TW202030327A - Ornithine decarboxylase variants，methods for producing putrescine using the same, polynucleotide, microorganism, method for preparing polyamine, and composition for preparing polyamide - Google Patents

Abstract

The present application relates to: an ornithine decarboxylase or a protein variant thereof; a polynucleotide encoding same; a microorganism containing same; and a method for producing putrescine by using same. The present invention achieves effects of increasing the productivity, production efficiency or production selectivity of putrescine, and reducing costs during putrescine purification by suppressing side reactions.

Description

Ornithine decarboxylase variant and method for producing butanediamine using it

This application relates to a variant of ornithine decarboxylase, a gene encoding the ornithine decarboxylase variant, microorganisms including the ornithine decarboxylase variant, and the synthesis of butanediamine using the same.

This application claims the benefit of priority based on Taiwan Patent Application No. 107147749 filed on December 28, 2018, and all the contents disclosed in the documents of this patent application are included as part of this specification.

Putrescine (1,4-diamino-butane) is a substance that causes rotten organisms to produce malodor, but it can be used to synthesize 4,6-nylon, so it is industrially produced. At present, more than 10,000 tons of butanediamine are produced annually due to petroleum resources, but there is a problem of unstable supply and demand of raw materials due to frequent changes in petroleum prices. In addition, there is a problem of environmental pollution due to the production of large amounts of toxic substances during the production process.

In order to solve this problem, the synthesis of bio-derived butanediamine is being actively studied recently. For example, research is under way to synthesize butanediamine from ornithine derived from organisms or to mass-produce butanediamine from microorganisms using sugar.

Regarding the method of producing butanediamine from microorganisms, various biological engineering methods to increase the production of butanediamine have been used. The above method may be the following: for example, using a promoter to regulate the activity of an enzyme involved in the biosynthesis of butanediamine; overexpression of antiporters to easily release butanediamine to the outside of the cell; or blocking the decomposition of butanediamine route of. Among them, it is known that regulating the activity of enzymes involved in the biosynthesis of butanediamine in microorganisms greatly contributes to increasing the production of butanediamine.

Ornithine decarboxylase is an enzyme that cleaves the terminal carboxyl group of ornithine to synthesize butanediamine, and it is one of the enzymes that performs an important role in the biosynthesis of butanediamine. However, ornithine decarboxylase not only synthesizes butanediamine from ornithine acid, but also has the activity of synthesizing lysine into 1,5-diamino-pentane (side reaction), so its activity is improved In this case, it will produce pentane diamine together with butane diamine and reduce the yield of butane diamine. The above-mentioned pentamethylene diamine can also cause many problems when refining butanediamine. Specifically, in the process of purifying microbial culture broth by distillation, due to the combination of butanediamine (H ₂ N(CH ₂ ) ₄ NH ₂ ) and pentane diamine (H ₂ N(CH ₂ ) ₅ NH ₂ ) The structure is very similar, so it takes a lot of cost and time to selectively refine the above-mentioned microorganism culture solution.

Therefore, when the activity of ornithine decarboxylase is to be adjusted, it is very important to maintain the activity of synthesizing butanediamine from ornithine and to reduce the activity of synthesizing pentanediamine from lysine (side reaction).

Therefore, the inventors discovered a novel ornithine decarboxylase, and completed the application by confirming that the synthetic activity of pentane diamine is low and the synthesis activity of butanediamine is high among the above-mentioned ornithine decarboxylase.

[The problem to be solved by the invention]

This application provides an ornithine decarboxylase or a variant thereof.

In addition, this application provides a polynucleotide encoding the above-mentioned ornithine decarboxylase or a variant thereof.

This application provides a microorganism that produces butanediamine including the above-mentioned ornithine decarboxylase or a variant thereof.

Another object of the present application is to provide a method for producing butanediamine including the step of culturing the above-mentioned microorganism in a medium.

Another object of the present application is to provide a method for increasing the purity of butanediamine including the step of culturing the above-mentioned microorganism in a medium.

Another object of the present application is to provide a method for increasing the ratio of butanediamine to pentamethylenediamine that includes the step of culturing the above-mentioned microorganism in a medium.

Moreover, another objective of the present application is to provide a use of the above-mentioned butanediamine to synthesize polyamide polymers. [Means to solve the problem]

If this is specifically described, it will be as follows. On the other hand, each of the descriptions and embodiments disclosed in this application can also be applied to the other descriptions and embodiments of each. That is, all combinations of various elements disclosed in this application belong to the scope of this application. In addition, the scope of this application is not limited to the specific description described below.

One embodiment of the present application provides a variant of ornithine decarboxylase, which has an amino acid sequence of SEQ ID NO: 1 including more than one amino acid substituted butanediamine production activity.

Specifically, this application provides a protein variant. In the amino acid sequence of SEQ ID NO:1, i) the alanine as the 713th amino acid is substituted with other amino acids and/or ii) The glutamic acid, which is the 698th amino acid, is substituted with another amino acid. The above amino acid substitution may include: i) Alanine as the 713th amino acid is selected from leucine, isoleucine, valine, arginine, aspartic acid, tryptophan and gluten The amino acid in the amino acid is substituted; and/or ii) the glutamic acid as the 698th amino acid is substituted by aspartic acid.

In this application, the term "butanediamine" is a substance produced by the decarboxylation reaction of ornithine acid or the hydrolysis of spermine. Although it is also present in decay, it is widely distributed in organisms as a normal component. As a kind of polyamine, it has the function of forming ribosomes, promoting cell growth or promoting ribonucleic acid (RNA) synthesis. In particular, it is an important raw material for the production of polyamide 4 and polyamide 6 including nylon 4 and nylon 6, and is a substance that still needs to be mass-produced.

Butanediamine can be produced by using ornithine as a substrate. In addition, after synthesizing ornithine using a substance that becomes a precursor of ornithine acid as a substrate, butanediamine can be produced from the ornithine acid. As long as the synthesis of ornithine acid can be easily selected by the manufacturer, it can be used without limitation.

In this application, the term "ornithine" is a basic amino acid that plays an important role in the ornithine cycle, especially L-ornithine is widely found in plants, animals, and microorganisms. Generally, in organisms with ornithine acid cycle, it is related to element production and plays an important role in metabolism. In addition, it can mutually transform with arginine, glutamine, and proline in the living body to transfer keto acid, glyoxylic acid and amino groups. As a substrate for the production of amine (butanediamine) by ornithine decarboxylase, it is synthesized into polyamines. In the present invention, L-ornithine is particularly useful as a substrate for ornithine decarboxylase.

In this application, the term "ornithine decarboxylase (ODC)" is an enzyme that promotes the following reaction formula as the first step in the synthesis of polyamines and the last step in the production route of putrescine . In this application, ornithine decarboxylase can be mixed with ornithine decarboxylase. ODC uses L-ornithine as a substrate to produce butanediamine, and Pyridoxal phosphate (PLP) acts as a co-factor.

[Reaction formula]

L-ornithine>=>butanediamine+CO ₂

Figure 1 shows the chemical reaction formula of the process of synthesizing butanediamine using ornithine as a substrate using ornithine decarboxylase. In addition, it indicates the side reaction of ornithine decarboxylase that needs to be inhibited, that is, the cadaverine synthesis pathway.

In this application, the method for obtaining ODC (ornithine decarboxylase) can be applied to various methods well known in the art. As an example of this method, a bioinformatics method can be used based on gene synthesis technology including codon optimization in a way that can obtain enzymes with high efficiency in microorganisms that are generally widely used for enzyme expression, and a large amount of genomic information of microorganisms. It is obtained by screening useful enzyme resources and is not limited to this.

In this application, SEQ ID NO: 1 refers to the amino acid sequence of ornithine decarboxylase with butanediamine production activity. The amino acid sequence of SEQ ID NO: 1 can be obtained from the GenBank of the National Center for Biotechnology Information (NCBI), which is a well-known database. As an example, ornithine decarboxylase can be derived from Lactobacillus sp., Saccharomyces sp. or Escherichia coli (E. coli), specifically, it can be derived from Lactobacillus sp. saerimneri), but it is not limited to this, as long as it is an amino acid sequence of a protein having the same activity as the protein including the above-mentioned amino acid sequence, it may be included without limitation. In addition, as the ornithine decarboxylase with butanediamine production activity in the present application, a protein including the amino acid sequence of SEQ ID NO:1 is described, but it is not excluded from the amino acid sequence of SEQ ID NO:1 A meaningless addition to the sequence before and after the acid sequence, a mutation that can occur naturally, or a silent mutation thereof, as long as it has the same or corresponding activity as the protein including the amino acid sequence of SEQ ID NO:1 , The industry understands that it is equivalent to the protein with the activity of butanediamine in this application. Specifically, for example, the protein with butanediamine production activity of the present application may be the amino acid sequence of SEQ ID NO:1, or 80%, 85%, 90%, 95%, 96%, 97 %, 98% or 99% homology or identity of the amino acid sequence of the protein. In addition, as long as it is an amino acid sequence that has such homology or identity and exhibits functions corresponding to the above-mentioned protein, it should be understood that a protein has an amino acid sequence in which a part of the sequence is deleted, deformed, substituted, or added. It is also included in the scope of the protein to be the subject of mutation in this application.

That is, in the present application, even if "protein or polypeptide having the amino acid sequence described in a specific SEQ ID NO" or "protein or polypeptide including the amino acid sequence described in the specific SEQ ID NO" is described, as long as In order to have the same or corresponding activity as the polypeptide comprising the amino acid sequence of the corresponding SEQ ID NO, it should be understood that the protein with a part of the sequence deletion, modification, substitution or addition of the amino acid sequence can also be used in This application. For example, as long as the "polypeptide including the amino acid sequence of SEQ ID NO: 1" is a sequence corresponding to SEQ ID NO: 1 or a sequence having the same or corresponding activity, it should be understood that it may belong to "including A polypeptide of the amino acid sequence of SEQ ID NO:1". For example, as long as it has the same or corresponding activity to the above-mentioned mutant protein, the addition of sequences that do not change the function of the protein before and after the above-mentioned amino acid sequence, naturally occurring mutations, and silent mutations are not excluded. ) Or reservation substitution, and it should be understood that when there is such a sequence addition or mutation, it also falls within the scope of this application.

In this application, the term "conservative substitution" refers to the substitution of an amino acid by another amino acid with similar structure and/or chemical properties. The aforementioned variants may still retain more than one biological activity, and have, for example, more than one retention substitution. Such amino acid substitutions can generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residue. For example, the positively charged (basic) amino acids of the amino acids with electrically charged amino acid include arginine, lysine and histidine, and the negatively charged (acidic) Amino acids include glutamine and aspartic acid. Among the amino acids with uncharged amino acid, the nonpolar amino acid includes glycine, alanine, and valeric acid. Amino acid, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline, polar (polar) or hydrophilic (hydrophilic) amino acids include serine, threonine, Cysteine, tyrosine, asparagine, and glutamic acid. The aromatic amino acids in the non-polar amino acids include amphetamine, tryptophan and tyrosine.

