Method for producing recombinant microorganism and compound

Abstract

To provide a genetically modified microorganism with reduced production of by-products.SOLUTION: Provided is a recombinant microorganism having a hexamethylenediamine production pathway and comprising one or more genetic modifications that inhibit at least one of ornithine decarboxylase and lysine decarboxylase.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing a recombinant microorganism and a compound.

Background Art

[0002] Hexamethylenediamine (CAS No. 124-09-4), also known as 1,6-diaminohexane or 1,6-hexanediamine, is an important chemical used as a raw material for polyamide resins and the like.

[0003] In the chemical product manufacturing process, raw materials derived from fossil fuels that have been conventionally used are feared to be depleted and are considered to be a cause of global warming. Therefore, in recent years, a conversion to renewable raw materials such as biomass-derived raw materials has been desired. As part of the conversion to renewable raw materials, a technique using a microorganism whose metabolism has been modified by genetic recombination is expected as a new manufacturing process for chemical products. For example, it has been suggested that hexamethylenediamine can be produced by a genetically modified microorganism into which an exogenous nucleic acid has been introduced (Patent Document 1).

[0004] On the other hand, in a manufacturing process using a microorganism, the production of by-products such as compounds originally produced by the microorganism that are different from the target compound and compounds produced by side reactions from metabolic intermediates has become a problem. Specifically, the production of by-products contributes to an increase in the load and cost in the separation and purification processes.

[0005] Escherichia coli, which is one of the typical host microorganisms used in the production process of chemical products, originally produces diamine compounds such as putrescine (CAS No. 110-60-1; 1,4-diaminobutane, 1,4-butanediamine) and cadaverine (CAS No. 462-94-2; 1,5-diaminobutane, 1,5-pentanediamine) (Non-Patent Document 1). As a method for separating hexamethylenediamine, which is the target compound, from a microbial culture solution, for example, a method has been proposed in which carbonates or carbamates are formed from diamine compounds in the culture solution, and after converting these salts into free bases and carbon dioxide, solvent extraction is performed (Patent Document 2). However, in this separation method, in addition to the target compound hexamethylenediamine, the above-mentioned by-product diamine compounds, namely putrescine and cadaverine, may also be extracted.

[0006] In gene recombinant microorganisms having a hexamethylenediamine production pathway, although several gene modifications have been proposed that can improve the hexamethylenediamine yield (Patent Document 1, Patent Document 3), nothing has been mentioned about the decarboxylase involved in the by-production reaction of putrescine and cadaverine.

[0007] Therefore, in the production of hexamethylenediamine using microorganisms, by-products such as putrescine and cadaverine may also be produced simultaneously. Since these diamine by-products are difficult to separate because they have a similar structure to hexamethylenediamine and may increase the load in the separation and purification processes, the development of technologies for reducing the contamination of these diamine by-products has been desired.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Literature

[0009]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a recombinant microorganism in which the production of by-products is reduced.

Means for Solving the Problems

[0011] As a result of intensive studies, the present inventors have found that, in a microorganism having a hexamethylenediamine production pathway, by introducing one or more genetic modifications that suppress at least one of endogenous ornithine decarboxylase and lysine decarboxylase, the production of putrescine and / or cadaverine, which are by-products, is reduced, and thus the present invention has been completed. Furthermore, unexpectedly, it has been found that hexamethylenediamine can be produced more efficiently from a carbon source as a raw material.

[0012] That is, the present invention provides the following: [1] A recombinant microorganism having a hexamethylenediamine production pathway and comprising one or more genetic modifications that suppress at least one of ornithine decarboxylase and lysine decarboxylase; [2] The recombinant microorganism according to [1], belonging to a genus selected from the group consisting of the genera Escherichia, Bacillus, Corynebacterium, Arthrobacter, Brevibacterium, Clostridium, Zymomonas, Pseudomonas, Burkholderia, Streptomyces, Rhodococcus, Synechocystis, Alkalihalobacillus, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, and Aspergillus; [3] The recombinant microorganism according to [1] or [2], wherein the recombinant microorganism is Escherichia coli; [4] The genetic modification is · A modification that suppresses the expression of at least one of the endogenous gene encoding ornithine decarboxylase and the endogenous gene encoding lysine decarboxylase, and · A modification in which the enzyme activity of at least one of the ornithine decarboxylase and the lysine decarboxylase is reduced as compared with the non-mutant strain The recombinant microorganism according to any one of [1] to [3], which is one or more selected from the above; [5] The ornithine decarboxylase is (A) (A-1) DNA consisting of a base sequence selected from SEQ ID NOs: 123 and 124, (A-2) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence selected from SEQ ID NOs: 123 and 124 and encodes a protein having ornithine decarboxylase activity (A-3) A DNA encoding a protein consisting of a nucleotide sequence selected from nucleotide sequences 123 and 124 and having a sequence identity of 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more, and having ornithine decarboxylase activity, (A-4) A DNA encoding a protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added with respect to the amino acid sequence of the protein encoded by the nucleotide sequence selected from nucleotide sequences 123 and 124, and having ornithine decarboxylase enzyme activity, or (A-5) A protein encoded by a DNA consisting of a degenerate isomer of the nucleotide sequence selected from nucleotide sequences 123 and 124, or (B)(B-1) A protein consisting of an amino acid sequence selected from amino acid sequences 119 and 120, (B-2) A protein having an amino acid sequence having a sequence identity of 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence selected from amino acid sequences 119 and 120, and having ornithine decarboxylase activity, or (B-3) A protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added with respect to the amino acid sequence selected from amino acid sequences 119 and 120, and having ornithine decarboxylase activity, [1] The recombinant microorganism according to any one of [1] to [4]; [6] The lysine decarboxylase is (C)(C-1) A DNA consisting of a nucleotide sequence selected from nucleotide sequences 125 and 126, DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence selected from SEQ ID NOs: 125 and 126 and that encodes a protein having lysine decarboxylase activity, (C-3) DNA consisting of a base sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the base sequence selected from SEQ ID NOs: 125 and 126 and that encodes a protein having lysine decarboxylase activity, (C-4) A base sequence encoding a protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added with respect to the amino acid sequence of the protein encoded by the base sequence selected from SEQ ID NOs: 125 and 126, and that encodes a protein having lysine decarboxylase enzyme activity, or (C-5) DNA consisting of a degenerate isomer of the base sequence selected from SEQ ID NOs: 125 and 126 a protein encoded thereby, or (D) (D-1) a protein consisting of the amino acid sequence selected from SEQ ID NOs: 121 and 122, (D-2) a protein having an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence selected from SEQ ID NOs: 121 and 122 and that has lysine decarboxylase activity, or (D-3) a protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added with respect to the amino acid sequence selected from SEQ ID NOs: 121 and 122 and that has lysine decarboxylase activity, [1] to [5] The recombinant microorganism according to any one of paragraphs; [7] The recombinant microorganism according to any one of [1] to [6], wherein the ornithine decarboxylase is a protein encoded by at least one selected from the group consisting of the speC gene and the speF gene; [8] The recombinant microorganism according to any one of [1] to [7], wherein the ornithine decarboxylase is a protein encoded by the speC gene; [9] The recombinant microorganism according to any one of [1] to [8], wherein the lysine decarboxylase is a protein encoded by at least one selected from the group consisting of the cadA gene and the ldcC gene;

[10] The recombinant microorganism according to any one of [1] to [9], wherein the lysine decarboxylase is a protein encoded by the cadA gene;

[11] A method for producing hexamethylenediamine, comprising a culturing step of culturing the recombinant microorganism according to any one of [1] to

[10] .

Advantages of the Invention

[0013] According to the present invention, a genetically recombinant microorganism with reduced production of by-products can be provided.

Brief Description of the Drawings

[0014]

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Mode for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described in detail. It should be noted that the present invention is not limited to the following embodiments, and various conditions can be changed and modified within the scope of the gist thereof. In addition, genetic operations such as DNA acquisition, vector preparation, and transformation described in this specification can be carried out by methods described in known documents such as Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience), and Experimental Notes on Genetic Engineering (Yodosha, Takaki Tamura), unless otherwise specified. Unless otherwise specified in this specification, nucleotide sequences are described from the 5' direction to the 3' direction. In this specification, the terms "polypeptide" and "protein" are used interchangeably. The term "genetically modified microorganism" is also simply referred to as "recombinant microorganism".

[0016] In this specification, a numerical range indicated by "~" indicates a range including the numerical values described before and after "~" as the minimum value and the maximum value, respectively. In the numerical ranges described stepwise in this specification, the upper limit value or the lower limit value of a certain stepwise numerical range can be arbitrarily combined with the upper limit value or the lower limit value of another stepwise numerical range.

[0017] In this specification, the terms "endogenous" or "endogenous" are used to mean that a host microorganism that has not been modified by genetic recombination has the gene or the protein (typically an enzyme) encoded thereby, regardless of whether it is functionally expressed to such an extent that it can promote a dominant biochemical reaction within the host cell. The terms "endogenous" and "endogenous" are used interchangeably in this specification.

[0018] As used herein, the terms "foreign" or "exogenous" are used to mean introducing a gene or nucleic acid sequence according to the present invention into a host when the host microorganism before genetic recombination does not have the gene to be introduced by the present invention, does not substantially express the enzyme encoded by the gene, and encodes the amino acid sequence of the enzyme with a different gene but does not express comparable endogenous enzyme activity after genetic recombination. The terms "exogenous" and "extrinsic" are used interchangeably herein.

