阅读说明：本技术 新型多肽及使用其生产基于鸟氨酸的产物的方法 (Novel polypeptide and method for producing ornithine-based product using same ) 是由 金善慧 朴寿真 李京珉 罗景洙 李红仙 裵贤贞 沈知炫 梁荣烈 严惠媛 李孝炯 于 2018-06-14 设计创作，主要内容包括：本申请涉及具有输出基于鸟氨酸的产物的能力的新型多肽,以及使用其生产基于鸟氨酸的产物的方法。(The present application relates to novel polypeptides having the ability to export an ornithine-based product, and methods of using the same to produce an ornithine-based product.)

1. A polypeptide having an ability to export an ornithine-based product, wherein the glycine residue at position 77 of the N-terminus of the amino acid sequence of a protein exported from the ornithine-based product consists of the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2 is substituted with another amino acid.

2. The polypeptide of claim 1, wherein the glycine at position 77 is substituted with an alanine or an arginine.

3. The polypeptide of claim 1, wherein the polypeptide consists of an amino acid sequence represented by any one of SEQ ID NO 3 to SEQ ID NO 6.

4. A polynucleotide encoding said polypeptide having the ability to export an ornithine-based product of any one of claims 1 to 3.

5. A vector comprising the polynucleotide of claim 4.

6. A microorganism of the genus Corynebacterium (genus Corynebacterium) producing putrescine, said microorganism comprising a polypeptide according to any one of claims 1 to 3 or having enhanced activity thereof.

7. The microorganism of claim 6, wherein the microorganism is Corynebacterium glutamicum (Corynebacterium glutamicum).

8. The microorganism of claim 6, wherein an Ornithine Decarboxylase (ODC) activity is further introduced.

9. The microorganism of claim 6, wherein the activity of at least one selected from the group consisting of: ornithine carbamoyltransferase (ArgF) and proteins involved in glutamate export.

10. The microorganism of claim 6, wherein the activity of at least one selected from the group consisting of: acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (argJ), acetylglutamate kinase (ArgB), and acetylornithine transaminase (ArgD).

11. The microorganism of claim 6, wherein the activity of putrescine acetyltransferase is further attenuated compared to its endogenous activity.

12. An arginine-producing microorganism of the genus corynebacterium comprising the polypeptide of any one of claims 1 to 3 or having enhanced activity thereof.

13. The microorganism of claim 12, wherein the activity of at least one selected from the group consisting of: acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (argJ), acetylglutamate kinase (ArgB), and acetylornithine transaminase (ArgD).

14. The microorganism of claim 12, wherein the activity of at least one selected from the group consisting of: ornithine carbamoyltransferase (ArgF), argininosuccinate synthase (argG), argininosuccinate lyase (argH), aspartate ammonia lyase and/or aspartate aminotransferase.

15. The microorganism of claim 12, wherein the microorganism is corynebacterium glutamicum.

16. A method of producing putrescine, the method comprising:

(i) culturing a microorganism of the genus corynebacterium of any one of claims 6 to 11 in a culture medium; and

(ii) recovering putrescine from the microorganism or the culture medium obtained above.

17. A method of producing arginine, the method comprising:

(i) culturing a microorganism of the genus corynebacterium of any one of claims 12 to 15 in a culture medium; and

(ii) recovering arginine from the microorganism or the medium obtained above.

18. A microorganism producing an ornithine-based product, said microorganism comprising a polypeptide of any one of claims 1 to 3 or having enhanced activity thereof.

19. The microorganism of claim 18, wherein the activity of at least one selected from the group consisting of: acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (argJ), acetylglutamate kinase (ArgB), and acetylornithine transaminase (ArgD).

20. The microorganism of claim 18, wherein the microorganism is corynebacterium glutamicum.

21. A method for producing an ornithine-based product, the method comprising:

(i) culturing the microorganism of any one of claims 18 to 20 in a culture medium; and

(ii) recovering an ornithine-based product from the microorganism or the culture medium obtained above.

Technical Field

The present disclosure relates to novel polypeptides having the ability to export an ornithine-based product, and methods of using the same to produce an ornithine-based product.

