Microorganism having L-ornithine-producing ability and method for producing L-ornithine using same

Abstract

The present application relates to a recombinant microorganism of the genus Corynebacterium having an L-ornithine producing ability, comprising an amino acid transporter of the LysE / ArgO family derived from a Shewanella strain or a polynucleotide encoding an amino acid transporter of the LysE / ArgO family.

Description

Technical Field

[0001] The present disclosure relates to a microorganism capable of producing L-ornithine and a method for producing L-ornithine using the same. Background Art

[0002] L-amino acid is used in animal feed, human medicine and cosmetics industry, and some L-amino acid is produced by using the fermentation of microorganism. In order to improve the method of using microorganism to produce L-amino acid, the research using recombinant DNA technology has been carried out. For example, the L-amino acid production capacity of microorganism can be increased by expressing some genes that have been missing or weakened or by amplification of the gene related to L-amino acid biosynthesis. Especially, improving the L-amino acid output capacity of microorganism is considered to be the key technology (US10995378 B2) that increases L-amino acid production capacity. Therefore, it is possible to release the high concentration L-amino acid accumulated in microorganism by the gene that its L-amino acid output capacity has been disclosed by importing and amplifying, thereby greatly improving production capacity. Summary of the invention

[0003]

Technical issues

[0004] The present inventors have confirmed that when exogenous LysE / ArgO family amino acid transporters are introduced into microorganisms, their L-ornithine production capacity is increased compared to unmodified microorganisms, thereby completing the present disclosure.

[0005]

Technical solution

[0006] An object of the present disclosure is to provide a recombinant microorganism of the genus Corynebacterium having the ability to produce L-ornithine, wherein the recombinant microorganism comprises a LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the LysE / ArgO family amino acid transporter.

[0007] Another object of the present disclosure is to provide a method for producing L-ornithine, comprising the step of culturing a recombinant microorganism of the genus Corynebacterium having an L-ornithine-producing ability in a culture medium, wherein the recombinant microorganism comprises a LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the LysE / ArgO family amino acid transporter.

[0008]

Beneficial Effects

[0009] Compared to existing unmodified microorganisms, the microorganism of the present disclosure into which an exogenous LysE / ArgO family amino acid transporter is introduced can have an increased L-ornithine production capacity. DETAILED DESCRIPTION

[0010] The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is to say, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific description described below. In addition, many papers and patent documents are referenced and cited throughout the specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further illustrate the level and scope of the subject matter to which the present disclosure belongs.

[0011] One aspect of the present disclosure provides a recombinant microorganism of the genus Corynebacterium having an L-ornithine-producing ability, wherein the recombinant microorganism comprises a LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the LysE / ArgO family amino acid transporter.

[0012] As used herein, 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. In other words, the LysE / ArgO family amino acid transporter refers to a protein having lysine and / or arginine export activity.

[0013] The amino acid sequence of the LysE / ArgO family amino acid transporter can be obtained from Genbank (i.e., a known database) of NCBI or the like.

[0014] For example, the LysE / ArgO family amino acid transporter of the present disclosure may be derived from a microorganism.

[0015] For another example, the LysE / ArgO family amino acid transporter of the present disclosure may be an exogenous protein that is not endogenously present in a microorganism of the genus Corynebacterium.

[0016] For yet another example, the LysE / ArgO family amino acid transporter of the present disclosure may be derived from a microorganism. The microorganism can specifically be derived from a microorganism of the genus Shewanella, more specifically, can 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, more specifically, can be derived from Shewanella corallii or Shewanella Oneidensis, but is not limited thereto.

[0017] For another example, the amino acid sequence of the LysE / ArgO family amino acid transporter disclosed in the present invention can be WP_115137742.1 derived from coral Shewanella or WP_011072781.1 derived from Oneida Lake Shewanella MR-1, but is not limited thereto. Obviously, proteins having LysE / ArgO family amino acid transporter activity derived from various microorganisms of the genus Shewanella are included.

[0018] As used herein, the term "Shewanella microorganism" is a marine bacterium that can survive in both aerobic and anaerobic environments and is known as a bacterium having a function of inducing reduction and precipitation of toxic metals such as chromium and uranium.

[0019] In the present disclosure, the LysE / ArgO family amino acid transporter derived from a Shewanella strain may have, include, or consist of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or may essentially consist of the amino acid sequence.

[0020] In the present disclosure, the LysE / ArgO family amino acid transporter derived from a Shewanella strain may 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 thereto. In addition, it is obvious that any protein having an amino acid sequence including deletion, modification, substitution, conservative substitution or addition in part of the sequence may also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein comprising the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 3.

[0021] Examples thereof include those having sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence that do not alter the function of the protein of the present disclosure.

[0022] "Conservative substitutions" refer 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 polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

[0023] As used herein, the term "homology" or "identity" refers to 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" are often used interchangeably.

[0024] The sequence homology or identity of conservative polynucleotides or polypeptides is determined by standard comparison algorithms, and the default gap penalty set up by the used program can be used together. Basically, homologous or identical sequences are usually able to hybridize with all or part of the sequence under medium or high stringency conditions. Obviously, hybridization also includes hybridization with polynucleotides containing universal codons or codons that take into account codon degeneracy.

[0025] Known computer algorithms (such as the "FASTA" program) can be used to determine whether any two polynucleotide or polypeptide sequences have homology, similarity or identity, for example using the default parameters in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the homology, similarity or identity can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is implemented in the following programs: the Needleman program (version 5.0.0 or later) in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (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; Diego, 1994, and [CARILLO et al.,] (1988) SIAM J Applied Math 48:1073). For example, BLAST of the National Center for Biotechnology Information, or ClustalW can be used to determine the homology, similarity or identity.

