TWI756604B - Ornithine decarboxylase variants, methods for producing putrescine using the same, polynucleotide, microorganism, method for preparing polyamine, and composition for preparing polyamide - Google Patents

Abstract

This application relates to an ornithine decarboxylase or protein variant, ed. A polynucleotide encoding the ornithine decarboxylase or protein variant, a microorganism including the same, a method for producing butanediamine using the same, a method for preparing polyamide, and a composition for preparing polyamide. The present invention achieves the following effects: increasing the productivity, production efficiency or production selectivity of butanediamine, suppressing side reactions and reducing the cost of purifying butanediamine.

Description

Ornithine decarboxylase variant and method for producing butanediamine using it Method, polynucleotide, microorganism, preparation method of polyamide and composition for preparation of polyamide

The present application relates to an ornithine decarboxylase variant, a gene encoding the ornithine decarboxylase variant, a microorganism 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, all content disclosed in the documents of this patent application being incorporated as a part of this specification.

Butanediamine (putrescine, 1,4-diamino-butane) is a substance that causes putrefaction of organisms to produce odor, but it can be used to synthesize 4,6-nylon, and thus is produced industrially. At present, more than 10,000 tons of butanediamine is produced annually due to petroleum resources, but there is a problem of unstable supply and demand of raw materials due to frequent fluctuations in petroleum prices. In addition, there is The problem of environmental pollution is caused by the production of a large amount of toxic substances in the production process.

In order to solve such a problem, the synthesis of bio-derived butanediamine is being actively studied recently. For example, studies are being conducted on methods for synthesizing butanediamine from ornithine derived from organisms or for mass-producing butanediamine from microorganisms using sugars.

As a method for producing butanediamine from microorganisms, various biological engineering methods for increasing the production of butanediamine have been used. Such methods may be as follows: for example, using a promoter to modulate the activity of an enzyme involved in the biosynthesis of butanediamine; overexpressing an antiporter to readily release butanediamine extracellularly; or blocking the breakdown of butanediamine route of. Among them, it is known that regulation of the activity of an enzyme involved in the biosynthesis of butanediamine in microorganisms greatly contributes to an increase in the production of butanediamine.

Ornithine decarboxylase, as an enzyme that cleaves the terminal carboxyl group of ornithine to synthesize butanediamine, is one of the enzymes that performs an important role in the biosynthesis of butanediamine. However, ornithine decarboxylase not only synthesizes butanediamine from ornithine, but also has the activity of synthesizing 1,5-diamino-pentane from lysine at the same time (a side reaction), so its activity is improved. In the case of butanediamine, pentamethylenediamine will be formed together with butanediamine and the yield of butanediamine will be reduced. The above-mentioned pentamethylene diamine also causes many problems when purifying butanediamine. Specifically, in the process of purifying the microbial culture liquid by the distillation method, due to the difference between butanediamine (H2N( _CH2 ) _4NH2 ) and pentamethylenediamine ₍ H2N _{( CH2 ) 5NH2 )} Since the structures are very similar, it takes a lot of time and money to selectively purify the above-mentioned microorganism culture liquid.

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 reduce the activity (side reaction) of synthesizing pentamethylenediamine from lysine.

Therefore, the inventors of the present invention discovered a novel ornithine decarboxylase by identifying It is considered that the synthetic activity of pentamethylenediamine is low and the synthetic activity of butanediamine is high in the above-mentioned ornithine decarboxylase, and the present application is completed.

The present application provides an ornithine decarboxylase or a variant thereof.

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

The present application provides a microorganism for producing butanediamine comprising the above-mentioned ornithine decarboxylase or a variant thereof.

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

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

Yet another object of the present application is to provide a method of increasing the ratio of butanediamine to pentamethylenediamine comprising the step of culturing the above-mentioned microorganism in a medium.

Furthermore, another object of the present application is to provide a use of the above-mentioned butanediamine to synthesize a polyamide-based polymer.

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

One embodiment of the present application provides an ornithine decarboxylase variant having butanediamine production activity including one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

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

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

Butanediamine can be produced by a method using ornithine as a substrate. In addition, after synthesizing ornithine using a substance that becomes a precursor of ornithine as a substrate, butanediamine can be produced from the ornithine. As long as the synthesis of ornithine 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 having an ornithine cycle, it plays an important role in metabolism in relation to element production. In addition, it can interact with arginine, glutamine in vivo The acid and proline are converted into each other, and the keto acid, glyoxylic acid and amine group are transferred. A polyamine is synthesized from it as a substrate to generate an amine (butanediamine) by ornithine decarboxylase. In the present invention, in particular, L-ornithine can be used 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 path of butanediamine (putrescine) . In the present application, ornithine decarboxylase may be used in combination 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

Fig. 1 shows the chemical reaction formula of the process of synthesizing butanediamine with ornithine as a substrate by ornithine decarboxylase. In addition, the side reaction of ornithine decarboxylase to be inhibited, that is, a cadaverine synthesis pathway is shown.

In the present application, the method of obtaining ODC (ornithine decarboxylase) can apply various methods well known in the art. As an example of this method, bioinformatics methods can be utilized based on gene synthesis techniques including codon-optimized gene synthesis techniques and a large amount of genomic information of microorganisms in such a way that enzymes can be obtained with high efficiency in microorganisms generally widely used for enzyme expression. However, it is obtained by a method of screening useful enzyme resources, but is not limited thereto.

In this application, SEQ ID NO: 1 refers to the amino acid sequence of ornithine decarboxylase having butanediamine production activity. The amino acid sequence of the above-mentioned SEQ ID NO: 1 can be obtained from the National Center for Biotechnology Information (National Center for Biotechnology Information), which is a well-known database. Center for Biotechnology Information, NCBI) of the GenBank (GenBank) its sequence. As an example, ornithine decarboxylase can be derived from Lactobacillus sp., Saccharomyces sp. or Escherichia coli (E. coli), specifically, Lactobacillus saerimneri), but not limited thereto, and can be included without limitation 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. In addition, as the ornithine decarboxylase having 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 Insignificant addition sequences before and after the acid sequence, naturally occurring mutations, or silent mutations thereof, as long as they have the same or corresponding activities as the proteins including the amino acid sequence of SEQ ID NO: 1. , the industry understands that it corresponds to a protein having the activity of butanediamine of the present application. Specifically, for example, the protein with butanediamine production activity of the present application may be composed of the amino acid sequence of SEQ ID NO: 1, or have 80%, 85%, 90%, 95%, 96%, 97% therewith %, 98%, or more than 99% homology or identity of amino acid sequences composed of proteins. In addition, as long as it is an amino acid sequence having such homology or identity and showing the function corresponding to the above-mentioned protein, it should be understood that a protein having a partial sequence deletion, modification, substitution or addition of the amino acid sequence should be understood. It is also included in the scope of the protein that is the subject of mutation in the present application.

That is, in the present application, even if "a protein or polypeptide having the amino acid sequence described in a specific SEQ ID NO" or "a protein or polypeptide including the amino acid sequence described in a specific SEQ ID NO" is described, as long as In the case of having the same or corresponding activity as a polypeptide comprising the amino acid sequence of the corresponding SEQ ID NO, it should be understood that a protein having a partial 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 "including Polypeptide of the amino acid sequence of SEQ ID NO: 1". For example, as long as it has the same or corresponding activity as the above-mentioned variant protein, it is not excluded to add a sequence that does not change the function of the protein before and after the above-mentioned amino acid sequence, a naturally occurring mutation, and its latent mutation (silent mutation). ) or retention substitutions, and it should be understood that in the case of such sequence additions or mutations, it is also within the scope of this application.

In this application, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and/or chemical properties. Such variants may still retain more than one biological activity, and have, for example, one or more retaining substitutions. Such amino acid substitutions can typically occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. For example, positively charged (basic) amino acids in amino acids with electrically charged side chains include arginine, lysine, and histidine, and negatively charged (acidic) Amino acids include glutamic acid and aspartic acid, non-polar amino acids in amino acids with uncharged side chains (uncharged amino acid) include glycine, alanine, valine Amino acid, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline, polar or hydrophilic amino acids including serine, threonine, Cysteine, tyrosine, asparagine and glutamic acid, the aromatic amino acids in the above non-polar amino acids include phenylalanine, tryptophan and tyrosine.

In this application, the term "variant" refers to more than one A protein whose amino acid differs from the recited sequence in conservative substitution and/or modification, but retains the functions or properties of the protein. Variants differ from an identified sequence by several amino acid substitutions, deletions, or additions. Generally, one or more amino acids in the amino acid sequence of the protein described above can be mutated, and the variant can be identified by evaluating the properties of the mutated protein. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein. In addition, a portion of the variants may include variants in which one or more portions such as the N-terminal leader sequence or the transmembrane domain have been removed. Other variants may include variants in which a portion is removed from the N- and/or C-terminus of a mature protein. The above term "variant" may use terms such as variant, variant, variant protein, variant polypeptide, variant (expressed in English as modification, modified protein, modified polypeptide, mutant, mutein, divergent, variant, etc.), as long as it is based on variant The terms used in the meaning of , are not limited to this. For the purposes of this application, the aforementioned variants may be, but are not limited to, those with increased activity of the mutated protein over the native wild-type or non-mutated protein.

