity derived from various Shewanella microorganisms.

[0017] In the present application, the term "Shewanella microorganism" is one of marine bacteria, which can inhabit both oxygenated and oxygen-free environmental conditions, and is known as a bacterium having a function of inducing the reduction of toxic metals such as chromium and uranium and precipitating them.

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

[0019] In the present application, the LysE / ArgO family amino acid transporter protein derived from a Shewanella strain can include the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or an amino acid sequence 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 therewith. Also, it is obvious that a protein having such homology or identity and having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added, as long as it shows an efficacy corresponding to the protein containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, is also included within the scope of the present application.

[0020] For example, it is a case where an addition or deletion of a sequence that does not change the function of the protein of the present application, a naturally occurring mutation, a silent mutation, or a conservative substitution is present at the N-terminus, C-terminus, and / or inside of the amino acid sequence.

[0021] The "conservative substitution" means substituting 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. Usually, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0022] In the present application, the terms "homology" or "identity" mean the degree of similarity between two given amino acid sequences or nucleotide sequences and can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0023] The homology or identity of a conserved polynucleotide or polypeptide sequence is determined by standard sequence algorithms and the default gap penalties established by the programs used are available. Substantially, homologous or identical sequences generally hybridize with all or part of the sequence under moderately or highly stringent conditions. It is obvious that hybridization also includes hybridization with a polynucleotide containing codons that take into account general codons or codon degeneracy in polynucleotides.

[0024] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as, for example, in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Or it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as performed by 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 versions) (including the 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 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 of the National Center for Biotechnology Information Database.

[0025] The homology, similarity or identity of a polynucleotide or polypeptide can be determined by, for example, comparing sequence information using a GAP computer program such as that known in Smith and Waterman, Adv. Appl. Math (1981) 2:482, for example, as in Needleman et al. (1970), J Mol Biol. 48:443. Briefly, the GAP program can be defined as the total number of symbols in the shorter of two sequences, minus the number of similar aligned symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program can include (1) a binary comparison matrix (with values of 1 for identity and 0 for non-identity) and a weighted comparison matrix as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al(1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (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 and 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 of the present application may be a gene named lysE and / or argO.

[0027] As an example, the lysE gene and / or the argO gene may be a polynucleotide encoding WP_115137742.1 derived from Shewanella coralii or WP_011072781.1 derived from Shewanella oneidensis MR-1, but is not necessarily limited thereto. It is obvious that it includes the lysE gene and / or the argO gene derived from various Shewanella microorganisms encoding a protein having LysE / ArgO family amino acid transporter activity.

[0028] In the present application, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are covalently linked in a long chain, and is a DNA or RNA chain of a certain length or more. More specifically, it means a polynucleotide fragment encoding the protein.

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

[0030] The polynucleotide of the present application may be subjected to various modifications in the coding region within the range that does not change the amino acid sequence of the LysE / ArgO family amino acid transporter protein, taking into account the degeneracy of codons or the codons preferred in the organism in which the LysE / ArgO family amino acid transporter protein is to be expressed. Specifically, the polynucleotide of the present application has, comprises, or consists of, or may be essentially composed of, but is not limited to, a nucleotide sequence having 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more homology or identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0031] In addition, the polynucleotide of the present application may be included without limitation as long as it is a probe produced from a known gene sequence, for example, a sequence capable of hybridizing under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions where polynucleotides with high homology or identity, i.e., polynucleotides with homology or identity 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 hybridize with each other, and polynucleotides with lower homology or identity do not hybridize with each other, or the washing conditions of ordinary Southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically, the salt concentration and temperature corresponding to 68°C, 0.1×SSC, 0.1% SDS, and washing once, specifically, two to three times can be enumerated.

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

[0033] Specifically, polynucleotides having homology or identity with the polynucleotides of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C. Also, the Tm value may be 60°C, 63°C or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art according to the purpose.

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

[0035] In the present application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and microorganisms in which a specific mechanism has been weakened or strengthened due to causes such as insertion of an exogenous gene or enhancement or weakening of the activity of an endogenous gene, and may be a microorganism including a genetic modification for the production of a target polypeptide, protein or product.