[0026] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48: 443, for example, as described in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. In summary, the GAP program can be defined as the value obtained by dividing the number of similar aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program may include (1) a binary comparison matrix (including a value of 1 for identity and a value of 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix), as disclosed in 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 open penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0027] The gene encoding the LysE / ArgO family amino acid transporter of the present disclosure may be a gene named lysE and / or argO.

[0028] For example, the lysE gene and / or argO gene can be a polynucleotide encoding WP_115137742.1 derived from coral Shewanella or WP_011072781.1 derived from Oneida Lake Shewanella MR-1, but is not necessarily limited thereto. It is apparent that the present disclosure includes lysE genes and / or argO genes derived from various microorganisms of the genus Shewanella, which encode proteins having LysE / ArgO family amino acid transporter activity.

[0029] As used herein, the term "polynucleotide" is a polymer of nucleotides in which nucleotide monomers are linked by covalent bonds into a long chain, and refers to a DNA or RNA chain of a predetermined or longer length, and more specifically, refers to a polynucleotide fragment encoding the protein.

[0030] The polynucleotide encoding the LysE / ArgO family amino acid transporter of the present disclosure may include a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. In one embodiment of the present disclosure, the polynucleotide of the present disclosure may have or include the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In addition, the polynucleotide of the present disclosure may consist of or substantially consist of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Specifically, the LysE / ArgO family amino acid transporter may be encoded by a polynucleotide shown in the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0031] In the polynucleotides of the present disclosure, various modifications may be made in the coding region, taking into account the degeneracy of codons or preferred codons in an organism intended to express the LysE / ArgO family amino acid transporter of the present disclosure, as long as the amino acid sequence of the LysE / ArgO family amino acid transporter is not changed. Specifically, the polynucleotides of the present disclosure may have or include 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, or may consist of or be substantially composed of 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 sequence of SEQ ID NO: 2 or SEQ ID NO: 4, but are not limited thereto.

[0032] In addition, the polynucleotides of the present disclosure may include probes prepared from known gene sequences, for example, any sequence that is not limited, as long as it is a sequence that can hybridize with all or part of the complementary sequence of the polynucleotide sequence of the present disclosure under stringent conditions. "Stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second 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). Examples thereof include conditions under which polynucleotides having higher homology or identity, i.e., polynucleotides 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, or 99% or more homology or identity hybridize with each other, whereas polynucleotides having lower homology or identity do not hybridize with each other, or conditions in which washing is performed once, specifically, 2 to 3 times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically, 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, which are general washing conditions for Southern hybridization.

[0033] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are allowed depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present disclosure may also include substantially similar nucleic acid sequences and isolated nucleic acid fragments that are complementary to the entire sequence.

[0034] Specifically, the hybridization conditions including the hybridization step with a Tm value of 55° C. and the above conditions can be used to detect polynucleotides having homology or identity with the polynucleotides disclosed herein. In addition, the Tm value can be 60° C., 63° C. or 65° C., but is not limited thereto, and those skilled in the art can make appropriate adjustments according to the purpose.

[0035] The appropriate stringency for hybridizing such polynucleotides depends on the length of the polynucleotides and the degree of complementation, and these variables are well known in the art (eg, J. Sambrook et al., supra).

[0036] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it can be a microorganism in which a specific mechanism is weakened or enhanced due to the insertion of exogenous genes or the enhancement or reduction of the activity of endogenous genes, and it can be a microorganism comprising a genetic modification for producing a target polypeptide, protein or product.

[0037] The strain disclosed herein may be a microorganism naturally having the ability to produce L-ornithine, or a microorganism prepared by providing the ability to produce L-ornithine to a strain that does not have the ability to produce L-ornithine. For example, the strain may be a microorganism in which the activity of ornithine carbamoyltransferase subunit F (ArgF) and / or arginine repressor protein (ArgR) is weakened; and / or the endogenous LysE protein is missing. For another example, the strain may be a microorganism in which a LysE / ArgO family amino acid transporter or a polynucleotide encoding the same derived from the Shewanella strain disclosed herein is introduced, and thus the L-ornithine export capacity is improved, but is not limited thereto.

[0038] The strain of the present disclosure may be a microorganism in which the L-ornithine production capacity is increased compared to a Corynebacterium strain or a Corynebacterium wild-type strain that does not contain a LysE / ArgO family amino acid transporter derived from the Shewanella strain of the present disclosure. Specifically, the strain of the present disclosure may be a recombinant microorganism into which a LysE / ArgO family amino acid transporter derived from Coral Shewanella or a LysE / ArgO family amino acid transporter derived from Oneida Lake Shewanella is introduced, which has an increased activity compared to the endogenous activity. Compared to the endogenous activity, the strain has an increased activity of the LysE / ArgO family amino acid transporter, and thus its L-ornithine export capacity is improved, thereby having an increased L-ornithine production capacity. In other words, compared to the endogenous activity, the strain of the present disclosure may have an increased activity of the LysE / ArgO family amino acid transporter.

[0039] For example, "an unmodified microorganism into which the LysE / ArgO family amino acid transporter derived from a Shewanella strain has not been introduced" is a target strain for comparing whether the L-ornithine production capacity is increased, which may be a Corynebacterium glutamicum strain having L-ornithine production capacity, in which the serine at position 55 of the N-terminus of the endogenous ArgF amino acid sequence is replaced by a stop codon, and the glutamic acid at position 47 of the N-terminus of the endogenous ArgR amino acid sequence is replaced by a stop codon; or a Corynebacterium glutamicum strain in which the endogenous LysE is deleted, but is not limited thereto.