Background

The substance ornithine, widely present in plants, animals and microorganisms, is biosynthesized from glutamic acid and is used as a precursor in the biosynthesis of putrescine, citrulline and proline. Further, ornithine plays an important role in the excretion pathway of urea produced from amino acids or ammonia through the ornithine cycle in the in vivo metabolism of higher animals. Ornithine is effective in enhancing muscle growth and reducing body fat, and thus is used as a nutritional supplement, and also as a medicine for improving liver cirrhosis and liver dysfunction. Known methods for producing ornithine include treatment of milk casein (milk casein) with a digestive enzyme and methods using transformed microorganisms of Escherichia coli (E.coli) or Corynebacterium (genus Corynebacterium) (Korean patent No. 10-1372635; T.Gotoh et al, Bioprocess biosystem. Eng., 33: 773-.

Putrescine (or 1, 4-butanediamine) is a very important starting material for the production of polyamide-4 and polyamide-6, including nylon-4 and nylon-6, and can be produced on an industrial scale by hydrogenation of succinonitrile, which is produced from acrylonitrile by addition of hydrogen cyanide. The synthetic routes to these chemicals require non-renewable petrochemicals as starting materials. In addition, high temperatures and pressures associated with the use of expensive catalyst systems, as well as relatively complex preparation steps and equipment, are also required. Therefore, as an alternative to chemical production processes, there is a need for processes for producing putrescine from renewable biomass-derived carbon sources. Recently, studies using environmentally friendly microorganisms have been continuously conducted in order to produce industrially useful polyamines (putrescine) at high concentrations (Qian ZG et al, Biotechnol Bioeng, 104: 651-.

Meanwhile, NCgl2522 has been identified as a gene having the ability to export putrescine (Korean patent No. 2014-0115244). However, in order to produce putrescine in higher yield, there is still a need to develop proteins with improved ability to export putrescine, which are able to export putrescine more efficiently from a putrescine-producing strain.

L-arginine has been widely used in medicine as a liver function promoter, a brain function promoter, and as a component of various amino acid supplements, L-arginine has drawn much attention in the food industry as a food additive for fish cakes and health drinks, and as a salt substitute for hypertensive patients, studies using microorganisms have been continuously conducted in order to produce arginine at high concentrations that is industrially usable, and examples thereof include a method using a microorganism-induced mutant strain from the genus Brevibacterium (genebrevibacterium) or Corynebacterium, which is a glutamic acid-producing strain, or a method using an amino acid-producing strain, the growth of which is improved by cell fusion, and, at the same time, lysE of a microorganism belonging to the genus Corynebacterium, which has the ability to export L-lysine, has also been shown to export the same basic amino acid L-arginine (Bellmann A et al, Microbiology, 147:1765-1774, 2001).

Disclosure of Invention

[ problem ] to

The present inventors have made extensive efforts to develop variants of export proteins capable of further improving productivity by enhancing the ability to export an ornithine product, and as a result, confirmed that the ability to export an ornithine-based product is enhanced when a modification is introduced at a specific site of the amino acid sequence of NCgl2522 protein. Therefore, they have found that putrescine or arginine, which is an ornithine-based product, can be produced in high yield by introducing a protein variant into a putrescine or arginine producing strain, thereby completing the present invention.

[ solution ]

It is an object of the present disclosure to provide novel polypeptides having the ability to export an ornithine-based product.

It is another object of the present disclosure to provide polynucleotides encoding the polypeptides, and vectors comprising the polynucleotides.

It is a further object of the present disclosure to provide a microorganism producing an ornithine based product comprising or having enhanced activity of a polypeptide.

It is yet another object of the present disclosure to provide a method for producing an ornithine-based product comprising:

(i) culturing a microorganism of the genus corynebacterium that produces an ornithine-based product in a culture medium; and

(ii) recovering an ornithine-based product from the microorganism or culture medium obtained as described above.

[ advantageous effects ]

The polypeptide having the ability to export an ornithine-based product of the present disclosure shows excellent activity of exporting the ornithine-based product, and thus, when such activity is introduced into a microorganism producing the ornithine-based product, the ability to produce the ornithine-based product can be further improved.

