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

Abstract

A production method of one embodiment, said production method being for producing a 4-(aminomethyl)cyclohexane-1-carboxylic acid from 1,4-bis(aminomethyl)cyclohexane, comprises using a protein that comprises an amino acid sequence having 60% or more identity with an amino acid sequence represented by one of SEQ ID NOS: 1-4 and 103-105 and has aminotransferase activity, and a protein that comprises an amino acid sequences having 50% or more identity with an amino acid sequence represented by one of SEQ ID NOS: 19-22, 35-46 and 127-139 and has aldehyde dehydrogenase activity.

Description

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

[0001] The present invention relates to a protein used in the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid, a method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid using the protein, and an efficient method for producing trans-4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0002] 4-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA) is an artificially synthesized amino acid developed as an antiplasmin agent (Non-Patent Document 1). AMCHA exists as cis- and trans-stereoisomers. Of these, Non-Patent Document 2 confirmed that the trans-isomer has antiplasmin activity. The trans-isomer, trans-4-aminomethylcyclohexane-1-carboxylic acid (t-AMCHA), is also known as tranexamic acid (TXA), and is used as a hemostatic and anti-inflammatory agent for the prevention and treatment of bleeding due to its antiplasmin activity (Non-Patent Documents 3 and 4).

[0003] Known methods for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid are mainly chemical synthesis methods. For example, Patent Document 1 discloses a method for producing trans 4-(aminomethyl)cyclohexane-1-carboxylic acid through a three-step chemical synthesis reaction, which involves using 4-(chloromethyl)benzoic acid as a starting substrate, generating 4-(aminomethyl)cyclohexane-1-carboxylic acid through an amination reaction and a hydrogenation reaction, and then subsequently generating 4-(aminomethyl)cyclohexane-1-carboxylic acid through an isomerization reaction.

[0004] Chemical synthesis methods, including those mentioned above, require reactions at high temperatures and pressures, which pose problems such as high energy costs and a heavy environmental burden.

[0005] An alternative to chemical synthesis is a production method using microorganisms. Patent Document 2 discloses a method for producing trans-4-(aminomethyl)cyclohexane-1-carboxylic acid by contacting a mixture of cis and trans isomers of 1,4-bis(aminomethyl)-cyclohexane with a microorganism belonging to the genus Corynebacterium or Nocardia. However, because this method is a conversion reaction using a microorganism isolated from nature, the productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid is low, and the enzyme that produces the active form has not been identified.

[0006] CN114014768A Japanese Unexamined Patent Publication No. 63-152991

[0007] Keio Journal of Medicine (1962)11, 8, 105-115Keio Journal of Medicine (1964) 18, 4, 177-185European Journal of Haematology (2020) 104, 2, 79-87Journal of Trauma and Acute Care Surgery (2019) 86, 1, 101-107

[0008] An object of the present invention is to provide a method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid based on an enzymatic reaction using 1,4-bis(aminomethyl)cyclohexane as a substrate, and a protein derived from a microorganism that can be used in the enzymatic reaction.

[0009] Until now, the reaction pathway for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 1,4-bis(aminomethyl)cyclohexane was unknown. Therefore, the present inventors assumed a two-step enzymatic reaction pathway involving aminotransferase and aldehyde dehydrogenase, and selected proteins that may have the desired activity. They expressed and purified these proteins in an Escherichia coli strain and performed an enzymatic reaction, and found that 4-(aminomethyl)cyclohexane-1-carboxylic acid could be produced from 1,4-bis(aminomethyl)cyclohexane, leading to the completion of the present invention.

[0010] The present invention relates to, for example, the following inventions: [1] A protein having transamination activity that transfers the amino group of 1,4-bis(aminomethyl)cyclohexane to another compound to produce 4-(aminomethyl)cyclohexane-1-carbaldehyde, and consisting of an amino acid sequence having 60% or more identity to the amino acid sequence represented by any one of SEQ ID NOS: 1 to 4 and 103 to 105. [2] DNA encoding the protein described in [1]. [3] Recombinant DNA comprising the DNA described in [2]. [4] A recombinant cell comprising the DNA described in [2] or obtained by transforming a host cell with the recombinant DNA described in [3]. [5] A protein having aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, and consisting of an amino acid sequence having 50% or more identity to 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-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, and consisting of an amino acid sequence having 60% or more identity to the amino acid sequences represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139. [7] DNA encoding the protein according to [5] or [6]. [8] Recombinant DNA comprising the DNA according to [7]. [9] A recombinant cell comprising the DNA according to [7] or obtained by transforming a host cell with the recombinant DNA according to [8].

[10] A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid from cis and / or trans 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-carbaldehyde in the presence of an aldehyde dehydrogenase.

[11] The 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].

[12] The method according to

[10] or

[11] , wherein alanine dehydrogenase and / or NADH oxidase are co-present in part or all of the method.

[13] The production method according to any one of

[10] to

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

[14] The production method according to any one of

[10] to

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

[15] The production method according to any one of

[10] to

[13] , in which part or all of the above process is carried out under neutral or basic conditions.

[16] The production method according to

[15] , in which part or all of the above process is carried out in the presence of a secondary amine.

[17] An aldehyde dehydrogenase protein having aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid.

[18] The protein according to

[17] , which is a benzaldehyde dehydrogenase and has aldehyde dehydrogenase activity of oxidizing the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid.

[19] The protein according to

[17] or

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

[20] The protein according to

[17] or

[18] , which consists of an amino acid sequence having 50% or more identity to 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 an aldehyde dehydrogenase activity that produces 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carbaldehyde, and consisting of an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 20, and containing at least one amino acid group selected from the following (1) to (26) when aligned with the amino acid sequence represented by SEQ ID NO: 20:(1) The amino acid residue at 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 at position 104 is a glycine residue (G). (5) The amino acid residue at position 109 is a glycine residue (G). (6) The amino acid residue at position 110 is an isoleucine residue (I). (7) The amino acid residue at 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 at position 124 is a leucine residue (L). (10) The amino acid residue at position 134 is a valine residue (V). (11) The amino acid residue at position 144 is a valine residue (V). (12) The amino acid residue at position 160 is a phenylalanine residue (F). (13) The amino acid residue at 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 at position 319 is an aspartic acid residue (D). (16) The amino acid residue at position 333 is an aspartic acid residue (D). (17) The amino acid residue at position 365 is a glutamine residue (Q). (18) The amino acid residue at 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 at position 423 is a leucine residue (L). (21) The amino acid residue at position 434 is a valine residue (V). (22) The amino acid residue at position 440 is a proline residue (P). (23) The amino acid residue at position 443 is a cysteine ​​residue (C). (24) The amino acid residue at position 445 is a proline residue (P). (25) The amino acid residue at position 466 is a serine residue (S). (26) The amino acid residue at position 467 is an isoleucine residue (I).

[22] DNA encoding the protein according to any one of

[17] to

[21] .

[23] Recombinant DNA comprising the DNA according to

[22] .

[24] A recombinant cell comprising the DNA according to

[22] or obtained by transforming a host cell with the recombinant DNA according to

[23] .

[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] .

[26] The production method according to

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

[27] The production method according to

[25] or

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

[28] The production method according to

[25] or

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

[29] The production method according to any one of

[25] to

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

[30] The method according to any one of

[25] to

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

[0011] The protein having transamination activity and the protein having aldehyde dehydrogenase activity of the present invention enable the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid using 1,4-bis(aminomethyl)cyclohexane as a substrate. Furthermore, by utilizing the substrate specificity of the protein having aldehyde dehydrogenase activity depending on the stereostructure of the target 4-(aminomethyl)cyclohexane-1-carboxylic acid, it is possible to selectively produce a large amount of a compound having the desired structure even when a mixture of cis and trans isomers is used as a substrate.

[0012] According to the production method of the present invention, 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced efficiently while reducing production costs and without requiring high temperatures and pressures. Furthermore, by introducing a keto acid and / or a coenzyme or a regeneration system thereof into the reaction system, the enzymatic reaction can be made more efficient, thereby improving the productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid. Furthermore, when a mixture of cis and trans isomers of 1,4-bis(aminomethyl)cyclohexane is used as a substrate, the aldehyde dehydrogenase of the present invention, which selectively acts on trans 4-(aminomethyl)cyclohexane-1-carbaldehyde, can be used, and the reaction can be carried out under neutral or basic conditions. This promotes the isomerization of the intermediate 4-(aminomethyl)cyclohexane-1-carbaldehyde from the cis to the trans isomer, thereby enabling the production of trans 4-(aminomethyl)cyclohexane-carboxylic acid (tranexamic acid) in high yield and with high selectivity by dynamic kinetic resolution.

[0013] 5A and 5B show a scheme for producing cis- or trans-4-(aminomethyl)cyclohexane-1-carbaldehyde and cis- or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid from cis- or trans-1,4-bis(aminomethyl)cyclohexane in one embodiment.

[0034] FIG. 5B is a schematic diagram showing the construction of an aminotransferase (AT) expression plasmid (pQE80L-PpAT8) in Example 1.

[0035] FIG. 5C is a schematic diagram showing the construction of an AT and aldehyde dehydrogenase (ALDH) co-expression plasmid (pET28a-PatA-XylC) in Example 7.

[0036] FIG. 5A and 5B show the alignment of the amino acid sequences of aldehyde dehydrogenase in Example 11. 5A and 5B show the results of alignment of the amino acid sequences of aldehyde dehydrogenases in Example 11.

[0014] 1. Compounds and Enzyme Reactions 4-(aminomethyl)cyclohexane-1-carboxylic acid (4-(aminomethyl)cyclohexanecarboxylic acid; hereinafter, sometimes referred to as "AMCHA"), the target compound of the present invention, is an unnatural amino acid. 4-(aminomethyl)cyclohexane-1-carboxylic acid exists in two forms: the cis-4-(aminomethyl)cyclohexane-1-carboxylic acid (cis-4-(aminomethyl)cyclohexanecarboxylic acid) and the trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (trans-4-(aminomethyl)cyclohexanecarboxylic acid). Of these, trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is also called tranexamic acid (hereinafter sometimes referred to as "TXA"). Unless otherwise specified herein, "tranexamic acid" or "TXA" refers to 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.

[0015] The enzymatic reaction in the present invention comprises a two-step reaction as shown below. In the first step (step (i)), the substrate compound 1,4-bis(aminomethyl)cyclohexane reacts with a keto acid (e.g., pyruvic acid), which is a compound capable of donating a carbonyl group, and is converted by an aminotransferase (AT) into the intermediate 4-(aminomethyl)cyclohexane-1-carbaldehyde and a compound that accepts an amino group (alanine when pyruvic acid is used as the keto acid). In the second step (step (ii)), the intermediate 4-(aminomethyl)cyclohexane-1-carbaldehyde is converted into NAD(P). ＋It is converted to 4-(aminomethyl)cyclohexane-1-carboxylic acid and NAD(P)H by aldehyde dehydrogenase (ALDH), which uses 4-(aminomethyl)cyclohexane-1-carboxylic acid as a coenzyme (Figure 1).

[0016] In addition, when pyruvate is used as a compound to provide a carbonyl group in the first step, the produced alanine can be converted (regenerated) into pyruvate by alanine dehydrogenase, and in the second step, the produced NAD(P)H can be converted into NAD(P) by NAD(P)H oxidase. ＋ In addition, NAD(P) can be converted (regenerated) into NAD(P) by electrically oxidizing NAD(P)H. ＋ It is possible to regenerate the

[0017] The substrate compound 1,4-bis(aminomethyl)cyclohexane may be a mixture of cis and trans isomers or either the trans or cis isomer. The intermediate 4-(aminomethyl)cyclohexane-1-carbaldehyde may also be a mixture of cis and trans isomers or either the trans or cis isomer.

[0018] 2. Proteins and DNAs of the Present Invention (1) Aminotransferases of the Present Invention and DNAs Encoding the Same Aminotransferases (also referred to as aminotransferases; ATs, or transaminases) are a general term for enzymes that catalyze the reaction between amino acids and α-keto acids in biochemistry. In this specification, "having transamination activity" refers to the activity of catalyzing the reaction of transferring an amino group from a compound having an amino group to another compound, converting it into a carbonyl group.

[0019] The protein having transamination activity of the present invention is a protein that has the activity of transferring the amino group of 1,4-bis(aminomethyl)cyclohexane to another compound and producing 4-(aminomethyl)cyclohexane-1-carbaldehyde. That is, it is a protein that has the activity of mediating the reaction from 1,4-bis(aminomethyl)cyclohexane to 4-(aminomethyl)cyclohexane-1-carbaldehyde, and specifically, it is a protein that has the activity of removing the amino group from 1,4-bis(aminomethyl)cyclohexane and producing 4-(aminomethyl)cyclohexane-1-carbaldehyde. In this specification, these proteins are collectively referred to as the "transamination enzyme of the present invention" or the "AT of the present invention."

[0020] In one embodiment, the AT is a protein having an amino acid sequence that has 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 to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105, and having transamination activity.