In addition, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a polypeptide can be conjugated to a signal (or leader) sequence at the N-terminus of a protein that is involved in the transfer of the protein either co-translationally or post-translationally. In addition, the above-mentioned polypeptides can be conjugated with other sequences or linkers so that the polypeptides can be identified, purified or synthesized.

The protein variants of the present application may be ornithine decarboxylase variants. At In this application, the term "ornithine decarboxylase variant" may be used in conjunction 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 can be any one or more amino acids in the 713th and 698th amino acids in the amino acid sequence of SEQ ID NO: 1 are replaced by amino acids different from the amino acids before substitution. adult.

The above-mentioned "substituted with another amino acid" is not limited as long as it is an amino acid different from the amino acid before substitution. For example, alanine, which can be included as the 713th amino acid of 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 alanine which is the 713th amino acid of the amino acid sequence of SEQ ID NO: 1 is substituted with other amino acid residues other than alanine, or glutamine which is the 698th amino acid The acid is substituted with amino acid residues other than glutamic acid, without limitation. On the other hand, in the present application, when it is expressed as "a specific amino acid that has been substituted", even if it is not indicated to be substituted by other amino acids, it should be understood that it is caused by a different amino acid from the amino acid before the 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 with another amino acid, and/or ii) as the 698th amino acid Variants of amino acids in which glutamic acid is substituted by other amino acids. The above-mentioned substitution by other amino acids may be as follows: i) Alanine as the 713th amino acid is selected from the group consisting of leucine, isoleucine, valine, arginine, aspartic acid, tryptamine amino acid substitution in acid and glutamic acid; and/or ii) glutamic acid as the 698th amino acid is substituted with aspartic acid. More specifically, the above variant may be i) as the 713th The alanine of the amino acid is substituted with an amino acid selected from the group consisting of leucine, isoleucine, valine, arginine, aspartic acid, tryptophan and glutamic acid, and/or ii ) as a variant in which the 698th amino acid has glutamic acid replaced by aspartic acid.

In the amino acid sequence of SEQ ID NO: 1, i) alanine as the 713th amino acid is selected from the group consisting of leucine, isoleucine, valine, arginine, aspartic acid, color Amino acid substitutions in amino acid and glutamic acid, and/or ii) Variants in which glutamic acid as the 698th amino acid is substituted with aspartic acid may include those selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 4, SEQ ID NO: 4, SEQ ID NO: 4 Any amino acid sequence of ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 23, specifically, may necessarily consist of 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, any amino acid sequence of SEQ ID NO: 19 to SEQ ID NO: 23, but not limited thereto.

The above variant may be as follows: the substitution of other amino acids at positions corresponding to the 713th and/or the 698th position of SEQ ID NO: 1 and having at least one amino acid sequence with the amino acid sequence of SEQ ID NO: 1 is included. 80%, 90%, 95%, 96%, 97%, 98% or 99% or more and less than 100% sequence homology, and have 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 the amino acid sequence with the above-mentioned amino acid. The amino acid sequence has more than 80% homology or identity to the amino acid sequence, but is not limited thereto. Specifically, the variants of the present application may include amino acid sequences related to any of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 19 to SEQ ID NO: 23 Polypeptides having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity. In addition, as long as the Amino acid sequences that have such homology or identity and exhibit the corresponding efficacy to the above-mentioned proteins have, except for the 713th or 698th amino acid position, a part of the sequence deleted, deformed, substituted or added. Proteins of the resulting amino acid sequence are also included within the scope of this application.

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

Conserved polynucleotide or polypeptide sequence homology or identity is determined by standard alignment algorithms, together with preset gap penalties established by the program used. Substantially, homologous or identical sequences generally can be moderately or highly stringent along at least about 50%, 60%, 70%, 80% or 90% of the entire sequence or entire-length. Hybridization under stringent conditions. Hybridization also contemplates polynucleotides that contain degenerate codons in place of codons in the polynucleotide.

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

For example, as published in Smith and Waterman, Adv. Appl. Math (1981) 2:482, using, for example, Needleman et al. (1970), J Mol Biol. 48: 443's GAP computer program compares sequence information, whereby homology, similarity or identity of polynucleotides or polypeptides can be determined. Briefly, the GAP formula is defined as the value obtained by dividing the number of similarly arranged symbols (ie, nucleotides or amino acids) by the total number of symbols of the shorter of the two sequences. Preset parameters for the GAP program may include: (1) a binary comparison matrix (with values of 1 for consistency and 0 for non-coincidence), and as in Schwartz and Dayhoff, eds., protein Atlas Of Protein Sequence And Structure, revealed by National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res.) 14: A weighted comparison matrix of 6745 (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each slot and an additional 0.10 penalty for each symbol in each slot ( or void opening penalty of 10, void expansion penalty of 0.5); and (3) no penalty for end voids. Thus, as used in this application, the terms "homology" or "identity" refer to the relationship between sequences.

In addition, homology, similarity or identity of any two polynucleotide or polypeptide sequences can be confirmed by comparing the sequences by Southern hybridization experiments under defined stringent conditions, where appropriate hybridization conditions are corresponding Within the technical scope, it can be carried out by methods well known to the artisan (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FMAusubel et al. al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

In the present application, the term "ornithine decarboxylase variant" may be used in combination with variant polypeptides of ornithine decarboxylase, ornithine decarboxylase protein variants, ornithine decarboxylase, which have the ability to produce butanediamine. protein variant polypeptide, ornithine decarboxylase variant polypeptide, ornithine decarboxylase variant, ornithine decarboxylase protein variant, variant ornithine decarboxylase, variant ornithine decarboxylase protein etc. mixed. In addition, the above-mentioned 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 positions 713 and/or 698 in the amino acid sequence of SEQ ID NO: 1, even if the amino acid is added or deleted in SEQ ID NO: 1. Amino acid sequences, as long as they are substituted variants of amino acids at positions corresponding to amino acids 713 and/or 698 from the N-terminus of SEQ ID NO: 1, are included in the scope of this application .

With regard to amino acid residue positions, the term "corresponding to" as disclosed in this application refers to the amino acid residue at the recited position in the protein or peptide, or is similar to the recited residue in the protein or peptide , identical or homologous amino acid residues. A "corresponding region" as used in this application generally refers to a similar position in a related or reference protein.

The variant of the above-mentioned ornithine decarboxylase protein is that the 713th and/or the 698th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted by other amino acids, which may include SEQ ID NO: 1 The amino acid sequence of or a variant ornithine decarboxylase protein having enhanced activity compared to a pre-mutated ornithine decarboxylase from a wild-type microorganism. The variant of the ornithine decarboxylase protein as described above is meant to have at least 80%, 85%, 90%, 95%, 96%, 97%, 80%, 85%, 90%, 95%, 96%, 97%, The amino acid variation at the position corresponding to the 713th or 698th position of SEQ ID NO: 1 among the 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 is composed of leucine, isoleucine, valine, arginine, aspartic acid , tryptophan or glutamic acid substitution, and/or ii) glutamic acid as the 698th amino acid is substituted by aspartic acid.

Specifically, the ornithine decarboxylase variant can be in the amino acid sequence of SEQ ID NO: 1, i) alanine as the 713th amino acid is composed of leucine, isoleucine, valeric acid amino acid, arginine, aspartic acid, tryptophan or glutamic acid, and/or ii) glutamic acid as the 698th amino acid is substituted by aspartic acid, and may have Those with enhanced activity compared to a protein comprising the amino acid sequence of SEQ ID NO: 1 above or a premutated ornithine decarboxylase protein from a wild-type microorganism.

For the purposes of this application, in the case of microorganisms comprising variants of the above-described ornithine decarboxylase proteins, characterized by increased production of butanediamine, increased purity of butanediamine, or increased selection of butanediamine production sex. The protein variants of the present application are characterized in that they have gene regulatory activity to compare with the natural wild-type or non-variant Ornithine decarboxylase increases butanediamine production capacity, butanediamine purity, or butanediamine production selectivity. In particular, it is significant in that the synthesis of cadaverine, which is one of the side reactions of ornithine decarboxylase, can be inhibited by microorganisms into which the protein variant of the present application is introduced, and the production of butanediamine can be increased.

Yet another embodiment of the present application provides a polynucleotide encoding a variant of the above-described ornithine decarboxylase protein.

Ornithine decarboxylase proteins comprising the amino acid sequence of SEQ ID NO: 1 and variants thereof are described above.