[0019] The genetically modified microorganism according to the present invention has the ability to produce hexamethylenediamine. With respect to the microorganism of the present invention, "having the ability to produce hexamethylenediamine" means a microorganism in which hexamethylenediamine is produced at any stage of the process of producing hexamethylenediamine using the microorganism. Specifically, the culture solution obtained by culturing the microorganism may contain hexamethylenediamine, or a precursor of hexamethylenediamine, such as a carboxylic acid compound, an aldehyde compound and / or a carbonyl compound, is added to the medium, and hexamethylenediamine may be produced by culturing a microorganism that converts the hexamethylenediamine precursor into hexamethylenediamine. "Microorganisms having the ability to produce hexamethylenediamine" include microorganisms having one or more of these properties.

[0020] Furthermore, when it is said that the recombinant microorganism according to the present invention "has the ability to produce hexamethylenediamine", it means that the microorganism has a production pathway for hexamethylenediamine. In the present invention, with respect to a certain compound, "having a production pathway" means that the genetically modified microorganism according to the present invention expresses an amount of enzyme sufficient for each reaction step of the production pathway of the compound to proceed, and can biosynthesize the compound. That is, the recombinant microorganism according to the present invention expresses an amount of enzyme sufficient for each reaction step of the production pathway of hexamethylenediamine to proceed, and can biosynthesize hexamethylenediamine. The recombinant microorganism of the present invention may be one using a host microorganism that originally has the ability to produce hexamethylenediamine, or may be one obtained by modifying a host microorganism that originally does not have the ability to produce the compound so as to have the ability to produce hexamethylenediamine.

[0021] The genetically modified microorganism of the present invention includes one or more genetic modifications that suppress at least one of endogenous ornithine decarboxylase and lysine decarboxylase. That is, the recombinant microorganism of the present invention is one obtained by performing one or more genetic modifications that suppress at least one of endogenous ornithine decarboxylase and lysine decarboxylase on a host microorganism having a hexamethylenediamine production pathway.

[0022] The host microorganism of the present invention is not particularly limited and may be either a prokaryote or a eukaryote. Any of those already isolated and preserved, those newly isolated from nature, and those genetically modified can be arbitrarily selected. The host microorganism belongs to, for example, the genus Escherichia, the genus Bacillus, the genus Corynebacterium, the genus Arthrobacter, the genus Brevibacterium, the genus Clostridium, the genus Zymomonas, the genus Pseudomonas, the genus Burkholderia, the genus Streptomyces, the genus Rhodococcus, the genus Synechocystis, the genus Alkalihalobacillus, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Yarrowia, the genus Candida, the genus Pichia, or the genus Aspergillus. The host microorganism preferably belongs to the genus Escherichia, and more preferably is Escherichia coli.

[0023] Therefore, the recombinant microorganism according to the present invention belongs to, for example, the genus Escherichia, the genus Bacillus, the genus Corynebacterium, the genus Arthrobacter, the genus Brevibacterium, the genus Clostridium, the genus Zymomonas, the genus Pseudomonas, the genus Burkholderia, the genus Streptomyces, the genus Rhodococcus, the genus Synechocystis, the genus Alkalihalobacillus, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Yarrowia, the genus Candida, the genus Pichia, or the genus Aspergillus. The recombinant microorganism according to the present invention preferably belongs to the genus Escherichia, and more preferably Escherichia coli.

[0024] In the present specification, with respect to an enzyme, "including one or more genetic modifications that suppress the enzyme" means that at least a modification that suppresses the expression of the gene encoding the enzyme has been performed. The "one or more genetic modifications that suppress the enzyme" include, in addition to the modification that suppresses the expression of the gene encoding the enzyme, a modification that reduces the activity of the enzyme. That is, the recombinant microorganism of the present invention has been modified such that the expression of the gene encoding at least one of the enzymes ornithine decarboxylase and lysine decarboxylase is suppressed or the activity of the enzyme is reduced with respect to the non-mutant strain (for example, the host microorganism).

[0025] The host microorganism has multiple genes encoding each of ornithine decarboxylase and lysine decarboxylase, and there may be multiple genes encoding enzymes that exhibit activity against the same substrate. Therefore, even when modifications are made to reduce activity, the activity of the enzyme may be maintained. When it is stated that the enzyme "comprises one or more genetic modifications that suppress it," this includes cases where, despite modifications being made with the aim of reducing activity, the activity of the enzyme is maintained. In the present invention, with respect to a gene encoding an enzyme, "suppression of expression" shall include "reduction of expression." Further, with respect to an enzyme, "suppression of activity" is synonymous with "suppression of function," "reduction of function," and "reduction of activity," and these terms may be used interchangeably. Additionally, with respect to a microorganism, the terms "non-mutant strain" and "non-reducing strain" may be used interchangeably.

[0026] In a preferred embodiment, one or more genetic modifications that suppress at least one of the endogenous ornithine decarboxylase and lysine decarboxylase are · a modification that suppresses the expression of at least one of the endogenous gene encoding ornithine decarboxylase and the endogenous gene encoding lysine decarboxylase, and · a modification that reduces the activity of at least one of ornithine decarboxylase and lysine decarboxylase and are one or more selected from these.

[0027] In a more preferred embodiment, one or more genetic modifications that suppress at least one of the endogenous ornithine decarboxylase and lysine decarboxylase are · a modification that suppresses the expression of at least one of the endogenous gene encoding ornithine decarboxylase and the endogenous gene encoding lysine decarboxylase, or · a modification that reduces the activity of at least one of ornithine decarboxylase and lysine decarboxylase and are as follows.

[0028] The recombinant microorganism according to the present invention containing the above genetic modification is a genetically modified microorganism modified such that the enzyme activity of at least one of ornithine decarboxylase and lysine decarboxylase is reduced as compared with the non-mutant strain.

[0029] Modification to suppress the expression of an enzyme can be achieved, for example, by reducing the expression of the gene encoding the enzyme. More specifically, a decrease in gene expression may mean a decrease in the transcription amount (mRNA amount) of the gene and / or a decrease in the translation amount (protein amount) of the gene. A decrease in gene expression includes the case where the gene is not expressed at all.

[0030] The decrease in gene expression may be, for example, a decrease in the transcription amount, a decrease in the translation amount, or a combination thereof. The decrease in the transcription amount can be achieved, for example, by modifying transcription regulatory regions such as the promoter region and operator region of the enzyme gene. The decrease in the transcription amount of a gene can be evaluated by methods well known to those skilled in the art, and examples include quantitative RT-PCR method and Northern blotting method. The transcription amount of a gene may be decreased to 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0% as compared with a non-decreasing strain, for example.

[0031] Methods for reducing translation include, for example, methods of modifying translation regulatory regions such as ribosome binding sites (RBS), and methods of suppressing translation by inserting riboswitch regions upstream of genes. A riboswitch refers to RNA that selectively binds to a specific small molecule compound, and the small molecule compound is called a ligand. In the absence of the ligand, it forms a secondary structure by RNA base pairs, affecting the nucleic acids around the riboswitch. In particular, when a ribosome binding site is included downstream of the riboswitch, it prevents the ribosome from approaching the ribosome binding site, thereby preventing the translation of the mRNA of the gene located further downstream. On the other hand, in the presence of the ligand, through the dissolution of the secondary structure accompanying ligand binding, the ribosome can approach the ribosome binding site. Therefore, when the ligand is not added, the mRNA of the gene is not translated, and the expression of the target gene is suppressed. The reduction in the translation amount of a gene can be evaluated by methods well-known to those skilled in the art, for example, Western blotting. The translation amount of a gene may be reduced to 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0% compared to, for example, a non-reducing strain.

[0032] Moreover, modifications that reduce the activity of an enzyme can also be achieved by disrupting the gene encoding the enzyme. Disruption of an enzyme gene means that the gene is modified so that a protein with enzyme activity is not expressed, including cases where no protein is produced at all and cases where a protein with reduced or lost enzyme activity is produced. For example, the reduction in enzyme activity may be reduced to 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the enzyme activity of a non-reducing strain.

[0033] Modifications that reduce the activity of the enzyme can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome. Furthermore, the entire gene, including the sequences before and after the gene on the chromosome, may be deleted. As long as a reduction in enzyme activity can be achieved, the region to be deleted may be any of the N-terminal region, internal region, and C-terminal region.

[0034] In addition, disruption of the enzyme gene can also be achieved, for example, by introducing an amino acid substitution (missense mutation), introducing a stop codon (nonsense mutation), and introducing a frameshift mutation that adds or deletes 1 to 2 bases into the coding region of the enzyme gene on the chromosome.

[0035] Furthermore, disruption of the enzyme gene can also be achieved by inserting another sequence into the coding region of the gene on the chromosome. Examples of other sequences include antibiotic resistance genes and transposons, but are not particularly limited as long as they reduce enzyme activity.

[0036] Disruption of the enzyme gene can utilize methods using homologous recombination, such as the method using the Red recombinase of λ-phage (Datsenko, Kirill A., and Barry L. Wanner. “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.” Proceedings of the National Academy of Sciences 97.12 (2000): 6640-6645.), the method using a suicide vector containing a temperature-sensitive replication origin (Blomfield et al., Molecular microbiology 5.6 (1991): 1447-1457.), and the method using the CRISPR-Cas9 system (Jiang, Yu, et al. “Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.” Appl. Environ. Microbiol. 81.7 (2015): 2506-2514.), etc., but is not limited to these methods.

