RU2817284C1 - Microorganism for producing putrescine and method of producing putrescine using same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a microorganism of the genus Corynebacterium, having the ability to produce putrescine, in which N-acetyltransferase activity, originating from strain of Corynebacterium, and activity of acetylornithine deacetylase (argE), originating from E. coli are introduced. Also disclosed is a method of producing putrescine using said microorganism, a composition for producing putrescine containing said microorganism, and use of said microorganism for producing putrescine.

EFFECT: invention enables to obtain putrescine in high output.

13 cl, 2 dwg, 11 tbl, 5 ex

Description

TECHNICAL FIELD

The present disclosure relates to a putrescine-producing microorganism and a method for producing putrescine using it.

BACKGROUND ART

Putrescine, which is the raw material for nylon, is produced mainly by chemical processes using petroleum compounds as raw materials. Specifically, putrescine is produced by adding hydrogen cyanide to acrylonitrile to produce succinonitrile, followed by hydrogenation. This chemical method is effective and economically feasible, but has the disadvantage of harming the environment. Therefore, due to increasing environmental regulations and the above, there is a need to produce alternative substances through biological routes.

In this regard, methods for obtaining high concentrations of putrescine through the transformation of E. coli and microorganisms of the genus Corynebacterium have been disclosed (Morris et al., J Biol. Chem. 241: 13, 3129-3135, 1996; WO 06/005603; WO 09/125924 ; Qian ZD et al., Biotechnol. Bioeng. 104: 4, 651-662, 2009; Schneider et al., Appl. Biotechnol. 88: 4, 859-868, 2010 Biotechnol. 91: 17-30, 2011). In addition, various methods for producing putrescine using microorganisms are known (US 13/992242, US 14/372000, US 14/373265, EP 2236613 B1 and US 8497098 B2).

Among the proteins associated with the putrescine biosynthetic pathway, argJ - bifunctional ornithine acetyltransferase/N-acetylglutamate synthase - is an enzyme that can carry out the reaction of two substances: acetyl-CoA and N-acetylornithine, and has functions as ornithine acetyltransferase (L-glutamate-N- acetyltransferase) and N-acetylglutamate synthase, and has the functions of both ornithine acetyltransferase and N-acetylglutamate synthase. The argJ enzyme may reduce byproduct levels and reduce the burden of putrescine production because one enzyme mediates two enzymatic reactions. However, argJ activity can be inhibited by intermediate metabolites such as ornithine (Sakanyan V, Petrosyan R, Lecocq M, Boyen A, Legrain C, Demarez M, Hallet J, Glansdorff N: Genes and enzymes of the acetyl cycle of arginine biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early steps of the arginine pathway. Microbiology 1996, 142:99-108), leading to reduced biosynthetic efficiency of putrescine.

N-acetyltransferase present in Corynebacterium glutamicum has the ability to produce N-acetyl-L-glutamate in the presence of acetyl-CoA and glutamate. However, this N-acetyltransferase has been reported to have at least 9.43 times higher specific activity than argJ (A new type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum. "BMC genomics 2013(14) p. 713).

DESCRIPTION OF THE INVENTION

Technical problem

The inventors of the present invention made intensive efforts to increase the production of putrescine in microorganisms and, as a result, confirmed that the introduction of N-acetyltransferase activity originating from a strain of the genus Corynebacterium and acetyl ornithine acetylase activity originating from E. coli into a microorganism of the genus Corynebacterium resulted in to increase the production of putrescine and thus completed the present invention.

Technical solution

According to one aspect of the present invention, there is provided a microorganism of the genus Corynebacterium having the ability to produce putrescine into which N-acetyltransferase activity derived from a strain of the genus Corynebacterium and acetylornithine deacetylase (argE) activity derived from E. coli are introduced.

According to another aspect of the present invention, there is provided a method for producing putrescine, comprising culturing in a medium a microorganism of the genus Corynebacterium having the ability to produce putrescine, into which an N-acetyltransferase activity originating from a strain of the genus Corynebacterium and an acetylornithine deacetylase (argE) activity originating from E. coli are introduced.

According to yet another aspect of the present disclosure, there is provided a composition for producing putrescine, comprising a microorganism of the genus Corynebacterium having the ability to produce putrescine, into which an N-acetyltransferase activity derived from a strain of the genus Corynebacterium and an acetylornithine deacetylase (argE) activity derived from E. coli are introduced.

Beneficial effects

A microorganism of the genus Corynebacterium having the ability to produce putrescine into which N-acetyltransferase activity originating from a strain of the genus Corynebacterium and acetylornithine deacetylase (argE) activity originating from E. coli are introduced according to the present invention can be cultivated to produce putrescine in high yield compared with an existing unmodified microorganism.

Brief description of graphic materials

In FIG. Figure 1 schematically shows the pathway for the biosynthesis of putrescine and ornithine in the microorganism of the genus Corynebacterium (A) and the pathway for the biosynthesis of putrescine and ornithine in E. coli (B).

In FIG. 2 schematically shows the biosynthesis pathway of putrescine and ornithine in a microorganism of the genus Corynebacterium with improved ability to produce putrescine and ornithine by enhancing Coryne Ncgl2644 and introducing E. coli argE.

Detailed Description of the Invention

The present invention will be specifically described as follows. Each description and embodiment in the present disclosure may also apply to other descriptions and embodiments. That is, all combinations of various elements in the present invention are included within the scope of the present invention. Moreover, the scope of the present invention is not limited to the specific description given below.

According to one aspect of the present invention, there is provided a microorganism of the genus Corynebacterium having the ability to produce putrescine into which N-acetyltransferase activity derived from a strain of the genus Corynebacterium and acetylornithine deacetylase (argE) activity derived from E. coli are introduced.

The term "putrescine" as used herein refers to the organic four-carbon diamine compound which has the molecular formula NH ₂ (CH ₂ ) ₄ NH ₂ and is also called 1,4-diaminobutane or butanediamine.

The term "N-acetyltransferase", as used here, refers to an enzyme (EC 2.3.1.5) that catalyzes the transfer of an acetyl group from acetyl-CoA to arylamine, arylhydroxylamine and arylhydrazine. This enzyme can catalyze the transfer of acetyl between arylamines without CoA and may have the ability to produce N-acetyl-L-glutamate in the presence of acetyl-CoA and glutamate. This enzyme is known to have broad specificity for aromatic amines.

In the present invention, the N-acetyltransferase may belong to, but is not limited to, the GCN5-related N-acetyltransferase (GNAT) family.

The N-acetyltransferase may have the amino acid sequence of SEQ ID NO: 1, consist of, but is not limited to the amino acid sequence of SEQ ID NO: 1, or contain the amino acid sequence as shown in SEQ ID NO: 1. The sequence of SEQ ID NO: 1 can be confirmed in the well-known NCBI (US National Center for Biotechnology Information) GenBank database.

In particular, the N-acetyltransferase may have an amino acid sequence of SEQ ID NO: 1 and/or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or greater homology or identity with SEQ ID NO: 1. It is also apparent that even an N-acetyltransferase having an amino acid sequence with a deletion, modification, substitution or addition in part thereof may be within the scope of the present invention, provided that the amino acid sequence has such homology or identity and exhibits function corresponding to N-acetyltransferase. That is, notwithstanding the description as “a protein or polypeptide containing an amino acid sequence as shown in a particular sequence number” or “a protein or polypeptide consisting of an amino acid sequence as shown in a particular sequence number” in the present invention, any protein consisting of amino acid sequence with a deletion, modification, substitution or addition in part thereof may also be used in the present invention, provided that the protein has an activity identical to or corresponding to that of the polypeptide consisting of the amino acid sequence of the corresponding sequence number. For example, it will be appreciated that any polypeptide that has an activity identical to or corresponding to "a polypeptide containing the amino acid sequence of SEQ ID NO: 1" may fall within the scope of "a polypeptide containing the amino acid sequence of SEQ ID NO: 1".

In the present disclosure, the gene encoding the N-acetyltransferase may be derived from a strain of the genus Corynebacterium, and in particular may be derived from Corynebacterium glutamicum, but any strain of the genus Corynebacterium that can express the gene encoding the N-acetyltransferase is not particularly limited. The gene may contain a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, and more specifically may contain, but is not limited to, the nucleotide sequence of SEQ ID NO: 2. The nucleotide sequence of SEQ ID NO: 2 can be obtained from the known GenBank database and is named Ncgl2644. The term “gene containing the nucleotide sequence of SEQ ID NO: 2” may be used interchangeably with the term “polynucleotide containing the nucleotide sequence of SEQ ID NO: 2”, or “gene or polynucleotide having the nucleotide sequence of SEQ ID NO: 2”, or “gene or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2."

The term "polynucleotide" as used herein refers to a polymer of nucleotides extended in length by the covalent bonding of nucleotide monomers and, generally, to a strand of DNA or RNA of a specified length and, more specifically, may mean a polynucleotide fragment encoding a variant . A polynucleotide may be described as a gene when the polynucleotide is a collection of polynucleotides capable of performing functions. In the present invention, the terms "polynucleotides" and "genes" may be used interchangeably.

In particular, due to codon degeneracy or by considering the codons preferred by the microorganism in which the polypeptide can be expressed, the polynucleotide of the present invention may be variously modified in its coding region within a range in which the amino acid sequence of the polypeptide is not changed. In particular, any polynucleotide sequence that encodes an N-acetyltransferase consisting of the amino acid sequence of SEQ ID NO: 1 may be included, without limitation.

In addition, any probe that can be derived from a known gene sequence may be included, for example, without limitation, any sequence that can hybridize with a complementary sequence to part or all of a nucleotide sequence under stringent conditions may be included to encode an N-acetyltransferase consisting of an amino acid sequence SEQ ID NO: 1. The term “stringent conditions” refers to conditions that allow specific hybridization of polynucleotides. Such conditions are well known in the art. For example, these conditions may include conditions in which genes having high homology or identity, such as genes having at least 40%, in particular at least 90%, more particularly at least 95% -th, even more specifically, at least 97% and even more specifically, at least 99% homology or identity, hybridize with each other, and genes having less homology or identity do not hybridize with each other; or typical washing conditions for Southern hybridization, i.e. conditions where the washing is carried out once, more specifically two or three times, with a salt concentration and temperature corresponding to 60°C, 1xSSC (sodium chloride and citrate solution), 0.1% SDS (sodium dodecyl sulfate), specifically at 60°C, 0.1×SSC and 0.1% SDS, and more specifically at 68°C, 0.1×SSC, 0.1% SDS.

Hybridization requires that two nucleic acids have complementary sequences, although there may be mismatches between nucleotides, depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in relation to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the present invention may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments complementary to the entire sequence.

In particular, polynucleotides having homology or identity can be detected using the hybridization conditions described above, including a hybridization step at a T _m value of 55°C. In addition, the T _m value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be suitably adjusted by one skilled in the art according to the purpose.

The appropriate degree of stringency for polynucleotide hybridization may depend on the lengths of the polynucleotides and the degree of complementarity of the polynucleotides, and its parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

The term "homology" or "identity" as used herein refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.

The homology or identity of conserved polynucleotides or polypeptides can be determined using standard alignment algorithms and can share default gap penalties set by the program being used. Substantially homologous or identical sequences can generally hybridize to each other, in whole or in part, under moderate to very stringent conditions. Obviously, hybridization also includes hybridization with a polynucleotide containing regular codons or codons taking into account the degeneracy of codons in the polynucleotide.