In the present application, the term "polynucleotide" is used as a polymer of nucleotides of nucleotide monomers (monomers) long linked by covalent bonds to form a chain of nucleotides, which is a fixed length or more. Deoxyribonucleic Acid (DNA) or RNA strands, more specifically, refers to polynucleotide fragments encoding the above variants.

The polynucleotide encoding the ornithine decarboxylase variant of the present application can 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, a speC, odc, spel or speF gene, which can be derived from Lactobacillus, Saccharomyces or Escherichia coli (Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli .coli), but is not subject to this restriction. In addition, the above-mentioned gene can be any amino acid sequence encoding 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 The base sequence of , more specifically, may include 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 thereto.

Specifically, the polynucleotides of the present application are due to the degeneracy of codons Various modifications can be made to the coding region within a range that does not alter the amino acid sequence of the polypeptide in consideration of the codons preferred by the organism in which the polypeptide is to be expressed (degeneracy). Specifically, as long as it is a polynucleoside encoding a variant of ornithine decarboxylase protein in which the 713th and/or 698th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted by other amino acids acid sequences can be included without limitation.

In addition, the 713th and 713th amino acid sequences of SEQ ID NO: 1 and /or the sequence of the ornithine decarboxylase protein having butanediamine producing activity in which the 698th amino acid is substituted by other amino acids may be included without limitation. The above-mentioned "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically documented in the literature (eg, J. Sambrook et al., homologs). For example, the following conditions can be mentioned: intergenic hybridization with high homology or identity, 40% or more, specifically 90% or more, more specifically 95% or more, and more specifically 97% or more , In particular, intergenic hybridization with more than 99% homology or identity, and the conditions of non-hybridization between genes with homology or identity lower than it; or ordinary southern hybridization (southern hybridization) cleaning conditions , that is, equivalent to 60°C, 1×SSC, 0.1% Sodium Dodecyl Sulfonate (SDS), specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68 ℃, 0.1×SSC, and 0.1% SDS salt concentration and temperature under the conditions of one wash, specifically two to three times.

Hybridization requires that the two nucleic acids have complementary sequences even though a mismatch between bases may occur depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine and thymus Pyrimidine is complementary, cytosine is complementary to guanine. Thus, the present application can include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

Specifically, polynucleotides having homology or identity can be probed using hybridization conditions including a hybridization step at a Tm value of 55°C using the conditions described above. In addition, although the said Tm value may be 60 degreeC, 63 degreeC, or 65 degreeC, it is not limited to this, It can adjust suitably by a manufacturer according to the objective.

Appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, parameters well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

Yet another embodiment of the present application provides a vector comprising a polynucleotide encoding a variant of ornithine decarboxylase.

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

The term "vector" as used in this application refers to a polynucleotide comprising a polynucleotide encoding the above-mentioned polypeptide of interest operably linked to appropriate regulatory sequences in such a manner that the polypeptide of interest can be expressed in a suitable host. DNA preparations of base sequences. Such regulatory sequences may include a promoter that can initiate transcription, any operator sequence to regulate such transcription, a sequence encoding an appropriate messenger RNA (mRNA, mRNA) ribosome binding site, and regulation of transcription and decoding 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 carriers include: natural state or recombinant plastids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR, pUC, pBluescriptII, etc. can be used as plastid vectors. class, pGEM class, pTZ class, pCL class and pET class, etc. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.

As an example, the intrachromosomal polynucleotide encoding the polypeptide of interest can be inserted into the chromosome by a vector for intracellular chromosome insertion. The above-mentioned polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. It may further include a selection marker for confirming the above-mentioned chromosomal insertion. Screening markers are used to screen cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule, using selectable tables that confer, for example, drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface polypeptides. type logo. In an environment treated with a selective agent, only cells expressing the selectable marker survive or exhibit different phenotypic traits, and thus transformed cells can be screened.

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

In the present application, the terms "microorganisms including variant polypeptides" or "microorganisms including ornithine decarboxylase variants" can be used as long as they are microorganisms that can produce butanediamine including the protein variants of the present application. , but not limited to this. For example, microorganisms comprising protein variants of the present application may be native wild-type microorganisms A recombinant microorganism that expresses the protein variant of the present application in an organism or a butanediamine-producing microorganism, thereby increasing the production capacity of butanediamine, the production purity of butanediamine, or the production selectivity of butanediamine. The above-mentioned recombinant microorganism may be a microorganism whose production capacity, production purity, or production selectivity of butanediamine is increased compared to a natural wild-type microorganism or a non-transformed microorganism, but is not limited thereto.

Specifically, the above-mentioned microorganisms are microorganisms that express ornithine decarboxylase variants including one or more amino acid variations within the amino acid sequence of SEQ ID NO: 1, and the above-mentioned amino acid variations may include starting from the N-terminus. The 713th and/or the 698th amino acid is substituted with other amino acids. 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 by another amino acid and has the production activity of butanediamine, But not limited to this.

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

In the present application, the term "expressed/expressed" protein refers to a form in which a target protein is introduced into a microorganism or deformed so as to be expressed in a microorganism. When the above-mentioned target protein is a protein existing in a microorganism, it refers to a state in which the activity is relatively intensified or before deformation. For the purposes of this application, a "target protein" may be a variant of the above-described ornithine decarboxylase protein having the ability to produce butanediamine.

Specifically, "introduction of a protein" refers to expressing the activity of a specific protein not originally possessed by a microorganism, or expressing an activity that is improved over the intrinsic activity or the activity before modification of the above-mentioned protein. For example, a polynucleotide encoding a specific protein can be introduced into a chromosome of a microorganism, or a vector including a polynucleotide encoding a specific protein can be introduced into a microorganism to exhibit its activity. In addition, "enhanced activity" means that the activity is less The intrinsic activity or pre-modification activity of a specific protein possessed by an organism is increased. The above-mentioned "intrinsic activity" refers to the activity of a specific protein originally possessed by the parent strain before transformation when the microorganism is transformed due to genetic variation caused by natural or human factors.

Specifically, the potentiation of the activity of the present application can be achieved by any one or more methods selected from the group consisting of, but not limited to: cells that increase the gene encoding the protein variant of the present application A method for introducing variation into the expression regulatory sequence of the gene encoding the above-mentioned protein variant; the method for replacing the expression regulatory sequence of the gene encoding the ornithine decarboxylase protein variant with a highly active sequence; to encode the above-mentioned protein variant A method in which the gene of the protein variant replaces the gene encoding the wild-type protein of ornithine decarboxylase on the chromosome; the mutation is also introduced into the gene encoding the above-mentioned ornithine decarboxylase protein in a manner to enhance the activity of the protein variant. methods; and methods of introducing protein variants into microorganisms.

In the above, increasing the number of copies of a gene is not particularly limited, but can be performed in a form operably linked to a vector, or by insertion into a chromosome in a host cell. Specifically, a vector in which a polynucleotide encoding the protein of the present application is operably linked and capable of replicating and functioning independently of the host can be introduced into a host cell. Alternatively, a vector in which the above-described polynucleotide is operably linked and inserted into the chromosome of the host cell can be introduced into the chromosome of the host cell. The above-described polynucleotides can be inserted into chromosomes by any method known in the art, such as homologous recombination.

Morphing the expression regulatory sequence in a manner that increases the expression of the polynucleotide is not particularly limited, but can be modified by deletions, insertions, non-retained or retained substitutions, or a combination thereof. Nucleic acid sequence-induced sequence variation is performed to further enhance the activity of the above-described expressive regulatory sequence, or by replacement with a nucleic acid sequence with greater activity. The above-mentioned expression regulation sequences are not particularly limited thereto, and may include promoters, operator sequences, sequences encoding ribosome binding sites, sequences regulating termination of transcription and decoding, and the like.

A strong promoter may be connected to the upper part of the above-mentioned polynucleotide expression unit instead of the original promoter, and it is not limited to this. Examples of 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 (Korea Registered Patent No. 10-1783170), O2 promoter (Korea Registered Patent No. 10-1632642), tkt promoter and yccA promoter, etc., but not limited to this.

The modification of the polynucleotide sequence on the chromosome is not particularly limited, and the above-mentioned polynucleotide can be further strengthened by inducing the variation in the expression regulatory sequence of the nucleic acid sequence by deletion, insertion, non-retention or retention substitution or a combination thereof. activity of the sequence, or by replacing it with a polynucleotide sequence modified in a way that has greater activity.

The introduction and enhancement of the protein activity as described above can be performed by increasing the activity or concentration of the corresponding protein, typically to a minimum of 1%, 10%, 25%, 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, maximum 1000% or 2000%, but not limited thereto.

In the present application, a microorganism comprising the above-mentioned ornithine decarboxylase variant or a polynucleotide encoding the above-mentioned ornithine decarboxylase variant can be obtained by including The recombinant microorganism prepared by transforming the above-mentioned polynucleotide vector is not limited to this.