Detailed Description

The present disclosure will be described in detail below. Meanwhile, the descriptions and embodiments disclosed in the present disclosure may be applied to other descriptions and embodiments, respectively. That is, all combinations of the various elements disclosed herein are within the scope of the present disclosure. In addition, the scope of the present disclosure should not be limited by the specific description set forth below.

To achieve the above objects, one aspect of the present disclosure provides a novel polypeptide having the ability to export an ornithine-based product, in which the 77 th glycine residue from the N-terminus of the amino acid sequence of the ornithine-based product export protein is substituted with another amino acid.

As used herein, an ornithine-based product export protein refers to a protein that functions in the extracellular export of a product biosynthesized from ornithine as a precursor, and specifically refers to a protein that functions in the extracellular export of putrescine or arginine. More specifically, it may be NCgl2522 protein disclosed in korean patent application laid-open No. 2014-0115244. For example, the NCgl2522 protein may consist of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, but may include any sequence having the same activity as the protein without limitation, and the sequence information thereof may be obtained from GenBank of the known database NCBI.

The novel polypeptide having the ability to export an ornithine-based product of the present disclosure has a feature in which the glycine residue at position 77 of the N-terminus of the amino acid sequence of the protein exported from the ornithine-based product is substituted with other amino acids, and thus has an improved ability to export an ornithine-based product compared to a non-modified polypeptide (specifically, a polypeptide having a glycine residue at position 77). The polypeptide having the ability to export an ornithine-based product may be, for example, those in which glycine at position 77 in the amino acid sequence of the ornithine-based product export protein is replaced with alanine or arginine. Specifically, the polypeptide may be a polypeptide consisting of the amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO:6, or an amino acid sequence having homology or identity with 70% or more, 80% or more, specifically 85% or more, more specifically 90% or more, even more specifically 95% or more, and even more specifically 99% or more thereof (as long as it has the ability to export an ornithine-based product by substituting glycine at position 77 with other amino acid), but is not limited thereto. In addition, it should be construed that, as an amino acid sequence having such homology or identity, an amino acid sequence in which a partial sequence is deleted, modified, substituted or added falls within the scope of the present disclosure as long as the amino acid sequence has a biological activity substantially the same as or corresponding to that of a polypeptide consisting of an amino acid sequence of any one of SEQ ID NO:3 to SEQ ID NO: 6.

As used herein, the term "ornithine-based product" refers to a substance that can be biosynthesized from ornithine as a precursor. Specifically, examples of the substance that can be produced by ornithine cycle include putrescine, citrulline, proline and arginine, but the substance is not limited thereto as long as it can be biosynthesized from ornithine as a precursor. For example, the ornithine based product may be putrescine and arginine. In addition, any substance that can be synthesized from ornithine as a precursor and exported by a novel polypeptide (having the ability to export an ornithine-based product of the present disclosure) can be included without limitation.

Another aspect of the disclosure provides polynucleotides encoding polypeptides having the ability to export an ornithine-based product.

The polynucleotide may include a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs 3 to 6, or a polypeptide having homology or identity with 70% or more, 80% or more, specifically 85% or more, more specifically 90% or more, even more specifically 95% or more, and even more specifically 99% or more thereof, but is not limited thereto as long as it has activity similar to that of the polypeptide (having the ability to output an ornithine-based product). In addition, it is apparent that, due to codon degeneracy, a polynucleotide capable of being translated into a protein consisting of the amino acid sequence of SEQ ID NO. 1 or a protein having homology or identity thereto may also be included. Alternatively, probes that can be prepared from known gene sequences, such as any sequence that hybridizes to a sequence complementary to all or part of a nucleotide sequence under stringent conditions to encode a protein having the activity of the protein consisting of the amino acid sequence of SEQ ID NO:1, may be included without limitation.

