Recombinant microorganism containing a novel lysine decarboxylase that produces pentamethylenediamine and method for producing pentamethylenediamine using the same

Abstract

This specification relates to recombinant microorganisms containing a decarboxylase enzyme protein derived from E. coli Nissle and having pentamethylenediamine (PMDA) production activity, as well as their uses.

Description

Technical Field

[0001] Cross-reference of related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0011856 filed on January 25, 2024, and all the contents disclosed in the literature of the Korean Patent Application are included herein as part of this specification.

[0002] This specification relates to a recombinant microorganism containing a decarboxylase enzyme protein derived from E. coli Nissle and having the production activity of pentamethylenediamine (PMDA), and its use.

Background Art

[0003] PMDA (Pentamethylenediamine) is a C5 diamine and is a substance used as a monomer for nylon PA56 and PA510. Using the amino acid lysine as a raw material, PMDA can be produced by a biological method through an enzymatic conversion of lysine decarboxylase. The lysine decarboxylases studied so far are roughly classified into two types, CadA and LdcC, and it is known that LdcC shows a tendency to tolerate higher pH better than CadA.

[0004] An E. coli strain was used for the production of PMDA. However, at present, there is a need to search for strains to improve the efficiency of the PMDA production industrial process. Non-pathogenic E. coli Nissle (EcN) has been revealed to have no enterotoxin, cytotoxin, pathogenic adhesion factor, serum resistance, antibiotic resistance gene, etc., and is known as a relatively safe host strain in terms of LMO issue in the production of target products. When EcN is used as a host strain, PMDA can be safely produced in terms of LMO issue.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Against the above background, we completed the present invention by confirming the excellent PMDA production activity of recombinant microorganisms into which lysine decarboxylase (LDC) derived from E. coli Nissle has been introduced.

[0006] One example provided herein is a recombinant microorganism containing (and / or expressing) a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle and having pentamethylenediamine (PMDA) production activity.

[0007] Other examples provided herein include recombinant vectors comprising polynucleotides encoding lysine decarboxylase enzyme proteins derived from the microorganism E. coli Nissle.

[0008] Other examples provided herein include compositions for pentamethylenediamine production comprising the recombinant microorganism, a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle, a polynucleotide encoding the enzyme protein, and / or the recombinant vector.

[0009] Other examples herein provide applications of recombinant microorganisms containing (and / or expressing) lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle for pentamethylenediamine production.

[0010] Other examples herein provide applications for recombinant vectors containing polynucleotides encoding lysine decarboxylase enzyme proteins derived from the microorganism E. coli Nissle for pentamethylenediamine production.

[0011] Other examples herein provide uses for recombinant microorganisms containing (and / or expressing) a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle for pentamethylenediamine production, and / or recombinant vectors containing polynucleotides encoding a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle for pentamethylenediamine production.

[0012] Another example provided herein is a method for producing pentamethylenediamine, comprising the step of reacting the recombinant microorganism with a substrate. [Means for solving the problem]

[0013] An example provided herein is a recombinant microorganism comprising a lysine decarboxylase (LDC) enzyme protein and / or a polynucleotide encoding a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle.

[0014] Another example provided herein is a method for producing recombinant microorganisms having pentamethylenediamine production activity, comprising the step of introducing a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle, a polynucleotide encoding the protein, and / or a recombinant vector containing the polynucleotide into a microorganism (host cell).

[0015] The lysine decarboxylase enzyme protein can be expressed by the LdcC gene and / or CadA gene, and the polynucleotide encoding the lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle may be, but is not limited to, the LdcC gene and / or CadA gene of E. coli Nissle.

[0016] Other examples provided herein include recombinant vectors comprising polynucleotides encoding lysine decarboxylase enzyme proteins derived from the microorganism E. coli Nissle.

[0017] Other examples provided herein include compositions for pentamethylenediamine production, comprising one or more selected from the group consisting of the recombinant microorganism, a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle, a polynucleotide encoding the protein, and a recombinant vector containing the polynucleotide.

[0018] Another example provided herein is a method for producing pentamethylenediamine, comprising the step of reacting the recombinant microorganism with a substrate.