[0040] For example, compared to the parent strain before modification or the unmodified microorganism, the recombinant strain with increased productivity can 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, about 20.3% or more (the upper limit is not particularly limited, but can be, for example, about 200% or less, about 300% or less, about 300% or less, about 300% or less, about 300% or less, about 300% or less, about 300% or less, about 300% or less, about 300% or less, about 300% or less, about 300% or less, about 300% or less, about 300% or less, about 300% or more ... The L-ornithine production capacity of the strain is 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), but the increase is not limited thereto, as long as the production capacity has an increase of + value compared with the production capacity of the following microorganisms: the parent strain before modification, the unmodified microorganism, or the microorganism into which the LysE / ArgO family amino acid transporter derived from the genus Shewanella strain or the polynucleotide encoding the same is not introduced, but it is not limited thereto. In another example, the recombinant strain with increased L-ornithine-producing ability may have an increased L-ornithine-producing ability of 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, about 1.203 times or more (the upper limit is not particularly limited, but may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less) compared to the L-ornithine-producing ability of the parent strain before modification, an unmodified microorganism, or a microorganism into which the LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the same is not introduced (but not limited thereto).

[0041] As used herein, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, and may be a wild-type strain or a natural strain itself, or may be a strain before the trait is changed due to genetic variation caused by natural or artificial factors. In addition, the microorganism may refer to a strain into which the LysE / ArgO family amino acid transporter derived from a Shewanella strain described herein has not been introduced or has not been introduced. In addition to the introduction of the LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the same, the unmodified microorganism of the present disclosure does not exclude strains containing the introduction or modification of another protein or another gene.

[0042] As used herein, the term "unmodified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "unaltered strain", "unmodified strain", "unaltered microorganism" or "reference microorganism".

[0043] The microorganism of the present disclosure may be a microorganism into which a LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the same has been introduced; or a microorganism (e.g., a recombinant microorganism) that has been genetically modified to introduce a LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the same has been introduced, but is not limited thereto. "Endogenous activity" refers to the activity of a specific polypeptide originally present in a parent strain or a wild-type or unmodified microorganism before the trait is changed when the trait is changed due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification".

[0044] In another embodiment of the present disclosure, the microorganism of the present disclosure 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 serrata. flavescens), specifically, Corynebacterium glutamicum.

[0045] In the microorganism of the present disclosure, in addition to introducing the LysE / ArgO family amino acid transporter derived from the Shewanella strain of the present disclosure, the activity of the LysE / ArgO family amino acid transporter can be enhanced by a known method.

[0046] As used herein, the term "enhancement" of polypeptide (e.g., protein specified by the name of each enzyme) activity refers to an increase in the activity of the polypeptide compared to its endogenous activity. Enhancement can be used interchangeably with terms such as activation, upregulation, overexpression and increase. In particular, activation, enhancement, upregulation, overexpression and increase can include two situations in which an activity that was not originally present or an activity enhancement is shown compared to the activity before endogenous activity or modification. "Endogenous activity" refers to the activity of a specific polypeptide originally present in a parent strain or unmodified microorganism before a trait is changed when the trait is changed due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification". The fact that the activity of a polypeptide is "enhanced", "upregulated", "overexpressed" or "increased" compared to the endogenous activity means that the activity of the polypeptide is improved compared to the activity and / or concentration (expression level) of the specific polypeptide originally present in the parent strain or unmodified microorganism before the trait is changed.

[0047] Enhancement can be achieved by introducing exogenous polypeptides or enhancing the activity and / or concentration (expression level) of endogenous polypeptides. The enhancement of polypeptide activity can be confirmed by an increase in the activity degree and expression level of the corresponding polypeptide or an increase in the amount of the corresponding polypeptide released product.

[0048] In order to enhance the activity of polypeptide, various methods well known in the art can be applied, as long as the activity of the desired polypeptide can be enhanced compared with the microbial activity before modification, the method is not limited. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used, which is a conventional method of molecular biology, but the method 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.).

[0049] Specifically, the activity enhancement of the polypeptide of the present disclosure may be:

[0050] 1) Increasing the copy number of the polynucleotide encoding the polypeptide in the cell;

[0051] 2) Replace the gene expression regulatory region on the chromosome encoding the polypeptide with a more active sequence;

[0052] 3) modifying the nucleotide sequence of the start codon or 5'-UTR region encoding a gene transcript, wherein the gene transcript encodes a polypeptide;

[0053] 4) Modify the amino acid sequence of a polypeptide to enhance the activity of the polypeptide;

[0054] 5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying a polynucleotide sequence of a polypeptide gene to encode a modified polypeptide to enhance the activity of the polypeptide);

[0055] 6) introducing an exogenous polypeptide expressing polypeptide activity or an exogenous polynucleotide encoding a polypeptide;

[0056] 7) Codon optimization of polynucleotides encoding polypeptides;

[0057] 8) analyzing the tertiary structure of the polypeptide to select and modify or chemically modify exposed sites; or

[0058] 9) A combination of two or more selected from 1) to 8), but not particularly limited thereto.

[0059] More specifically,

[0060] 1) Increasing the number of copies of the polynucleotide encoding the polypeptide in the cell can be achieved by introducing a vector into the host cell, the vector can replicate and function independently of the host, and the polynucleotide encoding the corresponding polypeptide is operably linked to the vector. Alternatively, this increase can be achieved by introducing one copy or two or more copies of the polynucleotide encoding the corresponding polypeptide into the chromosome of the host cell. The introduction into the chromosome can be carried out by introducing a vector capable of inserting the polynucleotide into the host cell chromosome, but is not limited thereto. The vector is as described above.

[0061] 2) Replacing the gene expression regulatory region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence showing strong activity may be, for example, a variation in the sequence due to deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacing it with a sequence showing stronger activity, thereby further enhancing 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, a sequence controlling transcription and translation termination, etc. For example, the replacement may be replacing the original promoter with a strong promoter, but is not limited thereto.

[0062] Examples of known strong promoters include CJ1 to CJ7 promoters (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 (US10584338 B2), O2 promoter (US10273491 B2), tkt promoter, yccA promoter, etc., but are not limited to these.