In this application, the term "variant" means that more than one amino acid is different from the recited sequence (the recited sequence) in conservative substitution and/or modification. , But retain the functions or properties of the aforementioned protein. The variant is different from the identified sequence due to several amino acid substitutions, deletions or additions. Generally, one or more amino acids in the amino acid sequence of the aforementioned protein can be deformed, and the characteristics of the deformed aforementioned protein can be evaluated to identify such variants. That is, compared with the native protein (native protein), the ability of the variant will increase, remain unchanged, or decrease. In addition, a part of the variant may include a variant obtained by removing one or more parts such as the N-terminal leader sequence or the transmembrane domain. Other variants may include variants obtained by removing a part from the N- and/or C-terminus of mature protein. The above-mentioned term "variant" can use terms such as variant, modified, mutated protein, variant polypeptide, and variant (in English, it means modification, modified protein, modified polypeptide, mutant, mutein, divergent, variant, etc.), as long as it is a variant The meaning of the terms used is not limited to this. For the purpose of this application, the aforementioned variant may be a variant protein whose activity is increased compared to a natural wild-type or non-deformed protein, but it is not limited thereto.

In addition, variants may include deletions or additions of amino acids that have the least impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of a protein that participates in the transfer of the protein co-translationally or post-translationally. In addition, the above-mentioned polypeptides can be conjugated with other sequences or linkers to confirm, refine or synthesize the polypeptides.

The protein variant of this application may be an ornithine decarboxylase variant. In this application, the term "ornithine decarboxylase variant" can be used with "variant ODC protein, ODC variant, variant ornithine decarboxylase, variant ornithine decarboxylase, variant ODC protein, ODC variant, variant ODC enzyme protein, variant ODC enzyme "etc. are mixed.

The above variant may be that any one or more of the 713th and 698th amino acids in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid different from the amino acid before substitution. Winner.

As long as it is an amino acid different from the amino acid before substitution, the above-mentioned "substitution with another amino acid" is not limited. For example, alanine, which may include the 713th amino acid as the amino acid sequence of SEQ ID NO:1, is composed of hydrophobic amino acids, basic amino acids, acidic amino acids, neutral Amino acid or aromatic amino acid substitution. That is, as long as the alanine, which is the 713th amino acid of the amino acid sequence of SEQ ID NO: 1, is substituted with an amino acid residue other than alanine, or the glutamine is the 698th amino acid The acid is substituted by amino acid residues other than glutamic acid without limitation. On the other hand, in this application, when it is expressed as "the specific amino acid has been substituted", even if it is not otherwise indicated that it is substituted by another amino acid, it should be understood that it is different from the amino acid before substitution. Amino acid substitutes.

Specifically, the above variant may be in the amino acid sequence of SEQ ID NO:1, i) the alanine as the 713th amino acid is substituted by another amino acid, and/or ii) as the 698th amino acid. A variant in which the glutamic acid of an amino acid is substituted by another amino acid. The above substitution by other amino acids may be the following: i) Alanine as the 713th amino acid is selected from leucine, isoleucine, valine, arginine, aspartic acid, tryptamine The amino acid in the acid and glutamic acid is substituted; and/or ii) the glutamic acid as the 698th amino acid is substituted with aspartic acid. More specifically, the above variant may be i) Alanine as the 713th amino acid is selected from leucine, isoleucine, valine, arginine, aspartic acid, tryptophan and The amino acid in the glutamic acid is substituted, and/or ii) a variant in which the glutamic acid of the 698th amino acid is substituted by aspartic acid.

In the amino acid sequence of SEQ ID NO: 1, i) alanine as the 713th amino acid is selected from leucine, isoleucine, valine, arginine, aspartic acid, The amino acid substitution of the amino acid and the glutamic acid, and/or ii) the variant in which the 698th amino acid is substituted by aspartic acid, may include a variant selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 23 of any amino acid sequence, specifically, may have to be from SEQ ID NO: 4, SEQ ID NO: 8, Any amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 23 constitutes (consisting essentially of), more specifically, may include SEQ ID NO: 4, SEQ ID NO: 8 , SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 23, any amino acid sequence, but it is not limited thereto.

The above-mentioned variants may be the following: at positions corresponding to the 713th and/or 698th positions of SEQ ID NO:1, the substitution of other amino acids is included, and the amino acid sequence of SEQ ID NO:1 has at least 80%, 90%, 95%, 96%, 97%, 98% or more than 99% and less than 100% sequence homology, and has butanediamine production activity.

In addition, the above-mentioned variant may include any amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 19 to SEQ ID NO: 23, or a combination with the above-mentioned amino acid sequence. The acid sequence has more than 80% homology or identity of the amino acid sequence, but it is not limited to this. Specifically, the variant of the present application may include any amino acid sequence in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 19 to SEQ ID NO: 23. A polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity. In addition, as long as it is an amino acid sequence that has such homology or identity and exhibits functions corresponding to the above-mentioned protein, it has a part of the sequence that is missing, deformed, excluding the 713th or 698th amino acid position. Proteins with substituted or added amino acid sequences are also included in the scope of this application.

In this application, the term "homology" or "identity" refers to the degree of correlation with two given amino acid sequences or base sequences, which can be expressed as a percentage. The terms homology and consistency are often used interchangeably.

The sequence homology or identity of the conserved polynucleotide or polypeptide is determined by standard permutation algorithms, and the preset gap penalty established by the program used can be used together. In essence, a homologous or identical sequence can usually be along the entire sequence or at least about 50%, 60%, 70%, 80%, or 90% of the entire length. Hybridization under stringent conditions. Hybridization also considers polynucleotides that contain degenerate codons instead of codons.

For example, the default parameters in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 can be used by well-known computer algorithms such as the "FASTA" program Determine whether any two polynucleotide or polypeptide sequences have homology, similarity or identity. Alternatively, you can use the Nederman program (EMBOSS: The European Molecular Biology Open Software Suite), such as the EMBOSS package, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or higher) implemented in the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.) 48 : 443-453) confirmed (including GCG 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) The Society for Industrial and Applied Mathematics (SIAM J Applied Math) 48: 1073). For example, BLAST or ClustalW of the National Center for Bioengineering Information Database can be used to determine homology, similarity or consistency.

For example, as announced in Smith and Waterman, Advances in Applied Mathematics (Adv. Appl. Math) (1981) 2:482, such as Needleman et al. (1970), J Mol Biol. 48: The 443 GAP computer program compares sequence information to determine the homology, similarity or identity of polynucleotides or polypeptides. Briefly, the GAP formula is defined as the number of similarly arranged symbols (ie, nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program can include: (1) Binary comparison matrix (contains a value of 1 for consistency and a value of 0 for non-consistency), and such as Schwartz and Dayhoff, eds., protein Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), disclosed by Gribskov et al (1986) Nucl. Acids Res.) 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) replacement matrix); (2) 3.0 penalty for each gap and 0.10 penalty for each symbol in each gap ( Or gap opening penalty 10, gap expansion penalty 0.5); and (3) no penalty for end gaps. Therefore, as the user in this application, the term "homology" or "identity" means the relevance between sequences.

In addition, you can confirm whether any two polynucleotide or polypeptide sequences have homology, similarity or identity by comparing sequences under defined stringent conditions by southern hybridization experiments. The defined appropriate hybridization conditions are corresponding Within the technical scope, methods well known to the industry can be used (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al. al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

In this application, the term "ornithine decarboxylase variant" can be used in conjunction with ornithine decarboxylase variant polypeptides, ornithine decarboxylase protein variants, ornithine decarboxylase capable of producing butanediamine. Protein variant polypeptide, ornithine decarboxylase variant polypeptide, ornithine decarboxylase variant, ornithine decarboxylase protein variant, variant ornithine decarboxylase, variant ornithine decarboxylase protein And so mixed. In addition, the aforementioned ornithine decarboxylase may be derived from Lactobacillus sp., Saccharomyces sp. or Escherichia coli (E. coli), but is not limited thereto.

The above-mentioned ornithine decarboxylase variants may include variations at the 713th and/or 698th positions in the amino acid sequence of SEQ ID NO:1, even those with addition or deletion of amino acids in SEQ ID NO:1 The amino acid sequence, as long as it is a variant of the amino acid substitution at the position corresponding to the amino acid No. 713 and/or No. 698 from the N-terminus of SEQ ID NO: 1, is included in the scope of the application .

Regarding the positions of amino acid residues, the term "corresponding to" disclosed in this application refers to the amino acid residues at the positions listed in the protein or peptide, or with the residues listed in the protein or peptide. Similar, identical or homologous amino acid residues. The "corresponding region" used in this application usually refers to a similar position in a related protein or a reference protein.

The above-mentioned ornithine decarboxylase protein variants are those in which the 713th and/or 698th amino acid in the amino acid sequence of SEQ ID NO: 1 are replaced by other amino acids, which may include SEQ ID NO: 1 The amino acid sequence or the mutant ornithine decarboxylase protein with enhanced activity compared with the mutant pre-ornithine decarboxylase from wild-type microorganisms. The above-mentioned ornithine acid decarboxylase protein variant refers to the amino acid sequence of SEQ ID NO: 1 described above having at least 80%, 85%, 90%, 95%, 96%, 97%, The amino acid at the 713th or 698th position corresponding to SEQ ID NO:1 among amino acids with 98% or more homology or identity.

The above-mentioned 713th and/or 698th amino acid variation can be i) Alanine as the 713th amino acid consists of leucine, isoleucine, valine, arginine, aspartic acid , Tryptophan or glutamic acid substitution, and/or ii) the glutamine as the 698th amino acid is substituted by aspartic acid.

Specifically, the aforementioned ornithine decarboxylase variant may be in the amino acid sequence of SEQ ID NO:1, i) alanine as the 713th amino acid is composed of leucine, isoleucine, and valerate. Amino acid, arginine, aspartic acid, tryptophan or glutamic acid substitution, and/or ii) the glutamic acid as the 698th amino acid is substituted by aspartic acid, and may have Compared with the protein including the amino acid sequence of SEQ ID NO: 1 above or the protein with enhanced activity of the mutant pre-ornithine decarboxylase protein from wild-type microorganisms.

For the purpose of this application, in the case of microorganisms including the above-mentioned ornithine decarboxylase protein variants, it is characterized by increasing the production of butanediamine, increasing the purity of butanediamine or increasing the production options of butanediamine Sex. The protein variant of the present application is characterized in that it has gene regulation activity to increase the production capacity of butanediamine, the purity of butanediamine or the selectivity of butanediamine production by a more natural wild-type or non-mutated ornithine decarboxylase. . In particular, it is significant in that it is possible to inhibit the synthesis of cadaverine, which is one of the side reactions of ornithine decarboxylase, by microorganisms into which the protein variant of the present application is introduced, and to increase the production of butanediamine.