"stringent conditions" refers to conditions that allow specific hybridization between polynucleotides, which are specifically disclosed in the literature (e.g., J.Sambrook et al.) stringent conditions may include conditions in which genes having high homology or identity (e.g., homology or identity of 80% or more, 85% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and even more specifically 99% or more) hybridize with each other, while genes having homology or identity lower than the above homology or identity do not hybridize with each other, or may include conventional washing conditions as Southern hybridization (i.e., washing once, specifically twice or three times corresponding to salt concentrations and temperatures of 1 × SSC and 0.1% SSC, specifically 60 ℃, 0.1 × and 0.1% SDS, more specifically 68 ℃, 0.1 × SSC and 0.1% SDS).

Although mismatches between bases are possible depending on the stringency of hybridization, hybridization requires that two nucleotides have complementary sequences. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the disclosure may also include isolated nucleic acid fragments that are complementary to the entire sequence as well as nucleic acid sequences substantially similar thereto.

Specifically, under the above conditions, the inclusion at T may be used_mThe hybridization conditions of the hybridization step at 55 ℃ are used to detect polynucleotides having homology. In addition, T_mThe value may be 60 ℃, 63 ℃, or 65 ℃, but is not limited thereto, and may be appropriately controlled by those skilled in the art according to the purpose thereof.

The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (Sambrook et al, supra, 9.50 to 9.51, 11.7 to 11.8).

As used herein, the term "homology" refers to the degree of identity between two given amino acid sequences or nucleotide sequences, and can be expressed as a percentage. In the present disclosure, homologous sequences having an activity identical or similar to the activity of a given amino acid sequence or nucleotide sequence may be represented by the term "% homology".

As used herein, the term "identity" refers to the degree of correlation between two given amino acid or nucleotide sequences, in some cases, identity is determined by the identity between strings of such sequences, for example, standard software for calculating parameters such as score, identity, and similarity (specifically, B L AST 2.0) or can be confirmed by comparing sequences via Southern hybridization experiments under defined stringent conditions (and the defined appropriate hybridization conditions are within the relevant technical field), and can be determined by methods well known to those skilled in the art (e.g., J.Sambrook et al, Molecular Cloning, A L analysis Manual,2 Edition, Cold Spring Harbor L analysis press, Aud Spring Harbor, New York, 1989; F.M.subel et al, Curr protocols in Molecular Biology, John & Yoy, Inc.).

The terms "homology" and "identity" are often used interchangeably with each other.

Sequence homology or identity for conserved polynucleotides or polypeptides can be determined by standard alignment algorithms and can be used with default gap penalties (default gap penalties) established by the program being used. Substantially homologous or identical polynucleotides or polypeptides are generally expected to hybridize to all or at least about 50%, about 60%, about 70%, about 80%, or about 90% of the full length of the polynucleotide or polypeptide of interest under medium or high stringency conditions. Polynucleotides containing degenerate codons instead of codons are also contemplated in the hybrid polynucleotides.

Whether any two polynucleotide or polypeptide sequences have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity to each other can be determined by known computer algorithms (such as The "FASTA" program) (e.g., Pearson ET al, (1988) Proc. Natl. Acad. Sci. USA 85:2444) using default parameters alternatively, The homology can be determined by using The EMBOSS package (EMBOSS: The European molecular Biology Open Sovie Suite, Rice ET al 2000, Trends Genet.16: 276. 277) (preferably version 5.0.0 or later) (GCG Package (Devereux, J. ET al, Nucleic Acids Research 34: 387 (1984)) performed by The New Schedule-coding program (American Association, USA: 94: USA; USA: 94: USA: Biocoding [ ASE ] No. 5: 94: USA ] using The biological Information [ ASE ] 94: 94, USA ] No. 94: USA # 12: USA ] algorithm [ ASE ] homology: FAS ] and No. 5: 94: USA # 12 [ ASE ] using The homology: 94: FAS # 12, USA ] algorithm [ 12, USA ] homology, USA # 12, USA ] and 76, USA [ 12 ] algorithms [ 12 ] homology [ 12, USA ] homology, USA ] algorithms (USA ] homology, USA ] and 76, USA ] algorithms [ 12, USA ] homology [ 12, USA ] algorithms [ 12, USA ] and 76, USA # algorithms (see [ 12 [ 17 [ 12, USA ] homology [.