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

Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, for example Needleman et al., (1970), J Mol Biol. 48:443, as reported in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines similarity as the number of aligned characters (i.e., nucleotides or amino acids) that are similar divided by the total number of characters in the shorter of the two sequences. Default parameters for the GAP program may include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and a weighted comparison matrix Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or EDNAFULL substitution matrix (EMBOSS NUC4.4 NCBI version); (2 ) a penalty of 3.0 for each space and an additional penalty of 0.10 for each character in each space (or a space opening penalty of 10 and a space expansion penalty of 0.5 and (3) no penalty for trailing spaces);

In one embodiment of the present invention, a variant of the present invention may have phytoene desaturase activity. In addition, a variant of the present invention may have the activity of increasing the ability to produce 5'-inosine monophosphate (IMP) compared to a wild-type polypeptide having phytoene desaturase activity.

The term "phytoene desaturase", as used here, is a polypeptide that converts colorless 15-cis-phytoene to light red lycopene in a biochemical pathway called the poly-trans pathway. In particular, the term "phytoene desaturase of the present invention" may be used interchangeably with phytoene desaturase or Crt1. In the present invention, the sequence of phytoene desaturase can be obtained from the known NCBI GenBank database. In particular, the phytoene desaturase may be, but is not limited to, a polypeptide having the activity of a phytoene desaturase encoded by crtl.

The term "acetylornithine deacetylase" as used herein refers to an enzyme (EC 3.5.1.16) that mediates the reaction involved in the production of acetic acid and ornithine by mediating the hydrolysis of acetylornithine. In the present invention, acetyl op nitinde acetylase may be argE, but is not limited to it.

argE may have the amino acid sequence of SEQ ID NO: 3, consist of, but is not limited to, the amino acid sequence of SEQ ID NO: 3, or contain the amino acid sequence as shown in SEQ ID NO: 3. The sequence of SEQ ID NO: 3 can be confirmed in the known GenBank NCBI database.

In the present disclosure, the gene encoding argE may be derived from bacteria, and in particular may be derived from E. coli, but any strain that can express the gene encoding argE is not particularly limited. The gene may contain, but is not limited to, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, and more specifically may contain the nucleotide sequence of SEQ ID NO: 4. The nucleotide sequence of SEQ ID NO: 4 can be obtained from the known GenBank database.

In the present disclosure, a microorganism of the genus Corynebacterium having the ability to produce putrescine may have an attenuated activity of bifunctional ornithine acetyltransferase/N-acetylglutamate synthase (argJ) derived from a strain of the genus Corynebacterium, but is not limited to this.

The term "bifunctional ornithine acetyltransferase/N-acetylglutamate synthase" as used herein refers to an enzyme that can carry out the conversion reactions of two substances: acetyl-CoA and N-acetylornithine, and this enzyme has functions as ornithine acetyltransferase (L-glutamate-N-acetyltransferase (EC 2.3.1.35)) and N-acetylglutamate synthase (EC 2.3.1.1). Ornithine acetyltransferase converts N2-acetyl-L-ornithine and L-glutamate to L-ornithine, and N-acetylglutamate synthase catalyzes the formation of N-acetylglutamate from glutamate and acetyl-CoA. In the present invention, the bifunctional ornithine acetyltransferase/N-acetylglutamate synthase may be, but is not limited to, argJ.

argJ may reduce the level of byproducts and reduce the burden of putrescine production, since one enzyme mediates two enzymatic reactions. However, argJ activity can be inhibited by metabolic intermediates such as ornithine, resulting in reduced efficiency of putrescine biosynthesis.

argJ may have the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7, consist of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7, or contain the amino acid sequence AS as shown in SEQ ID NO: 5 or SEQ ID NO: 7, but not limited to this. The sequence of SEQ ID NO: 5 or SEQ ID NO: 7 can be confirmed in the known GenBank NCBI database.

In the present disclosure, the gene encoding argJ may be derived from a strain of the genus Corynebacterium, and in particular may be obtained from Corynebacterium glutamicum, but any strain of the genus Corynebacterium that can express the gene encoding argJ is not particularly limited. In particular, the gene encoding the amino acid sequence of SEQ ID NO: 5 may be derived from, but is not limited to, Corynebacterium glutamicum ATCC13869. The gene may comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5, and more specifically may contain, but is not limited to, the nucleotide sequence of SEQ ID NO: 6. The nucleotide sequence of SEQ ID NO: 6 can be obtained from the known GenBank database.

In addition, the gene encoding the amino acid sequence of SEQ ID NO: 7 may be derived from, but is not limited to, Corynebacterium glutamicum ATCC13032. The gene may contain, but is not limited to, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7, and more specifically may contain the nucleotide sequence of SEQ ID NO: 8. The nucleotide sequence of SEQ ID NO: 8 can be obtained from the known GenBank database.

The term “microorganism having the ability to produce putrescine,” as used herein, refers to a microorganism that has the natural ability to produce putrescine, or to a microorganism obtained by endowing a parent strain that does not have the ability to produce putrescine with the ability to produce putrescine. In particular, the microorganism is a putrescine-producing microorganism into which N-acetyltransferase activity derived from a strain of the genus Corynebacterium and argE activity derived from E. coli are introduced, but is not limited to them.

In particular, “putrescine-producing microorganism” includes all of wild-type microorganisms or naturally occurring or artificially genetically modified microorganisms. More specifically, a putrescine-producing microorganism may be a microorganism in which a specific mechanism is weakened or enhanced due to the insertion of an exogenous gene or due to the enhancement or attenuation of the activity of its own gene, where the microorganism may have a genetic mutation or an enhanced ability to produce putrescine to produce target putrescine.

In particular, the term "introducing" an activity means that a gene not originally possessed by the microorganism is expressed in the microorganism and, thus, the microorganism exhibits the activity of a specific protein or exhibits increased or enhanced activity of the corresponding protein compared to its own activity or activity before modifications. For example, the term may indicate that a polynucleotide encoding a particular protein is introduced into the chromosome of a microorganism, or a vector containing a polynucleotide encoding a particular protein is introduced into a microorganism thereby demonstrating the activity of that particular protein.

The term "enhancing" the activity of a polypeptide, as used herein, means that the activity of the polypeptide is increased relative to its own activity. The term "amplification" can be used interchangeably with terms such as "activation", "up-regulation", "overexpression" and "amplification". In particular, “activation,” “amplification,” “upregulation,” “overexpression,” and “increase” may include both demonstrating activity that the polypeptide did not originally possess or demonstrating improved activity over its own activity or activity before modification. The term "intrinsic activity" means the activity of a particular polypeptide that was originally possessed by the parent strain before the trait changed, or by an unmodified microorganism when the trait is changed by genetic variation due to natural or artificial factors. This term may be used interchangeably with the term activity before modification. The fact that the activity of a polypeptide is “enhanced,” “up-regulated,” “overexpressed,” or “increased” relative to its own activity means that the activity of the polypeptide is improved relative to the activity and/or concentration (level of expression) of the particular polypeptide that the original one was possessed by the parent strain before the change in the trait or by an unmodified microorganism.

Enhancement can be achieved by introducing an exogenous polypeptide or increasing the activity and/or concentration (expression level) of an endogenous polypeptide. Increased activity of a polypeptide can be tested by increasing the activity level or expression level of the corresponding polypeptide, or by increasing the amount of product produced from the corresponding polypeptide.

Various methods well known in the art can be used to enhance the activity of a polypeptide, and the method is not limited as long as the activity of the target polypeptide can be enhanced relative to the microorganism prior to modification. In particular, genetic engineering and/or protein engineering, well known to those skilled in the art, which are traditional methods of molecular biology, can be used, but the method is not limited to them (for example, Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology . 2010, Vol. 2. 1-16; Sambrook et al., Molecular Cloning 2012 and the like).

In particular, increased activity of a polypeptide of the present invention may be indicated by:

1) an increase in the number of intracellular copies of the polynucleotide encoding this polypeptide;

2) replacement of the expression control region of the gene encoding a given polypeptide on the chromosome with a sequence having strong activity;

3) modification of the nucleotide sequence encoding the initiation codon or the 5'-UTR (5'-untranslated region) region of the transcript of the gene encoding this polypeptide;

4) modification of the amino acid sequence of a polypeptide to enhance the activity of this polypeptide;

5) modification of a polynucleotide sequence encoding a given polypeptide to enhance the activity of a given polypeptide (for example, modification of a polynucleotide sequence of a polypeptide gene so as to encode a polypeptide modified to enhance the activity of a given polypeptide);

6) introduction of an exogenous polypeptide demonstrating the activity of this polypeptide, or an exogenous polynucleotide encoding this polypeptide;

7) optimization of codons of the polynucleotide encoding this polypeptide;

8) modification or chemical modification of the exposed region selected by analysis of the tertiary structure of the polypeptide; or

9) a combination of two or more selected from paragraphs. (1) - (8), but is not specifically limited to this.

More specifically, the above points are described as follows.

Increasing the intracellular copy number of the polynucleotide encoding the polypeptide in (1) above can be achieved by introducing into a host cell a vector to which the polynucleotide encoding the corresponding polypeptide is operably linked and which can replicate and function independently of the host. Alternatively, expansion may be achieved by introducing one or more copies of a polynucleotide encoding the corresponding polypeptide into a chromosome in a host cell. Introduction into a chromosome can be accomplished by, but is not limited to, introducing into a host cell a vector capable of inserting a polynucleotide into a chromosome in the host cell. This vector is the same as described above.

Replacement on the chromosome of the expression control region of the gene encoding a given polypeptide with a sequence with strong activity in claim 2) may represent, for example, the occurrence of a modification in this sequence through deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with a sequence having stronger activity, so as to further enhance the activity of the expression control region. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a ribosome binding site coding sequence, a transcription and translation termination control sequence, and the like. For example, the replacement may be, but is not limited to, replacing the original promoter with a strong promoter.

Examples of known strong promoters may include CJ1-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 promoter (sm3) (US 10584338 B2), 02 promoter (US 10273491 B2), tkt promoter, uscA promoter and the like, but are not limited to them.

A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the transcript of the gene encoding the polypeptide in paragraph (3) may be, for example, a replacement with a nucleotide sequence encoding not the endogenous start codon, but another start codon that provides higher the rate of expression of the polypeptide, but is not limited to it.

Modification of the amino acid sequence or polynucleotide sequence in claims. (4) and (5) may be the occurrence of a modification in sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or in the polynucleotide sequence encoding the polypeptide, or substitution with an amino acid sequence or polynucleotide sequence so modified, to have greater activity, or an amino acid sequence or polynucleotide sequence modified to have increased activity, so as to enhance the activity of the polypeptide, but is not limited to them. In particular, the replacement may be accomplished by, but is not limited to, insertion of a polynucleotide into a chromosome via homologous recombination. The vector used may further include a selectable marker to verify insertion into the chromosome. The selectable marker is as described above.

The introduction of an exogenous polypeptide exhibiting the activity of the polypeptide in claim (6) may be the introduction into a host cell of an exogenous polynucleotide encoding a polypeptide exhibiting the same/similar activity to the polypeptide. The exogenous polynucleotide is not limited in its origin or sequence, provided that the exogenous polynucleotide exhibits identical/similar activity to the polynucleotide. Administration can be carried out by any known transformation method suitably selected by one skilled in the art, and the introduced polynucleotide is expressed in the host cell, and thus a polypeptide is produced and its activity can be enhanced.

The codon optimization of the polynucleotide encoding the polypeptide in (7) may be a codon optimization of the endogenous polynucleotide so as to enhance its transcription or translation in the host cell, or a codon optimization of the exogenous polynucleotide so as to achieve optimized transcription or translation in the host cell .

The modification or chemical modification of the exposed region selected by analyzing the tertiary structure of the polypeptide in claim (8) may be, for example, modification or chemical modification of the exposed region to be modified or chemically modified by comparing the sequence information of the polypeptide to be analyzed with a database that stores sequence information of known proteins to determine a template protein candidate according to sequence similarity and identify a structure based on the identified candidate.

Such an increase in the activity of a polypeptide may mean that the activity or concentration or level of expression of the corresponding polypeptide is increased relative to the activity or concentration of the polypeptide expressed in the wild-type microbial strain or the microbial strain before modification; or that the amount of product derived from the corresponding polypeptide increases, but is not limited to this.