[0036] The strain of the present application may be a strain that naturally has the ability to produce L-ornithine or a microorganism in which the ability to produce L-ornithine is imparted to a strain that does not have the ability to produce L-ornithine. As an example, it may be a microorganism in which the activity of ornithine carbamoyltransferase subunit F (ArgF) and / or arginine repressor (ArgR) is weakened; and / or a microorganism in which the endogenous LysE protein is deleted. As another example, it may be a microorganism in which the LysE / ArgO family amino acid transporter protein derived from the Shewanella strain of the present application or a polynucleotide encoding the same is introduced to improve the ability to excrete L-ornithine, but is not limited thereto.

[0037] The strain of the present application may be a microorganism in which the ability to produce L-ornithine is increased compared to a Corynebacterium strain or a wild-type Corynebacterium strain that does not contain the LysE / ArgO family amino acid transporter protein derived from the Shewanella strain of the present application. Specifically, the strain of the present application may be a recombinant microorganism into which a LysE / ArgO family amino acid transporter protein derived from Shewanella coralii or a LysE / ArgO family amino acid transporter protein derived from Shewanella oneidensis having increased activity compared to the endogenous activity is introduced. The strain may have an improved ability to produce L-ornithine by improving the ability to excrete L-ornithine due to an increase in the activity of the LysE / ArgO family amino acid transporter protein compared to the endogenous activity. That is, the strain of the present application may have an increased activity of the LysE / ArgO family amino acid transporter protein compared to the endogenous activity.

[0038] As an example, the target strain for comparing the presence or absence of an increase in the L-ornithine production ability, the "non-transformed microorganism into which the LysE / ArgO family amino acid transporter protein derived from a Shewanella strain has not been introduced" may be a Corynebacterium glutamicum strain having L-ornithine production ability in which the serine located at the 55th position from the N-terminus of the amino acid sequence of the endogenous ArgF is replaced with a stop codon and the glutamate located at the 47th position from the N-terminus of the amino acid sequence of the endogenous ArgR is replaced with a stop codon; or a Corynebacterium glutamicum strain in which the endogenous LysE is deleted, but is not limited thereto.

[0039] As an example, the recombinant strain with increased production ability may have an increase of about 1% or more, specifically, about 1% or more, about 2% or more, about 5% or more, about 10% or more, about 15% or more, about 18% or more, about 18.9% or more, about 19% or more, about 19.1% or more, about 19.7% or more, about 20% or more, or about 20.3% or more (there is no special limitation on the upper limit value, for example, it may be about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 15% or less) compared to the L-ornithine production ability of the parent strain or non-transformed microorganism before mutation, but is not limited thereto as long as it has an increase in the + value compared to the production ability of a microorganism into which the LysE / ArgO family amino acid transporter protein derived from the parent strain, non-transformed microorganism or Shewanella strain before mutation or the polynucleotide encoding the same has not been introduced. In another example, the recombinant strain with increased L-ornithine production ability may have an L-ornithine production ability that is about 1.01 times or more, about 1.02 times or more, about 1.05 times or more, about 1.10 times or more, about 1.15 times or more, about 1.18 times or more, about 1.189 times or more, about 1.19 times or more, about 1.191 times or more, about 1.197 times or more, about 1.20 times or more, or about 1.203 times or more (there is no special limitation on the upper limit value, for example, it may be about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) compared to a microorganism into which the LysE / ArgO family amino acid transporter protein derived from the parent strain, non-transformed microorganism or Shewanella strain before mutation or the polynucleotide encoding the same has not been introduced, but is not limited thereto.

[0040] In the present application, the term "non-modified microorganism" does not exclude strains including mutations that can occur naturally in microorganisms, but means a wild-type strain or a natural-type strain itself, or a strain before its traits are changed by genetic mutations due to natural or artificial factors. Further, the microorganism means a strain into which the LysE / ArgO family amino acid transporter protein derived from a Shewanella strain described in the present specification has not been introduced, or a strain before such introduction. The non-modified microorganism of the present application does not exclude strains including the introduction or modification of other proteins or other genes other than the introduction of the LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or the polynucleotide encoding the same.

[0041] In the present application, the term "non-modified microorganism" may be used interchangeably with "strain before modification", "microorganism before modification", "non-mutated strain", "non-modified strain", "non-mutated microorganism" or "reference microorganism".