Another embodiment of the present application provides a polynucleotide encoding a variant of the aforementioned ornithine decarboxylase protein.

The ornithine decarboxylase protein including the amino acid sequence of SEQ ID NO: 1 and its variants are as described above.

In this application, the term "polynucleotide" is used as a polymer of nucleotide monomers (monomers) that are linked by covalent bonds to form a chain of nucleotides longer than a fixed length. Deoxyribonucleic Acid (DNA) or RNA strands, more specifically, refer to polynucleotide fragments encoding the aforementioned variants.

The polynucleotide encoding the ornithine decarboxylase variant of the present application may be included without limitation as long as it is a polynucleotide sequence encoding the variant polypeptide having the production activity of butanediamine of the present application. In the present application, the gene encoding the amino acid sequence of the ornithine decarboxylase protein can be, for example, the speC, odc, spe1 or speF gene, and the aforementioned genes can be from Lactobacillus, Saccharomyces or Escherichia coli (Escherichia coli, E . coli), but not subject to this restriction. In addition, the above-mentioned gene may encode any amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 23 More specifically, the base sequence of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 24 to SEQ ID NO: 28 The sequence of any base sequence, but not limited to this.

Specifically, the polynucleotide of the present application can be within the range of not changing the amino acid sequence of the polypeptide due to the degeneracy of the codons or considering the codons preferred by the organisms that intend to express the above-mentioned polypeptides. Realize various deformations to the coding area. Specifically, as long as it is a polynucleoside encoding a variant of ornithine decarboxylase protein in which the 713th and/or 698th amino acid of the amino acid sequence of SEQ ID NO: 1 is substituted by another amino acid Acid sequences can be included without limitation.

In addition, as long as it is encoded under stringent conditions with a probe that can be prepared from a known gene sequence, such as the complementary sequence of the whole or part of the above-mentioned base sequence, the 713th and the 713th in the amino acid sequence of SEQ ID NO:1 /Or the sequence of the ornithine decarboxylase protein with butanediamine production activity in which the 698th amino acid is substituted by another amino acid, can be included without limitation. The above-mentioned "stringent conditions" refer to conditions that can achieve specific hybridization between polynucleotides. Such conditions are specifically described in the literature (for example, J. Sambrook et al., homology). For example, the following conditions can be cited: intergene hybridization with high homology or identity, having 40% or more, specifically 90% or more, more specifically 95% or more, and more specifically 97% or more , In particular, the hybridization between genes with more than 99% homology or identity, and the conditions for non-hybridization between genes whose homology or identity is lower; or the cleaning conditions of ordinary southern hybridization , Which is equivalent to 60℃, 1×SSC, 0.1% Sodium Dodecyl Sulfonate (SDS), specifically 60℃, 0.1×SSC, 0.1% SDS, more specifically 68 The conditions of washing once, specifically 2 to 3 times under the salt concentration and temperature of ℃, 0.1×SSC, 0.1% SDS.

Even though hybridization may cause mismatch between bases according to the stringency of hybridization, it requires that the two nucleic acids have complementary sequences. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with regard to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the present application may not only include substantially similar nucleic acid sequences, but also include isolated nucleic acid fragments that are complementary to the entire sequence.

Specifically, polynucleotides with homology or identity can be probed using hybridization conditions including a hybridization step at a Tm value of 55°C and using the above-mentioned conditions. In addition, the above-mentioned Tm value may be 60°C, 63°C, or 65°C, but it is not limited to this, and can be appropriately adjusted by the operator according to the purpose.

The appropriate stringency of a hybrid polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

Another embodiment of the present application provides a vector including a polynucleotide encoding an ornithine decarboxylase variant.

The ornithine decarboxylase including the amino acid sequence of SEQ ID NO:1, its variants, and the above-mentioned polynucleotide are as described above.

The term "vector" as used in this application refers to a polynucleotide that encodes the above-mentioned target polypeptide operably linked to an appropriate regulatory sequence in a manner that can express the target polypeptide in an appropriate host DNA preparation of base sequence. The above-mentioned regulatory sequence may include a promoter that can initiate transcription, any manipulation sequence used to regulate such transcription, a sequence encoding an appropriate messenger RNA (mRNA) ribosome binding site, and regulation of transcription and decoding The terminating sequence. After the vector is transformed into an appropriate host cell, it can replicate or function independently of the host genome, and can be integrated into the genome itself.

The carrier used in this application is not particularly limited, and any carrier known in the industry can be used. Examples of commonly used vectors include natural or recombinant plastids, mucilages, viruses, and bacteriophages. For example, as a phage vector or a mucin vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as a plastid vector, pBR, pUC, pBluescriptII can be used Class, pGEM class, pTZ class, pCL class and pET class, etc. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

As an example, the polynucleotide encoding the target polypeptide in the chromosome can be inserted into the chromosome by using a vector for intracellular chromosome insertion. The polynucleotide can be inserted into the chromosome by any method known in the industry, such as homologous recombination, but it is not limited to this. It may further include a selection marker to confirm whether the chromosome is inserted or not. Screening markers are used to screen cells transformed with vectors, that is, to confirm whether the target nucleic acid molecule has been inserted or not, and can use selectable tables that confer drug resistance, nutritional requirements, resistance to cytotoxic agents, or surface polypeptide performance Type logo. In an environment treated with a selective agent, only cells that exhibit screening markers or show different phenotypic traits survive, so transformed cells can be screened.

As another embodiment of the present application, the present application provides a microorganism that includes the above-mentioned ornithine decarboxylase or a variant thereof, or includes a polynucleotide encoding the above-mentioned enzyme to produce butanediamine.

In the present application, the term "microorganisms including variant polypeptides" or "microorganisms including ornithine decarboxylase variants" can be used as long as they include the protein variants of the present application and can produce butanediamine. , But not limited to this. For example, the microorganisms including the protein variants of the present application may be natural wild-type microorganisms or microbes that produce butanediamine that express the protein variants of the present application, so that the production capacity of butanediamine and the production of butanediamine Recombinant microorganisms with increased purity or selective production of butanediamine. The above-mentioned recombinant microorganisms may be microorganisms with increased production capacity, production purity, or butanediamine production selectivity compared to natural wild-type microorganisms or non-deformed microorganisms, but are not limited thereto.

Specifically, the above-mentioned microorganisms are microorganisms that exhibit ornithine decarboxylase variants that include more than one amino acid mutation in the amino acid sequence of SEQ ID NO:1, and the above-mentioned amino acid mutations may include those from the N-terminus. The 713th and/or 698th amino acid is replaced by another amino acid. In addition, the above-mentioned microorganism may be a microorganism expressing a variant polypeptide in which the 713th or 698th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid and has butanediamine production activity, But it is not limited to this.

The above-mentioned butanediamine, ornithine decarboxylase protein including the amino acid sequence of SEQ ID NO:1 and variants thereof are as described above.

In this application, the term "expressing/expressing" protein refers to a form in which the target protein is introduced into the microorganism or deformed in a manner expressed in the microorganism. When the above-mentioned target protein is a protein that exists in a microorganism, it refers to a state in which the activity is more intrinsic or strengthened before deformation. For the purpose of this application, the "target protein" may be a variant of the aforementioned ornithine decarboxylase protein with butanediamine production capacity.

Specifically, "introduction of protein" refers to exhibiting the activity of a specific protein that the microorganism does not originally possess, or exhibiting an activity that is higher than the intrinsic activity of the aforementioned protein or the activity before modification. For example, a polynucleotide encoding a specific protein may be introduced into a chromosome in a microorganism, or a vector including a polynucleotide encoding a specific protein may be introduced into a microorganism to exhibit its activity. In addition, "enhancement of activity" means that the activity is higher than the intrinsic activity of the specific protein possessed by the microorganism or the activity before modification. The above-mentioned "intrinsic activity" refers to the activity of a specific protein originally possessed by the parent strain before the transformation when the microorganism is transformed due to genetic variation caused by natural or human factors.

Specifically, the activity enhancement of the present application can be achieved by any one or more methods selected from the group consisting of the following methods, but is not limited to this: increase the cell of the gene encoding the protein variant of the present application A method of introducing a mutation into the expression regulatory sequence of a gene encoding the aforementioned protein variant; a method of replacing the gene expression regulatory sequence encoding an ornithine decarboxylase protein variant with a strongly active sequence; A method of replacing the gene of the protein variant with the gene encoding the wild-type protein of ornithine decarboxylase on the chromosome; and introducing the mutation into the gene encoding the aforementioned ornithine decarboxylase protein in a manner that enhances the activity of the aforementioned protein variant Methods; and methods for introducing protein variants into microorganisms.

In the above description, increasing the number of copies of the gene is not particularly limited, but it can be performed in a form that can be operably linked to a vector, or by being inserted into a chromosome in a host cell. Specifically, a vector can be introduced into a host cell in which a polynucleotide encoding the protein of the present application is linked in an operable manner and can be replicated and function independently of the host. Alternatively, a vector may be introduced into the chromosome of the host cell: the polynucleotide is operably linked and the polynucleotide may be inserted into the chromosome of the host cell. The polynucleotide can be inserted into the chromosome by any method known in the industry, such as homologous recombination.

There is no particular restriction on the modification of the expression regulating sequence in a manner that increases the expression of the polynucleotide, but it can be further enhanced by inducing sequence variation on the nucleic acid sequence by deletion, insertion, non-retention or retention substitution or a combination thereof It can be performed by regulating the activity of the sequence, or by replacing it with a nucleic acid sequence with stronger activity. The above-mentioned expression regulating sequences are not particularly limited thereto, and may include promoters, operating sequences, sequences encoding ribosome binding sites, sequences regulating the termination of transcription and decoding, and the like.

A powerful promoter can be connected to the upper part of the polynucleotide expression unit to replace the original promoter, and it is not limited to this. Examples of well-known powerful promoters include cj1 to cj7 promoters (Korea Registered Patent No. 10-0620092), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (Korean registered patent No. 10-1783170), O2 promoter (Korean registered patent No. 10-1632642), tkt promoter And yccA promoter, but not limited to this.

The deformation of the polynucleotide sequence on the chromosome is not particularly limited, and the nucleic acid sequence can be induced to express a regulatory sequence variation by deletion, insertion, non-reserved or reserved substitution or a combination thereof to further strengthen the aforementioned polynucleotide The activity of the sequence is performed, or it is performed by replacing it with a polynucleotide sequence that is improved in a way that has stronger activity.

The introduction and enhancement of protein activity as described above can be that the activity or concentration of the corresponding protein is generally increased to a minimum of 1%, 10%, 25%, etc., based on the activity or concentration of the protein in the wild-type or non-proteomorphic microorganism strain. 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000%, but not limited to this.

In the present application, a microorganism including the aforementioned ornithine decarboxylase variant or a polynucleotide encoding the aforementioned ornithine decarboxylase variant can be prepared by transforming a vector comprising the aforementioned polynucleotide Of recombinant microorganisms, but not limited to this.