[0037] Also, disruption of the enzyme gene may be performed by mutagenesis. Examples of mutagenesis include physical treatments such as X-ray treatment, ultraviolet treatment, and γ-ray treatment, as well as chemical treatments with mutagens such as N-methyl-N’-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate, but are not particularly limited as long as they reduce enzyme activity.

[0038] Ornithine decarboxylase is an enzyme that catalyzes the reaction of converting L-ornithine into putrescine and CO2. Ornithine decarboxylase is classified as enzyme number EC 4.1.1.17 and catalyzes an enzyme reaction belonging to the gene ontology (GO) term ID of GO:0004586.

[0039] As an example of ornithine decarboxylase, SpeC and SpeF in Escherichia coli are mentioned (Table 1), and they are encoded by the speC gene and the speF gene, respectively. The amino acid sequences of SpeC and SpeF are shown in SEQ ID NO: 119 and SEQ ID NO: 120, respectively, and the nucleotide sequences of the speC gene and the speF gene are shown in SEQ ID NO: 123 and SEQ ID NO: 124, respectively (FIGS. 14 and 15).

[0040]

Table 1

[0041] Lysine decarboxylase is an enzyme that catalyzes the reaction of converting lysine into cadaverine and CO2. Lysine decarboxylase is classified as Enzyme Commission number EC 4.1.1.18 and catalyzes an enzyme reaction belonging to the gene ontology (GO) term ID of GO:0008923.

[0042] As an example of lysine decarboxylase, CadA and LdcC in Escherichia coli are mentioned (Table 2), and they are encoded by the cadA gene and the ldcC gene, respectively. The amino acid sequences of CadA and LdcC are shown in SEQ ID NO: 121 and SEQ ID NO: 122, respectively, and the nucleotide sequences of the cadA gene and the ldcC gene are shown in SEQ ID NO: 125 and SEQ ID NO: 126, respectively (FIGS. 14 and 15).

[0043]

Table 2

[0044] In the present invention, the "non-decreased strain" refers to a strain in which no modification has been made to reduce the activity of the enzyme ornithine decarboxylase and / or lysine decarboxylase, and is also referred to as a "non-mutated strain". Examples of non-decreased strains include, but are not limited to, wild-type strains and reference strains of each microbial strain, and derivative strains such as strains obtained by breeding. For example, in Escherichia coli strains, the K-12 strain, B strain, C strain, and W strain, as well as derivative strains of these strains, such as the BL21(DE3) strain, W3110 strain, MG1655 strain, JM109 strain, DH5α strain, and HB101 strain, etc., can be mentioned, but are not limited to these.

[0045] Hereinafter, ornithine decarboxylase and lysine decarboxylase will be described with reference to sequence numbers, but the present invention is not intended to be limited only to aspects related to the genes specified by the following sequence numbers for these enzymes. Specifically, there are multiple endogenous ornithine decarboxylases in Escherichia coli, and among them, genes that have been identified so far include speC (sequence number 123) and speF (sequence number 124). The present invention includes not only aspects in which the genes specified by the sequence numbers are suppressed, but also aspects in which, in addition to the genes specified by the sequence numbers, any other ornithine decarboxylase genes are suppressed. The same applies to lysine decarboxylase, and it is not intended to be limited only to aspects related to the genes specified by the sequence numbers.

[0046] In one aspect, ornithine decarboxylase is (A-1) DNA consisting of a base sequence selected from sequence numbers 123 and 124, (A-2) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence selected from sequence numbers 123 and 124 and encodes a protein having ornithine decarboxylase activity, (A-3) A DNA consisting of a base sequence selected from SEQ ID NOs: 123 and 124 and having a sequence identity of 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more, and encoding a protein having ornithine decarboxylase activity, (A-4) A base sequence encoding a protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added with respect to the amino acid sequence of the protein encoded by the base sequence selected from SEQ ID NOs: 123 and 124, and encoding a protein having ornithine decarboxylase enzyme activity, or (A-5) A DNA consisting of a degenerate isomer of the base sequence selected from SEQ ID NOs: 123 and 124 It is a protein encoded by

[0047] In a preferred embodiment, ornithine decarboxylase is (A-1) A DNA consisting of the base sequence selected from SEQ ID NOs: 123 and 124 (A-2) A DNA consisting of a base sequence complementary to the base sequence selected from SEQ ID NOs: 123 and 124, hybridizing under stringent conditions, and encoding a protein having ornithine decarboxylase activity (A-3) A DNA consisting of a base sequence having a sequence identity of 90% or more with the base sequence selected from SEQ ID NOs: 123 and 124, and encoding a protein having ornithine decarboxylase activity (A-4) A base sequence encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted, and / or added with respect to the amino acid sequence of the protein encoded by the base sequence selected from SEQ ID NOs: 123 and 124, and encoding a protein having ornithine decarboxylase enzyme activity, or (A-5) A DNA consisting of a degenerate isomer of the base sequence selected from SEQ ID NOs: 123 and 124 is a protein encoded by

[0048] In a more preferred embodiment, ornithine decarboxylase is a protein encoded by DNA consisting of a base sequence selected from the base sequences of SEQ ID NOs: 123 and 124.

[0049] In the present specification, the "stringent conditions" are, for example, conditions of about "1xSSC, 0.1% SDS, 60 °C", and more stringent conditions are conditions of about "0.1xSSC, 0.1% SDS, 60 °C", and even more stringent conditions are conditions of about "0.1xSSC, 0.1% SDS, 68 °C".

[0050] In another embodiment, ornithine decarboxylase is a protein consisting of an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 119 and 120, or a protein having an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 119 and 120 and having ornithine decarboxylase activity, or a protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 119 and 120 and having ornithine decarboxylase activity.

[0051] In a preferred embodiment, ornithine decarboxylase is a protein consisting of an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 119 and 120, or a protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 119 and 120 and having ornithine decarboxylase activity, or It is a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted, and / or added with respect to the amino acid sequence selected from SEQ ID NOs: 119 and 120, and has ornithine decarboxylase activity.

[0052] In a more preferred embodiment, ornithine decarboxylase is a protein consisting of an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 119 and 120.

[0053] Ornithine decarboxylase is specifically a protein encoded by at least one selected from the group consisting of the speC gene and the speF gene, and preferably a protein encoded by the speC gene.

[0054] In one embodiment, lysine decarboxylase is (C-1) DNA consisting of a base sequence selected from SEQ ID NOs: 125 and 126, (C-2) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence selected from SEQ ID NOs: 125 and 126 and encodes a protein having lysine decarboxylase activity, (C-3) DNA consisting of a base sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the base sequence selected from SEQ ID NOs: 125 and 126 and encodes a protein having lysine decarboxylase activity, (C-4) A base sequence encoding a protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added with respect to the amino acid sequence of the protein encoded by the base sequence selected from SEQ ID NOs: 125 and 126, and encodes a protein having lysine decarboxylase enzyme activity, or (C-5) DNA consisting of a degenerate isomer of the base sequence selected from SEQ ID NOs: 125 and 126 is a protein encoded by

[0055] In a more preferred embodiment, the lysine decarboxylase is (C-1) DNA consisting of a base sequence selected from SEQ ID NOs: 125 and 126, (C-2) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence selected from SEQ ID NOs: 125 and 126 and encodes a protein having lysine decarboxylase activity, (C-3) DNA consisting of a base sequence having 90% or more sequence identity with the base sequence selected from SEQ ID NOs: 125 and 126 and encoding a protein having lysine decarboxylase activity, (C-4) a base sequence encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added with respect to the amino acid sequence of the protein encoded by the base sequence selected from SEQ ID NOs: 125 and 126, and encoding a protein having lysine decarboxylase enzyme activity, or (C-5) DNA consisting of a degenerate isomer of the base sequence selected from SEQ ID NOs: 125 and 126 is a protein encoded by

[0056] In a more preferred embodiment, the lysine decarboxylase is a protein encoded by DNA consisting of a base sequence selected from SEQ ID NOs: 125 and 126.

[0057] In another embodiment, the lysine decarboxylase is (D-1) a protein consisting of an amino acid sequence selected from SEQ ID NOs: 121 and 122, or (D-2) a protein having an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more or 99% or more sequence identity with the amino acid sequence selected from SEQ ID NOs: 121 and 122 and having lysine decarboxylase activity, or It is a protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted and / or added with respect to the amino acid sequence selected from SEQ ID NOs: 121 and 122, and has lysine decarboxylase activity.

[0058] In a preferred embodiment, the lysine decarboxylase is · a protein consisting of an amino acid sequence selected from SEQ ID NOs: 121 and 122, · a protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence selected from SEQ ID NOs: 121 and 122 and having lysine decarboxylase activity, or · a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added with respect to the amino acid sequence selected from SEQ ID NOs: 121 and 122 and having lysine decarboxylase activity.

[0059] In a more preferred embodiment, the lysine decarboxylase is a protein consisting of an amino acid sequence selected from SEQ ID NOs: 121 and 122.

