Method for producing 4-(aminomethyl) cyclohexane-1-carboxylic acid

Abstract

A production method according to one embodiment of the present invention is a production method for producing 4-(aminomethyl) cyclohexane-1-carboxylic acid from 1, 4-bis (aminomethyl) cyclohexane, protein having amino acid sequence having 60% or more consistency with the amino acid sequence represented by any one of SEQ ID NOS: 1-4 and 103-105 and having amino acid transfer activity, and protein having amino acid sequence having amino acid sequence represented by SEQ ID NOS: 19-22, 35-46 and 127-12, and protein having amino acid sequence represented by SEQ ID NOS: 19-22, 35-46 and 127-12 and a step for producing a protein having aldehyde dehydrogenase activity, said protein comprising an amino acid sequence having 50% or more consistency with the amino acid sequence represented by any one of the amino acid sequences (139, 139).

Description

Technical Field

[0001] The present invention relates to a protein used in the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid and a method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid using the protein. In addition, the present invention relates to a method for producing trans-4-(aminomethyl)cyclohexane-1-carboxylic acid with high efficiency. Background Art

[0002] 4-(Aminomethyl)cyclohexane-1-carboxylic acid (4-(aminomethyl)cyclohexanecarboxylic acid; AMCHA) is an artificially synthesized amino acid developed as a plasmin inhibitor (non-patent document 1). AMCHA has cis-type and trans-type stereoisomers. Among them, non-patent document 2 confirms that the trans-type has a plasmin inhibitory effect. Trans-4-aminomethylcyclohexane-1-carboxylic acid (t-AMCHA), which is a trans-type of 4-(aminomethyl)cyclohexane-1-carboxylic acid, is also called tranexamic acid (TXA). Due to its plasmin inhibitory effect, it can be used as a hemostatic agent and anti-inflammatory agent for the prevention and treatment of bleeding (non-patent documents 3-4).

[0003] As a method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid, chemical synthesis methods are mainly known. For example, Patent Document 1 discloses a method for producing a trans-isomer of 4-(aminomethyl)cyclohexane-1-carboxylic acid by using 4-(chloromethyl)benzoic acid as a starting substrate, performing an amination reaction and a hydrogenation reaction to generate 4-(aminomethyl)cyclohexane-1-carboxylic acid, and then performing an isomerization reaction.

[0004] The chemical synthesis methods mentioned above require reactions under high temperature and high pressure, and have the problems of high energy costs and heavy environmental load.

[0005] As an alternative to chemical synthesis, a manufacturing method using microorganisms can be cited. Patent document 2 discloses a method for producing trans-isomer 4-(aminomethyl)cyclohexane-1-carboxylic acid by contacting a microorganism belonging to the genus Corynebacterium or Nocardia with a mixture of the cis-isomer and the trans-isomer of 1,4-bis(aminomethyl)-cyclohexane. However, this method uses the conversion reaction of the microorganism itself isolated from nature, so the productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid is low, and the identification of the active main enzyme is not performed.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent document 1: CN114014768A

[0009] Patent Document 2: Japanese Patent Application Laid-Open No. 63-152991

[0010] Non-patent literature

[0011] Non-patent document 1: Keio Journal of Medicine (1962) 11, 8, 105-115

[0012] Non-patent document 2: Keio Journal of Medicine (1964) 18, 4, 177-185

[0013] Non-patent literature 3: European Journal of Haematology (2020) 104, 2, 79-87

[0014] Non-patent literature 4: Journal of Trauma and Acute Care Surgery (2019) 86, 1, 101-107 Summary of the invention

[0015] Problem that the invention aims to solve

[0016] An object of the present invention is to provide a method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid by an enzyme reaction using 1,4-bis(aminomethyl)cyclohexane as a substrate, and a protein derived from a microorganism that can be used in the enzyme reaction.

[0017] Methods used to solve problems

[0018] Until now, the reaction pathway for generating 4-(aminomethyl)cyclohexane-1-carboxylic acid from 1,4-bis(aminomethyl)cyclohexane has not been known. Therefore, the present inventors have conceived a two-step enzyme reaction pathway using aminotransferase and aldehyde dehydrogenase, and screened proteins that may have the target activity. It was found that 4-(aminomethyl)cyclohexane-1-carboxylic acid can be generated from 1,4-bis(aminomethyl)cyclohexane by expressing the protein in an Escherichia coli strain, purifying it, and performing an enzyme reaction, thereby completing the present invention.

[0019] The present invention relates to, for example, the following inventions.

[0020] [1] A protein having an amino group transfer activity of 1,4-bis(aminomethyl)cyclohexane to another compound to produce 4-(aminomethyl)cyclohexane-1-carbaldehyde, and comprising an amino acid sequence having an identity of 60% or more to the amino acid sequence represented by any one of SEQ ID NOs. 1 to 4 and 103 to 105.

[0021] [2] A DNA encoding the protein described in [1].

[0022] [3] A recombinant DNA comprising the DNA described in [2].

[0023] [4] A recombinant cell comprising the DNA described in [2], or obtained by transforming a host cell with the recombinant DNA described in [3].

[0024] [5] A protein having aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, and comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139.

[0025] [6] A protein having aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, and comprising an amino acid sequence having an identity of 60% or more to the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139.

[0026] [7] A DNA encoding the protein described in [5] or [6].

[0027] [8] A recombinant DNA comprising the DNA described in [7].

[0028] [9] A recombinant cell comprising the DNA described in [7], or obtained by transforming a host cell with the recombinant DNA described in [8].

[0029]

[10] A method for producing cis-isomer and / or trans-isomer 4-(aminomethyl)cyclohexane-1-carboxylic acid from cis-isomer and / or trans-isomer 1,4-bis(aminomethyl)cyclohexane, comprising:

[0030] (i) a step of producing 4-(aminomethyl)cyclohexane-1-carbaldehyde from 1,4-bis(aminomethyl)cyclohexane in the presence of an aminotransferase; and

[0031] (ii) A step of producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carboxaldehyde in the presence of aldehyde dehydrogenase.

[0032]

[11] The production method according to

[10] , wherein the aminotransferase is the protein according to [1], and the aldehyde dehydrogenase is the protein according to [5] or [6].

[0033]

[12] The production method according to

[10] or

[11] , comprising a step of allowing alanine dehydrogenase and / or NADH oxidase to coexist in part or all of the above method.

[0034]

[13] The production method according to any one of

[10] to

[12] , wherein trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is produced from cis-isomer and / or trans-isomer 1,4-bis(aminomethyl)cyclohexane.

[0035]

[14] The production method according to any one of

[10] to

[12] , wherein cis-4-(aminomethyl)cyclohexane-1-carboxylic acid is produced from cis-isomer and / or trans-isomer of 1,4-bis(aminomethyl)cyclohexane.

[0036]

[15] The production method according to any one of

[10] to

[13] , wherein part or all of the method is carried out under neutral or alkaline conditions.

[0037]

[16] The production method according to

[15] , wherein part or all of the method is carried out in the presence of a secondary amine.

[0038]

[17] A protein which is an aldehyde dehydrogenase and has an aldehyde dehydrogenase activity of oxidizing the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0039]

[18] The protein according to

[17] , which is a benzaldehyde dehydrogenase having an aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0040]

[19] The protein according to

[17] or

[18] , wherein the aldehyde dehydrogenase is an aldehyde dehydrogenase derived from bacteria of the genus Pseudomonas.

[0041]

[20] The protein according to

[17] or

[18] , which consists of an amino acid sequence that is 50% or more identical to the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139.

[0042]

[21] A protein having aldehyde dehydrogenase activity for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carboxaldehyde, the protein comprising an amino acid sequence having an identity of 50% or more to the amino acid sequence shown in SEQ ID NO: 20 and containing at least one of the following amino acid bases (1) to (26) when aligned with the amino acid sequence shown in SEQ ID NO: 20.

[0043] (1) The amino acid residue at position 35 is a glycine residue (G)

[0044] (2) The amino acid residue at position 65 is an alanine residue (A)

[0045] (3) The amino acid residue at position 82 is a leucine residue (L)

[0046] (4) The amino acid residue at position 104 is a glycine residue (G)

[0047] (5) The amino acid residue at position 109 is a glycine residue (G)

[0048] (6) The amino acid residue at position 110 is an isoleucine residue (I)

[0049] (7) The amino acid residue at position 117 is a glutamine residue (Q)

[0050] (8) The amino acid residue at position 122 is an alanine residue (A)

[0051] (9) The amino acid residue at position 124 is a leucine residue (L)

[0052] (10) The amino acid residue at position 134 is a valine residue (V)

[0053] (11) The amino acid residue at position 144 is a valine residue (V)

[0054] (12) The amino acid residue at position 160 is a phenylalanine residue (F)

[0055] (13) The amino acid residue at position 271 is a glycine residue (G)

[0056] (14) The amino acid residue at position 290 is an alanine residue (A)

[0057] (15) The amino acid residue at position 319 is an aspartic acid residue (D)

[0058] (16) The amino acid residue corresponding to position 333 is an aspartic acid residue (D)

[0059] (17) The amino acid residue corresponding to position 365 is a glutamine residue (Q)

[0060] (18) The amino acid residue at position 390 is an isoleucine residue (I)

[0061] (19) The amino acid residue at position 420 is an alanine residue (A)

[0062] (20) The amino acid residue at position 423 is a leucine residue (L)

[0063] (21) The amino acid residue at position 434 is a valine residue (V)

[0064] (22) The amino acid residue corresponding to position 440 is a proline residue (P)

[0065] (23) The amino acid residue corresponding to position 443 is a cysteine ​​residue (C)

[0066] (24) The amino acid residue corresponding to position 445 is a proline residue (P)

[0067] (25) The amino acid residue at position 466 is a serine residue (S)

[0068] (26) The amino acid residue at position 467 is an isoleucine residue (I)

[0069]

[22] A DNA encoding the protein described in any one of

[17] to

[21] .

[0070]

[23] A recombinant DNA comprising the DNA described in

[22] .

[0071]

[24] A recombinant cell comprising the DNA described in

[22] , or obtained by transforming a host cell with the recombinant DNA described in

[23] .

[0072]

[25] The production method according to

[10] , wherein the aminotransferase is the protein according to [1], and the aldehyde dehydrogenase is the protein according to any one of

[17] to

[21] .

[0073]

[26] The production method according to

[25] , wherein alanine dehydrogenase and / or NADH oxidase coexists in part or all of the above method.

[0074]

[27] The production method according to

[25] or

[26] , wherein trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is produced from cis-isomer and / or trans-isomer of 1,4-bis(aminomethyl)cyclohexane.

[0075]

[28] The production method according to

[25] or

[26] , wherein cis-4-(aminomethyl)cyclohexane-1-carboxylic acid is produced from cis-isomer and / or trans-isomer of 1,4-bis(aminomethyl)cyclohexane.

[0076]

[29] The production method according to any one of

[25] to

[28] , wherein part or all of the method is carried out under neutral or alkaline conditions.

[0077]

[30] The production method according to any one of

[25] to

[28] , wherein part or all of the method is carried out in the presence of a secondary amine.

[0078] Effects of the Invention

[0079] According to the protein having transamination activity and the protein having aldehyde dehydrogenase activity of the present invention, 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced using 1,4-bis(aminomethyl)cyclohexane as a substrate. In addition, by effectively utilizing the substrate specificity of the protein having aldehyde dehydrogenase activity according to the stereostructure of the target 4-(aminomethyl)cyclohexane-1-carboxylic acid, even when a mixture of cis-isomer and trans-isomer is used as a substrate, a compound having a target structure can be selectively produced in large quantities.

[0080] According to the manufacturing method of the present invention, 4-(aminomethyl)cyclohexane-1-carboxylic acid can be efficiently manufactured without high temperature and high pressure and when manufacturing cost is reduced. In addition, by introducing keto acid and / or coenzyme or their regeneration system into the reaction system, the enzyme reaction can be made more efficient, thereby the productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid can be improved. Moreover, in the case of using a mixture of cis-isomer and trans-isomer of 1,4-bis(aminomethyl)cyclohexane as a substrate, by using the aldehyde dehydrogenase of the present invention of 4-(aminomethyl)cyclohexane-1-carboxaldehyde that selectively acts on the trans-isomer and reacting under neutral or alkaline conditions, the isomerization reaction of cis-isomer to trans-isomer of 4-(aminomethyl)cyclohexane-1-carboxaldehyde as an intermediate can be promoted, and therefore 4-(aminomethyl)cyclohexane-carboxylic acid (tranexamic acid) of trans-isomer can be manufactured in high yield and high selectivity by dynamic kinetic resolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 A scheme for producing cis- or trans-isomer 4-(aminomethyl)cyclohexane-1-carboxaldehyde and cis- or trans-isomer 4-(aminomethyl)cyclohexane-1-carboxylic acid from cis- or trans-isomer 1,4-bis(aminomethyl)cyclohexane according to one embodiment is shown.

[0082] Figure 2 It is a schematic diagram showing the construction method of the aminotransferase (AT) expression plasmid (pQE80L-PpAT8) of Example 1.

[0083] Figure 3 The LC-MS chromatogram of the PatA enzyme reaction solution in Example 1 is shown (EIC: 142.10 m / z).

[0084] Figure 4Schematic diagram showing the construction method of the AT and aldehyde dehydrogenase (ALDH) co-expression plasmid (pET28a-PatA-XylC) in Example 7.

[0085] Figure 5A This is a diagram showing the results of the alignment of the amino acid sequences of aldehyde dehydrogenases in Example 11.

[0086] Figure 5B This is a diagram showing the results of the alignment of the amino acid sequences of aldehyde dehydrogenases in Example 11. DETAILED DESCRIPTION

[0087] 1. Compounds and enzyme reactions

[0088] 4-(aminomethyl)cyclohexane-1-carboxylic acid (hereinafter sometimes referred to as "AMCHA"), which is the target compound of the present invention, is an unnatural amino acid. 4-(aminomethyl)cyclohexane-1-carboxylic acid exists in cis-4-(aminomethyl)cyclohexane-1-carboxylic acid (cis-4-(aminomethyl)cyclohexanecarboxylic acid) which is a cis isomer and trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (trans-4-(aminomethyl)cyclohexanecarboxylic acid) which is a trans isomer. Among them, trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is also called tranexamic acid (hereinafter sometimes referred to as "TXA"). In the present specification, unless otherwise specified, "tranexamic acid" or "TXA" means trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, and cis-4-(aminomethyl)cyclohexane-1-carboxylic acid is referred to as cis-tranexamic acid or cis-TXA.

[0089] The enzyme reaction in the present invention includes two steps as shown below. In the first step (step (i)), 1,4-bis(aminomethyl)cyclohexane as a substrate compound reacts with a keto acid (e.g., pyruvate) as a compound capable of providing a carbonyl group, and is converted into 4-(aminomethyl)cyclohexane-1-carbaldehyde as an intermediate and a compound that accepts an amino group (alanine when pyruvate is used as the keto acid) by aminotransferase (AT). In the second step (step (ii)), the intermediate 4-(aminomethyl)cyclohexane-1-carbaldehyde is converted into NAD(P) by NAD(P) +It is converted into 4-(aminomethyl)cyclohexane-1-carboxylic acid and NAD(P)H( Figure 1 ).

[0090] In addition, when pyruvate is used as the compound for providing a carbonyl group in the first step, the generated alanine can be converted (regenerated) into pyruvate by alanine dehydrogenase, and in the second step, the generated NAD(P)H can be converted (regenerated) into NAD(P) by NAD(P)H oxidase. + In addition, NAD(P)H can be electrochemically oxidized to produce NAD(P) + regeneration( Figure 1 ).

[0091] The substrate compound 1,4-bis(aminomethyl)cyclohexane can be a mixture of cis and trans isomers, can be trans isomers, or can be cis isomers. The intermediate 4-(aminomethyl)cyclohexane-1-carbaldehyde can also be a mixture of cis and trans isomers, can be trans isomers, or can be cis isomers.

[0092] 2. Protein and DNA of the present invention

[0093] (1) Aminotransferase of the present invention and DNA encoding the same

[0094] Aminotransferase (also referred to as transaminase, aminotransferase, AT or transaminase) is a general term for enzymes that catalyze the reaction between amino acids and α-keto acids in biochemistry. In this specification, having transamination activity means the activity of catalyzing the reaction of transferring an amino group from a compound having an amino group to another compound to convert it into a carbonyl group.

[0095] The protein having amino transfer activity of the present invention is a protein having the activity of transferring the amino group of 1,4-bis(aminomethyl)cyclohexane to another compound to produce 4-(aminomethyl)cyclohexane-1-carboxaldehyde. That is, it is a protein having the activity of mediating the reaction from 1,4-bis(aminomethyl)cyclohexane to 4-(aminomethyl)cyclohexane-1-carboxaldehyde, specifically, it is a protein having the activity of removing the amino group from 1,4-bis(aminomethyl)cyclohexane to produce 4-(aminomethyl)cyclohexane-1-carboxaldehyde. In this specification, these proteins are collectively referred to as "aminotransferase of the present invention" or "AT of the present invention".

[0096] AT according to one embodiment is, for example, a protein composed of an amino acid sequence having 60% or more (preferably 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more or 98% or more) identity with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105 and having amino transfer activity.

[0097] AT in the present embodiment is a protein composed of an amino acid sequence shown in any one of sequence numbers 1 to 4 and 103 to 105. The protein having the amino acid sequence shown in sequence number 1 is an aminotransferase PpAT8 derived from Pseudomonas putida KT2440, the protein having the amino acid sequence shown in sequence number 2 is an aminotransferase PpAT2 derived from Pseudomonas putida KT2440, the protein having the amino acid sequence shown in sequence number 3 is an aminotransferase AsAT5 derived from Aeromonas salmonicida subsp. Salmonicida, and the protein having the amino acid sequence shown in sequence number 4 is an aminotransferase PatA derived from Escherichia coli K12MG1655. The proteins consisting of the amino acid sequences shown in SEQ ID NOs. 103 to 105 are homologous proteins of the proteins (PpAT8 or PatA) consisting of the amino acid sequences shown in SEQ ID NOs. 1 or 4 and derived from microorganisms listed in Table 3. It has not been known so far that these proteins have the activity of transferring the amino group of 1,4-bis(aminomethyl)cyclohexane to other compounds to produce 4-(aminomethyl)cyclohexane-1-carbaldehyde.