[0063] 3) Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of a gene transcript (which encodes a polypeptide) may be, for example, replacement with a nucleotide sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0064] 4) and 5) modification of the amino acid sequence or polynucleotide sequence may involve variation in the sequence due to deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence that is improved to exhibit stronger activity or an amino acid sequence or polynucleotide sequence that is improved to be more active, thereby enhancing the activity of the polypeptide, but is not limited thereto. Replacement may be specifically performed by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used herein may further comprise a selection marker for confirming chromosomal insertion.

[0065] 6) Introduction of exogenous polynucleotides expressing polypeptide activity may involve introduction of exogenous polynucleotides into host cells, wherein the polypeptide encoded by the exogenous polynucleotides expresses the same or similar activity as the polypeptide. There is no limitation on its source or sequence, as long as the exogenous polynucleotides express the same or similar activity as the polypeptide. The method used in the introduction may be carried out by appropriately selecting a known transformation method by a person skilled in the art. As the introduced polynucleotides are expressed in the host cells, polypeptides may be produced and their activity may be increased.

[0066] 7) Codon optimization of a polynucleotide encoding a polypeptide may involve codon optimization of an endogenous polynucleotide to enhance transcription or translation in a host cell, or codon optimization of an exogenous polynucleotide to achieve optimized transcription and translation in a host cell.

[0067] 8) Analyzing the tertiary structure of a polypeptide to select and modify or chemically modify an exposed site may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining template protein candidates based on the degree of sequence similarity, confirming the structure based thereon, and modifying or chemically modifying the exposed site to be modified or chemically modified.

[0068] Based on the activity or concentration of the polypeptide expressed in the wild type or microbial strain before modification, this enhancement of polypeptide activity can lead to an increase in the activity or concentration / expression level of the corresponding polypeptide, or an increase in the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0069] The modification of some or all of the polynucleotides in the microorganisms disclosed herein can be induced by: (a) homologous recombination using a vector for chromosome insertion in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) treatment with light (e.g., ultraviolet light and radiation) and / or chemicals, but not limited thereto. Methods for modifying some or all of the genes may include methods using DNA recombination technology. For example, a nucleotide sequence or vector comprising a nucleotide sequence homologous to the target gene can be introduced into the microorganism to cause homologous recombination, resulting in partial or complete deletion of the gene. The nucleotide sequence or vector to be introduced may include a dominant selection marker, but is not limited thereto.

[0070] The vector of the present disclosure may comprise a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a desired polypeptide, which is operably linked to a suitable expression regulatory region (or expression control sequence) so that the desired polypeptide can be expressed in a suitable host. The expression regulatory region may comprise a promoter capable of initiating transcription, any operon sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling transcription and translation termination. The vector may be transformed into a suitable host cell and then replicated or functioned independently of the host genome, or may be integrated into the genome itself.

[0071] The vector used in the present disclosure is not particularly limited, but any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors; and pDZ system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0072] For example, the polynucleotide encoding the desired polypeptide can be inserted into the chromosome by the vector for intracellular chromosome insertion. The polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. The carrier can further include a selection marker for confirming chromosome insertion. The selection marker is used to select cells transformed with the carrier, i.e., for confirming the insertion of the desired nucleic acid molecule, and can use a marker that grants a selectable phenotype (such as drug resistance, auxotrophy, cytotoxic agent resistance or surface polypeptide expression). In the environment of processing with a selection agent, only the cells expressing the selection marker survive or show other phenotypic traits, and therefore the cells transformed can be selected.

[0073] As used herein, the term "transformation" refers to the introduction of 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 converted polynucleotide can be expressed in the host cell, it can be located, regardless of the position by being inserted into the chromosome of the host cell or being located outside the chromosome. In addition, the polynucleotide comprises DNA and / or RNA encoding the desired polypeptide. As long as the polynucleotide can be introduced into the host cell and expressed, it can be introduced in any form. 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 required for autonomous expression. The expression cassette can generally include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably connected to the polynucleotide. The expression cassette can be in the form of an expression vector capable of self-replication. In addition, the polynucleotide can be introduced into the host cell in its own form and operably connected to the sequence required for expression in the host cell, but is not limited thereto.

[0074] Furthermore, the term "operably linked" means that a polynucleotide sequence is functionally linked to a promoter sequence, which initiates and mediates the transcription of the polynucleotide encoding the desired polypeptide of the present disclosure.

[0075] In yet another embodiment of the present disclosure, the microorganism of the present disclosure may be a microorganism in which the L-ornithine-producing ability is enhanced by additionally enhancing the activity of a portion of proteins in the L-ornithine biosynthesis pathway or by additionally weakening the activity of a portion of proteins in the L-ornithine degradation pathway.

[0076] Specifically, the microorganism of the present disclosure may be a microorganism in which the activity of endogenous ArgF is additionally weakened, or the argF gene encoding it is additionally deleted, or a microorganism in which the activity of endogenous ArgR is additionally weakened, or the argR gene encoding it is additionally deleted, and / or a microorganism in which the activity of LysE is additionally weakened, or the lysE gene encoding it is additionally deleted.

[0077] The amino acid sequence of ArgF, ArgR or LysE can be obtained from Genbank of NCBI, which is a known database, etc. For example, the amino acid sequence of ArgF of the present disclosure 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 of the present disclosure 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 ATCC13869 or an amino acid sequence having 80% or more sequence identity thereto, but is not limited thereto, and obviously includes proteins having ArgF, ArgR or LysE activity derived from various sources.

[0078] However, the weakening of ArgF, ArgR or LysE protein activity or the deletion of argF, argR and / or lysE genes is only an example and is not limited thereto. The microorganism of the present disclosure can be a microorganism in which the activity of proteins in various known L-ornithine biosynthetic pathways is enhanced or the activity of proteins in the degradation pathway is weakened.