The homology or identity of a polynucleotide or polypeptide can be determined by comparing the sequence information using, for example, the GAP computer program (e.g., Needleman et al, (1970), J Mol biol.48:443) as disclosed in Smith and Waterman, adv.Appl.Math (1981)2: 482). In summary, the GAP program defines homology or identity as the number of similar aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program may include: (1) unary comparison matrices (containing identity values Of 1 And non-identity values Of 0) And weighted comparison matrices such as Gribskov et al, (1986), Nucl. acids Res.14:6745 as disclosed in Schwartz And Dayhoff, eds., Atlas Of Protein Sequence And Structure, National biological research Foundation, pp.353-358 (1979); (2) a penalty of 3.0 per gap and an additional 0.10 penalty per symbol per gap (or a gap open penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used herein, the term "homology" or "identity" refers to a comparison between polypeptides or polynucleotides.

Yet another aspect of the invention provides a vector comprising a polynucleotide.

As used herein, the term "vector" refers to a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a polypeptide of interest, operably linked to suitable regulatory sequences, for expression of the polypeptide of interest in a suitable host. Regulatory sequences may include a promoter capable of initiating transcription, any operator sequence used to control transcription, sequences encoding suitable mRNA ribosome binding domains, and sequences which regulate termination of transcription and translation. After transformation into a suitable host cell, the vector may replicate or function independently of the host genome, and may be integrated into the host genome itself.

Examples of the vectors used in the present disclosure may include natural or recombinant plasmids, cosmids, viruses, and phages, for example, as phage vectors or cosmid vectors, pWE15, M13, MB L, MB L, ixi, ASHII, APII, t10, t11, Charon4A, Charon21A, and the like may be used, and as plasmid vectors, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pC L-based, pET-based, and the like may be used.

In embodiments, a polynucleotide encoding a polypeptide of interest in a chromosome may be replaced with a modified polynucleotide by a vector for chromosomal insertion in a cell. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art (e.g., homologous recombination), but is not limited thereto.

As used herein, the term "transformation" refers to the introduction of a vector comprising a polynucleotide encoding a polypeptide of interest into a host cell, such that the polynucleotideA process by which a polypeptide encoded by a nucleotide can be expressed in a host cell. It does not matter whether it is inserted into and located in the chromosome of the host cell or is located extrachromosomally, as long as the transformed polynucleotide can be expressed in the host cell, and both cases may be included. For example, electroporation, calcium phosphate (CaPO), can be performed₄) Precipitate, calcium chloride (CaCl)₂) Precipitation, microinjection, polyethylene glycol (PEG) technique, DEAE-dextran technique, cationic liposome technique, lithium acetate-DMSO technique, etc., but the method is not limited thereto. In addition, polynucleotides include DNA and RNA that encode a polypeptide of interest. The polynucleotide may be introduced in any form as long as it can be introduced into and expressed in a host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct that includes all the elements necessary for autonomous expression. The expression cassette can conventionally include a promoter, a terminator, a ribosome binding domain, and a stop codon operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. Alternatively, the polynucleotide may be introduced into the host cell as such and operably linked to the sequences necessary for its expression in the host cell, but is not limited thereto.

Further, as used above, the term "operably linked" refers to a functional linkage between the above-described gene sequences and the promoter sequence that initiates and mediates transcription of a polynucleotide encoding a polypeptide of interest of the present disclosure.

Yet another aspect of the present disclosure provides a microorganism producing an ornithine-based product, comprising a polypeptide having the ability to export the ornithine-based product or having enhanced activity thereof.

In particular, the present disclosure provides microorganisms of the genus corynebacterium that produce putrescine or arginine, including polypeptides having the ability to export an ornithine-based product or having enhanced activity thereof.

As used herein, the term "microorganism" includes all wild-type microorganisms, or naturally or artificially genetically modified microorganisms, and it may be microorganisms in which a specific mechanism is attenuated or enhanced due to insertion of an exogenous gene, or enhancement or attenuation of activity of an endogenous gene.

