Recombinant microorganism comprising novel lysine decarboxylase and producing pentamethylenediamine and method for producing pentamethylenediamine by using same

Abstract

The present specification relates to a recombinant microorganism comprising an E. coli Nissle-derived decarboxylase enzyme protein and having pentamethylenediamine (PMDA)-producing activity, and a use thereof.

Description

Recombinant microorganism producing pentamethylenediamine including novel lysine decarboxylase and method for producing pentamethylenediamine using the same

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority from Republic of Korea Patent Application No. 10-2024-0011856, filed January 25, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present specification relates to a recombinant microorganism comprising a decarboxylase enzyme protein derived from E. coli Nissle and having pentamethylenediamine (PMDA) production activity, and to a use thereof.

[0004] PMDA (Pentamethylenediamine) is a C5 diamine used as a monomer for nylon PA56 and PA510. PMDA can be produced biologically using the amino acid lysine as a raw material through an enzyme called lysine decarboxylase. Two main types of lysine decarboxylase have been studied to date: CadA and LdcC. LdcC is known to exhibit a tendency to withstand high pH better than CadA.

[0005] An E. coli strain was used for the above PMDA production. However, strain exploration is necessary to improve the efficiency of the PMDA production industrial process. The non-pathogenic E. coli Nissle (EcN) has been found to be free of enterotoxins, cytotoxins, pathogenic adhesion factors, serum resistance, and antibiotic resistance genes, making it a relatively safe host strain for target product production from LMO issues. Therefore, when EcN is used as a host strain, PMDA production is possible safely from LMO issues.

[0006]

[0007] Under the above background, the present invention was completed by confirming the excellent PMDA production activity of a recombinant microorganism into which lysine decarboxylase (LDC) derived from E. coli Nissle was introduced.

[0008] An example of the present disclosure provides a recombinant microorganism comprising (and / or expressing) a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism and having pentamethylenediamine (PMDA) production activity.

[0009] Another example of the present disclosure provides a recombinant vector comprising a polynucleotide encoding a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism.

[0010] Another example of the present specification provides a composition for producing pentamethylenediamine, comprising the recombinant microorganism, the lysine decarboxylase enzyme protein derived from the E. coli Nissle microorganism, a polynucleotide encoding the enzyme protein, and / or the recombinant vector.

[0011] Another example of the present disclosure provides the use of a recombinant microorganism comprising (and / or expressing) a lysine decarboxylase enzyme protein from an E. coli Nissle microorganism for the production of pentamethylenediamine.

[0012] Another example of the present disclosure provides the use of a recombinant vector comprising a polynucleotide encoding a lysine decarboxylase enzyme protein from an E. coli Nissle microorganism for the production of pentamethylenediamine.

[0013] Another example of the present disclosure provides the use of a recombinant microorganism comprising (and / or expressing) a lysine decarboxylase enzyme protein from an E. coli Nissle microorganism for producing pentamethylenediamine and / or a recombinant vector comprising a polynucleotide encoding a lysine decarboxylase enzyme protein from an E. coli Nissle microorganism for producing pentamethylenediamine for preparing a composition for producing pentamethylenediamine.

[0014] Another example of the present disclosure provides a method for producing pentamethylenediamine, comprising the step of reacting the recombinant microorganism with a substrate.

[0015] An example of the present disclosure provides a recombinant microorganism comprising a lysine decarboxylase (LDC) enzyme protein derived from an E. coli Nissle microorganism and / or a polynucleotide encoding a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism.

[0016] Another example of the present specification provides a method for producing a recombinant microorganism having pentamethylenediamine production activity, comprising the step of introducing into a microorganism (host cell) a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism, a polynucleotide encoding the protein, and / or a recombinant vector comprising the polynucleotide.

[0017] The above lysine decarboxylase enzyme protein can be expressed by the LdcC gene and / or the CadA gene, and the polynucleotide encoding the lysine decarboxylase enzyme protein derived from the E. coli Nissle microorganism can be, but is not limited to, the LdcC gene and / or the CadA gene of E. coli Nissle.

[0018] Another example of the present disclosure provides a recombinant vector comprising a polynucleotide encoding a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism.