[0079] As used herein, the term "reduction" of polypeptide activity has all concepts covering reduced activity or loss of activity compared to endogenous activity. Reduction can be used interchangeably with terms such as inactivation, lack, downregulation, reduction, decrease, weakening, etc.

[0080] The attenuation may also include: due to the variation of the polynucleotide encoding the polypeptide, the activity of the polypeptide itself is reduced or disappears compared with the original polypeptide activity of the microorganism; due to the inhibition of gene expression of the polynucleotide encoding the polypeptide or the inhibition of translation into polypeptide, the overall polypeptide activity level and / or concentration (expression level) in the cell is lower than that of the natural strain; the polynucleotide is not expressed at all; and / or even when the polynucleotide is expressed, the polypeptide activity does not exist. "Inactivation, lack, reduction, downregulation, reduction or weakening" of polypeptide activity compared with its endogenous activity means that the activity of the polypeptide is reduced compared with the activity of the original specific polypeptide of the parent strain or the unmodified microorganism before the trait change.

[0081] The attenuation of the 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 (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.).

[0082] Specifically, attenuating the activity of the polypeptide of the present disclosure can be achieved by:

[0083] 1) Deletion of all or part of the gene encoding the polypeptide;

[0084] 2) modifying the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

[0085] 3) Modifying the amino acid sequence constituting the polypeptide to eliminate or weaken the activity of the polypeptide (for example, deleting / replacing / adding one or more amino acids in the amino acid sequence);

[0086] 4) Modifying a gene sequence encoding a polypeptide to eliminate or weaken the activity of the polypeptide (for example, deleting / replacing / adding one or more nucleotides in the nucleotide sequence of the polypeptide gene to encode the modified polypeptide, thereby eliminating or weakening the activity of the polypeptide);

[0087] 5) modifying the nucleotide sequence of the start codon or 5'-UTR region encoding a gene transcript, wherein the gene transcript encodes a polypeptide;

[0088] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to a gene transcript encoding a polypeptide;

[0089] 7) adding a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide upstream of the Shine-Dalgarno sequence to form a secondary structure that prevents ribosomes from attaching;

[0090] 8) adding the promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

[0091] 9) A combination of two or more selected from 1) to 8), but not particularly limited thereto.

[0092] For example,

[0093] 1) Deletion of part or all of a gene encoding a polypeptide may involve deletion of all polynucleotides encoding an endogenous target polypeptide in a chromosome, replacement with a polynucleotide lacking some nucleotides, or replacement with a marker gene.

[0094] 2) Modification of the expression control region (or expression control sequence) may involve mutations in the expression control region (or expression control sequence) by deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof, or replacement with a sequence having weaker activity. The expression control region includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling transcription and translation termination, but is not limited thereto.

[0095] 3) and 4) modification of the amino acid sequence or polynucleotide sequence may involve mutation of the sequence through deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, thereby reducing the activity of the polypeptide, but is not limited thereto. For example, the expression of a gene may be inhibited or reduced by introducing a mutation into a polynucleotide sequence to form a stop codon, but is not limited thereto.

[0096] 5) Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of a gene transcript (which encodes a polypeptide) may involve, for example, replacing the endogenous start codon with a nucleotide sequence encoding another start codon having a lower polypeptide expression rate, but is not limited thereto.

[0097] 6) Introduction of antisense oligonucleotides (e.g., antisense RNA) that complementarily bind to gene transcripts encoding polypeptides can be found in, 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].

[0098] 7) A sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide is added upstream of the Shine-Dalgarno sequence to form a secondary structure that prevents ribosomes from attaching, which may involve making the mRNA unable to be translated or reducing its translation rate.

[0099] Furthermore, 8) adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) may involve making the antisense nucleotide complementary to the gene transcript encoding the polypeptide, thereby attenuating the activity.

[0100] Yet another aspect of the present disclosure provides a method for producing L-ornithine, the method comprising the step of culturing a recombinant microorganism of the genus Corynebacterium having an ability to produce L-ornithine in a culture medium, the recombinant microorganism comprising a LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the LysE / ArgO family amino acid transporter.

[0101] The method for producing L-ornithine of the present disclosure may include the step of culturing a microorganism in a culture medium, the microorganism being genetically modified to contain a LysE / ArgO family amino acid transporter derived from the Shewanella strain of the present disclosure or a polynucleotide encoding the same.

[0102] As used herein, the term "culture" refers to growing the microorganism of the present disclosure under appropriately adjusted environmental conditions. The culture procedure of the present disclosure can be carried out according to suitable culture media or culture conditions known in the art. Those skilled in the art can easily adjust such culture procedures according to the selected microorganism. Specifically, the culture can be batch type, continuous type and / or fed-batch type, but is not limited thereto.

[0103] As used herein, "culture medium" refers to a mixture containing nutrients required for culturing the microorganisms of the present disclosure as the main components, wherein the culture medium provides nutrients including water, growth factors, etc. necessary for survival and growth. Specifically, for the culture medium and other culture conditions for culturing the microorganisms of the present disclosure, any culture medium used for conventional culture of microorganisms can be used without particular limitation. However, the microorganisms of the present disclosure can be cultured in a general culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc. under aerobic conditions, while controlling temperature, pH, etc.

[0104] Specifically, a culture medium of a microorganism of the genus Corynebacterium can be found in the document ["Manual of Methods for General Bacteriology" of the American Society of Bacteriology (Washington, DC, USA, 1981)].

[0105] In the present disclosure, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, 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. In addition, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn syrup may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and appropriate amounts of various other carbon sources may be used without restriction. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0106] 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., and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its degradation product, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0107] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and their corresponding sodium salts. For inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. may be used, and amino acids, vitamins and / or suitable precursors may also be included. These components or precursors may be added to the culture medium in batches or continuously. However, the present disclosure is not limited thereto.