[0019] The recombinant microorganism may contain (or express or introduce) a polynucleotide encoding lysine decarboxylase (LDC) enzyme protein and / or protein derived from the microorganism E. coli Nissle, and may have superior pentamethylenediamine production activity compared to microorganisms that do not contain this (e.g., non-mutated or wild-type microorganisms).

[0020] The present application will be described in more detail below.

[0021] Recombinant microorganisms with pentamethylenediamine production activity One example provided herein is a recombinant microorganism comprising a lysine decarboxylase (LDC) enzyme protein derived from the microorganism E. coli Nissle or a polynucleotide encoding a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle. The recombinant microorganism can express a lysine decarboxylase (LDC) enzyme protein derived from the microorganism E. coli Nissle.

[0022] More specifically, the recombinant microorganism may be one into which one or more selected from the group consisting of a lysine decarboxylase enzyme protein derived from E. coli Nissle microorganism, a polynucleotide encoding the protein, and a recombinant vector containing the polynucleotide are introduced. The recombinant microorganism may have the production activity of pentamethylenediamine (Pentamethylenediamine; PMDA) and / or may have an increased pentamethylenediamine production ability as compared with a microorganism (non-transformed or wild-type microorganism) that does not contain the lysine decarboxylase enzyme protein derived from the E. coli Nissle microorganism.

[0023] Another example of the present specification provides a method for producing a recombinant microorganism having the production activity of pentamethylenediamine, which includes the step of introducing a lysine decarboxylase enzyme protein derived from E. coli Nissle microorganism, a polynucleotide encoding the protein, and / or a recombinant vector containing the polynucleotide into a microorganism (host cell) (for example, a microorganism belonging to the genus Escherichia (specifically, E. coli Nissle, E. coli WL3110, E. coli XL1-Blue, E. coli BL21(DE3)), a microorganism belonging to the genus Corynebacterium (specifically, Corynebacterium glutamicum), or a microorganism belonging to the genus Saccharomyces (specifically, Saccharomyces cerevisiae)).

[0024] The recombinant microorganism may be one into which a polynucleotide encoding a lysine decarboxylase enzyme protein derived from E. coli Nissle microorganism (for example, LdcC gene and / or CadA gene) is introduced. The lysine decarboxylase enzyme protein can be expressed by the LdcC gene and / or CadA gene, but is not limited thereto.

[0025] As used herein, "activity enhancement" or "enhanced activity" not only includes the introduction of new activity or the increase in the activity of the protein itself to derive an effect greater than the original function, but also includes an increase in gene expression, an increase in intrinsic gene activity, amplification of an endogenous gene from internal or external factors, deletion of a regulatory factor for suppressing the gene expression, an increase in gene copy number, introduction of an external gene, modification of an expression regulatory sequence, and / or replacement or modification of a promoter and an increase in enzyme activity due to an intragenic mutation, etc. It can mean a state in which the activity of the microorganism after the operation is increased compared to the activity of the microorganism before the operations such as those described above are performed.

[0026] As used herein, enhancement of the activity of a protein or an enzyme (e.g., lysine decarboxylase derived from E. coli Nissle microorganism) can be achieved by applying various methods well-known in the art. The methods for enhancing or increasing the activity of the protein and / or enzyme can involve applying various methods well-known in the art. For example, but not limited thereto, methods for increasing the copy number of the nucleotide sequence encoding the enzyme (or protein) such as additionally inserting a polynucleotide containing the nucleotide sequence encoding the enzyme into the chromosome, introducing the polynucleotide into a vector system, etc., methods for replacing a promoter capable of expressing the polynucleotide with a strong promoter, methods for introducing a mutation into the promoter, and methods for mutating the enzyme (or protein) into an enzyme (or protein) with strong activity by gene mutation, etc. may be included.

[0027] The lysine decarboxylase enzyme protein derived from the E. coli Nissle microorganism can be a foreign enzyme protein. As used herein, "foreign" can mean not inherently present but introduced from the outside by a normal method such as a recombinant method, etc., and can mean derived from another strain or cell of different or the same species.

[0028] The recombinant microorganism may be one in which the activity of the lysine decarboxylase enzyme derived from the microorganism E. coli Nissle is enhanced.