[0042] The microorganism of the present application may be, but is not limited to, a microorganism into which the LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or the polynucleotide encoding the same has been introduced; or a microorganism (for example, a recombinant microorganism) genetically modified so that the LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or the polynucleotide encoding the same is introduced. The "intrinsic activity" means the activity of a specific polypeptide originally possessed by the parental strain, wild-type or non-modified microorganism before the change in traits when the traits are changed by genetic mutations due to natural or artificial factors. This may be used interchangeably with "activity before modification".

[0043] As another example of the present application, the microorganism of the present application may be Corynebacterium stationis, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, specifically, it may be Corynebacterium glutamicum.

[0044] In addition to the introduction of the LysE / ArgO family amino acid transporter protein derived from the Shewanella genus strain of the present application, the activity of the LysE / ArgO family amino acid transporter of the microorganism of the present application can be enhanced by known methods.

[0045] In this application, the "enhancement" of the activity of a term, polypeptide (including proteins identified by the name of each enzyme as an example thereof) means that the activity of the polypeptide increases compared to the intrinsic activity. The said enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, increase, etc. Here, activation, enhancement, up-regulation, overexpression, and increase all include indicating an activity that was not originally present or an activity improved compared to the intrinsic activity or the activity before modification. The said "intrinsic activity" means the activity of a specific polypeptide that the parent strain or non-modified microorganism before the trait change originally had when the trait changes due to genetic mutations caused by natural or artificial factors. This may be used interchangeably with the "activity before modification". That the activity of a polypeptide is "enhanced", "up-regulated", "overexpressed" or "increased" compared to the intrinsic activity means that it is improved compared to the activity and / or concentration (expression level) of the specific polypeptide that the parent strain or non-modified microorganism before the trait change originally had.

[0046] The said enhancement can be achieved by introducing an exogenous polypeptide or through enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether the activity of the said polypeptide is enhanced can be confirmed from an increase in the activity level, expression level of the said polypeptide or the amount of the product excreted from the said polypeptide.

[0047] The enhancement of the activity of the said polypeptide can be achieved by applying various methods well-known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to the microorganism before modification. Specifically, it may utilize genetic engineering and / or protein engineering well-known to ordinary technicians in the industry, which are routine methods in molecular biology, but is not limited thereto (for example, 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 activity of the polypeptide of the present application is 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence having a strong activity; 3) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying the polynucleotide sequence encoding the polypeptide so that the polypeptide activity is enhanced (for example, modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so that the polypeptide activity is enhanced); 6) introducing a foreign polypeptide showing the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide, selecting and modifying the exposed site or chemically modifying it; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0049] More specifically, The increase in the intracellular copy number of the polynucleotide encoding the polypeptide in the above 1) may be achieved by introducing into the host cell a vector that is replicated and functional regardless of the host and to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be achieved by introducing one copy or two or more copies of the polynucleotide encoding the polypeptide into the chromosome in the host cell. The introduction into the chromosome is performed by introducing into the host cell a vector that inserts the polynucleotide into the chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0050] Exchanging the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the said 2) polypeptide with a sequence having strong activity may, for example, cause mutations in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof so as to further enhance the activity of the said expression regulatory region, or may be an exchange with a sequence having stronger activity. The said expression regulatory region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, it may be to exchange the original promoter with a strong promoter, but is not limited thereto.

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

[0052] Modifying the base sequence encoding the start codon of the gene transcript encoding the said 3) polypeptide or the 5'UTR region may, for example, be to substitute it with a base sequence encoding another start codon with a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0053] The modifications of the amino acid sequences or polynucleotide sequences in the above (4) and (5) may be deletions, insertions, non-conservative or conservative substitutions, or combinations thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, resulting in sequence mutations so as to enhance the activity of the polypeptide, or may be exchanges with amino acid sequences or polynucleotide sequences improved to have stronger activity or amino acid sequences or polynucleotide sequences improved to increase activity, but are not limited thereto. Specifically, the exchange may be performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may further contain a selection marker for confirming the presence or absence of chromosomal insertion.