[0108] In addition, in the process of cultivating the microorganism of the present disclosure, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture medium in an appropriate manner. In addition, defoamers such as fatty acid polyethylene glycol esters can be used to suppress foam formation during cultivation. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain the aerobic state of the culture medium, or gas can not be injected, or nitrogen, hydrogen or carbon dioxide gas can be injected to maintain the anaerobic and microaerobic state of the culture medium, but it is not limited thereto.

[0109] In the culture of the present disclosure, the culture temperature may be maintained at 20° C. to 45° C., specifically, at 25° C. to 40° C., and the culture may be performed for about 10 hours to about 160 hours, but is not limited thereto.

[0110] L-ornithine produced by the culture of the present disclosure may be released into the medium, or may be retained in the cells.

[0111] The method for producing L-ornithine of the present disclosure may further include, for example, before the culturing step, the step of preparing the microorganism of the present disclosure, preparing a culture medium for culturing the microorganism, or a combination thereof (regardless of the order, in any order).

[0112] The method for producing L-ornithine of the present disclosure may further include a step of recovering L-ornithine from the culture medium produced by the culture (the culture medium in which the culture has been performed) or the cultured microorganism. A recovery step may be further included after the culture step.

[0113] The recovery can include methods for culturing microorganisms according to the present disclosure, such as batch, continuous or fed-batch type cultivation, collecting the desired L-ornithine by using suitable methods known in the art. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic treatment, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, etc.), HPLC and combinations of these methods can be used, and the desired L-ornithine can be recovered from the culture medium or microorganism by using suitable methods known in the art.

[0114] In addition, the method for producing L-ornithine disclosed herein may further include a purification step. Purification may be performed by using a suitable method known in the art. In an exemplary embodiment, when the method for producing L-ornithine disclosed herein includes a recovery step and a purification step, the recovery step and the purification step may be performed in a continuous or discontinuous manner without regard to the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0115] The microorganism of the present disclosure may be a microorganism in which L-ornithine-producing ability is enhanced by additionally enhancing the activity of a part of proteins in the L-ornithine biosynthesis pathway or by additionally weakening the activity of a part of proteins in the L-ornithine degradation pathway.

[0116] For example, the microorganism of the present disclosure may be a microorganism in which the activity of endogenous ArgF is additionally weakened, or the argF gene encoding it is additionally deleted, or a microorganism in which the activity of endogenous ArgR is additionally weakened, or the argR gene encoding it is additionally deleted, and / or a microorganism in which the activity of LysE is additionally weakened, or the lysE gene encoding it is additionally deleted.

[0117] In the methods of the present disclosure, the Shewanella strain, LysE / ArgO family amino acid transporter, polynucleotide, vector and microorganism are as described in other aspects.

[0118] Yet another aspect of the present disclosure provides a composition for producing L-ornithine, comprising: a recombinant microorganism of the genus Corynebacterium, the recombinant microorganism comprising a LysE / ArgO family amino acid transporter derived from a strain of the genus Shewanella or a polynucleotide encoding the LysE / ArgO family amino acid transporter; a culture in which the microorganism is cultured; or a combination thereof.

[0119] The composition of the present disclosure may further comprise any suitable excipients commonly used in compositions for producing L-ornithine, and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0120] Still another aspect of the present disclosure provides a method for preparing a recombinant microorganism of the genus Corynebacterium for producing L-ornithine, the method comprising the step of introducing a LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the LysE / ArgO family amino acid transporter.

[0121] Still another aspect of the present disclosure provides use of a recombinant microorganism of the genus Corynebacterium in producing L-ornithine, wherein the recombinant microorganism is introduced with a LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the LysE / ArgO family amino acid transporter.

[0122] The Shewanella strain, LysE / ArgO family amino acid transporter, introduction, Corynebacterium microorganism, etc. are as described in other aspects.

[0123] [Mode for carrying out the invention]

[0124] The present disclosure will be described in more detail below by way of exemplary embodiments. However, the following exemplary embodiments are only for illustrating preferred embodiments of the present disclosure, and therefore are not intended to limit the scope of the present disclosure thereto. At the same time, those skilled in the art or similar technical fields to which the present disclosure belongs can fully understand and easily implement technical matters not described in this specification.

[0125] Example 1: Screening of proteins with L-ornithine export activity and construction of plasmids

[0126] The membrane protein LysE / ArgO family amino acid transporter (WP_115137742.1) (SEQ ID NO: 1) derived from Shewanella corallii A687 and the membrane protein LysE / ArgO family amino acid transporter (WP_011072781.1) (SEQ ID NO: 3) derived from Shewanella Oneida Lake MR-1 were selected as proteins with high L-ornithine export activity, and in order to amplify the nucleotide sequence of the gene encoding it, information on the gene encoding the membrane protein and the nucleotide sequence around it was obtained from NIH GenBank (NC_004347.2). Based on the corresponding sequence information, DNA synthesis was performed (Cosmogenetech, South Korea).

[0127] The vectors for introducing the selected membrane proteins from coral Shewanella A687 and Oneida Lake Shewanella MR-1 into L-ornithine production strains were constructed respectively. First, ANU34435.1 (one of the transposases present in the wild-type Corynebacterium glutamicum ATCC13869 (NZ_CP016335.1) genome) was used as an insertion site. The genome of wild-type Corynebacterium glutamicum ATCC 13869 was used as a template, and the homologous recombination A arm was amplified using primers SEQ ID NO:11 and 12, and the homologous recombination B arm was amplified using primers SEQ ID NO:13 and 14.