Modification of part or all of a polynucleotide in a microorganism of the present invention can be induced by: (a) homologous recombination using a vector for insertion into a chromosome in a microorganism or genome editing using a genetically modified nuclease (eg CRISPR-Cas9) and/or (b) light treatment such as, but is not limited to, ultraviolet light and radiation, and/or chemicals. The method of modifying part or all of a gene may include a method using recombinant DNA technology. For example, by introducing a nucleotide sequence or a vector containing a nucleotide sequence homologous to a target gene into a microorganism to cause homologous recombination, part or all of the gene can be deleted. The introduced nucleotide sequence or vector may contain, but is not limited to, a dominant selection marker.

Such an increase in protein activity may mean that the activity or concentration of the corresponding protein is increased relative to, but is not limited to, the activity or concentration of the protein expressed in the wild-type microbial strain or in the microbial strain before modification. The term "pre-modification strain" or "pre-modification microorganism" as used herein does not exclude a strain containing a mutation that may occur naturally in the microorganism and refers to the native strain itself or the strain before a change in trait due to a genetic mutation. caused by artificial factors. In the present invention, the change in trait may be the introduction of N-acetyltransferase activity originating from a strain of the genus Corynebacterium and argE activity originating from E. coli. The terms "strain before modification" or "microorganism before modification" may be used interchangeably with the terms "unmutated strain", "unmodified strain", "unmutated microorganism", "unmodified microorganism" or "reference microorganism".

In the present invention, the reference microorganism is not particularly limited, as long as the reference microorganism produces putrescine, and a mutated strain having an increased ability to produce putrescine compared with the wild-type microorganism is also included without limitation. Examples thereof may include, but are not limited to, wild-type Corynebacterium glutamicum ATCC13032 wild-type strain, wild-type Corynebacterium glutamicum ATCC13869 strain, or strains in which one or more than one genetic modification is added to the above strains so as to enhance the putrescine biosynthetic pathway.

The one or more than one genetic modification may be, for example, any one or more than one genetic modification selected from: enhancing a gene in the putrescine biosynthetic pathway; overexpression of putrescine operon activity; improving the supply and efficacy of the putrescine precursor; improvement of putrescine export; and attenuation of, but not limited to, the activity of a gene in a competitive pathway, a regulator in the directional pathway of the putrescine operon, or a putrescine importer gene, and putrescine importer and lysis genes.

Genetic modification of gene enhancement in the putrescine biosynthetic pathway may be, for example, but is not limited to, introducing a gene (speC) encoding ornithine decarboxylase (ODC) derived from wild type E. coli W3110 into the chromosome.

Genetic modification of the overexpression activity of the putrescine operon can be, for example, but is not limited to, replacing the promoter of the argCJBD gene cluster encoding enzymes involved in the synthesis of ornithine from glutamate.

Genetic modification to improve putrescine export and attenuate the activity of a gene in a competing pathway, a regulator in the putrescine operon targeting pathway, or a putrescine importer gene and putrescine importer and lysis genes may be, for example, deletion of the gene (argF) encoding ornithine carbamoyltransferase and the gene (NCgl1221) , encoding a glutamate exporter involved in glutamate export on the chromosome; or inactivation of the activity of the protein NCgl1469, identified as, but not limited to, the histone acetyltransferase HPA2 and related acetyltransferase.

A strain having one or more than one genetic modification may be, for example, strain CC01-0064 having the ability to produce putrescine, in which: in ATCC 13032, a gene (argF) encoding an ornithine carbamoyltransferase and a gene (NCgl1221) encoding a glutamate exporter , involved in glutamate export, were deleted; a gene (speC) encoding ornithine decarboxylase (ODC) derived from wild-type E. coli W3110 was introduced into the chromosome; and the promoter of the gene cluster argCJBD, encoding enzymes involved in the synthesis of ornithine from glutamate (KCCM11138P, Korean patent publication No. 2012-0064046), or strain CC01-0063, in which the activity of the protein NCgl1469, defined as histone acetyltransferase HPA2, and related acetyltransferase in strain CC01-0064 (KCCM11240P, Korean patent publication No. 10-2013-0082478), but is not limited to them.

Alternatively, the putrescine-producing microorganism may have attenuated argJ activity derived from, but is not limited to, a strain of the genus Corynebacterium.

The term "attenuation" of a protein, as used herein, has a meaning encompassing all of a decrease in activity or lack of activity compared to its own activity. The term "attenuation" can be used interchangeably with inactivation, deficiency, down-regulation, reduction, decrease, attenuation, or the like.

Attenuation may also include: a case where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide possessed by the parent microorganism due to a mutation or the like of the polynucleotide encoding the polypeptide; the case when the entire activity and/or concentration (expression level) of the polypeptide in the cell is lower compared to the native strain due to inhibition of expression of the gene of the polynucleotide encoding this polypeptide, or inhibition of translation into the polypeptide; the case where polynucleotide expression is not achieved; and/or the case where the polypeptide has no activity despite expression of the polynucleotide. The term "intrinsic activity" refers to the activity of a particular polypeptide originally possessed by the parent strain before modification or by a wild-type microorganism or an unmodified microorganism when the trait is altered due to a genetic mutation caused by natural or man-made factors. This term can be used interchangeably with the term activity before modification. The terms "inactivation, deficiency, reduction, down-regulation, reduction or attenuation" of the activity of a polypeptide compared to its own activity mean that the activity of the polypeptide is reduced compared to the activity of the particular polypeptide originally possessed by the parent strain before transformation or by the unmodified microorganism.

Attenuation of the activity of a polypeptide can be accomplished by any method known in the art, without limitation, and can be achieved by using various methods well known in the art (eg, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing Int J Mol Sci 2014; 15(2):2773-2793 and Sambrook et al., Molecular Cloning 2012, etc.).

In the present disclosure, the protein that is the target of attenuation, i.e. the target protein may be, but is not limited to, argJ.

1) Deletion of part or all of the gene encoding a given polypeptide;

2) modifying the expression control region (or expression control sequence) so as to reduce the expression of the gene encoding the polypeptide;

3) modifying the amino acid sequence (eg, deletion/substitution/addition of at least one amino acid in a given amino acid sequence) constituting the polypeptide so as to eliminate or weaken the activity of the polypeptide;

4) modification of the sequence of a gene encoding a polynucleotide to eliminate or weaken the activity of the polypeptide (for example, deletion/replacement/addition of at least one nucleic acid nucleotide in the nucleic acid nucleotide sequence of the polypeptide gene so as to encode a polypeptide modified to eliminate or weaken the activity of the polypeptide );

5) modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the transcript of the gene encoding the polypeptide;

6) introduction of an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the transcript of the gene encoding the given polypeptide;

7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide, so as to form a secondary structure that makes it impossible for the ribosome to attach;

8) adding a promoter, which is subject to reverse transcription, to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

9) a combination of two or more than two selected from paragraphs. (1) - (8), but not specifically limited to them.

For example, they are described as follows.

Deletion of part or all of the gene encoding the polypeptide in paragraph (1) may be the elimination of the entire polynucleotide encoding the endogenous target protein in the chromosome, replacement with a polynucleotide with the deletion of some nucleotides, or replacement with a marker gene.

The modification of the expression control region (or expression control sequence) in (2) may be the occurrence of a mutation in the expression control region (or expression control sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or substitution with a sequence having more weak activity. The expression control region includes, but is not limited to, a promoter, an operator sequence, a ribosome binding site coding sequence, and a transcription and translation termination control sequence.

A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the transcript of the gene encoding the polypeptide in paragraph (5) may be, for example, a replacement with a nucleotide sequence encoding not the endogenous start codon, but another start codon having a higher rate polypeptide expression, but is not limited to this.

Modification of the amino acid sequence or polynucleotide sequence in claims. (4) and (5) may be the occurrence of a sequence modification by deletion, insertion, non-conservative or conservative substitution, or combination thereof, in the amino acid sequence of a polypeptide or polynucleotide sequence encoding the polypeptide, or substitution with an amino acid sequence or polynucleotide sequence modified so that have less activity, or an amino acid sequence or polynucleotide sequence modified to have less activity, so as to reduce the activity of the polypeptide, but is not limited to this. For example, gene expression can be inhibited or attenuated by introducing a mutation into the polynucleotide sequence to produce a stop codon.

With regard to the introduction of an antisense oligonucleotide (for example, antisense RNA), complementarily binding to the transcript of the gene encoding the polypeptide in paragraph (6), one can refer, for example, to the literature [Weintraub, N. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews -Trends in a Genetics, Vol. 1(1) 1986].

Adding a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide before the Shine-Dalgarno sequence so as to form a secondary structure that prevents the attachment of ribosomes in paragraph (7) may make translation of the mRNA impossible or reduce the rate of its translation.

The addition of a promoter that is subject to reverse transcription to the 3' end of the open reading frame (ORF) of the gene encoding the polypeptide (reverse transcription engineering, RTE) in paragraph (8) can make the antisense nucleotide complementary to the transcript of the gene encoding the polypeptide , thereby weakening the activity of the polypeptide.

The putrescine-producing microorganism may be any microorganism that can produce putrescine by introducing N-acetyltransferase activity originating from a strain of the genus Corynebacterium and argE activity originating from E. coli through the above-described method. For the purposes of the present invention, a putrescine-producing microorganism is a microorganism having an enhanced ability to produce the target putrescine, into which N-acetyltransferase activity originating from a strain of the genus Corynebacterium and argE activity originating from E. coli are introduced through the above-described method, and the activity argJ derived from a strain of the genus Corynebacterium is attenuated, and the microorganism may be, but is not limited to, a genetically modified microorganism or a recombinant microorganism. In the present disclosure, the term “putrescine-producing microorganism” may be used interchangeably with the term “putrescine-producing microorganism” or “microorganism having the ability to produce putrescine.”

Examples of this microorganism may include microorganisms belonging to the genus Corynebacterium, the genus Escherichia, the genus Enterbacter, the genus Erwinia, the genus Serratia, the genus Providencia and the genus Brevibacterium, and in particular, it may be a microorganism of the genus Corynebacterium.

More specifically, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium test udinoris, Corynebacterium flavescens or the like , and may be Corynebacterium glutamicum, and any microorganism belonging to the genus Corynebacterium may be included without limitation.

According to another aspect of the present invention, there is provided a method for producing putrescine, comprising cultivating in a medium a microorganism of the genus Corynebacterium having the ability to produce putrescine, comprising cultivating in a medium a microorganism of the genus Corynebacterium having the ability to produce putrescine, into which N-acetyltransferase activity derived from a strain of the genus Corynebacterium is introduced, and E. coli-derived acetylornithine deacetylase (argE) activity. N-acetyltransferase, acetylornithine deacetylase, putrescine and microorganism are as described above.

The Corynebacterium genus may have, but is not limited to, reduced bifunctional ornithine acetyltransferase/N-acetylglutamate synthase (argJ) activity derived from a Corynebacterium genus strain.

This method can be easily determined by one skilled in the art under optimized culture conditions and enzymatic activity conditions known in the art. In particular, the step of cultivating the microorganism is not particularly limited, but the step can be carried out by a known batch culture method, continuous culture method, fed-batch culture method or the like. The culture conditions may not be particularly limited, but adjustment to a suitable pH (e.g., pH 5 to pH 9, particularly pH 6 to pH 8, and most particularly pH 6.8) may be achieved by using an alkaline compound (e.g. sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg phosphoric acid or sulfuric acid), and aerobic conditions can be maintained by introducing oxygen or an oxygen-containing gas mixture into the culture. The culture temperature may be maintained at 20 to 45° C., and in particular 25 to 45° C., and the culture may be carried out for, but not limited to, about 10 to 160 hours. Putrescine produced by culture may be released into the medium or may remain in the cells.