[0098] AT in another embodiment is a mutant protein or homologous protein of a protein composed of the amino acid sequence shown in any one of the above-mentioned sequence numbers 1 to 4 and 103 to 105, and is a protein composed of an amino acid sequence having an identity of more than 60% (preferably more than 70%, more than 75%, more than 80%, more than 90%, more than 93%, more than 95% or more than 98%) with the amino acid sequence shown in any one of the sequence numbers 1 to 4 and 103 to 105 and having amino transfer activity.

[0099] In this specification, mutant protein refers to a protein obtained by artificially deleting or replacing amino acid residues in a protein as a basis, or artificially inserting or adding amino acid residues in the protein. In this specification, homologous protein refers to a protein possessed by organisms existing in nature, and is a group of proteins whose evolutionary origin is derived from the same protein. Homologous proteins are similar in structure and function to each other.

[0100] In the mutant protein, the deletion, substitution, insertion or addition of amino acids may be a deletion, substitution, insertion or addition of 1 to 20 amino acids at any position in the amino acid sequence, for example, it may be a deletion, substitution, insertion or addition of 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid.

[0101] The amino acid to be replaced, inserted or added may be either natural or non-natural. As natural amino acids, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine ​​etc. can be listed.

[0102] Examples of amino acids that can be substituted for each other are shown below. Amino acids included in the same group can be substituted for each other.

[0103] Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, o-methylserine, tert-butylglycine, tert-butylalanine, cyclohexylalanine

[0104] Group B: Aspartic acid, Glutamic acid, Isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid

[0105] Group C: Asparagine, Glutamine

[0106] Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid

[0107] Group E: Proline, 3-hydroxyproline, 4-hydroxyproline

[0108] Group F: serine, threonine, homoserine

[0109] Group G: Phenylalanine, Tyrosine

[0110] The amino acid sequence of a mutant protein or homologous protein of a protein composed of the amino acid sequence shown in any one of sequence numbers 1 to 4 and 103 to 105 has an identity of at least 60%, preferably 70%, 75%, 80%, 90% or 93%, further preferably 95% or more, and most preferably 98% or more relative to the amino acid sequence shown in any one of sequence numbers 1 to 4 and 103 to 105.

[0111] The identity (also called homology) of amino acid sequences and base sequences can be determined using the algorithms BLAST [Pro. Nat. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] developed by Karlin and Altschul. Based on the algorithm BLAST, programs called BLASTN and BLASTX were developed [J. Mol. Biol., 215, 403 (1990)]. When BLASTN is used to analyze base sequences based on BLAST, the parameters are set to, for example, Score=100 and wordlength=12. In addition, when BLASTX is used to analyze amino acid sequences based on BLAST, the parameters are set to, for example, score=50 and wordlength=3. When using BLAST and Gap ped BLAST programs, the default parameters of each program are used. The specific means of these analysis methods are well known.

[0112] The presence of transamination activity can be confirmed, for example, by the following method. First, a recombinant DNA containing a DNA encoding a protein for which the above-mentioned activity is to be confirmed is prepared by the method described below. Then, a microorganism such as Escherichia coli is transformed with the recombinant DNA and the obtained microorganism is cultured, and 1,4-bis(aminomethyl)cyclohexane and pyruvic acid are added to the culture medium to generate 4-(aminomethyl)cyclohexane-1-carboxaldehyde. Finally, 4-(aminomethyl)cyclohexane-1-carboxaldehyde is detected in the bacteria and in the culture supernatant using a conventional analytical method such as HPLC, thereby confirming that the target protein has transamination activity.

[0113] The DNA encoding AT in one embodiment is a DNA encoding a protein having an amino acid sequence having an identity of 60% or more (preferably 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more) with the amino acid sequence shown in any one of SEQ ID NOs. 1 to 4 and 103 to 105 and having transamination activity. In the present specification, the DNA may be a gene or a part of a gene.

[0114] The AT-encoding DNA of the present embodiment is a base sequence encoding a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs. 1 to 4 and 103 to 105, and an example thereof includes a DNA consisting of a base sequence represented by any one of SEQ ID NOs. 5 to 8 and 108 to 110.

[0115] The DNA encoding AT in another embodiment is a DNA encoding a mutant protein or a homologous protein of a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105. The DNA encoding the mutant protein or the homologous protein may have 1 to 50 bases deleted, substituted, inserted or added at any position of the base sequence represented by any one of SEQ ID NOs: 5 to 8 and 108 to 110, for example, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 base deleted, substituted, inserted or added. In addition, the DNA encoding the mutant protein or homologous protein is preferably composed of a base sequence having an identity of at least 60% or more, preferably 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or 93% or more, further preferably 95% or more, and most preferably 98% or more to the base sequence shown in any one of sequence numbers 5 to 8 and 108 to 110, and is more preferably composed of a base sequence shown in any one of sequence numbers 5 to 8 and 108 to 110.

[0116] The DNA consisting of the base sequence shown in any one of sequence numbers 5 to 8 and 108 to 110 or the DNA of a homologous protein with a known coding sequence can be amplified by PCR using appropriate primers using genomic DNA as a template. At this time, the end of the primer may contain a restriction enzyme site or the like for cloning into a DNA sequence in an expression vector. For homologous proteins, probes or primers can also be designed based on retrieval, and DNA encoding homologous proteins can be obtained using a microorganism having the DNA. DNA encoding mutant proteins can be obtained by error-prone PCR, site-directed mutagenesis using PCR, or commercially available site-directed mutagenesis kits, etc. In addition, the DNA encoding AT of one embodiment can also be prepared by chemical synthesis based on the base sequence of the determined DNA using an NTS M series DNA synthesizer manufactured by Japan TECHNOSERVICE Co., Ltd., etc. to prepare the target DNA.

[0117] Here, the expression level of the protein encoded by the DNA can be increased by base substitution of the DNA of one embodiment so as to use the codons most suitable for expression in the host cell. Information on the codon usage frequency in the host cell can be obtained from a public database.

[0118] The DNA encoding AT in one embodiment may also be a DNA that hybridizes under stringent conditions with a DNA consisting of a complementary base sequence of a base sequence represented by any one of SEQ ID NOs. 5 to 8 and 108 to 110. Hybridization refers to a step in which a DNA hybridizes with a DNA having a specific base sequence or a portion of the DNA. Therefore, the base sequence of the DNA that hybridizes with the DNA having a specific base sequence or a portion of the DNA may be a DNA of a length that is useful as a probe for Northern or Southern blot analysis or that can be used as an oligonucleotide primer for PCR analysis.

[0119] Examples of DNA used as a probe include DNA having at least 100 bases, preferably 200 bases or more, and more preferably 500 bases or more. Examples of DNA used as a primer include DNA having at least 10 bases, preferably 15 bases or more.

[0120] The method of DNA hybridization experiment is well known. For example, the hybridization conditions can be determined and the experiment can be performed according to Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press (2012)), Methods for General and Molecular Bacteriology (ASM Press (1994)), Immunology methods manual (Academic press (1997)) and various other standard textbooks.

[0121] In addition, DNA hybridized under stringent conditions can also be obtained according to the instructions attached to commercially available hybridization kits. Commercially available hybridization kits include, for example, a random primer DNA labeling kit (manufactured by Roche Diagnostics) that prepares probes by the random primer method and hybridizes under stringent conditions.

[0122] The above-mentioned stringent conditions can be exemplified by the following conditions: the filter membrane fixed with DNA and the probe DNA are incubated overnight at 42°C in a solution containing 50% formamide, 5×SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhart's solution, 10% dextran sulfate and 20 μg / L denatured salmon sperm DNA, and then the filter membrane is washed in a 0.2×SSC solution at about 65°C.

[0123] The above-mentioned various conditions can also be set by adding or changing the blocking reagent used to suppress the background of the hybridization experiment. In order to make the conditions suitable, the addition of the above-mentioned blocking reagent may be accompanied by the change of the hybridization conditions.

[0124] Examples of DNAs that can hybridize under the above-mentioned stringent conditions include, for example, DNAs composed of a base sequence that has an identity of at least 60% or more, preferably 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 93% or more, further preferably 95% or more, and most preferably 98% or more, with the base sequence represented by any one of sequence numbers 5 to 8 and 108 to 110 when calculated using the above-mentioned BLAST, FASTA or other programs.

[0125] (2) Aldehyde dehydrogenase of the present invention and DNA encoding the same

[0126] Aldehyde dehydrogenases (ALDH) are enzymes in biochemistry that use NAD(P) + A general term for an enzyme group that coenzymes catalyze the oxidation reaction of aldehydes to carboxylic acids. In the present specification, the aldehyde dehydrogenase activity refers to the activity of catalyzing the reaction of oxidizing the aldehyde group of a compound having an aldehyde group to convert it into a carboxyl group.

[0127] The protein having aldehyde dehydrogenase activity of the present invention is a protein having the activity of oxidizing the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid. In other words, it is a protein having the activity of catalyzing the reaction from 4-(aminomethyl)cyclohexane-1-carboxaldehyde to 4-(aminomethyl)cyclohexane-1-carboxylic acid. In this specification, these proteins are collectively referred to as "aldehyde dehydrogenase of the present invention" or "ALDH of the present invention".

[0128] The ALDH of one embodiment is, for example, a protein consisting of an amino acid sequence having 50% or more or 60% or more (preferably 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more) identity with the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139, and having aldehyde dehydrogenase activity.

[0129] The ALDH of the present embodiment may be a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139. The protein having the amino acid sequence represented by SEQ ID NO: 19 is γ-aminobutyraldehyde dehydrogenase PatD derived from Escherichia coli K12MG1655, the protein having the amino acid sequence represented by SEQ ID NO: 20 is benzaldehyde dehydrogenase XylC derived from Pseudomonas putida CSV86, the protein having the amino acid sequence represented by SEQ ID NO: 21 is phenylacetaldehyde dehydrogenase StyD derived from Pseudomonas putida S12, and the protein having the amino acid sequence represented by SEQ ID NO: 22 is 4-hydroxybenzaldehyde dehydrogenase PchA derived from Pseudomonas putida NCIMB 9866. The proteins consisting of the amino acid sequences shown in SEQ ID NOs: 35 to 46 and 127 to 139 are homologous proteins of the proteins (PatD, XylC or StyD) consisting of the amino acid sequences shown in SEQ ID NOs: 19, 20 or 21 and derived from microorganisms described in Tables 13 and 20. It has not been known so far that these proteins have the activity of oxidizing the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0130] ALDH in another embodiment is a mutant protein or homologous protein of a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139, and is a protein consisting of an amino acid sequence having 50% or more or 60% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139, and having aldehyde dehydrogenase activity. The amino acid sequence of the mutant protein or homologous protein has at least 50% or more or 60% or more, preferably 70% or more, 75% or more, 80% or more, or 85% or more, more preferably 90% or more, further preferably 93% or more or 95% or more, and most preferably 98% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139.

[0131] The presence of aldehyde dehydrogenase activity can be confirmed, for example, by the following method. First, a recombinant DNA containing a DNA encoding a protein for which the activity is to be confirmed is prepared by the method described below. Then, a microorganism such as Escherichia coli is transformed with the recombinant DNA and the obtained microorganism is cultured, and 4-(aminomethyl)cyclohexane-1-carboxaldehyde and NAD(P) are added to the culture medium. + Finally, by detecting 4-(aminomethyl)cyclohexane-1-carboxylic acid in the cells and in the culture supernatant using a conventional analytical method such as HPLC, it can be confirmed that the target protein has aldehyde dehydrogenase activity.

[0132] ALDH according to another embodiment may be a protein having aldehyde dehydrogenase activity to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carboxaldehyde and may be classified into any one or more of the following 1) to 6).

[0133] 1) A protein consisting of an amino acid sequence having 71% or more identity with the amino acid sequence shown in SEQ ID NO: 19

[0134] 2) A protein consisting of an amino acid sequence having 68% or more identity with the amino acid sequence shown in SEQ ID NO: 20

[0135] 3) Aldehyde dehydrogenase derived from Pseudomonas, a protein consisting of an amino acid sequence having 50% or more identity with the amino acid sequence shown in SEQ ID NO: 20

[0136] 4) A protein consisting of an amino acid sequence having 63% or more identity with the amino acid sequence shown in SEQ ID NO: 21

[0137] 5) A protein consisting of an amino acid sequence having 56.5% or more identity with the amino acid sequence shown in SEQ ID NO: 40

[0138] 6) A protein consisting of an amino acid sequence having 63.9% or more identity with the amino acid sequence shown in SEQ ID NO: 41

[0139] Examples of such proteins having aldehyde dehydrogenase activity include proteins consisting of an amino acid sequence represented by any one of SEQ ID NOs: 19 to 21, 35 to 41, 44 to 46, 127 to 131, 133 to 135, 138, and 139.

[0140] In another embodiment, the ALDH is preferably an aldehyde dehydrogenase derived from bacteria of the genus Pseudomonas.

[0141] Examples of Pseudomonas bacteria include Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas sp., Pseudomonas sp. MAP12, Pseudomonas fluorescens, Pseudomonas syringae, Pseudomonas amygdali, Pseudomonas oryzihabitans, Pseudomonas maltophilia, Pseudomonas trivialis, Pseudomonas savastanoi, and Pseudomonas stutzeri.

[0142] ALDH according to another embodiment is benzaldehyde dehydrogenase, which is a protein having aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0143] The benzaldehyde dehydrogenase of this embodiment has an aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid. Benzaldehyde dehydrogenase is a type of aldehyde dehydrogenase, and is a general term for enzymes that catalyze the oxidation reaction of benzaldehyde to benzoic acid.

[0144] The benzaldehyde dehydrogenase of the present embodiment may be derived from a microorganism expressing benzaldehyde dehydrogenase in addition to the benzaldehyde dehydrogenase derived from the Pseudomonas bacteria described above. Examples of the microorganism expressing benzaldehyde dehydrogenase include Hydrogenophaga aromaticivorans, Alteromonas, Tepidiphilus succinatimandens, Halomonas cupida, Glaciimonas immobilis, Paraburkholderia unamae, Marinobacter salsuginis, Aromatoleum toluclasticum, Burkholderia sp., Burkholderia sp. D7, Bacillus subtilis, Acinetobacter guillouiae, Polaromonas sp., Bacillus sp. sp.), Chloroflexi bacterium, Halioxenophilus aromaticivorans, Novosphingobium aromaticivorans, Oceanobacillusiheyensis, Sphingobium chungbukense, Streptomyces violaceoruber, Rhodococcus aetherivorans, Xanthomonas campestris, Streptomyces calvus, Prauserella muralis, Acinetobacter calcoaceticus, Xanthomonas campestris, Acidovorax sp., Streptomyces sp., etc.

[0145] The benzaldehyde dehydrogenase of one embodiment may be a protein consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 20, 40, 41, 43, 127 to 137, and 139. In addition, the benzaldehyde dehydrogenase of another embodiment may be a mutant protein or a homologous protein of the protein consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 20, 40, 41, 43, 127 to 137, and 139, and may be a protein consisting of an amino acid sequence having 50% or more or 60% or more (preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity with the amino acid sequence as shown in any one of SEQ ID NOs: 20, 40, 41, 43, 127 to 137, and 139, and having aldehyde dehydrogenase activity.

[0146] The benzaldehyde dehydrogenase according to another embodiment may be a protein having aldehyde dehydrogenase activity to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carboxaldehyde and may be classified into any one or more of the following 7) to 12).

[0147] 7) A protein consisting of an amino acid sequence having 71% or more identity with the amino acid sequence shown in SEQ ID NO: 19

[0148] 8) A protein consisting of an amino acid sequence having 68% or more identity with the amino acid sequence shown in SEQ ID NO: 20

[0149] 9) Aldehyde dehydrogenase derived from Pseudomonas sp., and a protein consisting of an amino acid sequence having 50% or more identity with the amino acid sequence shown in SEQ ID NO: 20

[0150] 10) A protein consisting of an amino acid sequence having 63% or more identity with the amino acid sequence shown in SEQ ID NO: 21

[0151] 11) A protein consisting of an amino acid sequence having 56.5% or more identity with the amino acid sequence shown in SEQ ID NO: 40

[0152] 12) A protein consisting of an amino acid sequence having 63.9% or more identity with the amino acid sequence shown in SEQ ID NO: 41

[0153] Examples of such benzaldehyde dehydrogenases include proteins consisting of an amino acid sequence represented by any one of SEQ ID NOs: 20, 40, 41, 127 to 131, 133 to 135, and 139.

[0154] ALDH in another embodiment may be a protein having aldehyde dehydrogenase activity for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carboxaldehyde, and comprising an amino acid sequence having 50% or more (preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, or 98% or more) identity with the amino acid sequence set forth in SEQ ID NO: 20, and containing at least one of the following amino acid groups (1) to (26) when aligned with the amino acid sequence set forth in SEQ ID NO: 20.