[0060] The lysine decarboxylase is specifically a protein encoded by at least one selected from the group consisting of the cadA gene and the ldcC gene, and preferably a protein encoded by the cadA gene.

[0061] In the recombinant microorganism according to the present invention, by further expressing an enzyme that catalyzes the reaction steps of the hexamethylenediamine production pathway, a recombinant microorganism having the hexamethylenediamine production pathway can be obtained.

[0062] In one embodiment, the recombinant microorganism according to the present invention is · a gene encoding 3-oxoadipyl-CoA thiolase · A gene encoding 3-hydroxyadipyl-CoA dehydrogenase, · A gene encoding 3-hydroxyadipyl-CoA dehydratase, · A gene encoding 2,3-dehydroadipyl-CoA reductase, · A gene encoding adipyl-CoA hydrolase, · A gene encoding adipic acid CoA transferase, · A gene encoding phosphoadipyl transferase, · A gene encoding adipic acid kinase, · A gene encoding dehydrogenase, · A gene encoding carboxylic acid reductase, · A gene encoding transaminase (aminotransferase), · A gene encoding CoA transferase, and · A gene encoding acid thiol ligase, further comprises at least one selected from the group consisting of.

[0063] · 3-oxoadipyl-CoA thiolase catalyzes the reaction of step A in Figure 1, · 3-hydroxyadipyl-CoA dehydrogenase catalyzes the reaction of step B in Figure 1, · 3-hydroxyadipyl-CoA dehydratase catalyzes the reaction of step C in Figure 1, · 2,3-dehydroadipyl-CoA reductase catalyzes the reaction of step D in Figure 1, · The combination of phosphoadipyl transferase and adipic acid kinase, adipyl-CoA hydrolase, or adipic acid CoA transferase catalyzes the reaction of step E in Figure 1, · Dehydrogenase catalyzes the reaction of at least one of steps F and M in Figure 1, · Carboxylic acid reductase catalyzes the reaction of at least one of steps G, I, and K in Figure 1, · Transaminase (aminotransferase) catalyzes the reaction of at least one of steps H, L, and N in Figure 1, ·CoA transferase or acid thiol ligase catalyzes the reaction of step J. Catalyze.

[0064] In a preferred embodiment, the recombinant microorganism according to the present invention · A gene encoding 3-oxoadipyl CoA thiolase, · A gene encoding 3-hydroxyadipyl CoA dehydrogenase, · A gene encoding 3-hydroxyadipyl CoA dehydratase, · A gene encoding 2,3-dehydroadipyl CoA reductase, · A gene encoding carboxylic acid reductase, and · A gene encoding a transaminase (aminotransferase) and further comprises at least one selected from the group consisting of.

[0065] When the genetically modified microorganism of the present invention expresses an enzyme in a sufficient amount to catalyze the reaction steps of the hexamethylenediamine production pathway without the introduction of a foreign gene, the reaction may proceed by the enzyme encoded by the endogenous gene.

[0066] An example of the hexamethylenediamine production pathway that the genetically modified microorganism of the present invention may have is shown in FIG. 1.

[0067] Hereinafter, the enzymes that catalyze each reaction included in the hexamethylenediamine production pathway will be described below with reference to FIG. 1.

[0068] In step A of Figure 1, succinyl-CoA and acetyl-CoA condense and are converted to 3-oxoadipyl-CoA. Examples of enzymes that can catalyze this conversion include β-ketothiolase. Further, for example, enzymes classified in the group such as EC 2.3.1.9 (acetoacetyl-CoA thiolase), EC 2.3.1.16 (3-ketoacyl-CoA thiolase), and EC 2.3.1.174 (3-oxoadipyl-CoA thiolase) can be exemplified as enzymes that may have activity for this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion, and for example, 3-oxoadipyl-CoA thiolase can be mentioned. In one aspect, PaaJ derived from Escherichia coli consisting of the amino acid sequence set forth in SEQ ID NO: 1 is used.

[0069] In step B of Figure 1, 3-oxoadipyl-CoA is converted to 3-hydroxyadipyl-CoA. Examples of enzymes that can catalyze this conversion include oxidoreductases classified in EC 1.1.1.o (o is an integer). For example, enzymes classified in the group such as EC 1.1.1.35 (3-hydroxyacyl-CoA dehydrogenase), EC 1.1.1.36 (acetoacetyl-CoA dehydrogenase), EC 1.1.1.157 (3-hydroxybutanoyl-CoA dehydrogenase), EC 1.1.1.211 (long-chain 3-hydroxyacyl-CoA dehydrogenase), and EC 1.1.1.259 (3-hydroxypimeloyl-CoA dehydrogenase) can be exemplified as enzymes that may have activity for this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion, and for example, it is 3-hydroxyadipyl-CoA dehydrogenase. In one aspect, PaaH derived from Escherichia coli consisting of the amino acid sequence set forth in SEQ ID NO: 2 is used.

[0070] In step C of FIG. 1, 3-hydroxyadipyl-CoA is converted to 2,3-dehydroadipyl-CoA. Examples of enzymes that can catalyze this conversion include hydrolases classified as EC 4.2.1.p (p is an integer). For example, enzymes classified in groups such as EC 4.2.1.17 (enoyl-CoA hydratase), EC 4.2.1.55 (3-hydroxybutanoyl-CoA dehydratase), and EC 4.2.1.74 (long-chain enoyl-CoA hydratase) can be exemplified as enzymes that may have activity towards this conversion. The enzyme used in the present invention is not limited as long as it has activity towards this conversion, and for example, it is 3-hydroxyadipyl CoA dehydratase. In one aspect, PaaF derived from Escherichia coli consisting of the amino acid sequence set forth in SEQ ID NO: 3 is used (FIG. 2). Further, as another aspect, as the enzyme, the nucleotide sequence (783 bp) of a PCR amplicon using the nucleotides shown in SEQ ID NO: 4 and SEQ ID NO: 5 as primers and the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, PaaF (L3) of 3-hydroxyadipyl CoA dehydratase encoded thereby is used (FIG. 2). The Burkholderia sp. LEBP-3 strain (hereinafter also abbreviated as "L3 strain") was applied for deposit to the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) on December 4, 2020 (accession number: NITE ABP-03334) and has been internationally deposited as "deposit number: NITE BP-03334".

[0071] Here, as the DNA polymerase used for preparing the PCR amplicon, those known in the art are used. For example, Taq DNA polymerase, hot start-adjusted DNA polymerase, proofreading DNA polymerase having 3'-5' exonuclease activity separate from polymerase activity, etc. can be mentioned, but are not limited thereto. In a preferred embodiment of the preparation conditions of the PCR amplicon, specific nucleotides are used at 1 μM each as the forward primer and the reverse primer, and PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio Inc.) is used as the enzyme, and heat treatment is performed at 98 °C for 10 seconds, annealing at 55 °C for 15 seconds, and extension at 72 °C for 5 seconds / kb in a volume of 25 μL for 30 cycles for preparation.

[0072] In step D of FIG. 1, 2,3-dehydroadipyl-CoA is converted to adipyl-CoA. Examples of enzymes that can catalyze this conversion include oxidoreductases classified as EC 1.3.1.q (q is an integer). For example, EC 1.3.1.8 (acyl-CoA dehydrogenase (NADP + )), EC 1.3.1.9 (enoyl-ACP reductase (NADH)), EC 1.3.1.38 (trans-2-enoyl-CoA reductase (NADP + )) (Ter), EC 1.3.1.44 (trans-2-enoyl-CoA reductase (NAD + ))), EC 1.3.1.86 (crotonyl-CoA reductase), EC 1.3.1.93 (long-chain acyl-CoA reductase), and enzymes classified in the group such as EC 1.3.1.104 (enoyl-ACP reductase (NADPH)) can be exemplified as enzymes that can have activity against this conversion. The enzyme used in the present invention is not limited as long as it has activity against this conversion, but for example, it is 2,3-dehydroadipyl CoA reductase.

[0073] The 2,3-dehydroadipyl-CoA reductase used in the present invention is not particularly limited. As typical enzymes, for example, the enzyme DcaA derived from Acinetobacter baylyi (Kallscheuer, Nicolai, et al. ”Improved production of adipate with Escherichia coli by reversal of β-oxidation.“ Applied microbiology and biotechnology 101.6 (2017): 2371-2382.), enoyl-CoA reductases derived from biological species such as Candida tropicalis, Euglena gracilis, Clostridium beijerinckii, and Yarrowia lipolytica (Special Table 2011-512868) have been reported to have 2,3-dehydroadipyl-CoA reductase activity and can also be used in the present invention. Further, as another aspect, as the enzyme, MmgC(L3) of 2,3-dehydroadipyl-CoA reductase encoded by the nucleotide sequence (1155 bp) of a PCR amplicon using the chromosome DNA of Burkholderia sp. L3 strain with the nucleotides shown in SEQ ID NO: 76 and SEQ ID NO: 77 (Figure 9) as primers can be used.

[0074] Also, for example, enzymes derived from biological species such as Candida auris, Kluyveromyces marxianus, Pichia kudriavzevii, Thermothelomyces thermophilus, Thermothielavioides terrestris, Chaetomium thermophilum, Podospora anserina, Purpureocillium lilacinum, and Pyrenophora teres can be used, and preferably at least one kind of enzyme consisting of the amino acid sequences described in SEQ ID NOs: 6 to 14 is used (Figure 3).