In addition, in the culture medium to be used, sugars and carbohydrates (for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oils and fats (for example, soybean oil, sunflower oil, peanut butter and coconut oil), fatty acids (eg palmitic acid, stearic acid and linoleic acid), alcohols (eg glycerin and ethanol), organic acids (eg acetic acid) and the like, alone or in combination, but the carbon source is not limited to them. The nitrogen source can be nitrogen-containing organic compounds (such as peptone, yeast extract, meat extract, malt extract, liquid corn extract, soybean meal and urea) or inorganic compounds (such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate ) and the like, alone or in combination, but the source of nitrogen is not limited to them. As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, their corresponding sodium salt and the like can be used, alone or in mixture, but the phosphorus source is not limited to them. In addition, the medium may also contain other metal salts (eg magnesium sulfate or ferrous sulfate) and essential growth promoting substances such as amino acids and vitamins.

The method may further include, but is not limited to, isolating putrescine from the culture medium or microorganism after the culture step.

As for the method for isolating putrescine produced in the culture step, the target putrescine can be isolated from the culture using a suitable method known in the art according to the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC (high performance liquid chromatography) and/or the like can be used, and the target putrescine can be isolated from the medium or microorganism by using a suitable method known in the art.

The method for isolating putrescine may further include a purification step. The purification step can be carried out using a suitable method known in the art. Therefore, the isolated putrescine may be in a purified form or may be a microorganism-fermented liquid containing putrescine.

According to yet another aspect of the present disclosure, there is provided a composition for producing putrescine comprising a microorganism of the genus Corynebacterium having the ability to produce putrescine, into which an N-acetyltransferase activity derived from a strain of the genus Corynebacterium and an acetylornithine deacetylase (argE) activity derived from E. coli are introduced.

N-acetyltransferase, acetylornithine deacetylase, putrescine and microorganism are as described above.

The Corynebacterium genus may have, but is not limited to, reduced bifunctional ornithine acetyltransferase/N-acetylglutamate synthase (argJ) activity derived from a Corynebacterium genus strain.

The composition for producing putrescine may include, without limitation, an element capable of introducing N-acetyltransferase activity derived from a strain of the genus Corynebacterium and acetylornithine deacetylase (argE) activity derived from E. coli. In particular, the element may be in a form contained in a vector such that a gene is expressed that is functionally associated with the host cell into which the element is introduced, and the form is as described above.

The N-acetyltransferase derived from a strain of the genus Corynebacterium may contain the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto, as described above.

The N-acetyltransferase derived from a strain of the genus Corynebacterium may contain the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto, as described above.

Acetylornithine deacetylase derived from E. coli may contain the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity thereto, as described above.

The composition of the present invention may further contain any suitable excipient that is commonly used in compositions for producing amino acids, and examples of the excipient may be, but are not limited to, a preservative, humectant, dispersing agent, suspending agent, buffer, stabilizer or isotonic agent.

According to another aspect of the present disclosure, the use of a composition for the production of putrescine is provided.

According to yet another aspect of the present disclosure, the use of a microorganism of the genus Corynebacterium having the ability to produce putrescine is provided for the production of putrescine, wherein the microorganism is introduced with N-acetyltransferase activity originating from a strain of the genus Corynebacterium and acetylornithine deacetylase (argE) activity originating from E. coli .

Method for carrying out the invention

Below, the present disclosure will be described in detail with reference to typical embodiments. However, it will be apparent to one skilled in the art that these exemplary embodiments are provided for purposes of illustration only and are not intended to limit the scope of the present disclosure.

Example 1: Putrescine-producing ability of an argJ gene-enhanced strain derived from Coryne

1-1. Generation of a strain having argJ derived from ATCC13869. enhanced in the transposon gene of the putrescine-producing strain. based on ATCC13032

To enhance the argJ gene, encoding the bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, originating from a strain of the genus Corynebacterium, in the putrescine-producing strain based on Corynebacterium glutamicum ATCC 13032, the argJ gene was introduced into the transposon gene of this strain.

pDZTn (WO 2009/125992) was used as a transformation vector that allows the introduction of a gene into the chromosome by using the transposon gene region of a microorganism of the genus Corynebacterium. In addition, and as a promoter, the lysCPl promoter (WO 2009/096689, SEQ ID NO: 9), obtained by modifying the promoter of the lysC-asd operon gene derived from Corynebacterium glutamicum, was used.

Specifically, the pDZTn vector was constructed as follows. To obtain the transposon gene, nucleotide sequence information about the complete nucleotide sequence of the transposon gene (NCBI accession number NC 003450, NCgI1021) originating from Corynebacterium glutamicum ATCC13032 was obtained from NIH GenBank, and based on this information, two pairs were synthesized primers (Table 1, SEQ ID NO: 10-13).

PCR (polymerase chain reaction) was carried out using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, together with a pair of primers SEQ ID NO: 10 and 13. PfuUltra™ High Fidelity DNA Polymerase (Stratagene) was used as the polymerase, and the following PCR conditions were set : 30 cycles of denaturation at 96°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 1 min.

As a result, two pairs of transposon genes (Tn-A, Tn-B), including promoter regions of approximately 500 bp, were obtained as PCR products. Tn-A (SEQ ID NO: 14) was amplified using SEQ ID NO: 10 and 11 as primers, and Tn-B (SEQ ID NO: 15) was amplified using SEQ ID NO: 12 and 13 as primers. The amplification products were cloned into the pDZ vector, pretreated with the restriction enzyme SalI, using the BD In-Fusion (BD) kit, thereby obtaining the pDZTn vector. Between these two amplified products, a number of restriction enzyme recognition sites were included, artificially inserted during primer design.

The lysCP1 promoter was obtained as follows. PCR was carried out using chromosomal DNA of strain CA01-0135 (WO 2009-096689, KCCM10919P) as a template, obtained by transforming Corynebacterium glutamicum KFCC10881 with a vector containing the lysCP1 promoter, together with the following primers SEQ ID NO: 16 and 17. The polymerase was used PfuUltra™ High Fidelity DNA Polymerase (Stratagene), and the following PCR conditions were set: 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s.

The sequences of the primers used are shown in Table 2 below.

As a result, the lysCP1 promoter region (SEQ ID NO: 9) was obtained as a PCR product.

To obtain the argJ gene, a pair of primers SEQ ID NO: 18 and 19 was prepared to obtain homologous recombinant fragments of the argJ open reading frame (ORF) based on the nucleotide sequence (SEQ ID NO: 6) of the argJ gene derived from Corynebacterium glutamicum ATCC 13869.

PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC 13869 as a template, together with a pair of primers SEQ ID NO: 18 and 19. PfuUltra™ High Fidelity DNA Polymerase (Stratagene) was used as the polymerase, and the following PCR conditions were set: 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min and 30 s.

The sequences of the primers used are shown in Table 3 below.

As a result, fragments of the argJ gene of approximately 1.2 kb. amplified as PCR products. These fragments were subjected to electrophoresis on a 0.8% agarose gel, and bands of the desired size were eluted and purified.

The previously obtained vector pDZTn was treated with the restriction enzyme XhoI, and the corresponding PCR products (lysCP1 promoter region and argJ gene fragments) obtained above were cloned by fusion into a vector using the in-Fusion® HD cloning kit (Clontech), and then the resulting plasmid named pDZTn-lysCP1-argJ.

Then the PDZTN-LYSCP1-ARGJ plasmid was introduced in the SS01-0163 (US 9677099, KCCM11240P) through electroption with a transformer, where the strain was obtained by removing the activity of the NCGL1469 protein, defined as a Histonacetyl, NRA2, and a related to Aza Tiltransferase in the SS01-0064 strain ( US 9890404, KCCM11138P) having the ability to produce putrescine, which was obtained by deleting the gene encoding ornithine carbamoyltransferase (argF) and the gene (NCgl1221) encoding the glutamate exporter involved in the export of glutamate, introducing the gene (speC) encoding ornithine decarboxylase (ODC) occurring from wild-type E. coli strain 3110 into the chromosome and replacing the promoter of the ArgCJBD gene cluster, encoding enzymes involved in the synthesis of ornithine from glutamate, into wild-type Corynebacterium glutamicum ATCC13032. The transformant was plated and cultured in BHIS plate medium (37 g/L brain heart extract, 91 g/L sorbitol, and 2% agar) containing 25 μg/mL kanamycin and X-gal (5-bromo-4-chloro -3-indoline-D-galactoside), with the formation of colonies. By selecting white colonies from the colonies formed in this way, transformant strains were selected into which the plasmid pDZTn-lysCPl-argJ was introduced.

Selected strains were cultured with shaking in SM medium (glucose (10 g/l), polypeptone (10 g/l), yeast extract (5 g/l), beef extract (5 g/l), NaCl (2.5 g/l). l), urea (2 g/l) and pH 6.8) at 30°C for 8 hours. Each cell culture was then serially diluted from 10 ^-4 to 10 ^-10 and then plated and cultured on solid media containing X-gal to form colonies. By selecting white colonies appearing at a relatively low frequency among the colonies formed, a strain was finally selected into which the gene encoding argJ was introduced through a second crossover.

PCR was performed on the final selected strain using the primer pair SEQ ID NOs: 18 and 19 to confirm that the gene encoding argJ had been introduced, and the mutant strain of Corynebacterium glutamicum was named CC01-0163 Tn:lysCP1-argJ.

1-2. Evaluation of the putrescine-producing ability of a Coryne-based strain enhanced with the Coryne-derived argJ gene

To investigate the effect on putrescine production when the Corynebacterium-derived argJ gene was enhanced in a putrescine-producing strain, the ability to produce putrescine was compared with the mutant Corynebacterium glutamicum strain obtained in Example 1-1.

Specifically, the mutant strain of Corynebacterium glutamicum (CC01-0163 Tn:lysCP1-argJ) obtained in Example 1-1 was sown on CM plate medium (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5 % beef extract, 0.25% NaCl, 0.2% urea, 100 μl 50% NaOH, 2% agar, pH 6.8, based on 1 l) containing 1 mM arginine, and cultured at 30°C for 24 hours.

Approximately one platinum loop of each strain cultured in this manner was inoculated into 25 ml _of titration medium (8% glucose, 0.25% soy protein, 0.50% corn extract solids, 4% ( _NH4 ) _2SO4.0 , 1% KH ₂ PO ₄ , 0.05% MgSO ₄ ⋅7H ₂ O, 0.15% urea, 100 μg biotin, 3 mg thiamine hydrochloride, 3 mg calcium pantothenic acid, 3 mg nicotinamide, 5% CaCO ₃ , on base 1 l) and then cultured with shaking at 30°C and 200 rpm for 98 hours. Then, 1 mM arginine was added to the medium to cultivate each strain. The concentration of putrescine obtained from each crop was measured and the results are shown in Table 4 below.

As a result, as shown in Table 4, putrescine production increased by 6% in a mutant strain of Corynebacterium glutamicum in which Corynebacterium-derived argJ was enhanced.

Example 2: Introduction of E. coli-derived argA and argE into a putrescine-producing strain and its ability to produce putrescine

2-1. Generation of strain by co-injection as argA. so is argE. originating from E. coli into the transposon gene of the putrescine-producing strain. based on ATCC 13032

To investigate whether the ability to produce putrescine was improved by inserting argA encoding E. coli-derived N-acetylglutamate synthase and argE encoding E. coli-derived acetylornithine deacetylase into the putrescine-producing Corynebacterium glutamicum strain ATCC 13032 (CC01-0163), argA and argE were introduced into the transposon gene of this strain.

For this purpose, a pair of primers SEQ ID NO: 21 and 22 was obtained to obtain homologous recombinant fragments in the argA ORF region from the nucleotide sequence (SEQ ID NO: 20) of the argA gene originating from E. coli, and a pair of primers SEQ ID NO: 23 and 24 to obtain homologous recombinant fragments in the argE ORF region from the nucleotide sequence (SEQ ID NO: 4) of the argE gene.