In this application, the term "transformation" refers to introducing a vector including a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it may include both inserted into the chromosome of the host cell or located outside the chromosome. In addition, the aforementioned polynucleotides include DNA and RNA encoding the target protein. The aforementioned polynucleotide can be introduced in any form as long as it can be introduced into a host cell and expressed. For example, the above-mentioned polynucleotide can be introduced into a host cell in the form of an expression cassette as a gene structure including all elements required for self-expression. The performance cassette may generally include a promoter connected to the polynucleotide in an operable manner, a transcription termination signal, a ribosome binding site, and a translation termination signal. The above-mentioned performance cassette may be in the form of a performance vector that can realize self-replication. In addition, the above-mentioned polynucleotide may be introduced into a host cell in its own form and linked to a sequence required for expression in an operable manner in the host cell, and is not limited thereto.

In addition, in the above-mentioned content, the term "linked in an actionable manner" means that the polynucleotide encoding the target polypeptide of the present application starts transcription and mediates the promoter sequence to be functionally linked to the aforementioned gene sequence.

The term "non-modified microorganism" in this application refers to the natural strain itself, a microorganism that does not include the protein variant of the present application, or a vector that does not include the polynucleotide encoding the protein variant of the present application. Of microorganisms.

As long as the "microorganism" in the present application is a microorganism capable of producing butanediamine, it may include any of prokaryotic microorganisms and eukaryotic microorganisms.

In this application, the term "microorganisms that produce butanediamine" refers to wild-type microorganisms that naturally have butanediamine production capacity, or by introducing wild-type or variants into non-butanediamine production capacity or obvious production capacity Microorganisms with lower mother strain but with butadiamine production capacity. Specifically, it includes all microorganisms that cause genetic deformation naturally or artificially. As microorganisms that are weakened or strengthened by a specific mechanism due to insertion of external genes, enhancement of the activity of internal genes, or inactivation, etc., it can be used for the production of target diacetyl A microorganism that undergoes genetic mutation or enhanced activity due to amines. For the purpose of this application, the microorganisms of this application include the protein variants of this application, and therefore can increase the production capacity, production purity or production selectivity of the target butanediamine. Specifically, the microorganism of the present application may be a microorganism in which a part of genes in the butanediamine biosynthesis pathway is strengthened or weakened, or a part of the genes in the butanediamine decomposition pathway is strengthened or weakened. For the purpose of this application, a microorganism that produces butanediamine may refer to a microorganism with the following characteristics: including the above-mentioned ornithine decarboxylase variant, and therefore the yield of butanediamine to be produced from the carbon source in the culture medium, and The purity of amine or the selectivity of butanediamine production is increased compared with wild-type or non-deformed microorganisms. In this application, the above-mentioned "microorganisms producing butanediamine" can be mixed with "microorganisms capable of producing butanediamine" or "microorganisms producing butanediamine".

The above-mentioned microorganism producing butanediamine may be a recombinant microorganism. The aforementioned recombinant microorganism is as described above.

The microorganisms that produce the above-mentioned butanediamine are not particularly limited as long as they can produce butanediamine. Specifically, they may belong to the genus Corynebacterium, Escherichia, and Enterbacter. ), Erwinia (Erwinia), Serratia, Providencia, and Brevibacterium. More specifically, they may be microorganisms belonging to Corynebacterium ( Corynebacterium) or Escherichia (Escherichia) genus microorganisms.

More specifically, the microorganism of the genus Escherichia may be Escherichia coli, and the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum or Corynebacterium amine-producing (Corynebacterium ammoniagenes), Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium callunae, Corynebacterium deserti stationis), Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, Corynebacterium imitans ), Corynebacterium testudinoris (Corynebacterium testudinoris) or Corynebacterium flavescens (Corynebacterium flavescens), etc., and it can be Corynebacterium glutamicum, but it can include without limitation those that can introduce or strengthen ornithine Decarboxylase proteins to increase the production of butanediamine, the purity of butanediamine, and the production of butanediamine is selective for microorganisms of the genus Corynebacterium or Escherichia.

In the present application, the mother strain of a microorganism that produces butanediamine deformed to express ornithine decarboxylase protein or a variant of the aforementioned protein is not particularly limited as long as it is a microorganism that produces butanediamine.

Although the microorganisms of the genus Corynebacterium do not have a biosynthetic pathway for butanediamine, if ornithine decarboxylase (ODC) is introduced from the outside, butanediamine can be synthesized.

In addition, the above-mentioned microorganisms that produce butanediamine are not particularly limited, and may be the following: ornithine carbamoyltransfrase (ArgF) involved in the synthesis of arginine in ornithine, and glutamine The excreted protein (NCgl1221) is inactivated.

In addition, the above-mentioned microorganisms that produce butanediamine are not particularly limited. For example, they may be the following: in order to strengthen the biosynthesis pathway from glutamate to ornithine, convert glutamate to acetylglutamate (N-acetylglutamate ) Acetoglutamate synthase, or ornithine acetyltransferase (ArgJ), which converts acetoglutamate to ornithine, converts acetoglutamate to acetoglutamate phosphate (N -Acetylglutamate kinase (ArgB) of acetylglutamyl phosphate, acetylglutamate-phosphate reductase (ArgC) that converts acetylglutamyl phosphate into N-acetylglutamate semialdehyde (N-acetylglutamate semialdehyde) , The activity of acetylornithine transaminase (ArgD), which converts acetylornithine semialdehyde into N-acetylornithine, is stronger than its intrinsic activity, so it can be used as a raw material for the biosynthesis of butanediamine The productivity of ornithine is improved.

In addition, the above-mentioned microorganisms that produce butanediamine are not particularly limited, and may further be Corynebacterium microorganisms that have weakened butanediamine acetyltransferase activity and have the ability to produce butanediamine. In addition, the microorganism that produces the above-mentioned butanediamine is not particularly limited, and may be those that enhance the activity of butanediamine to excrete proteins, but it is not limited thereto.

In this application, the term "enhance/increase" includes all concepts that the activity is increased compared to the intrinsic activity.

The enhancement or increase of such gene activity can be achieved by applying various methods well known in the art. As an example of the above method, it may include any one or more methods selected from the group consisting of the following methods, and can also be achieved by a combination thereof, but is not particularly limited to the above example: increase the number of intracellular copies of genes; Methods of introducing mutations into gene expression regulatory sequences; methods of replacing gene expression regulatory sequences with sequences with strong activity; methods of introducing mutations into corresponding genes to enhance gene activity; and methods of introducing foreign genes into microorganisms.

In this application, the term "inactivation" includes all concepts that the activity is weakened or inactive compared to the intrinsic activity.

The inactivation of such gene activity can be achieved by applying various methods well known in the art. As an example of the above method, there are the following methods, which can also be achieved by a combination thereof, but are not particularly limited to the above example: including the case where the activity of the above gene is removed, and the whole or part of the gene on the chromosome is deleted Method; a method of replacing the gene encoding the aforementioned protein on the chromosome with a gene mutated in a manner that reduces the activity of the corresponding protein; a method of introducing a mutation into the expression regulatory sequence of the gene on the chromosome encoding the aforementioned protein; The method of replacing the expression regulatory sequence of the gene with a weaker or inactive sequence (for example, a method of replacing the promoter of the aforementioned gene with a promoter weaker than the intrinsic promoter); making the gene on the chromosome encoding the aforementioned protein The method of deleting the whole or part of the gene; the method of introducing the transcript of the gene complementary to the above-mentioned chromosome and preventing the translation of the above-mentioned mRNA into protein (for example, antisense RNA); for the gene encoding the above-mentioned protein A method of artificially appending a sequence complementary to the SD sequence at the front end of the SD sequence to form a secondary structure so that the ribosome cannot be attached; and reverse transcription to the open reading frame (ORF) of the corresponding sequence Reverse transcription engineering (Reverse transcription engineering, RTE) method, etc., with a promoter attached to the 3'end. In this application, the term "intrinsic activity" refers to the activity of a specific protein originally possessed by the mother strain before the transformation when the microorganism is transformed due to genetic variation caused by natural or human factors.

As yet another embodiment of the present application, there is provided a method for producing butanediamine including the step of culturing the above-mentioned microorganism that produces butanediamine in a medium.

The above-mentioned butanediamine, ornithine decarboxylase including the amino acid sequence of SEQ ID NO:1, its variants, protein expression and microorganisms are as described above.

In this application, the term "cultivation" refers to the growth of the above-mentioned microorganisms under appropriately adjusted environmental conditions. The cultivation process of this application can be realized by the appropriate medium and cultivation conditions known in the industry. The industry can easily adjust and use this kind of culture process according to the selected strain. Specifically, the above-mentioned culture may be batch type, continuous type and fed-batch type, but it is not limited thereto.

In this application, the term "medium" refers to a substance mixed with nutrients required for cultivating the above-mentioned microorganisms as main components, including water that is indispensable for survival and development to supply nutrients and growth factors. Specifically, as long as the medium and other culture conditions used to cultivate the microorganisms of the present application are medium for cultivating common microorganisms, any medium can be used without particular limitation, but may contain appropriate carbon sources, nitrogen sources, The microorganism of the present application is cultivated in a common medium of phosphorus, inorganic compounds, amino acids and/or vitamins, etc., under aerobic conditions by adjusting temperature, pH, etc.

In the above method, the step of culturing the above microorganism is not particularly limited, and can be performed by a well-known batch culture method, continuous culture method, fed-batch culture method, and the like. At this time, the culture conditions are not particularly limited, and alkaline compounds (for example, sodium hydroxide, potassium hydroxide, or amines) or acidic compounds (for example, phosphoric acid or sulfuric acid) can be used to adjust an appropriately large pH value (for example, pH 5 to pH 9). , Specifically pH 6 to pH 8, and most specifically pH 6.8), oxygen or oxygen-containing gas mixture can be introduced into the culture to maintain aerobic conditions. The culture temperature may be maintained at 20°C to 45°C, specifically 25°C to 40°C, and the culture may be about 10 hours to 160 hours, but is not limited thereto. The butanediamine produced by the above-mentioned culture can be secreted into the medium or left in the cells.

Furthermore, the culture medium used can be used alone or in combination with sugars and carbohydrates (for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oils and fats (for example, soybean oil, sunflower oil) , Peanut oil and coconut oil), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid) as carbon sources, but are not limited to this. Nitrogen-containing organic compounds (for example, egg whites, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea) or inorganic compounds (for example, ammonium sulfate, ammonium chloride, phosphoric acid) can be used alone or in combination. Ammonium, ammonium carbonate, and ammonium nitrate) are used as nitrogen supply sources, but are not limited thereto. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salt can be used alone or in combination as the phosphorus supply source, but it is not limited thereto. In addition, the culture medium may include other metal salts (for example, magnesium sulfate or iron sulfate), amino acids, and essential growth promoting substances such as vitamins.