As used herein, the term "microorganism of the genus Corynebacterium" may specifically be Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), Brevibacterium lactofermentum (Brevibacterium lactofermentum), Brevibacterium flavum (Brevibacterium flavum), Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes), Corynebacterium valium (Corynebacterium efficiens), and the like, but is not limited thereto. More specifically, the microorganism of the genus corynebacterium of the present disclosure may be corynebacterium glutamicum, the cell growth and survival of which are hardly affected even when exposed to high concentrations of putrescine or arginine.

As used herein, the term "microorganism of the genus corynebacterium that produces an ornithine-based product" refers to a microorganism of the genus corynebacterium that has the ability to produce an ornithine-based product, either naturally or via modification. The microorganism of the genus corynebacterium that produces the ornithine-based product may be, but is not particularly limited to, a microorganism modified so that an activity of at least one selected from the group consisting of: for example, acetylglutamate synthase (agj) which converts glutamate into N-acetylglutamate, or ornithine acetyltransferase (ArgJ) which converts N-acetylornithine into ornithine, acetylglutamate kinase (ArgB) which converts N-acetylglutamate into N-acetylglutamyl phosphate, acetyl- γ -glutamyl-phosphate reductase (ArgC) which converts N-acetylglutamyl phosphate into N-acetylglutamate semialdehyde, and acetylornithine transaminase (ArgD) which converts N-acetylglutamate semialdehyde into N-acetylornithine are used to enhance the biosynthetic pathway from glutamate to ornithine, thereby improving ornithine productivity.

The term "microorganism of the genus corynebacterium which produces putrescine or arginine" as used herein refers to a microorganism of the genus corynebacterium which has the ability to produce putrescine or arginine, either naturally or via modification. Microorganisms of the genus Corynebacterium produce arginine without producing putrescine, but the productivity of arginine is significantly low. Thus, as used herein, a microorganism of the genus Corynebacterium which produces putrescine or arginine refers to a native strain itself, or a microorganism of the genus Corynebacterium in which an exogenous gene involved in the production mechanism of putrescine or arginine, or the activity of an endogenous gene is enhanced or attenuated to have an increased production ability of putrescine or arginine.

In addition, the putrescine-producing microorganism may be a microorganism further modified such that the activity of at least one selected from the group consisting of: ornithine carbamoyltransferase (ArgF) involved in the synthesis of arginine from ornithine, protein involved in the export of glutamic acid, and acetyltransferase which acetylates putrescine; and/or may be a microorganism modified such that Ornithine Decarboxylase (ODC) activity is introduced.

Further, the arginine producing microorganism may be a microorganism further modified such that the activity of at least one selected from the group consisting of: ornithine carbamoyltransferase (ArgF), argininosuccinate synthase (argG), argininosuccinate lyase (argH), aspartate ammonia lyase, and aspartate aminotransferase, which are involved in the synthesis of arginine from ornithine.

As used herein, the term "enhancement" of the activity of a protein means that the activity of the protein is introduced, or enhanced, as compared to its endogenous activity. By "introduction" of activity is meant that the activity of a particular polypeptide not originally possessed by the microorganism is naturally or artificially expressed.

As used herein, the term "increase" in the activity of a protein as compared to its endogenous activity means that the activity of the protein is increased as compared to the endogenous activity of the protein possessed by the microorganism or as compared to the activity prior to transformation. "endogenous activity" means the activity of a specific protein originally possessed by a parent strain or unmodified microorganism prior to transformation thereof when the trait of the microorganism is altered by genetic modification due to natural or artificial factors, and it may be used interchangeably with the activity prior to transformation.

Specifically, the activity enhancement in the present disclosure may be performed by the following method, but the method is not limited thereto:

1) methods of increasing the copy number of a polynucleotide encoding a protein;

2) methods of modifying an expression regulatory sequence such that expression of the polynucleotide is increased;

3) a method of modifying a polynucleotide sequence on a chromosome so that the activity of the protein is enhanced;

4) a method of introducing an exogenous polynucleotide or a modified polynucleotide exhibiting the activity of a protein, wherein codons of the above-mentioned polynucleotide have been optimized; and

5) a method of modifying to enhance activity by a combination of the above methods.