[0019] Another example of the present specification provides a composition for producing pentamethylenediamine, comprising at least one selected from the group consisting of the recombinant microorganism, a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism, a polynucleotide encoding the protein, and a recombinant vector comprising the polynucleotide.

[0020] Another example of the present disclosure provides a method for producing pentamethylenediamine, comprising the step of reacting the recombinant microorganism with a substrate.

[0021] The recombinant microorganism may have superior pentamethylenediamine production activity compared to a microorganism (e.g., an unmodified or wild-type microorganism) that does not contain (or expresses or introduces) a lysine decarboxylase (LDC) enzyme protein derived from an E. coli Nissle microorganism and / or a polynucleotide encoding the protein.

[0022]

[0023] Hereinafter, the present application will be described in more detail.

[0024]

[0025] Recombinant microorganism having pentamethylenediamine production activity

[0026] An example of the present disclosure provides a recombinant microorganism comprising a lysine decarboxylase (LDC) enzyme protein derived from an E. coli Nissle microorganism or a polynucleotide encoding the lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism. The recombinant microorganism can express the lysine decarboxylase (LDC) enzyme protein derived from an E. coli Nissle microorganism.

[0027] More specifically, the recombinant microorganism may be one into which at least one selected from the group consisting of a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism, a polynucleotide encoding the protein, and a recombinant vector comprising the polynucleotide may be introduced. The recombinant microorganism may have pentamethylenediamine (PMDA) production activity and / or have increased pentamethylenediamine production ability compared to a microorganism (unmodified or wild-type microorganism) that does not contain the lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism.

[0028] Another example of the present specification provides a method for producing a recombinant microorganism having pentamethylenediamine producing activity, comprising the step of introducing a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism, a polynucleotide encoding the protein, and / or a recombinant vector comprising the polynucleotide into a microorganism (host cell) (e.g., a microorganism of the genus Escherichia (specifically, E. coli Nissle, E. coli WL3110, E. coli XL1-Blue, E. coli BL21 (DE3)), a microorganism of the genus Corynebacterium (specifically, Corynebacterium glutamicum), or a microorganism of the genus Saccharomyces (specifically, Saccharomyces cerevisiae)).

[0029] The recombinant microorganism may be one into which a polynucleotide encoding a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism (e.g., an LdcC gene and / or a CadA gene) has been introduced. The lysine decarboxylase enzyme protein may be expressed by the LdcC gene and / or the CadA gene, but is not limited thereto.

[0030] In this specification, “enhanced activity” or “enhanced activity” may include not only a case where the activity of the protein itself is newly introduced or increased to produce an effect beyond its original function, but also a state where the activity of the microorganism after manipulation is increased compared to the activity of the microorganism before manipulation, such as increased gene expression, increased endogenous gene activity, amplification of endogenous genes from internal or external factors, deletion of inhibitory regulatory factors of the gene expression, increased gene copy number, introduction of genes from outside, modification of expression regulatory sequences, and / or replacement or modification of promoters and increased enzyme activity due to mutations in genes.

[0031] In the present specification, the enhancement of the activity of a protein or enzyme (e.g., lysine decarboxylase derived from E. coli Nissle microorganism) can be achieved by applying various methods well known in the art. Methods for enhancing or increasing the activity of the protein and / or enzyme can be applied by various methods well known in the art. For example, but not limited to, a method of increasing the copy number of a base sequence encoding an enzyme (or protein) by additionally inserting a polynucleotide including a base sequence encoding the enzyme (or protein) into a chromosome, a method of introducing the polynucleotide into a vector system, a method of replacing a promoter capable of expressing a polynucleotide with a strong promoter, a method of introducing a mutation into a promoter, and a method of mutating an enzyme (or protein) with a strong activity by genetic mutation, etc.

[0032] The above-mentioned E. coli Nissle-derived lysine decarboxylase enzyme protein may be a foreign enzyme protein. As used herein, "foreign" may mean that it is not endogenously present, but is introduced from the outside through conventional methods such as recombinant methods as mentioned above, and may mean that it is derived from another strain or cell of a different or identical species.