The method of recovering the butanediamine produced in the above-mentioned culture step of the present application can collect the target amino acid from the culture solution by an appropriate method known in the art according to the culture method. For example, centrifugal separation, filtration, anion exchange chromatography, crystallization and high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) etc. can be used, and an appropriate method known in the art can be used to recover the target from the culture medium or microorganism. Butanediamine. The method for recovering the above-mentioned butanediamine may further include a refining step.

As another embodiment of the present application, there is provided a method for improving the purity of butanediamine including the step of cultivating the above-mentioned butanediamine-producing microorganism. In addition, there is provided a method of increasing the ratio of butanediamine to pentamethylenediamine including the step of cultivating the above-mentioned butanediamine-producing microorganism. The above-mentioned butanediamine and microorganisms are as described above.

As another embodiment of the present application, there is provided a use of butanediamine prepared by culturing the above-mentioned butanediamine-producing microorganism to prepare polyamide. In addition, there is provided a polyamide preparation composition including a microorganism that produces the above-mentioned butanediamine. The above-mentioned butanediamine and microorganisms are as described above.

The microorganisms that produce the above-mentioned butanediamine include microorganisms comprising the following ornithine decarboxylase: including the polypeptide of SEQ ID NO:1, or a) the 713th, b) the 698th or c) of SEQ ID NO:1 The positions corresponding to the 713th and 698th positions include amino acid substitutions. The polypeptide in SEQ ID NO:1 includes a polypeptide having a sequence homology of at least 80% and less than 100%. In addition, the step of cultivating the above-mentioned butanediamine-producing microorganism includes the step of cultivating a microorganism including the following ornithine decarboxylase: including the polypeptide of SEQ ID NO:1, or the 713th, a) of SEQ ID NO:1, b) The 698th or c) The positions corresponding to the 713th and 698th positions include amino acid substitutions. The polypeptide of SEQ ID NO:1 includes those with at least 80% sequence homology and less than 100% Peptides.

The above-mentioned polyamides are used as materials for various materials, and are excellent in heat resistance, chemical resistance, etc. due to the hydrogen bonds between the amide bonds, and therefore have been developed as materials for various materials. For example, the above-mentioned polyamide may be a fiber raw material, specifically, a nylon raw material. Polyamide fibers have excellent characteristics in terms of high strength, abrasion resistance, flexibility, gloss characteristics, and vividness of dyeing. Therefore, they can be used for leg wear such as stockings, inner wear, and sports. Clothing products such as sports wear. In addition, the above-mentioned polyamide can be a raw material for pharmaceuticals, surfactants, films, plastics, and the like. For example, in the case of using polyamide to prepare a film, it can achieve excellent optical properties and mechanical properties, as well as flexibility. Therefore, it can be used as a material for various molded products. The above polyamide film can be applied to a display substrate , Display protective film, touch panel, window cover of folding equipment, etc. [Invention Effect]

The ornithine decarboxylase of the present application has the effect of increasing the productivity or production efficiency of butanediamine and suppressing side reactions. In particular, the present application has the effect of hindering the synthesis of pentanediamine, which is one of the side reactions of ornithine decarboxylase, and thus achieves the effect of simplifying the purification/separation process of butanediamine and reducing production costs.

In addition, this application can realize various utilizations of polymer precursors, pharmaceuticals, chemical additives, etc. by mass production of butanediamine.

Hereinafter, the present application will be described in more detail through examples. However, the above-mentioned embodiments are used to exemplarily describe the present application, and the scope of the present application is not limited to the above-mentioned embodiments, which is obvious to those with common sense in the technical field to which the present application belongs.

Example 1. Comparison of the activity of ornithine decarboxylase from various sources The reactivity of the substrates of ornithine decarboxylase from four microorganisms was compared. Based on chicken-derived Lactobacillus (Lactobacillus saerimneri (inducible)), Saccharomyces cerevisiae (inducible), Escherichia coli (E.coli (constitutive)), Escherichia coli (E.coli (inducible)) The wild-type ornithine decarboxylase is the target, which is labeled ODC_Lb, ODC_Sc, ODC_Ec, ODC_Ef. After inserting the gene corresponding to the above enzyme into the pET24ma vector, E. coli BL21 (DE3) was used to express the protein under the conditions of 0.1 mM Isopropyl Thiogalactoside (IPTG) and 18°C. After that, the initial reaction rate was compared with 10% cell extract at 45°C. Compare the case of using 4 mM ornithine as the substrate and the case of using 4 mM lysine as the substrate.

Figure 2 shows the activity of ODC enzymes from the above four microorganisms. Specifically, it means ornithine decarboxylase activity that produces butanediamine using ornithine as a substrate, and lysine decarboxylase activity that produces pentane diamine using lysine as a substrate. The activities of lysine decarboxylase are all similar, but the ornithine decarboxylase from Lactobacillus (ODC_Lb) has the best activity. That is, in ODC_Lb, the lysine decarboxylase activity relative to the ornithine decarboxylase activity, that is, the generation ratio of side reactions is the lowest, and therefore the variants were prepared based on the above-mentioned content.

Example 2. Selection of the mutation position of ornithine decarboxylase The crystal structure of ornithine decarboxylase of Lactobacillus is known, and the tunnel through which the substrate enters and exits the enzyme can be predicted by analyzing the structure. In order to select the functional residues that perform saturation mutation in the predicted tunnel part, multiple sequence alignment using sequence information from bioinformatics is performed, from the ornithine decarboxylase amine used in the present invention. The positions of A696, V702, A713, and E698 were selected as the mutation positions for the N-terminus of the base acid sequence.

Residues that store amino acid residues at specific positions in the protein structure play a very important role in the structure and function of the above-mentioned protein. In particular, they are more likely to play a direct role in the catalytic process, so they are excluded. Is a variant residue.

Example 3. Functional residues of ornithine decarboxylase embodiment performs saturation variation and explore variants variants saturated (saturation mutagenesis) is specified position refers to a gene into various changes in the nucleotide sequence. Saturation mutation refers to inserting the NNK codon (codon) in the primer of the complementary sequence of the template strand to replace the sequence to be mutated to insert the mutation by polymerase chain reaction (PCR). At this time, in the NNK codon, N refers to A, T, G, and C of nucleotides, and K refers to T, G.

After using NNK codons to perform saturation mutation on the selected functional residues, perform screening on the variant library. All libraries are screened once and twice by whole cell reaction. The above-mentioned whole-cell reaction refers to a reaction in which cells including a specific enzyme are crushed and the cell contents are used, or the whole cell is used without isolating and purifying the enzyme. A screening is performed by the whole cell reaction of ornithine acid, and a variant that shows similar or faster activity when compared with the wild type is selected as the change in absorbance. The secondary screening is performed by whole-cell lysine reaction, and the variants selected in the first screening are selected if their reactivity to lysine is lower than that of the wild type.

For the selected variant enzymes, the specific activity when ornithine or lysine is used as the substrate is measured. Specific activity refers to the unit activity of pure protein that is purified by enzymes to remove impurities and other proteins. It is usually expressed as the number of units per 1 mg in units of the amount of enzyme that promotes 1 μmol of matrix changes in 1 minute. Specifically, ornithine decarboxylase derived from wild-type and variants of Lactobacillus was transformed into E. coli BL21 (DE3) and expressed in a culture volume of 50 mL using IPTG as an inducer, and then a Ni-NTA tube The column only refines pure protein. First, after protein expression, the cells are crushed by a sonic crusher, and the cell extract is obtained after centrifugal separation. The cell extract was put into a column equilibrated with 50 mM phosphate buffer (pH 8.0) added with 300 mM sodium chloride and combined with nickel resin (resin) at 0°C 1 hour. After that, the proteins that failed to bind to the resin were discarded, and other proteins that were not specifically bound were removed by Tris buffer including 50 mM imidazole. Finally, a Tris buffer containing 250 mM imidazole was used to dissolve only the desired protein. In order to remove imidazole from the eluted protein, a desalting process using a filter column is performed to obtain only active protein in the end. Thereafter, the Bradford protein quantification kit was used to determine the protein quality, and the same amount of protein was used to determine the specific activity.

The specific activity of ornithine decarboxylase derived from wild-type and variants of Lactobacillus when ornithine or lysine is used as a substrate is measured by HPLC (High-performance liquid chromatography) analysis method. The reaction was performed at 50°C for 30 minutes to 300 minutes, and the average value of three experiments was obtained. The initial reaction rate when a conversion yield of 10% to 25% is shown is determined. Using a cation exchange column, the mobile phase includes 0.6 g/L citric acid, 4 g/L tartaric acid, 1.4 g/L ethylenediamine, 5% methanol and 95% water. When the pH buffer used is at pH 5.0, use citric-sodium citrate buffer. The specific activity of ornithine decarboxylase of wild type and variants is shown in Figure 3.

As shown in Figure 3(a) and Figure 3(b), the wild-type and variants (A696E, V702G, A713L, A696E/A713L, V702G/A713L, A696E/V702G/A713L, E698D and E698D/A713L) enzyme Specific activity, the results confirmed that the above functional residues (A696, V702, A713, E698) are located in the active site (active site) or matrix close to the tunnel.

Specifically, when ornithine was used as a substrate, it was confirmed that the specific activity of the variants (A696E, V702G, A713L, A696E/A713L, V702G/A713L, A696E/V702G/A713L, E698D, E698D/A713L) was relative to The specific activities of the wild type were 19.9%, 4.3%, 89.4%, 12.8%, 4.9%, 0.1%, 75.6%, 74.4% (Figure 3(a)).

When using lysine as a substrate, the ratio of the specific activity of the variant (A696E, V702G, A713L, A696E/A713L, V702G/A713L, A696E/V702G/A713L, E698D, E698D/A713L) to the wild-type enzyme The activities were 16.9%, 0.6%, 42.4%, 4.4%, 0.9%, 0.7%, 50.8%, 29.2%, and it was confirmed that side reactions were suppressed (Figure 3(b)).

Example 4. Confirmation of the kinetic coefficients of the functional residues of ornithine decarboxylase. More detailed confirmation of the variants of ornithine decarboxylase used in the above example 3 that have a specific activity of 70% or more Features of A713L, E698D and E698D/A713L. In order to compare the kinetic parameters of the above variant and wild type, lysine with various concentration conditions was used. The kinetic coefficient uses matrix solutions with different concentrations to represent the matrix affinity and matrix conversion capacity values of enzymes.

Specifically, in order to confirm the kinetic coefficient of lysine of wild-type and variant ornithine decarboxylase that has completed protein purification, a lysine concentration of 0.45 mM to 140 mM is used. The pH buffer is pH5.0 citrate buffer (citric-sodium citrate buffer), and the reaction volume is 200μl. The analysis was performed by the above-mentioned HPLC analysis method as indicated, and the average value of three experiments was obtained. The kinetic coefficients of wild-type and variant lysine decarboxylase are shown in Figure 4.