The increase in the copy number of the polynucleotide in the above method 1) may be performed in a form in which the polynucleotide is operably linked to a vector, or by insertion into a chromosome of a host cell, but is not particularly limited thereto. Specifically, it can be performed by operably linking a polynucleotide encoding a protein of the present disclosure to a vector (which can replicate and function independently of a host cell), and introducing it into the host cell. Alternatively, it may be carried out by operably linking the polynucleotide to a vector capable of inserting the polynucleotide into the chromosome of the host cell and introducing it into the host cell, for use in a method of increasing the copy number of the polynucleotide in the chromosome of the host cell.

Next, the modification of the expression regulatory sequence in method 2) such that the expression of the polynucleotide is increased may be performed by deletion, insertion or non-conservative or conservative substitution of the nucleic acid sequence, or a combination thereof to induce the modification of the sequence, thereby further enhancing the activity of the expression regulatory sequence, or by substitution with a nucleic acid sequence having stronger activity, but is not particularly limited thereto. In addition, the expression regulatory sequence may include a promoter, an operator sequence, a sequence encoding a ribosome binding domain, a sequence regulating termination of transcription and translation, and the like, but is not particularly limited thereto.

Examples of the strong promoter include CJ7 promoter (korean patent No. 0620092 and international publication No. WO2006/065095), lysCP1 promoter (international publication No. WO2009/096689), EF-Tu promoter, groE L promoter, aceA or aceB promoter, etc., but the strong promoter is not limited thereto, further, the modification of the polynucleotide sequence on the chromosome in method 3) may be performed by inducing the modification of the expression regulatory sequence by deletion, insertion or non-conservative or conservative substitution of the nucleic acid sequence, or a combination thereof, thereby further enhancing the activity of the polynucleotide sequence, or by replacing the polynucleotide sequence with the modified polynucleotide sequence to have stronger activity, but not particularly limited thereto.

In addition, the introduction of the exogenous polynucleotide sequence in method 4) may be performed by introducing an exogenous polynucleotide encoding a protein exhibiting the same or similar activity to the above-described protein, or a modified polynucleotide in which codons of the exogenous polynucleotide have been optimized, into a host cell. The exogenous polynucleotide may be used without limitation of its origin or sequence as long as it exhibits the same or similar activity as that of the protein. Further, the exogenous polynucleotide may be introduced into the host cell after its codon optimization, thereby achieving optimized transcription and translation in the host cell. The introduction can be performed by a person skilled in the art by selecting suitable transformation methods known in the art, and the protein can be produced when the introduced polynucleotide is expressed in a host cell, thereby increasing its activity.

Finally, the modification method of the method 5) for enhancing the activity by the combination of the methods 1) to 4) may be performed by applying at least one of the following methods in combination: increasing the copy number of a polynucleotide encoding a protein, modifying an expression regulatory sequence such that expression of the polynucleotide is increased, modifying a polynucleotide sequence on a chromosome, and modifying an exogenous polynucleotide displaying the activity of the protein or a codon-optimized modified polynucleotide thereof.

As used herein, the term "attenuation" of the activity of a protein includes a reduction in activity or the complete absence of activity as compared to its endogenous activity.

The reduction of protein activity can be achieved by various methods known in the art. Examples of methods include: a method of deleting part or all of a gene encoding a protein on a chromosome (including a case where the activity of the protein is eliminated); a method of reducing the enzyme activity by replacing a gene encoding a protein on a chromosome with a mutated gene; a method of introducing a modification into an expression regulatory sequence of a gene encoding a protein on a chromosome; a method of replacing an expression regulatory sequence of a gene encoding a protein with a sequence having weak activity or no activity (for example, a method of replacing a promoter of a gene with a promoter weaker than an endogenous promoter); a method of deleting a part or all of a gene encoding a protein on a chromosome; a method of introducing an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to a gene transcript on a chromosome to inhibit translation of mRNA into protein; artificially adding a sequence complementary to the upstream of the SD sequence of the gene encoding the enzyme to form a secondary structure, thereby making the attachment of the ribosome impossible; and a Reverse Transcription Engineering (RTE) method in which a promoter is added to the 3' end of an Open Reading Frame (ORF) of the corresponding sequence so as to be reverse transcribed, or a combination thereof, but is not particularly limited thereto.