[0021] The AT of this embodiment is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105. The protein having the amino acid sequence represented by SEQ ID NO: 1 is the aminotransferase PpAT8 derived from Pseudomonas putida KT2440, the protein having the amino acid sequence represented by SEQ ID NO: 2 is the aminotransferase PpAT2 derived from Pseudomonas putida KT2440, the protein having the amino acid sequence represented by SEQ ID NO: 3 is the aminotransferase AsAT5 derived from Aeromonas salmonicida subsp. Salmonicida, and the protein having the amino acid sequence represented by SEQ ID NO: 4 is the aminotransferase PatA derived from Escherichia coli K12 MG1655. 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 sequence shown in SEQ ID NO: 1 or 4, which are derived from the microorganisms listed in Table 3. These proteins have not been known to have the activity of transferring the amino group of 1,4-bis(aminomethyl)cyclohexane to another compound to produce 4-(aminomethyl)cyclohexane-1-carbaldehyde.

[0022] In another embodiment, the AT is a mutant protein or a homologous protein of a protein consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105, which consists of an amino acid sequence having 60% or more identity (preferably 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more) to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105, and which has transamination activity.

[0023] As used herein, a mutant protein refers to a protein obtained by artificially deleting or substituting amino acid residues in a parent protein, or artificially inserting or adding amino acid residues into the protein. As used herein, a homologous protein refers to a group of proteins found in organisms that exist in nature and that are derived from the same protein in evolutionary origin. Homologous proteins are similar to one another in structure and function.

[0024] In a mutant protein, deletion, substitution, insertion, or addition of amino acids may mean that 1 to 20 amino acids have been deleted, substituted, inserted, or added at any position in the amino acid sequence, and for example, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid may have been deleted, substituted, inserted, or added.

[0025] The amino acids to be substituted, inserted, or added may be natural or non-natural. Natural amino acids include 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, and L-cysteine.

[0026] Examples of amino acids that can be substituted for each other are shown below. Amino acids in the same group can be substituted for each other. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3-hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine

[0027] The amino acid sequence of a mutant protein or a homologous protein of a protein consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105 has at least 60% or more, preferably 70% or more, 75% or more, 80% or more, 90% or more, or 93% or more, more preferably 95% or more, and most preferably 98% or more identity to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105.

[0028] The identity (also called homology) of amino acid sequences or nucleotide sequences can be determined using the algorithm BLAST by Karlin and Altschul [Pro. Nat. Acad. Sci. USA, 90, 5873 (1993)] or FASTA [Methods Enzymol., 183, 63 (1990)]. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing a nucleotide sequence using BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

[0029] The presence of transamination activity can be confirmed, for example, by the following method. First, recombinant DNA containing DNA encoding the protein whose activity is to be confirmed is prepared by the method described below. Next, a microorganism such as Escherichia coli is transformed with the recombinant DNA, and the resulting microorganism is cultured. 1,4-bis(aminomethyl)cyclohexane and pyruvic acid are added to the medium to produce 4-(aminomethyl)cyclohexane-1-carbaldehyde. Finally, 4-(aminomethyl)cyclohexane-1-carbaldehyde can be detected within the cells or in the culture supernatant using a common analytical method such as HPLC, thereby confirming that the target protein has transamination activity.

[0030] In one embodiment, the DNA encoding AT is a DNA consisting of an amino acid sequence having 60% or more identity (preferably 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more) to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105, and encoding a protein having transamination activity. In this specification, the DNA may be a gene or a part of a gene.

[0031] The DNA encoding the AT of this 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 examples thereof include DNA consisting of a base sequence represented by any one of SEQ ID NOs: 5 to 8 and 108 to 110.

[0032] In another embodiment, the DNA encoding AT is 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 homologous protein may have 1 to 50 bases deleted, substituted, inserted or added at any position in the base sequence represented by any one of SEQ ID NOs: 5 to 8 and 108 to 110, and may have, 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. Furthermore, the DNA encoding the mutant protein or homologous protein preferably consists of a base sequence that has at least 60% identity, preferably 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 93% or more, more preferably 95% or more, and most preferably 98% or more identity to the base sequence represented by any one of SEQ ID NOs: 5 to 8 and 108 to 110, and more preferably consists of a base sequence represented by any one of SEQ ID NOs: 5 to 8 and 108 to 110.

[0033] DNA consisting of any one of the nucleotide sequences represented by SEQ ID NOs: 5 to 8 and 108 to 110, or DNA encoding a homologous protein whose sequence is known, may be amplified by PCR using appropriate primers with genomic DNA as a template. In this case, the primers may contain a DNA sequence for cloning into an expression vector, such as a restriction enzyme site, at the end. Homologous proteins can also be obtained by designing probes or primers based on the search and using a microorganism containing the DNA to obtain DNA encoding the homologous protein. DNA encoding mutant proteins can be obtained by error-prone PCR, PCR-based site-directed mutagenesis, or a commercially available site-directed mutagenesis kit. Furthermore, DNA encoding the AT of one embodiment can also be prepared by chemical synthesis using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd., or the like, based on the determined DNA sequence.

[0034] Here, by substituting bases in the DNA of one embodiment so that the codons are optimal for expression in the host cell, the expression level of the protein encoded by the DNA can be improved. Information on codon usage in host cells is available through public databases.

[0035] In one embodiment, the DNA encoding AT may also be DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 5 to 8 and 108 to 110. Hybridization refers to the process in which DNA hybridizes to DNA having a specific base sequence or a part of said DNA. Therefore, the base sequence of the DNA having the specific base sequence or the DNA that hybridizes to a part of said DNA may be 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.

[0036] Examples of DNA used as a probe include DNA of at least 100 bases or more, preferably 200 bases or more, and more preferably 500 bases or more, and examples of DNA used as a primer include DNA of at least 10 bases or more, and preferably 15 bases or more.

[0037] Methods for DNA hybridization experiments are well known, and hybridization conditions can be determined and experiments can be performed according to, for example, 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 many other standard textbooks.

[0038] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions provided with a commercially available hybridization kit, such as the Random Primed DNA Labeling Kit (manufactured by Roche Diagnostics), which prepares a probe by the random prime method and hybridizes under stringent conditions.

[0039] The above-mentioned stringent conditions include, for example, incubating a DNA-immobilized filter and probe DNA in a solution containing 50% formamide, 5x SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / L of denatured salmon sperm DNA at 42°C overnight, followed by washing the filter in a 0.2x SSC solution at about 65°C.

[0040] The various conditions described above can also be achieved by adding or changing blocking reagents used to suppress background in hybridization experiments. The addition of blocking reagents described above may be accompanied by changes in hybridization conditions to suit the conditions.

[0041] Examples of DNA that can hybridize under the above-mentioned stringent conditions include DNA consisting of a base sequence that has 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, more preferably 95% or more, and most preferably 98% or more identity to the base sequence represented by any one of SEQ ID NOs: 5 to 8 and 108 to 110, when calculated using a program such as BLAST or FASTA.

[0042] (2) Aldehyde dehydrogenase of the present invention and DNA encoding the same Aldehyde dehydrogenase (ALDH) is a enzyme that converts NAD(P) into NAD(P) in biochemistry. ＋Aldehyde dehydrogenase is a general term for a group of enzymes that catalyze the oxidation reaction of aldehydes to carboxylic acids using a coenzyme. As used herein, having aldehyde dehydrogenase activity refers to the activity of catalyzing the reaction of oxidizing the aldehyde group of a compound that has an aldehyde group and converting it to a carboxy group.

[0043] 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-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid. That is, it is a protein having the activity of catalyzing the reaction from 4-(aminomethyl)cyclohexane-1-carbaldehyde 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."

[0044] In one embodiment, the ALDH is 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 to the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139, for example, and having aldehyde dehydrogenase activity.

[0045] The ALDH of this 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 gamma-aminobutyraldehyde dehydrogenase PatD derived from Escherichia coli K12 MG1655, 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 sequence shown in SEQ ID NO: 19, 20, or 21, which are derived from the microorganisms listed in Table 13 and Table 20. These proteins have not been known to have the activity of oxidizing the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0046] In another embodiment, the ALDH is a mutant protein or a homologous protein of a protein consisting of the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139, which has 50% or more or 60% or more identity to the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139 and has 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, even more preferably 93% or more or 95% or more, and most preferably 98% or more identity to the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139.

[0047] The presence of aldehyde dehydrogenase activity can be confirmed, for example, by the following method. First, a recombinant DNA containing DNA encoding the protein whose activity is to be confirmed is prepared by the method described below. Next, a microorganism such as Escherichia coli is transformed with the recombinant DNA, and the resulting microorganism is cultured. 4-(aminomethyl)cyclohexane-1-carbaldehyde and NAD(P) are added to the medium. ＋ Finally, 4-(aminomethyl)cyclohexane-1-carboxylic acid is detected in the cells or in the culture supernatant using a general analytical method such as HPLC, thereby confirming that the target protein has aldehyde dehydrogenase activity.

[0048] In a further embodiment, the ALDH has an aldehyde dehydrogenase activity that produces 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carbaldehyde, and may be a protein classified into any one or more of the following 1) to 6): 1) a protein consisting of an amino acid sequence that is 71% or more identical to the amino acid sequence represented by SEQ ID NO: 19; 2) a protein consisting of an amino acid sequence that is 68% or more identical to the amino acid sequence represented by SEQ ID NO: 20; 3) a protein that is an aldehyde dehydrogenase derived from the genus Psudomonas and that is 50% or more identical to the amino acid sequence represented by SEQ ID NO: 20; 4) a protein consisting of an amino acid sequence that is 63% or more identical to the amino acid sequence represented by SEQ ID NO: 21; 5) a protein consisting of an amino acid sequence that is 56.5% or more identical to the amino acid sequence represented by SEQ ID NO: 40; and 6) a protein consisting of an amino acid sequence that is 63.9% or more identical to the amino acid sequence represented by SEQ ID NO: 41. Examples of proteins having such 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.

[0049] In yet another embodiment, the ALDH is preferably an aldehyde dehydrogenase derived from a bacterium of the genus Pseudomonas.

[0050] Examples of bacteria of the genus Pseudomonas include Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas sp., and Pseudomonas sp. MAP12, Pseudomonas fluorescens, Pseudomonas syringae, Pseudomonas amygdali, Pseudomonas oryzihabitans, Pseudomonas maltophilia, Pseudomonas trivialis, Pseudomonas savastanoi, Pseudomonas stutzeri, and the like.

[0051] In another embodiment, ALDH is a benzaldehyde dehydrogenase, which is a protein having aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0052] The benzaldehyde dehydrogenase of this embodiment has aldehyde dehydrogenase activity of oxidizing the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid. Benzaldehyde dehydrogenase is a type of aldehyde dehydrogenase, and is a general term for a group of enzymes that catalyze the oxidation reaction of benzaldehyde to benzoic acid.

[0053] The benzaldehyde dehydrogenase of this embodiment may be benzaldehyde dehydrogenase derived from a microorganism that expresses benzaldehyde dehydrogenase, in addition to the above-mentioned Pseudomonas bacteria. Examples of microorganisms that express 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. , Chloroflexi bacterium, Halioxenophilus aromaticivorans, Novosphingobium aromaticivorans, Oceanobacillus iheyensis, Sphingobium chungbukense, Streptomyces violaceoruber, Rhodococcus atherivorans, Xanthomonas campestris, Streptomyces calvus, Prauserella muralis, Acinetobacter calcoaceticus, Xanthomonas campestris, Acidovorax sp., Streptomyces sp., and the like.

[0054] In one embodiment, the benzaldehyde dehydrogenase may be a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 20, 40, 41, 43, 127 to 137, and 139. In another embodiment, the benzaldehyde dehydrogenase may be a mutant protein or a homologous protein of the protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 20, 40, 41, 43, 127 to 137, and 139, which protein has an amino acid sequence that is 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) identical to the amino acid sequence represented by any one of SEQ ID NOs: 20, 40, 41, 43, 127 to 137, and 139, and which protein has aldehyde dehydrogenase activity.

[0055] In a further embodiment, the benzaldehyde dehydrogenase has an aldehyde dehydrogenase activity that produces 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carbaldehyde, and may be a protein classified into any one or more of the following 7) to 12): 7) a protein consisting of an amino acid sequence that is 71% or more identical to the amino acid sequence represented by SEQ ID NO: 19. 8) a protein consisting of an amino acid sequence that is 68% or more identical to the amino acid sequence represented by SEQ ID NO: 20. 9) a protein that is an aldehyde dehydrogenase derived from the genus Psudomonas and that is 50% or more identical to the amino acid sequence represented by SEQ ID NO: 20. 10) a protein consisting of an amino acid sequence that is 63% or more identical to the amino acid sequence represented by SEQ ID NO: 21. 11) a protein consisting of an amino acid sequence that is 56.5% or more identical to the amino acid sequence represented by SEQ ID NO: 40. 12) a protein consisting of an amino acid sequence that is 63.9% or more identical to the amino acid sequence represented by SEQ ID NO: 41. 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.

[0056] In another further embodiment, the ALDH is a protein having aldehyde dehydrogenase activity of producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carbaldehyde, and consisting of an amino acid sequence having 50% or more identity (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) to the amino acid sequence represented by SEQ ID NO: 20, and comprising at least one amino acid group selected from the following (1) to (26) when aligned with the amino acid sequence represented by SEQ ID NO: 20:(1) The amino acid residue at 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 at position 104 is a glycine residue (G). (5) The amino acid residue at position 109 is a glycine residue (G). (6) The amino acid residue at position 110 is an isoleucine residue (I). (7) The amino acid residue at 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 at position 124 is a leucine residue (L). (10) The amino acid residue at position 134 is a valine residue (V). (11) The amino acid residue at position 144 is a valine residue (V). (12) The amino acid residue at position 160 is a phenylalanine residue (F). (13) The amino acid residue at 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 at position 319 is an aspartic acid residue (D). (16) The amino acid residue at position 333 is an aspartic acid residue (D). (17) The amino acid residue at position 365 is a glutamine residue (Q). (18) The amino acid residue at 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 at position 423 is a leucine residue (L). (21) The amino acid residue at position 434 is a valine residue (V). (22) The amino acid residue at position 440 is a proline residue (P). (23) The amino acid residue at position 443 is a cysteine ​​residue (C). (24) The amino acid residue at position 445 is a proline residue (P). (25) The amino acid residue at position 466 is a serine residue (S). (26) The amino acid residue at position 467 is an isoleucine residue (I).