[0033] The above recombinant microorganism may have enhanced activity of lysine decarboxylase enzyme derived from E. coli Nissle microorganism.

[0034] The nucleic acid (gene) sequence or amino acid sequence provided herein may include a modification by conventional mutagenesis, such as directed evolution and / or site-directed mutagenesis, within the scope of maintaining their original function or desired function. In one example, a polynucleotide or polypeptide “comprises, has, or consists of a particular nucleic acid sequence or amino acid sequence” may mean that the polynucleotide or polypeptide (i) essentially comprises the particular nucleic acid sequence or amino acid sequence, or (ii) essentially comprises or consists of an amino acid sequence that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology with the particular nucleic acid sequence or amino acid sequence and maintains its original function and / or desired function. In the present specification, the desired function may mean the function of increasing or imparting pentamethylenediamine production ability of a microorganism.

[0035] The nucleic acid sequence described herein may be modified in various ways in the coding region within a range that does not change the amino acid sequence and / or function of the protein expressed from the coding region, taking into account the codons preferred in the microorganism that is to express the protein due to the degeneracy of the codons.

[0036] As used herein, the term "identity" refers to the degree of identity between a given nucleic acid sequence or amino acid sequence, and may be expressed as a percentage (%). Homology for nucleic acid sequences can be determined, for example, using the BLAST algorithm based on the literature or the FASTA algorithm based on Pearson. Based on the BLAST algorithm, programs called BLASTN and BLASTX have been developed.

[0037] In one specific example, the lysine decarboxylase enzyme protein derived from the E. coli Nissle microorganism may have an amino acid sequence of SEQ ID NO: 1, or 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 sequence, and may have pentamethylenediamine production activity. The polynucleotide encoding the lysine decarboxylase enzyme protein derived from the E. coli Nissle microorganism may have a nucleic acid sequence of SEQ ID NO: 2, or 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 sequence.

[0038] The recombinant microorganism may be, but is not limited to, a microorganism of the genus Escherichia (specifically, E. coli Nissle, E. coli WL3110, E. coli XL1-Blue, E. coli BL21 (DE3)), a microorganism of the genus Corynebacterium (specifically, Corynebacterium glutamicum), or a microorganism of the genus Saccharomyces (specifically, Saccharomyces cerevisia).

[0039] Another example provides a recombinant vector comprising a polynucleotide encoding a lysine decarboxylase enzyme protein derived from the E. coli Nissle microorganism.

[0040] The polynucleotide encoding the lysine decarboxylase enzyme protein derived from the above E. coli Nissle microorganism may be, but is not limited to, the LdcC gene and / or the CadA gene.

[0041] A microorganism containing the above recombinant vector or into which the recombinant vector has been introduced may have pentamethylenediamine (PMDA) production activity, but is not limited thereto.

[0042] As used herein, "vector" refers to any medium for cloning and / or transferring bases into a host cell. A vector may be a replicating unit (replicon) capable of binding to another DNA fragment and causing replication of the bound fragment. The term "replicating unit" may refer to any genetic unit (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in a living body, i.e., is capable of replicating under its own control. In the present invention, the vector is not particularly limited as long as it is replicable in a host, and any vector known in the art may be used.

[0043] The vector used for producing the above recombinant vector, specifically the vector used for introducing a 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 a natural or recombinant state. For example, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon4A, and / or Charon21A, etc. may be used as the phage vector or cosmid vector, and pKE vector, pHCMS vector, pHCP vector, pCES vector, pCG vector, pDZ vector, pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL type, and / or pET type, etc. may be used as the plasmid vector. The usable vector is not particularly limited, and any known expression vector can be used. In one example, the vector may be one that is inserted into the vector and irreversibly fuses the transferred gene into the genome of the host cell, thereby allowing long-term, stable gene expression within the cell. Such a vector may include transcription and translation expression control sequences that enable the gene to be expressed in the selected host. The expression control sequences may include any operator sequence for regulating transcription, and / or sequences for regulating the termination of transcription and translation. In one example, the initiation and termination codons may generally be considered part of the nucleic acid sequence encoding the protein of interest, and must be functional in the subject when the genetic construct is administered, and may be in frame with the coding sequence. In addition, if the expression vector is replicable, it may include an origin of replication. In addition, it may also appropriately include an enhancer, an untranslated region at the 3' end of the gene of interest, a selectable marker (e.g., an antibiotic resistance marker), and / or a replicable unit.The vector may be self-replicating or integrated into the host genomic DNA. For example, each component within the vector must be operably linked to one another, and the linkage of these component sequences may be accomplished by ligation at convenient restriction enzyme sites, or, if such sites do not exist, by using synthetic oligonucleotide adaptors or linkers according to conventional methods.