In this application, the term "transformation" refers to the introduction of a vector comprising 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. The transformed polynucleotide can include both intrachromosomal location or extrachromosomal location into the host cell as long as it can be expressed in the host cell. In addition, the above-mentioned polynucleotide includes DNA and RNA encoding the target protein. The above-mentioned polynucleotide can be introduced in any form as long as it can be introduced into and expressed in a host cell. 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 expression cassette may generally include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The above-mentioned presentation cassette can be in the form of a presentation carrier 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, but is not limited to this.

In addition, in the above content, the term "operably linked" means that a promoter sequence that initiates transcription and mediates the polynucleotide encoding the polypeptide of interest of the present application is functionally linked to the above-mentioned gene sequence.

The term "non-transformed microorganism" as used herein refers to the native strain itself, a microorganism that does not include the protein variant of the present application, or is not transformed with a vector that includes a polynucleotide encoding the protein variant of the present application of microorganisms.

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

In this application, the term "microorganism producing butanediamine" refers to a A butanediamine-producing wild-type microorganism, or a microorganism having butanediamine-producing ability by introducing a wild-type or a variant into a parent strain having no butanediamine-producing ability or a significantly lower production ability. Specifically, it includes all microorganisms that cause genetic deformation naturally or artificially, as microorganisms whose specific mechanisms are weakened or enhanced due to insertion of external genes, enhancement of the activity of internal genes, or inactivation, etc. Microorganisms with genetic variation or enhanced activity due to amines. For the purposes of the present application, the microorganisms of the present application include the protein variants of the present application, and thus may increase the production capacity, production purity, or production selectivity of butanediamine of interest. Specifically, the microorganism of the present application may be a microorganism in which a part of genes in the butanediamine biosynthesis pathway is enhanced or weakened, or a part of genes in the butanediamine decomposition pathway is enhanced or weakened. For the purposes of this application, a butanediamine-producing microorganism may refer to a microorganism characterized by including the ornithine decarboxylase variants described above, and thus the production of butanediamine to be generated from the carbon source in the culture medium, the production of butanediamine The purity of the amine or the selectivity for the production of butanediamine is increased compared to wild-type or non-transformed microorganisms. In the present application, the above-mentioned "microorganisms producing butanediamine" may be used in combination with "microorganisms having the ability to produce butanediamine" or "microorganisms producing butanediamine".

The above-mentioned butanediamine-producing microorganism may be a recombinant microorganism. The above-mentioned recombinant microorganisms are as described above.

The type of the microorganism that produces the above-mentioned butanediamine is not particularly limited as long as it can produce butanediamine. ), Erwinia (Erwinia), Serratia (Serratia), Providencia (Providencia) and Brevibacterium (Brevibacterium) genera microorganisms, more specifically, may belong to Corynebacterium ( Corynebacterium) or Escherichia (Escherichia) microorganisms.

More specifically, the microorganism of the genus Escherichia can be Escherichia coli, and the microorganism of the genus Corynebacterium can be Corynebacterium glutamicum, Corynebacterium amine-producing bacteria. (Corynebacterium ammoniagenes), Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stasis stationis), Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, Corynebacterium imitans ), Corynebacterium testudinoris (Corynebacterium testudinoris) or Corynebacterium flavescens (Corynebacterium flavescens), etc., and can be Corynebacterium glutamicum (Corynebacterium glutamicum), but can include without limitation those that can introduce or fortify ornithine Microorganisms of the genus Corynebacterium or Escherichia that increase the yield of butanediamine, the purity of butanediamine, and the production selectivity of butanediamine by decarboxylase protein.

In the present application, the parent strain of a butanediamine-producing microorganism modified to express an ornithine decarboxylase protein or a variant of the protein is not particularly limited as long as it is a butanediamine-producing microorganism.

Microorganisms belonging to the genus Corynebacterium do not have a biosynthetic pathway for butanediamine, but they can synthesize butanediamine when ornithine decarboxylase (ODC) is introduced from outside.

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

In addition, the above-mentioned microorganisms that produce butanediamine are not particularly limited, and may be, for example, the following: in order to strengthen the biosynthetic pathway from glutamic acid to ornithine, glutamic acid is converted into acetylglutamate (N-acetylglutamate) ) of acetoglutamate synthase, or ornithine acetyltransferase (ArgJ), which converts acetonitrile to ornithine, and acetonitrile to acetonitrile phosphate (N -acetylglutamyl phosphate), acetylglutamate kinase (ArgB), and acetylglutamate-phosphate reductase (ArgC), which converts acetylglutamyl phosphate to N-acetylglutamate semialdehyde (N-acetylglutamate semialdehyde) , The activity of acetonitrile transaminase (ArgD), which converts acetonitrile semialdehyde into acetylornithine (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 increases.

In addition, the above-mentioned microorganism for producing butanediamine is not particularly limited, and may further be a microorganism of the genus Corynebacterium that has a butanediamine-producing ability with weakened butanediamine acetyltransferase activity. Furthermore, the microorganisms producing the above-mentioned butanediamine are not particularly limited, and may be an activity enhancer of butanediamine-excreted proteins, but are not limited thereto.

In this application, the term "enhancing/increasing" is intended to include all concepts of increased activity compared to intrinsic activity.

Such enhancement or increase in gene activity can be achieved by applying various methods well known in the art. Examples of the above-mentioned methods 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 are not particularly limited to the above-mentioned examples: increasing the intracellular copy number of a gene; A method for introducing mutation into the expression regulatory sequence of a gene; replacing the gene expression regulatory sequence with a highly active one A method of introducing a strong sequence; a method of introducing a mutation into a corresponding gene to enhance the activity of the gene; and a method of introducing a foreign gene into a microorganism.

In this application, the term "inactivation" includes all concepts that are less active than intrinsically active or inactive.

Inactivation of such gene activity can be achieved by applying various methods well known in the art. Examples of the above-mentioned methods include the following methods, which can also be achieved by a combination thereof, but are not particularly limited to the above-mentioned examples: deletion of the entire or a part of the gene on the chromosome, including the case of removing the activity of the above-mentioned gene method; a method for replacing a gene encoding the above-mentioned protein on a chromosome with a gene mutated in a manner that reduces the activity of the corresponding protein; a method for introducing mutation into the expression regulatory sequence of the gene on the chromosome encoding the above-mentioned protein; the method for encoding the above-mentioned protein A 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 above-mentioned gene with a promoter that is weaker than the intrinsic promoter); making the gene on the chromosome encoding the above-mentioned protein A method for deletion in whole or in part; a method for introducing an antisense oligonucleotide (for example, antisense RNA) that is complementary to the above-mentioned mRNA and translating into a protein by introducing a transcript of the gene that is complementary to the above-mentioned mRNA; in a gene encoding the above-mentioned protein A method of artificially attaching a sequence complementary to the SD sequence to the front of the SD sequence to form a secondary structure to prevent ribosomes from attaching; Reverse transcription engineering (RTE) method of adding a promoter in the form of a 3' end. In the present application, the term "intrinsic activity" refers to the activity of a specific protein originally possessed by the parent strain before transformation when the microorganism is transformed due to genetic variation caused by natural or human factors.

As another embodiment of the present application, there is provided a A butanediamine production method of the step of culturing the above-mentioned microorganisms producing butanediamine.

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

In this application, the term "culturing" refers to growing the above-mentioned microorganisms under appropriately regulated environmental conditions. The culturing process of the present application can be realized by appropriate medium and culturing conditions known in the industry. This cultivation process can be easily adapted by the practitioner depending on the strain of choice. Specifically, the above-mentioned culturing can be batch type, continuous type and fed-batch type, but is not limited thereto.

In the present application, the term "medium" refers to a substance in which nutrients necessary for culturing the above-mentioned microorganisms are mixed as main components, and supplies nutrients, development factors, etc., including water indispensable for survival and development. Specifically, as long as the medium and other culture conditions for culturing the microorganisms of the present application are those used for culturing ordinary microorganisms, any medium can be used without particular limitation, and any medium may be used that contains an appropriate carbon source, nitrogen source, The microorganism of the present application is cultivated in a common medium containing phosphorus, inorganic compounds, amino acids, and/or vitamins, etc., under aerobic conditions by adjusting the temperature, pH, and the like.

In the above-mentioned method, the step of culturing the above-mentioned microorganism is not particularly limited, and can be performed by a known batch culture method, continuous culture method, fed-batch culture method, or the like. At this time, the culture conditions are not particularly limited, and a basic compound (for example, sodium hydroxide, potassium hydroxide, or amine) or an acidic compound (for example, phosphoric acid or sulfuric acid) can be used to adjust an appropriately large pH value (for example, pH5 to pH9) , specifically pH 6 to pH 8, most specifically pH 6.8), oxygen or oxygen-containing gas mixtures can be introduced into the culture to maintain aerobic conditions. The culturing temperature may be maintained at 20°C to 45°C, specifically 25°C to 40°C, and may be cultured for 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 remain in the cells.

