Recombinant microorganism capable of producing diamine and method for producing diamine

Abstract

[Problem] To provide: with respect to the production of a diamine, a recombinant microorganism and a method for manufacturing a diamine, that realize a simplified process and / or a reduction in effluent treatment costs; and a halophilic and / or alkalophilic recombinant microorganism in which N-acetyl diamine production is suppressed. [Solution] The present invention provides a halophilic and / or alkalophilic recombinant microorganism having a diamine producing ability, wherein the diamine is represented by the formula NH2CH2(CH2)nCH2NH2 (in the formula, n is an integer between 0 and 10), the halophilic and / or alkalophilic recombinant microorganism resulting from modification of a halophilic and / or alkalophilic host microorganism so as to impart a diamine-producing ability; and a halophilic and / or alkalophilic recombinant microorganism having an amine producing ability, the recombinant microorganism having one or more genetic modifications for suppressing an N-acetylation enzyme which N-acetylates a diamine compound and generates an N-acetyl diamine compound.

Description

[Technical field]

[0001] The present invention relates to a recombinant halophilic and / or alkalophilic microorganism capable of producing diamine, which is an industrially useful compound, and a method for producing diamine using said recombinant microorganism.The present invention also relates to a recombinant halophilic and / or alkalophilic microorganism in which N-acetyldiamine production is suppressed. [Background technology]

[0002] In recent years, disasters and climate change caused by global warming have led to a strong demand for the development of sustainable processes aimed at coexistence with and environmental conservation of the global environment. Among these, there are high expectations for bioprocesses, which are substance production processes that use renewable raw materials and utilize biological reactions. To date, fermentation production processes for various chemical products have been developed. For example, fermentation production processes for polyols used as polyurethane raw materials, polyester raw materials, plasticizer raw materials, pharmaceutical intermediates, etc. have been proposed.

[0003] Diamines, along with dicarboxylic acids such as adipic acid, are industrially important compounds as raw materials for polyamides (such as PA66 (6,6-nylon)).

[0004] For example, 1,6-hexamethylenediamine (also called 1,6-diaminohexane or hexamethylenediamine) has the molecular formula C 6 H 12 N 2Hexamethylenediamine is a compound having the formula: Hexamethylenediamine is widely used in fiber and resin applications as a raw material for nylon 66 (PA66) and is expected to be in demand worldwide. Hexamethylenediamine is also used as a urethane raw material via isocyanate, and as an intermediate for agricultural chemicals and medicines. Hexamethylenediamine is synthesized by obtaining adiponitrile through hydrocyanation of butadiene, electrolytic dimerization of acrylonitrile, or nitrification of adipic acid, and then subjecting the adiponitrile to hydrogenation using nickel or the like as a catalyst (Non-Patent Document 1). Hexamethylenediamine is industrially produced by this method, but the hydrogenation reaction is carried out after adiponitrile is first synthesized.

[0005] As a method for producing hexamethylenediamine using a microorganism, a method for producing diamine from intracellular dicarboxylic acid, aminocarboxylic acid, dialdehyde, etc. by combining exogenous enzymes, such as carboxylic acid decarboxylase and aminotransferase, has been reported (Patent Document 1 and Patent Document 2). Patent Document 1 predicts and illustrates enzyme genes that are expected to improve yield by deletion and / or destruction in a host microorganism modified to have a hexamethylenediamine production pathway, based on an in silico metabolic simulation. However, there is no mention of by-products derived from intermediates in the hexamethylenediamine production pathway and a method for suppressing them. Patent Document 2 describes a method for producing hexamethylenediamine by an enzyme reaction pathway via 6-hydroxyhexanoic acid. However, there is no mention of the generation of by-products derived from intermediates in the hexamethylenediamine production pathway newly constructed by genetic recombination, and a method for suppressing them.

[0006] 1,5-Pentamethylenediamine (also known as 1,5-diaminopentane or cadaverine), molecular formula C 5 H 14 N 21,5-Pentamethylenediamine is a compound having the formula: 1,5-pentamethylenediamine is a compound that is expected to be in demand for fiber and resin applications as a raw material for nylon (PA56). PA56 fiber has the same strength and heat resistance as PA66, and is highly hygroscopic and releases moisture, so new market developments are expected. 1,5-Pentamethylenediamine is also attracting attention as a raw material for urethane via isocyanate, and as an intermediate for agricultural chemicals and pharmaceuticals.

[0007] While an efficient method for chemically synthesizing 1,5-pentamethylenediamine from petroleum raw materials has not yet been established, it is known that it can be easily produced in biosynthesis by enzymatic decarboxylation of L-lysine. PA56 resin, which is made from biomass-derived 1,5-pentamethylenediamine, is attracting industrial attention as a bioplastic from the perspective of reducing the environmental burden (Non-Patent Document 2).

[0008] Fermentative production processes for diamines from renewable raw materials have also been proposed. For example, 1,5-pentamethylenediamine and hexamethylenediamine are examples of amine compounds that can be produced by fermentation, and these can be used as raw materials for polymer production in the production of chemical products (Patent Documents 3 to 7 and Non-Patent Document 3).

[0009] As a method for producing 1,5-pentamethylenediamine using a microorganism, a method is known in which Escherichia coli in which lysine decarboxylase is highly expressed is cultured, and 1,5-pentamethylenediamine is obtained by reacting the precursor lysine with the enzyme and decarboxylating it (Patent Document 8). Also proposed is a method for producing 1,5-pentamethylenediamine from glucose by culturing a coryneform bacterium in which the activity of the lysine decarboxylase gene has been increased and the activity of a gene that plays an important role in lysine biosynthesis has been decreased (Patent Document 9).

[0010] On the other hand, as a method for separating and purifying diamine from a culture solution, for example, a method is known in which an alkaline solution such as sodium hydroxide is added to liberate 1,5-pentamethylenediamine, and then an appropriate solvent is used to extract the diamine (Patent Documents 10, 11, and 12). In addition, a method has been proposed in which an aqueous solution of 1,5-pentamethylenediamine carbonate is pyrolyzed to separate crude 1,5-pentamethylenediamine and carbon dioxide, and then the diamine is purified by distillation (Patent Document 13). Furthermore, in the fermentation production of diamine by a microorganism, it has been proposed to separate (dissociate) the free base of the diamine and carbon dioxide from diamine carbonates and / or diamine carbamates neutralized with carbon dioxide added from the outside or generated by metabolism, and then extract the diamine with an organic solvent (Patent Document 14).

[0011] However, the above-mentioned methods proposed so far have the following disadvantages. First, regarding the fermentative production of diamines by microorganisms, for example, when lysine is converted to 1,5-pentamethylenediamine, the pH in the medium rises, but the Escherichia coli and coryneform bacteria used in the methods described in Patent Documents 8 and 9 can generally only grow at about pH 7 to 8, and their growth is significantly inhibited in an environment of pH 9 or higher. For this reason, when such microorganisms are used as catalysts, it is necessary to appropriately add an acid or alkali solution to control the pH within a range that does not inhibit the growth of the microorganisms, which makes the production process complicated.

[0012] Next, regarding the purification process of diamine, the methods described in Patent Documents 11 and 14 use polar organic solvents such as chloroform and hexane for extraction, but many organic solvents are harmful and are not suitable for handling. In addition, low extraction efficiency significantly affects production costs. Furthermore, if the organic solvent used is recovered to reduce production costs, the production process becomes complicated. In addition, in the method described in Patent Document 12, purification is performed by a crystallization method, but the crystallization rate is 40 to 45%, and a high yield cannot be expected.

[0013] In the method described in Patent Document 10, a large amount of salt is generated as a by-product in the diamine liberation step. For example, when 50 g / L of cadaverine is produced by culturing while maintaining neutrality while adding sulfuric acid, a maximum of about 100 g / L of sodium sulfate is generated as a by-product due to the presence of sulfate ions, which are counter ions of cadaverine. It is highly likely that a large amount of cost will be required to treat the wastewater containing this by-product. Furthermore, the method described in Patent Document 14 does not mention the handling of the aqueous phase containing inorganic salts after diamine solvent extraction, but this method may also require a large amount of cost for wastewater treatment.

[0014] Thus, there is room for further improvement in the existing diamine production methods. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] JP 2015-146810 A [Patent Document 2] JP 2017-544854 A [Patent Document 3] JP 2012-188407 A [Patent Document 4] Special Publication No. 2012-525856 [Patent Document 5] Special Publication No. 2016-538870 [Patent Document 6] Special Publication No. 2016-501031 [Patent Document 7] Special Publication No. 2017-533734 [Patent Document 8] Patent No. 5553394 [Patent Document 9] Patent No. 5210295 [Patent Document 10] Patent No. 5646345 [Patent Document 11] Patent No. 4196620 [Patent Document 12] JP 2005-6650 A [Patent Document 13] Patent No. 5930594 [Patent Document 14] Special Publication No. 2018-500911 [Non-patent literature]

[0016] [Non-Patent Document 1] Process Economics Program Report 31B (IHS market) [Non-Patent Document 2] Tsuge, Y. et al.,Engineering cell factories for producing building block chemicals for bio-polymer synthesis.,Microb. Cell Fact.,Vol.,15,19(2016) [Non-Patent Document 3] A Novel Process For Cadaverine Bio-Production Using a Consorsium of Two Engineering Escherichia Coli, Wang j Et Al.Frontiers in Microbiology(2018) Summary of the Invention [Problem to be solved by the invention]

[0017] In view of the above-mentioned current situation, an object of the present invention is to provide a recombinant microorganism and a method for producing a diamine, which can realize at least one of simplification of the process and reduction in wastewater treatment costs in the production of a diamine.

[0018] A further object of the present invention is to provide a recombinant halophilic and / or alkaliphilic microorganism in which N-acetyldiamine production is suppressed. [Means for solving the problem]

[0019] In order to solve the above problems, the inventors conducted intensive research and found that the recombinant microorganism of the present invention, which is obtained by modifying a host microorganism having at least one of halophilic and alkalophilic properties so as to have diamine production ability, can achieve at least one of process simplification and reduction in wastewater treatment costs in diamine production.

[0020] That is, the present invention provides: [1] A recombinant halophilic and / or alkaliphilic microorganism capable of producing diamines, The diamine has the formula: NH 2 CH 2 (CH 2 ) n CH 2 NH 2 (wherein n is an integer of 0 to 10), A recombinant microorganism in which a halophilic and / or alkaliphilic host microorganism has been modified to have the ability to produce diamines; [2] The recombinant microorganism according to [1], wherein n is 3 or 4; [3] The recombinant microorganism according to [2], wherein the host microorganism has been modified by one or more genetic manipulations to induce overproduction of L-lysine decarboxylase (EC 4.1.1.18); [4] The genetic manipulation is any one of the following (A), (B), (C), (D), and (E): (A) introducing an exogenous gene encoding the L-lysine decarboxylase into the host microorganism; (B) increasing the copy number of the endogenous L-lysine decarboxylase gene in the host microorganism; (C) introducing a mutation into an expression regulatory region of the endogenous L-lysine decarboxylase gene in the host microorganism; (D) replacing the expression regulatory region of the endogenous gene for L-lysine decarboxylase in the host microorganism with an exogenous regulatory region capable of high expression; and (E) deleting a regulatory region of the endogenous gene for L-lysine decarboxylase in the host microorganism; The recombinant microorganism according to [3], wherein the recombinant microorganism is one or more operations selected from the group consisting of: [5] A nucleotide sequence having 85, 90, 92, 95, 98 or 99% or more homology to the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or The recombinant microorganism according to any one of [2] to [4], which contains a nucleotide sequence having 85, 90, 92, 95, 98, or 99% or more homology to a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 3; [6] The recombinant microorganism according to any one of [2] to [5], which has been further modified by mutation or genetic recombination to improve lysine-producing ability; [7] The recombinant microorganism according to [6], wherein the mutation operation or the genetic recombination operation is an operation for removing feedback inhibition of at least one of aspartokinase III (EC 2.7.2.4) and 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7); [8] The recombinant microorganism according to any one of [1] to [7], wherein the host microorganism is selected from the group consisting of Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis; [9] The recombinant microorganism according to any one of [1] to [8], wherein the host microorganism is Bacillus pseudofirmus;

[10] Using the recombinant microorganism according to any one of [1] to [9], 2 CH 2 (CH 2 ) n CH 2 NH 2 (wherein n is an integer of 0 to 10);

[11] The method according to

[10] , wherein n in the formula is 3 or 4;

[12] The production method according to

[10] or

[11] , comprising a culture step of culturing the recombinant microorganism in a medium containing 5 mass% or more of an inorganic salt to obtain a culture solution containing a diamine;

[13] A culturing step of culturing the recombinant microorganism to obtain a culture solution containing the microorganism; a reaction step of contacting the culture solution and / or the bacterial cells with an aqueous solution containing 5% by mass or more of an inorganic salt and lysine to obtain a reaction solution containing 1,5-pentanediamine; The method according to any one of

[10] to

[12] ,

[14] The method according to

[12] or

[13] , further comprising a step of removing the recombinant microorganism from the culture medium or reaction solution;

[15] The production method according to

[14] , further comprising a step of concentrating the culture solution or reaction solution.