The sequences of these primers are shown in Table 5.

The lysCP1 promoter region was obtained by PCR using the chromosome of strain KCCM10919P as a template, together with the primer pair SEQ ID NO: 16 and 17, in the same manner as in Example 1-1.

To obtain the argA gene, PCR was performed using the chromosome of E. coli strain W3110 as a template, together with a pair of primers SEQ ID NO: 21 and 22. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase, and the following PCR conditions were set : 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min and 30 s.

As a result, fragments of the argA gene measuring approximately 1.6 kb. amplified as PCR products, and these fragments were cloned by fusion together with the lysCP1 promoter region into the pDZTn vector obtained in Example 1-1 in the same way as in Example 1-1. The resulting plasmid was named pDZTn-lysCP1-argA.

The vector pDZTn obtained in Example 1-1 was treated with the restriction enzyme Xhol, and the corresponding PCR products obtained above (lysCP1 promoter region and argA gene fragment) were cloned by fusion into a vector in the same way as in Example 1-1, and the resulting plasmid was then named pDZTn-lysCP1-argA.

To obtain the argE gene, PCR was performed using the chromosome of E. coli strain W3110 as a template, together with the primer pair SEQ ID NO: 23 and 24, in the same manner as above. PfuUltra™ High Fidelity DNA Polymerase (Stratagene) was used as the polymerase, and the following PCR conditions were set: 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min and 30 s.

As a result, fragments of the argE gene measuring approximately 1.4 kb. amplified as PCR products, and these fragments were cloned by fusion together with the previously obtained lysCP1 promoter region into the pDZTn vector obtained in Example 1-1 in the same way as in Example 1-1. The resulting plasmid was named pDZTn-lysCP1-argE.

Then, the plasmid pDZTn-lysCP1-argA was introduced into CC01-0163 by electrophoresis to obtain transformant strains, and the transformant strains into which the plasmid pDZTn-lysCP1-argA was introduced were selected in the same way as in Example 1-1.

The selected strains were subjected to a second crossover to finally select a strain into which the gene encoding argA was introduced. PCR was performed on the final selected strain using the primer pair SEQ ID NOs: 21 and 22 to confirm that the gene encoding argA had been introduced, and the mutant strain of Corynebacterium glutamicum was named CC01-0163 Tn:lysCP1-argA.

To introduce argE into the resulting mutant strain of Corynebacterium glutamicum, into which argA was introduced, the previously constructed pDZTn-lysCPl-argE was transformed into CC01-0163 Tn:lysCP1-argA in the same way as above, and PCR was carried out on the finally selected strain, together with the primer pair SEQ ID NOs: 23 and 24, to confirm that argE was introduced into the transposon.

The mutant strain of Corynebacterium glutamicum selected in this way was named CC01-0163 Tn:lysCP1-argA Tn:lysCPl-argE.

2-2. Evaluation of the putrescine-producing ability of a putrescine-producing strain of the genus Corynebacterium into which E. coli-derived argA and argE were introduced

To study the effect on putrescine production of introducing argA and argE derived from E. coli into a putrescine-producing strain, the ability to produce putrescine was compared with the mutant strain Corynebacterium glutamicum obtained in Example 2-1.

Each of the mutant strain Corynebacterium glutamicum (CC01-0163 Tn:lysCP1-argA Tn:lysCP1-argE) obtained in Example 2-1 and the parent strain (CC01-0163) were cultivated in the same way as in Example 1-2, and the concentration of putrescine produced from each culture was measured. The results are shown in Table 6 below.

As a result, as shown in Table 6, putrescine production was increased by 9.8% in the mutant strain of Corynebacterium glutamicum into which the E. coli-derived genes argA and argE were introduced.

Example 3: Putrescine-producing ability of a strain enhanced with the Coryne-derived gene Ncgl2644

3-1. Preparation of a strain in which Ncgl2644, derived from ATCC13869, was introduced into the transposon gene of a putrescine-producing strain based on ATCC13032

To investigate whether the ability to produce putrescine was improved by enhancing the Ncgl2644 gene encoding the GCN5-related N-acetyltransferase (GNAT) family N-acetyltransferase derived from Corynebacterium glutamicum ATCC 13869 in the putrescine-producing strain (CC01-0163) based on Corynebacterium glutamicum ATCC 13032 (CC01-0163), Ncgl2644 was introduced and amplified in the transposon gene of this strain.

For this purpose, a pair of primers SEQ ID NO: 25 and 26 was prepared to obtain homologous recombinant fragments of the Ncgl2644 ORF region from the nucleotide sequence (SEQ ID NO: 2) of the Ncgl2644 gene originating from Corynebacterium.

Primer sequences are shown in Table 7.

To obtain the Ncgl2644 gene, PCR was performed using the chromosome of Corynebacterium glutamicum strain ATCC13869 as a template, together with a pair of primers SEQ ID NO: 25 and 26. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase, and the following PCR conditions were set: 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. As a result, fragments of the Ncgl2644 gene of approximately 1 kb. amplified as PCR products, and these fragments were cloned by fusion, together with the lysCP1 promoter region obtained in Example 2-1, into the pDZTn vector obtained in Example 1-1, in the same way as in Example 1-1. The resulting plasmid was named pDZTn-lysCP1-Ncgl2644.

Then, this plasmid pDZTn-lysCP1-Ncgl2644 was introduced into CC01-0163 by electrophoresis to obtain transformant strains, and transformant strains into which the plasmid pDZTn-lysCP1-Ncgl2644 was introduced were selected in the same way as in Example 1-1.

The selected strains were subjected to a second crossover to finally select a strain into which the gene encoding Ncgl2644 was introduced. PCR was performed on this final selected strain using the primer pair SEQ ID NOs: 25 and 26 to confirm that the gene encoding Ncgl2644 had been introduced, and this mutant strain of Corynebacterium glutamicum was named CC01-0163 Tn:lysCP1-Ncgl2644.

3-2. Evaluation of the putrescine-producing ability of a putrescine-producing strain of the genus Corynebacterium in which Ncgl2644 was enhanced. originating from Coryne

To investigate the effect on putrescine production by enhancing the Corynebacterium-derived gene Ncgl2644 in a putrescine-producing strain, the ability to produce putrescine was compared with the mutant Corynebacterium glutamicum strain obtained in Example 3-1.

Specifically, each of the mutant strain Corynebacterium glutamicum (CC01-0163) obtained in Example 3-1 and the parental strain (CC01-0163) were cultured in the same way as in Example 1-2, and the concentration of putrescine obtained from every culture. The results are shown in Table 8 below.

As a result, as shown in Table 8, the ability to produce putrescine was reduced when Ncgl2644 derived from Corynebacterium was upregulated. This may be due to a bottleneck in this biosynthetic pathway caused by an increase in the concentration of acetyl glutamate, an intermediate metabolite in the putrescine production pathway. Therefore, the effect of Ncgl2644 on putrescine production was assessed through additional examples given below.

Example 4: Ability to produce putrescine from a strain with deletion of the argJ gene and amplification of Ncgl2644

4-1. Generation of a strain in which argJ is deleted in a putrescine-producing strain based on ATCC13032

A strain was obtained in which the argJ gene, derived from Corynebacterium glutamicum ATCC13032, was deleted in a putrescine-producing strain based on Corynebacterium glutamicum ATCC13032 (CC01-0163).

For this purpose, a pair of primers SEQ ID NO: 27 and 28 was obtained to obtain homologous recombinant fragments of the N-terminal region of argJ and a pair of primers SEQ ID NO: 29 and 30 to obtain homologous recombinant fragments of the C-terminal region of argJ from the nucleotide sequence (SEQ ID NO : 8) argJ gene, derived from Corynebacterium glutamicum ATCC13032.

The sequences of these primers are shown in Table 9.

To obtain homologous recombinant fragments of the C-terminal region and N-terminal region of argJ, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, together with the primer pair SEQ ID NO: 27 and 28, and the primer pair SEQ ID NO: 29 and 30 PfuUltra™ High Fidelity DNA Polymerase (Stratagene) was used as the polymerase, and the following PCR conditions were set: 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C. for 30 s. As a result, the PCR fragments of the N-terminal region and the C-terminal region were amplified as PCR products, respectively, and these fragments were subjected to electrophoresis on a 0.8% agarose gel, and the bands with the desired sizes were eluted and purified.

The resulting fragments of the N-terminal region were treated with restriction enzymes BamHI and SalI, and the resulting fragments of the C-terminal region were treated with restriction enzymes SalI and XbaI. The processed fragments were cloned into the pDZ vector treated with the restriction enzymes BamHI and XbaI to construct the plasmid pDZ-1'argJ(K/O).

The constructed plasmid pDZ-1'argJ(K/O) was introduced into Corynebacterium glutamicum CC01-0163 by electrophoresis to obtain a transformant, and strains with introduced pDZ-1'NCglargJ(K/O) were selected in the same way as in Example 1- 1.

From the selected strains, a Corynebacterium glutamicum strain having weakened putrescine productivity was finally selected by deleting the gene encoding argJ, and a mutant Corynebacterium glutamicum strain with weakened putrescine export ability was named CC01-0163ΔargJ.

The Ncgl2644 gene was transformed into the resulting strain CC01-0163 ΔargJ in the same way as in Example 3-1, obtaining strain CC01-0163 ΔargJ Tn:lysCP1-Ncgl2644.

4-2. Evaluation of the putrescine-producing ability of a putrescine-producing strain of the genus Corynebacterium in which argJ was deleted. originating from Coryne, and enhanced by Ncgl2644

To investigate the effect on putrescine production by deletion of the Corynebacterium-derived argJ gene and amplification of the Corynebacterium-derived Ncgl2644 gene in a putrescine-producing strain, the ability to produce putrescine was compared with the mutant Corynebacterium glutamicum strain obtained in Example 4-1.

Specifically, each of the mutant strain Corynebacterium glutamicum (CC01-0163 ΔargJ Tn:lysCP1-Ncgl2644) and the parental strain (CC01-0163 ΔargJ) were cultured in the same way as in Example 1-2, and the concentration of putrescine produced from each culture. The results are shown in Table 10 below.

As a result, as shown in Table 10, the argJ deletion strain had difficulty in being able to produce putrescine, and even though Ncgl2644 capable of generating acetyl glutamate was introduced, putrescine production was low due to the lack of an enzyme that can deacetylate the acetylornithine intermediate metabolite generated after administration. Therefore, the strain co-enhanced with Ncgl2644 and argE was evaluated for the ability to produce putrescine through additional working examples.

Example 5: Putrescine-producing ability of strains enhanced with the Coryne-derived Ncgl2644 gene and with the introduction of the E. coli-derived argE gene

5-1. Obtaining strains enhanced by the Ncgl2644 gene. from putrescine-producing strains. based on ATCC 13032

Strain CC01-0163 Tn:lysCP1-Ncgl2644, obtained in Example 3-1, and strain CC01-0163 ΔargJ Tn:lysCP1-Ncgl2644, obtained in Example 4-1, were introduced with argE, encoding acetyl ornithine acetylase, capable of deacetylating acetylornithine, originating from E. coli W3110, argE.

Specifically, the plasmid pDZTn-lysCP1-argE constructed in Example 2-1 was transformed into strain CC01-0163 Tn:lysCP1-Ncgl2644 and strain CC01-0163 ΔargJ Tn:lysCP1-Ncgl2644 in the same manner as in Example 2-1 , to obtain transformants, and transformant strains into which the plasmid pDZTn-lysCP1-argE was introduced were selected in the same way as in Example 1-1.