[0044]

[0045] Composition for producing pentamethylenediamine

[0046] Another example of the present specification provides a composition for producing pentamethylenediamine, comprising at least one selected from the group consisting of the recombinant microorganism, a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism, a polynucleotide encoding the enzyme protein, and the recombinant vector.

[0047] The above recombinant microorganism may mean one or more selected from the group consisting of the recombinant microorganism itself (cells), and cultures, fragments, and extracts thereof, but is not limited thereto.

[0048] The above composition may additionally comprise a substrate. The substrate is present in an amount of 50 to 500 g / L, 50 to 400 g / L, 50 to 350 g / L, 50 to 320 g / L, 50 to 300 g / L, 50 to 250 g / L, 50 to 220 g / L, 50 to 200 g / L, 50 to 150 g / L, 50 to 120 g / L, 50 to 100 g / L, 80 to 500 g / L, 80 to 400 g / L, 80 to 350 g / L, 80 to 320 g / L, 80 to 300 g / L, 80 to 250 g / L, 80 to 220 g / L, 80 to 200 g / L, 80 to 150 g / L, 80 to 120 g / L, 80 to 100 g / L, 100 to 500 g / L, 100 to 400 g / L, 100 to 350 g / L, 100 to 320 g / L, 100 to 300 g / L, 100 to 250 g / L, 100 to 220 g / L, 100 to 200 g / L, 100 to 150 g / L, 100 to 120 g / L, 150 to 500 g / L, 150 to 400 g / L, 150 to 350 g / L, 150 to 320 g / L, 150 to 300 g / L, 150 to 250 g / L, 150 to 220 g / L, 150 to 200 g / L, 180 to 500 g / L, 180 to 400 g / L, 180 to 350 g / L, 180 to 320 g / L, 180 to 300 g / L, 180 to 250 g / L, 180 to 220 g / L, 180 to 200 g / L, 200 to 500 g / L, 200 to 400 g / L, 200 to 350 g / L, 200 to 320 g / L, 200 to 300 g / L, 200 to 250 g / L, 200 to 220 g / L, 250 to 500 g / L, 250 to 400 g / L, 250 to 350 g / L, 250 to 320 g / L, 250 to 300 g / L, 280 to 500 g / L,280 to 400 g / L, 280 to 350 g / L, 280 to 320 g / L, 280 to 300 g / L, 300 to 500 g / L, 300 to 400 g / L, 300 to 350 g / L, 300 to 320 g / L, for example, but not limited to, 100 g / L, 200 g / L or 300 g / L.

[0049] The above substrate may be, but is not limited to, lysine, carbon source, nitrogen source, phosphorus and / or trace element components.

[0050] In the present specification, “lysine” may mean lysine alone and / or lysine including a salt thereof, and specifically, may be at least one selected from the group consisting of lysine, lysine-HCl, lysine-H2SO4, etc., but is not limited thereto.

[0051] The carbon source may include glucose, glycerol, and / or molasses. In addition, monosaccharides, oligosaccharides, polysaccharides, single-carbon substrates, or mixtures thereof may be exemplified. For example, monosaccharides such as glucose and fructose; oligosaccharides such as sucrose, maltose, or lactose; polysaccharides such as starch (starch) or cellulose; and single-carbon substrates such as methanol, formaldehyde, or formate may be exemplified. In addition, 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; Examples of fatty acids include, but are not limited to, propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, palmatic acid, stearic acid, and linoleic acid; and oils (specifically, vegetable oils) such as soybean oil, sunflower oil, peanut oil, and coconut oil. These substances may be used individually or as a mixture.