[16] The method according to

[15] , further comprising a pH adjustment step of adjusting the pH of the concentrated culture solution or reaction solution to 12 or more after concentrating the culture solution or reaction solution in the concentration step;

[17] The production method according to any one of

[12] to

[16] , further comprising a separation step of separating the culture solution or reaction solution into a phase containing a diamine and an aqueous phase containing the inorganic salt;

[18] The method according to any one of

[12] to

[17] , wherein in the separation step, an organic solvent is added to the culture solution or reaction solution to separate into a phase containing the diamine and the organic solvent and an aqueous phase;

[19] The method according to

[17] or

[18] , wherein an alkaline compound is not added in the separation step;

[20] The method according to any one of

[12] to

[19] , wherein the inorganic salt is sodium carbonate and / or sodium sulfate;

[21] The diamine produced in the culture step and / or the reaction step is in the form of one or more selected from the group consisting of carbonate, bicarbonate, bisbicarbonate, carbamate, and biscarbamate; The method according to any one of

[12] to

[20] , wherein in the concentrating step, the diamine in the form of a salt is converted into a free base and carbon dioxide, and the carbon dioxide is separated;

[22] The method according to any one of

[18] to

[21] , wherein the organic solvent is at least one selected from the group consisting of hexane, butanol, and 2-ethyl-1-hexanol;

[23] The method according to any one of

[12] to

[22] , wherein the diamine is produced by fermentation of at least one of sugar, carbon dioxide, synthetic gas, methanol, and amino acids by the recombinant microorganism;

[24] The method according to any one of

[17] to

[23] , further comprising reusing the separated aqueous phase containing the inorganic salt as a culture solution;

[25] The method according to any one of

[12] to

[24] , further comprising a purification step of purifying the diamine obtained.

[0021] Furthermore, the present inventors have conducted intensive research to solve the above-mentioned further problems. Specifically, the present inventors have discovered the use of a halophilic and / or alkaliphilic recombinant microorganism in order to simplify the diamine production process and reduce wastewater treatment costs as described above, and by using a halophilic and / or alkaliphilic recombinant microorganism as a host microorganism, it has become possible to simplify the pH control step during the production of 1,5-pentamethylenediamine and to reuse, as a medium, the highly salt-containing water generated in the release step of 1,5-pentamethylenediamine. However, it has been found that, in the production process of 1,5-pentamethylenediamine by the halophilic and / or alkaliphilic recombinant microorganism, N-acetylcadaverine, in which the amino group at the N-terminus of 1,5-pentamethylenediamine is acetylated, is by-produced.

[0022] The N-acetylation of 1,5-pentamethylenediamine is catalyzed by an acetyltransferase present in microbial cells. In Corynebacterium, an N-acetyltransferase of 1,5-pentamethylenediamine has been identified, and the N-acetylation of 1,5-pentamethylenediamine has been inhibited by disrupting the gene encoding the enzyme (Patent Document 9 and Japanese Patent No. 5960604).

[0023] However, a homology search was performed on the genome information of recombinant halophilic and / or alkaliphilic microorganisms, revealing that there was no homolog of the gene. Therefore, it was predicted that an acetyltransferase phylogenetically different from that of Corynebacterium truncatum catalyzes the acetylation of 1,5-pentamethylenediamine in recombinant halophilic and / or alkaliphilic microorganisms.

[0024] Therefore, in order to suppress the acetylation of diamine in the production of 1,5-pentamethylenediamine by halophilic and / or alkaliphilic recombinant microorganisms, it was first necessary to identify the enzyme that catalyzes the acetylation of 1,5-pentamethylenediamine.

[0025] As a result of the above-mentioned intensive investigations, the present inventors succeeded in identifying a gene encoding an enzyme that catalyzes the acetylation of 1,5-pentamethylenediamine from the genome sequence of a halophilic and / or alkalophilic host microorganism, and modified the gene to suppress the production of the by-product N-acetyldiamine in the halophilic and / or alkalophilic host microorganism.

[0026] That is, the present invention further provides the following:

[26] A recombinant halophilic and / or alkaliphilic microorganism capable of producing diamines, a recombinant microorganism comprising one or more genetic modifications that inhibit an N-acetyltransferase that N-acetylates a diamine compound to produce an N-acetyldiamine compound;

[27] The genetic modification is a modification that inhibits expression of an endogenous gene encoding the N-acetyltransferase, or the recombinant microorganism according to

[26] , which is modified to reduce the activity of the N-acetyltransferase;

[28] The recombinant microorganism according to

[26] or

[27] , wherein the ability to produce an N-acetyldiamine compound is suppressed or eliminated as compared with the ability of a non-mutant strain not containing the genetic modification;

[29] The recombinant microorganism according to any one of

[26] to

[28] , wherein the gene encoding the N-acetyltransferase is the yjbC gene;

[30] The N-acetyltransferase (A) (A-1) consists of the amino acid sequence shown in SEQ ID NO: 23, or (A-2) consisting of an amino acid sequence having 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and having an enzyme activity for N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (A-3) 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 shown in SEQ ID NO: 23, and having an enzyme activity for N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (B) (B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24; (B-2) a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO:24 and encodes a protein having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound; (B-3) A base sequence having a sequence identity of 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 24, and DNA encoding a protein having an enzyme activity for N-acetylating a diamine compound to produce an N-acetyldiamine compound; (B-4) A DNA encoding a protein having an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids have been deleted, substituted, inserted, and / or added to the amino acid sequence of a protein encoded by the base sequence shown in SEQ ID NO:24, and which encodes a protein having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (B-5) DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 24 A recombinant microorganism according to any one of

[26] to

[29] , wherein the recombinant microorganism is encoded by

[31] The recombinant microorganism according to any one of

[26] to

[30] , wherein one or more genetic modifications are performed to suppress the N-acetyltransferase in a halophilic and / or alkaliphilic host microorganism.

[32] The recombinant microorganism according to any one of

[26] to

[31] , wherein the host microorganism is selected from the group consisting of Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis;

[33] The diamine compound is represented by the formula: NH 2 CH 2 (CH 2 ) n CH 2 NH 2 (wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10);

[34] The recombinant microorganism according to any one of

[26] to

[33] , wherein the diamine compound is cadaverine;

[35] A method for producing a diamine compound, comprising a culturing step of culturing the recombinant microorganism according to any one of

[26] to

[34] to obtain a culture and / or an extract of the culture of the recombinant microorganism;

[36] The method according to

[35] , further comprising a mixing step of mixing the culture and / or an extract of the culture with a substrate compound to obtain a mixed solution;

[37] The method according to

[35] or

[36] , further comprising a recovery step of recovering a diamine compound from the culture or the mixed liquid. Effect of the Invention

[0027] According to the present invention, in the production of diamine, it is possible to realize at least one of simplification of the process and reduction in wastewater treatment costs.

[0028] Furthermore, according to the present invention, in the production of diamine using a halophilic and / or alkaliphilic diamine-producing bacterium, the production of the by-product N-acetyldiamine can be suppressed. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 is a diagram showing an example of a hexamethylenediamine production pathway. [Figure 2A] FIG. 2A shows the amino acid sequence of each enzyme. [Figure 2B] FIG. 2B shows the amino acid sequence of each enzyme. [Diagram 3] FIG. 3 is a diagram showing the amino acid sequence of the yjbC enzyme of Bacillus psuedofirmus (SEQ ID NO: 23). [Figure 4] FIG. 4 is a diagram showing the nucleotide sequence of the Bacillus psuedofirmus yjbC gene (SEQ ID NO: 24). [Figure 5A] FIG. 5A shows the nucleotide sequences of the primers. [Figure 5B] FIG. 5B is a diagram showing the base sequences of the primers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The following describes in detail the embodiments for carrying out the present invention. The present invention is not limited to the following embodiments, and can be carried out in various modifications within the scope of the gist of the present invention. Furthermore, unless otherwise specified, the acquisition of DNA described in this specification, the preparation of vectors, and the genetic manipulations such as transformation 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 Genetic Engineering Experiment Notes (Yodosha, Takaaki Tamura). Unless otherwise specified in this specification, nucleotide sequences are described in the 5' to 3' direction. In this specification, the terms "polypeptide" and "protein" are used interchangeably. Furthermore, a "genetically modified microorganism" is also simply referred to as a "recombinant microorganism".

[0031] In this specification, a numerical range indicated using "~" indicates a range including the numerical values ​​before and after "~" as the minimum and maximum values, respectively. In the numerical ranges described in stages in this specification, the upper limit or lower limit of a numerical range of a certain stage can be arbitrarily combined with the upper limit or lower limit of a numerical range of another stage.

[0032] As used herein, the terms "endogenous" or "endogenous" are used to mean that a host microbial organism that has not been genetically modified possesses a referenced gene or the protein (typically an enzyme) encoded thereby, regardless of whether the host microbial organism functionally expresses the gene or the protein (typically an enzyme) encoded thereby to a degree sufficient to facilitate a dominant biochemical reaction within the host cell. The terms "endogenous" and "endogenous" are used interchangeably herein.

[0033] As used herein, the terms "foreign" or "exogenous" are used to mean the introduction of a gene or nucleic acid sequence into a host when the host microorganism does not have the gene to be introduced prior to genetic modification, does not substantially express the enzyme from that gene, and has the amino acid sequence of the enzyme encoded by a different gene but does not express the comparable endogenous enzyme activity after genetic modification. The terms "foreign" and "exogenous" are used interchangeably herein.

[0034] As used herein, the term "having diamine-producing ability" in relation to a microorganism refers to a microorganism that produces a diamine at any stage of the diamine production process using the microorganism. Specifically, the diamine may be contained in a culture solution obtained by culturing the microorganism, or a diamine may be produced by adding a diamine precursor, such as a carboxylic acid compound, an aldehyde compound, and / or a carbonyl compound, to a medium and culturing a microorganism that converts the diamine precursor to a diamine. "Microorganisms having diamine-producing ability" include microorganisms having one or more of these properties.

[0035] Furthermore, with respect to a recombinant microorganism, "having the ability to produce diamines" means that the microorganism has a production pathway for diamines. As used herein, with respect to a compound, when a microorganism "has a production pathway" means that the microorganism expresses sufficient amounts of enzymes for each reaction step in the production pathway for the compound to proceed, and is capable of biosynthesizing the compound. The recombinant microorganism of the present invention may be one that uses a host microorganism that is inherently capable of producing diamines, or may be one that has been modified to have diamine production ability from a halophilic and / or alkalophilic host microorganism that does not inherently have the ability to produce diamines.

[0036] In the context of the present invention, the diamine compound has the formula: NH 2 CH 2 (CH 2 ) n CH 2 NH 2In the formula, n is, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 2, 3, 4 or 5, more preferably 2, 3 or 4, and particularly preferably 3 or 4.

[0037] Examples of diamine compounds include ethylenediamine, propylenediamine, tetramethylenediamine (e.g., putrescine), pentamethylenediamine (e.g., cadaverine), hexamethylenediamine, and heptamethylenediamine. In a preferred embodiment, the diamine compound is hexamethylenediamine or cadaverine.

[0038] <1> Invention A: Recombinant microorganism capable of producing diamine and method for producing diamine The recombinant microorganism of the present invention is a microorganism having a diamine producing ability, and specifically, a host microorganism having at least one of halophilicity and alkalophilicity has been modified so as to have a diamine producing ability.

[0039] In nature, there exist microorganisms that have alkaliphilic properties that allow them to grow in an alkaline environment with a pH of 9 or higher, halophilic properties that allow them to grow in an environment with a NaCl concentration of 0.2M or higher, or a combination of both properties. The present inventors considered that if the above alkaliphilic microorganisms could be used for the production of diamines, they would be able to grow even if the pH rises due to an increase in the product concentration in the enzymatic conversion step or the culture step, and therefore pH adjustment by adding an acid solution could be omitted. In addition, if the above halophilic microorganisms could be used for the production of diamines, they considered that the high-concentration salt-containing water generated in the release step could be reused as a medium. Note that there have been no reports of the application of microorganisms having at least one of the properties of halophilicity and alkaliphilicity to the production of diamines.

[0040] The present inventors attempted to use a microorganism having at least one of the properties of halophilicity and alkalophilicity as a host microorganism and modify it to have the ability to produce diamine, and found that the obtained recombinant microorganism makes it possible to at least one of simplifying the process and reducing wastewater treatment costs.

[0041] In other words, the inventors have succeeded in imparting diamine production ability to halophilic and / or alkaliphilic microorganisms, and have discovered that by using such microorganisms, it is possible to grow the microorganisms without adjusting the pH, for example by adding an acid solution, even if the pH increases due to an increase in product concentration in the enzymatic conversion process or culture process, and further that, in a preferred embodiment, high-salt-containing water generated during the isolation of diamine can be reused as a culture medium.

[0042] In producing a recombinant microorganism according to an embodiment of the present invention, for example, a technique for introducing a foreign gene into the cells of a host microorganism having at least one of the properties of halophilicity and alkalophilicity, a technique for introducing an arbitrary foreign gene sequence into a genomic sequence, or a technique for removing an unnecessary gene sequence from a genomic sequence can be used.