[0029] The nucleic acid (gene) sequences or amino acid sequences provided herein may include those modified by conventional mutagenesis methods, such as direct evolution and / or site-directed mutagenesis, to the extent that they maintain their original or intended functions. For example, when a polynucleotide or polypeptide is said to "contain, have, or consist of a particular nucleic acid sequence or amino acid sequence," it can mean that the polynucleotide or polypeptide (i) essentially contains the particular nucleic acid sequence or amino acid sequence, or (ii) consists of or essentially contains an amino acid sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with the particular nucleic acid sequence or amino acid sequence, while maintaining its original and / or intended functions. In this specification, the aforementioned target function may mean a function that increases or imparts the pentamethylenediamine production capacity of microorganisms.

[0030] The nucleic acid sequences described herein may be modified in various ways within the coding region, taking into account codons preferred by microorganisms that are intended to express the protein by codon degeneracy, as long as the amino acid sequence and / or function of the protein expressed from the coding region are not altered.

[0031] In this specification, the term "identity" refers to the degree to which a given nucleic acid sequence or amino acid sequence matches, and may be expressed as a percentage (%). In the case of homology to nucleic acid sequences, it can be determined, for example, using the literature-based algorithms BLAST or Pearson's FASTA. Based on such algorithms as BLAST, programs called BLASTN and BLASTX have been developed.

[0032] In one specific example, the lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle may contain the amino acid sequence of Sequence ID No. 1, or have an amino acid sequence having 90% or more sequence homology, 95% or more sequence homology, 97% or more sequence homology, 99% or more sequence homology, 99.5% or more sequence homology, or 99.9% or more sequence homology with the aforementioned sequence, and may have pentamethylenediamine production activity. The polynucleotide encoding the lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle may contain the nucleic acid sequence of Sequence ID No. 2, or have a nucleic acid sequence having 90% or more sequence homology, 95% or more sequence homology, 97% or more sequence homology, 99% or more sequence homology, 99.5% or more sequence homology, or 99.9% or more sequence homology with the aforementioned sequence.

[0033] The recombinant microorganisms may, but are not limited to, microorganisms of the genus Escherichia (specifically, E. coli Nissle, E. coli WL3110, E. coli XL1-Blue, E. coli BL21(DE3)), microorganisms of the genus Corynebacterium (specifically, Corynebacterium glutamicum), or microorganisms of the genus Saccharomyces (specifically, Saccharomyces cerevisia).

[0034] Another example provides a recombinant vector containing a polynucleotide encoding a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle.

[0035] The polynucleotide encoding the lysine decarboxylase enzyme protein derived from the E. coli Nissle microorganism may be, but is not limited to, the LdcC gene and / or the CadA gene.

[0036] Microorganisms containing the recombinant vector, or into which the recombinant vector has been introduced, may, but are not limited to, have the activity to produce pentamethylenediamine (PMDA).

[0037] In this specification, “vector” means any medium for the cloning and / or transfer of bases to a host cell. A vector can be a replication unit (replicon) to which other DNA fragments bind, resulting in the replication of the bound fragment. A “replication unit” can mean any genetic unit (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as a self-unit of DNA replication in a living organism, that is, a self-regulating replicable unit. In this invention, the vector is not particularly limited as long as it is a replicable host, and any vector known in the art can be used.

[0038] The vector used in the preparation of the recombinant vector, specifically the vector used to introduce the polynucleotide encoding the lysine decarboxylase enzyme protein derived from the E. coli Nissle microorganism into the microorganism, may be a plasmid, cosmid, virus, and / or bacteriophage in its native state or in a recombinant state. For example, as a phage vector or cosmid vector, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon4A, and / or Charon21A can be used, and as a plasmid vector, pKE vector, pHCMS vector, pHCP vector, pCES vector, pCG vector, pDZ vector, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and / or pET series can be used. The available vectors are not particularly limited, and known expression vectors can be used. In one example, the vector may be designed so that the gene inserted and transmitted within the vector irreversibly fuses into the genome of a host cell, thereby ensuring stable and long-term gene expression within the cell. Such a vector may include transcriptional and decoded expression regulatory sequences that enable the gene to be expressed in a selected host. The expression regulatory sequences may include any operator sequences for regulating transcription and / or sequences for regulating the termination of transcription and decoding. In one example, the start and termination codons may generally be considered as part of the nucleic acid sequence encoding the target protein, must exert their effect in the organism when the gene construct is administered, and may be in frame with the coding sequence. In the case of a replicable expression vector, a replication origin may also be included. Other appropriate elements may include enhancers, the 3' untranslated region of the target gene, selection markers (e.g., antibiotic resistance markers), and / or replicable units. The vector may self-replicate or integrate into the host genomic DNA.For example, each component within the vector must be operably linked to one another, and the linking of these component sequences may be carried out by ligation at a convenient restriction enzyme site, or, if such a site is not present, by using a conventional synthetic oligonucleotide adapter or linker.