[0155] (1) The amino acid residue at position 35 is a glycine residue (G)

[0156] (2) The amino acid residue at position 65 is an alanine residue (A)

[0157] (3) The amino acid residue at position 82 is a leucine residue (L)

[0158] (4) The amino acid residue at position 104 is a glycine residue (G)

[0159] (5) The amino acid residue at position 109 is a glycine residue (G)

[0160] (6) The amino acid residue at position 110 is an isoleucine residue (I)

[0161] (7) The amino acid residue at position 117 is a glutamine residue (Q)

[0162] (8) The amino acid residue at position 122 is an alanine residue (A)

[0163] (9) The amino acid residue at position 124 is a leucine residue (L)

[0164] (10) The amino acid residue at position 134 is a valine residue (V)

[0165] (11) The amino acid residue at position 144 is a valine residue (V)

[0166] (12) The amino acid residue at position 160 is a phenylalanine residue (F)

[0167] (13) The amino acid residue at position 271 is a glycine residue (G)

[0168] (14) The amino acid residue at position 290 is an alanine residue (A)

[0169] (15) The amino acid residue at position 319 is an aspartic acid residue (D)

[0170] (16) The amino acid residue corresponding to position 333 is an aspartic acid residue (D)

[0171] (17) The amino acid residue corresponding to position 365 is a glutamine residue (Q)

[0172] (18) The amino acid residue at position 390 is an isoleucine residue (I)

[0173] (19) The amino acid residue at position 420 is an alanine residue (A)

[0174] (20) The amino acid residue at position 423 is a leucine residue (L)

[0175] (21) The amino acid residue at position 434 is a valine residue (V)

[0176] (22) The amino acid residue corresponding to position 440 is a proline residue (P)

[0177] (23) The amino acid residue corresponding to position 443 is a cysteine ​​residue (C)

[0178] (24) The amino acid residue corresponding to position 445 is a proline residue (P)

[0179] (25) The amino acid residue at position 466 is a serine residue (S)

[0180] (26) The amino acid residue at position 467 is an isoleucine residue (I)

[0181] Here, for example, "the amino acid residue at the position corresponding to position 35" means: when the amino acid sequence of the target protein is aligned with the amino acid sequence shown in sequence number 20, the amino acid residue at the position corresponding to the position of the amino acid residue at position 35 of sequence number 20 in the amino acid sequence of the target protein.

[0182] As shown in the Examples described below, it is suggested that the amino acid residues shown in (1) to (26) above are conserved amino acid residues in ALDH having particularly high specificity for trans-4-(aminomethyl)cyclohexane-1-carboxaldehyde. Therefore, it is considered that the amino acid sequence of the aldehyde dehydrogenase of the present embodiment has specificity for trans-4-(aminomethyl)cyclohexane-1-carboxaldehyde because it contains at least one amino acid group among (1) to (26). That is, the ALDH of the present embodiment can have an aldehyde dehydrogenase activity with high trans selectivity (also referred to as trans specificity). Here, high trans selectivity means that when ALDH is used to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, the trans ratio of 4-(aminomethyl)cyclohexane-1-carboxylic acid calculated using the calculation formula shown in Mathematical Formula 2 described below in the generated 4-(aminomethyl)cyclohexane-1-carboxylic acid containing tranexamic acid (TXA) and / or cis-TXA exceeds 50%, and particularly high trans specificity means that the above trans ratio is 70% or more. It should be noted that the term "selectivity" in the present application may be regarded as synonymous with "specificity", and the term "selection" may be regarded as synonymous with "specific".

[0183] The amino acid sequence of the aldehyde dehydrogenase of the present embodiment may contain at least one of the amino acid groups (1) to (26) above. For example, it may contain 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, or all 26 of them.

[0184] The alignment of amino acid sequences can be created using, for example, the well-known alignment program Clustal Omega. Clustal Omega is available, for example, from https: / / www.ebi.ac.uk / Tools / msa / clustalo / . When creating an alignment using Clustal Omega, for example, the default parameters can be used.

[0185] The aldehyde dehydrogenase of the present embodiment may be a protein having an aldehyde dehydrogenase activity and comprising an amino acid sequence having 50% or more (preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, or 98% or more) identity with the amino acid sequence represented by any one of SEQ ID NOs: 20, 40, 41, 127, 128, 130, 131, and 133.

[0186] The DNA encoding aldehyde dehydrogenase (ALDH) of the present embodiment is a DNA encoding a protein having the above-mentioned aldehyde dehydrogenase activity. The DNA encoding ALDH of one embodiment is a base sequence encoding a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139, and as an example thereof, a DNA consisting of a base sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152 can be cited.

[0187] The DNA encoding ALDH in another embodiment is a DNA encoding a mutant protein or a homologous protein of a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139. The DNA encoding the mutant protein or the homologous protein may have 1 to 50 bases deleted, substituted, inserted, or added at any position of the base sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152, for example, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 base deleted, substituted, inserted, or added. The DNA encoding ALDH according to one embodiment is preferably composed of a base sequence having at least 50% or 60% identity with the base sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152, preferably 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, further preferably 93% or more or 95% or more, and most preferably 98% or more, and more preferably consists of the base sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152.

[0188] The DNA encoding ALDH according to one embodiment may be a DNA that hybridizes under stringent conditions with a DNA consisting of a complementary base sequence to the base sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152. Examples of DNA that can hybridize under stringent conditions include a DNA consisting of a base sequence that has at least 50% or 60% identity, preferably 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, more preferably 93% or more, or 95% or more, and most preferably 98% or more identity to the base sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152, when calculated using a program such as BLAST or FASTA.

[0189] 3. Recombinant DNA of the present invention

[0190] The recombinant DNA of the present invention contains a DNA encoding the aminotransferase of the present invention and / or a DNA encoding the aldehyde dehydrogenase of the present invention. It may also contain a DNA encoding alanine dehydrogenase and / or a DNA encoding NADH oxidase described below. The recombinant DNA of the present invention may contain a DNA encoding only one enzyme in one recombinant DNA, or may contain a DNA encoding two or more enzymes in one recombinant DNA. In the case of two or more enzymes, each enzyme may be controlled by one promoter or by separate promoters.

[0191] The recombinant DNA of the present invention is a vector capable of autonomous replication in a host cell and / or a vector capable of integration into the chromosome of a host cell, and is a vector capable of transcribing the above DNA.

[0192] When prokaryotes such as bacteria are used as host cells, the recombinant DNA of the present invention preferably contains a promoter, a ribosome binding sequence and a transcription termination sequence in addition to the DNA encoding the above-mentioned various enzymes, and may further contain a gene that controls the promoter. Here, it is preferred to adjust the distance between the SD (Shine-Dalgarno) sequence as the ribosome binding sequence and the start codon to an appropriate distance, for example, 6 to 18 bases. In the recombinant DNA of the present invention, a transcription termination sequence is not necessarily required for the expression of the DNA encoding the above-mentioned various enzymes, but it is preferred to configure a transcription termination sequence immediately downstream of the structural gene.

[0193] The vector is not particularly limited as long as it is an appropriate DNA molecule for introducing a target DNA into a host, growing it, and expressing it. Not only plasmids but also artificial chromosomes, vectors using transposons, and cosmids can be used.

[0194] When a microorganism belonging to the genus Escherichia is used as a host cell for introducing the recombinant DNA of the present invention, examples of vectors include pColdI, pSTV28, pSTV29, pUC118 (all manufactured by Takara Bio), pMW118, pMW119, and pMW218 (all manufactured by NIPPON GENE), pET21a, pET28a, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5x (manufactured by NanoLab), pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Life Sciences), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE-60, pQE80L (all manufactured by QIAGEN), pET-3, pBluescriptII SK(+), pBluescriptII KS(-) (all manufactured by Agilent Technologies), pKYP10 (Japanese Patent Application Laid-Open No. 58-110600), pKYP200 [Agric.Biol.Chem., 48, 669 (1984)], pLSA1 [Agric.Biol.Chem., 53, 277 (1989)], pGEL1 [Proc.Natl.Acad.Sci., USA, 82, 4306 (1985)], pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, p6378-6385], pPE167 (Appl. Environ. Microbiol. 2007, 73: 6378-6385), pPAC31 (WO98 / 12343), pUC19 [Gene, 33, 103 (1985)], pPA1 (Japanese Patent Application Laid-Open No. 63-233798), etc.

[0195] As a promoter when using the above-mentioned vector, any promoter may be used as long as it functions in cells of microorganisms belonging to the genus Escherichia, and for example, promoters derived from Escherichia coli, bacteriophages, etc. such as trp promoter, gapA promoter, lac promoter, PL promoter, PR promoter, PSE promoter, etc. can be used. In addition, promoters artificially designed and modified such as a promoter in which two trp promoters are connected in series, a tac promoter, a trc promoter, a lacT5 promoter, a lacT7 promoter, and a let I promoter can also be used.

[0196] When coryneform bacteria are used as host cells for introducing the recombinant DNA of the present invention, examples of vectors include pCG1 (Japanese Patent Application Laid-Open No. 57-134500), pCG2 (Japanese Patent Application Laid-Open No. 58-35197), pCG4 (Japanese Patent Application Laid-Open No. 57-183799), pCG11 (Japanese Patent Application Laid-Open No. 57-134500), pCG116, pCE54, pCB101 (all see Japanese Patent Application Laid-Open No. 58-105999), pCE51, pCE52, pCE53 [all see Molecular and General Genetics, 196, 175 (1984)] and the like.

[0197] When the above-mentioned vector is used, any promoter may be used as long as it functions in cells of coryneform bacteria, and for example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, p674-679 (2000)] can be used.

[0198] When a yeast strain is used as a host cell for introducing the recombinant DNA of the present invention, examples of the vector include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0199] As a promoter when using the above-mentioned vector, any promoter may be used as long as it functions in cells of a yeast strain, and examples thereof include promoters such as PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0200] The recombinant DNA of the present invention can be prepared, for example, by using the In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio), or by subjecting a DNA fragment encoding a desired enzyme to restriction enzyme treatment and inserting it downstream of the promoter of the above-mentioned appropriate expression vector.

[0201] 4. Recombinant cells of the present invention

[0202] The recombinant cell of the present invention is a recombinant cell containing the DNA of the present invention, or a recombinant cell obtained by transforming a host cell with the recombinant DNA of the present invention. In addition, it may also contain a DNA encoding alanine dehydrogenase and / or a DNA encoding NADH oxidase described later. In the recombinant cell, the DNA of the present invention or the recombinant DNA may be integrated into the genome, or may exist in the form of a plasmid capable of autonomous replication, and contain the DNA of the present invention in a state capable of transcription. A host cell may contain only one DNA, or may contain two or more DNAs.

[0203] The host cell may be any of prokaryotes, yeast, animal cells, insect cells, plant cells, etc., preferably prokaryotes or yeast strains, more preferably prokaryotes belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwann yeast, Pichia, or Candida, and most preferably Escherichia coli BL21 codon plus, E. coli XL1-Blue, E. coli XL2-Blue (all manufactured by Agilent Technologies), E. coli BL21 (DE3) (manufactured by Novagene), E. coli BL21 (DE3) pLysS (manufactured by Merck Millipore), E. coli DH5α, E. coli HST08 Premium, E. coli HST02, E. coli HST04 dam- / dcm-, E. coli JM109, E. coli HB101, E. coli CJ236, E. coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio), Escherichia coli W, Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli ATCC9637, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354 or Pseudomonas sp. unspecified D-0110, or Saccharomyces cerevisiaecerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris or Candida utilis.

[0204] Examples of methods for introducing the recombinant DNA of the present invention in the form of a plasmid capable of autonomously replicating in host cells include a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], a protoplast method (Japanese Patent Application Laid-Open No. 63-248394), an electroporation method [Nucleic Acids Res., 16, 6127 (1988)], a spheroplast method [Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)], and a lithium acetate method [J. Bacteriol., 153, 163 (1983)].

[0205] As a method for introducing the recombinant DNA of the present invention into the chromosome of the host cell, for example, homologous recombination method can be cited. As a homologous recombination method, for example, a method using a homologous recombination plasmid can be cited, and the homologous recombination plasmid can be prepared by connecting with a plasmid DNA having a drug resistance gene that cannot replicate autonomously in the host cell to be introduced. As a method using homologous recombination commonly used in Escherichia coli, for example, a method for introducing recombinant DNA using the homologous recombination system of λ phage can be cited [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].

[0206] Furthermore, by using a selection method that utilizes the fact that Escherichia coli is made sucrose sensitive by integrating Bacillus subtilis levansucrase into the chromosome together with the recombinant DNA, or a selection method that utilizes the fact that Escherichia coli is made streptomycin sensitive by integrating the wild-type rpsL gene into Escherichia coli with a mutant rpsL gene having streptomycin resistance [Mol.Microbiol., 55, 137 (2005), Biosci.Biotechnol.Biochem., 71, 2905 (2007)], etc., it is possible to obtain Escherichia coli in which the target region on the chromosomal DNA of the host cell is replaced with the DNA of the present invention or the recombinant DNA.

[0207] 5. Manufacturing method of the present invention

[0208] The production method of the present invention is a production method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 1,4-bis(aminomethyl)cyclohexane, which comprises:

[0209] (i) a step of producing 4-(aminomethyl)cyclohexane-1-carbaldehyde from 1,4-bis(aminomethyl)cyclohexane in the presence of an aminotransferase; and

[0210] (ii) A step of producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carboxaldehyde in the presence of aldehyde dehydrogenase.

[0211] The production method of this embodiment includes a step of allowing alanine dehydrogenase and / or NADH oxidase to coexist in part or all of the method. In particular, it is preferred that alanine dehydrogenase and / or NADH oxidase coexist in the first step (step (i)), and NADH oxidase coexist in the second step (step (ii)).

[0212] When the substrate material (1,4-bis(aminomethyl)cyclohexane used in the first step or 4-(aminomethyl)cyclohexane-1-carboxaldehyde used in the second step) in each step of the production method of the present invention has a different stereostructure from the target product (4-(aminomethyl)cyclohexane-1-carboxaldehyde in the first step or 4-(aminomethyl)cyclohexane-1-carboxylic acid in the second step), it is preferred that a part or all of the production method of this embodiment is carried out under neutral or alkaline conditions. For example, it is preferred that a part or all of the steps of the first step and / or a part or all of the steps of the second step are carried out under neutral or alkaline conditions. When the reaction is carried out under neutral or alkaline conditions, the isomerization of the cis-isomer of 4-(aminomethyl)cyclohexane-1-carboxaldehyde as a reaction intermediate into the trans-isomer or the isomerization of the trans-isomer into the cis-isomer can be promoted. Isomerization is particularly easy under alkaline conditions above pH 9, and the isomerization of 4-(aminomethyl)cyclohexane-1-carboxaldehyde reaches equilibrium when the trans ratio is about 60%. Thus, for example, when tranexamic acid is the final target product, even when a cis-isomer or a mixture of a cis-isomer and a trans-isomer is used as a substrate, by using an aldehyde dehydrogenase with high trans selectivity described later, more trans-isomer 4-(aminomethyl)cyclohexane-1-carboxylic acid can be obtained, that is, a higher trans ratio of the product mixture can be obtained. Thus, tranexamic acid can be produced in high yield. On the other hand, when a trans-isomer is used as a substrate, by setting it to acidic (e.g., pH 4 to 6), more trans-isomer products can be obtained by preventing isomerization.

[0213] Neutral means that the pH of the reaction solution is about 7, alkaline means that the pH of the reaction solution is greater than 7 and is below 12, and neutral or alkaline means that the pH of the reaction solution is preferably 7 to 12, and more preferably 7 to 10. There is no particular limitation on the method of making it alkaline, and it can be achieved by adding an alkaline solution, urea, calcium carbonate, ammonia, etc. Part of the step is carried out under neutral or alkaline conditions, which means that it is set to neutral or alkaline from the beginning to the middle of the step, or from the middle to the end. Here, the beginning of the step refers to before the reaction solution contains an enzyme, and the end of the step refers to after the enzyme reaction is completed (the same applies below). When the cis-isomer is isomerized to the trans-isomer by neutrality or alkalinity, an isomerization rate of 20% or more, for example, 40% or more, 50% or more, or 60% or more can be obtained. In addition, when the trans-isomer is isomerized to the cis-isomer, an isomerization rate of 20% or more, for example, 30% or more or 35% or more can be obtained.

[0214] When the substrate material in each step of the production method of the present invention has a different stereostructure from the target product, it is preferred that a part or all of the reaction steps be carried out in the presence of a secondary amine. The presence of a secondary amine can promote the isomerization of 4-(aminomethyl)cyclohexane-1-carboxaldehyde. Examples of secondary amines include L-proline, pyrrolidine, pyrrolidine derivatives, trans-4-hydroxy-L-proline, and the like. The amount of secondary amine added to the reaction solution can be 0.01 to 500 mM, preferably 0.1 to 200 mM. By adding a secondary amine, an isomerization rate of 20% or more, for example, 60% or more, to a trans isomer can be obtained, or an isomerization rate of 20% or more, for example, 30% or more or 35% or more, to a cis isomer can be obtained.

[0215] (1) Steps for producing 4-(aminomethyl)cyclohexane-1-carbaldehyde

[0216] The production method of the present invention includes (i) a step of producing 4-(aminomethyl)cyclohexane-1-carboxaldehyde from 1,4-bis(aminomethyl)cyclohexane in the presence of an aminotransferase (hereinafter also referred to as step 1). As the aminotransferase, the aminotransferase of the present invention is preferably used. In the presence of an aminotransferase means that an aminotransferase that is active in a manner capable of catalyzing the reaction of (i) is present in the reaction system of (i). For example, the following steps are included: adding the aminotransferase of the present invention to the reaction system; or, allowing a cell that contains a DNA encoding the aminotransferase of the present invention and is capable of expressing the aminotransferase, or a cell that is capable of expressing the aminotransferase obtained by transforming a host cell with a recombinant DNA containing a DNA encoding the aminotransferase of the present invention to act. Here, allowing the cell to act refers to adding a substrate substance to the system in which the cell is cultured and acting on the protein expressed by the cell.

[0217] In step 1, a protein having amino transfer activity can be used as an enzyme source and the enzyme source and a reaction substrate can be present in an aqueous medium to generate and accumulate 4-(aminomethyl)cyclohexane-1-carboxaldehyde in the aqueous medium. Alternatively, cells having the ability to produce amino transferase can be cultured in a culture medium to generate and accumulate 4-(aminomethyl)cyclohexane-1-carboxaldehyde in the culture medium.

[0218] In the method using an enzyme source, the enzyme source may be a purified protein, or may be a culture obtained by culturing cells having an ability to produce a protein having a target activity in a culture medium, or a processed product of the culture.