[0128] To obtain the gene fragment encoding the membrane protein derived from Shewanella corallii A687 (SEQ ID NO: 2), the synthetic DNA was used as a template and the primer pair SEQ ID NO: 15 and 16 was used, and to obtain the gapA promoter, the genomic DNA of the wild-type Corynebacterium glutamicum ATCC 13032 (NC_006958.1) was used as a template and the primer pair SEQ ID NO: 19 and 20 was used to perform PCR at 95°C for 2 minutes, followed by 25 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, polymerization at 72°C for 1 minute, and then polymerization at 72°C for 5 minutes. At this time, Solg TM PCR was performed using Pfu-X DNA polymerase.

[0129] To obtain a gene fragment encoding a membrane protein derived from Oneida Lake Shewanella MR-1 (SEQ ID NO: 4), PCR was performed using the synthetic DNA as a template and a primer pair of SEQ ID NOs: 17 and 18 under the same conditions as above.

[0130] The amplified gapA promoter region, the gene fragment encoding the membrane protein derived from coral Shewanella A687, or the gene fragment encoding the membrane protein derived from Oneida Lake Shewanella MR-1, the homologous recombination arm gene fragment, and the vector pDCM2 (WO2021-187781A1) digested with SalI and BamHI restriction enzymes were connected using the Gibson assembly (DG Gibson et al., NATURE METHODS, Vol. 6, No. 5, May 2009, NEBuilder HiFi DNA Assembly Master Mix) method and transformed into Escherichia coli (E. coli) DH5α, which was plated on LB solid medium containing kanamycin (25 mg / l). In order to screen the colonies transformed with the vector containing the gene encoding the membrane protein derived from coral Shewanella A687 or the membrane protein derived from Oneida Lake Shewanella MR-1, PCR was performed using primers SEQ ID NO: 21 and 22. Plasmids were extracted from the screened colonies using a plasmid preparation kit (QIAGEN), and these plasmids were named pDCM2-PgapA-Sco and pDCM2-PgapA-Son, respectively.

[0131] The sequences of the primers used in this article are shown in Table 1 below.

[0132] [Table 1]

[0133]

[0134] Example 2: Preparation of L-ornithine producing strain and lysE deletion strain

[0135] In order to prepare an L-ornithine producing strain, a vector was constructed in which the amino acid sequence (SEQ ID NO: 5) of ArgF (ANU33618.1) derived from wild-type Corynebacterium glutamicum was replaced with a stop codon at the 55th serine from the N-terminus. The genome of wild-type Corynebacterium glutamicum ATCC 13869 was used as a template, and the homologous recombination A arm was amplified using primers SEQ ID NO: 23 and 24, and the homologous recombination B arm was amplified using primers SEQ ID NO: 25 and 26. Afterwards, a plasmid was obtained in the same manner as in Example 1, and the plasmid was named pDCM2-argF (S55*).

[0136] In order to prepare a strain having a further improved L-ornithine production capacity, a vector was constructed in which the amino acid sequence (SEQ ID NO: 7) derived from ArgR (ANU33619.1) of wild-type Corynebacterium glutamicum was replaced with a stop codon at position 47 from the N-terminus. The genome of wild-type Corynebacterium glutamicum ATCC 13869 was used as a template, and the homologous recombination A arm was amplified using a primer pair of SEQ ID NO: 27 and 28, and the homologous recombination B arm was amplified using a primer pair of SEQ ID NO: 29 and 30. Thereafter, a plasmid was obtained by the above method, and the plasmid was named pDCM2-argR (E47*).

[0137] With pDCM2-argF (S55 *) carrier, transform wild-type Corynebacterium glutamicum ATCC 13869 by electroporation (Appl.Microbiol.Biotechnol. (1999) 52:541-545), then carry out the second crossover process to obtain bacterial strain, wherein the aminoacid sequence of ArgF is replaced by the 55th serine from N-terminal by termination codon.Use primer to carry out PCR and sequencing analysis to SEQ ID NO:23 and 26, this primer pair can increase the adjacent region that comprises the corresponding gene insertion position, and confirmed corresponding genetic manipulation.The bacterial strain named after C.gl::argF * that obtains like this.

[0138] C.gl::argF* was transformed with pDCM2-argR(E47*) vector and a strain was obtained in the same manner as above, wherein the amino acid sequence of ArgR was replaced by a stop codon at position 47 from the N-terminus. PCR and sequencing analysis were performed using primers SEQ ID NO:27 and 30, which were able to amplify adjacent regions containing the corresponding gene insertion positions and confirmed the corresponding genetic manipulation. The strain thus obtained was named C.gl::argF*_argR*.

[0139] Next, in order to construct a vector (SEQ ID NO:9) for deleting the open reading frame (ORF) of wild-type Corynebacterium glutamicum LysE (ANU33473.1), to prepare LysE deletion strains, the genome of wild-type Corynebacterium glutamicum ATCC13869 was used as a template, and the homologous recombination A arm was amplified using primers SEQ ID NO:31 and 32, and the homologous recombination B arm was amplified using primers SEQ ID NO:33 and 34. In the same manner as in Example 1, the homologous recombination arm gene fragment was cloned into the vector pDCM2 cut with SalI and BamHI restriction enzymes, and transformed into Escherichia coli DH5α. In order to screen the transformed bacterium colonies, PCR was carried out using primers SEQ ID NO:21 and 22. Plasmid was extracted from the screened bacterium colonies using a plasmid preparation kit (QIAGEN), and the plasmid was named pDCM2-△lysE.

[0140] The pDCM2-△ lysE plasmid was transformed into the L-ornithine production strain C.gl::argF*_argR* by electroporation, and then a second crossover process was performed to obtain a strain in which the gene (lysE) encoding LysE was deleted. PCR and sequencing analysis were performed using primer pairs SEQ ID NO:31 and 34, which were able to amplify the adjacent region containing the corresponding gene insertion position and confirmed the corresponding genetic manipulation. The strain thus obtained was named C.gl::argF*_argR*_△ lysE.

[0141] The sequences of the primers used in this article are shown in Table 2 below.