[0057] Here, for example, "amino acid residue at a position corresponding to position 35" means an amino acid residue in the amino acid sequence of a target protein that is located at a position corresponding to the 35th amino acid residue in SEQ ID NO: 20 when the amino acid sequence of the target protein and the amino acid sequence represented by SEQ ID NO: 20 are aligned.

[0058] As shown in the Examples below, it has been suggested that the amino acid residues shown in (1) to (26) above are conserved in ALDHs that have particularly high specificity for trans-4-(aminomethyl)cyclohexane-1-carbaldehyde. Therefore, it is believed that the aldehyde dehydrogenase of this embodiment has specificity for trans-4-(aminomethyl)cyclohexane-1-carbaldehyde when the amino acid sequence thereof contains at least one of the amino acid groups (1) to (26) above. In other words, the ALDH of this embodiment can have aldehyde dehydrogenase activity with high trans selectivity (also referred to as trans specificity). Here, high trans selectivity means that, when 4-(aminomethyl)cyclohexane-1-carboxylic acid is produced using ALDH, the trans ratio of the resulting 4-(aminomethyl)cyclohexane-1-carboxylic acid containing tranexamic acid (TXA) and / or cis-TXA is greater than 50%, as calculated using the formula shown in Equation 2 below. In particular, high trans specificity means that the trans ratio is 70% or greater. In this application, "selectivity" is treated as synonymous with "specificity," and "selective" is treated as synonymous with "specific."

[0059] The amino acid sequence of the aldehyde dehydrogenase of this embodiment may contain at least one of the amino acid groups (1) to (26) above, and may include, for example, 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.

[0060] An alignment of amino acid sequences can be created using, for example, the known alignment program Clustal Omega. Clustal Omega can be accessed, for example, at https: / / www.ebi.ac.uk / Tools / msa / clustalo / . When creating an alignment using Clustal Omega, for example, default values ​​can be used as parameters.

[0061] The aldehyde dehydrogenase of this embodiment may be a protein consisting of an amino acid sequence that has 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 to the amino acid sequence represented by any one of SEQ ID NOs: 20, 40, 41, 127, 128, 130, 131, and 133, and that has aldehyde dehydrogenase activity.

[0062] The DNA encoding the aldehyde dehydrogenase (ALDH) of this embodiment is a DNA encoding a protein having the above-mentioned aldehyde dehydrogenase activity. In one embodiment, the DNA encoding the ALDH is a nucleotide 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 examples thereof include DNA consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152.

[0063] In another embodiment, the DNA encoding ALDH is 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 homologous protein may have 1 to 50 bases deleted, substituted, inserted, or added at any position in the base sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152, and may have, 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 one embodiment, the DNA encoding ALDH preferably comprises a base sequence having 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, and more preferably comprises a base sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152.

[0064] In one embodiment, the DNA encoding ALDH may be DNA that hybridizes under stringent conditions to DNA consisting of a base sequence complementary to any one of the base sequences represented by SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152. Examples of DNA that can hybridize under stringent conditions include 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 any one of the base sequences represented by SEQ ID NOs: 23 to 26, 47 to 58, and 140 to 152, when calculated using a program such as BLAST or FASTA.

[0065] 3. Recombinant DNA of the Present Invention The recombinant DNA of the present invention comprises DNA encoding the aminotransferase of the present invention and / or DNA encoding the aldehyde dehydrogenase of the present invention. It may further comprise DNA encoding the alanine dehydrogenase described below and / or DNA encoding the NADH oxidase. The recombinant DNA of the present invention may contain DNA encoding only one type of enzyme in a single recombinant DNA, or may contain DNA encoding multiple types of enzymes. In the case of multiple types of enzymes, each enzyme may be controlled by a single promoter or by separate promoters.

[0066] 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 capable of transcribing the DNA.

[0067] When prokaryotes such as bacteria are used as host cells, the recombinant DNA of the present invention preferably further comprises a promoter, a ribosome binding sequence, and a transcription termination sequence in addition to the DNA encoding the various enzymes described above, and may further comprise a gene that controls the promoter. Here, it is preferable to adjust the distance between the Shine-Dalgarno sequence, which is the ribosome binding sequence, and the initiation 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 various enzymes described above, but it is preferable to place the transcription termination sequence immediately downstream of the structural gene.

[0068] The vector is not particularly limited as long as it is a suitable DNA molecule for introducing, propagating, and expressing the target DNA into a host, and not only plasmids but also, for example, artificial chromosomes, vectors using transposons, and cosmids may be used.

[0069] When a microorganism belonging to the genus Escherichia is used as a host cell into which the recombinant DNA of the present invention is introduced, examples of the vector include pColdI, pSTV28, pSTV29, pUC118 (all manufactured by Takara Bio Inc.), pMW118, pMW119, pMW218 (all manufactured by Nippon Gene Co., Ltd.), pET21a, pET28a, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore), pMAL-c5x (New England Biolabs), pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Biosciences), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE-60, pQE80L (all manufactured by Qiagen), pET-3, pBluescript II SK(+), pBluescript II KS(-) (all manufactured by Agilent Technologies), pKYP10 (Japanese Patent Laid-Open Publication 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 (JP-A-63-233798), and the like.

[0070] When using the above vectors, any promoter may be used as long as it functions in the cells of a microorganism belonging to the genus Escherichia, and examples of promoters that can be used include promoters derived from Escherichia coli or phages, such as the trp promoter, gapA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter. Artificially designed and modified promoters such as a promoter consisting of two trp promoters in tandem, the tac promoter, the trc promoter, the lacT5 promoter, the lacT7 promoter, and the let I promoter can also be used.

[0071] When a coryneform bacterium is used as a host cell into which the recombinant DNA of the present invention is introduced, examples of the vector include pCG1 (Japanese Patent Laid-Open No. 57-134500), pCG2 (Japanese Patent Laid-Open No. 58-35197), pCG4 (Japanese Patent Laid-Open No. 57-183799), pCG11 (Japanese Patent Laid-Open No. 57-134500), pCG116, pCE54, pCB101 (all Japanese Patent Laid-Open No. 58-105999), pCE51, pCE52, and pCE53 [all Molecular and General Genetics, 196, 175 (1984)].

[0072] When the above vector is used, any promoter may be used as long as it functions in the cells of coryneform bacteria. For example, the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, pp. 674-679 (2000)] can be used.

[0073] When a yeast strain is used as a host cell into which the recombinant DNA of the present invention is introduced, examples of vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0074] When using the above vectors, any promoter may be used as long as it functions in the cells of a yeast strain, and examples include the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0075] The recombinant DNA of the present invention can be produced, for example, using In-Fusion™ HD Cloning Kit (Takara Bio Inc.), or by treating a DNA fragment encoding the desired enzyme with a restriction enzyme and inserting it downstream of the promoter of the appropriate expression vector described above.

[0076] 4. Recombinant Cells of the Present Invention The recombinant cells of the present invention contain the DNA of the present invention or are recombinant cells obtained by transforming a host cell with the recombinant DNA of the present invention. They may further contain DNA encoding alanine dehydrogenase and / or DNA encoding NADH oxidase, as described below. In the recombinant cells, the DNA of the present invention or recombinant DNA may be integrated into the genome or may exist as an autonomously replicating plasmid, with the DNA of the present invention contained in a transcriptional state. A single host cell may contain only one type of DNA, or two or more types of DNA.

[0077] The host cell may be any of prokaryotes, yeast, animal cells, insect cells, plant cells, etc., but is preferably a prokaryote or yeast strain, more preferably a prokaryote belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast strain belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and most preferably Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli BL21(DE3) (Novagen), Escherichia coli BL21(DE3)pLysS (Merck Millipore), Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm-, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio Inc.), 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 ATCC 13869, Corynebacterium acetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354, or Pseudomonas sp. D-0110, or yeast strains such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, or Candida utilis can be mentioned. ,

[0078] Methods for introducing the recombinant DNA of the present invention into host cells as an autonomously replicable plasmid include, for example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Laid-Open Publication No. 63-248394), the electroporation method [Nucleic Acids Res., 16, 6127 (1988)], the spheroplast method [Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)], and the lithium acetate method [J. Bacteriol., 153, 163 (1983)].

[0079] Methods for introducing the recombinant DNA of the present invention into the chromosome of a host cell include, for example, homologous recombination. Examples of homologous recombination include a method using a plasmid for homologous recombination, which can be prepared by ligating with plasmid DNA carrying a drug resistance gene that cannot autonomously replicate in the host cell to be introduced. Examples of methods using homologous recombination that are frequently used in Escherichia coli include a method in which recombinant DNA is introduced using the homologous recombination system of lambda phage [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].

[0080] Furthermore, E. coli in which a target region on the chromosomal DNA of a host cell has been replaced with the DNA of the present invention or recombinant DNA can be obtained using a selection method that utilizes the fact that E. coli becomes sucrose sensitive due to Bacillus subtilis levansucrase that has been incorporated onto the chromosome together with the recombinant DNA, or a selection method that utilizes the fact that E. coli becomes streptomycin sensitive by incorporating a wild-type rpsL gene into E. coli that has a mutant rpsL gene that is resistant to streptomycin [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)].

[0081] 5. Production Method of the Present Invention 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, and includes the steps of: (i) producing 4-(aminomethyl)cyclohexane-1-carbaldehyde from 1,4-bis(aminomethyl)cyclohexane in the presence of an aminotransferase; and (ii) producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carbaldehyde in the presence of an aldehyde dehydrogenase.

[0082] The production method of this embodiment includes the coexistence of alanine dehydrogenase and / or NADH oxidase in part or all of the method. In particular, the coexistence of alanine dehydrogenase and / or NADH oxidase is preferred in the first step (step (i)), and the coexistence of NADH oxidase is preferred in the second step (step (ii)).

[0083] When the substrate substance in each step of the production method of the present invention (1,4-bis(aminomethyl)cyclohexane used in the first step or 4-(aminomethyl)cyclohexane-1-carbaldehyde used in the second step) has a different stereostructure from the target product in each step (4-(aminomethyl)cyclohexane-1-carbaldehyde in the first step or 4-(aminomethyl)cyclohexane-1-carboxylic acid in the second step), it is preferable that part or all of the production method of this embodiment be carried out under neutral or basic conditions. For example, it is preferable that part or all of the steps in the first step and / or part or all of the steps in the second step be carried out under neutral or basic conditions. Carrying out the reaction under neutral or basic conditions promotes isomerization of the reaction intermediate 4-(aminomethyl)cyclohexane-1-carbaldehyde from the cis isomer to the trans isomer or from the trans isomer to the cis isomer. Isomerization is particularly likely under basic conditions of pH 9 or higher, and the isomerization of 4-(aminomethyl)cyclohexane-1-carbaldehyde reaches equilibrium at a trans ratio of approximately 60%. For example, when tranexamic acid is the final target product, even when the cis isomer or a mixture of cis and trans isomers is used as the substrate, by using an aldehyde dehydrogenase with high trans selectivity, as described below, it is possible to obtain a larger amount of trans 4-(aminomethyl)cyclohexane-1-carboxylic acid, i.e., a higher trans ratio in the product mixture. This allows tranexamic acid to be produced in high yield. On the other hand, when the trans isomer is used as the substrate, isomerization can be prevented by making the solution acidic (e.g., pH 4 to 6), thereby allowing a larger amount of the trans product to be obtained.

[0084] Neutrality refers to a reaction solution with a pH of around 7. Basicity refers to a reaction solution with a pH greater than 7 but ranging from 12. Neutrality or basicity refers to a reaction solution with a pH of preferably 7 to 12, more preferably 7 to 10. The method for achieving basicity is not particularly limited, and can be achieved by adding an alkaline solution, urea, calcium carbonate, ammonia, or the like. A part of the process being carried out under neutral or basic conditions means that the process is neutral or basic from the beginning to the middle, or neutral or basic from the middle to the end. Here, the beginning of the process refers to before the enzyme is added to the reaction solution, and the end of the process refers to after the enzymatic reaction has ended (the same applies hereinafter). When isomerizing a cis isomer to a trans isomer under neutral or basic conditions, an isomerization rate of 20% or more, for example, 40% or more, 50% or more, or 60% or more, can be achieved. Furthermore, when isomerizing a trans isomer to a cis isomer, an isomerization rate of 20% or more, for example, 30% or more, or 35% or more, can be achieved.