From the selected strains, Corynebacterium glutamicum strains with introduced argE were finally selected, and the prepared Corynebacterium glutamicum strains CC01-0163 Tn:lysCP1-Ncgl2644 Tn:lysCP1-argE and CC01-0163 ΔargJ Tn:lysCP1-Ncgl2644 Tn:lysCP1-argE were obtained. CC01-0163 ΔargJ Tn:lysCPl-Ncgl2644 Tn:lysCP1-argE was named Corynebacterium glutamicum CC01-1425. Corynebacterium glutamicum CC01-1425 (CC01-0163 ΔargJ Tn:lysCP1-Ncgl2644 Tn:lysCP1-argE) deposited (KCCM12774P) July 24, 2020.

5-2. Evaluation of the putrescine-producing ability of a putrescine-producing strain of the genus Corynebacterium in which the Ncgl2644 and argE genes were enhanced

To examine the effect on putrescine production by enhancing Corynebacterium-derived Ncgl2644 and introducing Corynebacterium-derived argE into the putrescine-producing strain, the putrescine-producing ability of the mutant Corynebacterium glutamicum strain obtained in Example 5-1 was compared.

In particular, each of the mutant strains of Corynebacterium glutamicum (CC01-0163 Tn:lysCP1-Ncgl2644 Tn:lysCP1-argE and CC01-0163 AargJ Tn:lysCPl-Ncgl2644 Tn:lysCP1-argE) and the parent strain (CC01-0163) were cultivated with the same method as in Example 1-2, and measured the concentration of putrescine produced from each culture. These results are shown in Table 11 below.

As shown in Table 11, co-administration of Ncgl2644 and argE resulted in significantly improved putrescine production compared to strain CC01-0163. In particular, strain CC01-0163 ΔargJ Tn:lysCPl-Ncgl2644 Tn:lysCP1-argE with eliminated argJ activity demonstrated the ability to produce putrescine, improved by 36.6% compared to strain CC01-0163, and strain CC01-0163 Tn:lysCP1- Ncgl2644 Tn:lysCP1-argE with retained argJ activity exhibited a poor ability to produce putrescine compared to the strain with eliminated argJ activity, but had only a 30.8% improvement in putrescine production ability compared to strain CC01-0163. In addition, strain CC01-0163 Tn:lysCP1-Ncgl2644 Tn:lysCP1-argE with remaining argJ activity exhibited significantly higher putrescine production compared to the Corynebacterium glutamicum mutant strain co-injected with E. coli-derived argA and argE. demonstrating similar functions to Ncgl2644 (Table 6).

The results of the present Examples confirmed that deletion of argJ and co-expression of Ncgl2644 and argE resulted in a greater ability to produce putrescine compared to no deletion of argJ or deletion of argJ and introduction of E. coli-derived argA and argE, which exhibit similar functions, into Ncgl2644.

As set forth above, one skilled in the art to which the present disclosure pertains will appreciate that the present invention may be embodied in other specific forms without departing from its technical spirit or essential characteristics. Therefore, the exemplary embodiments described above should be construed as exemplary and non-limiting of the present invention. The scope of the present invention should be understood to mean that all changes or modifications derived from the definitions and scope of the claims, and their equivalents, are included within the scope of this invention.