[0052] Examples of the nitrogen sources include, but are not limited to, peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may also be used individually or as a mixture.

[0053] Examples of the above-mentioned ingredients include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or their corresponding sodium-containing salts. Furthermore, the growth medium may include, but is not limited to, metal salts necessary for growth, such as magnesium sulfate or iron sulfate, or essential growth substances, such as amino acids and vitamins. The above-mentioned ingredients may be added to the culture in a suitable manner, either batchwise or continuously, during the culturing process.

[0054] The above composition may additionally include, but is not limited to, 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 30 μM, 1 to 20 μM, 1 to 15 μM, 1 to 12 μM, 5 to 1,000 μM, 5 to 800 μM, 5 to 600 μM, 5 to 500 μM, 5 to 400 μM, 5 to 300 μM, 5 to 200 μM, 5 to 100 μM, 5 to 80 μM, 5 to It may be included at a concentration of, but is not limited to, 60 μM, 5 to 50 μM, 5 to 30 μM, 5 to 20 μM, 5 to 15 μM, 5 to 12 μM, 8 to 1,000 μM, 8 to 800 μM, 8 to 600 μM, 8 to 500 μM, 8 to 400 μM, 8 to 300 μM, 8 to 200 μM, 8 to 100 μM, 8 to 80 μM, 8 to 60 μM, 8 to 50 μM, 8 to 30 μM, 8 to 20 μM, 8 to 15 μM or 8 to 12 μM, for example, 10 μM.

[0055]

[0056] Pentamethylenediamine production method

[0057] Another example of the present disclosure provides a method for producing pentamethylenediamine, comprising the step of reacting the recombinant microorganism with a substrate.

[0058] The above recombinant microorganism may refer to one or more selected from the group consisting of the recombinant microorganism itself (cells), and cultures, fragments, and extracts thereof, but is not limited thereto.

[0059] The substrate is at least one selected from the group consisting of a culture of the recombinant microorganism, a cell of the recombinant microorganism, a lysate of the cell, and an extract thereof, in a volume-based amount of 50 to 500 g / L, 50 to 400 g / L, 50 to 350 g / L, 50 to 320 g / L, 50 to 300 g / L, 50 to 250 g / L, 50 to 220 g / L, 50 to 200 g / L, 50 to 150 g / L, 50 to 120 g / L, 50 to 100 g / L, 80 to 500 g / L, 80 to 400 g / L, 80 to 350 g / L, 80 to 320 g / L, 80 to 300 g / L, 80 to 250 g / L, 80 to 220 g / L, 80 to 200 g / L, 80 to 150 g / L, 80 to 120 g / L, 80 to 100 g / L, 100 to 500 g / L, 100 to 400 g / L, 100 to 350 g / L, 100 to 320 g / L, 100 to 300 g / L, 100 to 250 g / L, 100 to 220 g / L, 100 to 200 g / L, 100 to 150 g / L, 100 to 120 g / L, 150 to 500 g / L, 150 to 400 g / L, 150 to 350 g / L, 150 to 320 g / L, 150 to 300 g / L, 150 to 250 g / L, 150 to 220 g / L, 150 to 200 g / L, 180 to 500 g / L, 180 to 400 g / L, 180 to 350 g / L, 180 to 320 g / L, 180 to 300 g / L, 180 to 250 g / L, 180 to 220 g / L, 180 to 200 g / L, 200 to 500 g / L, 200 to 400 g / L, 200 to 350 g / L, 200 to 320 g / L, 200 to 300 g / L, 200 to 250 g / L, 200 to 220 g / L, 250 to 500 g / L, 250 to 400 g / L, 250 to 350 g / L,250 to 320 g / L, 250 to 300 g / L, 280 to 500 g / L, 280 to 400 g / L, 280 to 350 g / L, 280 to 320 g / L, 280 to 300 g / L, 300 to 500 g / L, 300 to 400 g / L, 300 to 350 g / L, 300 to 320 g / L, for example, 100 g / L, 200 g / L or 300 g / L, but is not limited thereto.