Furthermore, the culture medium to be 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 (for example, palmitic acid, stearic acid, and linoleic acid), alcohols (for example, glycerol and ethanol), and organic acids (for example, acetic acid), etc. as carbon supply sources, but not limited to this. Nitrogen-containing organic compounds (eg, egg whites, yeast extract, gravy, malt extract, corn steep liquor, soybean meal, and urea), or inorganic compounds (eg, ammonium sulfate, ammonium chloride, phosphoric acid), can be used alone or in combination ammonium, ammonium carbonate, and ammonium nitrate) etc. are used as nitrogen supply sources, but are not limited thereto. As the phosphorus supply source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium-containing salts corresponding thereto, and the like may be used alone or in combination, but are not limited thereto. Additionally, the medium may include other metal salts (eg, magnesium sulfate or ferric sulfate), amino acids, and essential growth promoting substances such as vitamins.

In the method for recovering the butanediamine produced in the above-mentioned culturing step of the present application, the target amino acid can be collected from the culture solution by an appropriate method known in the art according to the culturing method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, and high performance liquid chromatography (HPLC), etc. can be used, and the target can be recovered from the culture medium or microorganisms using appropriate methods known in the art Butanediamine. The method for recovering the above-mentioned butanediamine may further include a refining step.

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

As yet another embodiment of the present application, there is provided a use of butanediamine prepared by culturing a microorganism producing the above-mentioned butanediamine for preparing polyamide. in addition, Provided is a polyamide preparation composition comprising a microorganism producing 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: the polypeptide comprising SEQ ID NO: 1, or a) No. 713, b) No. 698 or c) of SEQ ID NO: 1 The positions corresponding to the 713th and 698th positions include amino acid substitutions, and the polypeptide in SEQ ID NO: 1 includes a polypeptide having sequence homology of at least 80% or more and less than 100%. In addition, the step of culturing the above-mentioned butanediamine-producing microorganism includes the step of culturing a microorganism comprising ornithine decarboxylase comprising the polypeptide of SEQ ID NO: 1, or the 713th item in a) of SEQ ID NO: 1, b) the 698th or c) positions corresponding to the 713th and 698th include amino acid substitutions, and the polypeptide in SEQ ID NO: 1 includes at least 80% or more and less than 100% sequence homology peptide.

The above-mentioned polyamides are used for various materials because they are excellent in heat resistance, chemical resistance, etc. due to hydrogen bonds between amide bonds, and thus 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, softness, gloss properties, and dyeing clarity, so they can be used for leg wear such as stockings, inner wear, sportswear, etc. Apparel products such as sports wear. In addition, the above-mentioned polyamide can be used as a raw material for pharmaceuticals, surfactants, films, plastics, and the like. For example, when a film is prepared from polyamide, it can achieve excellent optical properties and mechanical properties, and also has flexibility, so it can be used as a material for various molded products, and the above-mentioned polyamide film can be applied to display substrates , Display protective film, touch panel, window cover of folding equipment, etc.

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 inhibiting the synthesis of pentamethylenediamine, which is one of the side reactions of ornithine decarboxylase, thus achieving the effect of simplifying the purification/separation process of butanediamine and reducing production costs.

In addition, the present application can realize various utilizations of polymer precursors, pharmaceuticals, chemical additives, and the like by mass-producing butanediamine.

FIG. 1 is a schematic diagram showing the synthesis of butanediamine using ornithine decarboxylase using ornithine as a substrate in the present application. In addition, the synthetic route of pentamethylenediamine, which is a side reaction of ornithine decarboxylase to be inhibited, is shown.

Fig. 2 shows the activity of ornithine decarboxylase from various sources confirmed, and shows the relative activity of the reactivity when ornithine is used as a substrate and the reactivity (side reaction) when lysine is used as a substrate. ODC_Lb is from Lactobacillus hen (inducible), ODC_Sc is from Saccharomyces cerevisiae (inducible), ODC_Ec is from Escherichia coli (constitutive), and ODC_Ef is from Escherichia coli (inducible).

Fig. 3 is a graph showing the comparison of the quantification of (a) ornithine specific activity and (b) lysine specific activity by wild-type ornithine decarboxylase derived from purified Lactobacillus and the following variants, respectively : The 696th alanine of wild-type ornithine decarboxylase is replaced by glutamic acid (A696E); the 702nd valine of wild-type ornithine decarboxylase is replaced by glycine (V702G); wild-type ornithine The 713th alanine of acid decarboxylase is replaced by leucine (A713L); the 696th and 713th alanine of wild-type ornithine decarboxylase are replaced by glutamic acid and leucine (A696E/A713L) ); wild type bird The 702nd valine and 713th alanine of amino acid decarboxylase are replaced by glycine and leucine (V702G/A713L); the 696th alanine and 702nd valine of wild-type ornithine decarboxylase amino acid and the 713th alanine were replaced by glutamic acid, glycine and leucine (A696E/V702G/A713L); the 698th glutamic acid of wild-type ornithine decarboxylase was replaced by aspartic acid ( E698D); the 698th glutamic acid and 713th alanine of wild-type ornithine decarboxylase were 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 purified Lactobacillus and the following variants: 698th gluten of wild-type ornithine decarboxylase Amino acid is replaced by aspartic acid (E698D); 713 alanine of wild-type decarboxylase is replaced by leucine (A713L); 698 glutamic acid and 713 of 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) When the synthesis of butanediamine was quantified based on ornithine substrate, wild-type ornithine decarboxylase derived from purified Lactobacillus was reacted in a buffer solution with a concentration of 0.37 M (refer to ●), wild-type ornithine was reacted with When the variant in which the 713th alanine of decarboxylase was substituted by leucine was reacted in a buffer of 0.37M concentration (refer to ○), and when wild-type ornithine decarboxylase was reacted in a buffer of 0.1M concentration (refer to ◆ ), and when the variant in which the 713th alanine of the wild-type decarboxylase was substituted with leucine was reacted in a 0.1 M concentration buffer (refer to ◇). (b) When quantifying pentamethylenediamine synthesis using lysine as a substrate, and reacting wild-type ornithine decarboxylase derived from purified Lactobacillus in 0.37 M buffer solution (refer to ●), wild-type ornithine decarboxylase was reacted with When the variant in which the 713th alanine of decarboxylase was substituted by leucine was reacted in a buffer of 0.37M concentration (refer to ○), and when wild-type ornithine decarboxylase was reacted in a buffer of 0.1M concentration (refer to ◆ ), making wild-type ornithine decarboxylase The 713th variant of alanine substituted by leucine was reacted in 0.1M 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 Lactobacillus chicken origin (inducible), and ODC_Saccharomyces cerevisiae is from Saccharomyces cerevisiae (inducible).

Hereinafter, the present application will be described in more detail by way of examples. However, the above-mentioned embodiments are used to illustrate the present application, and the scope of the present application is not limited to the above-mentioned embodiments, which are obvious to those skilled in the technical field to which the present application belongs.

Example 1. Activity comparison of ornithine decarboxylase from various sources

The reactivity of ornithine decarboxylase substrates from four microorganisms was compared. With Lactobacillus saerimneri (inducible), Saccharomyces cerevisiae (inducible), Escherichia coli (E.coli (constitutive)), Escherichia coli (E.coli (inducible)) The wild-type ornithine decarboxylase was used as the target, which were marked as ODC_Lb, ODC_Sc, ODC_Ec, and ODC_Ef, respectively. After inserting the gene corresponding to the above enzyme into the pET24ma vector, the protein was expressed using Escherichia coli BL21 (DE3) under the conditions of 0.1 mM Isopropyl Thiogalactoside (IPTG) and 18°C. Thereafter, initial reaction rates were compared using 10% cell extracts at 45°C. The case of using 4 m of ornithine as the substrate was compared with the case of using 4 mM of lysine as the substrate, respectively.

Figure 2 shows the activity of ODC enzymes from the above four microorganisms. Specifically, it shows the ornithine decarboxylase activity of producing butanediamine using ornithine as a substrate, and the lysine decarboxylase activity of producing pentamethylenediamine using lysine as a substrate. The lysine decarboxylase activities were all similar, but the ornithine decarboxylase activity of Lactobacillus-derived ornithine decarboxylase (ODC_Lb) was the best. That is, in ODC_Lb, the lysine decarboxylase activity relative to the ornithine decarboxylase activity, that is, the generation ratio of the side reaction was the lowest, so a variant was prepared based on the above.

Example 2. Selection of variant positions of ornithine decarboxylase

Knowing the crystal structure of Lactobacillus ornithine decarboxylase, the tunneling of substrates into and out of the enzyme can be predicted by analyzing the structure. To select the functional residues in the predicted tunnel moiety that perform saturation variation, multiple sequence alignments using sequence information from bioinformatics were performed, from the ornithine decarboxylase amine utilized in the present invention. The positions of A696, V702, A713, and E698 were selected as the variation positions at the N-terminus of the amino acid sequence.