[0043] That is, the following technologies are included: (1) A technique for transforming a host microorganism having halophilic, alkalophilic, or both properties by combining a promoter capable of expression in the host cell with a gene involved in diamine production, (2) The above-mentioned technique (1), characterized in that the previous technique uses a plasmid vector that is stably replicated in the cells of a microorganism having halophilic, alkalophilic, or both properties; (2') The above-mentioned technique (2), in which the plasmid vector to be used in the previous technique is pUB110; (3) The above-mentioned technique (1), characterized in that the previous technique stably expresses a desired trait in the cells of a microorganism having halophilic, alkalophilic, or both properties; (4) A technique for arbitrarily modifying gene sequences on chromosomes by combining temperature-sensitive plasmid vectors and negative selection. (5) The technology of (4) above, characterized in that the technology stops replication of a plasmid carried by a microorganism or reduces the copy number of the plasmid carried by the microorganism at a temperature of 37°C or higher; (6) The above-mentioned (4) technology, characterized in that the growth of microorganisms carrying a specific gene is inhibited in the presence of substances such as sucrose and 4-chlorophenylalanine, thereby selecting microorganisms not carrying a specific gene (negative selection); (6') The technique of (6) above, wherein the specific gene targeted by the previous technique is the levansucrase gene (sacB gene); (6") The technology described above in (6) in which the specific gene targeted by the previous technology is a phenylalanine tRNA synthetase alpha subunit gene (pheS gene) derived from a host into which a mutation has been introduced into the base sequence.

[0044] In one preferred embodiment of the present invention, a high expression promoter and a foreign gene are carried in a plasmid vector and introduced into the cells of a microorganism having halophilic, alkalophilic, or both properties, thereby stably and highly expressing the foreign gene. In another preferred embodiment, a technique combining a temperature-sensitive plasmid vector and negative selection is used to arbitrarily edit the genome sequence carried by a microorganism having halophilic, alkalophilic, or both properties. The cell line into which the gene is introduced or the genome sequence is edited using this technique may be a wild-type strain in the usual sense, or may be an auxotrophic mutant strain or an antibiotic-resistant mutant strain derived from the wild-type strain. Furthermore, the cell line that can be used as the host cell of the present invention may already be transformed to have various marker genes related to the above-mentioned mutations. These techniques can provide properties that are useful for the production, maintenance, and / or management of the recombinant microorganism of the present invention.

[0045] In the present invention, alkalophilic microorganisms that can be used as host microorganisms are a type of extremophilic microorganisms that are diverse in distribution, and are a general term for microorganisms that can grow in environments with a pH of 9 or higher. These are classified into obligate alkalophilic microorganisms that can grow only in environments with a pH of 9 or higher, and facultative alkalophilic microorganisms that have an optimum growth pH of 9 or higher but can also grow at a pH of less than 9. Some of these can also grow in strongly alkaline environments with a pH of 12 or higher. All of these are alkalophilic microorganisms of the present invention.

[0046] In the present invention, halophilic microorganisms that can be used as host microorganisms are a general term for microorganisms that can cope with high salt stress. These are classified into non-halophilic bacteria, whose optimum growth salt concentration is 0 to 0.2 M sodium chloride, mild halophilic bacteria, whose optimum growth salt concentration is 0.2 to 0.5 M sodium chloride, moderate halophilic bacteria, whose optimum growth salt concentration is 0.5 to 2.5 M sodium chloride, and severe halophilic bacteria, whose optimum growth salt concentration is 2.5 to 5.2 M sodium chloride. All of these except non-halophilic bacteria are halophilic microorganisms of the present invention.

[0047] Microorganisms having halophilic, alkalophilic, or both properties that can be used as the host microorganism of the present invention include various microorganisms, non-limiting examples of which include bacteria of the genera Bacillus, Halomonas, Halobacteroides, Salinibacter, Alkaliferus, Clostridium, and Anaerobranca. The host microorganism of the present invention is preferably a bacterium of the genus Bacillus. Among the microorganisms of the genus Bacillus, Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis are preferred, and Bacillus psuedofirmus is more preferred.

[0048] In a preferred embodiment of the present invention, a host microorganism is modified to construct a new diamine biosynthetic pathway. For example, when the diamine is 1,5-pentamethylenediamine, a recombinant microorganism is obtained by introducing one or more L-lysine decarboxylase (EC 4.1.1.18) enzyme genes into the cells of the host microorganism to construct a new 1,5-pentamethylenediamine biosynthetic pathway. Here, L-lysine decarboxylase (EC 4.1.1.18) is an enzyme that catalyzes the reaction of decarboxylating L-lysine to produce 1,5-pentanediamine. Alternatively, a recombinant microorganism is obtained by inserting the enzyme gene sequence into the genome sequence of the host microorganism.

[0049] By such manipulation, overproduction of L-lysine decarboxylase (EC 4.1.1.18) is induced in the obtained recombinant microorganism. That is, the recombinant microorganism according to the present invention is modified by one or more genetic manipulations so as to induce overproduction of L-lysine decarboxylase (EC 4.1.1.18).

[0050] When the diamine is 1,5-pentamethylenediamine, the genetic manipulation may be, for example, one or more genetic manipulations selected from the group consisting of the following (A), (B), (C), (D) and (E). (A) introducing an exogenous gene encoding L-lysine decarboxylase into a host microorganism; (B) increasing the copy number of the endogenous L-lysine decarboxylase gene in the host microorganism; (C) introducing a mutation into the expression regulatory region of an endogenous L-lysine decarboxylase gene in a host microorganism; (D) replacing the expression regulatory region of the endogenous gene for L-lysine decarboxylase in the host microorganism with an exogenous regulatory region capable of high expression; and (E) Engineering a deletion of the regulatory region of the endogenous gene for L-lysine decarboxylase in a host microorganism.

[0051] Representative genes for L-lysine decarboxylase (EC 4.1.1.18) include cadA and ldcC of Escherichia coli. The amino acid sequence of the E. coli cadA enzyme is shown in SEQ ID NO: 1, and the base sequence of the E. coli cadA is shown in SEQ ID NO: 2. The amino acid sequence of the E. coli ldcC enzyme is shown in SEQ ID NO: 3, and the base sequence of the E. coli ldcC is shown in SEQ ID NO: 4.

[0052] [ka] TIFF0007672486000002.tif239168TIFF0007672486000003.tif47163

[0053] In a preferred embodiment of the present invention, a host microorganism is modified to construct a new diamine biosynthetic pathway. For example, when the diamine is hexamethylenediamine, a recombinant microorganism is obtained by introducing one or more enzyme genes into the cells of the host microorganism to construct a new hexamethylenediamine biosynthetic pathway. Alternatively, a recombinant microorganism is obtained by inserting the enzyme gene sequence into the genome sequence of the host microorganism. An example of a hexamethylenediamine production pathway that a microorganism may have is shown in Figure 1.

[0054] Examples of enzymes that catalyze each reaction step are given below.

[0055] In the conversion in step A of FIG. 1 (succinyl-CoA:acetyl-CoA acyltransferase, or 3-oxoadipyl-CoA thiolase), succinyl-CoA and acetyl-CoA are condensed to form 3-oxoadipyl-CoA. An example of an enzyme that can catalyze this conversion is β-ketothiolase. For example, enzymes classified into groups 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 can have activity in this conversion. The enzyme used in the present invention is not limited as long as it has activity in this conversion, and for example, PaaJ derived from Escherichia coli consisting of the amino acid sequence set forth in SEQ ID NO: 12 is used (FIG. 2).

[0056] In the conversion of step B in FIG. 1 (3-hydroxyadipyl-CoA dehydrogenase), 3-oxoadipyl-CoA is converted to 3-hydroxyadipyl-CoA. Examples of enzymes that can catalyze this conversion include oxidoreductases classified in the EC 1.1.1 group. For example, enzymes classified in groups 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 can have activity in this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion. For example, PaaH derived from Escherichia coli and consisting of the amino acid sequence set forth in SEQ ID NO: 13 is used (FIG. 2).

[0057] In the conversion in step C of FIG. 1 (3-hydroxyadipyl-CoA dehydratase), 3-hydroxyadipyl-CoA is converted to 2,3-dehydroadipyl-CoA. Examples of enzymes that can catalyze this conversion include hydrolyases classified into the EC 4.2.1 group. For example, enzymes classified into 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 can have activity in this conversion. The enzyme used in the present invention is not limited as long as it has activity in this conversion, and for example, PaaF derived from E. coli having the amino acid sequence set forth in SEQ ID NO: 14 is used (FIG. 2).

[0058] In the conversion of step D in FIG. 1 (2,3-dehydroadipyl-CoA reductase), 2,3-dehydroadipyl-CoA is converted to adipyl-CoA. Examples of enzymes that can catalyze this conversion include oxidoreductases classified in the EC 1.3.1 group. 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 + )), 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 EC 1.3.1.104 (enoyl-ACP reductase (NADPH)) can be exemplified as enzymes that may have activity in this conversion.

[0059] The 2,3-dehydroadipyl-CoA reductase used in the present invention may be, for example, an enzyme derived from any of the organisms selected from Candida auris, Kluyveromyces marxianus, Pichia kudriavzevii, Thermothelomyces thermophilus, Thermothielavioides terrestris, Chaetomium thermophilum, Podospora anserina, Purpureocillium lilacinum, and Pyrenophora teres. Preferably, an enzyme derived from Thermothelomyces thermophilus having the amino acid sequence set forth in SEQ ID NO: 15, an enzyme derived from Chaetomium thermophilum having the amino acid sequence set forth in SEQ ID NO: 16, and an enzyme derived from Candida tropicalis having the amino acid sequence set forth in SEQ ID NO: 17 are used (FIG. 2).

[0060] In the conversion of step E in FIG. 1, adipyl-CoA is converted to adipic acid. An example of an enzyme that can catalyze this conversion is a thioester hydratase classified in the EC 3.1.2 group. For example, enzymes classified in the EC 3.1.2.1 (acetyl-CoA hydratase) and EC 3.1.2.20 (acyl-CoA hydratase) groups can be exemplified as enzymes that can have activity in this conversion.

[0061] Another example of an enzyme that can catalyze the conversion in step E of Figure 1 is CoA-transferase classified into the EC 2.8.3 group. For example, enzymes classified into groups such as EC 2.8.3.5 (3-oxoacid CoA-transferase), EC 2.8.3.6 (3-oxoadipate CoA-transferase), and EC 2.8.3.18 (succinyl-CoA:acetate CoA-transferase) can be exemplified as enzymes that can have activity in this conversion.

[0062] Furthermore, as an example of another enzyme conversion that can catalyze the conversion in step E in Fig. 1, a pathway in which the adipyl group of adipyl-CoA is transferred to phosphate by an acyltransferase classified in the EC 2.3.1 group to generate adipyl phosphate, followed by dephosphorylation by a phosphotransferase classified in the EC 2.7.2 group, can be exemplified. For example, enzymes classified in groups such as EC 2.3.1.8 (phosphate acetyltransferase) and EC 2.3.1.19 (phosphate butyryltransferase) as acyltransferases, and enzymes classified in groups such as EC 2.7.2.1 (acetate kinase) and EC 2.7.2.7 (butanoate kinase) as phosphotransferases can be exemplified as enzymes that can have activity in this conversion.

[0063] In step F of FIG. 1, adipyl-CoA is converted to adipic semialdehyde. Examples of enzymes that can catalyze this conversion include those classified in the EC 1.2.1 group. For example, enzymes classified into groups such as EC 1.2.1.10 (acetaldehyde dehydrogenase (acetylation)), EC 1.2.1.17 (glyoxylate dehydrogenase (acylation)), EC 1.2.1.42 (hexadecanal dehydrogenase (acylation)), EC 1.2.1.44 (cinnamoyl-CoA reductase (acylation)), EC 1.2.1.75 (malonyl-CoA reductase (formation of malonic semialdehyde)), and EC 1.2.1.76 (succinic semialdehyde dehydrogenase (acylation)) can be exemplified as enzymes that may have activity in this conversion because they catalyze a conversion reaction in which CoA is eliminated to produce 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 and consisting of the amino acid sequence set forth in SEQ ID NO: 18 is used (FIG. 2).

[0064] In the conversions of steps G, I, K, and N in FIG. 1, a carboxyl group is converted to an aldehyde. An example of an enzyme that can catalyze this conversion is carboxyl acid reductase (CAR). For example, EC 1.2.1.30 (carboxyl acid reductase (NADP +)), EC 1.2.1.31 (L-aminoadipate semialdehyde dehydrogenase), EC 1.2.1.95 (L-2-aminoadipate reductase), and EC 1.2.99.6 (carboxylic acid reductase) can be exemplified as enzymes that may have activity in this conversion because they catalyze a conversion reaction that produces an aldehyde from a carboxylic acid, similar to this conversion. Typical examples of 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 for this conversion, but for example, at least one of the enzyme MaCar derived from Mycobacterium abscessus and consisting of the amino acid sequence set forth in SEQ ID NO: 19, and MaCar(m), a variant of MaCar consisting of the amino acid sequence set forth in SEQ ID NO: 20, is used, and more preferably MaCar(m) consisting of the amino acid sequence set forth in SEQ ID NO: 20 is used (Figure 2).