[0085] When the substrate substance and the target product in each step of the production method of the present invention have different stereostructures, it is preferable to carry out some or all of the reaction steps in the presence of a secondary amine. The presence of a secondary amine promotes the isomerization of 4-(aminomethyl)cyclohexane-1-carbaldehyde. Examples of secondary amines include L-proline, pyrrolidine, pyrrolidine derivatives, and trans-4-hydroxy-L-proline. The amount of secondary amine added to the reaction solution may be 0.01 to 500 mM, preferably 0.1 to 200 mM. The addition of a secondary amine can achieve a cis-to-trans isomerization rate of 20% or more, for example, 60% or more, or a trans-to-cis isomerization rate of 20% or more, for example, 30% or more or 35% or more.

[0086] (1) Step of Producing 4-(aminomethyl)cyclohexane-1-carbaldehyde The production method of the present invention includes (i) a step of producing 4-(aminomethyl)cyclohexane-1-carbaldehyde from 1,4-bis(aminomethyl)cyclohexane in the presence of an aminotransferase (hereinafter also referred to as the first step). The aminotransferase is preferably the aminotransferase of the present invention. "In the presence of an aminotransferase" means that an active aminotransferase is present in the reaction system of (i) so as to catalyze the reaction of (i). For example, the method includes a step of adding the aminotransferase of the present invention to the reaction system, or a step of allowing cells containing DNA encoding the aminotransferase of the present invention and capable of expressing the aminotransferase, or recombinant cells capable of expressing the aminotransferase obtained by transforming a host cell with recombinant DNA containing DNA encoding the aminotransferase of the present invention, to act on the cells. Here, "allowing the cells to act" means adding a substrate substance to a cell culture system and allowing it to act on the protein expressed by the cells.

[0087] In the first step, a protein having transamination activity may be used as an enzyme source, and the enzyme source and a reaction substrate may be present in an aqueous medium to produce and accumulate 4-(aminomethyl)cyclohexane-1-carbaldehyde in the aqueous medium. Alternatively, cells capable of producing the aminotransferase may be cultured in a medium to produce and accumulate 4-(aminomethyl)cyclohexane-1-carbaldehyde in the culture.

[0088] In the method using an enzyme source, the enzyme source may be a purified protein, or may be a culture obtained by culturing cells capable of producing a protein having the desired activity in a medium, or a processed product of the culture.

[0089] The culture or a processed culture product contains a protein having the desired activity as an enzyme source. Examples of processed culture products include a concentrate of the culture, a dried culture, bacterial cells obtained by centrifuging 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 ultrasonically 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, an immobilized product of the bacterial cells, and an enzyme preparation obtained by extraction from the bacterial cells.

[0090] In the method using the enzyme source in the first step, when a purified protein is used as the 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 the enzyme source, the amount of the enzyme source varies depending on the specific activity of the enzyme source, but may be, for example, 5 to 1000% by weight, preferably 10 to 400% by weight, in terms of wet cell weight, relative to 1,4-bis(aminomethyl)cyclohexane.

[0091] Examples of aqueous media include water, buffer solutions such as phosphate, carbonate, acetate, borate, citrate, Tris, 2-morpholinoethanesulfonic acid (hereinafter referred to as MES), 3-morpholinopropanesulfonic acid (hereinafter referred to as MOPS), and N-cyclohexyl-2-aminoethanesulfonic acid (hereinafter referred to as CHES), alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, and amides such as acetamide. The culture medium of the microorganism used as the enzyme source can also be used as the aqueous medium.

[0092] As described in 1. above, the reaction of producing 4-(aminomethyl)cyclohexane-1-carbaldehyde from 1,4-bis(aminomethyl)cyclohexane is an enzymatic reaction catalyzed by aminotransferase (a protein with transamination activity) in the presence of a compound capable of donating a carbonyl group. Therefore, in the first step, it is desirable to have a compound capable of donating a carbonyl group coexist.

[0093] Examples of compounds capable of providing a carbonyl group include keto acids, and preferred examples include pyruvic acid, α-ketoglutaric acid, and α-ketobutyric acid.

[0094] The keto acid may be of any origin as long as it serves as a substrate for the aminotransferase of the present invention. A culture of a microorganism capable of producing a keto acid or a processed product of the culture may be used as is, or a keto acid recovered from the culture or a processed product of the culture may be used.

[0095] Furthermore, when culturing a microorganism in a medium to produce and accumulate 4-(aminomethyl)cyclohexane-1-carbaldehyde, the microorganism may be a microorganism capable of producing a keto acid. Examples of microorganisms capable of producing a keto acid include microorganisms in which the gene for the pyruvate dehydrogenase complex involved in pyruvate decomposition in vivo has been deleted or weakened, microorganisms in which the lactate dehydrogenase gene has been deleted or weakened, and microorganisms in which the pyruvate oxidase gene has been deleted or weakened. Here, "attenuating" means reducing the expression level of the target gene (here, the gene for the pyruvate dehydrogenase complex).

[0096] When a keto acid is added in the first step, the amount added may be in a molar ratio of 0.01 to 100, preferably 0.01 to 60, relative to 1,4-bis(aminomethyl)cyclohexane.

[0097] In the first step, when pyruvic acid is used as the keto acid, it is preferable to use alanine dehydrogenase (AlaDH). As explained in 1. above, AlaDH is a keto acid that decomposes NAD. ＋ AlaDH is an enzyme that can catalyze the conversion (regeneration) of the produced alanine to pyruvic acid using AlaDH as a coenzyme. Therefore, by allowing AlaDH to coexist, pyruvic acid is reused, and 4-(aminomethyl)cyclohexane-1-carbaldehyde can be produced efficiently with a small amount of raw material. In addition, when AlaDH is coexisted, the NAD required for the reaction is ＋It is desirable to add

[0098] AlaDH is one of the enzymes that metabolize alanine, aspartic acid, and glutamic acid (alanine + water + NAD ＋ ) and (pyruvic acid + NH ３ +NADH + H ＋ In the first step of the method for producing tranexamic acid of the present invention, alanine produced when pyruvic acid is used as the keto acid can be converted to pyruvic acid.

[0099] The AlaDH is not particularly limited, and may be, for example, AlaDH derived from Bacillus subtilis, Alkalihalobacillus pseudofirmus, or Staphylococcus aureus. Specific examples include 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).

[0100] The coexistence of AlaDH may mean that a culture of cells expressing AlaDH or a processed product of said culture is added to the enzyme reaction in the first step, that cells expressing AlaDH are co-cultured, or that DNA encoding AlaDH is contained in the recombinant cells.

[0101] The amount of AlaDH may be 0.01 to 100% by weight, preferably 0.1 to 50% by weight, based on the alanine produced by the transamination reaction. When a culture or a processed product of the culture is used as the enzyme source, the amount of the enzyme source varies depending on the specific activity of the enzyme source, but may be, for example, 5 to 1000% by weight, preferably 10 to 400% by weight, based on the wet cell weight of 1,4-bis(aminomethyl)cyclohexane.＋ It is desirable to add NAD ＋ The amount of the compound added may be in the range of 0.01 to 100, preferably 0.01 to 60, molar ratio relative to the alanine produced in the transamination reaction.

[0102] The substrate substance 1,4-bis(aminomethyl)cyclohexane may be either the trans isomer, the cis isomer, or a mixture thereof, with a mixture of the trans and cis isomers being preferred due to the availability of the raw material. Furthermore, the target 4-(aminomethyl)cyclohexane-1-carbaldehyde may be either the trans isomer, the cis isomer, or a mixture thereof. When the resulting 4-(aminomethyl)cyclohexane-1-carbaldehyde is to be supplied to the production of tranexamic acid, it is preferred that it be the trans isomer. In the mixture of trans and cis isomers, the ratio of the trans isomer to the cis isomer is not important, but the ratio of the trans isomer is preferably greater than 50%.

[0103] (2) Step of Producing 4-(aminomethyl)cyclohexane-1-carboxylic Acid The production method of the present invention includes (ii) a step of producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carbaldehyde in the presence of aldehyde dehydrogenase (hereinafter also referred to as the second step). Preferably, the production method includes a step of treating 4-(aminomethyl)cyclohexane-1-carbaldehyde with the protein having aldehyde dehydrogenase activity of the present invention, a cell containing DNA encoding the protein having aldehyde dehydrogenase activity of the present invention, or a recombinant cell obtained by transforming a host cell with recombinant DNA containing DNA encoding the protein having aldehyde dehydrogenase activity of the present invention.

[0104] In the second step, a protein having aldehyde dehydrogenase activity may be used as an enzyme source, and the enzyme source and a reaction substrate may be present in an aqueous medium to produce and accumulate 4-(aminomethyl)cyclohexane-1-carboxylic acid in the aqueous medium. Alternatively, a microorganism capable of producing aldehyde dehydrogenase may be cultured in a medium to produce and accumulate 4-(aminomethyl)cyclohexane-1-carboxylic acid in the culture.

[0105] As mentioned in 1. above, the reaction of producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carbaldehyde requires the coenzyme NAD(P). ＋ This is an enzymatic reaction catalyzed by aldehyde dehydrogenase (a protein with aldehyde dehydrogenase activity) in the presence of NAD as a coenzyme. ＋ NAD prefers ＋ Type, NADP ＋ NADP prefers ＋ type, NAD ＋ and NADP ＋ Therefore, it is preferable to use a coenzyme that is highly available to the aldehyde dehydrogenase used.

[0106] NAD(P) ＋ The amount of may be in a molar ratio of 0.01 to 100, preferably 0.01 to 60, relative to 4-(aminomethyl)cyclohexane-1-carbaldehyde.

[0107] Furthermore, as a method for supplying a coenzyme, a microorganism capable of producing a coenzyme can be used in a host expressing aldehyde dehydrogenase. This is expected to further improve the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid. Examples of microorganisms capable of producing a coenzyme include those capable of producing NAD(P) in vivo. ＋ Microorganisms in which genes involved in decomposition have been deleted or weakened, NAD(P) ＋ Microorganisms in which genes involved in the production (e.g., pncB, nadA, nadB) have been enhanced or introduced, ＋A microorganism in which a gene (e.g., nadR) that suppresses the expression or function of a gene product involved in the production of NAD(P) is deleted or weakened, or ＋ Examples include microorganisms in which genes involved in decomposition (for example, mazG, nudC) have been deleted or weakened.

[0108] 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 the enzyme source, the amount of the enzyme source varies depending on the specific activity of the enzyme source, but may be, for example, 5 to 1000% by weight, preferably 10 to 400% by weight, in terms of wet cell weight, relative to 4-(aminomethyl)cyclohexane-1-carbaldehyde.

[0109] In the second step, it is preferable to use NAD(P)H oxidase (NOX).

[0110] NAD(P)H oxidase is a metabolic enzyme that converts NAD(P)H into NAD(P)H. ＋ +O ２ ) to (NAD(P) ＋ +H ２ O ２ or H ２ The oxidoreductase catalyzes the reaction of converting 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 into NAD(P). ＋ Therefore, in the presence of NAD(P)H oxidase, NAD(P) ＋ is recycled, and 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced efficiently using a small amount of raw materials.

[0111] The 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, or NADH oxidase SmNOX (accession number: WP_002268044.1) derived from Lactococcus lactis-derived NADH oxidase LlNOX (accession number: CAL97012.1), and Enterococcus feacalis-derived NADH oxidase EfNOX (accession number: WP_002361833.1).

[0112] The coexistence of NAD(P)H oxidase may mean adding a culture of cells expressing NAD(P)H oxidase or a processed product of the culture, or co-culturing cells expressing NAD(P)H oxidase, or incorporating DNA encoding NAD(P)H oxidase into the recombinant cells.

[0113] The amount of NAD(P)H oxidase may be 0.001 to 100% by weight, preferably 0.01 to 50% by weight, based on the total amount of NAD(P)H produced when alanine dehydrogenase is used in the first step and NAD(P)H produced by ALDH in the second step. When a culture or a processed product of the culture is used as the enzyme source, the amount of the enzyme source varies depending on the specific activity of the enzyme source, and may be, for example, 0.5 to 1000% by weight, preferably 1 to 400% by weight, based on the wet cell weight of 1,4-bis(aminomethyl)cyclohexane.

[0114] Instead of using NAD(P)H oxidase, NAD(P)H is electrically oxidized to NAD(P). ＋ It can also be played back.

[0115] The substrate substance, 4-(aminomethyl)cyclohexane-1-carbaldehyde, may be either a trans isomer, a cis isomer, or a mixture thereof. When the substrate and the target compound have different conformations, it is preferable that part or all of the process be carried out in a neutral or basic environment, and further, it is preferable that part or all of the process be carried out in the presence of a secondary amine. When the substrate and the target compound have the same conformation, it is desirable that part or all of the process be carried out in an acidic environment.

[0116] The target 4-(aminomethyl)cyclohexane-1-carboxylic acid may be a cis-isomer, a trans-isomer, or a mixture thereof. When the target is a cis-isomer (i.e., cis-tranexamic acid), the protein having aldehyde dehydrogenase activity in the second step is preferably an enzyme capable of accepting a cis-isomer substrate, such as any of SEQ ID NOS: 19, 21, 22, 35-37, 39, 44-46, 132, 134, 135, and 138, or a mutant or homologous protein thereof. When the target is a trans-isomer (i.e., tranexamic acid), the protein having aldehyde dehydrogenase activity in the second step is preferably an enzyme having high trans-selectivity, such as any of SEQ ID NOS: 20, 38, 40, 41, 127, 128, 129, 130, 131, 133, and 139, or a mutant or homologous protein thereof. Among these, it is more preferable to use enzymes with particularly high trans specificity, such as those represented by SEQ ID NOs: 20, 40, 41, 127, 128, 130, 131, and 133, or mutant or homologous proteins thereof. To obtain a mixture of cis and trans isomers, any aldehyde dehydrogenase of the present invention may be used. The trans isomer ratio in the mixture of cis and trans isomers is preferably 30% or more, for example, 40% or more, 50% or more, 60% or more, or 70% or more.