[0039] Composition for pentamethylenediamine production Other examples provided herein include compositions for pentamethylenediamine production, comprising one or more selected from the group consisting of the recombinant microorganism, the lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle, the polynucleotide encoding the enzyme protein, and the recombinant vector.

[0040] The recombinant microorganism may mean, but is not limited to, one or more species selected from the group consisting of the recombinant microorganism itself (bacterial cells), its cultures, lysates, and extracts.

[0041] The composition may additionally contain a substrate. The substrate may be present in concentrations of 50-500 g / L, 50-400 g / L, 50-350 g / L, 50-320 g / L, 50-300 g / L, 50-250 g / L, 50-220 g / L, 50-200 g / L, 50-150 g / L, 50-120 g / L, 50-100 g / L, 80-500 g / L, 80-400 g / L, 80-350 g / L, 80-320 g / L, 80-300 g / L, 80-250 g / L, 80-220 g / L, and 80 g / L based on the total volume of the composition. ~200g / L, 80~150g / L, 80~120g / L, 80~100g / L, 100~500g / L, 100~400g / L, 100~350g / L, 100~320g / L, 100~300g / L, 100~250g / L, 100~220g / L, 100~200g / L, 100~150g / L, 100~120g / L, 150~500g / L, 150~400g / L, 150~350g / L, 150~320g / L, 150~300g / L, 150~250g / L, 150~220g / L, 150~200g / L, 180~500g / L, 180~400g / L, 180~350g / L, 180~320g / L, 180~300g / L, 180~250g / L, 18 0~220g / L, 180~200g / L, 200~500g / L, 200~400g / L, 200~350g / L, 200~320g / L, 200~300g / L, 200~250g / L, 200~220g / L, 250~5 It may be included in concentrations of 00g / L, 250-400g / L, 250-350g / L, 250-320g / L, 250-300g / L, 280-500g / L, 280-400g / L, 280-350g / L, 280-320g / L, 280-300g / L, 300-500g / L, 300-400g / L, 300-350g / L, 300-320g / L, for example, 100g / L, 200g / L, or 300g / L, but is not limited to these concentrations.

[0042] The substrate may, but is not limited to, lysine, a carbon source, a nitrogen source, a phosphorus source, and / or trace element components.

[0043] In this specification, "lysine" may mean lysine alone and / or lysine containing salts thereof, and may specifically be, but not limited to, one or more selected from the group consisting of lysine, lysine-HCl, lysine-H2SO4, etc.

[0044] The carbon source may include glucose, glycerol, and / or molasses. Other examples include monosaccharides, oligosaccharides, polysaccharides, single-carbon substrates, or mixtures thereof. For example, monosaccharides such as glucose and fructose; oligosaccharides such as sucrose, maltose, or lactose; polysaccharides such as starch or cellulose; and single-carbon substrates such as methanol, formaldehyde, or formate. Examples of other substances that can be used include, but are not limited to, lower alcohols such as ethanol, propanol, and butanol; polyhydric alcohols such as glycerol; organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid, and gluconic acid; fatty acids such as propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, palmitic acid, stearic acid, and linoleic acid; and oils such as bean oil, sunflower oil, peanut oil, and coconut oil (specifically, vegetable oils). These substances can be used individually or in mixtures.

[0045] Examples of the nitrogen sources include, but are not limited to, peptone, yeast extract, meat juice, malt extract, corn maceration, soybean meal, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The nitrogen sources can also be used individually or in mixtures.