As shown in Figure 4, it was confirmed that the k _cat value of the lysine acid of the variant (A713L) was 2.16 times lower than that of the wild type. K _cat lysine was confirmed due to the decrease of the value of k _cat variants (A713L) is / K _M value of 1.93 times compared to the wild-type reduction. As a result, it was confirmed that the variant (A713L) can reduce the activity of lysine decarboxylase. It was confirmed that the k _cat values of lysine acid of variants E698D and E698D/A713L were also reduced by 2.08 times and 2.59 times, respectively, and the k _cat /K _M values of lysine acid were confirmed to be reduced by 1.28 times and 1.67 times. That is, it was confirmed that the aforementioned variants can reduce side reactions.

Example 5. Analysis of the characteristics of the variant ornithine decarboxylase. The ornithine decarboxylase (A713L), which is the variant with the highest specific activity of ornithine among the variants, has the effect of butanediamine or pentanediamine. The impact of generation. A case where a high concentration of 51.5 g/L (0.39 M) ornithine was used as a substrate and a case where a concentration of 2.57 g/L (17.6 mM) lysine was used as a substrate were performed respectively. In order to set the appropriate reaction conditions for the reaction under the two substrate conditions, the buffer concentration was performed under the two conditions (0.1 M or 0.37 M) to make the pH appropriate.

Specifically, 0.1 mg of wild-type and variant enzymes that completed protein purification were used in the reaction. Use 0.39 M ornithine or 17.6 mM lysine as the matrix, and use 0.1 M or 0.37 M citric-sodium citrate buffer (pH 5.0) as the buffer. Using 0.1 mM PLP coenzyme, the reaction was carried out at 50°C with a reaction volume of 2 mL. The results are shown in Fig. 5.

Figure 5(a) is the case where 51.5 g/L (0.39 M) ornithine acid is used as the substrate. In the case of converting ornithine to butanediamine, when 0.37 M buffer is used (refer to ● and ○ in Figure 5(a)), 33.0 are generated from the wild type and the mutant (A713L) after 4 hours. g/L and 31.6 g/L butanediamine. In addition, when using a low-concentration buffer (0.1 M) (refer to ◆ and ◇ in Figure 5(a)), 7 hours later, the wild-type and variant (A713L) produced 20.2 g/L and 20.7 g/L, respectively Butanediamine. It was confirmed that the butanediamine production capacity of the wild-type and variant (A713L) was similar, and it was confirmed that the use of a high-concentration buffer (0.37 M) is beneficial to the reactivity.

Figure 5(b) shows the case where 2.57 g/L (17.6 mM) lysine is used as the substrate. In the case of converting lysine into pentanediamine, when using 0.37 M buffer (refer to ● and ○ in Figure 5(b)), 0.03 is generated from the wild type and the mutant (A713L) after 4 hours. g/L and 0.007 g/L pentanediamine. In addition, when using 0.1 M buffer (refer to ◆ and ◇ in Figure 5(b)), the side reaction of pentane diamine increased, and after 7 hours, 0.59 g/m of the wild-type and variant (A713L) were generated respectively. L and 0.38 g/L pentanediamine were used as side reactions. As a result, it was confirmed that the side reaction of the self-mutant (A713L) to produce pentane diamine was suppressed, and it was confirmed that the above-mentioned side reaction suppression effect was more prominent when a high concentration buffer (0.37 M) was used.

Example 6. Comparative determination of the expression level of recombinant ODC genes in coryneform strains to express the recombinant genes of ornithine decarboxylase ODC_Lb, ODC_Sc, ODC_Ec, ODC_Ef from the four microorganisms mentioned in Example 1. The production method is as follows.

Specifically, the genetic information of Lactobacillus from chicken (ACCESSION no. P43099), Saccharomyces cerevisiae (ACCESSION no. J02777.1) and E. coli str. K-12 (ACCESSION no. BAA35349) are used to specify the genes in Table 1. After the sequence is amplified by the gene coding region of the ornithine decarboxylase gene by PCR, the PCR product is treated with restriction enzymes and inserted into the plastids.

oclc_Lb_R （SEQ ID NO:33）

odc_Sc_F （SEQ ID NO:34）

cdc_Sc_R （SEQ ID NO:35）

Odc_Ec_F （SEQ ID NO:36）

Odc_Ec_R （SEQ ID NO:37）

Odc_Ef_F （SEQ ID NO:38）

Odc_Ef_R （SEQ ID NO:39）

[Table 1] Introduction Primer sequence odc_Lb_F (SEQ ID NO: 32)

oclc_Lb_R (SEQ ID NO: 33)

odc_Sc_F (SEQ ID NO: 34)

cdc_Sc_R (SEQ ID NO: 35)

Odc_Ec_F (SEQ ID NO: 36)

Odc_Ec_R (SEQ ID NO: 37)

Odc_Ef_F (SEQ ID NO: 38)

Odc_Ef_R (SEQ ID NO: 39)

The recombinant gene can be produced in a way that can additionally translate His-tag at the C-terminal of the protein. E. coli DH5α was used as a host strain for DNA manipulation, and E. coli BL21 (DE3) was used as a host strain for expressing the C-terminal His6-tag ODC gene. Recombinant Escherichia coli BL21 was grown in 50 mL of LB medium containing 50 μg/mL of kangmycin at 37°C. When the culture solution reached 0.8 under the OD ₆₀₀ condition, 0.2 mM IPTG was added to the culture solution. After induction of protein expression at 18°C to 30°C, cells were harvested. The cells are resuspended in the lysis buffer and subjected to ultrasonic treatment to destroy the cells. The obtained recombinant ODC was refined into Quiagen (Hilden, Germany) Ni-NTA agarose resin at 4°C. Centriplus YM-30 (Millipore, Bedford, MA) was used together with a molecular mass cut off of 100 kDa to obtain recombinant protein. The performance results are shown in Figure 6.

If the results of induction conditions at 30℃ are analyzed on the Sodium Dodecyl Sulfonate Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel, it can be confirmed that the recombinant ODC_Lb and The performance of ODC_Ec is higher than ODC_Sc and ODC_Ef. However, in the case of ODC_Ec, it was confirmed that the soluble protein mass was significantly reduced when the temperature was expressed. Furthermore, when performing expression at 37°C, the soluble protein mass of ODC_Ec was further reduced.

In coryneform strains, the ODC_Lb and ODC_Ec genes are expressed and the amount of soluble protein expressed as a comparative evaluation. The CJ7 promoter (KCCM10617, Korean Registered Patent No. 10-0620092) was introduced before the start codons of the ODC_Lb gene and ODC_Ec gene. First, in order to obtain a gene including the CJ7 promoter sequence, PCR using the primer pair specified in Table 2 was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template. The PCR reaction was carried out by repeating the following process 30 times: modification at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 30 seconds.

Co-CJ7_3 （SEQ ID NO:41）

[Table 2] Introduction Primer sequence Co-CJ7_5 (SEQ ID NO: 40)

Co-CJ7_3 (SEQ ID NO: 41)

After electrophoresis using a 1.5% agarose gel, it was confirmed that the PCR nucleic acid product had a size of 400 base pairs (bp). Use PCR prep kit (GeneAll, Seoul) to refine the obtained PCR products. BamHI and XbaI were put into the purified PCR product and pSCEC vector solution sample, and subjected to restriction enzyme treatment due to the reaction at 37°C for 4 hours. After electrophoresis on 1.5% agarose gel, the 400bp size was cut PCR nucleic acid product band and vector size band, and then use Gel prep kit (GeneAll, Seoul) to obtain nucleic acid fragments. After ligating the purified CJ7 promoter fragments of 1 mg each with the vector using T4 ligase, electrophoration was performed on the E. coli DH5α strain. Electroporation was applied at 2500 V. The recovered strains were smeared on an LB plate medium containing 50 µg/L spectinomycin and cultured at 37°C for 1 day, and then strains showing resistance were selected. After screening 18 strains and performing colony PCR with SEQ ID NO: 9 and SEQ ID NO: 10, PCR products with a size of 400 bp can be confirmed. According to the colony PCR results, the production of pSCEC_cj7 with CJ7 promoter was confirmed.

Based on the obtained pSCEC_cj7 vector and the primers shown in Table 3, the two genes ODC_Lb and ODC_Ec were amplified by PCR.

Co-ODC_Lb_3 （SEQ ID NO:43）

Co-ODC_Ec_5 （SEQ ID NO:44）

Co-ODC_Ec_3 （SEQ ID NO:45）

[table 3] Introduction Primer sequence Co-ODC_Lb_5 (SEQ ID NO: 42)

Co-ODC_Lb_3 (SEQ ID NO: 43)

Co-ODC_Ec_5 (SEQ ID NO: 44)

Co-ODC_Ec_3 (SEQ ID NO: 45)

The obtained PCR product and pSCEC_cj7 vector were processed by restriction enzymes XbaI and SalI. The nucleic acid treated with restriction enzymes was gel-prepared, and the ODC_Lb, ODC_Ec, and pSCEC_cj7 nucleic acid fragments were ligated and inserted into E. coli DH5α strain. PSCEC_cj7_ODC_Lb and pSCEC_cj7_ODC_Ec were obtained from the selected strains whose insertion was confirmed, and electrophoration was performed on the Corynebacterium genus KCCM11240P, which produces butanediamine, at 2500 V, respectively.

Spread the above strains on BHIS plate medium (Braine heart infusion 37 g/l, sorbitol 91 g/l, agar 2%) containing 50 µg/L spectinomycin to cultivate, thereby Form colonies. It has been confirmed that the selected strains can be used in CM medium (glucose 10 g/L, polypeptone 10 g/L, and yeast extract) containing 50 µg/L spectinomycin. 5 g/L, beef extract 5 g/L, NaCl 2.5 g/L, Urea 2 g/L, pH 6.8) for shaking culture. After culturing 3 mL of the produced two kinds of Corynebacterium glutamicum mutant strains, they were centrifuged to obtain bacterial cells. After pulverizing the cells of the obtained bacterial cells by an ultrasonic treatment method, they are centrifuged to obtain a solution containing soluble proteins.

The ODC_Lb and ODC_Ec proteins including His-tag were purified by Ni-NTA Spin Columns (Hilden, Germany). Use Nano drop to obtain and measure the protein concentration. The recombinant protein concentrations calculated based on the measured values were ODC_Lb: 1.282 g/L and ODC_Ec: 0.039 g/L. It was confirmed that ODC_Lb in the coryneform strain was 30 times more soluble protein than ODC_Ec.

It can be confirmed that when E. coli and coryneform strains are used as hosts to express ODC_Lb under moderate temperature conditions, a highly soluble protein can be produced due to high expression levels and normal protein wrinkles.

Example 7. Preparation of rod-shaped butanediamine producing strain with variant ornithine decarboxylase introduced and measurement of butanediamine production capacity In order to study the production and production of butanediamine by the ornithine decarboxylase variant of this application As a result of the influence of the above-mentioned ornithine decarboxylase variants, strains of microorganisms of the genus Corynebacterium with improved butanediamine production capacity were produced.