[0054] The introduction of a foreign polynucleotide showing the activity of the above (6) polypeptide may be the introduction of a foreign polynucleotide encoding a polypeptide showing the same / similar activity as the above polypeptide into a host cell. The origin and sequence of the foreign polynucleotide are not limited as long as it shows the same / similar activity as the above polypeptide. The method used for the introduction is appropriately selected by those skilled in the art from known transformation methods, and the introduced polynucleotide is expressed in the host cell to generate a polypeptide, and its activity increases.

[0055] The codon optimization of the polynucleotide encoding the above (7) polypeptide is such that the endogenous polynucleotide is codon-optimized so that transcription or translation increases in the host cell, or the codons of the foreign polynucleotide are optimized so that optimized transcription and translation are performed in the host cell.

[0056] Analyzing the tertiary structure of the polypeptide described in 8) and selecting and modifying the exposed sites or chemically modifying them may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins, determining candidates for the template protein according to the degree of sequence similarity, confirming the structure based on this, and selecting and modifying or chemically modifying the exposed sites to be modified.

[0057] Such enhancement of polypeptide activity may be an increase in the activity or concentration expression level of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or unmodified microbial strain, or an increase in the amount of the product produced from the polypeptide, but is not limited thereto.

[0058] In the microorganism of the present application, partial or total modification of the polynucleotide may be induced by (a) homologous recombination using a chromosomal insertion vector in the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light such as ultraviolet rays and radiation and / or chemical substances, but is not limited thereto. The method for partial or total modification of the gene may include a method by 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 cause homologous recombination, resulting in partial or total deletion of the gene. The injected nucleotide sequence or vector may contain a dominant selection marker, but is not limited thereto.

[0059] The vector of the present application can include a DNA product containing the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to an expression regulatory region (or expression regulatory sequence) suitable for expressing the target polypeptide in a suitable host. The expression regulatory region can include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate or function regardless of the host genome and is integrated into the genome itself.

[0060] The vector used in the present application is not particularly limited, and any vector known in the art can be utilized. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. can be used as phage vectors or cosmid vectors, and pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0061] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for chromosomal insertion within a cell. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of the chromosomal insertion may further be included. The selection marker is for selecting cells transformed with the vector, that is, for confirming the presence or absence of the insertion of the target nucleic acid molecule, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to a cytotoxic agent, or expression of a surface polypeptide is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that transformed cells can be selected.

[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. As long as the transformed polynucleotide can be expressed in the host cell, it can include all of them regardless of whether it is inserted into the host cell chromosome or located extrachromosomally. Further, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for autonomous expression. The expression cassette can usually 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 be in the form of an expression vector capable of self-replication. Further, the polynucleotide may be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell, and is not limited thereto.

[0063] Also, the term "operably linked" as used above means that the promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target polypeptide of this application is functionally linked to the polynucleotide sequence.

[0064] As another example of this application, the microorganism of this application may be a microorganism in which the activity of a part of the protein in the L-ornithine biosynthetic pathway is further enhanced, or the activity of a part of the protein in the L-ornithine degradation pathway is further weakened, resulting in enhanced L-ornithine production ability.

[0065] Specifically, the microorganism of the present application may be a microorganism in which the activity of endogenous ArgF is further weakened, or the argF gene encoding the same is further deleted, a microorganism in which the activity of endogenous ArgR is further weakened, or the argR gene encoding the same is further deleted, and / or a microorganism in which the activity of LysE is further weakened, or the lysE gene encoding the same is further deleted.

[0066] The amino acid sequences of the aforementioned ArgF, ArgR, or LysE can be obtained from known databases such as Genebank of NCBI. As an example, the amino acid sequence of ArgF of the present 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 therewith. 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 therewith. 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 therewith, but is not limited thereto. It is obvious that proteins having ArgF, ArgR, or LysE activities from various sources are included.

[0067] However, the weakening of the ArgF, ArgR, and / or LysE protein activities or the deletion of the argF, argR, and / or lysE genes is not limited to these as an example. The microorganism of the present application may be a microorganism in which the protein activities of various known L-ornithine biosynthetic pathways are enhanced or the protein activities of the degradation pathways are weakened.

[0068] In the present application, the term "attenuation" of polypeptide activity encompasses all cases where the activity is decreased or absent compared to the intrinsic activity. The said attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, attenuation, etc.