[0065] Furthermore, carboxylic acid reductase can be converted to an active holoenzyme by phosphopantetheinylation (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007)). Phosphopantetheinylation is catalyzed by phosphopantetheinyl transferase (PT). Enzymes capable of catalyzing this reaction include, for example, enzymes classified in 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. Methods for increasing the activity of phosphopantetheinyl transferase include, but are not limited to, a method of introducing an exogenous phosphopantetheinyl transferase gene and a method of enhancing the expression of an endogenous phosphopantetheinyl transferase gene. The enzymes used in the present invention are not limited as long as they have phosphopantetheinyl group transfer activity, but typical examples include EntD from Escherichia coli, Sfp from Bacillus subtilis, Npt from Nocardia iowensis (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007)), and Lys5 from 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, but for example, Npt from Nocardia iowensis consisting of the amino acid sequence set forth in SEQ ID NO: 21 is used (FIG. 2).

[0066] The conversions in steps J, M, P, and R in FIG. 1 are transamination reactions. Examples of enzymes that can catalyze this conversion include transaminases (aminotransferases) classified in the EC 2.6.1 group. For example, enzymes classified into groups such as EC 2.6.1.19 (4-aminobutanoate-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 can have activity in this conversion. The enzymes used in the present invention are not particularly limited as long as they have the conversion activity of each step. For example, YgjG, a putrescine aminotransferase from Escherichia coli that has been reported to transaminate cadaverine and spermidine (Samsonova, et al., BMC microbiology 3.1 (2003): 2.), SpuC, a putrescine aminotransferase from the genus Pseudomonas (Lu et al., Journal of bacteriology 184.14 (2002): 3765-3773.; Galman et al., Green Chemistry 19.2 (2017): 361-366.), GabT, a GABA aminotransferase from Escherichia coli, and PuuE may be used. Furthermore, ω-transaminases derived from species such as Ruegeria pomeroyi, Chromobacterium violaceum, Arthrobacter citreus, Sphaerobacter thermophilus, Aspergillus fischeri, Vibrio fluvialis, Agrobacterium tumefaciens, and Mesorhizobium loti have been reported to have transamination activity toward diamine compounds such as 1,8-diaminooctane and 1,10-diaminodecane, 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 enzyme used in the present invention is not limited as long as it has activity for this conversion, and for example, the enzyme YgjG derived from Escherichia coli having the amino acid sequence set forth in SEQ ID NO: 22 may be used (FIG. 2). Typical amino group donors include, but are not limited to, L-glutamic acid, L-alanine, and glycine.

[0067] The genes encoding the above-mentioned enzymes that can be used in the present invention may be derived from microorganisms other than those exemplified, or may be artificially synthesized, as long as they are capable of expressing substantial enzyme activity within the host microbial cells.

[0068] Furthermore, the enzyme genes that can be used for the purposes of the present invention may have all mutations that can occur in nature, as well as mutations and modifications that have been artificially introduced, so long as they can express substantial enzyme activity in the host microbial cells. For example, it is known that there are extra codons for various codons that code for specific amino acids. Therefore, alternative codons that will ultimately be translated into the same amino acid may also be used in the present invention. In other words, since the genetic code is degenerate, multiple codons can be used to code for a specific amino acid, and therefore the amino acid sequence can be coded by any set of similar DNA oligonucleotides. Only one member of the set is identical to the gene sequence of the native enzyme, but even a mismatched DNA oligonucleotide can hybridize to the native sequence under appropriate stringent conditions (e.g., hybridize at 3xSSC, 68°C, wash at 2xSSC, 0.1% SDS, and 68°C), and the DNA encoding the native sequence can be identified and isolated, and such genes can also be used in the present invention. In particular, since most organisms are known to preferentially use a specific subset of codons (optimal codons) (Gene, Vol. 105, pp. 61-72, 1991, etc.), performing "codon optimization" depending on the host microorganism may also be useful in the present invention.

[0069] When the diamine is 1,5-pentamethylenediamine, in a preferred embodiment, the recombinant microorganism comprises a nucleotide sequence having 85, 90, 92, 95, 98 or 99% or more homology, preferably 92, 95, 98 or 99% or more homology, to the nucleotide sequence shown in SEQ ID NO: 2 or 4, or the recombinant microorganism comprises a nucleotide sequence having 85, 90, 92, 95, 98 or 99% or more homology, preferably 92, 95, 98 or 99% or more homology, to a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 3.

[0070] For example, a stable and high level of enzyme activity can be obtained by introducing the above-mentioned diamine synthase gene into a host microbial cell as an "expression cassette". Preferably, a more stable and high level of enzyme activity can be obtained by inserting the gene sequence of the "expression cassette" into the genome sequence of the host microbial cell.

[0071] Here, in the present specification, the term "expression cassette" refers to a nucleotide sequence containing a nucleic acid sequence that regulates transcription and translation functionally linked to a nucleic acid to be expressed or a gene to be expressed. Typically, the expression cassette of the present invention contains a promoter sequence 5' upstream from the coding sequence, a terminator sequence 3' downstream, and optionally further conventional regulatory elements in a functionally linked state, and in such a case, the nucleic acid to be expressed or the gene to be expressed is introduced into a host microorganism.

[0072] A promoter is defined as a DNA sequence that allows RNA polymerase to bind to DNA and initiate RNA synthesis, regardless of whether it is a constitutive or inducible promoter. A strong promoter is a promoter that initiates mRNA synthesis at a high frequency, and is preferably used in the present invention. For example, in Bacillus pseudophyllum, promoter regions for S-Layer protein synthesis enzymes, sigma factors (e.g., rpoD, etc.), glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase), lactate dehydrogenase, and glutamic acid decarboxylase A can be used.

[0073] The expression cassette described above is incorporated into a vector consisting of, for example, a plasmid, a phage, a transposon, an IS element, a phasmid, a cosmid, or linear or circular DNA, and then introduced into a host microorganism. In the present invention, plasmids and phages are preferred. These vectors may be autonomously replicating in the host microorganism, or may be inserted into a chromosome and replicated. Suitable plasmids include, for example, pUB110, pC194, and pBD214 for bacilli such as the genus Bacillus.

[0074] The expression cassette described above is preferably inserted into a chromosome, compared to a plasmid or a phage. In order to maintain the plasmid in the host microorganism, some selective pressure is required, and generally, an antibiotic corresponding to the antibiotic resistance marker carried by the plasmid must be added to the medium. Even if selective pressure is present, if the gene expressed on the plasmid is unnecessary for the host microorganism or is a burden on the growth of the host microorganism, there is a possibility that the gene may be mutated or deleted by the action of an enzyme endogenously held by the host microorganism, which often makes stable production of the substance difficult.

[0075] Usable plasmids and the like include those described above, as well as those described in "Cloning Vectors", Elsevier, 1985. An expression cassette can be introduced into a vector by conventional methods including fragment amplification by PCR, excision with an appropriate restriction enzyme, cloning, and various ligations.

[0076] After the vector having the expression cassette of the present invention is constructed as described above, the vector can be introduced into a host microorganism using conventional cloning and transfection methods such as conjugative transfer, coprecipitation, protoplast fusion, electroporation, retroviral transfection, etc. Examples of such methods are described in "Current Protocols in Molecular Biology", F. Ausubel et al., Publ. Wiley Interscience, New York, 1997, or Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0077] Furthermore, by including a temperature-sensitive replication origin sequence, a negative selection gene, and homologous sequences on both sides of the region to be edited in the genome sequence of the host microorganism in the sequence of the plasmid vector, a foreign gene can be introduced into the genome sequence of the host microorganism or an unnecessary gene can be removed. Preferably, the temperature-sensitive replication origin sequence is the replication origin used in the pE194ts vector, and the negative selection gene is the sacB gene or a mutant pheS gene.

[0078] In the process of culturing the host microorganism into which the above-described vector has been introduced, edited forms in which the genome sequence has been modified by changing the temperature and medium composition can be selected. The conditions for selecting edited forms are preferably a culture temperature of 37 to 43° C. or higher, a medium containing 10% or more sucrose, or a medium containing 1 mM or more 4-chlorophenylalanine.

[0079] In a preferred embodiment, when the diamine to be produced is 1,5-pentamethylenediamine, the host microorganism is further modified by a mutation or genetic recombination operation to improve the lysine production ability, i.e., the resulting recombinant microorganism further includes a mutation or genetic recombination operation to improve the lysine production ability. The mutation or genetic recombination operation is, for example, a procedure to release feedback inhibition against at least one of aspartokinase III (EC 2.7.2.4) and 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7).

[0080] Aspartokinase III is an enzyme that catalyzes the reaction of converting aspartic acid and adenosine triphosphate (ATP) into 4-phosphoaspartic acid and adenosine diphosphate. It is generally recognized that this enzyme is feedback inhibited by L-lysine. In a mutant enzyme modified to be free from feedback inhibition, the protein structure is changed so that L-lysine does not bind, and the enzyme activity is maintained even in the presence of lysine, so that microorganisms expressing this enzyme are known to produce a high amount of lysine. Hereinafter, as an example of an effective mutant, aspartokinase III (lysC) derived from Eschericia coli will be described, but the gene used in the present invention is not limited to this. Examples of mutant lysC that is not feedback inhibited by L-lysine include, but are not limited to, those in which the 352nd threonine residue in the amino acid sequence is replaced with an isoleucine residue, and those in which the 253rd threonine residue is replaced with an arginine residue.

[0081] 4-Hydroxy-tetrahydrodipicolinate synthase is an enzyme that catalyzes the reaction of converting pyruvate and aspartic acid semialdehyde to (2S,4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinate and water. It is generally recognized that this enzyme is feedback inhibited by L-lysine. In a mutant enzyme modified to be free from feedback inhibition, the protein structure is changed so that L-lysine does not bind, and the enzyme activity is maintained even in the presence of lysine, so that microorganisms expressing this enzyme are known to produce a high amount of lysine. Below, 4-hydroxy-tetrahydrodipicolinate synthase (dapA) derived from Eschericia coli will be described as an example of an effective mutant, but the gene used in the present invention is not limited to this. Mutant dapA that is not subject to feedback inhibition by L-lysine includes, but is not limited to, those in which the 81st alanine residue in the amino acid sequence is replaced with a valine residue, those in which the 84th glutamic acid residue is replaced with a threonine residue, and those in which the 118th histidine residue is replaced with an arginine residue or a tyrosine residue.

[0082] The transformant or genome edited product obtained as described above is cultured and maintained under conditions suitable for the growth and / or maintenance of the transformant for the production of diamine. For example, when 1,5-pentanediamine is produced, a transformant transformed with a vector having an expression cassette of an exogenous L-lysine decarboxylase gene (each expression cassette may be placed on a separate or same vector), or a genome edited product in which an expression cassette of an exogenous L-lysine decarboxylase gene is integrated into the genome sequence of a host microorganism, is cultured and maintained under conditions suitable for the growth and / or maintenance of the transformant for the production of 1,5-pentanediamine. Suitable medium compositions, culture conditions, and culture times for transformants derived from various host microbial cells can be easily determined by those skilled in the art.

[0083] The medium may be a natural, semi-synthetic, or synthetic medium containing one or more carbon sources, nitrogen sources, inorganic salts, vitamins, and optionally trace elements, vitamins, etc. However, it goes without saying that the medium used must adequately meet the nutritional requirements of the transformant to be cultured.

[0084] Carbon sources include D-glucose, sucrose, lactose, fructose, maltose, oligosaccharides, polysaccharides, starch, cellulose, rice bran, blackstrap molasses, fats and oils (e.g., soybean oil, sunflower oil, peanut oil, coconut oil, etc.), fatty acids (e.g., palmitic acid, linoleic acid, linolenic acid, etc.), alcohols (e.g., glycerol, ethanol, etc.), and organic acids (e.g., acetic acid, lactic acid, succinic acid, etc.). It may also be biomass containing D-glucose. Suitable biomass includes corn decomposition liquid and cellulose decomposition liquid. Other carbon sources include sugars, carbon dioxide, synthetic gas, methanol, and amino acids. These carbon sources can be used individually or as a mixture.

[0085] When biomass-derived raw materials are used, the resulting diamine can be clearly distinguished from synthetic raw materials derived from, for example, petroleum, natural gas, or coal by measuring the biobased carbon content based on Carbon-14 (radioactive carbon) analysis specified in ISO 16620-2 or ASTM D6866.

[0086] Nitrogen sources include nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour, and urea), or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, sodium nitrate, ammonium nitrate, etc.) These nitrogen sources can be used individually or in mixtures.

[0087] In addition, if the transformant expresses a useful additional trait, for example, if it has an antibiotic resistance marker, the medium may contain the corresponding antibiotic, thereby reducing the risk of contamination by unwanted bacteria during fermentation. Examples of antibiotics include, but are not limited to, ampicillin, kanamycin, chloramphenicol, tetracycline, erythromycin, streptomycin, and spectinomycin.