[0142] [Table 2]

[0143]

[0144] Example 3: Preparation of strains into which exogenous LysE / ArgO family amino acid transporter genes are introduced

[0145] In order to prepare a strain into which an exogenous membrane protein gene is introduced, the pDCM2-PgapA-Sco and pDCM2-PgapA-Son vectors constructed in Example 1 were 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, respectively, by electroporation, and then a second crossover process was performed to obtain a strain into which PgapA-Sco was inserted and a strain into which PgapA-Son was inserted. PCR and sequencing analysis were performed using primers SEQ ID NO: 26 and 29, which were able to amplify the adjacent region containing the corresponding gene insertion position, and the corresponding genetic manipulation was confirmed. 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.

[0146] Example 4: Comparison of L-ornithine production capacity between strains into which exogenous LysE / ArgO family amino acid transporters were introduced

[0147] Wild-type Corynebacterium glutamicum ATCC 13869 and the strain prepared in Example 3 and the strain prepared in Example 2 were cultured by the following method to compare cell mass, sugar consumption, and L-ornithine production capacity.

[0148] First, each strain was inoculated into a 250 ml corner-baffled flask containing 25 ml of seed culture medium and cultured at 30° C. with shaking at 200 rpm for 20 hours. 1 ml of seed culture was inoculated into a 250 ml corner-baffled flask containing 24 ml of production culture medium and cultured at 33° C. with shaking at 200 rpm for 42 hours. After the culture was completed, the yield of L-ornithine was measured using HPLC, and the results are shown in Tables 3 and 4 below.

[0149] <Seed culture medium (pH 7.0)>

[0150] 20g glucose, 10g peptone, 5g yeast extract, 1.5g urea, 4g KH 2 PO 4 8gK 2 HPO 4 、0.5gMgSO 4 7H 2O, 0.1 mg biotin, 1 mg thiamine HCl, 22 mg calcium pantothenate, 2 mg niacinamide (based on 1 liter of distilled water)

[0151] <Production medium (pH 7.0)>

[0152] 50g raw sugar, 25g (NH 4 ) 2 SO 4 , 1g yeast extract, 0.55g KH 2 PO 4 、0.6gMgSO 4 7H 2 O, 0.2g L-arginine, 0.9mg biotin, 4.5mg thiamine HCl, 4.5mg calcium pantothenate, 30mg niacinamide, 9mg MnSO 4 , 9mg FeSO 4 , 0.45mg ZnSO 4 , 0.45 mg CuSO 4 、30g CaCO 3 (Based on 1L distilled water)

[0153] [Table 3]

[0154]

[0155]

[0156] [Table 4]

[0157]

[0158] As shown in Table 3, compared with the C.gl::argF*_argR*_△lysE strain lacking lysE, the C.gl::argF*_argR*_△lysE_PgapA-Sco strain (wherein the gene encoding the membrane protein LysE / ArgO family amino acid transporter derived from coral Shewanella A687 as an exogenous membrane protein is introduced into the lysE-deficient strain) showed significantly increased L-ornithine production capacity and L-ornithine yield. In addition, compared with C.gl::argF*_argR*, the C.gl::argF*_argR*_PgapA-Sco strain (wherein the membrane protein derived from coral Shewanella A687 is introduced) showed increased L-ornithine production capacity and L-ornithine yield.

[0159] As shown in Table 4, compared with the C.gl::argF*_argR*_△lysE strain lacking lysE, the C.gl::argF*_argR*_△lysE_PgapA-Son strain (wherein the gene encoding the membrane protein LysE / ArgO family amino acid transporter derived from Oneida Lake Shewanella MR-1 is introduced into the lysE-deficient strain as an exogenous membrane protein) showed significantly increased L-ornithine production capacity and L-ornithine yield. In addition, compared with C.gl::argF*_argR*, the C.gl::argF*_argR*_PgapA-Son strain (wherein the membrane protein derived from Oneida Lake Shewanella MR-1 was introduced) showed increased L-ornithine production capacity and L-ornithine yield.

[0160] Based on the above description, it will be appreciated by those skilled in the art that the present disclosure may be implemented in other specific forms without departing from its technical essence or essential features. In this regard, the above embodiments should be understood to be illustrative rather than restrictive in all respects. The scope of the present disclosure should be understood as the meaning and scope of the appended claims rather than the specific embodiments, and all changes or modifications derived from equivalent concepts fall within the scope of the present disclosure.

Claims

1. A recombinant microorganism of the genus Corynebacterium having an ability to produce L-ornithine, the recombinant microorganism comprising a LysE / ArgO family amino acid transporter derived from a Shewanella strain or a polynucleotide encoding the LysE / ArgO family amino acid transporter. 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 of claim 1, wherein the Shewanella strain is a Shewanella coralliensis or Shewanella Oneida Lake strain. The microorganism according to claim 1 , wherein the activity of ornithine carbamoyltransferase subunit F is further attenuated.

5. The microorganism according to any one of claims 1 to 4, wherein the activity of the arginine repressor protein is further attenuated. The microorganism according to claim 1 , wherein the microorganism is Corynebacterium glutamicum.

7. A method for producing L-ornithine, comprising the step of culturing a recombinant microorganism of the genus Corynebacterium having an ability to produce L-ornithine in a culture medium, wherein the recombinant microorganism comprises a LysE / ArgO family amino acid transporter derived from a strain of the genus Shewanella or a polynucleotide encoding the LysE / ArgO family amino acid transporter. 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 of claim 7, wherein the Shewanella strain is a Shewanella coralliensis or Shewanella Oneida Lake strain.

10. The method according to claim 7, wherein the activity of ornithine carbamoyltransferase subunit F in the microorganism is further attenuated.

11. The method according to any one of claims 7 to 10, wherein the activity of the arginine repressor protein in the microorganism is further attenuated.

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

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