[0117] As used herein, the term "highly trans-selective enzyme (ALDH)" refers to an enzyme in which the trans isomer ratio of 4-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA) produced by a two-stage reaction using a mixture of cis and trans isomers of 1,4-bis(aminomethyl)cyclohexane exceeds 50% (e.g., 51% or more, 55% or more, 60% or more, 65% or more, 70% or more), as calculated in the Examples described below. Among these, enzymes with a trans isomer ratio of 70% or more are considered to have particularly high trans selectivity. More specifically, the trans ratio of AMCHA can be calculated by multiplying the ratio of the amount of AMCHA produced by trans to the total amount of AMCHA produced by trans and cis, by 100. These highly trans-specific enzymes (ALDH) can produce tranexamic acid in high yield, and are therefore beneficial in the production of tranexamic acid.

[0118] The first and second steps may be carried out separately or simultaneously in the same system, in which case the various enzymes may be contained in cells or may be purified proteins, or may be partly contained in cells and partly purified proteins.

[0119] The cells can be cultured according to a conventional method. The medium for culturing the cells may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the cells and allows efficient cell culture.

[0120] In the method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid by fermentation, it is desirable to add the substrate 1,4-bis(aminomethyl)cyclohexane to the medium. 1,4-bis(aminomethyl)cyclohexane may be added to the medium before the culture or may be added to the culture solution during the culture.

[0121] Alternatively, instead of adding 1,4-bis(aminomethyl)cyclohexane to the 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-mentioned cells.

[0122] The carbon source may be any that can be utilized by the cells, 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.

[0123] Examples of nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as 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 hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria, and digested products thereof.

[0124] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0125] In a method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid by fermentation, pyruvic acid, α-ketoglutaric acid, α-ketobutyric acid, NAD, and the like, which are substrates for aminotransferase or aldehyde dehydrogenase, are produced. ＋ , NADP ＋ Alternatively, a secondary amine, preferably L-proline, a pyrrolidine derivative, or the like may be added to the medium in order to induce isomerization of 4-(aminomethyl)cyclohexane-1-carbaldehyde.

[0126] Alternatively, instead of adding the above compounds, these compounds may be supplied by co-culturing a microorganism capable of producing them with the cells or by co-expressing the microorganism with the enzymes.

[0127] Cultivation is preferably carried out under aerobic conditions, such as shaking culture or submerged aeration and 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 cultivation 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.

[0128] If necessary, antibiotics such as ampicillin or kanamycin may be added to the medium during culture. When culturing recombinant cells transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as necessary.

[0129] For example, when a microorganism transformed with an expression vector using a lac promoter is cultured, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium, and when a microorganism transformed with an expression vector using a trp promoter is cultured, indoleacrylic acid or the like may be added to the medium.

[0130] By the above-mentioned cultivation, 4-(aminomethyl)cyclohexane-1-carboxylic acid, preferably tranexamic acid, can be produced by producing 4-(aminomethyl)cyclohexane-1-carboxylic acid in the culture or in the cells. Quantitation of 4-(aminomethyl)cyclohexane-1-carboxylic acid can be carried out using LCMS (for example, an analytical apparatus LCMS-8040 manufactured by Shimadzu Corporation).

[0131] 4-(aminomethyl)cyclohexane-1-carboxylic acid can be collected from the culture by a combination of a conventional ion exchange resin method, a precipitation method, and other known methods. When 4-(aminomethyl)cyclohexane-1-carboxylic acid accumulates within the cells, the cells can be disrupted by ultrasonication or the like, and the cells can be removed by centrifugation. From the resulting supernatant, 4-(aminomethyl)cyclohexane-1-carboxylic acid can be collected by the ion exchange resin method or the like.

[0132] [Analysis Example] Analysis and quantification of 1,4-bis(aminomethyl)cyclohexane, 4-(aminomethyl)cyclohexane-1-carbaldehyde, or 4-(aminomethyl)cyclohexane-1-carboxylic acid 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 carried out according to the procedures shown below.

[0133] The reaction solution containing the enzyme after the enzymatic reaction, the reaction solution containing the microorganism after the resting cell reaction, or the culture solution containing the microorganism after the culture was centrifuged, and the supernatant was collected. 1,4-bis(aminomethyl)cyclohexane, 4-(aminomethyl)cyclohexane-1-carbaldehyde, or 4-(aminomethyl)cyclohexane-1-carboxylic acid contained in the supernatant was analyzed using an LCMS-8040 (Shimadzu Corporation). The following analytical conditions enable separation of cis- and trans-isomers of 4-(aminomethyl)cyclohexane-1-carbaldehyde and 4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0134] [Analysis conditions] Column: Develosil™ ODS-HG 5 μm 2.0 × 150 mm (Nomura Chemical Co., Ltd.) Column temperature: 40°C Mobile phase: (Mobile phase A) 5 mmol / L heptafluorobutyric acid in water (Mobile phase B) 5 mmol / L heptafluorobutyric acid in acetonitrile Mixing ratio of mobile phase A and B: (0-5 min) 100:0 (5-25 min) 100:0 to 50:50 (25-30 min) 50:50 (30-31 min) 50:50 to 100:0 (31-35 min) 100:0 Flow rate: 0.25 mL / min Detection: Positive mode Detected ion: (1,4-bis(aminomethyl)cyclohexane) 143.1 > 67.0 m / z (4-(aminomethyl)cyclohexane-1-carbaldehyde) 142.1 m / z (4-(aminomethyl)cyclohexane-1-carboxylic acid) 158.2>95.0 m / z

[0135] The following examples are provided in detail, but the present invention is not limited to these examples.

[0136] Example 1: Search for an enzyme (AT) with transamination activity for 1,4-bis(aminomethyl)cyclohexane (1) Selection of AT to be evaluated Until now, no enzyme with transamination activity that transfers the amino group of 1,4-bis(aminomethyl)cyclohexane to produce 4-(aminomethyl)cyclohexane-1-carbaldehyde has been known. To search for an enzyme with transamination activity toward the unnatural compound 1,4-bis(aminomethyl)cyclohexane, the present inventors targeted 33 enzymes annotated as aminotransferases in the genome of Pseudomonas putida KT2440, a strain of Pseudomonas putida that can grow in harsh environments (Applied Microbiology and Biotechnology, 2020, 104:7745-7766), whose genome information is publicly available. Based on preliminary tests using transamination activity as an indicator, they selected PpAT8 (SEQ ID NO: 1) and PpAT2 (SEQ ID NO: 2) as enzymes that may have the desired activity. Furthermore, Aeromonas salmonicida subsp. , a homologous enzyme with 65% identity to PpAT8, was identified. The aminotransferase AsAT5 (SEQ ID NO: 3) derived from Salmonicida was also evaluated. In addition, the putrescine aminotransferase PatA (Journal of Bacteriology, 2012, 194, 15:4080-4088) (SEQ ID NO: 4) derived from Escherichia coli K12 MG1655 was also evaluated, as it has been reported to have transamination activity toward putrescine and cadaverine, which, like 1,4-bis(aminomethyl)cyclohexane, have two amino groups per molecule.

[0137] (2) Construction of AT-expressing strains PCR was performed using DNAs consisting of the base sequences shown in "Primer set" in Table 1 as a primer set and DNAs listed in "Template" in Table 1 as templates to obtain various amplified DNA fragments. Various chromosomal DNAs were prepared by conventional methods. All gene sequences used in this example were obtained from the National Center for Biotechnology Information database (https: / / www.ncbi.nlm.nih.gov / ). All PCR reactions in this example were performed using PrimeSTAR™ MAX DNA Polymerase (Takara Bio Inc.) according to the manufacturer's instructions.

[0138] Using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 9 and 10 as a primer set and plasmid pQE80L (QIAGEN) containing a T5 promoter and an N-terminal His tag sequence as a template, a vector fragment of approximately 4.7 kb was obtained. The 5' end of the nucleotide sequence represented by SEQ ID NO: 9 contains a sequence complementary to the 5' end of the nucleotide sequences represented by SEQ ID NOs: 11, 13, 15, and 17, and the 5' end of the nucleotide sequence represented by SEQ ID NO: 10 contains a sequence complementary to the 5' end of the nucleotide sequences represented by SEQ ID NOs: 12, 14, 16, and 18.

[0139] The various amplified DNA fragments and vector fragments obtained above were ligated using an In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids expressing various ATs, pQE80L-PpAT8 (FIG. 2), pQE80L-PpAT2, pQE80L-AsAT5, and pQE80L-PatA. Escherichia coli BL21(DE3) was transformed with the resulting AT expression plasmids pQE80L-PpAT8, pQE80L-PpAT2, pQE80L-AsAT5, and pQE80L-PatA to construct recombinant E. coli containing the various plasmids.

[0140] (3) Expression of AT and Enzyme Purification The various recombinant E. coli strains obtained in (2) above were inoculated into test tubes containing 2 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking at 30°C for 16 hours. The culture was inoculated into a 250 mL Erlenmeyer flask containing 40 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking at 30°C for 2 hours. After that, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the culture was further cultured with shaking at 30°C for 5 hours. The culture was centrifuged to obtain wet bacterial cells. The enzymes were purified from the wet cells using TALON™ Metal Affinity Resin (Clontech) to obtain His-tagged recombinant purified enzymes PpAT8, PpAT2, AsAT5 and PatA.

[0141] (4) Evaluation of transamination activity toward 1,4-bis(aminomethyl)cyclohexane. The enzymatic activity of various ATs was evaluated using the amount of 4-(aminomethyl)cyclohexane-1-carbaldehyde produced as an index for each purified enzyme purified in (3). 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), 50 mM MOPS (pH 7.8), 10 mM magnesium chloride, 1 mM dithiothreitol (DTT), 10 mM pyruvate, 0.1 mM pyridoxal phosphate (PLP), and a 10 mM mixture of 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. As a negative control, a reaction was carried out in the same manner using a reaction solution to which an equal volume of MOPS buffer was added instead of the purified enzyme solution.

[0142] After the reaction was completed, the reaction mixture was diluted and centrifuged, and the reaction products and remaining substrates contained in the supernatant were analyzed by LCMS-8040 (Figure 3). The peaks of the trans and cis isomers of the product 4-(aminomethyl)cyclohexane-1-carbaldehyde were comprehensively determined based on their elution positions and m / z values, and the amounts of each product were expressed as peak areas (area values). The results are shown in Table 2.

[0143] It was confirmed that 4-(aminomethyl)cyclohexane-1-carbaldehyde was produced and accumulated only when PpAT8, PpAT2, AsAT5, or PatA was added to the reaction. Among these, PatA produced the least amount of remaining substrate and produced the most 4-(aminomethyl)cyclohexane-1-carbaldehyde. This indicates that putrescine aminotransferase PatA from Escherichia coli K12 MG1655 has high transamination activity for 1,4-bis(aminomethyl)cyclohexane.

[0144] <Evaluation of the transamination activity of AT homologs toward 1,4-bis(aminomethyl)cyclohexane> (5) Search for AT homolog enzymes in a database Using the amino acid sequences (SEQ ID NOS: 5 and 8) of PpAT8 and PatA, which were obtained in Example 1 as ATs active toward 1,4-bis(aminomethyl)cyclohexane, as queries, enzymes with 40 to 85% identity to each query enzyme were searched for using the homology search function of BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), a protein sequence database of the National Center for Biotechnology Information. The amino acid sequences (SEQ ID NOS: 103 to 107) of the enzymes extracted by the above procedure and the nucleotide sequences encoding each enzyme (SEQ ID NOS: 108 to 112) are shown in Table 3.

[0145] (6) Construction of AT Homolog-Expressing Strains To obtain gene fragments of each AT homolog enzyme listed in Table 3, PCR reactions were performed using the templates and primers 1 and 2 shown in Table 4. Various chromosomal DNAs were prepared by standard methods. In this reaction, the 5' end of the nucleotide sequence represented by SEQ ID NO:9 contains a nucleotide sequence complementary to the 5' end of each nucleotide sequence represented by Primer 1 in Table 4, and the 5' end of the nucleotide sequence represented by SEQ ID NO:10 contains a nucleotide sequence complementary to the 5' end of each nucleotide sequence represented by Primer 2 in Table 4.

[0146] Each AT expression plasmid was constructed by ligating the amplified DNA fragment obtained by the PCR described above with the vector fragment of pQE80L prepared in Example 1 using an In-Fusion™ HD Cloning Kit (Takara Bio Inc.). Each of the obtained expression plasmids was used to transform Escherichia coli BL21 (DE3), thereby constructing recombinant E. coli having each AT expression plasmid.

[0147] (7) Evaluation of transamination activity of AT homologs toward 1,4-bis(aminomethyl)cyclohexane. Using recombinant E. coli carrying each AT expression plasmid obtained in (6) above, purified His-tagged recombinant AT enzymes were obtained in the same manner as in (3).