[0060] The above substrate may be, but is not limited to, a lysine complex, a carbon source, a nitrogen source, a phosphorus source and / or a trace element component.

[0061] The above method for producing pentamethylenediamine may additionally include, but is not limited to, a step of reacting at least one selected from the group consisting of a recombinant microorganism, a culture of the recombinant microorganism, a cell of the recombinant microorganism, a lysate of the cell, and an extract 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 30 μM, 1 to 20 μM, 1 to 15 μM, 1 to 12 μM, 5 to 1,000 μM, 5 to 800 μM, 5 to 600 μM, 5 to 500 μM, 5 to 400 μM, 5 to 300 μM, 5 to 200 μM, 5 to 100 μM, 5 to 80 μM, 5 to It can react at a concentration of, but is not limited to, 60 μM, 5 to 50 μM, 5 to 30 μM, 5 to 20 μM, 5 to 15 μM, 5 to 12 μM, 8 to 1,000 μM, 8 to 800 μM, 8 to 600 μM, 8 to 500 μM, 8 to 400 μM, 8 to 300 μM, 8 to 200 μM, 8 to 100 μM, 8 to 80 μM, 8 to 60 μM, 8 to 50 μM, 8 to 30 μM, 8 to 20 μM, 8 to 15 μM or 8 to 12 μM, for example, 10 μM.

[0062] The above method for producing pentamethylenediamine may additionally include a step of culturing the recombinant microorganism.

[0063] The above method for producing pentamethylenediamine may further include a step of isolating and / or purifying pentamethylenediamine from a culture (or culture medium). The culture may be a culture obtained by reacting at least one selected from the group consisting of a recombinant microorganism, a culture of the recombinant microorganism, cells of the recombinant microorganism, a lysate of the cells, and extracts thereof with a substrate, but is not limited thereto.

[0064] The present invention can produce pentamethylenediamine using a recombinant microorganism containing a decarboxylase enzyme protein derived from E. coli Nissle.

[0065] Figure 1 shows a vector map of the pKE112 vector used for introducing a lysine decarboxylase enzyme gene derived from E. coli Nissle.

[0066] Figure 2 is a graph showing the results of confirming the pentamethylenediamine (PMDA) production activity of microorganisms depending on whether or not a lysine decarboxylase enzyme gene derived from E. coli Nissle was introduced.

[0067] Figure 3 is a graph showing the results of confirming the pentamethylenediamine (PMDA) production activity according to the strain derived from the lysine decarboxylase enzyme introduced into the recombinant microorganism or the substrate concentration used.

[0068] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited to the following examples.

[0069]

[0070] Examples 1 and 2. Production of recombinant microorganisms containing lysine decarboxylase enzyme genes derived from E. coli Nissle

[0071] To confirm the pentamethylenediamine (PMDA) production activity of lysine decarboxylase (LDC) derived from E. coli Nissle, the lysine decarboxylase enzyme gene was cloned.

[0072] Specifically, to insert the E. coli Nissle-derived lysine decarboxylase enzyme gene (NCBI Accession Number: AXY47745.1) expressed by the ldcC (lysine decarboxylase) gene into the pKE112 vector, pKE112 was digested using restriction enzymes KpnI / HindIII (New England Biolabs). The sequence information of the restriction enzymes used is shown in Table 1 below. The vector map of the pKE112 vector is shown in Fig. 1.

[0073] Species sequence (5' -> 3') Sequence number KpnIGGTACC7HindIIIAAGCTT8BamHIGGATCC9SbfICCTGCAGG10

[0074] The above E. coli Nissle-derived lysine decarboxylase was cloned by digestion using KpnI / HindIII and then ligation into the pKE112 vector (pKE112_EcNLdcC).

[0075] The amino acid sequence (SEQ ID NO: 1) and gene sequence (SEQ ID NO: 2) information of the lysine decarboxylase enzyme derived from E. coli Nissle are shown in Table 2 below.

[0076]

[0077] The above cloned pKE112_EcNLdcC vector was used in the E. coli Nissle microorganism (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.) by the heat shock method (Example 1) and E. Each of the coliWL3110 microorganisms (WL3110 microorganism used in Korean Patent Publication No. 2009-0018781) (Example 2) was transformed.