[0069] The said attenuation includes cases where the activity of the polypeptide itself is decreased or removed compared to the activity of the polypeptide originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, etc., inhibition of gene expression of the polynucleotide encoding it or inhibition of translation into the polypeptide, etc., resulting in the overall polypeptide activity level and / or concentration (expression level) in the cell being lower than that of the wild-type strain, cases where the expression of the polynucleotide is not carried out at all, and / or cases where, despite the expression of the polynucleotide, the polypeptide has no activity. The fact that the activity of the polypeptide is "inactivated, deficient, decreased, down-regulated, reduced, attenuated" compared to the intrinsic activity means that it is lower than the activity of the specific polypeptide originally possessed by the parental strain or non-transformed microorganism before the transformation.

[0070] Such attenuation of polypeptide activity can be carried out by any method known in the art, but is not limited thereto, and can be achieved by applying various methods well known in the art (for example, 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 attenuation of the polypeptide activity in the present application is 1) deletion of all or part of the gene encoding the polypeptide; 2) Modification of the expression regulatory region (or expression regulatory sequence) so that the expression of the gene encoding the polypeptide is decreased; 3) Modification of the amino acid sequence constituting the polypeptide (for example, deletion / substitution / addition of one or more amino acids in the amino acid sequence) so that the activity of the polypeptide is removed or weakened; 4) Modification of the gene sequence encoding the polypeptide (for example, deletion / substitution / addition of one or more nucleobases in the nucleobase sequence of the polypeptide gene so as to encode a polypeptide modified so that the activity of the polypeptide is removed or weakened) so that the activity of the polypeptide is removed or weakened; 5) Modification of the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide; 6) Introduction of an antisense oligonucleotide (for example, 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 in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide in order to form a secondary structure that prevents ribosome attachment; 8) Addition of a promoter 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); or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

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

[0073] In addition, the modification of the aforementioned (2) expression regulatory region (or expression regulatory sequence) may be a mutation occurring in the expression regulatory region (or expression regulatory sequence) due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or may be an exchange with a sequence having a 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 regulating the termination of transcription and translation.

[0074] The modification of the amino acid sequence or polynucleotide sequence in the aforementioned (3) and (4) may be a 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 a mutation occurring in the sequence, or an exchange with an amino acid sequence or polynucleotide sequence improved to have a weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, by introducing a mutation in the polynucleotide sequence to form a stop codon, the expression of the gene can be inhibited or weakened, but is not limited thereto.

[0075] The modification of the start codon of the gene transcript encoding the polypeptide in the aforementioned (5) or the base sequence encoding the 5'UTR region may be, for example, a substitution with a base sequence encoding another start codon having a lower polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0076] For the introduction of an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide in the aforementioned (6), reference can be made 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] In order to form a secondary structure that cannot be attached to the ribosome, the addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide may make mRNA translation impossible or may reduce the rate.

[0078] In addition, the addition of a promoter transcribed in the opposite direction (Reverse transcription engineering, RTE) to the 3'-end of the ORF (open reading frame) of the gene sequence encoding the polypeptide may create an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide and may weaken the activity.

[0079] Another aspect of the present application provides a method for producing L-ornithine, which includes culturing a Corynebacterium recombinant microorganism having the ability to produce L-ornithine in a medium, the microorganism containing a LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or a polynucleotide encoding the LysE / ArgO family amino acid transporter protein.

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

[0081] In the present application, the term "culturing" means growing the microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application is carried out according to appropriate media and culturing conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art according to the selected microorganism. Specifically, the culturing may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0082] In this application, the term "medium" means a substance mixed mainly with nutrients necessary for culturing the microorganisms of this application, and supplies water, which is essential for survival and growth, as well as nutrients and growth factors. Specifically, the medium and other culture conditions used for culturing the microorganisms of this application can be any medium used for culturing ordinary microorganisms without special restrictions. However, the microorganisms of this application can be cultured by adjusting temperature, pH, etc. under aerobic conditions in an ordinary medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0083] Specifically, the culture medium 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 D.C., USA, 1981)].

[0084] In this application, examples of the carbon source include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose; sugar alcohols such as mannitol, sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) are used, and other appropriate amounts of carbon sources can be used in various ways without limitation. These carbon sources can be used alone or in combination of two or more, and are not limited thereto.