[0219] The culture or the processed product of the culture contains a protein having a target activity as an enzyme source. Examples of the processed product of the culture include a concentrated product of the culture, a dried product of the culture, bacterial cells obtained by centrifugation of the culture, a dried product of the bacterial cells, a freeze-dried product of the bacterial cells, a surfactant-treated product of the bacterial cells, an ultrasonic-treated product of the bacterial cells, a mechanically ground product of the bacterial cells, a solvent-treated product of the bacterial cells, an enzyme-treated product of the bacterial cells, a protein fraction of the bacterial cells, a fixed product of the bacterial cells, or an enzyme standard product extracted from the bacterial cells.

[0220] In the method using the above enzyme source in step 1, when a purified protein is used as an enzyme source, the amount of the protein having transamination activity may be 0.01 to 100% by weight, preferably 0.1 to 50% by weight, relative to 1,4-bis(aminomethyl)cyclohexane. When a culture or a processed product of the culture is used as an enzyme source, the amount of the enzyme source varies depending on the specific activity of the enzyme source, for example, 5 to 1000% by weight, preferably 10 to 400% by weight, relative to 1,4-bis(aminomethyl)cyclohexane, based on the wet cell weight.

[0221] As the aqueous medium, water, phosphate, carbonate, acetate, borate, citrate, Tris, 2-morpholinoethanesulfonic acid (hereinafter referred to as MES), 3-morpholinopropanesulfonic acid (hereinafter referred to as MOPS), N-cyclohexyl-2-aminoethanesulfonic acid (hereinafter referred to as CHES) and other buffers, alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, amides such as acetamide, etc. can be listed. In addition, the culture solution of the microorganism used as the enzyme source can also be used as the aqueous medium.

[0222] As described in 1. above, the reaction of producing 4-(aminomethyl)cyclohexane-1-carbaldehyde from 1,4-bis(aminomethyl)cyclohexane is an enzyme reaction catalyzed by aminotransferase (protein having transamination activity) in the presence of a compound capable of donating a carbonyl group. Therefore, it is preferred that a compound capable of donating a carbonyl group coexists in the first step.

[0223] Examples of the compound capable of donating a carbonyl group include keto acids, and preferably pyruvic acid, α-ketoglutaric acid, α-ketobutyric acid, and the like.

[0224] The source of keto acid is not limited as long as it can serve as a substrate for the aminotransferase of the present invention. A culture of a microorganism capable of producing keto acid or a processed product of the culture may be used directly, or keto acid recovered from the culture or a processed product of the culture may be used.

[0225] In addition, when culturing a microorganism in a culture medium to generate and accumulate 4-(aminomethyl)cyclohexane-1-carboxaldehyde, a microorganism having the ability to produce keto acid can be used as the microorganism. The microorganism having the ability to produce keto acid can include, for example, a microorganism in which the gene of the pyruvate dehydrogenase complex that participates in the decomposition of pyruvate in vivo is deleted or weakened, a microorganism in which the lactate dehydrogenase gene is deleted or weakened, a microorganism in which the pyruvate oxidase gene is deleted or weakened, etc. Here, "weakened" refers to a reduction in the expression level of the target gene (here, the gene of the pyruvate dehydrogenase complex).

[0226] When the keto acid is added in the first step, the amount thereof added may be 0.01 to 100, preferably 0.01 to 60, in terms of molar ratio relative to 1,4-bis(aminomethyl)cyclohexane.

[0227] In the reaction step of step 1, when pyruvic acid is used as the keto acid, it is preferred that alanine dehydrogenase (AlaDH) be present. As described in 1. above, AlaDH is a catalytic enzyme that can utilize NAD + As a coenzyme, AlaDH is an enzyme that catalyzes the conversion (regeneration) of the generated alanine into pyruvate. Therefore, by making AlaDH coexist, pyruvate can be reused, and 4-(aminomethyl)cyclohexane-1-carbaldehyde can be efficiently produced with a small amount of raw materials. In addition, when AlaDH is coexistent, it is preferable to add NAD + .

[0228] AlaDH is one of the enzymes for alanine / aspartate / glutamate metabolism, which catalyzes (alanine + water + NAD + ) and (pyruvate + NH3 + NADH + H + In the first step of the method for producing tranexamic acid of the present invention, when pyruvic acid is used as the keto acid, the generated alanine can be converted into pyruvic acid.

[0229] AlaDH is not particularly limited and may be, for example, AlaDH derived from Bacillus subtilis, Alkalihalobacillus pseudofirmus, or Staphylococcus aureus. Specifically, it includes alanine dehydrogenase BsAlaDH derived from Bacillus subtilis 168 (accession number: WP_003243280.1), alanine dehydrogenase ApAlaDH derived from Alkalihalobacillus pseudofirmus (accession number: WP_012957376.1), and alanine dehydrogenase SaAlaDH derived from Staphylococcus aureus (accession number: WP_000689998.1).

[0230] Regarding the co-presence of AlaDH, a culture of cells expressing AlaDH or a processed product of the culture may be added to the enzyme reaction in the first step, cells expressing AlaDH may be co-cultured, or the recombinant cells may contain a DNA encoding AlaDH.

[0231] The amount of AlaDH may be 0.01 to 100% by weight, preferably 0.1 to 50% by weight, relative to alanine produced in the amino transfer reaction. When a culture or a processed product of the culture is used as an enzyme source, the amount of the enzyme source varies depending on the specific activity of the enzyme source, for example, 5 to 1000% by weight, preferably 10 to 400% by weight, relative to 1,4-bis(aminomethyl)cyclohexane in terms of wet bacterial weight. In addition, it is preferred to add NAD + About NAD + The amount of addition may be 0.01 to 100, preferably 0.01 to 60, in terms of molar ratio relative to alanine produced in the transamination reaction.

[0232] 1,4-bis(aminomethyl)cyclohexane as substrate can be any one of trans-isomer, cis-isomer or a mixture thereof, and is preferably a mixture of trans-isomer and cis-isomer from the ease of obtaining raw materials. In addition, the target 4-(aminomethyl)cyclohexane-1-carboxaldehyde can be any one of trans-isomer, cis-isomer or a mixture thereof. When the generated 4-(aminomethyl)cyclohexane-1-carboxaldehyde is supplied to the manufacture of tranexamic acid, it is preferably a trans-isomer. In a mixture of trans-isomer and cis-isomer, the ratio of trans-isomer to cis-isomer is not limited, but it is preferred that the ratio of trans-isomer exceeds 50%.

[0233] (2) Step of producing 4-(aminomethyl)cyclohexane-1-carboxylic acid

[0234] The production method of the present invention comprises the step (ii) of producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carboxaldehyde in the presence of an aldehyde dehydrogenase (hereinafter also referred to as the second step). Preferably, the step comprises allowing the protein having aldehyde dehydrogenase activity of the present invention, a cell containing a DNA encoding the protein having aldehyde dehydrogenase activity of the present invention, or a recombinant cell obtained by transforming a host cell with a recombinant DNA containing a DNA encoding the protein having aldehyde dehydrogenase activity of the present invention to act on 4-(aminomethyl)cyclohexane-1-carboxaldehyde.

[0235] In the second step, a protein having aldehyde dehydrogenase activity can be used as an enzyme source and the enzyme source and a reaction substrate can be present in an aqueous medium to produce and accumulate 4-(aminomethyl)cyclohexane-1-carboxylic acid in an aqueous medium. Alternatively, a microorganism having the ability to produce aldehyde dehydrogenase can be cultured in a culture medium to produce and accumulate 4-(aminomethyl)cyclohexane-1-carboxylic acid in the culture.

[0236] As described in 1. above, the reaction of generating 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carboxaldehyde is carried out in the presence of NAD(P) as a coenzyme. + In the presence of aldehyde dehydrogenase (a protein having aldehyde dehydrogenase activity), an enzyme reaction is catalyzed. Aldehyde dehydrogenase includes + NAD as a coenzyme + Type, prefer NADP + NADP as coenzyme + Type, capable of utilizing NAD + and NADP + These two are types of coenzymes. Therefore, it is preferable to allow a coenzyme with high utilization of the aldehyde dehydrogenase to be used to coexist.

[0237] As NAD(P) + The amount of 4-(aminomethyl)cyclohexane-1-carbaldehyde may be 0.01 to 100, preferably 0.01 to 60, in terms of molar ratio.

[0238] In addition, as a method for supplying a coenzyme, a microorganism having the ability to produce a coenzyme can be used as a host for expressing aldehyde dehydrogenase. As a result, it is expected that the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid will be further improved. Examples of microorganisms having the ability to produce a coenzyme include, for example, + Microorganisms whose genes for decomposition are missing or weakened, or genes involved in NAD(P) are enhanced or introduced + Genes (e.g., pncB, nadA, nadB) produced by microorganisms, inhibiting the above-mentioned NAD(P) +Microorganisms in which a gene (e.g., nadR) that controls the expression or function of a gene product produced is defective or weakened, or in which a gene involved in NAD(P) + Microorganisms with defective or weakened decomposition genes (e.g., mazG, nudC).

[0239] The amount of aldehyde dehydrogenase may be 0.01 to 100% by weight, preferably 0.1 to 50% by weight, relative to 1,4-bis(aminomethyl)cyclohexane. When a culture or a processed product of the culture is used as an enzyme source, the amount of the enzyme source varies depending on the specific activity of the enzyme source, for example, 5 to 1000% by weight, preferably 10 to 400% by weight, relative to 4-(aminomethyl)cyclohexane-1-carbaldehyde in terms of wet bacterial weight.

[0240] It is preferred that NAD(P)H oxidase (NOX) coexists in the second step.

[0241] NAD(P)H oxidase is one of the metabolic enzymes that catalyzes the conversion of (NAD(P)H+H + +O2) is converted to (NAD(P) + +H2O2 or H2O). The NAD(P)H produced in the first and second steps of the method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid of the present invention can be converted into NAD(P) + Therefore, by allowing NAD(P)H oxidase to coexist, NAD(P) + It can be reused, and 4-(aminomethyl)cyclohexane-1-carboxylic acid can be efficiently produced with a small amount of raw materials.

[0242] NOX is not particularly limited and may be, for example, NOX derived from Bacillus subtilis, Streptococcus mutans, Lactococcus lactis, or Enterococcus feacalis. Specifically, NADH oxidase BsNOX (accession number: NP_389836.1) derived from Bacillus subtilis 168, NADH oxidase SmNOX (accession number: WP_002268044.1) derived from Streptococcus mutans, NADH oxidase LlNOX (accession number: CAL97012.1) derived from Lactococcus lactis, and NADH oxidase EfNOX (accession number: WP_002361833.1) derived from Enterococcus faecalis can be cited.

[0243] Regarding the coexistence of NAD(P)H oxidase, a culture of cells expressing NAD(P)H oxidase or a processed product of the culture may be added, or cells expressing NAD(P)H oxidase may be co-cultured, or the above-mentioned recombinant cells may contain DNA encoding NAD(P)H oxidase.

[0244] The amount of NAD(P)H oxidase may be 0.001 to 100% by weight, preferably 0.01 to 50% by weight, relative to the total amount of NAD(P)H generated when alanine dehydrogenase is used in step 1 and NAD(P)H generated by ALDH in step 2. When a culture or a processed product of the culture is used as an enzyme source, the amount of the enzyme source varies depending on the specific activity of the enzyme source, for example, 0.5 to 1000% by weight, preferably 1 to 400% by weight, relative to 1,4-bis(aminomethyl)cyclohexane in terms of wet cell weight.

[0245] Instead of allowing NAD(P)H oxidase to coexist, NAD(P)H may be electrochemically oxidized to generate NAD(P) + regeneration.

[0246] 4-(aminomethyl)cyclohexane-1-carboxaldehyde as the substrate can be any one of the trans-isomer, cis-isomer or a mixture thereof. When the substrate and the target compound have different stereostructures, part or all of the method is preferably carried out in a neutral or alkaline environment, and further part or all of the method is preferably carried out in the presence of a secondary amine. When the substrate and the target compound have the same stereostructure, part or all of the method is preferably carried out in an acidic environment.

[0247] The above-mentioned target 4-(aminomethyl)cyclohexane-1-carboxylic acid may be any of the cis-isomer, the trans-isomer or a mixture thereof. When the cis-isomer (i.e., cis-tranexamic acid) is the target, as the protein having aldehyde dehydrogenase activity in the second step, it is preferred to use an enzyme that can accept a cis-isomer substrate of one embodiment, any of sequence numbers 19, 21, 22, 35 to 37, 39, 44 to 46, 132, 134, 135 and 138, or a mutant protein or homologous protein thereof. When the trans-isomer (i.e., tranexamic acid) is the target, as the protein having aldehyde dehydrogenase activity in the second step, it is preferred to use an enzyme having high trans selectivity of one embodiment, any of sequence numbers 20, 38, 40, 41, 127, 128, 129, 130, 131, 133 and 139, or a mutant protein or homologous protein thereof. Among them, it is more preferable to use an enzyme having particularly high trans-specificity, any of SEQ ID NOs: 20, 40, 41, 127, 128, 130, 131 and 133, or a mutant protein or homologous protein thereof. When a mixture of cis-isomer and trans-isomer is targeted, any aldehyde dehydrogenase of the present invention can be used. The trans-isomer ratio of the mixture of cis-isomer and trans-isomer is preferably 30% or more, for example 40% or more, 50% or more, 60% or more, or 70% or more.

[0248] In this specification, the enzyme (ALDH) with high trans selectivity refers to the trans ratio of 4-(aminomethyl) cyclohexane-1-carboxylic acid (AMCHA) calculated in the embodiment described later by using the cis-isomer and trans-isomer mixture of 1,4-bis(aminomethyl) cyclohexane to generate more than 50% (e.g., more than 51%, more than 55%, more than 60%, more than 65%, more than 70%) enzyme, wherein, the enzyme with the trans ratio of more than 70% is used as the enzyme with high trans selectivity. More specifically, the trans ratio of AMCHA can be calculated by multiplying the ratio of the trans-isomer production amount of AMCHA relative to the total amount of the trans-isomer production amount of AMCHA and the cis-isomer production amount of AMCHA by 100. These trans-specific high enzymes (ALDH) can manufacture tranexamic acid with high yield, so it can be said that it is beneficial in the manufacture of tranexamic acid.

[0249] Step 1 and step 2 may be performed separately or simultaneously in the same system. In this case, the various enzymes may be contained in cells or may be purified proteins, or some may be contained in cells and some may be purified proteins.

[0250] The cells can be cultured according to conventional methods. As a medium for culturing the cells, any natural medium or synthetic medium can be used as long as it contains a carbon source, a nitrogen source, and inorganic salts that can be assimilated by the cells and can efficiently culture the cells.

[0251] In the method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid by fermentation, 1,4-bis(aminomethyl)cyclohexane as a substrate is preferably added to the culture medium. 1,4-bis(aminomethyl)cyclohexane may be added to the culture medium before culture or to the culture medium during culture.

[0252] Alternatively, instead of adding 1,4-bis(aminomethyl)cyclohexane to the culture medium, 1,4-bis(aminomethyl)cyclohexane may be supplied by co-culturing a microorganism capable of producing 1,4-bis(aminomethyl)cyclohexane with the above cells.

[0253] The carbon source may be any carbon source as long as it can be assimilated by the cell, and examples thereof include carbohydrates such as glucose, fructose, sucrose, molasses containing these, glycerol, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.

[0254] Examples of nitrogen sources include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacteria and their digests, etc.

[0255] Examples of the inorganic salt include potassium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0256] In the method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid by fermentation, pyruvate, α-ketoglutarate, α-ketobutyrate, NAD + , NADP + In order to induce isomerization of 4-(aminomethyl)cyclohexane-1-carbaldehyde, a secondary amine, preferably L-proline, a pyrrolidine derivative, or the like may be added to the culture medium.

[0257] Alternatively, instead of adding the above compounds, the above compounds may be supplied by co-culturing microorganisms capable of producing the above compounds with the above cells and co-expressing the above enzymes.

[0258] Cultivation is usually preferably carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40° C., and the culture time is usually 5 hours to 7 days. The pH during the culture is preferably maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0259] In addition, during the culture, antibiotics such as ampicillin and kanamycin may be added to the medium as necessary. When culturing recombinant cells transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary.

[0260] For example, when culturing microorganisms transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactoside or the like may be added to the medium, and when culturing microorganisms transformed with an expression vector using a trp promoter, indoleacrylic acid or the like may be added to the medium.

[0261] By the above-mentioned culture, 4-(aminomethyl)cyclohexane-1-carboxylic acid is produced in the culture or in the microorganism, thereby, 4-(aminomethyl)cyclohexane-1-carboxylic acid, preferably tranexamic acid, can be produced. The quantification of 4-(aminomethyl)cyclohexane-1-carboxylic acid can be carried out using LCMS (e.g., an analysis device LCMS-8040 manufactured by Shimadzu Corporation).

[0262] Collecting 4-(aminomethyl)cyclohexane-1-carboxylic acid from the culture can be generally carried out by combining an ion exchange resin method, a precipitation method, or other known methods. It should be noted that, when 4-(aminomethyl)cyclohexane-1-carboxylic acid accumulates in the bacterial body, for example, the ion exchange resin method can be used to collect 4-(aminomethyl)cyclohexane-1-carboxylic acid from the supernatant obtained by crushing the bacterial body by ultrasound or the like and removing the bacterial body by centrifugation.

[0263] [Analysis Example] Analysis and Quantification of 1,4-bis(aminomethyl)cyclohexane, 4-(aminomethyl)cyclohexane-1-carboxaldehyde or 4-(aminomethyl)cyclohexane-1-carboxylic acid

[0264] In the Examples, analysis and quantification of 1,4-bis(aminomethyl)cyclohexane, 4-(aminomethyl)cyclohexane-1-carbaldehyde, or 4-(aminomethyl)cyclohexane-1-carboxylic acid were performed by the following procedures.