[0046] Examples of the phosphorus source include, but are not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or corresponding sodium-containing salts. The growth medium may also contain, but is not limited to, metal salts such as magnesium sulfate or ferrous sulfate necessary for growth, or essential growth substances such as amino acids and vitamins. The above raw materials can be added to the culture in a batch or continuous manner in a manner appropriate to the culture process.

[0047] The composition may, but is not limited to, additionally contain PLP. The PLP may be 1-1,000 μM, 1-800 μM, 1-600 μM, 1-500 μM, 1-400 μM, 1-300 μM, 1-200 μM, 1-100 μM, 1-80 μM, 1-60 μM, 1-50 μM, 1-30 μM, 1-20 μM, 1-15 μM, 1-12 μM, 5-1,000 μM, 5-800 μM, 5-600 μM, 5-500 μM, 5-400 μM, 5-300 μM, 5-200 μM, 5-100 μM, 5-80 μM, It may be included in concentrations of 5-60 μM, 5-50 μM, 5-30 μM, 5-20 μM, 5-15 μM, 5-12 μM, 8-1,000 μM, 8-800 μM, 8-600 μM, 8-500 μM, 8-400 μM, 8-300 μM, 8-200 μM, 8-100 μM, 8-80 μM, 8-60 μM, 8-50 μM, 8-30 μM, 8-20 μM, 8-15 μM, or 8-12 μM, for example, at a concentration of 10 μM, but is not limited to these.

[0048] Method for producing pentamethylenediamine Another example provided herein is a method for producing pentamethylenediamine, comprising the step of reacting the recombinant microorganism with a substrate.

[0049] The recombinant microorganism may mean, but is not limited to, one or more species selected from the group consisting of the recombinant microorganism itself (bacterial cells), its cultures, lysates, and extracts.

[0050] The substrate is selected from the group consisting of cultures of the recombinant microorganism, cells of the recombinant microorganism, lysates of the cells, and extracts thereof, based on a volume of one or more of these, in the following ranges: 50-500 g / L, 50-400 g / L, 50-350 g / L, 50-320 g / L, 50-300 g / L, 50-250 g / L, 50-220 g / L, 50-200 g / L, 50-150 g / L, 50-120 g / L, 50-100 g / L, 80-500 g / L, 80-400 g / L, 80-350 g / L, 80~320g / L, 80~300g / L, 80~250g / L, 80~220g / L, 80~200g / L, 80~150g / L, 80~120g / L, 80~100g / L, 100~500g / L, 100~400g / L, 100~35 0g / L, 100~320g / L, 100~300g / L, 100~250g / L, 100~220g / L, 100~200g / L, 100~150g / L, 100~120g / L, 150~500g / L, 150~400g / L, 150~ 350g / L, 150~320g / L, 150~300g / L, 150~250g / L, 150~220g / L, 150~200g / L, 180~500g / L, 180~400g / L, 180~350g / L, 180~320g / L, 18 0~300g / L, 180~250g / L, 180~220g / L, 180~200g / L, 200~500g / L, 200~400g / L, 200~350g / L, 200~320g / L, 200~300g / L, 200~250g / L, The reaction can be carried out at concentrations of 200-220 g / L, 250-500 g / L, 250-400 g / L, 250-350 g / L, 250-320 g / L, 250-300 g / L, 280-500 g / L, 280-400 g / L, 280-350 g / L, 280-320 g / L, 280-300 g / L, 300-500 g / L, 300-400 g / L, 300-350 g / L, 300-320 g / L, for example, 100 g / L, 200 g / L, or 300 g / L, but is not limited to these concentrations.

[0051] The substrate may, but is not limited to, a lysine complex, a carbon source, a nitrogen source, a phosphorus source, and / or trace element components.