[0085] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. are used as organic nitrogen sources. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0086] As the phosphorus source, primary potassium phosphate, secondary potassium phosphate, or sodium-containing salts corresponding thereto may be included. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. are used, and in addition, amino acids, vitamins and / or appropriate precursors may be included. These components or precursors can be added to the medium in a batch or continuous manner. However, it is not limited thereto.

[0087] In addition, during the culture of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. Also, during the culture, the generation of bubbles can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. Further, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or in order to maintain the anaerobic and microaerobic states, without injecting gas, or nitrogen, hydrogen or carbon dioxide gas can be injected, and it is not limited thereto.

[0088] In the culture of the present application, the culture temperature can be maintained at 20 to 45 °C, specifically 25 to 40 °C, and can be cultured for about 10 to 160 hours, but it is not limited thereto.

[0089] The L-ornithine produced by culturing in the present application is secreted into the culture medium or remains intracellular.

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

[0091] The method for producing L-ornithine in the present application can further include the step of recovering L-ornithine from the culture medium (the medium in which culturing has been performed) or the cultured microorganism by the culturing. The step of recovering can further be included after the culturing step.

[0092] The recovery may be to collect the target L-ornithine by using an appropriate method known in the art, such as a batch, continuous, or fed-batch culture method, etc., for the method of culturing the microorganism of the present application. For example, centrifugation, filtration, treatment with a crystallization protein precipitant (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and other various chromatographies, HPLC, or a combination of these methods can be used, and the target L-ornithine can be recovered from the culture medium or the microorganism by using an appropriate method known in the art.

[0093] Also, the method for producing L-ornithine in the present application may further include a purification step. The purification can be performed by using an appropriate method known in the art. In one example, when the method for producing L-ornithine in the present application includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously regardless of the procedure, or can be performed simultaneously or integrated into one step, but are not limited thereto.

[0094] The microorganism of the present 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, resulting in enhanced L-ornithine production ability.

[0095] As an example, the microorganism of the present application may be a microorganism in which the activity of endogenous ArgF is further weakened, or the argF gene encoding the same is further deleted, the activity of endogenous ArgR is further weakened, or the argR gene encoding the same is further deleted, and / or the activity of LysE is further weakened, or the lysE gene encoding the same is further deleted.

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

[0097] Another aspect of the present application provides a composition for L-ornithine production, comprising a Corynebacterium recombinant microorganism containing 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 medium in which it is cultured; or a combination thereof.

[0098] The composition of the present application may further contain any suitable excipient commonly used in compositions for L-ornithine production, and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents, etc., but are not limited thereto.

[0099] Another aspect of the present application provides a method for producing a Corynebacterium recombinant microorganism 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 the present application provides a use of a recombinant microorganism of the genus Corynebacterium into which a LysE / ArgO family amino acid transporter protein derived from a Shewanella strain or a polynucleotide encoding the LysE / ArgO family amino acid transporter protein has been introduced for L-ornithine production.

[0101] The Shewanella strain, LysE / ArgO family amino acid transporter protein, introduction, microorganism of the genus Corynebacterium, etc. are as described in the other aspects.

[0102] Hereinafter, the present application will be described in more detail with reference to experimental examples. However, the following examples are merely preferred embodiments for illustrating the present application, and thus are not intended to limit the scope of rights of the present application thereto. On the other hand, technical matters not described in this specification can be fully understood and easily carried out by an ordinary technician skilled in the technical field of the present application or a similar technical field.

[0103] Example 1: Selection of proteins with L-ornithine excretion activity and production of plasmids As proteins with high L-ornithine excretion activity, the LysE / ArgO family amino acid transporter (LysE / ArgO family amino acid transporter, WP_115137742.1) (SEQ ID NO: 1), which is a membrane protein derived from Shewanella corallii A687, and the LysE / ArgO family amino acid transporter (LysE / ArgO family amino acid transporter, WP_011072781.1) (SEQ ID NO: 3), which is a membrane protein derived from Shewanella oneidensis MR-1, were selected. To amplify the nucleotide sequences of the genes encoding these, information (NC_004347.2) on the genes encoding the membrane proteins and the surrounding nucleic acid sequences was obtained from the NIH GenBank of the National Institutes of Health, USA. DNA synthesis was performed based on the sequence information (Cosmo genetech, Korea).