[0075] In step E of FIG. 1, adipyl-CoA is converted to adipic acid. Examples of enzymes that can catalyze this conversion include thioester hydrolases classified as EC 3.1.2.r (r is an integer). For example, enzymes classified in groups such as EC 3.1.2.1 (acetyl-CoA hydrolase) and EC 3.1.2.20 (acyl-CoA hydrolase) can be exemplified as enzymes that may have activity towards this conversion. The enzyme that can be used in the present invention is not limited as long as it has activity towards this conversion, but for example, it is adipyl-CoA hydrolase.

[0076] Also, as an example of another enzyme that can catalyze the reaction in step E of FIG. 1, CoA-transferases classified as EC 2.8.3.s (s is an integer) can also be mentioned. For example, enzymes classified in groups such as EC 2.8.3.5 (3-oxoacid CoA-transferase), EC 2.8.3.6 (3-oxoadipic acid CoA-transferase), and EC 2.8.3.18 (succinyl-CoA:acetyl-CoA-transferase) can be exemplified as enzymes that may have activity towards this conversion. The enzyme that can be used in the present invention is not limited as long as it has activity towards this conversion, but for example, it is adipic acid CoA-transferase.

[0077] Furthermore, as another example of enzymatic conversion that can catalyze the reaction of step E in FIG. 1, after transferring the adipyl group of adipyl-CoA to phosphate to generate adipyl phosphate by an acyltransferase classified as EC 2.3.1.t (where t is an integer), a pathway via dephosphorylation by a phosphotransferase classified as EC 2.7.2.u (where u is an integer) can also be exemplified. For example, as acyltransferases, enzymes classified in groups such as EC 2.3.1.8 (phosphate acetyltransferase) and EC 2.3.1.19 (phosphate butyryltransferase) can be exemplified as enzymes that may have activity towards this conversion. As phosphotransferases, enzymes classified in groups such as EC 2.7.2.1 (acetate kinase) and EC 2.7.2.7 (butyrate kinase) can be exemplified as enzymes that may have activity towards this conversion. The enzymes that can be used in the present invention are not limited as long as they have activity towards this conversion, but for example, they are adipyl phosphate transferase and adipate kinase.

[0078] In steps F and M of FIG. 1, adipyl-CoA is converted to adipic acid semialdehyde. Examples of enzymes that can catalyze this conversion include enzymes classified as EC 1.2.1.v (where v is an integer). For example, enzymes classified in groups such as EC 1.2.1.10 (acetaldehyde dehydrogenase (acylating)), EC 1.2.1.17 (glyoxylate dehydrogenase (acylating)), EC 1.2.1.42 (hexadecanal dehydrogenase (acylating)), EC 1.2.1.44 (cinnamoyl-CoA reductase (acylating)), EC 1.2.1.75 (malonyl-CoA reductase (malonic semialdehyde forming)), EC 1.2.1.76 (succinic semialdehyde dehydrogenase (acylating)) can be exemplified as enzymes that may have activity for this conversion as well, since they catalyze a conversion reaction that eliminates CoA and generates an aldehyde, similar to this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion. For example, sucD derived from Clostridium kluyveri consisting of the amino acid sequence described in SEQ ID NO: 17 is used (FIG. 4).

[0079] In the reactions of steps G, I, and K in FIG. 1, a carboxyl group is converted to an aldehyde. Examples of enzymes that can catalyze this conversion include, for example, Carboxylic Acid Reductase (CAR). For example, EC 1.2.1.30 (carboxylic acid reductase (NADP +)), enzymes classified in the group such as EC 1.2.1.31 (L-aminoadipic acid semialdehyde dehydrogenase), EC 1.2.1.95 (L-2-aminoadipic acid reductase), and EC 1.2.99.6 (carboxylic acid reductase) can catalyze a conversion reaction that generates an aldehyde from a carboxylic acid, similar to this conversion. Therefore, they can be exemplified as enzymes that may also have activity against this conversion. Typical examples of the biological species from which the enzymes are derived include, but are not limited to, Nocardia iowensis, Nocardia asteroides, Nocardia brasiliensis, Nocardia farcinica, Segniliparus rugosus, Segniliparus rotundus, Tsukamurella paurometabola, Mycobacterium marinum, Mycobacterium neoaurum, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium chelonae, Mycobacterium immunogenum, Mycobacterium smegmatis, Serpula lacrymans, Heterobasidion annosum, Coprinopsis cinerea, Aspergillus flavus, Aspergillus terreus, Neurospora crassa, and Saccharomyces cerevisiae. The enzyme used in the present invention is not limited as long as it has activity against this conversion. For example, at least one enzyme consisting of the amino acid sequence described in any of SEQ ID NOs: 15 to 19 may be used. Preferably, at least one of MaCar, an enzyme derived from Mycobacterium abscessus consisting of the amino acid sequence described in SEQ ID NO: 17, and MaCar(m), a mutant of MaCar consisting of the amino acid sequence described in SEQ ID NO: 19, is used. More preferably, MaCar(m) consisting of the amino acid sequence described in SEQ ID NO: 19 is used (Figure 4).

[0080] Furthermore, carboxylic acid reductase can be converted into an active holoenzyme by being phosphopantetheinylated (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007)). Phosphopantetheinylation is catalyzed by phosphopantetheinyl transferase (PT). Examples of enzymes that can catalyze this reaction include enzymes classified as EC 2.7.8.7. Therefore, the microorganism of the present invention may be further modified so that the activity of phosphopantetheinyl transferase is increased. Examples of methods for increasing the activity of phosphopantetheinyl transferase include, but are not limited to, methods of introducing a foreign phosphopantetheinyl transferase gene and methods of enhancing the expression of an endogenous phosphopantetheinyl transferase gene. The enzymes used in the present invention are not limited to these as long as they have phosphopantetheinyl transfer activity. Typical enzymes include, for example, EntD of Escherichia coli, Sfp of Bacillus subtilis, Npt of Nocardia iowensis (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007)), and Lys5 of Saccharomyces cerevisiae (Ehmann et al., Biochemistry 38.19 (1999): 6171-6177.). The enzymes used in the present invention are not limited as long as they have activity for this conversion. For example, at least any one of the enzymes consisting of the amino acid sequences described in SEQ ID NOs: 20 to 23 may be used, and preferably, Npt of Nocardia iowensis derived from Nocardia iowensis consisting of the amino acid sequence described in SEQ ID NO: 21 is used (Figure 5).

[0081] The reactions in steps H, L, and N of FIG. 1 are aminotransfer reactions. Examples of enzymes that can catalyze this conversion include transaminases (aminotransferases) classified as EC 2.6.1.w (where w is an integer). For example, enzymes classified in groups such as EC 2.6.1.19 (4-aminobutanoic acid-2-oxoglutarate transaminase), EC 2.6.1.29 (diamine transaminase), and EC 2.6.1.48 (5-aminovalerate transaminase) can be exemplified as enzymes that may also be active for this conversion. The enzyme used in the present invention is not particularly limited as long as it has the conversion activity for each step. For example, YgjG, which is putrescine aminotransferase of Escherichia coli reported to transfer the amino groups of cadaverine and spermidine (Samsonova., et al., BMC microbiology 3.1 (2003): 2.), SpuC, which is putrescine aminotransferase of the genus Pseudomonas (Lu et al., Journal of bacteriology 184.14 (2002): 3765-3773., Galman et al., Green Chemistry 19.2 (2017): 361-366.), GABA aminotransferase GabT of Escherichia coli, and PuuE may be used.Furthermore, ω - transaminases derived from biological species such as Ruegeria pomeroyi, Chromobacterium violaceum, Arthrobacter citreus, Sphaerobacter thermophilus, Aspergillus fischeri, Vibrio fluvialis, Agrobacterium tumefaciens, Mesorhizobium loti have also been reported to have amino group transfer activity to diamine compounds such as 1,8 - diaminooctane and 1,10 - diaminedecane, and may be used in the present invention (Sung et al., Green Chemistry 20.20 (2018): 4591 - 4595., Sattler et al., Angewandte Chemie 124.36 (2012): 9290 - 9293.). The enzymes used in the present invention are not limited as long as they have activity for this conversion. For example, at least any one of the enzymes consisting of the amino acid sequences described in SEQ ID NOs: 24 to 28 may be used (Figure 5). Typical amino group donors include, but are not limited to, L - glutamic acid, L - alanine, and glycine.

[0082] In the reaction of step J in Figure 1, CoA is added to the carboxyl group. Examples of enzymes that can catalyze this conversion include CoA transferases classified as EC 2.8.3.y (y is an integer) and acid thiol ligases classified as EC 6.2.1.z (z is an integer). The enzymes used in the present invention are not limited as long as they have activity for this conversion.

[0083] The gene encoding the above - mentioned enzyme that can be used in the present invention may be derived from organisms other than the exemplified ones or may be artificially synthesized, as long as it can express substantial enzyme activity in the host microbial cell.

[0084] The recombinant microorganism according to the present invention may be one in which any gene is appropriately disrupted in the host microorganism. Disruption of the target gene is carried out by a method known in the art.