[0148] A 0.1 mL reaction mixture (pH 7.8) containing 0.07 mg of each purified enzyme, 50 mM MOPS (pH 7.8), 1 mM magnesium chloride, 1 mM DTT, 1 mM pyruvate, 0.1 mM PLP, and 1 mM cis- and trans-isomer mixture of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%) was prepared and reacted at 30°C and 400 rpm for 24 hours. A negative control reaction mixture was prepared by adding an equal volume of MOPS buffer instead of the purified enzyme, and a positive control reaction mixture was prepared using the purified enzyme PatA.

[0149] After the reaction was completed, the reaction product was analyzed in the same manner as in (4). The results are shown in Table 5.

[0150] PcAT, PaAT, and HeAT1 were confirmed to have transamination activity toward 1,4-bis(aminomethyl)cyclohexane and to produce and accumulate 4-(aminomethyl)cyclohexane-1-carbaldehyde. Of these, PaAT was found to have activity comparable to that of PatA. Furthermore, it was suggested that PpAT8 homologs with 60% or more identity to PpAT8 would have the desired activity.

[0151] Example 2 Effect of pH on the Production Process of 4-(aminomethyl)cyclohexane-1-carbaldehyde (1) Evaluation of the Effect of Solution pH on Transamination Reaction The effect of pH conditions (pH 7.0 or pH 9.0) on the supply of 4-(aminomethyl)cyclohexane-1-carbaldehyde, an intermediate for 4-(aminomethyl)cyclohexane-1-carboxylic acid, was evaluated.

[0152] A 0.1 mL 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 pyruvic acid, 0.1 mM PLP, and 1 mM cis- or trans-1,4-bis(aminomethyl)cyclohexane as a reaction substrate was prepared, and the reaction was carried out at 30°C and 400 rpm for 24 hours.

[0153] After completion of the reaction, 4-(aminomethyl)cyclohexane-1-carbaldehyde contained in the supernatant of the reaction solution was analyzed using an LCMS-8040 (Shimadzu Corporation). The trans ratio of 4-(aminomethyl)cyclohexane-1-carbaldehyde (intermediate trans ratio) was calculated using the calculation formula shown in Equation 1 based on the Area value of the produced 4-(aminomethyl)cyclohexane-1-carbaldehyde. In the formula, the trans isomer means trans-4-(aminomethyl)cyclohexane-1-carbaldehyde, and the cis isomer means cis-4-(aminomethyl)cyclohexane-1-carbaldehyde. The results are shown in Table 6.

[0154] Whether the substrate was cis or trans, approximately 95% or more of the 4-(aminomethyl)cyclohexane-1-carbaldehyde produced at pH 7.0 had the same stereochemistry as the added substrate. On the other hand, at pH 9.0, 30% or more of the 4-(aminomethyl)cyclohexane-1-carbaldehyde produced had the isomeric structure of the added substrate.

[0155] This result suggests that in the transamination reaction of 1,4-bis(aminomethyl)cyclohexane, the conformation of the resulting 4-(aminomethyl)cyclohexane-1-carbaldehyde changes depending on the pH of the reaction solution.

[0156] The above conformational change was thought to be due to (i) the action of aminotransferase or (ii) keto-enol tautomerism of the aldehyde group. Regarding (ii), 4-(aminomethyl)cyclohexane-1-carbaldehyde contains an aldehyde group, and it is generally known that aldehyde groups interconvert between the keto and enol forms in the presence of an acidic or basic catalyst (keto-enol tautomerism). The conversion of the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde from the keto form to the enol form involves the transfer of a hydrogen atom on the α-carbon, forming a carbon-carbon double bond with cyclohexane, and thus eliminating the distinction between cis and trans forms. Therefore, it was predicted that an isomerization reaction of 4-(aminomethyl)cyclohexane-1-carbaldehyde would occur in the presence of an acid or base, independently of the aminotransferase-catalyzed reaction. Therefore, the following test was conducted to confirm whether the above conformational change is dependent on aminotransferase.

[0157] (2) Evaluation of pH-Dependent Isomerization of 4-(Aminomethyl)cyclohexane-1-carbaldehyde According to the procedure described in (1) above, (i) a reaction solution in which the trans intermediate ratio was 2.1% was prepared by reacting the purified PatA enzyme with the cis substrate at pH 7.0, and (ii) a reaction solution in which the trans intermediate ratio was 95.3% was prepared by reacting the purified PatA enzyme with the trans substrate at pH 7.0. The enzyme was removed from each reaction solution using an Amicon™ Ultra 0.5 mL, 3K (Millipore), and the flow-through fraction was collected. To 10 μL of each flow-through fraction, 90 μL of any of the following buffers was added: 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 the mixture was reacted at 30° C. and 400 rpm for 20 hours.

[0158] To evaluate the effect of adding a secondary amine on isomerization, the reaction was also carried out under the same conditions except that 50 mM L-proline was added to the above composition. After the reaction was completed, the reaction product was analyzed in the same manner as in (1) above, and the trans intermediate ratio was calculated. The results are shown in Table 7.

[0159] As can be seen from Table 7, whether the substrate was a cis or trans isomer, almost no isomerization of the intermediate 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 higher. In particular, isomerization of the intermediate proceeded significantly under basic conditions of pH 9 to 10. Furthermore, it was observed that the isomerization of 4-(aminomethyl)cyclohexane-1-carbaldehyde reached equilibrium at a trans ratio of approximately 60%.

[0160] Furthermore, under the proline addition conditions, almost no change was observed between pH 4 and 6, but at pH 7, isomerization of the intermediate tended to proceed, and at pH 9, isomerization of the intermediate was further promoted.

[0161] These results demonstrate that isomerization of 4-(aminomethyl)cyclohexane-1-carbaldehyde occurs under neutral or basic conditions, regardless of the presence or absence of enzymes. Furthermore, the isomerization reaction under neutral or basic conditions is accelerated in the presence of secondary amines such as L-proline.

[0162] The above results suggest that the conformational pattern of the reaction product can be controlled by changing the pH conditions in accordance with the conformation of the substrate used.

[0163] Example 3 Evaluation of Aldehyde Dehydrogenase (ALDH) Activity (1) Selection of ALDH to be Evaluated Until now, no enzyme was known that has aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexanecarboxylic acid.

[0164] In the present invention, based on the results of Example 1, the NAD1 gene derived from Escherichia coli K12 MG1655, which is involved in the metabolism of putrescine and cadaverine like PatA, was identified. ＋ The NADPH-dependent gamma-aminobutyraldehyde dehydrogenase PatD (FEBS Letters, 2005, 579:4107-4112) was selected as the ALDH to be evaluated. In addition, considering that the substrate, 4-(aminomethyl)cyclohexane-1-carbaldehyde, has a bulky and low polarity cyclohexane ring structure, the NADPH-dependent gamma-aminobutyraldehyde dehydrogenase from Pseudomonas putida CSV86, which has been reported to have ALDH activity against a substrate with a bulky and low polarity benzene ring structure, although the steric structure is different, was selected as the ALDH to be evaluated. ＋ NAD-dependent benzaldehyde dehydrogenase XylC (Arch. Microbiol., 2011, 193:553-563), derived from Pseudomonas putida S12 ＋ NADP-dependent phenylacetaldehyde dehydrogenase StyD (Archives of Biochemistry and Biophysics, 2017, 616:47-58) and NADP-dependent phenylacetaldehyde dehydrogenase from Pseudomonas putida NCIMB 9866＋ The ALDH to be evaluated was PchA-dependent 4-hydroxybenzaldehyde dehydrogenase (Appl. Microbiol. Biotechnol., 2014, 98:1349-1356).

[0165] (2) Construction of ALDH-expressing strains The amino acid sequences of the ALDHs (PatD, XylC, StyD, and PchA) selected in (1) are shown in SEQ ID NOs: 19 to 22. The nucleotide sequences encoding these enzymes are shown in SEQ ID NOs: 23 to 26, respectively. Strains expressing these enzymes were constructed as follows.

[0166] PCR was carried out using the DNA shown in "Template" in Table 8 as a template and DNA consisting of the base sequences shown in "Primer set" in Table 8 as a primer set, and each DNA fragment was amplified.

[0167] Chromosomal DNA of Escherichia coli K12 MG1655 was prepared by conventional methods. DNA represented by SEQ ID NO:24 is the nucleotide sequence of the gene encoding XylC derived from Pseudomonas putida CSV86 represented by SEQ ID NO:20 and was prepared by artificial synthesis. DNA represented by SEQ ID NO:25 is the nucleotide sequence of the gene encoding StyD derived from Pseudomonas putida S12 represented by SEQ ID NO:21 and was prepared by artificial synthesis. DNA represented by SEQ ID NO:26 is the nucleotide sequence of the gene encoding PchA derived from Pseudomonas putida NCIMB 9866 represented by SEQ ID NO:22 and was prepared by artificial synthesis.

[0168] The 5' end of the base sequence represented by SEQ ID NO: 9 contains a sequence complementary to the 5' ends of SEQ ID NOs: 27, 29, 31, and 33, and the 5' end of the base sequence represented by SEQ ID NO: 10 contains a sequence complementary to the 5' ends of the base sequences represented by SEQ ID NOs: 28, 30, 32, and 34.

[0169] The various amplified DNA fragments obtained above were ligated to the pQE80L vector fragment prepared in Example 1 using an In-Fusion HD Cloning Kit (Takara Bio Inc.) to construct plasmids expressing various ALDHs, namely, pQE80L-PatD, pQE80L-XylC, pQE80L-StyD, and pQE80L-PchA.

[0170] Escherichia coli BL21 (DE3) was transformed with the ALDH-expressing plasmids pQE80L-PatD, pQE80L-XylC, pQE80L-StyD, and pQE80L-PchA obtained above, to construct E. coli strains carrying various plasmids.

[0171] (3) Expression of ALDH and Enzyme Purification Using the strain obtained in (2) above, wet cells were obtained in the same manner as in Example 1, and the enzymes were purified to obtain His-tagged recombinant purified enzymes PatD, XylC, StyD, and PchA.

[0172] (4) Evaluation of productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid by ALDH in combination with AT <NAD as a coenzyme for ALDH ＋ 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 pyruvic acid, 0.1 mM PLP, 2 mM nicotinamide adenine dinucleotide (NAD ＋ ), and 1 mM of a mixture of cis and trans isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%) were prepared, and the reaction was carried out at 30°C and 400 rpm for 24 hours. As a negative control, a reaction mixture to which an equal volume of MOPS buffer was added instead of the purified enzyme was also used, and the reaction was carried out in the same manner.

[0173] After completion of the reaction, the reaction product was analyzed by the same method as in Example 1. Furthermore, to confirm whether tranexamic acid (TXA) or cis-TXA could be selectively produced from the 4-(aminomethyl)cyclohexane-1-carboxylic acid produced, the trans ratio of 4-(aminomethyl)cyclohexane-1-carboxylic acid (also referred to as the AMCHA trans ratio or AMCHA trans rate) was calculated based on the amounts of TXA and cis-TXA produced using the calculation formula shown in Equation 2 below. The concentration (mM) of each compound was calculated by comparison with the Area value of a standard sample of known concentration.

[0174] First, Table 9 shows the results when a mixture of cis and trans isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%) was used as the substrate.

[0175] Table 9 shows that by combining PatA with PatD, XylC, or StyD, 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced and accumulated using 1,4-bis(aminomethyl)cyclohexane as a substrate.

[0176] Furthermore, because a mixture of cis and trans isomers was used as the substrate, it was expected that the product, 4-(aminomethyl)cyclohexane-1-carboxylic acid, would also be a mixture of cis and trans isomers. However, in reality, the trans ratio of 4-(aminomethyl)cyclohexanecarboxylic acid produced varied depending on the type of ALDH. These results suggest that ALDH has substrate selectivity. In particular, when XylC was used, the trans ratio of 4-(aminomethyl)cyclohexanecarboxylic acid was high, indicating that it can react selectively with the trans isomer of 4-(aminomethyl)cyclohexane-1-carbaldehyde. Therefore, XylC can be said to be a useful ALDH when attempting to efficiently produce TXA from a substrate that is a mixture of cis and trans isomers.

[0177] Next, Table 10 shows the results when only the trans isomer of 1,4-bis(aminomethyl)cyclohexane was used as the substrate.

[0178] The results in Table 10 show that when a trans isomer was used as a substrate, TXA was produced in all ALDHs, but cis-TXA was hardly detected. Therefore, when a trans isomer substrate was used, TXA could be produced in high yield in all ALDHs discovered here, demonstrating that the use of a trans isomer or a substrate rich in the trans isomer is desirable as a substrate for TX production. It was also found that these ALDHs can accept trans isomer substrates.

[0179] 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.

[0180] The purified enzyme PatA and various ALDHs (PatD, XylC, or StyD) obtained in the same manner as above were each 0.07 mg, and the resulting solution was diluted with 50 mM MOPS (pH 7.8), 10 mM magnesium chloride, 1 mM DTT, 10 mM pyruvate, 0.1 mM PLP, 2 mM NAD ＋ A 0.1 mL reaction solution containing 10 mM of cis-1,4-bis(aminomethyl)cyclohexane was prepared, and the reaction was carried out at 30°C and 400 rpm for 24 hours. The results are shown in Table 11.

[0181] Table 11 shows that when the cis isomer was used as a substrate, the proportion of cis-TXA produced was high for both ALDHs. Furthermore, the low production of cis-TXA with XylC indicated that XylC does not readily accept cis substrates.