Residues containing amino acid residues at specific positions in the protein structure play a very important role in structure and function in the above-mentioned proteins, especially in the catalytic process. for the variant residues.

Example 3. Saturation mutation of functional residues of ornithine decarboxylase and exploration of variants

Saturation mutation (saturation mutagenesis) refers to changes in various nucleotide sequences introduced into a given position of a gene. Saturation mutation refers to inserting a NNK codon (codon) into a primer bound to the complementary sequence of the template strand instead of the sequence to be mutated to insert mutation by polymerase chain reaction (PCR). In this case, in the NNK codon, N refers to A, T, G, C, K refers to T, G.

After performing saturation mutations on selected functional residues using NNK codons, screening of the variant library was performed. All libraries were subjected to primary and secondary screening by whole cell reactions. The whole-cell reaction described above refers to a reaction in which cells containing a specific enzyme are pulverized and cell contents are used, or whole cells are used without separating and purifying the enzyme. A single screen was performed by ornithine whole cell reaction, and variants showing similar or faster activity when compared to wild type were selected as changes in absorbance. Secondary screening was performed by whole cell reaction with lysine, and variants screened in the primary screening were selected if their reactivity to lysine was lower than that of the wild type.

For the screened variant enzymes, the specific activity was determined using ornithine or lysine as substrates. Specific activity refers to the unit activity of pure protein from which impurities and other proteins have been removed by enzymatic purification, and is usually expressed as a unit per 1 mg in terms of the amount of enzyme that promotes 1 μmol of substrate change in 1 minute. Specifically, the Lactobacillus-derived wild-type and mutant ornithine decarboxylase was transformed into Escherichia coli BL21 (DE3) and expressed in a culture volume of 50 mL using IPTG as an inducer, and then using a Ni-NTA column. Only pure protein is refined. First, after protein expression, the cells are pulverized by a sonic pulverizer, and a cell extract is obtained after centrifugation. The cell extract was placed into a column equilibrated with 50 mM phosphate buffer (pH 8.0) supplemented with 300 mM sodium chloride and bound to nickel resin for 1 hour at 0°C. Thereafter, proteins that failed to bind to the resin were discarded and other proteins bound nonspecifically were removed by a Tris buffer including 50 mM imidazole. Finally, only the desired protein was eluted using Tris buffer including 250 mM imidazole. In order to remove imidazole from the eluted protein, a desalting process using a filter column is performed to finally obtain only the active protein. After this, use brad The Bradford Protein Quantitation Kit determines protein amount and uses the same amount of protein to determine specific activity.

The specific activity of Lactobacillus-derived wild-type and mutant ornithine decarboxylase was measured by HPLC (High-performance liquid chromatography) analysis method using ornithine or lysine as a substrate. 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 was determined at which conversion yields from 10% to 25% were exhibited. A cation exchange column was used, and the mobile phase included 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 was pH 5.0, a citrate-sodium citrate buffer was used. The specific activities of ornithine decarboxylase of the wild type and the variant are shown in FIG. 3 .

As shown in Figures 3(a) and 3(b), wild-type and variant (A696E, V702G, A713L, A696E/A713L, V702G/A713L, A696E/V702G/A713L, E698D and E698D/A713L) enzymes were confirmed As a result, it was confirmed that the above-mentioned functional residues (A696, V702, A713, E698) were all located in the active site (active site) or the substrate near the tunnel.

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

Ratio of specific activity of variant (A696E, V702G, A713L, A696E/A713L, V702G/A713L, A696E/V702G/A713L, E698D, E698D/A713L) enzymes to wild-type enzymes when using lysine as substrate activity respectively Suppression of side reactions was confirmed at 16.9%, 0.6%, 42.4%, 4.4%, 0.9%, 0.7%, 50.8%, and 29.2% ( FIG. 3( b )).

Example 4. Confirmation of kinetic coefficients of functional residues of ornithine decarboxylase

The properties of variants A713L, E698D, and E698D/A713L having a specific activity of 70% or more among the ornithine decarboxylase variants used in Example 3 were confirmed in more detail. In order to compare the kinetic parameters (Kinetic parameters) of the variant and the wild type, lysine at various concentrations was used. Kinetic coefficients use substrate solutions with different concentrations to express the substrate affinity and substrate conversion capacity values of enzymes.

Specifically, in order to confirm the kinetic coefficient of lysine of the wild-type and variant ornithine decarboxylases that have completed protein purification, lysine concentrations of 0.45 mM to 140 mM were used. The pH buffer was carried out in a reaction volume of 200 μl using a pH 5.0 citrate-sodium citrate buffer. The analysis was carried out by the above-mentioned HPLC analysis method as indicated, and the average value of the three experiments was obtained. Kinetic coefficients of lysine decarboxylase of wild type and variants are shown in FIG. 4 .

As shown in FIG. 4 , the k _cat value of lysine of the variant (A713L) was confirmed to be 2.16 times lower than that of the wild type. It was confirmed that the k _cat /KM value of lysine in the variant ( _A713L ) was 1.93-fold lower than that of the wild type due to the decrease in the k _cat value. As a result, it was confirmed that the variant (A713L) reduced lysine decarboxylase activity. The k _cat values of lysine of the variants E698D and E698D/ _A713L were also confirmed to be reduced by 2.08 times and 2.59 times, respectively, and the k _cat /KM values of lysine were confirmed to be reduced by 1.28 times and 1.67 times. That is, it was confirmed that the above variant reduces side reactions.

Example 5. Characterization of variant ornithine decarboxylase

Among the variants to be studied, the bird with the highest ornithine specific activity The effect of amino acid decarboxylase (A713L) on the production of butanediamine or pentamethylenediamine. The case where ornithine at a high concentration of 51.5 g/L (0.39 M) was used as the substrate and the case where lysine at a concentration of 2.57 g/L (17.6 mM) was used as the substrate were respectively performed. In order to set the appropriate reaction conditions when performing the reaction under both substrate conditions, pH-appropriate buffer concentrations were performed under both conditions (0.1 M or 0.37 M).

Specifically, 0.1 mg of protein-purified wild-type and mutant enzymes were used in the reaction. 0.39M ornithine or 17.6mM lysine was used as matrix, and 0.1M or 0.37M citrate buffer (pH 5.0) was used as buffer. Reactions were performed at 50°C using 0.1 mM PLP coenzyme in a 2 mL reaction volume. The results are shown in FIG. 5 .

Figure 5(a) is the case where 51.5 g/L (0.39 M) ornithine was used as a substrate. In the case of converting ornithine to butanediamine, when a 0.37M buffer was used (refer to ● and ○ in Fig. 5(a) ), 33.0% were generated from the wild type and the mutant (A713L) after 4 hours, respectively. g/L and 31.6 g/L butanediamine. In addition, when a low-concentration buffer (0.1M) was used (see ◆ and ◇ in Fig. 5(a)), the wild-type and the variant (A713L) produced 20.2 g/L and 20.7 g/L, respectively, after 7 hours. Butanediamine. The wild-type and variant (A713L) were confirmed to have similar butanediamine productivity, and the use of high concentration buffer (0.37M) was confirmed to be beneficial for reactivity.

Figure 5(b) is the case where 2.57 g/L (17.6 mM) of lysine was used as a matrix. When lysine was converted into pentamethylenediamine, when 0.37M buffer was used (refer to ● and ○ in Fig. 5(b)), 0.03 was generated from the wild type and the mutant (A713L) after 4 hours, respectively. g/L and 0.007g/L of pentamethylenediamine. In addition, when a 0.1 M buffer was used (see ◆ and ◇ in Fig. 5(b)), the side reaction to generate pentamethylene diamine increased, and 0.59 g/mol of pentamethylene diamine was generated from the wild type and the mutant (A713L) after 7 hours, respectively. L and 0.38 g/L pentamethylene diamine as a side reaction. Thereby, it was confirmed that the side reaction of pentamethylenediamine from the variant (A713L) was suppressed, and it was confirmed that the effect of suppressing the side reaction was more prominent when a high-concentration buffer solution (0.37M) was used.

Example 6. Comparative determination of expression levels of recombinant ODC genes in coryneform strains

The production methods for expressing the recombinant genes of ornithine decarboxylase ODC_Lb, ODC_Sc, ODC_Ec, and ODC_Ef from the four microorganisms mentioned in Example 1 are as follows.

Specifically, the genes shown in Table 1 were determined using the genetic information of Lactobacillus gallis (ACCESSION no. P43099), Saccharomyces cerevisiae (ACCESSION no. J02777.1), and Escherichia coli str. After amplifying the gene coding region of the ornithine decarboxylase gene by PCR, the PCR product was treated with restriction enzymes and inserted into the plastid.