[0085] In addition, the amino acid sequence of the above enzyme or the nucleotide sequence of the gene encoding the enzyme that can be used in the present invention may have all mutations that can occur in nature, and / or artificially introduced mutations and modifications, as long as substantial enzyme activity can be expressed in the host microorganism cell. For example, mutations such as deletions, substitutions, insertions and additions may be contained. For example, it may contain an amino acid sequence in which one or more, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 7, particularly preferably 1 to 5, and even more particularly preferably 1 to 3 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of the above enzyme.

[0086] In addition, it is known that there are redundant codons for various codons encoding specific amino acids, so alternative codons that will ultimately be translated into the same amino acid may also be used in the present invention. That is, since the genetic code is degenerate, multiple codons can be used to encode a particular amino acid, so the amino acid sequence can be encoded by any one set of similar DNA oligonucleotides. Since most organisms are known to preferentially use a subset of specific codons (optimal codons) (Gene, Vol. 105, pp. 61-72, 1991, etc.), "codon optimization" according to the host microorganism may also be useful in the present invention.

[0087] Therefore, the genetically modified microorganism according to the present invention may include a nucleotide sequence having a sequence identity of, for example, 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the nucleotide sequence of the above enzyme gene, provided that it can express substantial enzyme activity. Alternatively, it may include a gene encoding an amino acid sequence having a sequence identity of, for example, 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence of the above enzyme.

[0088] In the present specification, the percentage (%) of "sequence identity" of a comparative amino acid sequence with respect to a reference amino acid sequence is defined as the percentage of amino acid residues in the comparative sequence that are identical to the amino acid residues in the reference sequence when the sequences are aligned so that the identity between these two sequences is maximized, and gaps are introduced into one or both of the two sequences if necessary. At this time, conservative substitutions are not considered as part of the sequence identity. The sequence identity can be determined by using publicly available computer software, for example, by using an alignment search tool such as BLAST (Basic Local Alignment Search Tool). A person skilled in the art can determine appropriate parameters for obtaining the maximum alignment of the comparative sequences in the alignment. The "sequence identity" of a nucleotide sequence can also be determined by a similar method.

[0089] In the present invention, by introducing the above C6 compound biosynthetic enzyme gene into a host microbial cell as an "expression cassette", more stable and high-level enzyme activity can be obtained. In the present specification, the "expression cassette" means a nucleotide containing a nucleic acid to be expressed or a nucleic acid sequence that regulates transcription and translation functionally linked to the gene to be expressed. Typically, the expression cassette of the present invention contains, in a functionally linked state, a promoter sequence upstream of the coding sequence at the 5'-end, a terminator sequence downstream of the 3'-end, and optionally, additional normal regulatory elements. In such a case, the nucleic acid to be expressed or the gene to be expressed is introduced into a host microorganism.

[0090] A promoter is defined as a DNA sequence that binds RNA polymerase to DNA and initiates RNA synthesis, regardless of whether it is a constitutive expression promoter or an inducible expression promoter. A strong promoter is a promoter that initiates mRNA synthesis at a high frequency and is also preferably used in the present invention. In Escherichia coli, the lac system, trp system, tac or trc system, the major operator and promoter regions of λ phage, the control region of fd coat protein, promoters for glycolytic enzymes (e.g., 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase), glutamate decarboxylase A, serine hydroxymethyltransferase, the promoter region of T7 phage-derived RNA polymerase, etc. can be used. In Corynebacterium glutamicum, the HCE (high-level constitutive expression) promoter, cspB promoter, sodA promoter, elongation factor (EF-Tu) promoter, etc. can be used. As terminators, the T7 terminator, rrnBT1T2 terminator, lac terminator, etc. can be used. In addition to promoter and terminator sequences, examples of other regulatory elements that can be mentioned are selection markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in "Gene Expression Technology: Methods in Enzymology 185", Academic Press (1990).

[0091] The expression cassette described above is incorporated into a vector consisting of, for example, a plasmid, phage, transposon, IS element, fosmid, cosmid, or linear or circular DNA, etc., and inserted into a host microorganism. Plasmids and phages are preferred. These vectors may be autonomously replicated in the host microorganism or replicated by the chromosome. Suitable plasmids are, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11 or pBdCI of Escherichia coli; pUB110, pC194 or pBD214 of Bacillus; pSA77 or pAJ667 of Corynebacterium, etc. Examples of Bacillus such as Bacillus genus include pUB110, pC194 or pBD214. Other plasmids and the like that can be used are described in "Gene Cloning and DNA analysis 7th edition", Wiley-Blackwell (2016). The introduction of the expression cassette into the vector can be carried out by conventional methods including excision, cloning and ligation with appropriate restriction enzymes. Each expression cassette may be arranged on one vector or on two or more vectors.

[0092] After constructing the vector having the expression cassette of the present invention as described above, conventional methods can be used as the techniques applicable when introducing the vector into a host microorganism. For example, the calcium chloride method, electroporation method, conjugation transfer method, protoplast fusion method, etc. can be mentioned, but are not limited thereto, and a method suitable for the host microorganism can be selected.

[0093] The genetically modified microorganism obtained as described above is cultured and maintained under conditions suitable for its growth and / or maintenance for the production of the target compound. Suitable medium compositions, culture conditions and culture times for transformants derived from various host microorganism cells can be easily set and selected by those skilled in the art.

[0094] Therefore, a second aspect of the present invention relates to a method for producing a target compound, including a culturing step of culturing the aforementioned recombinant microorganism. Specifically, the production method includes a culturing step of culturing the aforementioned recombinant microorganism to obtain a culture of the recombinant microorganism and / or an extract of the culture. The target compound is preferably hexamethylenediamine.

[0095] In the culturing step, a culture containing cells is obtained by culturing the recombinant microorganism in a medium containing a carbon source and a nitrogen source. The genetically recombinant microorganism of the present invention is cultured under conditions suitable for compound production and the growth and maintenance of the microorganism, and suitable medium composition, culture time, and culture conditions can be easily set by those skilled in the art.

[0096] Examples of the carbon source include D-glucose, sucrose, lactose, fructose, maltose, oligosaccharides, polysaccharides, starch, cellulose, rice bran, molasses, oils and fats (such as soybean oil, sunflower oil, peanut oil, coconut oil, etc.), fatty acids (such as palmitic acid, linoleic acid, oleic acid, linolenic acid, etc.), alcohols (such as glycerol, ethanol, etc.), organic acids (such as acetic acid, lactic acid, succinic acid, etc.), corn hydrolysis solution, and cellulose hydrolysis solution. Preferably, it is D-glucose, sucrose or glycerol. These carbon sources can be used individually or as a mixture.

[0097] Compounds produced using biomass-derived raw materials can be clearly distinguished from synthetic raw materials derived from, for example, petroleum, natural gas, and coal by measuring the bio-based carbon content based on the Carbon-14 (radioactive carbon) analysis specified in ISO16620-2 or ASTM D6866.

[0098] As the nitrogen source, nitrogen-containing organic compounds (such as peptone, casamino acid, tryptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean powder, amino acid, and urea, etc.) or inorganic compounds (such as aqueous ammonia solution, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, sodium nitrate, ammonium nitrate, etc.) can be mentioned. These nitrogen sources can be used individually or as a mixture.

[0099] In addition, when the recombinant microorganism expresses a useful additional trait, for example, when it has a resistance marker to an antibiotic, the corresponding antibiotic may be included in the medium. Thereby, the risk of contamination by miscellaneous bacteria during culturing is reduced. Examples of antibiotics include, but are not limited to, β-lactam antibiotics such as ampicillin, aminoglycoside antibiotics such as kanamycin, macrolide antibiotics such as erythromycin, tetracycline antibiotics, chloramphenicol, etc.

[0100] When the host microorganism cannot assimilate the above carbon sources such as cellulose and polysaccharides, known genetic engineering techniques such as introducing foreign genes into the host microorganism can be used to adapt it to the production of the target compound using these carbon sources. Examples of foreign genes include, for example, cellulase genes and amylase genes.

[0101] The culturing may be in a batch mode or a continuous mode. Also, in either case, it may be in a form of replenishing the additional carbon source, etc. at an appropriate time point during culturing. Furthermore, the culturing may be carried out while controlling conditions such as suitable temperature, oxygen concentration, pH, etc. The suitable culturing temperature for a transformant derived from a general microbial host cell is usually in the range of 15°C to 55°C, preferably 25°C to 40°C. When the host microorganism is aerobic, shaking (such as flask culturing, etc.), stirring / aeration (such as jar fermenter culturing, etc.) may be carried out to ensure an appropriate oxygen concentration during fermentation. These culturing conditions can be easily set by those skilled in the art.

[0102] The production method according to the present invention preferably further includes a mixing step of mixing the culture and / or an extract of the culture with a substrate compound to obtain a mixed solution.

[0103] In the culture and / or the mixed solution, as a result of the reaction, a diamine compound, which is the target compound, is produced. Therefore, in a more preferred embodiment, the production method according to the present invention further includes a recovery step of recovering the target compound, preferably hexamethylenediamine, from the culture or / and the mixed solution.

[0104] The step of recovering the target compound from the culture is performed by any method for separation and / or purification. For example, methods such as centrifugation, membrane filtration, membrane separation, crystallization, extraction, distillation, adsorption, phase separation, ion exchange, and various chromatographies can be mentioned, but are not limited thereto. Separation and / or purification may select one kind of method, or may be performed by combining a plurality of methods.