[0052] The method for producing pentamethylenediamine may, but is not limited to, include an additional step of reacting one or more selected from the group consisting of recombinant microorganisms, cultures of the recombinant microorganisms, cells of the recombinant microorganisms, lysates of the cells, and extracts thereof, with PLP. The PLP is 1 to 1,000 μM, 1 to 800 μM, 1 to 600 μM, 1 to 500 μM, 1 to 400 μM, 1 to 300 μM, 1 to 200 μM, 1 to 100 μM, 1 to 80 μM, 1 to 60 μM, 1 to 50 μM, 1 to 3 0μM, 1~20μM, 1~15μM, 1~12μM, 5~1,000μM, 5~800μM, 5~600μM, 5~500μM, 5~400μM, 5~300μM, 5~200μM, 5~100μM, 5~80μM Reactions can be carried out at concentrations of 5-60 μM, 5-50 μM, 5-30 μM, 5-20 μM, 5-15 μM, 5-12 μM, 8-1,000 μM, 8-800 μM, 8-600 μM, 8-500 μM, 8-400 μM, 8-300 μM, 8-200 μM, 8-100 μM, 8-80 μM, 8-60 μM, 8-50 μM, 8-30 μM, 8-20 μM, 8-15 μM, or 8-12 μM, for example, at a concentration of 10 μM, but are not limited to these.

[0053] The method for producing pentamethylenediamine may additionally include a step of culturing the recombinant microorganism.

[0054] The method for producing pentamethylenediamine may additionally include a step of separating and / or purifying pentamethylenediamine from a culture (or culture medium). The culture may be, but is not limited to, a culture obtained by reacting one or more selected from the group consisting of recombinant microorganisms, cultures of the recombinant microorganisms, cells of the recombinant microorganisms, lysates of the cells, and extracts thereof, with a substrate. [Effects of the Invention]

[0055] This invention allows for the production of pentamethylenediamine using recombinant microorganisms containing a decarboxylase enzyme protein derived from E. coli Nissle. [Brief explanation of the drawing]

[0056] [Figure 1] This figure shows the vector map of the pKE112 vector used for introducing the lysine decarboxylase enzyme gene derived from E. coli Nissle. [Figure 2] This graph shows the results of examining the pentamethylenediamine (PMDA) production activity of microorganisms depending on whether or not they have been introduced with the lysine decarboxylase enzyme gene derived from E. coli Nissle. [Figure 3] This graph shows the results of confirming the pentamethylenediamine (PMDA) production activity depending on the concentration of the lysine decarboxylase enzyme introduced into recombinant microorganisms or the substrate used. [Modes for carrying out the invention]

[0057] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited to the following examples.

[0058] Examples 1 and 2. Production of recombinant microorganisms containing the lysine decarboxylase enzyme gene derived from E. coli Nissle To confirm the pentamethylenediamine (PMDA) production activity of lysine decarboxylase (LDC) derived from E. coli Nissle, the lysine decarboxylase enzyme gene was cloned.

[0059] Specifically, to insert the lysine decarboxylase enzyme gene (NCBI Accession Number: AXY47745.1) derived from E. coli Nissle, expressed by the ldcC (lysine decarboxylase ldcC) gene, into the pKE112 vector, pKE112 was digested using the restriction enzyme KpnI / HindIII (New England Biolabs). The sequence information of the restriction enzyme used is shown in Table 1 below. The vector map of the pKE112 vector is shown in Figure 1. [Table 1]

[0060] The lysine decarboxylase derived from E. coli Nissle was degraded using KpnI / HindIII and then cloned during the ligation process into the pKE112 vector (pKE112_EcNLdcC).

[0061] The amino acid sequence (SEQ ID NO: 1) and gene sequence (SEQ ID NO: 2) of the lysine decarboxylase enzyme derived from E. coli Nissle are shown in Table 2 below. [Table 2]

[0062] The cloned pKE112_EcNLdcC vector was subjected to heat shock to E. coli Nissle microorganisms (Hwang, In Young, et al. "Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models." Nature communications 8.1 (2017):15028. Lan, Yi-Jun, et al. "Development of Escherichia coli Nissle 1917 derivative by CRISPR / Cas9 and application for gamma-aminobutyric acid (GABA) production in antibiotic-free system." Biochemical Engineering Journal 168 (2021):107952.) (Example 1) and E. coli Nissle 1917 microorganisms (Example 1) Each was transformed into the WL3110 microorganism (the WL3110 microorganism used in Korean Published Patent No. 2009-0018781) (Example 2).