[0088] When the host microorganism cannot assimilate the above carbon sources such as cellulose and polysaccharides, the host microorganism can be adapted to produce diamines using these carbon sources by applying known genetic engineering techniques such as introducing foreign genes into the host microorganism, such as cellulase genes and amylase genes.

[0089] The culture may be a batch type or a continuous type. In either case, the carbon source or the like may be additionally replenished at an appropriate time point during the culture. Furthermore, the culture should be continued while maintaining an appropriate temperature, oxygen concentration, pH, and the like. The appropriate culture temperature for a transformant derived from a general microbial host cell is usually in the range of 15°C to 50°C, preferably 25°C to 37°C. When the host microorganism is aerobic, shaking (flask culture, etc.) or stirring / aeration (jar fermenter culture, etc.) is required to ensure an appropriate oxygen concentration during fermentation. Those culture conditions can be easily set by those skilled in the art.

[0090] Another embodiment of the present invention relates to a method for producing a diamine using the recombinant microorganism described above. The method for producing a diamine includes, for example, the following steps:

[0091] (a) Culture process The method for producing diamine includes a culture step of culturing the recombinant microorganism according to the embodiment described above. The culture in this step produces a culture solution containing the bacterial cells. In this culture step, the recombinant microorganism may be cultured in a culture solution containing an inorganic salt. The inorganic salt is a hydrochloride, sulfate, phosphate, carbonate, hydrofluoride, etc. of a metal element, and examples thereof include sodium chloride, lithium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, and sodium carbonate, among which sodium sulfate and sodium carbonate are preferred. Although it is not intended to be limited by any theory, it is believed that when an inorganic salt is added to an aqueous solution, the strong hydration power of the inorganic salt fixes water molecules as hydration water, thereby reducing the amount of water molecules required for hydrating diamines, resulting in phase separation. The Hofmeister series is an index that indicates the strength of salting out of an inorganic salt, and a salt consisting of a combination of an anion and a cation (particularly a metal ion) that strongly induces salting out as shown in the Hofmeister series is preferred. Diamines are produced by culturing in a culture solution containing inorganic salts, but in the presence of high concentrations of inorganic salts, phase separation of the diamine aqueous solution occurs from the culture solution, and it is therefore believed that the inclusion of inorganic salts in the culture solution facilitates the separation of the diamine from the culture solution.

[0092] In this case, the recombinant composition is cultured in a culture medium containing, for example, 5% by mass or more of inorganic salts, preferably 10% by mass or more of inorganic salts. In another embodiment, the recombinant composition is cultured in a culture medium containing, for example, 5 to 20% by mass, preferably 10 to 20% by mass of inorganic salts.

[0093] The phase containing inorganic salts can be separated from the culture solution by a separation process described below and then reused as the culture solution. Wastewater containing high concentrations of salts is costly to treat, but reuse can reduce costs. It is also possible to reduce the load in the purification process, particularly the load of dehydration and concentration due to phase separation. For example, a prior art has been proposed in which a strong base is added to separate diamines. However, when a strong base is added, phase separation that occurs when inorganic salts are added does not occur, and the dehydration and concentration rate from the medium increases in diamine purification, and a solvent extraction process is also required. As described above, if inorganic salts are added to the culture solution, the load of dehydration and concentration can be reduced in the purification process, and an additional process for solvent extraction is not required.

[0094] (b) Reaction process This step is a step of contacting a precursor of a diamine with a recombinant microorganism, and producing the desired diamine from the diamine precursor. The contact with the diamine precursor may be carried out, for example, during or after the culturing step.

[0095] For example, if the diamine is 1,5-pentamethylenediamine, contacting the recombinant microorganism with lysine will cause the lysine decarboxylase produced by the recombinant microorganism to decarboxylate the lysine to produce 1,5-pentamethylenediamine.

[0096] In one embodiment, in this step, the culture solution and / or the bacterial cells obtained in the culturing step are contacted with an aqueous solution containing 5% by mass or more of an inorganic salt and lysine to obtain a reaction solution containing 1,5-pentanediamine. For example, in this step, the culture solution containing the bacterial cells obtained in the culturing step and / or the bacterial cells from which the supernatant has been removed by centrifugation or the like from the culture solution obtained in the culturing step are contacted with an aqueous solution containing an inorganic salt and lysine to obtain a reaction solution.

[0097] In another embodiment, the culturing step and the reaction step may be carried out in the same step. For example, when the diamine is 1,5-pentamethylenediamine, an aqueous solution containing an inorganic salt and lysine may be added to the culture solution in which the recombinant microorganism is cultured. In addition, for example, a bacterium that produces lysine by fermentation may be co-cultured with the recombinant microorganism according to the present invention. By co-culturing these, the lysine produced by the bacterium can be efficiently converted to 1,5-pentamethylenediamine by the lysine decarboxylase produced by the recombinant composition according to the present invention.

[0098] (Addition of inorganic salts) The inorganic salt may be present in the culture medium beforehand. That is, as explained in relation to the above-mentioned culture step, the recombinant microorganism according to the present invention may be cultured in a culture medium containing an inorganic salt. In this case, the recombinant composition is cultured in a culture medium containing, for example, 5% by mass or more of the inorganic salt, preferably 10% by mass or more of the inorganic salt. In another embodiment, the recombinant composition is cultured in a culture medium containing, for example, 5 to 20% by mass, preferably 10 to 20% by mass of the inorganic salt.

[0099] When an inorganic salt is added to the culture medium, it is added so that the concentration of the inorganic salt in the culture medium becomes 100 to 200 g / L, preferably 150 to 200 g / L, more preferably 160 to 200 g / L, and even more preferably 200 g / L.

[0100] Alternatively, when the diamine is 1,5-pentamethylenediamine, the inorganic salt may be contacted in the form of an aqueous solution together with lysine with the culture solution and / or the cells obtained in the culture step in the reaction step. In one embodiment, the inorganic salt may be added in both the culture step and the reaction step. In another embodiment, the inorganic salt may be added in one or more steps in the method for producing the diamine, as described below, in addition to the culture step and / or the reaction step.

[0101] The inorganic salt is sodium carbonate or sodium sulfate, more preferably sodium sulfate.

[0102] By adding an inorganic salt, in the separation step described below, a phase containing a diamine and an aqueous phase containing an inorganic salt can be separated, facilitating the separation of the diamine. In addition, when the recombinant microorganism according to the present invention is a microorganism having halophilic properties, the growth of the microorganism is not inhibited even when such a high concentration of inorganic salt is added, so that separation of the diamine-containing phase can be promoted without interfering with the production of the diamine, and the diamine can be easily isolated.

[0103] (c) Removal process The above production method may further include a removal step of removing the recombinant microorganism from the culture solution or reaction solution. This step is carried out, for example, by centrifugation and / or filtration after the diamine is produced in the culture step or reaction step. This step can remove solid matter such as bacteria contained in the culture solution or reaction solution. Furthermore, by using an ultrafiltration membrane during the filtration process, polymeric compounds having a molecular weight of any molecular weight or more, including polysaccharides and proteins, can be removed.

[0104] (d) Concentration process In this step, the culture solution or reaction solution is concentrated. The concentration step is carried out, for example, by concentrating the culture supernatant using an evaporator after removing the recombinant microorganism in the removal step. By concentrating the culture solution containing the diamine, the concentrations of the diamine and inorganic salt are increased, and it is expected that the separation efficiency can be further improved.

[0105] When the diamine is in the form of one or more selected from the group consisting of carbonate, bicarbonate, bisbicarbonate, carbamate, and biscarbamate produced in the culturing step and / or the reaction step, the diamine in the form of a salt is converted into a free base and carbon dioxide in the concentrating step, and carbon dioxide is separated. For example, when the diamine is 1,5-pentamethylenediamine, the 1,5-pentamethylenediamine in the form of one or more selected from the group consisting of carbonate, bicarbonate, bisbicarbonate, carbamate, and biscarbamate produced in the culturing step is converted into a free base and carbon dioxide in the concentrating step, and carbon dioxide is separated.

[0106] (e) pH adjustment process In the pH adjustment step, the pH of the culture solution or the reaction solution is adjusted to 12 or higher. For example, in this step, after concentrating the culture solution or the reaction solution in the concentration step, the pH of the concentrated culture solution or the reaction solution is adjusted to 12 or higher. Alternatively, it is confirmed that the pH is 12 or higher due to the increase in pH caused by the separation of carbon dioxide in the concentration step.

[0107] (f) Separation process In this step, a phase containing diamine is separated from the culture solution or reaction solution. The separation step does not include the addition of an alkaline compound. The alkaline compound here refers to a compound whose aqueous solution is basic and whose addition increases the pH value, particularly an inorganic alkaline compound. Examples of the alkaline compound include hydroxides of metal elements and inorganic substances that can accept hydrogen ions. Examples of the inorganic alkaline compound include potassium hydroxide, sodium hydroxide, calcium hydroxide, lithium hydroxide, ammonia, magnesium hydroxide, aluminum hydroxide, manganese hydroxide, iron hydroxide, cobalt hydroxide, copper hydroxide, zinc hydroxide, and barium hydroxide. By not adding an alkaline compound, costs can be reduced by not using an alkaline compound other than the medium components. In addition, the pH of the phase containing inorganic salts decreases after separating diamines caused by high pH, ​​making it possible to recycle the medium. When inorganic salts are contained in the culture solution or reaction solution, this separation step separates the phase containing diamines from the aqueous phase containing inorganic salts.

[0108] Phase separation may be performed by adding an organic solvent to the culture or reaction solution, or by contacting the culture or reaction solution with an organic solvent. The organic solvent is, for example, one or more selected from the group consisting of n-hexane, n-butanol, and 2-ethyl-1-hexanol. For example, when the diamine is 1,5-pentamethylenediamine, it is preferable to use n-butanol, and when the diamine is HMDA, it is preferable to use 2-ethyl-1-hexanol.

[0109] As described above, the phase separation of the diamines is promoted by the inorganic salts having high salt concentrations (e.g., sodium carbonate and sodium sulfate, etc.), but the phase separation may be promoted by adding an organic solvent. By adding an organic solvent, the extraction of the diamine from the aqueous phase to the organic phase is promoted, the migration rate to the organic phase is increased, and the yield can be further improved.

[0110] (g) Aqueous phase recovery and reuse process In this step, the phase containing inorganic salts (e.g., containing inorganic salts and water) is recovered and / or the recovered phase is reused as a culture medium. Since the recombinant microorganism of the present invention is halophilic, it is capable of producing diamines without inhibiting growth even in a culture medium containing inorganic salts. Furthermore, the inclusion of inorganic salts promotes phase separation, making it easier to separate diamines. Furthermore, although the treatment of water containing high concentrations of inorganic salts can be costly, the wastewater treatment costs can be reduced by reusing the water as a culture medium for microorganisms as described above.

[0111] (h) Purification process In this step, the diamine obtained from the culture is purified. Methods for purifying diamines, such as 1,5-pentanediamine, from cultures are known to those skilled in the art. In the case of a transformant or genome edited form of a prokaryotic microbial host cell, 1,5-pentanediamine is present in the culture supernatant or in the cells, but may be extracted from the cultured cells if necessary. When extracting from the cultured cells, for example, the culture is centrifuged to separate the supernatant from the cells, and the cells can be destroyed using a surfactant, an organic solvent, an enzyme, or the like while using a homogenizer. As a method for purifying the culture supernatant and, in some cases, the cell extract, there are deproteinization treatments using protein precipitation by pH adjustment or the like, removal of impurities by adsorption using activated carbon, removal of ionic substances by adsorption using ion exchange resins, etc., followed by extraction using a known solvent, distillation, or the like. Of course, it goes without saying that some steps may be deleted or additional purification steps such as chromatography may be performed depending on the purity desired for the product.

[0112] Yet another embodiment of the present invention also relates to a method for purifying a diamine from a culture obtained using the recombinant composition described above, which may comprise, alone or in combination, each of the steps described above for the method for producing the diamine.

[0113] In general, the growth of Escherichia coli and coryneform bacteria used in the conventional method was significantly inhibited in an environment with a pH of 9 or higher. For example, when the pH in the medium rises due to the conversion of lysine to 1,5-pentamethylenediamine, it was necessary to appropriately add an acid solution to control the pH to a range that does not inhibit the growth of the microorganism. However, since the recombinant microorganism according to the present invention has alkalophilicity that allows it to grow even in an alkaline environment, by using the microorganism for diamine production, it is not necessary to adjust the pH by adding an acid solution as in the conventional technology, and the complication of the diamine production process can be avoided. In addition, if the recombinant microorganism according to the present invention is halophilic, it can grow in a culture solution containing salt. Therefore, the wastewater containing salt added in the separation process can be reused for the culture of the microorganism, thereby reducing the cost of wastewater treatment. In addition, compared to the prior art in which a strong base is added to separate diamine, the addition of an inorganic salt to the culture solution reduces the load of dehydration and concentration in the purification process, and an additional process for solvent extraction is also unnecessary.