--->

LIST OF SEQUENCES

<110>CJ CheilJedang Corporation

<120> MICROORGANISM FOR PRODUCING PUTRESCINE AND PRODUCTION METHOD

PUTRECINE WITH ITS APPLICATION

<130>OPA21055

<150> KR 10-2020-0101894

<151> 2020-08-13

<160> 30

<170> KoPatentIn 3.0

<210> 1

<211> 335

<212>PRT

<213>Unknown

<220>

<223> N-acetyltransferase of the GNAT family of C. glutamicum

<400> 1

Met Thr Pro Ser Leu Pro Arg Phe Arg Ser Gln Lys Pro Ala Val Gly

1 5 10 15

Asp Arg Val Val Ala Arg Arg Arg Ile Pro Gly Ala Asn Val His Trp

20 25 30

Thr Asp Ala Ile Gly His Val Ile Gly Val Asp Pro Leu Val Val Arg

35 40 45

Pro Gln Ser Val Gly Gly Met Pro Ser Asp Ala Glu Glu Ile Val Ile

50 55 60

Pro Asp Asp Gln Leu Glu Val Ile Lys Ile Leu Ser Pro Arg Thr Ile

65 70 75 80

Arg Asn Ser Asp Ile Arg Ala Val Glu Val Ala Thr Ala Lys Ala Phe

85 90 95

Pro Gly Leu Val Asn Glu Trp His Asp Gly Trp Leu Leu Arg Ala Gly

100 105 110

Asp Gly Ile Ala Glu Arg Ser Asn Ser Ala Ser Pro Leu Gly Pro Ser

115 120 125

Val Gly Phe Glu Pro Val Pro Met Glu Asp Ile Ser Arg Phe Tyr Ala

130 135 140

Arg His Asp Leu Pro Val Lys Leu His Ile Pro Glu Arg Ile Gly Arg

145 150 155 160

Pro Ala Gln Lys Val Ile Asp Ala Asp Pro Gln Lys Trp Val Met Gly

165 170 175

Pro Glu Ile Leu Val Met Thr Lys Ser Leu Asp His Val Glu Ser His

180 185 190

Glu Leu Pro Gly Gly Leu Glu Phe Ser Val Asp Lys Gln Pro Asp Gln

195 200 205

Glu Trp Leu Gly Met Tyr His Phe Arg Gly Gln Ala Leu Pro Ala His

210 215 220

Ala Leu Glu Leu Leu Arg Thr Gln Ile Glu Gly Arg Met Gly Phe Gly

225 230 235 240

Arg Leu Thr Thr Pro Ala Gly Gln Thr Val Ala Ile Thr Arg Ala Thr

245 250 255

Ile Thr Ala Ala Glu Glu Arg Ile Phe Leu Gly Tyr Ser Ala Val Glu

260 265 270

Val Asp Pro Ala Phe Arg Arg Gln Gly Leu Gly Thr Ala Leu Gly Ser

275 280 285

Arg Ile Gln Glu Trp Gly Ala Glu Gln His Ala Gln Glu Ala Tyr Leu

290 295 300

Gln Val Val Ala His Asn Glu Ala Gly Ile Gly Leu Tyr Gln Lys Leu

305 310 315 320

Gly Phe Ser Glu His His Arg His Arg Tyr Ala Glu Arg Lys Phe

325 330 335

<210> 2

<211> 1008

<212> DNA

<213>Unknown

<220>

<223> N-acetyltransferase (Ncgl2644) of the GNAT family of C. glutamicum

<400> 2

atgacgccta gtcttccccg tttccgcagc cagaaacctg ccgtcggcga tcgtgttgtt 60

gcacgtcgcc ggattcctgg tgccaatgtg cattggacag atgccattgg ccatgtgatt 120

ggggtggatc cgttggtggt tcgcccgcag tcggttggtg ggatgccgtc ggatgcggaa 180

gaaattgtca ttcctgatga tcagcttgag gtgattaaga ttttgtcgcc gcgcaccatt 240

aggaattcgg atattcgtgc ggtggaggtt gccacggcga aggcctttcc ggggctggtc 300

aatgagtggc atgatggttg gctgctgcgt gccggtgatg gcattgcgga gcgttctaat 360

tctgcgtcgc cactcggccc aagtgttggt tttgagccgg taccgatgga ggatatttcg 420

cggttttacg caaggcacga tctccccgtg aagctgcaca ttccggagcg gattggtcgg 480

cctgcgcaga aagtcattga cgccgatccc cagaaatggg tgatgggccc ggagatttg 540

gtgatgacga aatctttgga ccatgtggag tcgcacgagt tgcccggtgg cctagaattt 600

agcgtcgata agcagcctga ccaggagtgg ctgggcatgt accatttccg cggacaggcg 660

ttgcccgctc acgcccttga gcttttgcgc acgcaaatcg agggccgcat ggggttcggg 720

cgcctgacca cgccggcggg gcaaaccgtc gcgatcacgc gcgccaccat cacggctgcg 780

gaggagcgca tatttttggg ctattcagcg gtcgaggtgg atcctgcttt tcgacgtcag 840

gggctgggca ccgcgctcgg ctcgcgcatc caggagtggg gcgccgagca acacgcacaa 900

gaggcatatc tccaggttgt cgcccataat gaagcaggta tcggcctgta tcaaaagctc 960

gggttcagtg aacaccaccg acaccggtac gccgaacgga aattctaa 1008

<210> 3

<211> 408

<212>PRT

<213>Unknown

<220>

<223> argE E. coli

<400> 3

Met Asp Lys Leu Leu Glu Arg Phe Leu Asn Tyr Val Ser Leu Asp Thr

1 5 10 15

Gln Ser Lys Ala Gly Val Arg Gln Val Pro Ser Thr Glu Gly Gln Trp

20 25 30

Lys Leu Leu His Leu Leu Lys Glu Gln Leu Glu Glu Met Gly Leu Ile

35 40 45

Asn Val Thr Leu Ser Glu Lys Gly Thr Leu Met Ala Thr Leu Pro Ala

50 55 60

Asn Val Pro Gly Asp Ile Pro Ala Ile Gly Phe Ile Ser His Val Asp

65 70 75 80

Thr Ser Pro Asp Cys Ser Gly Lys Asn Val Asn Pro Gln Ile Val Glu

85 90 95

Asn Tyr Arg Gly Gly Asp Ile Ala Leu Gly Ile Gly Asp Glu Val Leu

100 105 110

Ser Pro Val Met Phe Pro Val Leu His Gln Leu Leu Gly Gln Thr Leu

115 120 125

Ile Thr Thr Asp Gly Lys Thr Leu Leu Gly Ala Asp Asp Lys Ala Gly

130 135 140

Ile Ala Glu Ile Met Thr Ala Leu Ala Val Leu Gln Gln Lys Lys Ile

145 150 155 160

Pro His Gly Asp Ile Arg Val Ala Phe Thr Pro Asp Glu Glu Val Gly

165 170 175

Lys Gly Ala Lys His Phe Asp Val Asp Ala Phe Asp Ala Arg Trp Ala

180 185 190

Tyr Thr Val Asp Gly Gly Gly Val Gly Glu Leu Glu Phe Glu Asn Phe

195 200 205

Asn Ala Ala Ser Val Asn Ile Lys Ile Val Gly Asn Asn Val His Pro

210 215 220

Gly Thr Ala Lys Gly Val Met Val Asn Ala Leu Ser Leu Ala Ala Arg

225 230 235 240

Ile His Ala Glu Val Pro Ala Asp Glu Ser Pro Glu Met Thr Glu Gly

245 250 255

Tyr Glu Gly Phe Tyr His Leu Ala Ser Met Lys Gly Thr Val Glu Arg

260 265 270

Ala Asp Met His Tyr Ile Ile Arg Asp Phe Asp Arg Lys Gln Phe Glu

275 280 285

Ala Arg Lys Arg Lys Met Met Glu Ile Ala Lys Lys Val Gly Lys Gly

290 295 300

Leu His Pro Asp Cys Tyr Ile Glu Leu Val Ile Glu Asp Ser Tyr Tyr

305 310 315 320

Asn Met Arg Glu Lys Val Val Glu His Pro His Ile Leu Asp Ile Ala

325 330 335

Gln Gln Ala Met Arg Asp Cys Asp Ile Glu Pro Glu Leu Lys Pro Ile

340 345 350

Arg Gly Gly Thr Asp Gly Ala Gln Leu Ser Phe Met Gly Leu Pro Cys

355 360 365

Pro Asn Leu Phe Thr Gly Gly Tyr Asn Tyr His Gly Lys His Glu Phe

370 375 380

Val Thr Leu Glu Gly Met Glu Lys Ala Val Gln Val Ile Val Arg Ile

385 390 395 400

Ala Glu Leu Thr Ala Gln Arg Lys

405

<210> 4

<211> 1227

<212> DNA

<213> Unknown

<220>

<223> argE E. coli

<400> 4

atggataaac tacttgagcg atttttgaac tacgtgtctc tggataccca atcaaaagca 60

ggggtgagac aggttcccag cacggaaggc caatggaagt tattgcatct gctgaaagag 120

cagctcgaag agatggggct tatcaatgtg accttaagtg agaagggcac tttgatggcg 180

acgttaccgg ctaacgtccc tggcgatatc ccggcgattg gctttatttc tcatgtggat 240

acctcaccgg attgcagcgg caaaaatgtg aatccgcaaa ttgttgaaaa ctatcgcggt 300

ggcgatattg cgctgggtat cggcgatgaa gttttatcac cggttatgtt cccggtgctg 360

catcagctac tgggtcagac gctgattacc accgatggta aaaccttgtt aggtgccgat 420

gacaaagcag gtattgcaga aatcatgacc gcgctggcgg tattgcaaca gaaaaaaatt 480

ccgcatggtg atattcgcgt cgcctttacc ccggatgaag aagtgggcaa aggggcgaaa 540

cattttgatg ttgacgcctt cgatgcccgc tgggcttaca ctgttgatgg tggtggcgta 600

ggcgaactgg agtttgaaaa cttcaacgcc gcgtcggtca atatcaaaat tgtcggtaac 660

aatgttcatc cgggcacggc gaaaggagtg atggtaaatg cgctgtcgct ggcggcacgt 720

attcatgcgg aagttccggc ggatgaaagc ccggaaatga cagaaggcta tgaaggtttc 780

tatcatctgg cgagcatgaa aggcaccgtt gaacggggccg atatgcacta catcatccgt 840

gatttcgacc gtaaacagtt tgaagcgcgt aaacgtaaaa tgatggagat cgccaaaaaa 900

gtgggcaaag ggttacatcc tgattgctac attgaactgg tgattgaaga cagttactac 960

aatatgcgcg agaaagtggt tgagcatccg catattctcg atatcgccca gcaggcgatg 1020

cgcgattgcg atattgaacc ggaactgaaa ccgatccgcg gtggtaccga cggcgcgcag 1080

ttgtcgttta tgggattacc gtgcccgaac ctgttcactg gcggttacaa ctatcatggt 1140

aagcatgagt ttgtgactct ggaaggtatg gaaaaagcgg tgcaggtgat cgtccgtatt 1200

gccgagttaa cggcgcaacg gaagtaa 1227

<210> 5

<211> 388

<212>PRT

<213>Unknown

<220>

<223> argJ C. glutamicum 13869

<400> 5

Met Ala Lys Lys Gly Ile Thr Ala Pro Lys Gly Phe Val Ala Ser Ala

1 5 10 15

Thr Thr Ala Gly Ile Lys Ala Ser Gly Asn Pro Asp Met Ala Leu Val

20 25 30

Val Asn Gln Gly Pro Glu Phe Ser Ala Ala Ala Val Phe Thr Arg Asn

35 40 45

Arg Val Phe Ala Ala Pro Val Lys Val Ser Arg Glu Asn Val Ala Asp

50 55 60

Gly Gln Ile Arg Ala Val Leu Tyr Asn Ala Gly Asn Ala Asn Ala Cys

65 70 75 80

Asn Gly Leu Gln Gly Glu Lys Asp Ala Arg Glu Ser Val Ser His Leu

85 90 95

Ala Gln Asn Leu Gly Leu Glu Asp Ser Asp Ile Gly Val Cys Ser Thr

100 105 110

Gly Leu Ile Gly Glu Leu Leu Pro Met Asp Lys Leu Asn Thr Gly Ile

115 120 125

Asp Gln Leu Thr Ala Glu Gly Ala Leu Gly Asp Asn Gly Ala Ala Ala

130 135 140

Ala Lys Ala Ile Met Thr Thr Asp Thr Val Asp Lys Glu Thr Val Val

145 150 155 160

Phe Ala Asp Gly Trp Thr Val Gly Gly Met Gly Lys Gly Val Gly Met

165 170 175

Met Ala Pro Ser Leu Ala Thr Met Leu Val Cys Leu Thr Thr Asp Ala

180 185 190

Ser Val Thr Gln Glu Met Ala Gln Ile Ala Leu Ala Asn Ala Thr Ala

195 200 205

Val Thr Phe Asp Thr Leu Asp Ile Asp Gly Ser Thr Ser Thr Asn Asp

210 215 220

Thr Val Phe Leu Leu Ala Ser Gly Ala Ser Gly Ile Thr Pro Thr Gln

225 230 235 240

Asp Glu Leu Asn Asp Ala Val Tyr Ala Ala Cys Ser Asp Ile Ala Ala

245 250 255

Lys Leu Gln Ala Asp Ala Glu Gly Val Thr Lys Arg Val Ala Val Thr

260 265 270

Val Val Gly Thr Thr Asn Asn Glu Gln Ala Ile Asn Ala Ala Arg Thr

275 280 285

Val Ala Arg Asp Asn Leu Phe Lys Cys Ala Met Phe Gly Ser Asp Pro

290 295 300

Asn Trp Gly Arg Val Leu Ala Ala Val Gly Met Ala Asp Ala Asp Met

305 310 315 320

Glu Pro Glu Lys Ile Ser Val Phe Phe Asn Asp Gln Ala Val Cys Leu

325 330 335

Asp Ser Thr Gly Ala Pro Gly Ala Arg Glu Val Asp Leu Ser Gly Ala

340 345 350

Asp Ile Asp Val Arg Ile Asp Leu Gly Thr Ser Gly Glu Gly Gln Ala

355 360 365

Thr Val Arg Thr Thr Asp Leu Ser Phe Ser Tyr Val Glu Ile Asn Ser

370 375 380

Ala Tyr Ser Ser

385

<210> 6

<211> 1167

<212> DNA

<213>Unknown

<220>

<223> argJ C. glutamicum 13869

<400> 6

atggccaaaa aaggcattac cgcgccgaaa ggcttcgttg cttctgcaac gaccgcgggt 60

attaaagctt ctggcaatcc tgacatggcg ttggtggtta accaggtcc agagttttcc 120

gcagcggccg tgtttacacg caaccgagtt ttcgcagcgc ctgtgaaggt gagccgggag 180

aacgttgctg atggccagat cagggctgtt ttgtacaacg ctggtaatgc taatgcgtgt 240

aatggtctgc agggtgagaa ggatgctcgt gagtctgttt ctcatctagc tcaaaatttg 300

ggcttggagg attccgatat tggtgtgtgt tccactggtc ttattggtga gttgcttccg 360

atggataagc tcaatacagg tattgatcag ctgaccgctg agggcgcttt gggtgacaat 420

ggtgcagctg ctgccaaggc gatcatgacc actgacacgg tggataagga aaccgtcgtg 480

tttgctgatg gttggactgt cggcggaatg ggcaagggcg tgggcatgat ggcgccgtct 540

cttgccacca tgctggtctg cttgaccact gatgcatccg ttactcagga aatggctcag 600

attgcgctgg ctaatgctac ggccgttacg tttgacaccc tggatattga tggatcaacc 660

tccaccaatg acaccgtgtt cctgctggca tctggcgcta gcggaatcac cccaactcag 720

gatgaactca acgatgcggt gtacgcagct tgttctgata tcgcagcgaa gcttcaggct 780

gatgcagagg gggtgaccaa gcgcgttgct gtgacagtgg tgggaaccac caacaacgag 840

caggcgatca atgcggctcg cacggttgct cgtgacaatt tgttcaagtg cgcaatgttt 900

ggatctgatc caaactgggg tcgcgtgttg gctgcagtcg gcatggctga tgctgatatg 960

gaaccagaga agatttctgt gttcttcaat gatcaagcag tatgccttga ttccactggc 1020

gctcctggtg ctcgtgaggt ggatctttcc ggcgctgaca ttgatgtccg aattgatttg 1080

ggcaccagtg gggaaggcca ggcaacagtt cgaaccactg acctgagctt ctcctacgtg 1140

gagatcaact ccgcgtacag ctcttaa 1167

<210> 7

<211> 388

<212>PRT

<213> Unknown

<220>

<223> argJ C. glutamicum 13032

<400> 7

Met Ala Glu Lys Gly Ile Thr Ala Pro Lys Gly Phe Val Ala Ser Ala

1 5 10 15

Thr Thr Ala Gly Ile Lys Ala Ser Gly Asn Pro Asp Met Ala Leu Val

20 25 30

Val Asn Gln Gly Pro Glu Phe Ser Ala Ala Ala Val Phe Thr Arg Asn

35 40 45

Arg Val Phe Ala Ala Pro Val Lys Val Ser Arg Glu Asn Val Ala Asp

50 55 60

Gly Gln Ile Arg Ala Val Leu Tyr Asn Ala Gly Asn Ala Asn Ala Cys

65 70 75 80

Asn Gly Leu Gln Gly Glu Lys Asp Ala Arg Glu Ser Val Ser His Leu

85 90 95

Ala Gln Asn Leu Gly Leu Glu Asp Ser Asp Ile Gly Val Cys Ser Thr

100 105 110

Gly Leu Ile Gly Glu Leu Leu Pro Met Asp Lys Leu Asn Ala Gly Ile

115 120 125

Asp Gln Leu Thr Ala Glu Gly Ala Leu Gly Asp Asn Gly Ala Ala Ala

130 135 140

Ala Lys Ala Ile Met Thr Thr Asp Thr Val Asp Lys Glu Thr Val Val

145 150 155 160