[0063] Comparative Example 1. Production of microorganisms in which the lysine decarboxylase enzyme gene has not been transformed. A microorganism of E. coli Nissle used in Example 1 was prepared, in which the lysine decarboxylase enzyme gene had not been transformed (Comparative Example 1).

[0064] Comparative Examples 2 and 3. Production of recombinant microorganisms containing the lysine decarboxylase enzyme gene derived from wild-type E. coli. To confirm the pentamethylenediamine production activity of lysine decarboxylase derived from wild-type E. coli expressed by the cadA (lysine decarboxylase cadA) gene (NCBI accession number: UDE09507.1) and lysine decarboxylase derived from wild-type E. coli expressed by the ldcC gene (NCBI accession number: UDE09932.1), the lysine decarboxylase enzyme genes were cloned.

[0065] Specifically, the lysine decarboxylase enzyme gene expressed by the cadA gene was cloned into the pKE112 vector in substantially the same manner as pKE112_EcNLdcC, except that KpnI / BamHI (New England Biolabs) was used as the restriction enzyme to transform E. coli WL3110 (pKE112_EcCadA). Similarly, the lysine decarboxylase enzyme gene expressed by the ldcC gene was also cloned into the pKE112 vector in substantially the same manner as pKE112_EcNLdcC, except that KpnI / SbfI (New England Biolabs) was used as the restriction enzyme (pKE112_EcLdcC). The sequence information of the restriction enzymes used is shown in Table 1 above.

[0066] Table 3 below shows the amino acid sequence (SEQ ID NO: 3) and gene sequence (SEQ ID NO: 4) of the lysine decarboxylase enzyme derived from wild-type E. coli expressed by the cadA gene, and the amino acid sequence (SEQ ID NO: 5) and gene sequence (SEQ ID NO: 6) of the lysine decarboxylase enzyme derived from wild-type E. coli expressed by the ldcC gene. [Table 3-1] [Table 3-2]

[0067] The cloned pKE112_EcCadA (Comparative Example 2) and pKE112_EcLdcC (Comparative Example 3) vectors were each transformed into the same microorganism as the E. coli WL3110 microorganism used in Example 2 by the heat shock method.

[0068] Test Example 1. Measurement of pentamethylenediamine production activity of recombinant microorganisms containing a lysine decarboxylase enzyme gene derived from E. coli Nissle. The microorganisms of Example 1 and Comparative Example 1 prepared as described above were cultured for 16 hours at 30°C in MR medium containing 20 g / L glucose and 10 mg / L thiamine-HCl (6.67 g / L KH2PO4, 4 g / L (NH4)2HPO4, 0.8 g / L MgSO4·7H2O, 0.8 g / L citric acid, 5 mL trace metal solution). During the culture of the microorganisms of Example 1, the antibiotic ampicillin was added at a concentration of 50 μg / mL, and transformed microorganisms were selected by sorting the viable microorganisms.

[0069] The cultured microorganisms were centrifuged at 4°C, 4000 rpm, and for 15 minutes, and the cells obtained were subjected to OD (Oral Dissociation). 600 Lysine-HCl, diluted to a concentration of 50 in distilled water and titrated to pH 7.0, was added to a final concentration of 200 g / L. In addition, pyridoxal 5'-phosphate (PLP), which enhances the activity of the LDC enzyme, was added at a concentration of 10 μM to one group, and PLP was not added to the other group. The enzyme conversion reaction was carried out at 37°C for 120 hours.

[0070] The enzymatic conversion reaction described above was allowed to proceed, and the final production concentration (PMDA titer) of the produced pentamethylenediamine was measured by HPLC after the diethyl ethoxymethylenemalonate (DEEMM) reaction. The results are shown in Table 4 and Figure 2 below.

[0071] Specifically, an HPLC system equipped with a Variable Wavelength Detector (VWD) and a C18 column was used, and elution was performed in Mobile Phase A and Mobile Phase B. The flow rate was set to 1 mL / min, and the analysis was carried out at 284 nm. [Table 4]

[0072] The final production concentration of pentamethylenediamine was confirmed to be as follows: microorganisms transformed with the lysine decarboxylase enzyme gene from E. coli Nissle produced PMDA, while untransformed E. coli Nissle microorganisms did not produce PMDA. Furthermore, it was confirmed that PMDA production was higher when PLP was added.