[0265] The reaction solution containing the enzyme after the enzyme reaction, the reaction solution containing the microorganism after the static bacterial reaction, or the culture solution containing the microorganism after the culture is centrifuged to recover the supernatant. The 1,4-bis(aminomethyl)cyclohexane, 4-(aminomethyl)cyclohexane-1-carboxaldehyde, or 4-(aminomethyl)cyclohexane-1-carboxylic acid contained in the supernatant is analyzed using LCMS-8040 (manufactured by Shimadzu Corporation). The cis-trans isomers in 4-(aminomethyl)cyclohexane-1-carboxaldehyde and 4-(aminomethyl)cyclohexane-1-carboxylic acid can be separated by the following analysis conditions.

[0266] [Analysis conditions]

[0267] Column: Develosil (trademark) ODS-HG 5 μm 2.0×150 mm (Nomura Chemical Co., Ltd.)

[0268] Column temperature: 40°C

[0269] Mobile Phase:

[0270] (Mobile phase A) 5mmol / L heptafluorobutyric acid water

[0271] (Mobile phase B) 5mmol / L heptafluorobutyric acid acetonitrile

[0272] Mixing ratio of mobile phase A and B

[0273] (0~5 minutes)100:0

[0274] (5-25 minutes) 100:0 to 50:50

[0275] (25-30 minutes) 50:50

[0276] (30-31 minutes) 50:50 to 100:0

[0277] (31-35 minutes) 100:0

[0278] Flow rate: 0.25mL / min

[0279] Detection: Positive ion mode

[0280] Detection ions:

[0281] (1,4-bis(aminomethyl)cyclohexane) 143.1>67.0m / z

[0282] (4-(Aminomethyl)cyclohexane-1-carbaldehyde) 142.1m / z

[0283] (4-(Aminomethyl)cyclohexane-1-carboxylic acid) 158.2>95.0m / z

[0284] Although the details of the Examples are shown below, the present invention is not limited to these Examples.

[0285] Example

[0286] [Example 1] Search for an enzyme (AT) having transamination activity of 1,4-bis(aminomethyl)cyclohexane

[0287] (1) Selection of AT to be evaluated

[0288] Up to now, enzymes with transamination activity that can transaminize 1,4-bis(aminomethyl)cyclohexane to generate 4-(aminomethyl)cyclohexane-1-carboxaldehyde are still unknown. In order to explore enzymes with transamination activity for 1,4-bis(aminomethyl)cyclohexane as a non-natural compound, the inventors used 33 enzymes annotated as aminotransferases in the genome of Pseudomonas putida KT2440 (Applied Microbiology and Biotechnology, 2020, 104: 7745-7766), which can grow and reproduce in harsh environments, as the target, and selected enzymes PpAT8 (sequence number 1) and PpAT2 (sequence number 2) that may have the target activity through preliminary tests with transamination activity as an indicator. In addition, the aminotransferase AsAT5 (sequence number 3) from the salmonid Aeromonas salmonidida subspecies, which is a homologous enzyme with 65% consistency with PpAT8, was also evaluated. In addition, putrescine aminotransferase PatA (Journal of Bacteriology, 2012, 194, 15: 4080-4088) (SEQ ID NO: 4) derived from Escherichia coli K12 MG1655, which has been reported to have transamination activity on putrescine and cadaverine, which have two amino groups in one molecule like 1,4-bis(aminomethyl)cyclohexane, was evaluated.

[0289] (2) Preparation of AT expression strain

[0290] Using the DNA consisting of the base sequence shown in the "Primer Set" of Table 1 as the primer set and the DNA described in the "Template" of Table 1 as the template, PCR was performed to obtain each amplified DNA fragment. Various chromosomal DNAs were prepared by conventional methods. It should be noted that all the gene sequences used in this example were obtained from the database of the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / ). In addition, all PCRs in this example were performed using PrimeSTAR (trademark) MAX DNA polymerase (Takara Biotech) according to the instructions.

[0291] [Table 1]

[0292]

[0293] A PCR was performed using a DNA consisting of the base sequences shown in sequence numbers 9 and 10 as a primer set and a plasmid pQE80L (manufactured by QIAGEN) containing a T5 promoter and an N-terminal His tag sequence as a template to obtain a vector fragment of about 4.7 kb. It should be noted that the 5' end of the base sequence shown in sequence number 9 contains a sequence complementary to the 5' end of the base sequences shown in sequence numbers 11, 13, 15 and 17, and the 5' end of the base sequence shown in sequence number 10 contains a sequence complementary to the 5' end of the base sequences shown in sequence numbers 12, 14, 16 and 18.

[0294] The various amplified DNA fragments obtained above and the vector fragment were ligated using the In-Fusion HD Cloning Kit (manufactured by Takara Bio) to prepare plasmids pQE80L-PpAT8 ( Figure 2 ), pQE80L-PpAT2, pQE80L-AsAT5 and pQE80L-PatA. The obtained AT expression plasmids pQE80L-PpAT8, pQE80L-PpAT2, pQE80L-AsAT5 and pQE80L-PatA were used to transform Escherichia coli BL21 (DE3) to prepare recombinant Escherichia coli carrying various plasmids.

[0295] (3) AT expression and enzyme purification

[0296] The various recombinant Escherichia coli obtained in (2) above were inoculated into a test tube containing 2 mL of LB medium containing 100 mg / L of ampicillin, and cultured at 30°C for 16 hours under shaking. The culture solution was inoculated into a 250 mL conical flask containing 40 mL of LB medium containing 100 mg / L of ampicillin, and after shaking culture at 30°C for 2 hours, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and further cultured at 30°C for 5 hours under shaking. The culture solution was centrifuged to obtain wet cells. From the wet cells, TALON (trademark) Metal Affinity Resin (Clontech) was used for enzyme purification to obtain recombinant purified enzymes PpAT8, PpAT2, AsAT5 and PatA with His tags.

[0297] (4) Evaluation of the transamination activity of 1,4-bis(aminomethyl)cyclohexane

[0298] For each purified enzyme obtained by purification in (3), the enzyme activity of each AT was evaluated using the amount of 4-(aminomethyl)cyclohexane-1-carbaldehyde produced as an index.

[0299] A 0.1 mL reaction solution (pH 7.8) containing 0.07 mg of the purified enzyme (PpAT8, PpAT2, AsAT5 or PatA) obtained in (3) above, 50 mM MOPS (pH 7.8), 10 mM magnesium chloride, 1 mM dithiothreitol (DTT), 10 mM pyruvic acid, 0.1 mM pyridoxal phosphate (PLP) and 10 mM 1,4-bis(aminomethyl)cyclohexane cis- and trans-isomer mixture (trans ratio 45.7%) was prepared and reacted at 30° C. and 400 rpm for 24 hours. In addition, a reaction solution in which an equal amount of MOPS buffer was added instead of the purified enzyme solution as a negative control was reacted in the same manner.

[0300] After the reaction was completed, the reaction solution was diluted and then centrifuged, and the reaction product and residual substrate contained in the supernatant were analyzed by LCMS-8040 ( Figure 3 ). Each peak of the trans-isomer or cis-isomer of the product 4-(aminomethyl)cyclohexane-1-carboxaldehyde was comprehensively determined based on its elution position and m / z value, and the amount of each produced was expressed as the peak area (area value). The results are shown in Table 2.

[0301] [Table 2]

[0302]

[0303] Only when PpAT8, PpAT2, AsAT5 or PatA was added for reaction, the generation and accumulation of 4-(aminomethyl)cyclohexane-1-carboxaldehyde was confirmed. Among them, in the case of PatA, the residual substrate was small and the generation amount of 4-(aminomethyl)cyclohexane-1-carboxaldehyde was the largest. Therefore, it can be seen that putrescine aminotransferase PatA from Escherichia coli K12 MG1655 has high transamination activity for 1,4-bis(aminomethyl)cyclohexane.

[0304] <Evaluation of transamination activity of AT homologs towards 1,4-bis(aminomethyl)cyclohexane>

[0305] (5) Exploration of AT homologous enzymes in the database

[0306] The amino acid sequences of PpAT8 and PatA (SEQ ID NOs. 5 and 8) obtained as ATs with activity against 1,4-bis(aminomethyl)cyclohexane in Example 1 were used as queries, and the homology search function of the protein sequence database BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information was used to search for enzymes with a 40-85% identity with each query enzyme. The amino acid sequences (SEQ ID NOs. 103-107) of the enzymes extracted by the above operation and the base sequences (SEQ ID NOs. 108-112) encoding each enzyme are shown in Table 3.

[0307] [Table 3]

[0308]

[0309] (6) Preparation of AT homolog expression strain

[0310] In order to obtain the gene fragments of each AT homologous enzyme recorded in the above Table 3, a PCR reaction was performed using the templates, primers 1 and 2 shown in Table 4. Various chromosomal DNAs were prepared by conventional methods. At this time, the 5' end of the base sequence shown in sequence number 9 contains a base sequence complementary to the 5' end of the base sequence shown in each primer 1 in Table 4, and the 5' end of the base sequence shown in sequence number 10 contains a base sequence complementary to the 5' end of the base sequence shown in each primer 2 in Table 4.

[0311] [Table 4]

[0312]

[0313] The amplified DNA fragment obtained by the above PCR and the vector fragment of pQE80L prepared in Example 1 were ligated using the In-Fusion (trademark) HD ​​cloning kit (Takara Bio) to prepare each AT expression plasmid. Escherichia coli BL21 (DE3) was transformed with each expression plasmid to prepare a recombinant Escherichia coli having each AT expression plasmid.

[0314] (7) Evaluation of the transamination activity of AT homologs towards 1,4-bis(aminomethyl)cyclohexane

[0315] Using the recombinant E. coli obtained in (6) above and having each AT expression plasmid, the His-tagged recombinant AT purified enzyme was obtained by the same method as (3).

[0316] 0.1 mL of a reaction solution (pH 7.8) containing 0.07 mg of each purified enzyme obtained, 50 mM MOPS (pH 7.8), 1 mM magnesium chloride, 1 mM DTT, 1 mM pyruvic acid, 0.1 mM PLP, and 1 mM cis- and trans-isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%) was prepared and reacted at 30° C. and 400 rpm for 24 hours. In addition, a reaction solution in which an equal amount of MOPS buffer was added instead of the purified enzyme as a negative control and a reaction solution using the purified enzyme PatA as a positive control were also reacted in the same manner.

[0317] After the reaction was completed, the reaction product was analyzed by the same method as (4). The results are shown in Table 5.

[0318] [Table 5]

[0319]

[0320] It was confirmed that PcAT, PaAT, and HeAT1 have transamination activity on 1,4-bis(aminomethyl)cyclohexane, resulting in the production and accumulation of 4-(aminomethyl)cyclohexane-1-carboxaldehyde. Among them, PaAT has activity comparable to that of PatA. In addition, it was suggested that homologs of PpAT8 have the target activity when the identity with PpAT8 is 60% or more.

[0321] [Example 2] Effect of pH in the 4-(aminomethyl)cyclohexane-1-carbaldehyde production step

[0322] (1) Evaluation of the effect of solution pH on transamination reactions

[0323] The influence of pH conditions (pH 7.0 or pH 9.0) on the supply of 4-(aminomethyl)cyclohexane-1-carbaldehyde, which is an intermediate of 4-(aminomethyl)cyclohexane-1-carboxylic acid, was evaluated.

[0324] Prepare 0.1 mL of a reaction solution containing 0.07 mg of the purified PatA enzyme obtained in Example 1 (3), 50 mM MOPS (pH 7.0) or 50 mM CHES (pH 9.0), 1 mM magnesium chloride, 1 mM DTT, 1 mM pyruvate, 0.1 mM PLP, and 1 mM cis- or trans-1,4-bis(aminomethyl)cyclohexane as a reaction substrate, and react at 30°C and 400 rpm for 24 hours.

[0325] After the reaction was completed, LCMS-8040 (manufactured by Shimadzu Corporation) was used to analyze the 4-(aminomethyl)cyclohexane-1-carboxaldehyde contained in the supernatant of the reaction solution. The trans ratio (intermediate trans ratio) of 4-(aminomethyl)cyclohexane-1-carboxaldehyde was calculated based on the area value of the generated 4-(aminomethyl)cyclohexane-1-carboxaldehyde using the calculation formula shown in Mathematical Formula 1. The trans isomer in the formula refers to trans-4-(aminomethyl)cyclohexane-1-carboxaldehyde, and the cis isomer refers to cis-4-(aminomethyl)cyclohexane-1-carboxaldehyde. The results are shown in Table 6.

[0326] [Mathematical formula 1]

[0327] Intermediate trans ratio (%) = {area value of trans form / (area value of trans form + area value of cis form)}×100

[0328] [Table 6]

[0329]

[0330] It was shown that when either the cis-isomer or the trans-isomer substrate was used, at pH 7.0, about 95% or more of the generated 4-(aminomethyl)cyclohexane-1-carboxaldehyde had the same stereostructure as the added substrate. On the other hand, at pH 9.0, more than 30% of the generated 4-(aminomethyl)cyclohexane-1-carboxaldehyde had an isomeric structure of the added substrate.

[0331] This result suggests that the stereostructure of 4-(aminomethyl)cyclohexane-1-carbaldehyde generated in the transamination reaction of 1,4-bis(aminomethyl)cyclohexane changes depending on the pH of the reaction solution.

[0332] It is believed that the change in the above-mentioned stereostructure is caused by the action of (i) aminotransferase and (ii) keto-enol tautomerism of the aldehyde group. Regarding (ii), 4-(aminomethyl)cyclohexane-1-carboxaldehyde has an aldehyde group, and it is known that the aldehyde group usually converts between the keto form and the enol form in the presence of an acidic or alkaline catalyst (keto-enol tautomerism). It is believed that since the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde is converted from the keto form to the enol form, the hydrogen on the α-position carbon is transferred and a carbon-carbon double bond is generated between cyclohexane, so that the difference between the cis-isomer and the trans-isomer disappears. Therefore, it is predicted that in the presence of acid or alkali, the isomerization reaction of 4-(aminomethyl)cyclohexane-1-carboxaldehyde will occur independently of the reaction using aminotransferase. Therefore, in order to confirm whether the change in the above-mentioned stereostructure depends on aminotransferase, the following test was carried out.

[0333] (2) Evaluation of pH-dependent isomerization of 4-(aminomethyl)cyclohexane-1-carbaldehyde

[0334] According to the above (1), (i) a reaction solution in which the intermediate trans ratio reached 2.1% by reacting the purified PatA enzyme with the cis substrate at pH 7.0, and (ii) a reaction solution in which the intermediate trans ratio reached 95.3% by reacting the purified PatA enzyme with the trans substrate at pH 7.0 were prepared. The enzyme in each reaction solution was removed using Amicon (trademark) Ultra 0.5mL, 3K (Millipore), and the flow-through fraction was recovered. For each 10 μL flow-through fraction, add 90 μL of any one of 50 mM sodium acetate buffer (pH 4.0 or pH 5.0), 50 mM MES buffer (pH 6.0), 50 mM sodium phosphate buffer (pH 6.0 or pH 7.0), 50 mM MOPS buffer (pH 7.0), 50 mM CHES buffer (pH 9.0 or 10.0), or 50 mM sodium carbonate buffer (pH 9.0 or 10.0), and react at 30°C and 400 rpm for 20 hours.

[0335] In addition, in order to evaluate the effect of adding a secondary amine on isomerization, the reaction was also carried out in the same manner under the condition that 50 mM of L-proline was added to the above composition. After the reaction was completed, the reaction product was analyzed by the same method as in (1) above and the intermediate trans ratio was calculated. The results are shown in Table 7.

[0336] Table 7

[0337]

[0338] According to Table 7, when using either the cis-isomer or the trans-isomer substrate, the isomerization of the intermediate hardly occurred under acidic conditions of pH 4 to 6, but a change in the trans ratio of the intermediate was observed at pH 7 or above. In particular, under alkaline conditions of pH 9 to 10, the isomerization of the intermediate progressed significantly. In addition, it was observed that the isomerization of 4-(aminomethyl)cyclohexane-1-carbaldehyde reached equilibrium when the trans ratio was about 60%.

[0339] Furthermore, it was found that under the conditions of adding proline, almost no change was observed at pH 4 to 6, but a tendency for the isomerization of the intermediate to proceed was observed at pH 7, and at pH 9, the isomerization of the intermediate was further promoted.

[0340] The above indicates that isomerization of 4-(aminomethyl)cyclohexane-1-carbaldehyde occurs under neutral or alkaline conditions regardless of the presence or absence of an enzyme. It is also known that the isomerization reaction under neutral or alkaline conditions is promoted in the presence of a secondary amine such as L-proline.

[0341] The above results suggest that the stereochemical pattern of the reaction product can be controlled by changing the pH conditions according to the stereochemical structure of the substrate used.

[0342] [Example 3] Evaluation of aldehyde dehydrogenase (ALDH) activity

[0343] (1) Selection of ALDH to be evaluated

[0344] Until now, no enzyme having aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexanecarboxylic acid has been known.

[0345] In the present invention, based on the results of Example 1, NAD from Escherichia coli K12 MG1655, which participates in the metabolism of putrescine and cadaverine like PatA, was selected. + PatD (FEBS Letters, 2005, 579: 4107-4112) was selected as the ALDH to be evaluated. In addition, based on the fact that the substrate 4-(aminomethyl)cyclohexane-1-carbaldehyde has a bulky and low-polarity cyclohexane ring structure, NAD from Pseudomonas putida CSV86 was selected, which has been reported to have ALDH activity against substrates with different stereostructures but bulky and low-polarity benzene ring structures. + NAD from Pseudomonas putida S12 + Dependent phenylacetaldehyde dehydrogenase StyD (Archives of Biochemistry and Biophysics, 2017, 616: 47-58) and NADP from Pseudomonas putida NCIMB 9866 + The ALDH-dependent 4-hydroxybenzaldehyde dehydrogenase PchA (Appl. Microbiol. Biotechnol., 2014, 98: 1349-1356) was used as the evaluation object.