Specifically, as the microorganism of the genus Corynebacterium having the above-mentioned butanediamine-producing ability, the microorganism of the genus Corynebacterium having the ability to produce butanediamine disclosed in the patent application (Korea Patent Publication No. 2013-0082478) is used ( KCCM11240P). The above-mentioned Corynebacterium microorganism (KCCM11240P) with the ability to produce butanediamine is a microorganism prepared from Corynebacterium glutamicum ATCC13032 (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC( Ec): KCCM11138P (Korean Published Patent No. 2012-0064046)) a microorganism lacking NCgl1469.

A vector for replacing ornithine decarboxylase with a variant of ornithine decarboxylase derived from the above-mentioned Lactobacillus in the above-mentioned butanediamine producing microorganism is prepared. More specifically, the ODC_Lb_start (EcoRV)_5 and ODC_Lb_stop (MfeI)_3 primers disclosed in Table 4 below were used to compare the DNA of the Lactobacillus-derived ornithine decarboxylase variant prepared in Example 1 and Example 3. Perform amplification. Specifically, wild-type and variant (E698D, A713L) Lactobacillus ornithine decarboxylase were inserted into the prepared pET24ma vector and used as templates respectively, and the two primers L- disclosed in Table 4 below were used. odc_start (EcoRV)_5, L-odc_stop (MfeI)_3 execute PCR.

ODC_Lb_stop （Mfel）_3 （SEQ ID NO:47）

[Table 4] Introduction Primer sequence ODC_Lb_start (EcoRV)_5 (SEQ ID NO: 46)

ODC_Lb_stop (Mfel)_3 (SEQ ID NO: 47)

Treat the gene fragments amplified by PCR with EcoRV and MfeI restriction enzymes (37°C, 3 hours), and insert the gene fragments of wild-type and variant (E698D, A713L) ornithine decarboxylase from Lactobacillus To the pDZ-bioAD-P (CJ7) vector produced by the same method as that disclosed in Korean Patent Publication No. 2012-0064046. In the above method, EcoRV and MfeI restriction enzymes are used. The recombinant vectors for chromosome insertion (pDZ-ODC_Lb, pDZ-ODC_Lb_E698D, pDZ-ODC_Lb_A713L) prepared by the above method were confirmed by sequence analysis.

In order to obtain a coryneform strain with wild-type and variant ornithine decarboxylase from Lactobacillus inserted in the chromosome, the pDZ-ODC_Lb, pDZ-ODC_Lb_E698D, and pDZ-ODC_Lb_A713L recombinant vectors prepared above were respectively transformed by electroporation. After staining KCCM11240P strain, apply to BHIS plate medium (brain heart infusion 37 g/l, sorbitol 91 g/l, agar (agar) 2%, 1 L standard + kanamycin (kanamycin) 25 ug/ml) .

The successful insertion of the vector into the chromosome is determined by whether it shows blue in a solid medium containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). After shaking culture (30°C, 8 hours) in a nutrient medium, strains with a chromosome inserted once were serially diluted and smeared on a solid medium containing X-gal. Most of the colonies are blue. Conversely, white colonies can be screened at a low rate. The selected colonies can be obtained by cross over to the strain with the final Lactobacillus ornithine decarboxylase variant introduced into the chromosome . Finally, the strain was confirmed by sequence analysis of the variant. The confirmed strains were named KCCM11240P::ODC_Lb, KCCM11240P::ODC_Lb_E698D, KCCM11240P::ODC_Lb_A713L.

In order to confirm the effect of the introduction of wild-type and variant ornithine decarboxylase from Lactobacillus on the butanediamine production capacity of the butanediamine producing strain, the butanediamine production capacity was evaluated.

Specifically, the above-prepared strain was placed on a CM plate medium containing 1 mM arginine (glucose 1%, polyprotein 1%, yeast extract 0.5%, beef extract 0.5%, NaCl 0.25%, urea 0.2% , 50% NaOH 100 ul, 2% agar, pH 6.8, 1 L standard) after culturing at 30°C for 16 hours, inoculate about one type of platinum into 25 ml titer medium having the composition of Table 5 below, and then It was cultured with shaking at 200 rpm at 30°C for 24 hours. During fermentation, 1 mM arginine was added to the medium to cultivate all the strains produced.

[table 5] Composition Concentration/content (1 L standard) glucose 8% Soy protein 0.25% Corn steep liquor solids 0.5% (NH ₄ ) ₂ SO ₄ 4% Element 0.15% KH ₂ PO ₄ 0.1% MgSO ₄ -7H ₂ O 0.05% Biotin 100 ug Thiamine Hydrochloride 3000 ug thbrthdrexvbdr 3000 ug Nicotinamide 3000 ug CaCO ₃ 5%

As a result, as shown in Table 6, after 12 hours of culture, the strain introduced with the Lactobacillus-derived variant (A713L) ornithine decarboxylase was similar to the strain with E. coli ornithine decarboxylase (KCCM11240P). Compared with, butanediamine production increased by about 115%P. In addition, the strains introduced with A713L ornithine decarboxylase showed an increase of approximately 40% P compared to the strain with wild-type ornithine decarboxylase derived from Lactobacillus (KCCM11240P::ODC_Lb).

In addition, it can be seen that compared with KCCM11240P, the strain with A713L ornithine decarboxylase (KCCM11240P::ODC_Lb_A713L) produced pentane diamine due to side reactions during the production of butanediamine reduced by about 48% P, and it was found that the amount remained in the culture medium. It is estimated that the sugar consumption in the same time increases, and the productivity also increases.

[Table 6] Strain 12 hrs Insert (g/L) fold(%) Cad (ppm) fold(%) Residual glucose (g/L) KCCM11240P 1.3 100 39.427 100 41.7 KCCM11240P::ODC_Lb 2.0 154 26.196 66 37.3 KCCM11240P::ODC_Lb_A713L 2.8 215 20.367 52 29.0 KCCM11240P::ODC_Lb_E698D 2.7 208 23.243 59 29.8

The above results indicate that the introduction of a variant ornithine decarboxylase from Lactobacillus can not only produce butanediamine whose relative sugar consumption is higher than the existing concentration from butanediamine producing strains, but also reduce pentamethylenediamine. And the effect of improving productivity.

Example 8. Prediction of the influence of the saturation variation of the 713th amino acid residue of Lactobacillus ornithine decarboxylase In the selected variation, the 713th alanine of Lactobacillus ornithine decarboxylase is determined by leucine The acid-substituted functional residues (A713L) were replaced with amino acids other than alanine and leucine, and then introduced into the butanediamine production strain KCCM11240P to study the impact on butanediamine production.

Specifically, the following mutant strain was produced: instead of the wild-type ornithine decarboxylase derived from E. coli in the chromosome of a microorganism of the genus Corynebacterium (KCCM11240P) with improved butanediamine production capacity, the amine of SEQ ID NO:1 The base acid sequence is a variant in which the 713th amino acid from the N-terminus is substituted with another amino acid including a hydrophobic amino acid. More specifically, in order to separately produce valine, which is one of hydrophobic amino acids, arginine, which is one of basic amino acids, and aspartic acid, which is one of acidic amino acids, and neutral amino acids. One of the glutamic acid and the tryptophan-substituted vector of one of the aromatic amino acids, using the pDZ-ODC_Lb vector prepared in Example 7 as a template, using the above-mentioned Table 4 and the following Table 7 Primer to perform PCR. Perform a PCR on the front part (5') and the back part (3') centering on the site to be changed, and then perform a second PCR to match the two PCR fragments. For example, in the case of a variant (A713V) where the 713th amino acid is substituted from alanine to valine, the front part uses ODC_Lb_start (EcoRV)_5 and ODC_Lb_A713V_3 primers for amplification by PCR, and the back part uses ODC_Lb_A713V_5 and ODC_Lb_stop (MfeI)_3 primers are amplified by PCR. The two PCR fragments obtained by one PCR were used as templates for the second PCR, and PCR was performed using ODC_Lb_start (EcoRV)_5 and ODC_Lb_stop (MfeI)_3 primers. The finally obtained Lactobacillus ornithine decarboxylase variant A713V gene fragment was inserted into the pDZ-bioAD-P (CJ7) vector by the same method as in Example 7. The remaining variants A713R, A713D, A713W, and A713Q were also inserted into the pDZ-bioAD-P(CJ7) vector by PCR using the same method as above using the primers disclosed in Table 7. The prepared recombinant vectors for chromosome insertion (pDZ-ODC_Lb_A713V, pDZ-ODC_Lb_A713R, pDZ-ODC_Lb_A713D, pDZ-ODC_Lb_A713W, pDZ-ODC_Lb_A713Q) were confirmed by sequence analysis.

ODC_Lb_A713V_5 （SEQ ID NO:49）

ODC_Lb_A713R_3 （SEQ ID NO:50）

ODC_Lb_A713R_5 （SEQ ID NO:51）

ODC_Lb_A713D_3 （SEQ ID NO:52）

ODC_Lb_A713D_5 （SEQ ID NO:53）

ODC_Lb_A713Q_3 （SEQ ID NO:54）

ODC_Lb_A713Q_5 （SEQ ID NO:55）

ODC_Lb_A713W_3 （SEQ ID NO:56）

ODC_Lb_A713W_5 （SEQ ID NO:57）

[Table 7] Introduction Primer sequence ODC_Lb_A713V_3 (SEQ ID NO: 48)

ODC_Lb_A713V_5 (SEQ ID NO: 49)

ODC_Lb_A713R_3 (SEQ ID NO: 50)

ODC_Lb_A713R_5 (SEQ ID NO: 51)

ODC_Lb_A713D_3 (SEQ ID NO: 52)

ODC_Lb_A713D_5 (SEQ ID NO: 53)

ODC_Lb_A713Q_3 (SEQ ID NO: 54)

ODC_Lb_A713Q_5 (SEQ ID NO: 55)

ODC_Lb_A713W_3 (SEQ ID NO: 56)

ODC_Lb_A713W_5 (SEQ ID NO: 57)

In order to obtain a strain in which the 713th alanine of Lactobacillus ornithine decarboxylase inserted into the chromosome is substituted by other amino acids including hydrophobic amino acids, the method described in Example 7 can be used The pDZ-ODC_Lb_A713V, pDZ-ODC_Lb_A713R, pDZ-ODC_Lb_A713D, pDZ-ODC_Lb_A713W, and pDZ-ODC_Lb_A713Q recombinant vectors prepared above were respectively transfected into the KCCM11240P strain, and the final lactobacillus decarboxylase was introduced into the chromosome KCCM11240P strain. Variant strains. Finally, the strain was confirmed by sequence analysis of the variant. The confirmed strains were named KCCM11240P::ODC_Lb_A713V, KCCM11240P::ODC_Lb_A713R, KCCM11240P::ODC_Lb_A713D, KCCM11240P::ODC_Lb_A713Q, KCCM11240P::ODC_Lb_A713W.