[0105] The recombinant microorganism according to the present invention is a microorganism having a hexamethylenediamine production pathway and includes one or more genetic modifications that suppress at least one of ornithine decarboxylase and lysine decarboxylase. Therefore, diamine by-products generated in side reactions, namely putrescine and / or cadaverine, can be reduced. By suppressing side reactions and reducing by-products, hexamethylenediamine can be efficiently produced from a carbon source as a raw material. Furthermore, the load in separation and purification in the production process can be reduced, and the contamination of diamine by-products can be reduced. Therefore, by using the recombinant microorganism according to the present invention, a good production method of hexamethylenediamine, which is the target compound, is provided.

[0106] As described above, the embodiments for carrying out the present invention have been illustrated. However, the above embodiments are merely shown as examples and are not intended to limit the scope of the invention. The above embodiments can be implemented in various other forms, and various omissions, substitutions, and changes can be made without departing from the gist of the invention.

Example

[0107] Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.

[0108] Burkholderia sp. LEBP-3 strain (hereinafter also abbreviated as "L3 strain") was applied for deposit to the Patent Microorganism Depositary, National Institute of Technology and Evaluation (NPMD) on December 4, 2020, and has been internationally deposited with the accession number: NITE BP-03334.

[0109] <Construction of plasmid for gene disruption> For the disruption and insertion of Escherichia coli genes, homologous recombination using pHAK1 (deposited with the Patent Microorganism Depositary, Biotechnology Center, National Institute of Technology and Evaluation (NPMD) on March 18, 2019, with the accession number: NITE P-02919; international deposit number: NITE BP-02919) was performed. pHAK1 contains a temperature-sensitive mutant repA gene, a kanamycin resistance gene, and a levansucrase gene sacB derived from Bacillus subtilis. The levansucrase gene acts lethally on host microorganisms in the presence of sucrose. PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio Inc.) or KOD FX Neo (product name, manufactured by Toyobo Co., Ltd.) was used for the amplification of PCR fragments, and plasmid preparation was performed using Escherichia coli HST08 strain.

[0110] Using the genomic DNA of Escherichia coli BL21(DE3) strain as a template, a PCR product containing the 5' homologous region, coding region, and 3' homologous region of the target gene to be disrupted was obtained. The combinations of target genes and primer sequences are shown in Table 3 below. The nucleotide sequences of each primer are shown in Fig. 6.

[0111]

Table 3

[0112] Next, this PCR product was inserted into the pHAK1 plasmid fragment amplified using the primers of SEQ ID NOs: 49 and 50 using the In-Fusion HD cloning kit (product name, manufactured by Clontech) and circularized. It was transformed into Escherichia coli strain HST08, and the plasmid was extracted from the obtained transformant.

[0113] Using the pHAK1 plasmid inserted with the DNA fragments of the 5' homologous region, coding region, and 3' homologous region of the target gene to be disrupted extracted above as a template, PCR was performed using the primers described in Table 4 below, and a part or all of the coding region of the target gene to be disrupted was removed to obtain a pHAK1 plasmid fragment containing the 5' homologous region and 3' homologous region. The nucleotide sequences of each primer are shown in FIG. 7.

[0114]

Table 4

[0115] The obtained plasmid fragment was circularized by terminal phosphorylation and self-ligation. It was transformed into Escherichia coli strain HST08, and the plasmid was extracted from the obtained transformant to obtain a plasmid for gene disruption.

[0116] <Construction of Plasmid for Insertion of Hexamethylenediamine Production Pathway Enzyme Gene> The polynucleotide (SEQ ID NO: 71) encoding PaaJ (Ec) of Escherichia coli (SEQ ID NO: 1) was obtained by cloning from the genomic DNA of Escherichia coli strain W3110 (NBRC12713). The polynucleotide (SEQ ID NO: 72) encoding PaaH (Ec) of Escherichia coli (SEQ ID NO: 2) was obtained by cloning from the genomic DNA of Escherichia coli strain W3110 (NBRC12713). SEQ ID NOs: 71 and 72 are shown in FIG. 8.

[0117] The paaJ(Ec) and paaH(Ec) genes were used to design a plasmid for gene insertion such that they were inserted into the pflB gene region as an expression cassette. An expression cassette consisting of a promoter region (SEQ ID NO: 73), the paaJ(Ec) gene coding region, a linker sequence (SEQ ID NO: 74), the paaH gene coding region, and a terminator region (SEQ ID NO: 75) in this order was incorporated between the 5' homologous region and the 3' homologous region of the pflB gene coding region contained in the previously constructed pflB gene disruption plasmid. SEQ ID NOs: 73 to 75 are shown in Fig. 9.

[0118] The polynucleotide encoding PaaF(L3) of strain L3 was optimized for E. coli expression and obtained using the artificial gene synthesis service of Eurofins Genomics. The polynucleotide encoding MmgC(L3) of strain L3 was optimized for E. coli expression and obtained using the artificial gene synthesis service of Eurofins Genomics.

[0119] The optimized sequences of the paaF(L3) and mmgC(L3) gene coding regions were used to design a plasmid for gene insertion such that they were inserted into the yahK gene region as an expression cassette. An expression cassette consisting of a promoter region (SEQ ID NO: 78), the paaF(L3) gene coding region, a linker sequence (SEQ ID NO: 79), the mmgC(L3) gene coding region, and a terminator region (SEQ ID NO: 80) in this order was incorporated between the 5' homologous region and the 3' homologous region of the yahK gene coding region contained in the previously constructed yahK gene disruption plasmid. SEQ ID NOs: 78 to 80 are shown in Fig. 9.

[0120] The carboxylic acid reductase MaCar (SEQ ID NO: 17) of Mycobacterium abscessus was used with a mutant MaCar (m) in which a part of the amino acid sequence was substituted (SEQ ID NO: 19). The polynucleotide (SEQ ID NO: 81) (Figure 10) encoding MaCar (m) was optimized for the base sequence for E. coli expression and obtained using the artificial gene synthesis service of Eurofins Genomics. The polynucleotide (SEQ ID NO: 82) encoding Npt (SEQ ID NO: 21) of Nocardia iowensis was cloned from the genomic DNA of Nocardia iowensis JCM18299 strain (provided from RIKEN BRC via the Ministry of Education, Culture, Sports, Science and Technology National BioResource Project). The macar (m) and npt genes were designed for a gene insertion plasmid so as to be inserted into the ldhA gene region as an expression cassette. An expression cassette in which a promoter region (SEQ ID NO: 83), the macar (m) gene coding region, a linker sequence (SEQ ID NO: 84), the npt gene coding region, and a terminator region (SEQ ID NO: 85) were arranged in this order was incorporated between the 5' homologous region and the 3' homologous region of the ldhA gene coding region contained in the previously constructed ldhA gene disruption plasmid. SEQ ID NOS: 82 to 85 are shown in Figure 11.

[0121] <Construction of Modified E. coli Strains> By electroporation method (refer to "Genetic Engineering Experiment Notebook" published by Yodosha Co., Ltd., written by Takaki Tamura), after transforming Escherichia coli BL21(DE3) strain with a plasmid for disrupting a desired gene, it was spread on an LB agar medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar powder 15 g / L) containing 100 mg / L of kanamycin sulfate, and incubated overnight at 30 °C to obtain single colonies, thereby obtaining transformants. One platinum loop of this transformant was inoculated into 1 mL of an LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L) containing 100 mg / L of kanamycin sulfate, and shake culture was carried out at 30 °C. The obtained culture solution was spread on an LB agar medium containing 100 mg / L of kanamycin sulfate and incubated overnight at 42 °C. The resulting colonies have the plasmid inserted into the genome by single crossover. One platinum loop of the colonies was inoculated into 1 mL of an LB liquid medium and shake culture was carried out at 30 °C. The obtained culture solution was spread on an LB agar medium containing 20% sucrose and incubated at 30 °C for 2 days. Regarding the obtained colonies, it was confirmed by colony direct PCR using the primer sets shown in Table 5 that the desired gene was disrupted or inserted.

[0122] The above operations were repeated to construct an Escherichia coli strain containing multiple gene disruptions and gene insertions. The constructed Escherichia coli strains are shown in Table 6. In the table, genes described with Δ indicate that the enzyme gene is deficient, and "Gene Name A::Gene Name B" indicates that the Gene A region is replaced by the region containing Gene B. The sequences of each primer are shown in Figure 12.

[0123]

Table 5

[0124]

Table 6

[0125] <Liquid Culture of Modified Escherichia coli Strains (Reference Examples 1 to 3)> The mutant strains No. 060, 063, and 069 were each inoculated with one platinum loop into 2 mL of LB liquid medium (in a 14 mL round-bottom tube), and cultured with shaking at 37 °C for 24 hours to obtain precultures. 20 μL of the obtained preculture was inoculated into 2 mL of LB liquid medium (in a 14 mL round-bottom tube), and cultured with shaking at 37 °C for 24 hours.

[0126] After completion of the culture, the culture broth was separated into cells and supernatant by centrifugation, and the concentrations of putrescine and cadaverine in the supernatant were analyzed. The analysis was performed using an ion chromatograph. The conditions were as follows.