Phe Ala Asp Gly Trp Thr Val Gly Gly Met Gly Lys Gly Val Gly Met

165 170 175

Met Ala Pro Ser Leu Ala Thr Met Leu Val Cys Leu Thr Thr Asp Ala

180 185 190

Ser Val Thr Gln Glu Met Ala Gln Ile Ala Leu Ala Asn Ala Thr Ala

195 200 205

Val Thr Phe Asp Thr Leu Asp Ile Asp Gly Ser Thr Ser Thr Asn Asp

210 215 220

Thr Val Phe Leu Leu Ala Ser Gly Ala Ser Gly Ile Thr Pro Thr Gln

225 230 235 240

Asp Glu Leu Asn Asp Ala Val Tyr Ala Ala Cys Ser Asp Ile Ala Ala

245 250 255

Lys Leu Gln Ala Asp Ala Glu Gly Val Thr Lys Arg Val Ala Val Thr

260 265 270

Val Val Gly Thr Thr Asn Asn Glu Gln Ala Ile Asn Ala Ala Arg Thr

275 280 285

Val Ala Arg Asp Asn Leu Phe Lys Cys Ala Met Phe Gly Ser Asp Pro

290 295 300

Asn Trp Gly Arg Val Leu Ala Ala Val Gly Met Ala Asp Ala Asp Met

305 310 315 320

Glu Pro Glu Lys Ile Ser Val Phe Phe Asn Gly Gln Ala Val Cys Leu

325 330 335

Asp Ser Thr Gly Ala Pro Gly Ala Arg Glu Val Asp Leu Ser Gly Ala

340 345 350

Asp Ile Asp Val Arg Ile Asp Leu Gly Thr Ser Gly Glu Gly Gln Ala

355 360 365

Thr Val Arg Thr Thr Asp Leu Ser Phe Ser Tyr Val Glu Ile Asn Ser

370 375 380

Ala Tyr Ser Ser

385

<210> 8

<211> 1167

<212> DNA

<213>Unknown

<220>

<223> argJ C. glutamicum 13032

<400> 8

atggcagaaa aaggcattac cgcgccgaaa ggcttcgttg cttctgcaac gaccgcgggt 60

attaaagctt ctggcaatcc tgacatggcg ttggtggtta accaggtcc agagttttcc 120

gcagcggccg tgtttacacg taaccgagtt ttcgcagcgc ctgtgaaggt gagccgagag 180

aacgttgctg atggccagat cagggctgtt ttgtacaacg ctggtaatgc taatgcgtgt 240

aatggtctgc agggtgagaa ggatgctcgt gagtctgttt ctcatctagc tcaaaatttg 300

ggcttggagg attccgatat tggtgtgtgt tccactggtc ttattggtga gttgcttccg 360

atggataagc tcaatgcagg tattgatcag ctgaccgctg agggcgcttt gggtgacaat 420

ggtgcagctg ctgccaaggc gatcatgacc actgacacgg tggataagga aaccgtcgtg 480

tttgctgatg gttggactgt cggcggaatg ggcaagggcg tgggcatgat ggcgccgtct 540

cttgccacca tgctggtctg cttgaccact gatgcatccg ttactcagga aatggctcag 600

atcgcgctgg ctaatgctac ggccgttacg tttgacaccc tggatattga tggatcaacc 660

tccaccaatg acaccgtgtt cctgctggca tctggcgcta gcggaatcac cccaactcag 720

gatgaactca acgatgcggt gtacgcagct tgttctgata tcgcagcgaa gcttcaggct 780

gatgcagagg gtgtgaccaa gcgcgttgct gtgacagtgg tgggaaccac caacaacgag 840

caggcgatta atgcggctcg cactgttgct cgtgacaatt tgttcaagtg cgcaatgttt 900

ggatctgatc caaactgggg tcgcgtgttg gctgcagtcg gcatggctga tgctgatatg 960

gaaccagaga agatttctgt gttcttcaat ggtcaagcag tatgccttga ttccactggc 1020

gctcctggtg ctcgtgaggt ggatctttcc ggcgctgaca ttgatgtccg aattgatttg 1080

ggcaccagtg gggaaggcca ggcaacagtt cgaaccactg acctgagctt ctcctacgtg 1140

gagatcaact ccgcgtacag ctcttaa 1167

<210> 9

<211> 289

<212> DNA

<213>Unknown

<220>

<223> lysCP1

<400> 9

ccgatgctag ggcgaaaagc acggcgagca gattgctttg cacttgattc agggtagttg 60

actaaagagt tgctcgcgaa gtagcacctg tcacttttgt ctcaaatatt aaatcgaata 120

tcaatatatg gtctgtttat tggaacgcgt cccagtggct gagacgcatc cgctaaagcc 180

caggaaccc tgtgcagaaa gaaaacactc ctctggctag gtagacacag tttattgtgg 240

tagagttgag cgggtaactg tcagcacgta gatcgaaagg tgcacaaag 289

<210> 10

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> Tn-A-F

<400> 10

atcctctaga gtcgaccatc gctgacacca tctgcc 36

<210> 11

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Tn-A-R

<400> 11

gggcccacta gtctcgagtt caccgcggga gccaagcc 38

<210> 12

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Tn-B-F

<400> 12

ctcgagacta gtgggccctg gattccaagg ctacgcc 37

<210> 13

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> Tn-B-R

<400> 13

atgcctgcag gtcgaccctg aatggataag gcaccg 36

<210> 14

<211> 510

<212> DNA

<213> Unknown

<220>

<223> Tn-A

<400> 14

gcagacgcac tcgactacac ctccacctgc ccagaatgct cccaacctgg ggtgtttcgt 60

catcacaccc accggatgct cattgattta cccatcgtcg ggtttcccac caaactgttt 120

atccgtctac ctcgctaccg ctgcaccaac cccacatgta agcaaaagta tttccaagca 180

gaactaagct gcgctgacca cggtaaaaag gtcacccacc gggtcacccg ctggatttta 240

caacgccttg ctattgaccg gatgagtgtt cacgcaaccg cgaaagcact tgggctaggg 300

tgggatttaa cctgccaact agccctcgat atgtgccgtg agctggtcta taacgatcct 360

caccatcttg atggagtgta tgtcattggg gtggatgagc ataagtggtc acataatagg 420

gctaagcatg gtgatgggtt tgtcaccgtg attgtcgata tgaccgggca tcggtatgac 480

tcacggtgtc ctgcccggtt attagatgtc 510

<210> 15

<211> 479

<212> DNA

<213>Unknown

<220>

<223> Tn-B

<400> 15

aactcattcc ttctgctcgt cgcgtgatgg atccattcca tgttgtgcgg cttgctggtg 60

acaagctcac cgcctgccgg caacgcctcc agcgggagaa ataccagcgt cgtggtttaa 120

gccaggatcc gttgtataaa aaccggaaga ccttgttgac cacgcacaag tggttgagtc 180

ctcgtcagca agaaagcttg gagcagttgt gggcgtatga caaagactac ggggcgttaa 240

agcttgcgtg gcttgcgtat caggcgatta ttgattgtta tcagatgggt aataagcgtg 300

aagcgaagaa gaaaatgcgg accattattg atcagcttcg ggtgttgaag gggccgaata 360

aggaactcgc gcagttgggt cgtagtttgt ttaaacgact tggtgatgtg ttggcgtatt 420

tcgatgttgg tgtctccaac ggtccggtcg aagcgatcaa cggacggttg gagcatttg 479

<210> 16

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> lysCP1 promoter_F

<400> 16

gaatgagttc ctcgagccga tgctagggcg aaaa 34

<210> 17

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> lysCP1 promoter_R

<400> 17

ctttgtgcac ctttcgatct acgtgctgac agttac 36

<210> 18

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> argJ_F

<400> 18

gttccacatg gccaaaaaag gcattac 27

<210> 19

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> argJ_R

<400> 19

ttctttttaa gagctgtacg cggagttgat ctcc 34

<210> 20

<211> 1332

<212> DNA

<213>Unknown

<220>

<223> arg A E.coli

<400> 20

gtggtaaagg aacgtaaaac cgagttggtc gagggattcc gccattcggt tccctatatc 60

aatacccacc ggggaaaaac gtttgtcatc atgctcggcg gtgaagccat tgagcatgag 120

aatttctcca gtatcgttaa tgatatcggg ttgttgcaca gcctcggcat ccgtctggtg 180

gtggtctatg gcgcacgtcc gcagatcgac gcaaatctgg ctgcgcatca ccacgaaccg 240

ctgtatcaca agaatatacg tgtgaccgac gccaaaacac tggaactggt gaagcaggct 300

gcgggaacat tgcaactgga tattactgct cgcctgtcga tgagtctcaa taacacgccg 360

ctgcagggcg cgcatatcaa cgtcgtcagt ggcaatttta ttattgccca gccgctgggc 420

gtcgatgacg gcgtggatta ctgccatagc gggcgtatcc ggcggattga tgaagacgcg 480

atccatcgtc aactggacag cggtgcaata gtgctaatgg ggccggtcgc tgtttcagtc 540

actggcgaga gctttaacct gacctcggaa gagattgcca ctcaactggc catcaaactg 600

aaagctgaaa agatgattgg tttttgctct tcccagggcg tcactaatga cgacggtgat 660

attgtctccg aacttttccc taacgaagcg caagcgcggg tagaagccca ggaagagaaa 720

ggcgattaca actccggtac ggtgcgcttt ttgcgtggcg cagtgaaagc ctgccgcagc 780

ggcgtgcgtc gctgtcattt aatcagttat caggaagatg gcgcgctgtt gcaagagttg 840

ttctcacgcg acggtatcgg tacgcagatt gtgatggaaa gcgccgagca gattcgtcgc 900

gcaacaatca acgatattgg cggtattctg gagttgattc gcccactgga gcagcaaggt 960

attctggtac gccgttctcg cgagcagctg gagatggaaa tcgacaaatt caccattatt 1020

cagcgcgata acacgactat tgcctgcgcc gcgctctatc cgttcccgga agagaagatt 1080

ggggaaatgg cctgtgtggc agttcacccg gattaccgca gttcatcaag gggtgaagtt 1140

ctgctggaac gcattgccgc tcaggcgaag cagagcggct taagcaaatt gtttgtgctg 1200

accacgcgca gtattcactg gttccaggaa cgtggattta ccccagtgga tattgattta 1260

ctgcccgaga gcaaaaagca gttgtacaac taccagcgta aatccaaagt gttgatggcg 1320

gatttagggt aa 1332

<210> 21

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> argA ORF_F

<400> 21

gaaaggtgca caaagatggt aaaggaacgt aaaaccg 37

<210> 22

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> argA ORF_R

<400> 22

gcccactagt ctcgagcatg cggcgttgat tttg 34

<210> 23

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> argE ORF_F

<400> 23

gaaaggtgca caaagatgaa aaacaaatta ccgcc 35

<210> 24

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> argE ORF_R

<400> 24

gcccactagt ctcgaggttt gagtcactgt cggtcg 36

<210> 25

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Ncgl2644 ORF_F

<400> 25

gaggagatca aaacacatat gacgcctagt cttccccg 38

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Ncgl2644 ORF_R

<400> 26

ttagaatttc cgttcggcgt acc 23

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> argJ_N_F

<400> 27

cgggatccca cgcctgtctg gtcgc 25

<210> 28

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> argJ_N_R

<400> 28

acgcgtcgac ggatctaaag cggcgcttc 29

<210> 29

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> argJ_C_F

<400> 29

acgcgtcgac tccggcgctg acattgatgt cc 32

<210> 30

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> argJ_C_R

<400> 30

ctagtctaga gagctgcacc aggtagacg 29

<---

Claims (13)

1. A microorganism of the genus Corynebacterium , which has the ability to produce putrescine, into which N-acetyltransferase activity originating from a strain of the genus Corynebacterium and acetylornithine deacetylase (argE) activity originating from E. coli are introduced. 2. The microorganism of the genus Corynebacterium according to claim 1, wherein the N-acetyltransferase derived from a strain of the genus Corynebacterium contains the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto. 3. The microorganism of the genus Corynebacterium according to claim 1, wherein the acetylornithine deacetylase (argE) derived from E. coli contains the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity thereto. 4. The microorganism of the genus Corynebacterium according to claim 1, which additionally has a weakened activity of bifunctional ornithine acetyltransferase/N-acetylglutamate synthase (argJ) originating from a strain of the genus Corynebacterium , where the weakened activity is obtained through deletion of the argJ gene. 5. A microorganism of the genus Corynebacterium according to claim 1, which is Corynebacterium glutamicum . 6. A method for producing putrescine, comprising cultivating in a medium a microorganism of the genus Corynebacterium , which has the ability to produce putrescine, into which the activity of N-acetyltransferase originating from a strain of the genus Corynebacterium and the activity of acetylornithine deacetylase (argE) originating from E. coli are introduced. 7. The method according to claim 6, further comprising isolating putrescine from the culture medium or microorganism. 8. The method according to claim 6, wherein the N-acetyltransferase, derived from a strain of the genus Corynebacterium , has the amino acid sequence SEQ ID NO: 1. 9. The method of claim 6, wherein the E. coli -derived acetylornithine deacetylase has the amino acid sequence of SEQ ID NO: 3. 10. A composition for producing putrescine containing a microorganism of the genus Corynebacterium having the ability to produce putrescine, into which the activity of N-acetyltransferase originating from a strain of the genus Corynebacterium and the activity of acetylornithine deacetylase (argE) originating from E. coli and an excipient are introduced. 11. The composition of claim 10, wherein the N-acetyltransferase derived from a strain of the genus Corynebacterium contains the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto. 12. The composition of claim 10, wherein the E. coli -derived acetylornithine deacetylase comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity thereto. 13. The use of a microorganism of the genus Corynebacterium , which has the ability to produce putrescine, to produce putrescine, where the activity of N-acetyltransferase originating from a strain of the genus Corynebacterium and the activity of acetylornithine deacetylase (argE) originating from E. coli are introduced into this microorganism.