In order to confirm the effect of a variant of the form in which the 713th alanine acid introduced into Lactobacillus ornithine decarboxylase is replaced by other amino acids including hydrophobic amino acids on the butanediamine production capacity of the butanediamine producing strain, The production capacity of butanediamine was evaluated by the method as in Example 7 above.

[Table 8] Strain 12 hrs Insert (g/L) fold(%) Cad (ppm) fold(%) Residual glucose (g/L) KCCM11240P 1.3 100 39.024 100 40.9 KCCM11240P::ODC_Lb 2.0 154 27.283 70 37.6 KCCM11240P::ODC_Lb_A713L 2.8 215 20.069 51 29.8 KCCM11240P::ODC_Lb_A713V 2.2 169 24.123 62 31.6 KCCM11240P::ODC_Lb_A713R 2.5 192 22.961 59 25.9 KCCM11240P::ODC_Lb_A713D 2.3 177 23.615 61 33.4 KCCM11240P::ODC_Lb_A713Q 2.6 200 21.845 56 27.8 KCCM11240P::ODC_Lb_A713W 2.1 162 26.074 67 30.0

As a result, as shown in Table 8, the strain introduced with a variant of Lactobacillus ornithine decarboxylase in the form of substitution with other hydrophobic amino acids also had a higher yield of butanediamine from Escherichia coli than after 12 hours of cultivation. The wild-type ornithine decarboxylase strain (KCCM11240P) increased by an average of about 86% P. In addition, it showed an average increase of about 21% P compared to the strain with wild-type ornithine decarboxylase derived from Lactobacillus.

In addition, it can be seen that the production of pentamethylenediamine was reduced by about 41%P due to the side reaction during the production of butanediamine, and it was estimated from the concentration of glucose remaining in the culture solution that the sugar consumption in the same time increased, and the productivity also increased.

The above results show that, in addition to the 713th alanine of the Lactobacillus ornithine decarboxylase, which is replaced by leucine, it is replaced by valine and basic amine, which is one of the other hydrophobic amino acids. Arginine, one of the base acids, aspartic acid, one of the acidic amino acids, glutamic acid, one of the neutral amino acids, tryptophan, one of the aromatic amino acids Substitution, therefore, not only can produce butanediamine with a higher concentration than the existing concentration of sugar consumption in the butanediamine production strain, but also has the effects of reducing pentamethylenediamine and improving productivity.

Based on the above description, those in the technical field to which this application belongs should understand that this application can be implemented in other specific forms without changing its technical ideas or necessary features. In connection with this, it should be understood that the embodiments described above are illustrative in all aspects and have no restrictive meaning. The scope of this application should be interpreted as that all changes or deformations derived from the meaning, scope and equivalent concepts of the scope of patent application described below are included in the scope of this application, rather than the above detailed description.

no

Fig. 1 is a schematic diagram showing the synthesis of butanediamine using ornithine as a substrate and ornithine decarboxylase in this application. In addition, it shows a pentanediamine synthesis route which is a side reaction of ornithine decarboxylase to be inhibited. Figure 2 shows the activity of ornithine decarboxylase from various sources, showing the relative activity of the reactivity when ornithine is used as a substrate and the reactivity (side reaction) when lysine is used as the substrate. ODC_Lb is from Lactobacillus chicken (inducible), ODC_Sc is from Saccharomyces cerevisiae (inducible), ODC_Ec is from Escherichia coli (constitutive), and ODC_Ef is from Escherichia coli (inducible). Figure 3 is a graph showing the comparison of (a) ornithine specific activity and (b) lysine specific activity quantified using wild-type ornithine decarboxylase derived from purified Lactobacillus and the following variants respectively ：The 696th alanine of the wild-type ornithine decarboxylase is replaced by glutamine (A696E); the 702nd valine of the wild-type ornithine decarboxylase is replaced by glycine (V702G); the wild-type ornithine The 713th alanine of the acid decarboxylase was replaced by leucine (A713L); the 696th alanine and the 713th alanine of the wild-type ornithine decarboxylase were replaced by glutamine and leucine (A696E/A713L) ); the 702nd valine and 713th alanine of wild-type ornithine decarboxylase are replaced by glycine and leucine (V702G/A713L); the 696th alanine of wild-type ornithine decarboxylase , The 702nd valine and the 713th alanine were replaced by glutamine, glycine and leucine (A696E/V702G/A713L); the 698th glutamine of the wild-type ornithine decarboxylase Partic acid substitution (E698D); the 698th glutamine and 713th alanine of wild-type ornithine decarboxylase are replaced by aspartic acid and leucine (E698D/A713L). Figure 4 is a graph comparing the kinetic coefficients of lysine and ornithine using wild-type ornithine decarboxylase from refined Lactobacillus and the following variants: the 698th bran of wild-type ornithine decarboxylase The amino acid was replaced by aspartic acid (E698D); the 713th alanine of the wild-type decarboxylase was replaced by leucine (A713L); the 698th glutamine and the 713th of the wild-type ornithine decarboxylase Alanine is replaced by aspartic acid and leucine (E698D/A713L). Figure 5 is a graph comparing biotransformation reactions under various conditions. (A) The synthesis of butanediamine is quantitatively based on ornithine acid matrix, and wild-type ornithine decarboxylase from purified Lactobacillus is reacted in a 0.37 M concentration buffer (refer to ●). When the 713th variant of decarboxylase in which alanine is replaced by leucine is reacted in 0.37 M buffer (refer to ○), when wild-type ornithine decarboxylase is reacted in 0.1 M buffer (refer to ◆ ), when the wild-type decarboxylase variant of the 713th alanine replaced by leucine is reacted in a 0.1 M concentration buffer (refer to ◇). (B) When lysine is used as a substrate to quantitatively synthesize pentane diamine, and when wild-type ornithine decarboxylase from purified Lactobacillus is reacted in a 0.37 M concentration buffer (refer to ●), make wild-type ornithine When the 713th variant of decarboxylase in which alanine is replaced by leucine is reacted in 0.37 M buffer (refer to ○), when wild-type ornithine decarboxylase is reacted in 0.1 M buffer (refer to ◆ ). When the wild-type ornithine decarboxylase's 713th variant of alanine is replaced by leucine is reacted in a 0.1 M concentration buffer (refer to ◇). Figure 6 shows the expression levels of recombinant ornithine decarboxylase genes from four strains. ODC_e.coli_SpeC is from Escherichia coli (constitutive), ODC_e.coli_SpeF is from Escherichia coli (inducible), ODC_ Lactobacillus is from chicken Lactobacillus (inducible), ODC_ Saccharomyces cerevisiae is from Saccharomyces cerevisiae (inducible).

Claims (16)

A variant of ornithine decarboxylase, which includes an amino acid at a) 713th position, b) 698th position, or c) 713th position and 698th position corresponding to SEQ ID NO:1 Substitution, it has a sequence homology of at least 80% and less than 100% with the polypeptide of SEQ ID NO:1. The ornithine decarboxylase variant as described in item 1 of the scope of patent application, wherein the amino acid substitution at the 713th position is made by hydrophobic amino acids, basic amino acids, acidic amino acids other than alanine Amino acid, neutral amino acid or aromatic amino acid substitution. The ornithine decarboxylase variant as described in item 1 of the scope of patent application, wherein the amino acid at the 713th position is substituted with A713L, A713I, A713V, A713R, A713D, A713W or A713Q. The ornithine decarboxylase variant described in item 1 of the scope of patent application, wherein the amino acid at the 698th position is substituted with E698D. The ornithine decarboxylase variant described in item 1 of the scope of patent application, wherein the amino acid at the 713th position is substituted with A713L, A713I, A713V, A713R, A713D, A713W or A713Q, the 698th The amino acid at position is substituted with E698D. The ornithine decarboxylase variant as described in item 1 of the scope of the patent application, wherein the ornithine decarboxylase variant comprises a variant selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:19 to the polypeptide in SEQ ID NO:23. A polynucleotide encoding the ornithine decarboxylase variant according to any one of items 1 to 6 in the scope of the patent application. A microorganism comprising the following ornithine decarboxylase: comprising the polypeptide of SEQ ID NO:1, or at a) 713th position, b) 698th position or c) 713th position corresponding to SEQ ID NO:1 A polypeptide that includes amino acid substitutions at two positions and the 698th position and has a sequence homology of at least 80% and less than 100% relative to the polypeptide of SEQ ID NO:1. The microorganism described in item 8 of the scope of patent application, wherein the microorganism is of the genus Escherichia or Corynebacterium. A method for producing butanediamine, which includes culturing a microorganism containing ornithine decarboxylase, Wherein the ornithine decarboxylase includes the polypeptide of SEQ ID NO:1, or corresponding to SEQ ID NO:1 at a) the 713th position, b) the 698th position or c) the 713th position and the 698th position. A polypeptide that includes amino acid substitutions at three positions and has at least 80% or more and less than 100% sequence homology relative to the polypeptide of SEQ ID NO:1. The method for producing butanediamine as described in item 10 of the scope of patent application includes accumulating butanediamine in a culture medium. The method for producing butanediamine as described in item 10 of the scope of patent application includes recovering butanediamine from the cultured microorganism or culture medium. A method for increasing the purity of butanediamine, which includes culturing microorganisms containing ornithine decarboxylase, Wherein the ornithine decarboxylase includes the polypeptide of SEQ ID NO:1, or corresponding to SEQ ID NO:1 at a) the 713th position, b) the 698th position or c) the 713th position and the 698th position. A polypeptide that includes amino acid substitutions at each position and has a sequence homology of at least 80% and less than 100% relative to the polypeptide of SEQ ID NO:1. A method for increasing the ratio of butanediamine to pentamethylenediamine, which includes culturing a microorganism containing ornithine decarboxylase, Wherein the ornithine decarboxylase includes the polypeptide of SEQ ID NO:1, or corresponding to SEQ ID NO:1 at a) the 713th position, b) the 698th position or c) the 713th position and the 698th position. A polypeptide that includes amino acid substitutions at three positions and has at least 80% or more and less than 100% sequence homology relative to the polypeptide of SEQ ID NO:1. A preparation method of polyamide, which is a method for preparing polyamide by using butanediamine, said butanediamine is prepared by cultivating microorganisms including ornithine decarboxylase, and said ornithine decarboxylase includes SEQ ID NO:1 polypeptide, or a) corresponding to SEQ ID NO:1 at the 713th position, b) the 698th position or c) the 713th position and the 698th position including amino acid substitution and relative The polypeptide of SEQ ID NO:1 has a sequence homology of at least 80% and less than 100%. A composition for the preparation of polyamides, which comprises a microorganism comprising ornithine decarboxylase, said ornithine decarboxylase comprising the polypeptide of SEQ ID NO: 1 or a) corresponding to SEQ ID NO: 1 Positions, b) the 698th position or c) the 713th position and the 698th position include amino acid substitutions and have at least 80% or more and less than 100% relative to the polypeptide of SEQ ID NO:1 Polypeptides of sequence homology.

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