[0104] Vectors were respectively prepared for introducing the selected membrane proteins derived from Shewanella corallii A687 and the membrane proteins derived from Shewanella oneidensis MR-1 into the L-ornithine-producing strain. First, ANU34435.1, which is one of the transposases present inside the genome of wild-type Corynebacterium glutamicum ATCC 13869 (NZ_CP016335.1), was used as the insertion position. Using the genome of wild-type Corynebacterium glutamicum ATCC 13869 as a template, a homologous recombinant A arm was amplified using the primer pair of SEQ ID NOs: 11 and 12, and a homologous recombinant B arm was amplified using the primer pair of SEQ ID NOs: 13 and 14.

[0105] Using the synthesized DNA as a template to secure the gene fragment (SEQ ID NO: 2) encoding the membrane protein derived from Shewanella corallii A687, and using the genomic DNA (NC_006958.1) of wild-type Corynebacterium glutamicum ATCC 13032 as a template to secure the gapA promoter, PCR was performed using the primer pairs of SEQ ID NOs: 15 and 16 and SEQ ID NOs: 19 and 20 respectively. After denaturation at 95°C for 2 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 1 minute were repeated 25 times, and then polymerization was performed at 72°C for 5 minutes. At that time, Solg TM Pfu-X DNA polymerase was used for PCR.

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

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

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

[0109]

Table 1

[0110] Example 2: Production of L-ornithine-producing strains and lysE-deficient strains To produce an L-ornithine-producing strain, a vector was constructed by substituting the serine located at the 55th position from the N-terminus of the amino acid sequence of ArgF (ANU33618.1) (SEQ ID NO: 5) derived from wild-type Corynebacterium glutamicum 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. Thereafter, 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-producing ability, a vector was constructed by substituting the glutamate located at the 47th position from the N-terminus of the amino acid sequence of ArgR (ANU33619.1) (SEQ ID NO: 7) derived from wild-type Corynebacterium glutamicum 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. Thereafter, a plasmid was obtained by the method described above, and the plasmid was named pDCM2-argR(E47*).

[0112] After transforming wild-type Corynebacterium glutamicum ATCC 13869 with the pDCM2-argF(S55*) vector by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), a strain in which the serine located at the 55th position from the N-terminus of the amino acid sequence of ArgF was substituted with a stop codon was obtained through a secondary crossover process. PCR and nucleotide sequence analysis were performed using the primer pair of SEQ ID NOs: 23 and 26 that could amplify the adjacent site containing the position where the gene was inserted to confirm the genetic manipulation. The strain thus obtained was named C.gl::argF*.

[0113] The pDCM2-argR(E47*) vector was transformed into C.gl::argF*, and a strain was obtained in which the glutamate located at the 47th position from the N-terminus of the amino acid sequence of ArgR was replaced with a stop codon in the same manner as described above. PCR and nucleotide sequence analysis were performed using the primer pair of SEQ ID NO: 27 and 30 capable of amplifying the adjacent site containing the position where the gene was inserted 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, to construct a vector lacking the ORF (open reading frame) of wild-type Corynebacterium glutamicum LysE (ANU33473.1) (SEQ ID NO: 9), the genomic DNA of wild-type Corynebacterium glutamicum ATCC13869 was used as a template, and the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO: 31 and 32, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO: 33 and 34. The homologous recombinant arm gene fragment was cloned into the vector pDCM2 digested with SalI and BamHI restriction enzymes in the same manner as in Example 1 above and transformed into Escherichia coli DH5α. To select the transformed colonies, PCR was performed using the primer pair of SEQ ID NO: 21 and 22. The plasmid was extracted from the selected colonies using a plasmid prep kit (QIAGEN), and the plasmid was named pDCM2-△lysE.

[0115] After transforming the L-ornithine-producing strain C.gl::argF*_argR* with the pDCM2-△lysE plasmid by electroporation, a strain in which the gene (lysE) encoding LysE was deleted was obtained through a secondary crossover process. PCR and nucleotide sequence analysis were performed using the primer pair of SEQ ID NO: 31 and 34 capable of amplifying the adjacent site containing the position where the gene was inserted to confirm the genetic manipulation. The strain thus obtained was named C.gl::argF*_argR*_△lysE.