[0127] Ion Chromatograph Analysis Conditions Apparatus: ICS-3000 (manufactured by Dionex) Detector: Conductivity detector Column: IonPac CG19 (2×50 mm) / CS19 (2×250 mm) (manufactured by Thermo Scientific) Oven temperature: 30 °C Mobile phase: 8 mM methanesulfonic acid aqueous solution (A), 70 mM methanesulfonic acid aqueous solution (B) Gradient conditions: (A: 100%, B: 0%) - (10 min) - (A: 0%, B: 100%) - (held for 1 min) Flow rate: 0.28 mL / min Injection volume: 20 μL

[0128] The results are shown in Table 7. In strain No. 063 (Reference Example 2) in which the speC gene encoding ornithine decarboxylase of Escherichia coli was disrupted, it was confirmed that the concentration of putrescine in the culture broth decreased compared to strain No. 060 (Reference Example 1) in which the speC and cadA genes were not disrupted. Furthermore, in strain No. 069 (Reference Example 3) in which the cadA gene encoding lysine decarboxylase of Escherichia coli was disrupted, it was confirmed that the concentrations of putrescine and cadaverine in the culture broth decreased compared to Reference Examples 1 and 2.

[0129]

Table 7

[0130] <Construction of Enzyme Gene Expression Plasmid for Hexamethylenediamine Production Pathway> For the amplification of PCR fragments, PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio Inc.) or KOD FX Neo (product name, manufactured by Toyobo Co., Ltd.) was used. For the preparation of plasmids, Escherichia coli JM109 strain was used. For the optimization of nucleotide sequences, GeneArt GeneOptinizer (software name, manufactured by Thermo Fisher Scientific) or the artificial gene synthesis service of Eurofins Genomics was used.

[0131] Using the nucleotides shown in SEQ ID NO: 4 and the nucleotides shown in SEQ ID NO: 5 as primers, the polynucleotide encoding PaaF(L3) of strain L3, which is encoded by the nucleotide sequence of the PCR amplicon using the chromosomal DNA of strain L3 as a template, was optimized for the nucleotide sequence for expression in Escherichia coli and obtained using the artificial gene synthesis service of Eurofins Genomics. PCR was performed using the oligonucleotides of SEQ ID NOs: 106 and 107 as primers to obtain a PCR product containing the optimized sequence of the coding region of the paaF(L3) gene. Using pETDuet-1 (product name, manufactured by Merck) as a template, PCR was performed using the oligonucleotides of SEQ ID NOs: 108 and 109 as primers to obtain a pETDuet-1 fragment. A DNA fragment containing the coding region of paaF(L3) and the pETDuet-1 fragment were ligated using an In-Fusion HD cloning kit (product name, manufactured by Clontech). Escherichia coli JM109 strain was transformed, and plasmids were extracted from the obtained transformants. "paaF(L3)-pETDuet" was obtained as the PaaF(L3) expression plasmid. SEQ ID NOs: 106 to 109 are shown in Fig. 13.

[0132] The polynucleotide encoding Ter (SEQ ID NO: 9) of Thermothelomyces thermophilus was optimized for the nucleotide sequence for E. coli expression and obtained using the artificial gene synthesis service of Eurofins Genomics. PCR was performed using the oligonucleotides of SEQ ID NOs: 111 and 112 as primers to obtain a PCR product containing the coding region (SEQ ID NO: 110) of the ter gene. Using "paaF(L3)-pETDuet" as a template and the oligonucleotides of SEQ ID NOs: 113 and 114 as primers, PCR was performed to obtain a "paaF(L3)-pETDuet" fragment. A DNA fragment containing the coding region of the ter gene and the "paaF(L3)-pETDuet" fragment were ligated using an In-Fusion HD cloning kit (product name, manufactured by Clontech). Escherichia coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. "paaF(L3)-ter-pETDuet" was obtained as a PaaF(L3), Ter co-expression plasmid. SEQ ID NOs: 110 to 114 are shown in FIG. 13.

[0133] For YgjG (SEQ ID NO: 24) of Eshcherichia coli, a mutant YgjG(m) (SEQ ID NO: 115) with a partial substitution of the amino acid sequence was used (FIG. 13). The polynucleotide encoding YgjG(m) was subjected to PCR using the oligonucleotides of SEQ ID NOs: 117 and 118 as primers to obtain a PCR product containing the coding region of the ygjG(m) gene (SEQ ID NO: 116) (FIG. 13). Next, this PCR product was inserted between the restriction enzyme NcoI and HindIII cleavage sites of pACYCDuet-1 (product name, manufactured by Merck) using an In-Fusion HD cloning kit (product name, manufactured by Clontech). Escherichia coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. "ygjG(m)-pACYCDuet" was obtained as a YgjG(m) expression plasmid.

[0134] <Hexamethylenediamine production test (Comparative Example 1 and Example 1)> By the electroporation method, the Escherichia coli modified strain No. 060 or No. 069 was transformed with a hexamethylenediamine production pathway enzyme expression plasmid, and cultured at 37 °C for one day on an LB agar medium containing 50 mg / L of sodium ampicillin and 30 mg / L of chloramphenicol to form colonies. One platinum loop of colonies was inoculated into 2 mL of an LB liquid medium (14 mL round-bottom tube) containing 50 mg / L of sodium ampicillin and 30 mg / L of chloramphenicol, and cultured with shaking at 37 °C for 3 to 5 hours to obtain a preculture solution. 0.5 mL of the preculture solution was added to a 250 mL Jar culture apparatus (model name: Bio Jr. 8, manufactured by Biott) containing a synthetic medium (shown in the following table) containing 50 mg / L of sodium carbenicillin, 30 mg / L of chloramphenicol, and 0.02 mM of IPTG for main culture. The culture conditions were as follows: culture temperature 37 °C; culture pH 7.0; pH adjustment with 10% (w / v) aqueous ammonia; stirring at 750 rpm; aeration at 1 vvm. Glucose was supplemented at the end concentration of 35 g / L 22 hours after inoculation with the preculture solution, and cultured until 41 hours passed.

[0135]

Table 8

[0136] After the culture was completed, the culture solution was separated into cells and supernatant by centrifugation, and the concentrations of putrescine, cadaverine, hexamethylenediamine, and glucose in the supernatant were analyzed. The analysis of the concentrations of putrescine, cadaverine, and hexamethylenediamine was performed using an ion chromatograph, and the analysis of the glucose concentration was performed using a high-performance liquid chromatograph Prominence system (manufactured by Shimadzu Corporation) under the following conditions.

[0137] High-performance liquid chromatograph (HPLC) analysis conditions Detector: Differential refractive index detector Column: Shim-Pack Fast-OA(G), Fast-OA (manufactured by Shimadzu Corporation) Oven temperature: 40 °C Mobile phase: 8 mM methanesulfonic acid aqueous solution Flow rate: 0.6 mL / min Injection volume: 10 μL

[0138] The results are shown in Table 9. In the culture of transformant HMD2 (Example 1) in which the hexamethylenediamine production pathway enzyme gene was introduced into strain No. 069 in which the speC gene encoding Escherichia coli ornithine decarboxylase and the cadA gene encoding lysine decarboxylase were disrupted, compared with the culture of transformant HMD1 (Comparative Example 1) in which the speC and cadA genes were not disrupted, the concentrations of putrescine and cadaverine in the culture solution were reduced. In addition, the concentration of hexamethylenediamine in the culture solution and the yield with respect to glucose were improved.

[0139] [Table 9]

Industrial Applicability

[0140] According to the genetically modified microorganism of the present invention, an efficient production process of hexamethylenediamine can be provided, and its application to industrial-scale production is expected.

Claims

1. having a hexamethylenediamine production pathway, a recombinant microorganism comprising one or more genetic modifications that suppress at least one of ornithine decarboxylase and lysine decarboxylase.

2. The recombinant microorganism according to claim 1, wherein the recombinant microorganism belongs to a genus selected from the group consisting of the genus Escherichia, the genus Bacillus, the genus Corynebacterium, the genus Arthrobacter, the genus Brevibacterium, the genus Clostridium, the genus Zymomonas, the genus Pseudomonas, the genus Burkholderia, the genus Streptomyces, the genus Rhodococcus, the genus Synechocystis, the genus Alkalihalobacillus, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Yarrowia, the genus Candida, the genus Pichia, and the genus Aspergillus.

3. The recombinant microorganism according to claim 1 or 2, wherein the recombinant microorganism is Escherichia coli.

4. The genetic modification is ・ a modification that suppresses the expression of at least one of the endogenous gene encoding the ornithine decarboxylase and the endogenous gene encoding the lysine decarboxylase, and ・ a modification in which the enzyme activity of at least one of the ornithine decarboxylase and the lysine decarboxylase is reduced as compared to the non-mutant strain The recombinant microorganism according to any one of claims 1 to 3, which is one or more selected from the group consisting of.

5. The recombinant microorganism according to any one of claims 1 to 4, wherein the ornithine decarboxylase is a protein encoded by at least one selected from the group consisting of the speC gene and the speF gene.

6. The recombinant microorganism according to any one of claims 1 to 5, wherein the ornithine decarboxylase is a protein encoded by the speC gene.

7. The recombinant microorganism according to any one of claims 1 to 6, wherein the lysine decarboxylase is a protein encoded by at least one selected from the group consisting of the cadA gene and the ldcC gene.

8. The recombinant microorganism according to any one of claims 1 to 7, wherein the lysine decarboxylase is a protein encoded by the cadA gene.

9. A method for producing hexamethylenediamine, comprising a culturing step of culturing the recombinant microorganism according to any one of claims 1 to 8.

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