[0078]

[0079] Comparative Example 1. Production of a microorganism not transformed with the lysine decarboxylase enzyme gene.

[0080] The E. coli Nissle microorganism used in Example 1, in which the lysine decarboxylase enzyme gene was not transformed, was prepared (Comparative Example 1).

[0081]

[0082] Comparative Examples 2 and 3. Production of recombinant microorganisms containing wild-type E. coli-derived lysine decarboxylase enzyme genes

[0083] To confirm the pentamethylenediamine production activity of wild-type E. coli-derived lysine decarboxylase expressed by the cadA (lysine decarboxylasecadA) gene (NCBI accession number: UDE09507.1) and wild-type E. coli-derived lysine decarboxylase expressed by the ldcC gene (NCBI accession number: UDE09932.1), lysine decarboxylase enzyme genes were cloned.

[0084] Specifically, the enzyme gene for lysine decarboxylase expressed by the cadA gene was cloned into the pKE112 vector (pKE112_EcCadA) in a manner substantially identical to pKE112_EcNLdcC, except that KpnI / BamHI (New England Biolabs) was used as restriction enzymes to transform E. coliWL3110. In addition, the enzyme gene for lysine decarboxylase expressed by the ldcC gene was also cloned into the pKE112 vector (pKE112_EcLdcC) in a manner substantially identical to pKE112_EcNLdcC, except that KpnI / SbfI (New England Biolabs) was used as restriction enzymes. The sequence information of the restriction enzymes used is shown in Table 1 above.

[0085] The amino acid sequence (SEQ ID NO: 3) and gene sequence (SEQ ID NO: 4) information of the wild-type E. coli-derived lysine decarboxylase enzyme expressed by the cadA gene and the amino acid sequence (SEQ ID NO: 5) and gene sequence (SEQ ID NO: 6) information of the wild-type E. coli-derived lysine decarboxylase enzyme expressed by the ldcC gene are shown in Table 3 below.

[0086]

[0087]

[0088] The above-mentioned cloned pKE112_EcCadA (Comparative Example 2) and pKE112_EcLdcC (Comparative Example 3) vectors were each transformed into the same microorganism as the E. coliWL3110 microorganism used in Example 2 using the heat shock method.

[0089]

[0090] Test Example 1. Measurement of pentamethylenediamine production activity of recombinant microorganisms containing lysine decarboxylase enzyme genes derived from E. coli Nissle

[0091] The microorganisms of Example 1 and Comparative Example 1 prepared above were cultured in MR medium (6.67 g / L KH2PO4, 4 g / L (NH4)2HPO4, 0.8 g / L MgSO) containing 20 g / L glucose and 10 mg / L thiamine-HCl. 4· 7H2O, 0.8 g / L citric acid, 5 mL trace metal solution) at 30°C for 16 hours. In the microbial culture of Example 1, ampicillin antibiotic was added at a concentration of 50 μg / mL, and the transformed microorganisms were selected through the selection of surviving microorganisms.

[0092] The cultured microorganisms were centrifuged at 4℃, 4000 rpm, and 15 minutes, and the cells obtained through this were OD 600 =50 was diluted in distilled water, and lysine-HCl titrated to pH 7.0 was added to a final concentration of 200 g / L. In addition, pyridoxal 5'-phosphate (PLP), which assists the activity of LDC enzyme, was added to a concentration of 10 μM, and the group was divided into a group to which PLP was not added, and the enzyme conversion reaction was performed at 37°C for 120 hours.

[0093] The final production concentration (PMDA titer) of pentamethylenediamine produced by the above enzyme conversion reaction was measured through HPLC via diethyl ethoxymethylenemalonate (DEEMM) reaction, and the results are shown in Table 4 and Figure 2 below.

[0094] Specifically, HPLC was used with a Variable Wavelength Detector (VWD) and a C18 column, and elution was performed with Mobile Phase A and Mobile Phase B. The flow rate was set to 1 mL / min, and analysis was performed at 284 nm.