Recombinant genes are made in such a way that a His-tag can be additionally translated at the C-terminus of the protein. Escherichia coli DH5α was used as a host strain for DNA manipulation, and Escherichia coli BL21(DE3) was used as a host strain for expressing the C-terminal His6-tag ODC gene. Recombinant E. coli BL21 was grown at 37°C in 50 mL of LB medium containing 50 μg/mL of kanamycin. When the broth reached 0.8 at _OD600 , 0.2 mM IPTG was added to the broth. After induction of protein expression at 18°C to 30°C, cells were harvested. The cells were resuspended in lysis buffer and sonicated to disrupt the cells. The obtained recombinant ODC was refined into Ni-NTA agarose resin from Quiagen (Hilden, Germany) at 4°C. Recombinant proteins were obtained using Centriplus YM-30 (Millipore, Bedford, MA) along with a 100 kDa molecular mass cut off. The performance results are shown in Figure 6.

When the results of the induction conditions at 30°C were analyzed on a Sodium Dodecyl Sulfonate Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel (gel), it was confirmed that the recombinant ODC_Lb interacted with The performance of ODC_Ec is higher than that of ODC_Sc and ODC_Ef. However, in the case of ODC_Ec, it was confirmed that the amount of soluble protein was significantly reduced when the mesophilic condition was exhibited. Furthermore, the amount of soluble protein in ODC_Ec was further reduced when expressed at 37°C.

In the coryneform strain, the ODC_Lb and ODC_Ec genes were expressed and the amount expressed as a soluble protein was evaluated comparatively. The CJ7 promoter (KCCM10617, Korean Registered Patent No. 10-0620092) was introduced before the start codons of the ODC_Lb gene and the ODC_Ec gene. First, in order to obtain a gene including the CJ7 promoter sequence, PCR using the primer pairs indicated 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 procedures 30 times: modification at 95°C for 30 seconds, annealing at 55°C for 30 seconds and at 72°C Elongate for 30 seconds.

After electrophoresis using a 1.5% agarose gel, a PCR nucleic acid product with a size of 400 base pairs (bp) was confirmed. The obtained PCR product was purified using PCR prep kit (GeneAll, Seoul). BamHI and XbaI were put into the purified PCR product and pSCEC carrier solution sample, and subjected to restriction enzyme treatment by reacting at 37°C for 4 hours. The PCR nucleic acid product band and the vector-sized band were then used to obtain nucleic acid fragments using Gel prep kit (GeneAll, Seoul). After 1 mg of each purified CJ7 promoter fragment was ligated to the vector using T4 ligase, electrophoration was performed on Escherichia coli DH5α strain. Electroporation was applied at 2500V. The recovered strains were spread on LB plate medium containing 50 μg/L spectinomycin and cultured at 37° C. for 1 day, and then strains showing resistance were screened. After screening 18 strains and performing colony PCR with SEQ ID NO: 9 and SEQ ID NO: 10, a PCR product having a size of 400 bp was confirmed. The production of pSCEC_cj7 having the CJ7 promoter was confirmed from the results of colony PCR.

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

The obtained PCR product and pSCEC_cj7 vector were treated with restriction enzymes XbaI and SalI. The restriction enzyme-treated nucleic acid was subjected to gel preparation to ligate the ODC_Lb, ODC_Ec, and pSCEC_cj7 nucleic acid fragments, and inserted into E. coli DH5α strain. From the screened strains confirmed to be inserted, pSCEC_cj7_ODC_Lb and pSCEC_cj7_ODC_Ec were obtained, respectively, and electrophoration was performed on the butanediamine-producing Corynebacterium microorganism KCCM11240P at 2500 confirm V, respectively.

The above strains were smeared and cultured on BHIS plate medium (Braine heart infusion 37 g/l, sorbitol 91 g/l, agar 2%) including 50 μg/L spectinomycin, whereby colonies were formed. It was confirmed that the selected strains can be grown in CM medium containing 50μg/L spectinomycin (glucose 10g/L, polypeptone 10g/L, yeast extract 5g/L) , beef extract (beef extract) 5g/L, NaCl 2.5 g/L, urea (Urea) 2g/L, pH 6.8) for shaking culture. After culturing 3 mL of the prepared two types of Corynebacterium glutamicum mutant strains, centrifugation was performed to obtain bacterial cells. The obtained bacterial cells were cell-pulverized by ultrasonication, and then centrifuged to obtain a solution containing soluble proteins.

ODC_Lb and ODC_Ec proteins including His-tag were purified by Ni-NTA spin columns (Spin Columns) (Hilden, Germany). The protein concentration was measured by Nano drop acquisition. The recombinant protein concentrations calculated based on the measured values were ODC_Lb: 1.282 g/L, ODC_Ec: 0.039 g/L, respectively, and it was confirmed that soluble proteins with ODC_Lb more than 30 times higher than ODC_Ec in the coryneform strain were obtained. white matter.

It was confirmed that when ODC_Lb was expressed by using Escherichia coli and a coryneform strain as hosts under mesophilic conditions, a highly soluble protein could be produced due to the high expression level and normal protein folds.

Example 7. Preparation of rod-shaped butanediamine-producing strains introduced with variant ornithine decarboxylase and measurement of butanediamine production capacity

In order to study the effect of the ornithine decarboxylase variant of the present application on the production of butanediamine, a strain of the microorganism belonging to the genus Corynebacterium having improved butanediamine production ability was prepared by introducing the ornithine decarboxylase variant.

Specifically, as the microorganism of the genus Corynebacterium having the improved butanediamine-producing ability, the microorganism of the genus Corynebacterium having the ability to produce butanediamine disclosed in the patent application (Korean Laid-Open Patent Publication No. 2013-0082478 ) was used ( KCCM11240P). The above-mentioned Corynebacterium microorganism (KCCM11240P) having 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): A microorganism in which NCgl1469 is deleted in KCCM11138P (Korean Laid-Open Patent Publication No. 2012-0064046).

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

The gene fragment obtained by PCR amplification was treated with EcoRV and MfeI restriction enzymes (37°C, 3 hours), and the gene fragment of Lactobacillus-derived wild-type and variant (E698D, A713L) ornithine decarboxylase was inserted To the pDZ-bioAD-P(CJ7) vector produced by the same method as that disclosed in Korean Laid-Open Patent No. 2012-0064046. In the above method, EcoRV and MfeI restriction enzymes were used. The recombinant vectors for chromosomal insertion (pDZ-ODC_Lb, pDZ-ODC_Lb_E698D, pDZ-ODC_Lb_A713L) produced 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 into the chromosome, the recombinant vectors of pDZ-ODC_Lb, pDZ-ODC_Lb_E698D and pDZ-ODC_Lb_A713L prepared above were respectively transformed by electroporation. After staining to KCCM11240P strain, spread to BHIS plate medium (brain heart infusion 37g/l, sorbitol 91g/l, agar 2%, 1L standard + kanamycin 25ug/ml).

Successful insertion of the vector into the chromosome was judged by whether it showed blue color in the solid medium including X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). After shaking cultured in a nutrient medium (30° C., 8 hours), the primary chromosome-inserted strains were serially diluted and spread on a solid medium including X-gal. Most of the colonies are blue, conversely, white colonies can be screened at a low rate, and the screened Colonies can be obtained by secondary cross over to the strains into which the final Lactobacillus ornithine decarboxylase variant has been chromosomally introduced. Finally, strains were confirmed by sequence analysis of the variants. The confirmed strains were named KCCM11240P::ODC_Lb, KCCM11240P::ODC_Lb_E698D, KCCM11240P::ODC_Lb_A713L.

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

Specifically, the above strains were prepared in CM plate medium containing 1mM arginine (glucose 1%, polyprotein 1%, yeast extract 0.5%, beef extract 0.5%, NaCl 0.25%, urea 0.2%, After culturing at 30°C for 16 hours in 50% NaOH 100ul, agar 2%, pH 6.8, 1L reference), about one platinum was inoculated into 25 ml of titer medium having the composition of Table 5 below, which was then incubated at 30°C. Shaking at 200 rpm for 24 hours. All the produced strains were cultured by adding 1 mM arginine to the medium during fermentation.

As a result, as shown in Table 6, after culturing for 12 hours, the Lactobacillus-derived variant (A713L) ornithine decarboxylase-introduced strains were compared with Escherichia coli. Compared with the ornithine decarboxylase strain (KCCM11240P), the production of butanediamine increased by about 115%P. In addition, the A713L ornithine decarboxylase-introduced strain exhibited an increase of about 40% P compared to the strain (KCCM11240P::ODC_Lb) having the wild-type Lactobacillus-derived ornithine decarboxylase.

In addition, compared with KCCM11240P, it was found that the strain (KCCM11240P::ODC_Lb_A713L) into which the A713L ornithine decarboxylase was introduced was reduced by about 48%P due to the side reaction during the production of butanediamine, and the amount remaining in the culture solution was reduced by about 48%P. It is presumed that the sugar consumption in the same time increases, and the productivity also increases.