[0114] <2> Invention B: Recombinant halophilic and / or alkaliphilic microorganisms suppressing N-acetyldiamine production

[0115] The genetically modified microorganism of the present invention is a diamine-producing halophilic and / or alkalophilic host microorganism that has been subjected to one or more genetic modifications that suppress N-acetyltransferase.

[0116] In nature, there exist microorganisms that have alkaliphilic properties that allow them to grow in alkaline environments with a pH of 9 or higher, halophilic properties that allow them to grow in environments with a NaCl concentration of 0.2M or higher, or a combination of both properties. In the context of the present invention, alkaliphilic microorganisms that can be used as host microorganisms are a type of extreme environmental microorganisms that show a diverse distribution, and are a general term for microorganisms that can grow in environments with a pH of 9 or higher. These are classified into obligate alkaliphilic microorganisms that can grow only in environments with a pH of 9 or higher, and facultative alkaliphilic microorganisms that have an optimal growth pH of 9 or higher but can also grow in environments with a pH of less than 9. Some of these can grow in strong alkaline environments with a pH of 12 or higher. Any of these are alkaliphilic microorganisms and can be host microorganisms in the present invention.

[0117] In the present invention, halophilic microorganisms that can be used as host microorganisms are a general term for microorganisms that can cope with high salt stress. In terms of the classification of bacteria based on the optimum salt concentration for growth, these are classified into non-halophilic bacteria, whose optimum salt concentration for growth is 0 to 0.2 M sodium chloride, mild halophilic bacteria, whose optimum salt concentration for growth is 0.2 to 0.5 M sodium chloride, moderate halophilic bacteria, whose optimum salt concentration for growth is 0.5 to 2.5 M sodium chloride, and extreme halophilic bacteria, whose optimum salt concentration for growth is 2.5 to 5.2 M sodium chloride. Any of these are halophilic microorganisms and can be host microorganisms in the present invention.

[0118] Microorganisms having halophilic, alkalophilic, or both properties that can be used as the host microorganism of the present invention include various microorganisms, non-limiting examples of which include bacteria of the genera Bacillus, Halomonas, Halobacteroides, Salinibacter, Alkaliferus, Clostridium, and Anaerobranca. The host microorganism of the present invention is preferably a bacterium of the genus Bacillus. Among the microorganisms of the genus Bacillus, Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis are preferred, and Bacillus psuedofirmus is more preferred.

[0119] Escherichia coli and coryneform bacteria, which are generally used for the biosynthesis of compounds, are significantly inhibited in an environment with a pH of 9 or higher. For example, when the pH of the medium rises due to the conversion of lysine to 1,5-pentamethylenediamine, it is necessary to appropriately add an acid solution to control the pH within a range that does not inhibit the growth of the microorganism. However, since the recombinant microorganism according to the present invention has alkalophilicity that allows it to grow even in an alkaline environment, by using the microorganism for diamine production, it is not necessary to adjust the pH by adding an acid solution as in the past, and the complication of the diamine production process can be avoided. In addition, if the recombinant microorganism according to the present invention is halophilic, it can grow in a culture solution containing salt. Therefore, the wastewater containing salt added in the separation process of separating the phase containing diamine from the culture solution or mixed solution (reaction solution) can be reused for the cultivation of the microorganism according to the present invention, thereby reducing wastewater treatment costs.

[0120] By using a halophilic and / or alkaliphilic microorganism capable of producing diamine as a host microorganism and carrying out one or more genetic modifications to suppress N-acetyltransferase, it becomes possible to suppress the production of the by-product N-acetyldiamine in the resulting recombinant microorganism.

[0121] That is, by suppressing the production of N-acetyldiamine in a halophilic and / or alkaliphilic microorganism having diamine production ability, it is possible to suppress the production of N-acetyldiamine in the enzymatic conversion step or the culture step. In a preferred embodiment, the production of a diamine compound, which is a target compound, can be efficiently produced by suppressing the production of by-products.

[0122] As used herein, "N-acetyltransferase" refers to an enzyme that N-acetylates a diamine compound to produce an N-acetyldiamine compound. The term "N-acetyltransferase" is synonymous with the terms "N-acetyltransferase" and "N-acetyltransferase", and these terms are used interchangeably herein. N-acetyltransferase is an enzyme that catalyzes a reaction to acetylate the N-terminus of an amino acid, and in the diamine production pathway, it catalyzes a side reaction in which diamine is N-acetylated to produce N-acetyldiamine.

[0123] In the present invention, as described above, one or more genetic modifications that inhibit N-acetyltransferase are carried out in a halophilic and / or alkaliphilic host microorganism, and therefore, the recombinant microorganism of the present invention comprises one or more genetic modifications that inhibit N-acetyltransferase. a modification that inhibits expression of an endogenous gene encoding the N-acetyltransferase, or The modification reduces the activity of the N-acetyltransferase.

[0124] The inhibition of N-acetyltransferase in microorganisms can be determined, for example, by using an analytical method such as ion chromatography. -N-acetylated forms are not detected in the culture supernatant of the microorganism. - Cadaverine, the substrate, is not reduced when enzyme activity is measured using cell lysate, and -N-acetylated product is not detected This can be confirmed by at least one of the following:

[0125] In a preferred embodiment, the recombinant microorganism of the present invention has an ability to produce N-acetyldiamine compounds that is suppressed or eliminated as compared to the said production ability of a non-mutant strain that does not contain a genetic modification.

[0126] The one or more genetic modifications that inhibit N-acetyltransferase are modifications to inhibit the N-acetyldiamine biosynthetic pathway. The genetic modifications include, for example: in a host microorganism, deleting part or all of an endogenous gene encoding an N-acetyltransferase from the genomic sequence of said host microorganism; - Introducing a mutation into the gene sequence of N-acetyltransferase in the host microorganism, which causes the enzyme function to be deleted; -Introducing mutations such as substitutions, insertions, and deletions into the promoter and / or RBS sites of N-acetyltransferases This is carried out by one or more of the following:

[0127] Modifications for suppressing the N-acetyldiamine biosynthetic pathway include, for example: in a host microorganism, deleting part or all of an endogenous gene encoding an N-acetyltransferase from the genomic sequence of said host microorganism; - Introducing a mutation into the gene sequence of N-acetyltransferase in the host microorganism, which causes the enzyme function to be deleted; -Introducing mutations such as substitutions, insertions, and deletions into the promoter and / or RBS sites of N-acetyltransferases This is carried out by one or more of the following:

[0128] In the recombinant microorganism obtained by the above-mentioned manipulation, the production of N-acetyltransferase is suppressed. That is, the recombinant microorganism according to the present invention is modified by one or more genetic manipulations so as to suppress the production of N-acetyltransferase. Due to such modification, the recombinant microorganism according to the present invention preferably has a suppressed or eliminated ability to produce N-acetyldiamine compounds, compared to the said ability of a non-mutant strain not containing the above-mentioned genetic modification.

[0129] Specifically, the genetic manipulation may be, for example, one or more genetic manipulations selected from the group consisting of the following (A), (B), (C) and (D). (A) causing the host microorganism to be deleted of an endogenous gene encoding the N-acetyltransferase; (B) reducing the copy number of the endogenous gene for the N-acetyltransferase in the host microorganism; (C) introducing a mutation into an expression regulatory region of an endogenous gene for the N-acetyltransferase in the host microorganism; and (D) replacing the expression regulatory region of the endogenous gene for the N-acetyltransferase in the host microorganism with an exogenous regulatory region capable of low expression.

[0130] For gene modification, for example, a technique for introducing an arbitrary foreign gene sequence into the genomic sequence in the cells of a host microorganism, and a technique for removing an unnecessary gene sequence from the genomic sequence can be used.

[0131] Specifically, the following technologies are mentioned: (1) A chromosome that combines a temperature-sensitive plasmid vector and negative selection A technique for arbitrarily modifying the above gene sequences, and (2) A technology using CRISPR / CAS9 to arbitrarily modify gene sequences on chromosomes.

[0132] In the process of culturing the host microorganism into which the above-described vector has been introduced, a genetically modified strain in which the gene sequence of the chromosome has been modified by homologous recombination can be obtained by changing the temperature and medium composition. The conditions for obtaining a genetically modified strain are preferably a culture temperature of 37 to 43°C or higher and a medium containing 1 mM or more of 4-chlorophenylalanine.

[0133] The above-described vectors can be introduced into host microorganisms by conventional cloning and transfection methods, such as conjugation transfer, coprecipitation, protoplast fusion, electroporation, retrovirus transfection, etc. Examples of such methods are described in Gene Cloning and DNA Analysis, TA Brown, 2016, or Molecular Cloning: A Laboratory Manual (Fourth Edition): Sambrook et al., 2012.

[0134] A representative N-acetyltransferase gene is yjbC from Bacillus psuedofirmus. The amino acid sequence of the Bacillus pseudofilamus yjbC enzyme is shown in SEQ ID NO: 23, and the nucleotide sequence of the Bacillus pseudofilamus yjbC gene (BpOF4_01925 (GenBank: ADC48452)) is shown in SEQ ID NO: 24 (Figures 3 and 4).

[0135] In one embodiment, the N-acetyltransferase is (A-1) consists of the amino acid sequence shown in SEQ ID NO: 23; (A-2) consisting of 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 shown in SEQ ID NO: 23, and having an enzyme activity for N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (A-3) The antibody has an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 23, and has an enzyme activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound.

[0136] In a preferred embodiment, the N-acetyltransferase is (A-1) consists of the amino acid sequence shown in SEQ ID NO: 23; (A-2) consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and having an enzyme activity for N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (A-3) The amino acid sequence is formed by deleting, substituting, inserting and / or adding 1 to 10 amino acids relative to the amino acid sequence shown in SEQ ID NO: 23, and has an enzyme activity for N-acetylating a diamine compound to produce an N-acetyldiamine compound.

[0137] In a more preferred embodiment, the N-acetyltransferase consists of the amino acid sequence shown in (A-1) SEQ ID NO:23.

[0138] In another embodiment, the N-acetyltransferase is (B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24, (B-2) a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO:24 and encodes a protein having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound; (B-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 to the base sequence shown in SEQ ID NO: 24, and encoding a protein having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound; (B-4) A DNA encoding a protein having an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids have been deleted, substituted, inserted, and / or added to the amino acid sequence of a protein encoded by the base sequence shown in SEQ ID NO:24, and which encodes a protein having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (B-5) DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 24 is coded as

[0139] In a preferred embodiment, the N-acetyltransferase is (B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24, (B-2) a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO:24 and encodes a protein having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound; (B-3) DNA that consists of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 24 and encodes a protein having an enzyme activity that N-acetylates a diamine compound to produce an N-acetyldiamine compound; (B-4) A DNA encoding a protein having an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence of a protein encoded by the base sequence shown in SEQ ID NO:24, and which encodes a protein having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (B-5) DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 24 is coded as

[0140] As used herein, "stringent conditions" refers to conditions such as "1xSSC, 0.1% SDS, 60°C," more stringent conditions such as "0.1xSSC, 0.1% SDS, 60°C," and even more stringent conditions such as "0.1xSSC, 0.1% SDS, 68°C."

[0141] In a more preferred embodiment, the N-acetyltransferase is encoded by a DNA having the base sequence shown in SEQ ID NO:24 (B-1).

[0142] In this specification, the percentage (%) of "sequence identity" of a comparison amino acid sequence to a reference amino acid sequence is defined as the percentage of amino acid residues in the comparison sequence that are identical to those in the reference sequence when the sequences are aligned to maximize the identity between the two sequences and, if necessary, gaps are introduced into one or both of the two sequences. In this case, conservative substitutions are not considered as part of the sequence identity. Sequence identity can be determined by using publicly available computer software, for example, using an alignment search tool such as BLAST (registered trademark, hereinafter omitted) (Basic Local Alignment Search Tool). Those skilled in the art can determine appropriate parameters for obtaining maximum alignment of the comparison sequences in the alignment. The "sequence identity" of nucleotide sequences can also be determined by a similar method.

[0143] 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 diamine. Suitable medium compositions, culture conditions and culture times for transformants derived from various host microbial cells can be selected by those skilled in the art.

[0144] Therefore, a second aspect of the present invention relates to a method for producing a diamine compound, comprising culturing the recombinant microorganism described above. Specifically, the method comprises a culturing step of culturing the recombinant microorganism described above to obtain a culture and / or an extract of the culture of the recombinant microorganism.

[0145] The medium used in the culture step is as explained in Invention A above.

[0146] When biomass-derived raw materials are used, the diamine product can be clearly distinguished from synthetic raw materials by the measurement method described in Invention A.

[0147] In the case where the host microorganism cannot assimilate carbon sources such as cellulose and polysaccharides in the above-mentioned medium, the host microorganism can be adapted to the production of diamines using the carbon sources by subjecting the host microorganism to the genetic engineering techniques described in Invention A.

[0148] The culture format and conditions are as described in Invention A.

[0149] The production method of the present invention preferably further comprises a mixing step of mixing the culture and / or an extract of the culture with a substrate compound to obtain a mixed solution.

[0150] In the culture and / or the mixed solution, a diamine compound, which is a target compound, is produced as a result of the reaction. Therefore, in a more preferred embodiment, the production method of the present invention further includes a recovery step of recovering the diamine compound from the culture and / or the mixed solution.