[0095] Microorganism type PLP addition PMDA titer (g / L) Example 1-45.7+57.2 Comparative example 1-0+0

[0096] As a result of confirming the final production concentration of pentamethylenediamine, it was confirmed that the microorganism transformed with the lysine decarboxylase enzyme gene derived from E. coli Nissle produced PMDA, whereas the untransformed E. coli Nissle microorganism did not produce PMDA. In addition, it was confirmed that PMDA production was higher when PLP was added.

[0097]

[0098] Test Example 2. Measurement of pentamethylenediamine production activity according to lysine decarboxylase enzyme-derived strain and substrate concentration.

[0099] The microorganisms of Example 2 and Comparative Examples 2 and 3 were cultured in substantially the same manner as the culture method of the microorganism of Example 1 in Test Example 1.

[0100] The cultured microorganisms were centrifuged at 4℃, 4000 rpm, and 15 minutes, and the cells obtained through this were OD 600=50 was diluted in distilled water, and lysine-HCl titrated to pH 7.0 was added to a final concentration of 100, 200, or 300 g / L, and the enzyme conversion reaction was performed at 37°C for 120 hours.

[0101] The final production concentration (PMDA titer) of pentamethylenediamine produced by the above enzyme conversion reaction was measured using substantially the same method as in Test Example 1, and the results are shown in Table 5 and Figure 3 below.

[0102] Microorganism typeLysine-HCl final concentration (g / L)PMDA titer (g / L)Example 210036.9420073.13300105.24Comparative example 21001.662000.263001.13Comparative example 310014.0620012.293001.9

[0103] As a result of confirming the final production concentration of pentamethylenediamine, it was confirmed that the PMDA production of the microorganism transformed with E. coli Nissle-derived lysine decarboxylase was higher than that of the microorganism transformed with wild-type E. coli-derived lysine decarboxylase.

[0104] It was confirmed that the production of PMDA continued to increase as the concentration of Lysine-HCl increased in a microorganism transformed with lysine decarboxylase derived from E. coli Nissle (Example 2).

[0105] In addition, as a result of confirming the PMDA production amount of Comparative Examples 2 and 3, which are microorganisms transformed with lysine decarboxylase expressed by the cadA and ldcC genes, respectively, it was confirmed that the PMDA production amount of Comparative Example 3, which is a microorganism transformed with lysine decarboxylase expressed by the ldcC gene, was higher.

Claims

1. Containing a polynucleotide encoding a lysine decarboxylase (LDC) enzyme protein derived from E. coli Nissle microorganism or a lysine decarboxylase enzyme protein derived from E. coli Nissle microorganism, A recombinant microorganism having pentamethylenediamine (PMDA) production activity.

2. A recombinant microorganism, wherein a polynucleotide encoding a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism is introduced into the first paragraph.

3. A recombinant microorganism according to claim 1, wherein the lysine decarboxylase enzyme derived from the E. coli Nissle microorganism comprises the amino acid sequence of sequence number 1.

4. In the first paragraph, the recombinant microorganism is a microorganism of the genus Escherichia, a microorganism of the genus Corynebacterium, or a microorganism of the genus Saccharomyces.

5. A recombinant microorganism in the first paragraph, wherein the lysine decarboxylase enzyme protein is expressed by the ldcC gene.

6. A composition for producing pentamethylenediamine, comprising at least one selected from the group consisting of a recombinant microorganism of any one of claims 1 to 5, a lysine decarboxylase enzyme protein derived from an E. coli Nissle microorganism, a polynucleotide encoding the enzyme protein, and a recombinant vector comprising the polynucleotide.

7. A composition for producing pentamethylenediamine, wherein the composition further comprises a substrate in the sixth paragraph.

8. A composition for producing pentamethylenediamine, wherein the substrate is included in a concentration of 50 to 500 g / L based on the total volume of the composition in paragraph 7.

9. A composition for producing pentamethylenediamine, wherein in paragraph 7, the substrate is at least one selected from the group consisting of lysine, lysine-HCl, lysine-H2SO4, glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose.

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

11. A method for producing pentamethylenediamine in 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. A method for producing pentamethylenediamine in claim 10, wherein the substrate is at least one selected from the group consisting of lysine, lysine-HCl, and lysine-H2SO4.

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