[0073] Test Example 2. Measurement of pentamethylenediamine production activity according to the concentration of lysine decarboxylase enzyme-derived bacterial strains and substrates. The microorganisms of Example 2 and Comparative Examples 2 and 3 were cultured in substantially the same manner as the culture method for the microorganisms of Example 1 in Test Example 1.

[0074] The cultured microorganisms were centrifuged at 4°C, 4000 rpm, and for 15 minutes, and the cells obtained were subjected to OD (Oral Dissociation). 600 Lysine-HCl, diluted in distilled water to a concentration of 50 and titrated to pH 7.0, was added to achieve a final concentration of 100, 200, or 300 g / L, and the enzymatic conversion reaction was carried out at 37°C for 120 hours.

[0075] The enzyme conversion reaction was allowed to proceed, and the final production concentration (PMDA titer) of the produced pentamethylenediamine was measured in substantially the same manner as in Test Example 1. The results are shown in Table 5 and Figure 3 below. [Table 5]

[0076] After examining the final production concentration of pentamethylenediamine, we confirmed that microorganisms transformed with lysine decarboxylase derived from E. coli Nissle produced more PMDA than microorganisms transformed with lysine decarboxylase derived from wild-type E. coli.

[0077] We confirmed that microorganisms transformed with lysine decarboxylase derived from E. coli Nissle (Example 2) continued to increase in PMDA production as the concentration of lysine-HCl increased.

[0078] Furthermore, the PMDA production of Comparative Examples 2 and 3, which were microorganisms transformed with lysine decarboxylase expressed by the cadA and ldcC genes respectively, was examined, and it was confirmed that Comparative Example 3, which was a microorganism transformed with lysine decarboxylase expressed by the ldcC gene, produced a higher amount of PMDA.

Claims

1. It contains a polynucleotide encoding a lysine decarboxylase (LDC) enzyme protein derived from the microorganism E. coli Nissle or a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle. Recombinant microorganisms that have the activity to produce pentamethylenediamine (PMDA).

2. The recombinant microorganism according to claim 1, wherein a polynucleotide encoding a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle has been introduced.

3. The recombinant microorganism according to claim 1, wherein the lysine decarboxylase enzyme derived from the microorganism E. coli Nissle contains the amino acid sequence of SEQ ID NO:

1.

4. The recombinant microorganism according to claim 1, wherein the recombinant microorganism is a microorganism of the genus Escherichia, a microorganism of the genus Corynebacterium, or a microorganism of the genus Saccharomyces.

5. The recombinant microorganism according to claim 1, wherein the lysine decarboxylase enzyme protein is expressed by the ldcC gene.

6. A composition for pentamethylenediamine production comprising one or more selected from the group consisting of a recombinant microorganism according to any one of claims 1 to 5, a lysine decarboxylase enzyme protein derived from the microorganism E. coli Nissle, a polynucleotide encoding the enzyme protein, and a recombinant vector containing the polynucleotide.

7. The composition for pentamethylenediamine production according to claim 6, wherein the composition further comprises a substrate.

8. The composition for pentamethylenediamine production according to claim 7, wherein the substrate is contained in a concentration of 50 to 500 g / L based on the total volume of the composition.

9. The aforementioned substrates are lysine, lysine-HCl, and lysine-H ２ SO ４ (lysine-H ２ SO ４ The pentamethylenediamine production composition according to claim 7, which is one or more selected from the group consisting of glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose.

10. A method for producing pentamethylenediamine, comprising the step of reacting a recombinant microorganism according to any one of claims 1 to 5 with a substrate.

11. The method for producing pentamethylenediamine according to claim 10, wherein the substrate is reacted at a concentration of 50 to 500 g / L based on the volume of the culture of the recombinant microorganism.

12. The substrates are lysine, lysine-HCl, and lysine-H ２ SO ４ (lysine-H ２ SO ４ A method for producing pentamethylenediamine according to claim 10, wherein the selected material is one or more selected from the group consisting of ).

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