[0346] (2) Preparation of ALDH-expressing strain

[0347] The amino acid sequences of the ALDH (PatD, XylC, StyD, and PchA) selected in (1) are shown in SEQ ID NOs: 19 to 22. The base sequences encoding these enzymes are shown in SEQ ID NOs: 23 to 26, respectively. Strains expressing these enzymes were prepared as follows.

[0348] PCR was performed using the DNA described in "Template" in Table 8 as a template and the DNA consisting of the base sequence shown in "Primer Set" in Table 8 as a primer set to amplify each DNA fragment.

[0349] [Table 8]

[0350]

[0351] The chromosomal DNA of Escherichia coli K12 MG1655 was prepared by conventional methods. The DNA shown in sequence number 24 is the base sequence of the gene encoding XylC from Pseudomonas putida CSV86 shown in sequence number 20, and is prepared by artificial synthesis. The DNA shown in sequence number 25 is the base sequence of the gene encoding StyD from Pseudomonas putida S12 shown in sequence number 21, and is prepared by artificial synthesis. The DNA shown in sequence number 26 is the base sequence of the gene encoding PchA from Pseudomonas putida NCIMB9866 shown in sequence number 22, and is prepared by artificial synthesis.

[0352] The 5' end of the base sequence shown in SEQ ID NO: 9 contains a sequence complementary to the 5' end of SEQ ID NO: 27, 29, 31 and 33, and the 5' end of the base sequence shown in SEQ ID NO: 10 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO: 28, 30, 32 and 34.

[0353] The various amplified DNA fragments obtained above were ligated with the pQE80L vector fragment prepared in Example 1 using the In-FusionHD Cloning Kit (manufactured by Takara Bio) to prepare plasmids pQE80L-PatD, pQE80L-XylC, pQE80L-StyD and pQE80L-PchA expressing various ALDHs.

[0354] Escherichia coli BL21(DE3) was transformed with the ALDH-expressing plasmids pQE80L-PatD, pQE80L-XylC, pQE80L-StyD, and pQE80L-PchA obtained above, thereby preparing Escherichia coli cells harboring various plasmids.

[0355] (3) Expression and enzyme purification of ALDH

[0356] Using the strain obtained in (2) above, wet cells were obtained and enzymes were purified in the same manner as in Example 1, thereby obtaining purified recombinant enzymes PatD, XylC, StyD and PchA with a His tag.

[0357] (4) Evaluation of the productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid by ALDH in combination reaction with AT

[0358] <Using NAD + As a coenzyme of ALDH>

[0359] A solution containing 0.07 mg of the purified enzymes (PatD, XylC, StyD and PchA) obtained in (3) above, 0.07 mg of PatA obtained in Example 1 (3), 50 mM MOPS (pH 7.8), 1 mM magnesium chloride, 1 mM DTT, 1 mM pyruvate, 0.1 mM PLP, 2 mM nicotinamide adenine dinucleotide (NAD + ) and 1 mM cis- and trans-isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%), 0.1 mL of a reaction solution (pH 7.8) was reacted at 30°C and 400 rpm for 24 hours. In addition, a reaction solution in which an equal amount of MOPS buffer was added instead of purified enzyme as a negative control was also reacted in the same manner.

[0360] After reaction finishes, by the method same as embodiment 1, reaction product is analyzed.In addition, for the 4-(aminomethyl) cyclohexane-1-carboxylic acid generated, in order to confirm whether tranexamic acid (TXA) or cis-TXA can be selectively generated, the computing formula shown in the following mathematical formula 2 is used to calculate the trans ratio (also referred to as AMCHA trans ratio, AMCHA trans rate) of 4-(aminomethyl) cyclohexane-1-carboxylic acid based on the growing amount of TXA and cis-TXA.It should be noted that the concentration (mM) of each compound is calculated by comparing with the area value of the known standard substance of concentration.

[0361] [Mathematical formula 2]

[0362] AMCHA trans rate (%) = {TXA (mM) / (TXA (mM) + cis-TXA (mM))} × 100 First, the results when a mixture of cis and trans isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%) was used as a substrate are shown in Table 9.

[0363] [Table 9]

[0364]

[0365] ND: Below the detection limit (S / N<3)

[0366] Table 9 shows that the combination of PatA with PatD, XylC or StyD enables the production and accumulation of 4-(aminomethyl)cyclohexane-1-carboxylic acid using 1,4-bis(aminomethyl)cyclohexane as a substrate.

[0367] In addition, since a mixture of cis and trans isomers was used as the substrate, it was speculated that the resulting 4-(aminomethyl)cyclohexane-1-carboxylic acid was also a mixture of cis and trans isomers, but the actual result was that the trans ratio of the generated 4-(aminomethyl)cyclohexanecarboxylic acid was different depending on the type of ALDH. This result suggests that ALDH has substrate selectivity. In particular, when XylC is used, the trans ratio of 4-(aminomethyl)cyclohexanecarboxylic acid is large, indicating that it can selectively react with the trans-isomer 4-(aminomethyl)cyclohexane-1-carboxaldehyde. Therefore, it can be said that XylC is a useful ALDH when it is desired to efficiently generate TXA from a mixture substrate of cis and trans isomers.

[0368] Next, Table 10 shows the results when only the trans isomer of 1,4-bis(aminomethyl)cyclohexane was used as a substrate.

[0369] [Table 10]

[0370]

[0371] NQ: below the limit of quantification (S / N<10), ND: below the limit of detection (S / N<3)

[0372] According to the results in Table 10, when using the trans-isomer as a substrate, all ALDHs were able to confirm the generation of TXA, but cis-TXA was hardly detected. Therefore, when using a trans-isomer substrate, since all ALDHs found here were able to generate TXA at ​​a high yield, it was shown that it is preferable to use a trans-isomer or a trans-isomer-rich substrate as a substrate in TXA production. In addition, it can be seen that these ALDHs can accept trans-isomer substrates.

[0373] Next, the amount of 4-(aminomethyl)cyclohexanecarboxylic acid produced when only the cis-isomer of 1,4-bis(aminomethyl)cyclohexane was used as a substrate was evaluated.

[0374] Prepare a solution containing 0.07 mg of each of the purified enzyme PatA obtained in the same manner as above and various ALDHs (PatD, XylC or StyD), 50 mM MOPS (pH 7.8), 10 mM magnesium chloride, 1 mM DTT, 10 mM pyruvate, 0.1 mM PLP, and 2 mM NAD + The reaction mixture was stirred for 24 hours at 30° C. and 400 rpm. The results are shown in Table 11.

[0375] [Table 11]

[0376]

[0377] ND: Below the detection limit (S / N<3)

[0378] As can be seen from Table 11, when the cis-form is used as a substrate, the ratio of cis-TXA produced is high in all ALDHs. In addition, in the case of XylC, the production amount of cis-TXA is small, so XylC is not easy to accept the cis-form substrate.

[0379] Among them, TXA was produced at a high ratio only when XylC was used. It is speculated that this is because the reaction was carried out under weakly alkaline conditions of pH 7.8, so the intermediate isomerization under alkaline conditions shown in Example 2 occurred, and the intermediate of the trans body was produced even when the cis body substrate was added, resulting in a large amount of TXA accumulation. This suggests that when the goal is to produce TXA, by using XylC as an ALDH to selectively oxidize the intermediate of the trans body to produce TXA, and at the same time, the remaining cis body intermediate is dynamically isomerized to the trans body under neutral or alkaline conditions, it is theoretically possible to produce 100% TXA even when a mixed substrate of the cis body and the trans body is used.

[0380] <Using NADP + As a coenzyme of ALDH>

[0381] It is known that PchA, for which ALDH activity was not confirmed in the results of Table 6, utilizes NADP + Therefore, the purified enzyme PchA obtained in (3) above and XylC as a comparative object were used to evaluate the effect of NADP on the bioavailability of the enzyme. + Aldehyde oxidation activity of 4-(aminomethyl)cyclohexane-1-carbaldehyde when used as a coenzyme.

[0382] Prepare a reaction mixture to replace NAD + When 1 mM NADP was added + The same reaction as above was carried out with a reaction solution of . As substrate, a mixture of cis-isomer and trans-isomer (trans ratio 45.7%) was used. In addition, as a negative control, a reaction solution in which an equal amount of MOPS buffer was added instead of purified enzyme was also reacted in the same manner.

[0383] After the reaction was completed, the reaction product was analyzed by the same method as above. The results are shown in Table 12.

[0384] [Table 12]

[0385]

[0386] ND: Below the detection limit (S / N<3)

[0387] Table 12 confirmed that PchA can be activated by adding NADP + As a coenzyme, 4-(aminomethyl)cyclohexane-1-carboxylic acid is generated. In addition, although XylC is known to be NAD + It is a 4-aminomethylcyclohexane-1-carboxylic acid dependent enzyme, but it can also utilize NADP in the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid. + As a coenzyme.

[0388] [Example 4] Production of 4-(aminomethyl)cyclohexanecarboxylic acid using ALDH homologs

[0389] (1) Exploration of ALDH homologous enzymes in the database

[0390] The amino acid sequences (SEQ ID NOs. 19 to 21) of PatD, XylC, and StyD obtained as ALDH capable of generating 4-(aminomethyl)cyclohexane-1-carboxylic acid in Example 3 were used as queries, and the homology search function of the protein sequence database BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information was used to search for enzymes with 50 to 80% identity with each enzyme. The amino acid sequences (SEQ ID NOs. 35 to 46) of the enzymes extracted by the above operation and the base sequences (SEQ ID NOs. 47 to 58) encoding each enzyme are shown in Table 13. In Table 13, CkpatD, SepatD, CspatD, and PppatD are all proteins annotated as γ-aminobutyraldehyde dehydrogenase. SsBD, HaBD, and RrAD are proteins annotated as benzaldehyde dehydrogenase. PmsLAD is a protein annotated as phenylacetaldehyde dehydrogenase. Others are proteins annotated as aldehyde dehydrogenases or proteins of its family.

[0391] [Table 13]

[0392]

[0393] (2) Preparation of ALDH homolog expression strains

[0394] In order to obtain the gene fragments of the ALDH homologous enzymes listed in Table 13, a PCR reaction was carried out using the template and primers 1 and 2 shown in Table 14.

[0395] [Table 14]

[0396]

[0397] Various chromosomal DNAs are prepared by conventional methods. The DNA shown in sequence number 47 is a DNA in which the base sequence of the gene encoding ALDH from the ATCC BAA-895 strain of Citrobacter koseri shown in sequence number 35 is codon-optimized for expression in Escherichia coli, and is prepared by artificial synthesis. The DNA shown in sequence number 48 is a DNA in which the base sequence of the gene encoding ALDH from the SC-B67 strain of Salmonella enterica subsp. enterica serovar Choleraesuis str. shown in sequence number 36 is codon-optimized for expression in Escherichia coli, and is prepared by artificial synthesis. The DNA shown in sequence number 49 is a DNA in which the base sequence of the gene encoding ALDH from the SCRI1043 strain of Pectobacterium atrosepticum shown in sequence number 37 is codon-optimized for expression in Escherichia coli, and is prepared by artificial synthesis. The DNA shown in sequence number 52 is a DNA in which the base sequence of the gene encoding ALDH from the Halioxenophilus aromaticivorans strain shown in sequence number 40 was codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA shown in sequence number 53 is a DNA in which the base sequence of the gene encoding ALDH from the undetermined species (Sphingomonas sp.) TF3 strain shown in sequence number 41 was codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA shown in sequence number 56 is a DNA in which the base sequence of the gene encoding ALDH from the undetermined species LQ26 strain shown in sequence number 44 was codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA shown in sequence number 57 is a DNA in which the base sequence of the gene encoding ALDH from the Burkholderia lata strain shown in sequence number 45 was codon-optimized for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO: 58 is a DNA obtained by codon-optimizing the base sequence of the gene encoding ALDH derived from Paraburkholderia sp. 5N strain represented by SEQ ID NO: 46 for expression in Escherichia coli, and was prepared by artificial synthesis.

[0398] At this time, the base sequences shown in sequence number 9 and each primer 1 in Table 14, and the base sequences shown in sequence number 10 and each primer 2 in Table 14 contain complementary base sequences at their respective 5' ends. That is, the 5' end of the base sequence shown in sequence number 9 contains a base sequence complementary to the 5' end of the base sequence shown in each primer 1 in Table 14, and the 5' end of the base sequence shown in sequence number 10 contains a base sequence complementary to the 5' end of the base sequence shown in each primer 2 in Table 14.

[0399] The amplified DNA fragment obtained by the above PCR and the pQE80L vector fragment prepared in Example 1 were ligated using the In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio) to prepare each ALDH expression plasmid.

[0400] Escherichia coli BL21(DE3) was transformed using each expression plasmid obtained above to prepare Escherichia coli having each ALDH expression plasmid.

[0401] (3) Evaluation of the productivity of 4-(aminomethyl)cyclohexanecarboxylic acid, a homolog of ALDH

[0402] Using the E. coli obtained in (2) above and having the respective ALDH expression plasmids, the purified recombinant ALDH enzymes having a His tag were obtained by the same method as in Example 1.

[0403] Prepare a solution containing 0.07 mg of the purified enzyme obtained, 0.07 mg of PatA obtained in Example 1, 50 mM MOPS (pH 7.8), 1 mM magnesium chloride, 1 mM DTT, 1 mM pyruvic acid, 0.1 mM PLP, 1 mM NAD + 0.1 mL of a reaction solution (pH 7.8) containing a mixture of cis-isomer and trans-isomer of 1 mM 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%) was reacted at 30°C and 400 rpm for 24 hours. In addition, a reaction solution in which an equal amount of MOPS buffer was added instead of purified enzyme as a negative control and a reaction solution in which purified enzyme XylC was used as a positive control were also reacted in the same manner.

[0404] After the reaction was completed, the reaction product was analyzed by the same method as in Example 1. The results are shown in Table 15.

[0405] [Table 15]

[0406]

[0407] As shown in Table 15, when ALDH having a consistency of about 60% or more with PatD, XylC or StyD is used, 4-(aminomethyl)cyclohexane-1-carboxylic acid can also be generated. In addition, when CspatD, HaBD or SsBD is used, the trans ratio of the generated 4-(aminomethyl)cyclohexane-1-carboxylic acid is greater than 50%, and it can be seen that these enzymes are enzymes with high trans specificity. In particular, when HaBD or SsBD having a consistency of 70% or more with XylC is used, the trans ratio of the generated 4-(aminomethyl)cyclohexane-1-carboxylic acid is greater than 70%, and it can be seen that these enzymes react particularly trans-selectively like XylC, that is, they have a particularly high trans specificity.

[0408] [Example 5] Evaluation of TXA productivity based on introduction of coenzyme regeneration system

[0409] (1) Preparation of alanine dehydrogenase (AlaDH) expression strain

[0410] In order to obtain the gene fragment encoding BsAlaDH of alanine dehydrogenase from Bacillus subtilis 168 shown in SEQ ID NO: 123, PCR reaction was carried out using chromosomal DNA of Bacillus subtilis 168 prepared by a conventional method as a template and oligonucleotides having base sequences shown in SEQ ID NOs: 83 and 84 as a primer set to obtain the BsAlaDH gene fragment shown in SEQ ID NO: 124. The 5' end of the base sequence shown in SEQ ID NO: 9 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO: 83, and the 5' end of the base sequence shown in SEQ ID NO: 10 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO: 84.

[0411] The BsAlaDH fragment obtained by the above PCR and the pQE80L vector fragment prepared in Example 1 were ligated using In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio) to obtain an expression plasmid pQE80L-BsAlaDH.

[0412] The expression plasmid obtained above was used to transform Escherichia coli BL21(DE3) to obtain BL21(DE3) / pQE80L-BsAlaDH.

[0413] (2) Preparation of NADH oxidase (NOX) expression strain

[0414] In order to obtain the gene fragment of BsNOX encoding NADH oxidase from Bacillus subtilis 168 shown in sequence number 125, PCR reaction was carried out using chromosomal DNA of Bacillus subtilis 168 as a template and oligonucleotides having base sequences shown in sequence numbers 85 and 86 as a primer set to obtain the BsNOX gene fragment shown in sequence number 126. The 5' end of the base sequence shown in sequence number 9 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 85, and the 5' end of the base sequence shown in sequence number 10 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 86.

[0415] The BsNOX gene fragment obtained by the above PCR and the pQE80L vector fragment prepared in Example 1 were ligated using In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio) 5 to obtain the expression plasmid pQE80L-BsNOX.

[0416] The expression plasmid obtained above was used to transform Escherichia coli BL21(DE3) to obtain BL21(DE3) / pQE80L-BsNOX.

[0417] (3) Expression and enzyme purification of BsAlaDH and BsNOX

[0418] Using the BL21(DE3) / pQE80L-BsAlaDH and BL21(DE3) / pQE80L-BsNOX obtained above, the His-tagged recombinant enzyme-purified BsAlaDH and BsNOX were obtained in the same manner as in Example 1.

[0419] (4) Evaluation of TXA production activity after introduction of AlaDH and NOX

[0420] Prepare a solution containing 0.07 mg of PatA obtained in Example 1, 0.07 mg of XylC obtained in Example 2, 0.07 mg of BsAlaDH obtained in (3) above, 0.07 mg of BsNOX, 50 mM MOPS (pH 7.8), 1 mM magnesium chloride, 1 mM DTT, 1 mM pyruvic acid, 0.1 mM PLP, 2 mM NAD + A 0.1 mL reaction solution (pH 7.8) containing a 1 mM mixture of cis-isomer and trans-isomer of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%) was added and reacted at 30° C. and 400 rpm for 24 hours.

[0421] After the reaction was completed, the reaction product and the residual substrate were analyzed by the same method as in Example 1. The results are shown in Table 16.

[0422] [Table 16]

[0423]

[0424] According to Table 16, when the coenzyme regeneration system BsAlaDH and BsNOX were introduced, the substrate consumption was accelerated and the productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid was improved.