Specifically, the method of deleting part or all of a gene encoding a protein may be performed by replacing a polynucleotide encoding an endogenous target protein within a chromosome with a polynucleotide having a partially deleted nucleic acid sequence or a marker gene by a vector for inserting the chromosome into a microorganism. In embodiments, a method of deleting a gene by homologous recombination may be used, but is not limited thereto. In addition, as used herein, although the term "portion" may vary depending on the kind of polynucleotide and may be appropriately selected by those skilled in the art, it may specifically refer to 1 to 300 nucleotides, more specifically 1 to 100 nucleotides, and even more specifically 1 to 50 nucleotides, but is not particularly limited thereto.

In addition, the method of modifying an expression regulatory sequence may be performed by inducing a modification in the expression regulatory sequence by deletion, insertion, conservative or non-conservative substitution, or a combination thereof, thereby further decreasing the activity of the expression regulatory sequence; or by replacing the sequence with a nucleic acid sequence having a weaker activity. The expression regulatory sequence may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding domain, and a sequence regulating termination of transcription and translation.

Further, the method of modifying a gene sequence on a chromosome may be performed by inducing a modification in the gene sequence by deletion, insertion, conservative or non-conservative substitution, or a combination thereof, thereby further decreasing the activity of the protein; or by replacing the sequence with a gene sequence modified to have weaker activity or modified to have no activity at all, but is not limited thereto.

Yet another aspect of the present disclosure provides a method for producing an ornithine-based product comprising:

(i) culturing in a culture medium a microorganism that produces an ornithine-based product, the microorganism comprising a polypeptide having the ability to export the ornithine-based product or having enhanced activity thereof; and

(ii) recovering the ornithine-based product from the microorganism or culture medium obtained above.

In a specific embodiment, the present disclosure provides a method for producing putrescine, comprising:

(i) culturing in a culture medium a putrescine-producing microorganism comprising a polypeptide having the ability to export an ornithine-based product or having enhanced activity thereof; and

(ii) recovering putrescine from the microorganism or the culture medium obtained above.

In another embodiment, the present disclosure provides a method for producing L-arginine, comprising:

(i) culturing in a medium a microorganism producing L-arginine, the microorganism comprising a polypeptide having the ability to export an ornithine-based product or having enhanced activity thereof, and

(ii) l-arginine is recovered from the microorganism or the culture medium obtained above.

The microorganism having the ability to export an ornithine based product and/or producing an ornithine based product is as described above.

In the above method, the culture of the microorganism may be carried out by a known batch culture method, continuous culture method, fed-batch culture method, or the like, but is not particularly limited thereto. In particular, with respect to the culture conditions, the pH of the culture can be adjusted to a suitable pH (e.g., pH5 to 9, specifically pH6 to 8, and most specifically pH6.8) using a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid). In addition, oxygen or oxygen-containing gas mixtures can be injected into the culture to maintain aerobic conditions. The culture temperature may be maintained at 20 ℃ to 45 ℃, specifically 25 ℃ to 40 ℃, and the culture may be performed for about 10 hours to 160 hours, but the culture is not limited to the above. The putrescine produced by the culture may be secreted in the culture medium or may be maintained in the cell.

In addition, as a carbon source of the medium to be used, sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oils and fats (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), organic acids (e.g., acetic acid), and the like may be used alone or in combination, but are not limited thereto. As the nitrogen source, nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean powder, and urea), inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), and the like can be used alone or in combination, but not limited thereto. As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, corresponding sodium-containing salts thereof, and the like can be used alone or in combination, but are not limited thereto. In addition, the culture medium may contain necessary growth promoting substances, such as other metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, vitamins, and the like.

For example, centrifugation, filtration, anion exchange chromatography, crystallization, HP L C, and the like can be used, but are not limited thereto.