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

[0117] [Table 2]

[0118] Example 3: Production of strains into which foreign LysE / ArgO family amino acid transporter protein genes are introduced To produce strains into which foreign membrane protein genes were introduced, the pDCM2-PgapA-Sco vector and pDCM2-PgapA-Son vector prepared in Example 1 were each transformed into the L-ornithine-producing strain C.gl::argF*_argR* and the lysE-deficient strain C.gl::argF*_argR*_ΔlysE prepared in Example 2 by electroporation. After that, through a secondary crossing process, strains into which PgapA-Sco was inserted and strains into which PgapA-Son was inserted were obtained. PCR and nucleotide sequence analysis were performed using the primer pairs of SEQ ID NOs: 26 and 29 that can amplify the adjacent sites including the positions where the genes were inserted to confirm the genetic manipulation. The strains thus obtained were named C.gl::argF*_argR*_PgapA-Sco, C.gl::argF*_argR*_ΔlysE_PgapA-Sco, C.gl::argF*_argR*_PgapA-Son, and C.gl::argF*_argR*_ΔlysE_PgapA-Son, respectively.

[0119] Example 4: Comparison of L-ornithine production ability of strains into which foreign LysE / ArgO family amino acid transporter proteins are introduced Wild-type Corynebacterium glutamicum ATCC 13869, the strains prepared in Example 3, and the strains prepared in Example 2 were cultured by the following method, and the cell mass, sugar consumption ability, and L-ornithine production ability were compared.

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

[0121] <Seed medium (pH 7.0)> Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4·7H2O 0.5 g, biotin 0.1 mg, thiamine HCl 1 mg, calcium pantothenate 22 mg, nicotinamide 2 mg (based on 1 liter of distilled water)

[0122] <Production medium (pH 7.0)> Raw sugar 50 g, (NH4)2SO4 25 g, yeast extract 1 g, KH2PO4 0.55 g, MgSO4·7H2O 0.6 g, L-arginine 0.2 g, biotin 0.9 mg, thiamine hydrochloride 4.5 mg, calcium pantothenate 4.5 mg, nicotinamide 30 mg, MnSO4 9 mg, FeSO4 9 mg, ZnSO4 0.45 mg, CuSO4 0.45 mg, CaCO3 30 g (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 the gene encoding the LysE / ArgO family amino acid transporter protein, a membrane protein derived from Shewanella coralii A687, was introduced as a foreign membrane protein into the lysE-deficient strain had a significantly increased L-ornithine production ability and L-ornithine yield compared to the lysE-deficient strain C.gl::argF*_argR*_△lysE strain. In addition, the C.gl::argF*_argR*_PgapA-Sco strain into which the membrane protein derived from Shewanella coralii A687 was introduced showed an increased L-ornithine production ability and L-ornithine yield compared to C.gl::argF*_argR*.

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

[0127] From the above description, those skilled in the art to which this application pertains will be able to understand that this application can be implemented in other specific forms without changing its technical idea and essential features. In this regard, it should be understood that the embodiments described above are merely exemplary and not restrictive. The scope of this application should be construed as including the meaning and scope of the claims described below, and any changes or modifications derived from the equivalent concepts thereof are included in the scope of this application.

Claims

1. A recombinant microorganism of the genus Corynebacterium 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.

2. The microorganism according to claim 1, wherein the protein comprises an amino acid sequence having 80% 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 Shewanella strain is a Shewanella corallii or Shewanella oneidensis strain.

4. The microorganism according to claim 1, wherein the microorganism further has weakened activity of ornithine carbamoyltransferase subunit F.

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

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

7. A method for producing L-ornithine, comprising culturing a recombinant microorganism of the genus Corynebacterium having the ability to produce L-ornithine, which comprises 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.

8. The method according to claim 7, wherein the protein comprises an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO:

3.

9. The method according to claim 7, wherein the Shewanella strain is a Shewanella corallii or Shewanella oneidensis strain.

10. The method according to claim 7, wherein the microorganism further has weakened activity of ornithine carbamoyltransferase subunit F.

11. The method according to any one of claims 7 to 10, wherein the microorganism further has weakened activity of arginine repressor.

12. The method according to claim 7, wherein the microorganism is Corynebacterium glutamicum.

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