[0425] [Example 6] Production of 4-(aminomethyl)cyclohexane-1-carboxylic acid by resting cell reaction using AT-expressing strains and ALDH-expressing strains

[0426] Wet cells were obtained from the BL21(DE3) / pQE80L-PatA strain and the BL21(DE3) / pQE80L-XylC strain prepared in Example 1 by the same method as in Example 1(2). Xylene was added to the wet cells to a final concentration of 10 mL / L, and membrane treatment was performed at 30°C and 850 rpm for 30 minutes.

[0427] Prepare a mixture containing 50 g / L of wet cells after each membrane treatment, 50 mM MOPS (pH 7.8), 350 mM glucose, 50 mM of a mixture of cis- and trans-isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%), 50 mM magnesium chloride, and 50 mM NAD + 0.4 mL of the reaction solution was reacted at 30°C and 850 rpm for 24 hours.

[0428] After the reaction was completed, the reaction product was analyzed by the same method as in Example 1. As a result, the generation and accumulation of 3.9 mM TXA and 2.8 mM cis-TXA were confirmed. Therefore, it was confirmed that 4-(aminomethyl)cyclohexanecarboxylic acid can be generated even by a resting cell reaction using microbial cells expressing AT and ALDH, respectively.

[0429] [Example 7] Production of 4-(aminomethyl)cyclohexane-1-carboxylic acid based on resting cell reaction using AT and ALDH co-expressing strain

[0430] (1) Preparation of AT and ALDH co-expression strain

[0431] i) Construction of BL21(DE3) / pET28a-PatA-XylC strain

[0432] The chromosomal DNA of Escherichia coli K12 MG1655 was used as a template, and oligonucleotides having base sequences shown in sequence numbers 89 and 90 were used as a primer set to perform a PCR reaction to obtain the PatA fragment. Next, the base sequence shown in sequence number 24 was used as a template, and oligonucleotides having base sequences shown in sequence numbers 91 and 92 were used as a primer set to perform a PCR reaction to obtain the XylC fragment. It should be noted that the 5' end of the base sequence shown in sequence number 90 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 91.

[0433] A PCR reaction was carried out using the mixture of the PatA fragment and the XylC fragment obtained above as a template and oligonucleotides having base sequences shown in SEQ ID NOs: 89 and 92 as a primer set to obtain a DNA fragment (hereinafter referred to as PatA-XylC) formed by ligating the two fragments.

[0434] Furthermore, a PCR reaction was performed using a DNA consisting of the base sequences shown in sequence numbers 87 and 88 as a primer set and an expression vector pET28a (manufactured by Novagen) as a template to obtain a vector fragment of about 5.2 kb. It should be noted that the 5' end of the base sequence shown in sequence number 87 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 89, and the 5' end of the base sequence shown in sequence number 88 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 92.

[0435] The PatA-XylC fragment obtained above and the vector fragment were ligated using the In-Fusion (trademark) HD ​​cloning kit (Takara Bio) to obtain the expression plasmid pET28a-PatA-XylC ( Figure 4 ).

[0436] The expression plasmid obtained above was used to transform Escherichia coli BL21(DE3) to obtain the BL21(DE3) / pET28a-PatA-XylC strain.

[0437] ii) Preparation of BL21(DE3) / pET28a-XylC-PatA

[0438] The base sequence shown in SEQ ID NO: 24 was used as a template, and oligonucleotides having base sequences shown in SEQ ID NOs: 93 and 94 were used as a primer set to perform a PCR reaction to obtain the XylC fragment. The chromosomal DNA of Escherichia coli K12 MG1655 was used as a template, and oligonucleotides having base sequences shown in SEQ ID NOs: 95 and 96 were used as a primer set to perform a PCR reaction to obtain the PatA fragment. It should be noted that the 5' end of the base sequence shown in SEQ ID NO: 94 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO: 95.

[0439] The mixture of the XylC fragment and the PatA fragment obtained above was used as a template, and the oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 93 and 96 were used as a primer set to carry out a PCR reaction to obtain a DNA fragment (hereinafter referred to as XylC-PatA) formed by connecting the two fragments. It should be noted that the 5' end of the base sequence shown in SEQ ID NO: 87 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO: 93, and the 5' end of the base sequence shown in SEQ ID NO: 88 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO: 96.

[0440] The XylC-PatA fragment obtained above and the vector fragment obtained in i) above were ligated using In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio) to obtain the expression plasmid pET28a-XylC-PatA.

[0441] The expression plasmid obtained above was used to transform Escherichia coli BL21(DE3) to obtain BL21(DE3) / pET28a-XylC-PatA strain.

[0442] iii) Construction of BL21(DE3) / pUC19-PatA-XylC strain

[0443] The chromosomal DNA of Escherichia coli K12 MG1655 was used as a template and oligonucleotides having base sequences shown in SEQ ID NOs: 99 and 90 were used as a primer set to perform a PCR reaction to obtain the PatA fragment. Next, the base sequence shown in SEQ ID NO: 24 was used as a template and oligonucleotides having base sequences shown in SEQ ID NOs: 91 and 100 were used as a primer set to perform a PCR reaction to obtain the XylC fragment. It should be noted that the 5' end of the base sequence shown in SEQ ID NO: 90 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO: 91.

[0444] A PCR reaction was carried out using the mixture of the PatA fragment and the XylC fragment obtained above as a template and DNA consisting of the base sequences shown in SEQ ID NOs: 99 and 100 as a primer set to obtain a DNA fragment (hereinafter referred to as PatA-XylC) formed by connecting the two fragments.

[0445] A PCR reaction was performed using a DNA consisting of the base sequences shown in sequence numbers 97 and 98 as a primer set and an expression vector pUC19 (manufactured by NIPPON GENE) as a template to obtain a vector fragment of about 2.6 kb. It should be noted that the 5' end of the base sequence shown in sequence number 97 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 99, and the 5' end of the base sequence shown in sequence number 98 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 100.

[0446] The PatA-XylC fragment obtained above and the vector fragment were ligated using In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio) to obtain the expression plasmid pUC19-PatA-XylC.

[0447] The expression plasmid obtained above was used to transform Escherichia coli BL21(DE3) to obtain the BL21(DE3) / pUC19-PatA-XylC strain.

[0448] iv) Preparation of BL21(DE3) / pUC19-XylC-PatA strain

[0449] The DNA shown in SEQ ID NO: 24 was used as a template, and oligonucleotides having base sequences shown in SEQ ID NOs: 101 and 94 were used as a primer set to perform a PCR reaction to obtain the XylC fragment. Next, the chromosomal DNA of Escherichia coli K12 MG1655 was used as a template, and oligonucleotides having base sequences shown in SEQ ID NOs: 95 and 102 were used as a primer set to perform a PCR reaction to obtain the PatA fragment. It should be noted that the 5' end of the base sequence shown in SEQ ID NO: 94 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO: 95.

[0450] The mixture of the XylC fragment and the PatA gene fragment obtained above was used as a template, and the oligonucleotides of the base sequences shown in SEQ ID NOs. 101 and 102 were used as a primer set to carry out a PCR reaction to obtain a DNA fragment (hereinafter referred to as XylC-PatA) formed by connecting the two fragments. It should be noted that the 5' end of the base sequence shown in SEQ ID NO. 97 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO. 101, and the 5' end of the base sequence shown in SEQ ID NO. 98 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO. 102.

[0451] The XylC-PatA fragment obtained above and the pUC19 vector fragment obtained in iii) above were ligated using In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio) to obtain the expression plasmid pUC19-XylC-PatA.

[0452] The expression plasmid obtained above was used to transform Escherichia coli BL21(DE3) to obtain the BL21(DE3) / pUC19-XylC-PatA strain.

[0453] (2) Bacterial response using a strain co-expressing AT and ALDH

[0454] The productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid based on the resting cell reaction was evaluated using the BL21(DE3) / pET28a-PatA-XylC strain, BL21(DE3) / pET28a-XylC-PatA strain, BL21(DE3) / pUC19-PatA-XylC strain and BL21(DE3) / pUC19-XylC-PatA strain prepared in the above (1), as well as the BL21(DE3) strain as a negative control.

[0455] Each strain was inoculated into a test tube containing 2 mL of LB medium containing 30 mg / L kanamycin or 100 mg / L ampicillin, and cultured with shaking at 30°C for 16 hours. The culture solution was inoculated into a 250 mL conical flask containing 40 mL of LB medium containing 30 mg / L kanamycin or 100 mg / L ampicillin, and after shaking culture at 30°C for 2 hours, IPTG was added to a final concentration of 1 mM, and further cultured with shaking at 37°C for 5 hours. In addition, as a control, BL21 (DE3) was cultured in the same manner in a medium without kanamycin and ampicillin.

[0456] The culture solution after culturing was centrifuged to obtain wet cells, to which xylene was added so as to give a final concentration of 10 mL / L, and membrane treatment was performed at 30° C. and 850 rpm for 30 minutes.

[0457] Prepare a mixture containing 100 g / L of the above membrane-treated wet cells, 50 mM MOPS (pH 7.8), 350 mM glucose, 50 mM of a mixture of cis- and trans-isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%), 50 mM magnesium chloride, and 50 mM NAD + 0.4 mL of the reaction solution was reacted at 30°C and 850 rpm for 24 hours.

[0458] After the reaction was completed, the reaction product was analyzed by the same method as in Example 1. The results are shown in Table 17.

[0459] [Table 17]

[0460]

[0461] ND: Below the detection limit (S / N<3)

[0462] Table 17 confirmed that 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced even by a resting cell reaction using a strain co-expressing PatA and XylC.

[0463] Furthermore, NAD was removed from the reaction solution.+ The productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid in the case of was evaluated. The reaction conditions were as described above. The results are shown in Table 18.

[0464] [Table 18]

[0465]

[0466] ND: Below the detection limit (S / N<3)

[0467] When NAD+ was removed from the reaction solution, the amount of 4-(aminomethyl)cyclohexane-1-carboxylic acid produced decreased, suggesting that the supply of coenzymes is important in the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid by resting cell reaction.

[0468] [Example 8] Production of 4-(aminomethyl)cyclohexane-1-carboxylic acid by supplementary culture using a strain co-expressing AT and ALDH

[0469] The productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid by the addition culture method was evaluated using the BL21(DE3) / pUC19-PatA-XylC strain and BL21(DE3) / pUC19-XylC-PatA strain prepared in Example 7 and the BL21(DE3) strain as a negative control.

[0470] Each strain was inoculated into a test tube containing 2 mL of LB medium containing 100 mg / L of ampicillin and cultured with shaking at 30°C for 16 hours. The BL21 (DE3) strain was cultured in the same manner in a medium without ampicillin. The culture solution was inoculated into a large test tube containing 4 mL of LB medium containing 30 mg / L of 100 mg / L ampicillin, and after culturing at 30°C for 5 hours, a mixture of cis- and trans-isomers of 1,4-bis (aminomethyl) cyclohexane at a final concentration of 1 mM and 1 g / L (trans ratio 45.7%) was added, and further cultured at 37°C for 24 hours.

[0471] After the culture was completed, the reaction product was analyzed by the same method as in Example 1. The results are shown in Table 19.

[0472] [Table 19]

[0473]

[0474] Table 19 confirmed that TXA can also be produced by the addition culture method.

[0475] [Example 9] Production of 4-(aminomethyl)cyclohexanecarboxylic acid using ALDH homologs

[0476] (1) Exploration of ALDH homologous enzymes

[0477] Using the amino acid sequence of XylC (SEQ ID NO. 20) of ALDH with high selectivity for trans-isomer substrates in Example 3 as a query, the homology search function of the protein sequence database BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information was used to select those with a homology of 50% or more with XylC and a homology of less than 90% between homologous enzymes in the search results. The amino acid sequences of the enzymes extracted by the above operation (SEQ ID NOs. 127 to 139) are shown in Table 20.

[0478] [Table 20]

[0479]

[0480] ALDH homologs 1 to 11 and 13 in Table 20 are all proteins annotated as benzaldehyde dehydrogenases, and ALDH homolog 12 is a protein annotated as a protein of the aldehyde dehydrogenase family.

[0481] (2) Preparation of ALDH homolog expression strains

[0482] In order to obtain the gene fragments of each ALDH homologous enzyme described in Table 20, a PCR reaction was performed using the template and primers 1 and 2 shown in Table 21. At this time, the 5' end of the base sequence shown in SEQ ID NO: 9 contains a base sequence complementary to the 5' end of the base sequence shown in each primer 1 in Table 21, and the 5' end of the base sequence shown in SEQ ID NO: 10 contains a base sequence complementary to the 5' end of the base sequence shown in each primer 2 in Table 21.

[0483] [Table 21]

[0484]

[0485] The DNAs represented by SEQ ID NOs: 140 to 152 are DNAs obtained by codon-optimizing the base sequences of genes encoding ALDH homologous enzymes represented by SEQ ID NOs: 127 to 139 for expression in Escherichia coli, and were prepared by artificial synthesis.

[0486] The amplified DNA fragment obtained by the above PCR and the vector fragment of pQE80L prepared in Example 1 were ligated using the In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio) to prepare each ALDH expression plasmid. Escherichia coli BL21 (DE3) was transformed with each expression plasmid obtained above to prepare Escherichia coli carrying each ALDH expression plasmid.

[0487] (3) Evaluation of the productivity of 4-(aminomethyl)cyclohexanecarboxylic acid, a homolog of ALDH

[0488] Using the E. coli obtained in (2) above and having the respective ALDH expression plasmids, the purified recombinant ALDH enzymes carrying the His tag were obtained by the same method as in Example 1.

[0489] Prepare a solution containing 0.07 mg of the purified enzyme obtained, 0.07 mg of PatA obtained in Example 1, 50 mM CHES (pH 9.0), 10 mM magnesium chloride, 1 mM DTT, 1 mM pyruvic acid, 0.1 mM PLP, 1 mM NAD + The mixture was stirred for 24 hours at 30°C and 400 rpm. A reaction solution using purified enzyme XylC as a positive control was also reacted in the same manner.

[0490] After the reaction was completed, the reaction product was analyzed by the same method as in Example 1. The results are shown in Table 22.

[0491] Table 22

[0492]

[0493] According to Table 22, the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid was confirmed for all evaluated ALDH homologs 1 to 13. In particular, when ALDH homologs 1 to 5, 7 to 9, and 13 were used, significant production of 4-(aminomethyl)cyclohexane-1-carboxylic acid was confirmed. In addition, when ALDH homologs 1 to 5, 7, and 13 were used, the trans ratio of the produced 4-(aminomethyl)cyclohexane-1-carboxylic acid was greater than 50%, and it had high specificity for the trans substrate. Among them, the trans ratio of 4-(aminomethyl)cyclohexane-1-carboxylic acid was 70% or more for ALDH homologs 1, 2, 4, 5, and 7, and it was found that they had particularly high selectivity for the trans substrate comparable to XylC. Among them, in the case of ALDH homologs 4 and 5, the same amount of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid as XylC was produced.

[0494] [Example 10] Evaluation of TXA productivity by enhancing the supply of ketoacid and coenzyme

[0495] (1) Preparation of evaluation strains

[0496] i) Production of MG1655 / pQE80L-XylC-PatA

[0497] Using the base sequence shown in SEQ ID NO: 24 as a template and oligonucleotides of the base sequences shown in SEQ ID NOs: 179 and 94 as a primer set, a PCR reaction was performed to obtain the XylC fragment. Next, using the chromosomal DNA of Escherichia coli K12 MG1655 as a template and oligonucleotides of the base sequences shown in SEQ ID NOs: 95 and 180 as a primer set, a PCR reaction was performed to obtain the PatA fragment. It should be noted that the 5' end of the base sequence shown in SEQ ID NO: 94 contains a sequence complementary to the 5' end of the base sequence shown in SEQ ID NO: 95.

[0498] A PCR reaction was carried out using the mixture of the XylC fragment and the PatA fragment obtained above as a template and oligonucleotides having the base sequences shown in SEQ ID NOs: 179 and 180 as a primer set to obtain a DNA fragment (hereinafter referred to as XylC-PatA) in which the two fragments were connected.

[0499] Furthermore, a PCR reaction was performed using a DNA consisting of the base sequences shown in sequence numbers 181 and 182 as a primer set and an expression vector pQE80L (manufactured by QIAGEN) as a template to obtain a vector fragment of about 4.7 kb. It should be noted that the 5' end of the base sequence shown in sequence number 181 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 179, and the 5' end of the base sequence shown in sequence number 182 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 180.

[0500] The obtained XylC-PatA fragment and the vector fragment were ligated using In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio) to obtain the expression plasmid pQE80L-XylC-PatA. Escherichia coli MG1655 was transformed with the obtained expression plasmid to obtain the MG1655 / pQE80L-XylC-PatA strain.

[0501] ii) Preparation of MG1655 / pQE80L-PatA-BsAlaDH-XylC-BsNOX

[0502] The chromosomal DNA of Escherichia coli K12 MG1655 was used as a template, and oligonucleotides of the base sequences shown in sequence numbers 183 and 90 were used as a primer set to perform a PCR reaction to obtain the PatA fragment. Next, the base sequence shown in sequence number 24 was used as a template, and oligonucleotides of the base sequences shown in sequence numbers 91 and 30 were used as a primer set to perform a PCR reaction to obtain the XylC fragment. It should be noted that the 5' end of the base sequence shown in sequence number 90 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 91.

[0503] A PCR reaction was carried out using the mixture of the PatA fragment and the XylC fragment obtained above as a template and oligonucleotides having base sequences shown in SEQ ID NOs. 183 and 30 as a primer set to obtain a DNA fragment (hereinafter referred to as PatA-XylC) formed by ligating the two fragments.

[0504] Furthermore, a PCR reaction was performed using a DNA consisting of the base sequences shown in sequence numbers 181 and 182 as a primer set and an expression vector pQE80L (manufactured by QIAGEN) as a template to obtain a vector fragment of about 4.7 kb. It should be noted that the 5' end of the base sequence shown in sequence number 181 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 183, and the 5' end of the base sequence shown in sequence number 182 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 30.