[0182] Of these, TXA was produced at a relatively high rate only when XylC was used. This is presumably because this reaction was carried out under weakly basic conditions at pH 7.8, and therefore isomerization of the intermediate occurred under the basic conditions shown in Example 2, and even when a cis substrate was added, a trans intermediate was produced, resulting in a large accumulation of TXA. This suggests that, when the goal is to produce TXA, using XylC as ALDH selectively oxidizes the trans intermediate to produce TXA, while simultaneously dynamically isomerizing the remaining cis intermediate to the trans isomer under neutral or basic conditions. This suggests that, in theory, 100% TXA can be produced even when a mixture of cis and trans substrates is used.

[0183] <NADP as a coenzyme for ALDH ＋ In the results of Table 6 above, PchA in which ALDH activity was not confirmed was obtained using NADP as a coenzyme. ＋ It is known that PchA utilizes NADP as a coenzyme (Appl. Microbiol. Biotechnol., 2014, 98:1349-1356). Therefore, we used the purified enzyme PchA obtained in (3) above and XylC as a control. ＋ The aldehyde oxidation activity of 4-(aminomethyl)cyclohexane-1-carbaldehyde was evaluated using the above.

[0184] Among the above reaction solution compositions, NAD ＋ Instead of 1 mM NADP ＋ A reaction mixture containing cis- and trans-isomers (45.7% trans ratio) was used as the substrate. As a negative control, an equal volume of MOPS buffer was added instead of the purified enzyme, and the reaction was carried out in the same manner.

[0185] After the reaction was completed, the reaction product was analyzed in the same manner as described above. The results are shown in Table 12.

[0186] As shown in Table 12, PchA acts as a coenzyme for NADP ＋It was confirmed that 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced by adding XylC. ＋ Although it is known that 4-(aminomethyl)cyclohexane-1-carboxylic acid is an NADP-dependent enzyme, ＋ was also found to be usable as a coenzyme.

[0187] Example 4: Production of 4-(aminomethyl)cyclohexanecarboxylic acid using ALDH homologs (1) Search for ALDH homolog enzymes in a database Using the amino acid sequences (SEQ ID NOS: 19-21) of PatD, XylC, and StyD obtained in Example 3 as ALDHs capable of producing 4-(aminomethyl)cyclohexane-1-carboxylic acid as queries, enzymes with 50-80% identity to each enzyme were searched for using the homology search function of BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), a protein sequence database of the National Center for Biotechnology Information. The amino acid sequences (SEQ ID NOS: 35-46) of the enzymes extracted by the above procedure and the nucleotide sequences encoding each enzyme (SEQ ID NOS: 47-58) are shown in Table 13. In Table 13, CkpatD, SepatD, CspatD, and PppatD are all proteins annotated as gamma-aminobutyraldehyde dehydrogenase. SsBD, HaBD, and RrAD are proteins annotated as benzaldehyde dehydrogenase. PmsLAD is a protein annotated as phenylacetaldehyde dehydrogenase. The others are proteins annotated as aldehyde dehydrogenases or family proteins thereof.

[0188] (2) Construction of ALDH Homolog-Expressing Strains To obtain gene fragments of each ALDH homolog enzyme listed in Table 13 above, PCR reactions were carried out using the templates and primers 1 and 2 shown in Table 14.

[0189] Various chromosomal DNAs were prepared by conventional methods. The DNA represented by SEQ ID NO:47 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding ALDH derived from Citrobacter koseri ATCC BAA-895 strain represented by SEQ ID NO:35 for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO:48 is a DNA obtained by codon-optimizing the nucleotide sequence of the gene encoding ALDH derived from Salmonella enterica subsp. enterica serovar Choleraesuis str. SC-B67 strain represented by SEQ ID NO:36 for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO:49 is a DNA obtained by codon-optimizing the base sequence of the gene encoding ALDH derived from Pectobacterium atrosepticum SCRI1043 strain represented by SEQ ID NO:37 for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO:52 is a DNA obtained by codon-optimizing the base sequence of the gene encoding ALDH derived from Halioxenophilus aromaticivorans strain represented by SEQ ID NO:40 for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO:53 is a DNA obtained by codon-optimizing the base sequence of the gene encoding ALDH derived from Sphingomonas sp. TF3 strain represented by SEQ ID NO:41 for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO:56 is a DNA obtained by codon-optimizing the base sequence of the gene encoding ALDH derived from Pseudomonas sp. represented by SEQ ID NO:44 for expression in Escherichia coli, and was prepared by artificial synthesis. The DNA represented by SEQ ID NO:57 is a DNA obtained by codon-optimizing the base sequence of the gene encoding ALDH derived from the Burkholderia lata strain represented by SEQ ID NO:45 for expression in E. 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 the Paraburkholderia sp. 5N strain represented by SEQ ID NO:46 for expression in E. coli, and was prepared by artificial synthesis.

[0190] In this case, the nucleotide sequences represented by SEQ ID NO: 9 and each primer 1 in Table 14, and the nucleotide sequences represented by SEQ ID NO: 10 and each primer 2 in Table 14 contain complementary nucleotide sequences at their respective 5' ends. That is, the 5' end of the nucleotide sequence represented by SEQ ID NO: 9 contains a nucleotide sequence complementary to the 5' end of the nucleotide sequence represented by each primer 1 in Table 14, and the 5' end of the nucleotide sequence represented by SEQ ID NO: 10 contains a nucleotide sequence complementary to the 5' end of the nucleotide sequence represented by each primer 2 in Table 14.

[0191] The amplified DNA fragment obtained by the PCR and the vector fragment of pQE80L prepared in Example 1 were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.), to construct each ALDH expression plasmid.

[0192] Escherichia coli BL21 (DE3) was transformed with each of the expression plasmids obtained above to generate E. coli strains carrying each of the ALDH expression plasmids.

[0193] (3) Evaluation of productivity of 4-(aminomethyl)cyclohexanecarboxylic acid, an ALDH homologue. Each purified His-tagged recombinant ALDH enzyme was obtained in the same manner as in Example 1 using Escherichia coli carrying each ALDH expression plasmid obtained in (2) above.

[0194] 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 ＋ A 0.1 mL reaction mixture (pH 7.8) containing 1 mM of a mixture of cis and trans isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%) was prepared and reacted for 24 hours at 30°C and 400 rpm. A negative control reaction mixture was prepared by adding an equal volume of MOPS buffer instead of the purified enzyme, and a positive control reaction mixture was prepared by using the purified enzyme XylC.

[0195] After the reaction was completed, the reaction product was analyzed in the same manner as in Example 1. The results are shown in Table 15.

[0196] Table 15 shows that 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced even when an ALDH with approximately 60% or more identity to PatD, XylC, or StyD is used. Furthermore, when CspatD, HaBD, or SsBD was used, the trans ratio of the produced 4-(aminomethyl)cyclohexane-1-carboxylic acid was greater than 50%, indicating that these enzymes have high trans specificity. In particular, when HaBD or SsBD with 70% or more identity to XylC was used, the trans ratio of the produced 4-(aminomethyl)cyclohexane-1-carboxylic acid was greater than 70%, indicating that these enzymes, like XylC, react particularly trans-selectively, i.e., have high trans specificity.

[0197] Example 5 Evaluation of TXA productivity by introduction of a coenzyme regeneration system (1) Construction of an alanine dehydrogenase (AlaDH)-expressing strain To obtain a BsAlaDH gene fragment encoding the Bacillus subtilis 168-derived alanine dehydrogenase represented by SEQ ID NO: 123, a PCR reaction was performed using chromosomal DNA of Bacillus subtilis 168 prepared by a conventional method as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 83 and 84 as a primer set to obtain the BsAlaDH gene fragment represented by SEQ ID NO: 124. The 5' end of the nucleotide sequence represented by SEQ ID NO: 9 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 83, and the 5' end of the nucleotide sequence represented by SEQ ID NO: 10 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 84.

[0198] The BsAlaDH fragment obtained by the PCR and the vector fragment of pQE80L prepared in Example 1 were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pQE80L-BsAlaDH.

[0199] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain BL21(DE3) / pQE80L-BsAlaDH.

[0200] (2) Construction of NADH oxidase (NOX)-expressing strain To obtain a BsNOX gene fragment encoding the Bacillus subtilis 168-derived NADH oxidase represented by SEQ ID NO: 125, a PCR reaction was performed using the chromosomal DNA of Bacillus subtilis 168 as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 85 and 86 as a primer set to obtain the BsNOX gene fragment represented by SEQ ID NO: 126. The 5' end of the nucleotide sequence represented by SEQ ID NO: 9 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 85, and the 5' end of the nucleotide sequence represented by SEQ ID NO: 10 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 86.

[0201] The BsNOX gene fragment obtained by the above PCR and the vector fragment of pQE80L prepared in Example 1 were ligated in 5 parts using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pQE80L-BsNOX.

[0202] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain BL21(DE3) / pQE80L-BsNOX.

[0203] (3) Expression and Enzyme Purification of BsAlaDH and BsNOX Using the BL21(DE3) / pQE80L-BsAlaDH and BL21(DE3) / pQE80L-BsNOX obtained above, His-tagged recombinant enzyme-purified BsAlaDH and BsNOX were obtained in the same manner as in Example 1.

[0204] (4) Evaluation of TXA production activity with AlaDH and NOX introduced 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 pyruvate, 0.1 mM PLP, 2 mM NAD ＋ and 0.1 mL of a reaction solution (pH 7.8) containing 1 mM of a mixture of cis and trans isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%) was prepared, and the reaction was carried out at 30°C and 400 rpm for 24 hours.

[0205] After the reaction was completed, the reaction products and the remaining substrate were analyzed in the same manner as in Example 1. The results are shown in Table 16.

[0206] As can be seen from Table 16, when the coenzyme regeneration system BsAlaDH and BsNOX were introduced, substrate consumption increased and productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid improved.

[0207] Example 6 Production of 4-(aminomethyl)cyclohexane-1-carboxylic acid by resting cell reaction using an AT-expressing strain and an ALDH-expressing strain Wet cells were obtained in the same manner as in Example 1(2) for the BL21(DE3) / pQE80L-PatA strain and the BL21(DE3) / pQE80L-XylC strain constructed in Example 1. Xylene was added to the wet cells to a final concentration of 10 mL / L, and membrane treatment was carried out at 30°C and 850 rpm for 30 minutes.

[0208] 50 g / L of each membrane-treated wet bacterial cell, 50 mM MOPS (pH 7.8), 350 mM glucose, 50 mM cis- and trans-isomer mixture of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%), 50 mM magnesium chloride, and 50 mM NAD ＋ A 0.4 mL reaction solution containing the above was prepared, and the reaction was carried out at 30° C. and 850 rpm for 24 hours.

[0209] After completion of the reaction, the reaction products were analyzed in the same manner as in Example 1. As a result, the production and accumulation of 3.9 mM TXA and 2.8 mM cis-TXA was confirmed. Therefore, it was confirmed that 4-(aminomethyl)cyclohexanecarboxylic acid can be produced even by a resting cell reaction using microbial cells expressing AT and ALDH, respectively.

[0210] Example 7 Production of 4-(aminomethyl)cyclohexane-1-carboxylic acid by resting cell reaction using a strain co-expressing AT and ALDH (1) Construction of a strain co-expressing AT and ALDH A) Construction of BL21(DE3) / pET28a-PatA-XylC strain Using the chromosomal DNA of Escherichia coli K12 MG1655 as a template, a PCR reaction was performed using oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 89 and 90 as a primer set to obtain a PatA fragment. Subsequently, a PCR reaction was performed using the nucleotide sequence represented by SEQ ID NO: 24 as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 91 and 92 as a primer set to obtain a XylC fragment. Note that the 5' end of the nucleotide sequence represented by SEQ ID NO: 90 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 91.

[0211] Using a mixture of the PatA fragment and the XylC fragment obtained above as a template, a PCR reaction was carried out using oligonucleotides having the base sequences represented by SEQ ID NOs: 89 and 92 as a primer set to obtain a DNA fragment in which the two fragments were ligated (hereinafter referred to as PatA-XylC).

[0212] Furthermore, a PCR reaction was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 87 and 88 as a primer set and expression vector pET28a (Novagen) as a template to obtain a vector fragment of approximately 5.2 kb. The 5' end of the nucleotide sequence represented by SEQ ID NO: 87 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 89, and the 5' end of the nucleotide sequence represented by SEQ ID NO: 88 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 92.

[0213] The PatA-XylC fragment and the vector fragment obtained above were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pET28a-PatA-XylC (FIG. 4).

[0214] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain the BL21(DE3) / pET28a-PatA-XylC strain.

[0215] A) Construction of BL21(DE3) / pET28a-XylC-PatA Using the nucleotide sequence represented by SEQ ID NO:24 as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs:93 and 94 as a primer set, a PCR reaction was performed to obtain an XylC fragment. Using the chromosomal DNA of Escherichia coli K12 MG1655 as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs:95 and 96 as a primer set, a PCR reaction was performed to obtain a PatA fragment. The 5' end of the nucleotide sequence represented by SEQ ID NO:94 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO:95.

[0216] Using a mixture of the XylC fragment and PatA fragment obtained above as a template, PCR was performed using oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs:93 and 96 as a primer set to obtain a DNA fragment (hereinafter referred to as XylC-PatA) in which the two fragments were ligated. The 5' end of the nucleotide sequence represented by SEQ ID NO:87 contains a sequence complementary to the 5' end of SEQ ID NO:93, and the 5' end of the nucleotide sequence represented by SEQ ID NO:88 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO:96.