The results as described above show that, by introducing the variant ornithine decarboxylase from Lactobacillus, it is possible not only to produce butanediamine at a concentration higher than the existing concentration relative to sugar consumption from butanediamine-producing strains, but also to reduce pentamethylenediamine. and improve productivity.

Example 8. Prediction of the effect of saturation variation of amino acid residue 713 of Lactobacillus ornithine decarboxylase

In the selected variant, the functional residue (A713L) for the 713th alanine substituted by leucine for Lactobacillus ornithine decarboxylase was substituted with other amino acids than alanine and leucine, Then, it was introduced into butanediamine-producing strain KCCM11240P to study the effect on the production of butanediamine.

Specifically, the following mutant strains were prepared in place of E. coli derived in the chromosome of the microorganism of the genus Corynebacterium (KCCM11240P) having improved butanediamine productivity. The wild-type ornithine decarboxylase of Bacteria is substituted in the amino acid sequence of SEQ ID NO: 1 with other amino acids including hydrophobic amino acids. Variation in the form of the 713th amino acid from the N-terminus body. More specifically, in order to prepare valine, which is one of hydrophobic amino acids, arginine, which is one of basic amino acids, aspartic acid, which is one of acidic amino acids, and neutral amino acid, respectively. One of the glutamic acid, the tryptophan-substituted carrier of one of the aromatic amino acids, using the pDZ-ODC-Lb carrier prepared in Example 7 as a template and using the above-mentioned table 4 and the following table 7. Revealed primers to perform PCR. The first PCR is performed on the front part (5') and the rear part (3') with the site to be mutated as the center, and then the second PCR is performed to match the two PCR fragments. For example, in the case of the variation (A713V) in which the 713th amino acid is substituted from alanine to valine, the front part is amplified by PCR using primers ODC_Lb_start(EcoRV)_5 and ODC_Lb_A713V_3, and the rear part is ODC_Lb_A713V_5 and ODC_Lb_stop(MfeI)_3 primers were amplified by PCR. The two PCR fragments obtained by the primary PCR were used as templates for the secondary 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, A713Q other than that were also inserted into the pDZ-bioAD-P(CJ7) vector by PCR using the primers disclosed in Table 7 by the same method as above. The produced recombinant vectors for chromosomal 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.

[Table 7]

In order to obtain a strain in which the 713th alanine of Lactobacillus ornithine decarboxylase is chromosomally substituted by other amino acids including hydrophobic amino acids, the method of Example 7 can be used. The pDZ-ODC_Lb_A713V, pDZ-ODC_Lb_A713R, pDZ-ODC_Lb_A713D, pDZ-ODC_Lb_A713W, pDZ-ODC_Lb_A713Q recombinant vectors produced above were respectively transfected into the KCCM11240P strain and screened to obtain the final Lactobacillus ornithine decarboxylase introduced into the chromosome. mutant strains. Finally, the strains were confirmed by sequence analysis of the variants. 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 the variant in which the 713th alanine introduced into Lactobacillus ornithine decarboxylase was substituted with other amino acids including hydrophobic amino acids, on the butanediamine-producing ability of the butanediamine-producing strain, By the method as described above in Example 7 method to evaluate the production capacity of butanediamine.

As a result, as shown in Table 8, the production of butanediamine of the strain in which the Lactobacillus ornithine decarboxylase variant having been substituted with other amino acids including hydrophobicity was introduced was higher than that from Escherichia coli after culturing for 12 hours. The wild-type ornithine decarboxylase strain (KCCM11240P) had an average increase of about 86% P. In addition, the state exhibited an average increase of about 21% P compared to the strain having the wild-type ornithine decarboxylase from Lactobacillus.

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

The above results show that, except for the variant in which the 713th alanine of Lactobacillus ornithine decarboxylase is replaced by leucine, valine and basic amine, which are one of the other hydrophobic amino acids, are 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 Therefore, it is possible not only to produce butanediamine at a higher concentration than the conventional sugar consumption in the butanediamine-producing strain, but also to reduce pentamethylenediamine and improve productivity.

According to the above description, those in the technical field to which this application belongs should be able to It is understood that the present application can be implemented in other specific forms without changing its technical idea or essential features. In this connection, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning, scope and equivalent concepts of the scope of the application described below, rather than the above detailed description.

<110> CJ CheilJedang Corporation

<120> Ornithine decarboxylase variant, method for producing butanediamine using the same, polynucleotide, microorganism, method for producing polyamide, and composition for producing polyamide

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<223> DNA encoding amino acid sequence SEQ ID NO. 19

<400> 24

<210> 25

<211> 2196

<212> DNA

<213> Artificial sequences

<220>

<223> DNA encoding amino acid sequence SEQ ID NO. 20

<400> 25

<210> 26

<211> 2196

<212> DNA

<213> Artificial sequences

<220>

<223> DNA encoding amino acid sequence SEQ ID NO. 21

<400> 26

<210> 27

<211> 2196

<212> DNA

<213> Artificial sequences

<220>

<223> DNA encoding amino acid sequence SEQ ID NO. 22

<400> 27

<210> 28

<211> 2196

<212> DNA

<213> Artificial sequences

<220>

<223> DNA encoding amino acid sequence SEQ ID NO. 23

<400> 28

Claims (16)

An ornithine decarboxylase variant comprising an amino acid corresponding to a) the 713th position, b) the 698th position, or c) the 713th position and the 698th position of SEQ ID NO: 1 replace. The ornithine decarboxylase variant of claim 1, wherein the amino acid substitution at the 713th position is a hydrophobic amino acid other than alanine, a basic amino acid, an acidic Amino acid, neutral amino acid or aromatic amino acid substitution. The ornithine decarboxylase variant of claim 1, wherein the amino acid substitution at the 713th position is A713L, A713I, A713V, A713R, A713D, A713W or A713Q. The ornithine decarboxylase variant according to claim 1, wherein the amino acid substitution at the 698th position is E698D. The ornithine decarboxylase variant of claim 1, wherein the amino acid substitution at the 713th position is A713L, A713I, A713V, A713R, A713D, A713W or A713Q, and the 698th position The amino acid substitution at position is E698D. The ornithine decarboxylase variant according to claim 1, wherein the ornithine decarboxylase variant comprises a variant selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 9 Polypeptides in ID NO: 19 to SEQ ID NO: 23. A polynucleotide encoding the ornithine decarboxylase variant described in any one of items 1 to 6 of the application scope. A microorganism comprising the following ornithine decarboxylase: comprising the polypeptide of SEQ ID NO:1, or at a) the 713th position, b) the 698th position or c) the 713th position corresponding to SEQ ID NO:1 The amino acid substituted polypeptides are included at the 698th position and at the 698th position. The microorganism according to claim 8, wherein the microorganism is Escherichia or Corynebacterium. A method for producing butanediamine, comprising culturing a microorganism comprising an ornithine decarboxylase, wherein the ornithine decarboxylase comprises the polypeptide of SEQ ID NO: 1 or in a) corresponding to SEQ ID NO: 1 Polypeptides comprising amino acid substitutions at positions 713, b) 698 or c) 713 and 698. The method for producing butanediamine according to item 10 of the application scope, comprising accumulating butanediamine in a culture medium. The method for producing butanediamine as described in item 10 of the patent application scope, comprising recovering butanediamine from the cultured microorganism or culture medium. A method for increasing the purity of butanediamine, comprising culturing a microorganism comprising an ornithine decarboxylase, wherein the ornithine decarboxylase comprises the polypeptide of SEQ ID NO: 1, or in a corresponding to SEQ ID NO: 1 ) at position 713, b) at position 698, or c) at positions 713 and 698 comprising amino acid substitutions. A method of increasing the ratio of butanediamine to pentamethylenediamine, comprising culturing a microorganism comprising an ornithine decarboxylase, wherein the ornithine decarboxylase comprises the polypeptide of SEQ ID NO: 1, or in a manner corresponding to SEQ ID NO: 1 of a) 713th position, b) 698th position or c) th Amino acid substituted polypeptides are included at positions 713 and 698. A method for preparing polyamide, which is a method for preparing polyamide using butanediamine, the butanediamine is prepared by culturing a microorganism comprising ornithine decarboxylase, the ornithine decarboxylase comprising SEQ The polypeptide of ID NO: 1, or a polypeptide comprising amino acid substitutions at positions corresponding to a) 713, b) 698, or c) 713 and 698 of SEQ ID NO: 1 . A composition for the preparation of polyamide, comprising a microorganism comprising an ornithine decarboxylase, the ornithine decarboxylase comprising the polypeptide of SEQ ID NO: 1, or in a) section 713 corresponding to SEQ ID NO: 1 A polypeptide comprising amino acid substitutions at position 1, b) position 698, or c) position 713 and position 698.

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