[0505] The PatA fragment obtained above and the vector fragment were ligated using the In-Fusion (trademark) HD ​​cloning kit (Takara Bio) to obtain plasmid pQE80L-PatA-XylC. A PCR reaction was performed using the plasmid obtained above as a template and a DNA consisting of the base sequence shown in SEQ ID NOs. 184 and 182 as a primer set to obtain a DNA (hereinafter referred to as pQE80L-PatA) fragment.

[0506] The chromosomal DNA of Bacillus subtilis 168 was used as a template, and the oligonucleotides of the base sequences shown in sequence numbers 185 and 186 were used as a primer set to perform a PCR reaction to obtain a BsAlaDH gene fragment. The chromosomal DNA of Bacillus subtilis 168 was used as a template, and the oligonucleotides of the base sequences shown in sequence numbers 187 and 86 were used as a primer set to perform a PCR reaction to obtain a BsNOX gene fragment. It should be noted that the 5' end of the base sequence shown in sequence number 186 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 187.

[0507] A mixture of the BsAlaDH gene fragment and the NOX gene fragment obtained above was used as a template, and oligonucleotides with base sequences shown in SEQ ID NOs. 185 and 86 were used as a primer set to carry out a PCR reaction to obtain a DNA fragment (hereinafter referred to as BsAlaDH-BsNOX) formed by connecting the two fragments.

[0508] It should be noted that the 5' end of the base sequence shown in sequence number 185 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 184, and the 5' end of the base sequence shown in sequence number 86 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 182.

[0509] The pQE80L-PatA fragment and the BsAlaDH-BsNOX fragment obtained above were ligated using In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio) to obtain plasmid pQE80L-PatA-BsAlaDH-BsNOX.

[0510] A PCR reaction was carried out using the plasmid obtained above as a template and a DNA consisting of the base sequence shown in SEQ ID NOs: 188 and 189 as a primer set to obtain a DNA fragment (hereinafter referred to as pQE80L-PatA-BsAlaDH-BsNOX).

[0511] The XylC fragment was obtained by carrying out a PCR reaction using the base sequence represented by SEQ ID NO: 24 as a template and oligonucleotides represented by the base sequences represented by SEQ ID NOs: 190 and 94 as a primer set.

[0512] It should be noted that the 5' end of the base sequence shown in sequence number 188 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 190, and the 5' end of the base sequence shown in sequence number 189 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 94.

[0513] The pQE80L-PatA-BsAlaDH-BsNOX fragment and the XylC fragment obtained above were ligated using In-Fusion (trademark) HD ​​Cloning Kit (Takara Bio) to obtain the expression plasmid pQE80L-PatA-BsAlaDH-XylC-BsNOX.

[0514] The expression plasmid obtained above was used to transform Escherichia coli MG1655 to obtain the MG1655 / pQE80L-PatA-BsAlaDH-XylC-BsNOX strain.

[0515] iii) Construction of MG1655ΔaceE / pQE80L-patA-xylC-ald-nox strain

[0516] <Obtaining DNA fragments used as markers when genes are missing>

[0517] PCR was performed using DNA consisting of the base sequences shown in SEQ ID NOs: 191 and 192 as a primer set and pCatSac (Appl Environ Microbiol (2013) 79, 3033-3039) as a template to obtain a cat-sacB fragment containing the chloramphenicol resistance cat gene and the sucrose sensitivity sacB gene.

[0518] <Preparation of Escherichia coli in which pyruvate dehydrogenase activity is lost>

[0519] Escherichia coli deficient in DNA encoding pyruvate dehydrogenase (hereinafter referred to as aceE gene) was prepared by the following method. The genomic DNA of Escherichia coli MG1655 strain prepared by a conventional method was used as a template, and the DNA consisting of the base sequence shown in the "primer set" of Table 23 was used as a primer set to perform PCR to amplify each DNA fragment.

[0520] [Table 23]

[0521]

[0522] aceE upstream 1 and aceE upstream 2 include a region from the start codon of the aceE gene to about 1000 bp upstream of the start codon, and aceE downstream 1 and aceE downstream 2 include a region from about 50 bp downstream of the stop codon of the aceE gene to about 1000 bp.

[0523] A mixture of aceE upstream 1, aceE downstream 1 and cat-sacB fragments in an equimolar ratio was used as a template, and PCR was performed using a DNA consisting of the base sequence shown in sequence numbers 193 and 196 as a primer set to obtain a DNA fragment (hereinafter referred to as aceE::cat-sacB) consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the aceE gene peripheral region.

[0524] A substance obtained by mixing aceE upstream 2 and aceE downstream 2 in an equimolar ratio was used as a template, and PCR was performed using a DNA composed of the base sequence shown in sequence numbers 193 and 196 as a primer set to obtain a DNA (hereinafter referred to as ΔaceE) fragment composed of a sequence that does not contain aceE and in which aceE upstream and aceE downstream are directly connected.

[0525] The aceE::cat-sacB fragment was introduced into the MG1655 strain harboring the plasmid pKD46 [Datsenko, KA, Warner, BL, Proc. Natl. Acad. Sci., USA, Vol. 97, 6640-6645 (2000)] containing a gene encoding λ recombinase by electroporation, and transformants showing chloramphenicol resistance and sucrose sensitivity were obtained (transformants in which the aceE gene was replaced with aceE::cat-sacB).

[0526] The ΔaceE fragment was introduced into the transformant by electroporation to obtain a transformant showing chloramphenicol sensitivity and sucrose resistance (a transformant in which aceE::cat-sacB was replaced with ΔaceE). From these, a transformant showing ampicillin sensitivity (a transformant in which pKD46 was lost) was further obtained. The transformant was named MG1655ΔaceE.

[0527] <Obtaining expression plasmid>

[0528] Using the plasmid pQE80L-PatA-XylC obtained in ii) as a template and a DNA consisting of the base sequence shown in SEQ ID NOs: 199 and 182 as a primer set, PCR was performed to obtain a DNA (hereinafter referred to as pQE80L-PatA-XylC) fragment.

[0529] Using the BsAlaDH-BsNOX fragment obtained in ii) as a template and a DNA consisting of the base sequences shown in SEQ ID NOs: 200 and 86 as a primer set, PCR was performed to obtain a DNA (hereinafter referred to as BsAlaDH-BsNOX2) fragment.

[0530] It should be noted that the 5' end of the base sequence shown in sequence number 200 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 199, and the 5' end of the base sequence shown in sequence number 86 contains a sequence complementary to the 5' end of the base sequence shown in sequence number 182.

[0531] The pQE80L-PatA-XylC fragment obtained above and the BsAlaDH-BsNOX2 fragment were ligated using the In-Fusion (trademark) HD ​​cloning kit (Takara Bio) to obtain the expression plasmid pQE80L-PatA-XylC-BsAlaDH-BsNOX. The expression plasmid obtained above was used to transform Escherichia coli MG1655ΔaceE to obtain the MG1655ΔaceE / pQE80L-PatA-XylC-BsAlaDH-BsNOX strain.

[0532] (2) Resting cell reaction of strain introduced with coenzyme regeneration system

[0533] Using the MG1655 / pQE80L-XylC-PatA strain, MG1655 / pQE80L-PatA-BsAlaDH-XylC-BsNOX strain, and MG1655ΔaceE / pQE80L-PatA-XylC-BsAlaDH-BsNOX strain prepared in (1) above, the productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid based on the resting cell reaction was evaluated.

[0534] Each strain was inoculated into a test tube containing 2 mL of LB medium containing 100 mg / L of ampicillin, and cultured with shaking at 30°C for 16 hours. The culture solution was inoculated into a 250 mL conical flask containing 40 mL of LB medium containing 100 mg / L of ampicillin, and after shaking culture at 28°C for 2 hours, IPTG was added to a final concentration of 1 mM, and further cultured with shaking at 28°C for 24 hours. Among them, only in the case of the MG1655ΔaceE / pQE80L-PatA-XylC-BsAlaDH-BsNOX strain, the culture time until the addition of ITPG was set to 3.5 hours.

[0535] The culture solution after culturing was centrifuged to obtain wet bacterial cells, to which xylene was added so as to give a final concentration of 10 mL / L, and membrane treatment was performed at 30° C. and 850 rpm for 30 minutes.

[0536] Prepare a mixture containing 100 g / L of the above membrane-treated wet cells, 140 mM glucose, 50 mM of a mixture of cis- and trans-isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%), 50 mM magnesium chloride, 10 mM pyruvic acid, and 10 mM NAD + 0.2 mL of the reaction solution was reacted at 30°C and 850 rpm for 24 hours.

[0537] After the reaction was completed, the reaction product and the residual substrate were analyzed by the same method as in Example 1. The results are shown in Table 24.

[0538] [Table 24]

[0539]

[0540] ND: Below the detection limit (S / N<3)

[0541] According to Table 24, due to pyruvate and NAD + The introduction of the regeneration gene increased the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid and the consumption of the substrate 1,4-bis(aminomethyl)cyclohexane. In addition, due to the destruction of the pyruvate decomposition gene of the host Escherichia coli, the production of the target product and the consumption of the substrate increased, especially the increase in substrate consumption was significant. It is speculated that the destruction of the pyruvate decomposition gene promoted the transamination reaction of the substrate.

[0542] The above results show that, in the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid based on the resting cell reaction, it is effective to enhance the supply of keto acid and coenzyme by introducing genes for enzymes that regenerate keto acid and coenzyme or by shutting down the host's keto acid decomposition system.

[0543] [Example 11] Extraction of common amino acid residues in ALDH with high specificity for trans-4-(aminomethyl)cyclohexane-1-carbaldehyde

[0544] It can be said that the ALDH with a high trans isomer ratio of AMCHA produced by the reaction using a cis-trans isomer mixed substrate in Examples 3, 4, and 9 is useful in the production of AMCHA, particularly in the production of TXA. In order to find the characteristic amino acid residues of these ALDHs, the amino acid sequences were compared.

[0545] ALDHs (SEQ ID NOs. 20, 40, 41, 127, 128, 130, and 131) whose trans-isomer ratio of AMCHA produced by reaction using cis-trans isomer mixed substrate in Examples 3, 4, and 9 was 70% or more were extracted as ALDHs with particularly high specificity for the trans-isomer of 4-(aminomethyl)cyclohexane-1-carboxaldehyde. As a result, all of them were enzymes annotated as benzaldehyde dehydrogenases. Therefore, among the ALDHs annotated as benzaldehyde dehydrogenases used in Examples 3, 4, and 9, ALDHs (SEQ ID NOs. 134 and 135) whose trans-isomer ratio of AMCHA produced by reaction using cis-trans isomer mixed substrate was less than 50% were extracted as ALDHs that did not have specificity for the trans-isomer of 4-(aminomethyl)cyclohexane-1-carboxaldehyde.

[0546] It should be noted that, in order to eliminate the influence of the strength of enzyme activity, ALDH in Examples 3, 4 and 9 in which the AMCHA generated by the reaction using a cis-trans mixed substrate was less than 0.2 mM was excluded.

[0547] The amino acid sequence of ALDH extracted by the above operation was aligned using Clustal Omega ( Figure 5A and Figure 5B ). Furthermore, amino acid residues that are conserved in ALDH with high specificity for trans-4-(aminomethyl)cyclohexane-1-carboxaldehyde and are not conserved in ALDH that does not have specificity for trans-4-(aminomethyl)cyclohexane-1-carboxaldehyde were extracted. The extracted amino acid residues are shown in Table 25 with the amino acid residue numbers corresponding to XylC (SEQ ID NO: 20).

[0548] [Table 25]

[0549]

[0550] It was suggested that the amino acid residues shown in Table 25 are important for ALDH to have particularly high specificity for trans-4-(aminomethyl)cyclohexane-1-carbaldehyde.

Claims

1. A protein having an amino group transfer activity of 1,4-bis(aminomethyl)cyclohexane to another compound to produce 4-(aminomethyl)cyclohexane-1-carbaldehyde, and comprising an amino acid sequence having 60% or more identity with the amino acid sequence represented by any one of SEQ ID NOs. 1 to 4 and 103 to 105.

2. A DNA encoding the protein according to claim 1.

3. A recombinant DNA comprising the DNA according to claim 2.

4. A recombinant cell comprising the DNA according to claim 2, or obtained by transforming a host cell with the recombinant DNA according to claim 3.

5. A protein having aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, and comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139.

6. A protein having aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, and comprising an amino acid sequence having 60% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22 and 35 to 46.

7. A DNA encoding the protein according to claim 6. A recombinant DNA comprising the DNA according to claim 7.

9. A recombinant cell comprising the DNA according to claim 7, or obtained by transforming a host cell with the recombinant DNA according to claim 8.

10. A method for producing cis-isomer and / or trans-isomer 4-(aminomethyl)cyclohexane-1-carboxylic acid from cis-isomer and / or trans-isomer 1,4-bis(aminomethyl)cyclohexane, comprising: (i) a step of producing 4-(aminomethyl)cyclohexane-1-carbaldehyde from 1,4-bis(aminomethyl)cyclohexane in the presence of an aminotransferase; and (ii) A step of producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carboxaldehyde in the presence of aldehyde dehydrogenase.

11. The manufacturing method according to claim 10, wherein: The aminotransferase is the protein according to claim 1, and the aldehyde dehydrogenase is the protein according to claim 6.

12. The manufacturing method according to claim 10 or 11, wherein: Alanine dehydrogenase and / or NADH oxidase may coexist in a part or all of the above methods.

13. The manufacturing method according to claim 10 or 11, wherein: Production of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid from cis-isomer and / or trans-isomer 1,4-bis(aminomethyl)cyclohexane.

14. The manufacturing method according to claim 10 or 11, wherein: Cis-4-(aminomethyl)cyclohexane-1-carboxylic acid is produced from cis-isomer and / or trans-isomer of 1,4-bis(aminomethyl)cyclohexane.

15. The manufacturing method according to claim 10 or 11, wherein: Part or all of the method is carried out under neutral or alkaline conditions.

16. The manufacturing method according to claim 15, wherein: Part or all of the process is carried out in the presence of a secondary amine.

17. A protein which is an aldehyde dehydrogenase and has an aldehyde dehydrogenase activity of oxidizing an aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid. 18 . The protein according to claim 17 , which is benzaldehyde dehydrogenase and has aldehyde dehydrogenase activity of oxidizing the aldehyde group of 4-(aminomethyl)cyclohexane-1-carboxaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid.

19. The protein according to claim 17, wherein The aldehyde dehydrogenase is derived from bacteria of the genus Pseudomonas. 20 . The protein according to claim 17 , which consists of an amino acid sequence having 50% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139.

21. A protein having aldehyde dehydrogenase activity for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carboxaldehyde, the protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence set forth in SEQ ID NO: 20 and comprising at least one of the following amino acid groups [1] to [26] when aligned with the amino acid sequence set forth in SEQ ID NO: 20, [1] The amino acid residue corresponding to position 35 is a glycine residue (G), [2] The amino acid residue at position 65 is an alanine residue (A), [3] The amino acid residue at position 82 is a leucine residue (L), [4] The amino acid residue corresponding to position 104 is a glycine residue (G), [5] The amino acid residue corresponding to position 109 is a glycine residue (G), [6] The amino acid residue corresponding to position 110 is an isoleucine residue (I), [7] The amino acid residue corresponding to position 117 is a glutamine residue (Q), [8] The amino acid residue at position 122 is an alanine residue (A), [9] The amino acid residue corresponding to position 124 is a leucine residue (L), [10] The amino acid residue corresponding to position 134 is a valine residue (V), [11] The amino acid residue corresponding to position 144 is a valine residue (V), [12] The amino acid residue corresponding to position 160 is a phenylalanine residue (F), [13] The amino acid residue corresponding to position 271 is a glycine residue (G), [14] The amino acid residue at position 290 is an alanine residue (A), [15] The amino acid residue corresponding to position 319 is an aspartic acid residue (D), [16] The amino acid residue corresponding to position 333 is an aspartic acid residue (D), [17] The amino acid residue corresponding to position 365 is a glutamine residue (Q), [18] The amino acid residue corresponding to position 390 is an isoleucine residue (I), [19] The amino acid residue at position 420 is an alanine residue (A), [20] The amino acid residue corresponding to position 423 is a leucine residue (L), [21] The amino acid residue corresponding to position 434 is a valine residue (V), [22] The amino acid residue corresponding to position 440 is a proline residue (P), [23] The amino acid residue corresponding to position 443 is a cysteine ​​residue (C), [24] The amino acid residue corresponding to position 445 is a proline residue (P), [25] The amino acid residue corresponding to position 466 is a serine residue (S), [26] The amino acid residue corresponding to position 467 is an isoleucine residue (I).

22. A DNA encoding the protein according to any one of claims 5 and 17 to 21. A recombinant DNA comprising the DNA according to claim 22. A recombinant cell comprising the DNA according to claim 22.

25. A recombinant cell obtained by transforming a host cell with the recombinant DNA according to claim 23.

26. The manufacturing method according to claim 10, wherein: The aminotransferase is the protein according to claim 1, and the aldehyde dehydrogenase is the protein according to any one of claims 5 and 17 to 21.

27. The manufacturing method according to claim 26, wherein: Alanine dehydrogenase and / or NADH oxidase may coexist in a part or all of the above methods.

28. The manufacturing method according to claim 26, wherein: Production of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid from cis-isomer and / or trans-isomer 1,4-bis(aminomethyl)cyclohexane.

29. The manufacturing method according to claim 26, wherein: Cis-4-(aminomethyl)cyclohexane-1-carboxylic acid is produced from cis-isomer and / or trans-isomer of 1,4-bis(aminomethyl)cyclohexane.

30. The manufacturing method according to claim 26, wherein: Part or all of the method is carried out under neutral or alkaline conditions.

31. The manufacturing method according to claim 26, wherein: Part or all of the process is carried out in the presence of a secondary amine.

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