[0151] The production method of the present invention may include one or more steps selected from the steps described in Invention A.

[0152] As described above, the present invention relates to a recombinant halophilic and / or alkaliphilic microorganism capable of producing diamine, which comprises one or more genetic modifications that suppress N-acetyltransferase, which N-acetylates a diamine compound to produce an N-acetyldiamine compound. In culturing the recombinant microorganism of the present invention, the production of a by-product, an N-acetyldiamine compound, can be suppressed. The recombinant microorganism of the present invention has the ability to produce diamine, and can produce diamine while suppressing the production of by-products. Therefore, the target compound, diamine, can be efficiently obtained. Furthermore, the recombinant microorganism of the present invention is expected to be applied to the production of diamine compounds on an industrial scale.

[0153] Furthermore, when the recombinant microorganism according to the present invention is alkalophilic, by using the microorganism for diamine production, it is not necessary to adjust the pH by adding an acid solution, and it is possible to avoid complicating the diamine production process. Furthermore, when the recombinant microorganism according to the present invention is halophilic, it can grow in a culture solution containing salt, and the salt-containing wastewater added in the separation step of separating the diamine-containing phase from the culture solution or mixed solution (reaction solution) can be reused for culturing the microorganism, thereby reducing wastewater treatment costs.

[0154] Although the embodiment for carrying out the present invention has been described above, the embodiment is merely shown as an example and is not intended to limit the scope of the invention. The embodiment can be carried out in various other forms, and various omissions, substitutions, and modifications can be made without departing from the gist of the invention. EXAMPLES

[0155] <1> Invention A Hereinafter, the present invention A will be described based on examples, but the present invention A is not limited to these examples.

[0156] All PCRs shown in this example were performed using PrimeSTAR Max DNA Polymerase (product name, Takara Bio). Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis were transformed using electroporation. In the electroporation method, a 0.1 cm wide cuvette containing 1 μl of plasmid DNA together with 60 μl of competent cells was attached to a gene pulsar (Bio-Rad), and a pulse of 2.5 kV voltage, 200 Ω resistance, and 25 μF capacitance was applied to the cuvette. After 3 hours of recovery culture at 37°C, the cells were spread on 181 medium containing 10 μg / mL chloramphenicol to obtain transformants. The composition of 181 medium is shown in Table A-1.

[0157] [Table A-1]

[0158] Example A1: Construction of cadA gene expression plasmid for Bacillus pseudophyllum and obtaining transformants (Example A1-a) Cloning of the promoter region Bacillus pseudophyllum OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project) was cultured in 181 medium (2 ml) with shaking at 37°C. After the culture was completed, the cells were collected from the culture medium, and genomic DNA was extracted using Nucleo Spin Tissue (product name, manufactured by MACHEREY-NAGEL). The promoter region of the rpoD gene of Bacillus pseudophyllum (SEQ ID NO: 5) was PCR amplified (fragment 1) using a primer set having the sequences described in SEQ ID NO: 6 and SEQ ID NO: 7. The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), and 30 cycles. Plasmid pAL351 (deposited as NITE P-02918 at the National Institute of Technology and Evaluation, National Patent Microorganism Depository (NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on March 18, 2019, and then transferred to the international depository under the Budapest Treaty on July 28, 2020, and was assigned the accession number NITE BP-02918.) was PCR amplified with the primer set shown in SEQ ID NO: 8 and SEQ ID NO: 9 (fragment 2). The reaction conditions were 98 ° C (10 sec), 55 ° C (5 sec), 72 ° C (30 sec), 30 cycles. Fragment 2 was used as the vector side, and fragment 1 was ligated as an insert to construct pALP01.

[0159] (Example A1-b) Cloning of the cadA gene E. coli W3110 strain (NBRC12713) was cultured in LB medium (2 mL) at 37°C with shaking. After the culture was completed, the cells were collected from the culture medium, and genomic DNA was extracted using Nucleo Spin Tissue. Using the extracted genomic DNA as a template, PCR amplification was performed with a primer set having the sequences described in SEQ ID NO: 10 and SEQ ID NO: 11. The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), 30 cycles. The amplified fragment was ligated downstream of the promoter sequence of pALP01 to construct pAL328.

[0160] (Example A1-c) Obtaining transformants The pAL328 constructed in Example A1-b was Bacillus pseudophyllum OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Resource Project), Bacillus halodurans C-125 strain (JCM9153 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Resource Project), Bacillus marmaensis GMBE72 strain (JCM15719 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Resource Project) Transformed into, respectively, AKRM-1 strain, AKRM-2 strain, AKRM-3 strain was obtained.

[0161] On the other hand, in the control transformation, the plasmid pALP01, which does not contain the cadA gene, Bacillus pseudophyllum OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Resource Project), Bacillus halodurans C-125 strain (JCM9153 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Resource Project), Bacillus marmaensis GMBE72 strain (JCM15719 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Resource Project) Transformed into, respectively, AKRM-4 strain, AKRM-5 strain, AKRM-6 strain was obtained.

[0162] Furthermore, a transformant carrying the cadA gene expression cassette on the chromosome was also obtained for the Bacillus pseudofilamus OF4 strain. This strain, Bacillus pseudofilamus AKAL-001, was deposited at the National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary (NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on March 18, 2019 as NITE P-02920, and was subsequently transferred to an international deposit under the Budapest Treaty on July 28, 2020, and was assigned the accession number NITE BP-02920.

[0163] The sequence is shown below.

[0164] [ka]

[0165] Example A2: Cadaverine production by constructed strains (flask culture) Each of the obtained transformants was cultured on a 181 medium plate at 37° C. for 2 days to form colonies. 2 mL of 181 medium was placed in a 14 mL test tube, and a colony was inoculated from the plate using a platinum loop. The culture was carried out at 37° C. and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

[0166] 30 mL of BS medium (composition shown in Table A-2) was placed in a 150 mL Erlenmeyer flask, 0.3 mL of preculture solution was added, and main culture (cadaverine production test) was performed. The culture conditions were 37°C and 180 rpm. When culturing the AKRM-1 to AKRM-6 strains, 10 mg / L chloramphenicol was added to the medium for both preculture and main culture.

[0167] [Table A-2]

[0168] The above culture solution was centrifuged at 10,000g for 3 minutes to recover the supernatant, and the cadaverine concentration of the culture supernatant was measured. Specifically, CG-19 (guard column) and CS-19 (analysis column) (both trade names, manufactured by Thermo Fisher Scientific Co., Ltd.) were connected and ion chromatography analysis (detector: electrical conductivity, column temperature: 30°C, flow rate: 0.35mL / min, mobile phase: gradient of 8mM methanesulfonic acid aqueous solution → 70mM methanesulfonic acid aqueous solution) was performed to quantify the cadaverine concentration in the culture supernatant. The results of comparison of the cadaverine concentration between the transformants of the present invention (AKRM-1 strain, AKRM-2 strain, AKRM-3 strain, AKAL-001 strain) and the control strains (AKRM-4 strain, AKRM-5 strain, AKRM-6 strain) are shown in Table A-3.

[0169] Compared to the control strains (AKRM-4, AKRM-5, and AKRM-6), the transformants of the present invention (AKRM-1, AKRM-2, AKRM-3, and AKAL-001 strains) produced more cadaverine over the course of culture time.

[0170] [Table A-3]

[0171] Example A3: Cadaverine production by the constructed strain (fermentor culture) AKRM-1, AKRM-4, and AKAL-001 strains were cultured on 181 medium plates at 37°C for 2 days to form colonies. 100 mL of 181 medium was placed in a 500 mL Erlenmeyer flask, and colonies were inoculated from the plates using a platinum loop. Culture was performed at 37°C and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

[0172] A BS jar medium (shown in Table A-4) containing 20 g / L glucose was placed in a 10 L jar culture apparatus (model name: MDL-6C, manufactured by Marubishi Bioengineering Co., Ltd.), 100 mL of preculture solution was added, and main culture (cadaverine production test) was performed. When culturing the AKRM-1 and AKRM-4 strains, 10 mg / L of chloramphenicol was added to the medium for both preculture and main culture. The culture conditions were culture temperature: 37°C, culture pH: 7.5, alkali addition: 28% ammonia water, stirring speed: 700 rpm, and aeration speed: 0.1 vvm. In addition, feed medium (composition shown in Table A-5) was added successively from the middle of the culture so that the glucose concentration in the medium was 0 to 5 g / L. Sampling was performed over time during the culture, and the cadaverine concentration in the culture supernatant was quantified.

[0173] [Table A-4]

[0174] [Table A-5]

[0175] The results are shown in Table A-6. The AKRM-4 strain did not produce cadaverine, the cadaverine concentration did not increase in the AKRM-1 strain expressing the cadA gene on a plasmid, and the cadaverine concentration increased with the passage of culture time in the AKAL-001 strain expressing the cadA gene on the chromosome.

[0176] [Table A-6]

[0177] Example A4: Cadaverine production with increasing sodium sulfate concentration (flask culture) The AKAL-001 strain was cultured on a 181 medium plate at 37°C for 2 days to form colonies. 2 mL of 181 medium was placed in a 14 mL test tube, and a colony was inoculated from the plate using a platinum loop. The culture was carried out at 37°C and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

[0178] Two 150 mL Erlenmeyer flasks were prepared with 30 mL of BS medium (shown in Table A-2). One flask was added with 50 g / L of sodium sulfate as shown in Table A-2, and the other with 100 g / L of sodium sulfate. 0.3 mL of preculture solution was added and main culture (cadaverine production test) was performed. Culture conditions were 37°C and 180 rpm.

[0179] The above culture solution was centrifuged at 10,000g for 3 minutes to recover the supernatant, and the cadaverine concentration in the culture supernatant was measured by the method described in Example A2. The AKAL-001 strain produced cadaverine even in the presence of sodium sulfate at a high concentration of 100g / L, and the cadaverine concentration increased with the passage of culture time.

[0180] [Table A-7]

[0181] Example A5: Phase separation upon addition of salt compounds at various concentrations Cadaverine (1,5-diaminopentane, Fujifilm Wako Chemical) and sodium chloride, sodium sulfate, and sodium carbonate were added at various concentrations to the separation confirmation simulation liquid (composition shown in Table A-8) to prepare 5 mL of simulation liquid. After mixing with a vortex mixer, the state of phase separation after leaving it at room temperature for 10 minutes and the liquid volume of the aqueous phase and cadaverine phase were confirmed. In addition, the concentration of cadaverine in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the migration rate from the aqueous phase to the cadaverine phase was calculated.

[0182] [Table A-8]

[0183] The final solution composition and separation results are shown in Table A-9. In the phase separation column in the table, "x" indicates that no phase separation occurred, "o" indicates that the diamine phase formed was less than 10% of the total liquid volume when visually confirmed (for example, if the total liquid volume is 5 mL, the diamine phase was less than 0.5 mL), and "◎" indicates that the diamine phase formed was 10% or more of the total liquid volume when visually confirmed. This evaluation standard for phase separation is also applied to the following tables. As a result of the test, it was confirmed that diamine phase separation occurred under conditions where each inorganic salt was added at a high concentration.

[0184] [Table A-9]

[0185] Example A6: Phase Separation Properties of Cadaverine and Hexamethylenediamine (HMDA) Sodium sulfate and cadaverine (1,5-diaminopentane, Fujifilm Wako Chemical) or hexamethylenediamine (Fujifilm Wako Pure Chemical) were added at various concentrations to the separation confirmation simulation liquid (composition shown in Table A-8) to prepare 5 mL of simulation liquid. After mixing with a vortex mixer, the state of phase separation after leaving it at room temperature for 10 minutes and the liquid volume of the aqueous phase and cadaverine phase were confirmed. In addition, the concentration of cadaverine in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the migration rate from the aqueous phase to the cadaverine phase was calculated.

[0186] The final solution composition and separation results are shown in Table A-10. It was confirmed that in the presence of high concentrations of sodium sulfate, both cadaverine and hexamethylenediamine (HMDA) underwent phase separation.

[0187] [Table A-10]

[0188] Example A7: Solvent extractability (cadaverine) when sodium carbonate was added at various concentrations Sodium carbonate and cadaverine (1,5-diaminopentane, Fujifilm Wako Chemical) of various concentrations were added to the separation confirmation simulation liquid (composition shown in Table A-8) to prepare 5 mL of simulation liquid. An equal amount (5 mL) of n-butanol was added to the simulation liquid, mixed with a vortex mixer, and then allowed to stand at room temperature for 10 minutes. The cadaverine concentration in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the migration rate from the aqueous phase to the solvent phase was calculated.

[0189] The final solution composition and separation results are shown in Table A-11. It was confirmed that the extraction efficiency into the solvent improved with increasing sodium carbonate concentration.

[0190] [Table A-11]

[0191] Example A8: Solvent extractability when sodium sulfate is added at various concentrations (hexamethylenediamine) Sodium sulfate and hexamethylenediamine of various concentrations were added to the separation confirmation simulation liquid (composition shown in Table A-8) to prepare 5 mL of each simulation liquid. An equal amount (5 mL) of hexane or 2-ethyl-1-hexanol was added to the simulation liquid, mixed with a vortex mixer, and then allowed to stand at room temperature for 10 minutes. The concentration of hexamethylenediamine in the solvent phase was measured, and the migration rate from the aqueous phase to the solvent phase was calculated.

[0192] The final solution composition and separation results are shown in Table A-12. It was confirmed that the extraction efficiency to 2-ethyl-1-hexanol improved with increasing sodium sulfate concentration, while the extraction efficiency to n-hexane was very low.

[0193] [Table A-12]

[0194] Example A9: Phase separation in culture medium containing diamine Working Example A30 mL of the culture supernatant from the flask culture described in 4 was filtered through a 0.22 μm filter, and cadaverine (1,5-diaminopentane, Fujifilm Wako Chemical) or hexamethylenediamine (Fujifilm Wako Pure Chemical) was added to each concentration, and the mixture was concentrated twice using an evaporator (yield: 15 mL). After mixing the concentrated solution with a vortex mixer, the state of phase separation after leaving it at room temperature for 10 minutes and the liquid volume of the aqueous phase and cadaverine phase were confirmed. In addition, the cadaverine concentration in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the transition rate from the aqueous phase to the cadaverine phase was calculated. The final solution composition and separation results are shown in Table A-13. It was confirmed that when a high concentration of sodium sulfate was present in the culture solution, both cadaverine and hexamethylenediamine were phase separated.

[0195] [Table A-13]

[0196] Example A10: Cultivation using recycled aqueous phase after phase separation Working Example A 100 mL of the fermenter culture liquid (cultured for 63 hours) of the AKAL-001 strain described in 3 was collected, centrifuged, and the supernatant was collected. 100 mL of the culture supernatant was filtered through a 0.22 μm filter and concentrated twice using an evaporator (yield: 50 mL). Hexamethylenediamine (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to this concentrated liquid to a final concentration of 150 g / L. After mixing with a vortex mixer, the mixture was left to stand at room temperature for 10 minutes, and the formation of phase separation was confirmed. The aqueous phase was 20 mL, and the concentration of hexamethylenediamine was 48.5 g / L. This aqueous phase was collected with a pipette, and the pH was adjusted to 7.5 with sulfuric acid, and then 30 mL of distilled water was added to make up to 50 mL. This liquid and new BS medium (composition shown in Table A-14) were mixed at different ratios and prepared in a 150 mL Erlenmeyer flask.

[0197] [Table A-14]

[0198] Bacillus pseudophyllum OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project) was cultured on 181 medium plate at 37℃ for 2 days to form colonies. 2mL of 181 medium was placed in a 14mL test tube, and a colony was inoculated from the plate with a platinum loop. The medium was cultured at 37℃ and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

[0199] 0.1 mL of the preculture solution was added to the medium prepared in the flask, and main culture was performed. The culture conditions were 37°C and 180 rpm. After 49 hours of culture, the culture solution was sampled and the turbidity (OD600) was measured.

[0200] The results are shown in Table A-15. It was confirmed that the aqueous phase after phase separation could be reused as a medium.

[0201] [Table A-15]

[0202] From the results described in the above examples, it was confirmed that diamines could be produced using microorganisms with both halophilic and alkalophilic properties as hosts. It was also confirmed that diamines were phase-separated in the presence of high salt concentrations of sodium carbonate or sodium sulfate, and that the extraction efficiency during solvent extraction was improved.

[0203] By concentrating the culture supernatant containing the diamine, the concentration of the diamine and inorganic salt increases, and further improvement in separation efficiency can be expected. In addition, by using the recombinant microorganism according to the present invention, the step of adjusting the pH by adding an acid solution can be omitted. Furthermore, the high-concentration salt-containing water generated during the liberation of the diamine can be reused as a culture medium, making it possible to reduce wastewater treatment costs.

[0204] <2> Invention B Hereinafter, the present invention B will be described based on examples, but the present invention B is not limited to these examples.

[0205] All PCRs shown in this example were performed using PrimeSTAR Max DNA Polymerase (product name, Takara Bio). Transformation of Bacillus pseudofirmus was performed using the electroporation method. In the electroporation method, a 0.1 cm wide cuvette containing 1 μl of plasmid DNA together with 60 μl of competent cells was attached to a gene pulsar (Bio-Rad), and a pulse of 2.5 kV voltage, 200 Ω resistance, and 25 μF capacitance was applied to the cuvette. After 3 hours of recovery culture at 30°C, the cells were spread on 181 medium containing 10 μg / mL chloramphenicol to obtain transformants. The composition of 181 medium is shown in Table B-1.

[0206] [Table B-1]

[0207] Example B1: Obtaining a gene disruptant From the annotation information of Bacillus pseudophyllum OF4 strain published in the NCBI database, 39 genes encoding acetyltransferases were selected as candidate genes encoding diamine N-acetyltransferases. · BpOF4_11255 gene (GenBank: ADC50304), · BpOF4_16515 gene (GenBank: ADC51348), · BpOF4_18375 gene (GenBank: ADC51716), · BpOF4_16725 gene (GenBank: ADC51388), · BpOF4_18160-65 gene (GenBank: ADC51673-4), · BpOF4_18545 gene (GenBank: ADC51750), · BpOF4_19380 gene (GenBank: ADC51915), · BpOF4_00750 gene (GenBank: ADC48219), BpOF4_01925 gene (GenBank: ADC48452) The gene disruption strain was constructed by genetic manipulation into the chromosome via homologous recombination. The primer sequences used are shown in Figures 5A and 5B.

[0208] (Example B1-a) Preparation of gene disruption plasmid Bacillus pseudophyllum OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project) was cultured in 181 medium (2 ml) with shaking at 37°C. After the culture was completed, the cells were collected from the culture medium, and genomic DNA was extracted using Nucleo Spin Tissue (product name, manufactured by MACHEREY-NAGEL). The homologous region was amplified by PCR using the primer set with "A" and "B" at the end of the primer name, and the primer set with "C" and "D" at the end of the primer name (see Figure 5), to obtain fragments 1 and 2. The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), and 30 cycles.

[0209] Plasmid pAL351 (deposited at the National Institute of Technology and Evaluation, National Patent Microorganism Depository (NPMD), Biotechnology Center, on March 18, 2019. Accession number: NITE BP-02918) was amplified by PCR using a primer set with "E" and "F" at the end of the primer name (see Figure 5) to obtain fragment 3. The reaction conditions were 98 ° C (10 sec), 55 ° C (5 sec), 72 ° C (30 sec), 30 cycles. Fragment 3 was used as a vector, and fragments 1 and 2 were ligated as inserts to construct pAKNU01-09.

[0210] (Example B1-b) Obtaining transformants The pAKNU01 to 09 constructed in Example B1-a were transformed into the Bacillus pseudophyllum AKAL-001 strain (deposited at the National Institute of Technology and Evaluation, National Patent Microorganism Depositary (NPMD) at the Biotechnology Center. Accession number: NITE BP-02920) to obtain the AKALp-101 to AKALp-109 strains.

[0211] (Example B1-c) Obtaining a chromosomal insertion strain The AKALp-101 to AKALp-109 strains obtained in Example B1-b were cultured in 181 medium containing 10 μg / mL chloramphenicol at 30°C for one day, then diluted 100-fold and spread on 181 medium containing 10 μg / mL chloramphenicol and cultured at 43°C to obtain chromosomal insertion strains AKALp-201 to AKALp-209 in which the plasmid had been homologously recombined into the chromosome.

[0212] (Example B1-d) Obtaining a yjbC gene disruptant The AKALp-201 to AKALp-209 strains obtained in Example B1-c were cultured at 30°C for 1 day in 181 medium containing 10 μg / mL chloramphenicol, then diluted 100-fold and applied to 181 medium containing 5 mM 4-chlorophenylalanine, and cultured at 30°C to obtain gene-disrupted strains AKDNC-001 to AKDNC-009 in which the respective gene regions were removed by adding the plasmid to the chromosome. The deletion of the genes was confirmed by PCR using a primer set (see FIG. 5) with "G" and "H" added to the end of the primer name. The reaction conditions were 98°C (10 sec), 55°C (5 sec), 72°C (30 sec), and 30 cycles.

[0213] The constructed strains and their corresponding genes are shown in the table below.

[0214] [Table B-2]

[0215] Example B2: Evaluation of N-acetylcadaverine production ability in the constructed strain (fermentor culture) The AKAL-001 strain and the AKDNC-001 to 009 strains were cultured on a 181 medium plate at 37° C. for 1 day to form colonies. 2 mL of the 181 medium was placed in a 14 mL test tube, and a colony was inoculated from the plate using a platinum loop. The culture was carried out at 37° C. and 180 rpm until sufficient turbidity was obtained, and this was used as a preculture solution for main culture.

[0216] A BS jar medium (shown in Table B-3) containing 30 g / L glucose was placed in a 100 mL jar culture device (model name: Bio Jr.8, manufactured by Biot Co., Ltd.), 1 mL of preculture solution was added, and main culture was performed (cadaverine production test). The culture conditions were culture temperature: 37°C, culture pH: 7.5, alkali addition: 10% ammonia water, stirring speed: 750 rpm, and aeration speed: 0.1 vvm. Sampling was performed over time during the culture, and the turbidity of the bacterial cell in the culture solution and the diamine concentration in the culture supernatant were quantified. 24 hours after the start of culture, 6 ml of 50% glucose solution was added.

[0217] The above culture solution was centrifuged at 10,000g for 3 minutes to collect the supernatant, and the supernatant was then eluted. The cadaverine concentration and N-acetylcadaverine concentration were measured. Specifically, CG-19 (guard column) and CS-19 (analytical column) (both trade names, manufactured by Thermo Fisher Scientific Co., Ltd.) were connected to perform ion chromatography analysis (detector: electrical conductivity, column temperature: 30°C, flow rate: 0.35mL / min, mobile phase: gradient of 8mM methanesulfonic acid aqueous solution → 70mM methanesulfonic acid aqueous solution) to quantify the cadaverine concentration and N-acetylcadaverine concentration in the culture supernatant.

[0218] [Table B-3]

[0219] The cell density (OD) and cadaverine and N-acetylcadaverine concentrations (g / l) after 40 hours of culture are shown in Table B-4. Cadaverine and N-acetylcadaverine were detected in the culture supernatants of the AKAL-043 and AKDNC-001 to 008 strains. On the other hand, cadaverine was detected in the culture supernatant of the AKDNC-009 strain, but N-acetylcadaverine was not (Table B-4). The AKDNC-009 strain had a 14% increased cadaverine concentration compared to the AKAL-043 strain.

[0220] [Table B-4]

[0221] Regarding the results in Table B-4 above, it is understood that strains in which the amount of acetylcadaverine produced was 0.5 g / l or more lower than the non-mutant AKAL-001 strain have a lower activity to produce N-acetyldiamine compounds than the wild-type strain. [Industrial Applicability]

[0222] The present invention can be used for industrial fermentation production of diamines. Furthermore, since the present invention can suppress the production of by-products and efficiently produce diamines, it is expected that the present invention can be used for industrial-scale production of diamines by fermentation of the recombinant microorganism of the present invention.

Claims

1. A recombinant halophilic and / or alkaliphilic microorganism capable of producing cadaverine, The recombinant microorganism comprises one or more genetic modifications that inhibit an N-acetyltransferase that N-acetylates cadaverine to form N-acetylcadaverine; The suppression means that the ability to produce N-acetylcadaverine is lost compared to the ability of a non-mutant strain that does not contain the genetic modification, the genetic modification is a modification that suppresses expression of an endogenous gene encoding the N-acetyltransferase; The genetic modification is performed on a microorganism selected from the group consisting of Bacillus pseudofirmus, Bacillus halodurans and Bacillus marmarensis, and The N-acetyltransferase is (A) (A-1) consists of the amino acid sequence shown in SEQ ID NO: 23; (A-2) consisting of an amino acid sequence having 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and having an enzyme activity for N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (A-3) consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids have been deleted, substituted, inserted, and / or added to the amino acid sequence shown in SEQ ID NO:23, and having an enzyme activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (B) (B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24; (B-2) a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO:24 and encodes a protein having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound; (B-3) DNA consisting of a base sequence having 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the base sequence shown in SEQ ID NO:24, and encoding a protein having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound; (B-4) A DNA encoding a protein having an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids have been deleted, substituted, inserted, and / or added to the amino acid sequence of a protein encoded by the base sequence shown in SEQ ID NO:24, and which encodes a protein having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (B-5) Encoded by DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 24; Recombinant microorganisms.

2. The recombinant microorganism according to claim 1, wherein the gene encoding the N-acetyltransferase is the yjbC gene.

3. A method for producing cadaverine, comprising a culturing step of culturing the recombinant microorganism according to claim 1 or 2 to obtain a culture and / or an extract of the culture of the recombinant microorganism.

4. The method of claim 3 , further comprising a mixing step of mixing the culture and / or an extract of the culture with a substrate compound to obtain a mixed solution.

5. The method according to claim 3 , further comprising a recovery step of recovering cadaverine from the culture or the mixed solution.

Images