[0217] The XylC-PatA fragment obtained above and the vector fragment obtained in a) above were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pET28a-XylC-PatA.

[0218] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain the BL21(DE3) / pET28a-XylC-PatA strain.

[0219] c) Construction of BL21(DE3) / pUC19-PatA-XylC strain Using the chromosomal DNA of Escherichia coli K12 MG1655 as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 99 and 90 as a primer set, a PCR reaction was performed to obtain a PatA fragment. Subsequently, using the nucleotide sequence represented by SEQ ID NO: 24 as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 91 and 100 as a primer set, a PCR reaction was performed to obtain a XylC fragment. Note that the 5' end of the nucleotide sequence represented by SEQ ID NO: 90 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 91.

[0220] Using a mixture of the PatA fragment and the XylC fragment obtained above as a template, PCR was performed using DNAs consisting of the base sequences represented by SEQ ID NOs: 99 and 100 as a primer set to obtain a DNA fragment in which the two fragments were ligated (hereinafter referred to as PatA-XylC).

[0221] Using DNA consisting of the nucleotide sequences represented by SEQ ID NOs:97 and 98 as a primer set and expression vector pUC19 (manufactured by Nippon Gene Co., Ltd.) as a template, a PCR reaction was performed to obtain a vector fragment of approximately 2.6 kb. The 5' end of the nucleotide sequence represented by SEQ ID NO:97 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO:99, and the 5' end of the nucleotide sequence represented by SEQ ID NO:98 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO:100.

[0222] The PatA-XylC fragment and the vector fragment obtained above were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pUC19-PatA-XylC.

[0223] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain the BL21(DE3) / pUC19-PatA-XylC strain.

[0224] D) Construction of BL21(DE3) / pUC19-XylC-PatA strain Using the DNA represented by SEQ ID NO:24 as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs:101 and 94 as a primer set, a PCR reaction was performed to obtain a XylC fragment. Subsequently, using the chromosomal DNA of Escherichia coli K12 MG1655 as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs:95 and 102 as a primer set, a PCR reaction was performed to obtain a PatA fragment. Note that the 5' end of the nucleotide sequence represented by SEQ ID NO:94 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO:95.

[0225] A PCR reaction was performed using a mixture of the XylC fragment and the PatA gene fragment obtained above as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 101 and 102 as a primer set to obtain a DNA fragment (hereinafter referred to as XylC-PatA) in which the two fragments were linked. The 5' end of the nucleotide sequence represented by SEQ ID NO: 97 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 101, and the 5' end of the nucleotide sequence represented by SEQ ID NO: 98 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 102.

[0226] The XylC-PatA fragment obtained above and the pUC19 vector fragment obtained in c) above were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain an expression plasmid pUC19-XylC-PatA.

[0227] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain the BL21(DE3) / pUC19-XylC-PatA strain.

[0228] (2) Cell Reaction Using AT and ALDH Co-Expressing Strains The productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid in a 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 constructed in (1) above, as well as the BL21(DE3) strain as a negative control.

[0229] 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 was inoculated into a 250 mL Erlenmeyer flask containing 40 mL of LB medium containing 30 mg / L kanamycin or 100 mg / L ampicillin and cultured with shaking at 30°C for 2 hours. IPTG was then added to a final concentration of 1 mM, and the cultured strain was further cultured with shaking at 37°C for 5 hours. As a control, BL21(DE3) was cultured in the same manner in a medium lacking kanamycin and ampicillin.

[0230] After the culture, the culture medium was centrifuged to obtain wet cells, to which xylene was added to give a final concentration of 10 mL / L, and the wet cells were subjected to membrane treatment at 30° C. and 850 rpm for 30 minutes.

[0231] The mixture contained 100 g / L of the above-mentioned membrane-treated wet cells, 50 mM MOPS (pH 7.8), 350 mM glucose, 50 mM cis- and trans-isomer mixture of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%), 50 mM magnesium chloride, and 50 mM NAD. ＋ A 0.4 mL reaction solution containing the above was prepared, and the reaction was carried out at 30° C. and 850 rpm for 24 hours.

[0232] After the reaction was completed, the reaction product was analyzed in the same manner as in Example 1. The results are shown in Table 17.

[0233] From Table 17, it was confirmed that 4-(aminomethyl)cyclohexane-1-carboxylic acid could be produced by the resting cell reaction using the strain in which PatA and XylC were co-expressed.

[0234] From the reaction mixture, NAD ＋ The productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid was evaluated when the above was omitted. The reaction conditions were as described above. The results are shown in Table 18.

[0235] NAD from the reaction mixture ＋ When the coenzyme was removed, the amount of 4-(aminomethyl)cyclohexane-1-carboxylic acid produced decreased, suggesting that the supply of coenzymes is important for the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid by the resting cell reaction.

[0236] Example 8 Production of 4-(aminomethyl)cyclohexane-1-carboxylic acid by fed culture using a strain co-expressing AT and ALDH The productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid by fed culture was evaluated using the BL21(DE3) / pUC19-PatA-XylC strain and the BL21(DE3) / pUC19-XylC-PatA strain constructed in Example 7, and the BL21(DE3) strain as a negative control.

[0237] Each strain was inoculated into a test tube containing 2 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking at 30°C for 16 hours. The BL21(DE3) strain was similarly cultured in a medium without ampicillin. The culture was inoculated into a large test tube containing 4 mL of LB medium containing 30 mg / L or 100 mg / L ampicillin and cultured at 30°C for 5 hours. After incubation at 30°C for 5 hours, IPTG and a 1 g / L mixture of cis and trans isomers of 1,4-bis(aminomethyl)cyclohexane (trans ratio: 45.7%) were added to a final concentration of 1 mM, and the culture was continued at 37°C for an additional 24 hours.

[0238] After the cultivation was completed, the reaction products were analyzed in the same manner as in Example 1. The results are shown in Table 19.

[0239] From Table 19, it was confirmed that TXA can be produced even by the supplemented culture method.

[0240] Example 9: Production of 4-(aminomethyl)cyclohexanecarboxylic acid using ALDH homologs (1) Search for ALDH homolog enzymes Using the amino acid sequence of XylC (SEQ ID NO: 20), an ALDH with high selectivity for trans-isomer substrates as shown in Example 3, as a query, the homology search function of BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), a protein sequence database of the National Center for Biotechnology Information, was used to select enzymes that were 50% or more identical to XylC and had less than 90% identity with other homolog enzymes in the search results. The amino acid sequences (SEQ ID NOs: 127-139) of the enzymes extracted by the above procedure are shown in Table 20.

[0241] ALDH homologs 1 to 11 and 13 in Table 20 are all proteins annotated as benzaldehyde dehydrogenases, and ALDH homolog 12 is an aldehyde dehydrogenase family protein.

[0242] (2) Construction of ALDH Homolog-Expressing Strains In order to obtain gene fragments of each ALDH homolog enzyme listed in Table 20, PCR was performed using the templates and primers 1 and 2 shown in Table 21. In this reaction, the 5' end of the nucleotide sequence shown in SEQ ID NO: 9 contained a nucleotide sequence complementary to the 5' end of the nucleotide sequence shown in each primer 1 in Table 21, and the 5' end of the nucleotide sequence shown in SEQ ID NO: 10 contained a nucleotide sequence complementary to the 5' end of the nucleotide sequence shown in each primer 2 in Table 21.

[0243] The DNAs represented by SEQ ID NOs: 140 to 152 are DNAs in which the nucleotide sequences of the genes encoding the ALDH homolog enzymes represented by SEQ ID NOs: 127 to 139 have been codon-optimized for expression in Escherichia coli, and were prepared by artificial synthesis.

[0244] Each ALDH expression plasmid was constructed by ligating the amplified DNA fragment obtained by the PCR described above to the vector fragment of pQE80L prepared in Example 1 using an In-Fusion™ HD Cloning Kit (Takara Bio Inc.). Each of the expression plasmids obtained above was used to transform Escherichia coli BL21(DE3), thereby constructing E. coli strains carrying each ALDH expression plasmid.

[0245] (3) Evaluation of productivity of 4-(aminomethyl)cyclohexanecarboxylic acid, an ALDH homologue. Each purified His-tagged recombinant ALDH enzyme was obtained in the same manner as in Example 1 using Escherichia coli carrying each ALDH expression plasmid obtained in (2) above.

[0246] 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 ＋ A 0.1 mL reaction mixture (pH 7.8) containing 1 mM of a mixture of 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. A similar reaction was also carried out using purified enzyme XylC as a positive control.

[0247] After the reaction was completed, the reaction product was analyzed in the same manner as in Example 1. The results are shown in Table 22.

[0248] As shown in Table 22, the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid was confirmed in all ALDH homologs 1 to 13 evaluated. In particular, significant production of 4-(aminomethyl)cyclohexane-1-carboxylic acid was confirmed when ALDH homologs 1 to 5, 7 to 9, and 13 were used. Furthermore, the trans ratio of 4-(aminomethyl)cyclohexane-1-carboxylic acid produced when ALDH homologs 1 to 5, 7, and 13 were used was greater than 50%, demonstrating high specificity for the trans substrate. Among these, ALDH homologs 1, 2, 4, 5, and 7 in particular had a trans ratio of 4-(aminomethyl)cyclohexane-1-carboxylic acid of 70% or more, demonstrating particularly high selectivity for the trans substrate comparable to that of XylC. Of these, ALDH homologs 4 and 5 produced trans-4-(aminomethyl)cyclohexane-1-carboxylic acid in amounts equivalent to that of XylC.

[0249] Example 10 Evaluation of TXA productivity by enhancing keto acid and coenzyme supply (1) Construction of evaluation strains A) Construction of MG1655 / pQE80L-XylC-PatA Using the nucleotide sequence represented by SEQ ID NO:24 as a template and oligonucleotides represented by the nucleotide sequences represented by SEQ ID NOs:179 and 94 as a primer set, a PCR reaction was performed to obtain a XylC fragment. Subsequently, using the chromosomal DNA of Escherichia coli K12 MG1655 as a template and oligonucleotides represented by the nucleotide sequences represented by SEQ ID NOs:95 and 180 as a primer set, a PCR reaction was performed to obtain a PatA fragment. Note that the 5' end of the nucleotide sequence represented by SEQ ID NO:94 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO:95.

[0250] Using a mixture of the XylC fragment and the PatA fragment obtained above as a template, a PCR reaction was performed using oligonucleotides having the base sequences represented by SEQ ID NOs: 179 and 180 as a primer set to obtain a DNA fragment in which the two fragments were ligated (hereinafter referred to as XylC-PatA).

[0251] Furthermore, a PCR reaction was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NOs:181 and 182 as a primer set and expression vector pQE80L (QIAGEN) as a template to obtain a vector fragment of approximately 4.7 kb. The 5' end of the nucleotide sequence represented by SEQ ID NO:181 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO:179, and the 5' end of the nucleotide sequence represented by SEQ ID NO:182 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO:180.

[0252] The XylC-PatA fragment and vector fragment obtained above were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pQE80L-XylC-PatA. Escherichia coli MG1655 was transformed with the expression plasmid obtained above to obtain the MG1655 / pQE80L-XylC-PatA strain.

[0253] A) Construction of MG1655 / pQE80L-PatA-BsAlaDH-XylC-BsNOX Using the chromosomal DNA of Escherichia coli K12 MG1655 as a template, a PCR reaction was performed using oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 183 and 90 as a primer set to obtain a PatA fragment. Subsequently, a PCR reaction was performed using the nucleotide sequence represented by SEQ ID NO: 24 as a template and oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 91 and 30 as a primer set to obtain a XylC fragment. Note that the 5' end of the nucleotide sequence represented by SEQ ID NO: 90 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 91.

[0254] A PCR reaction was carried out using a mixture of the PatA fragment and the XylC fragment obtained above as a template and oligonucleotides having the base sequences represented by SEQ ID NOs: 183 and 30 as a primer set to obtain a DNA fragment in which the two fragments were ligated (hereinafter referred to as PatA-XylC).

[0255] Furthermore, a PCR reaction was performed using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 181 and 182 as a primer set and expression vector pQE80L (QIAGEN) as a template to obtain a vector fragment of approximately 4.7 kb. The 5' end of the nucleotide sequence represented by SEQ ID NO: 181 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 183, and the 5' end of the nucleotide sequence represented by SEQ ID NO: 182 contains a sequence complementary to the 5' end of the nucleotide sequence represented by SEQ ID NO: 30.

[0256] The PatA fragment and vector fragment obtained above were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain plasmid pQE80L-PatA-XylC. Using the plasmid obtained above as a template, a PCR reaction was performed using DNAs consisting of the base sequences represented by SEQ ID NOs: 184 and 182 as a primer set to obtain a DNA fragment (hereinafter referred to as pQE80L-PatA).

[0257] A PCR reaction was performed using the chromosomal DNA of Bacillus subtilis 168 as a template and oligonucleotides having the base sequences represented by SEQ ID NOs: 185 and 186 as a primer set to obtain a BsAlaDH gene fragment. A PCR reaction was performed using the chromosomal DNA of Bacillus subtilis 168 as a template and oligonucleotides having the base sequences represented by SEQ ID NOs: 187 and 86 as a primer set to obtain a BsNOX gene fragment. The 5' end of the base sequence represented by SEQ ID NO: 186 contains a sequence complementary to the 5' end of the base sequence represented by SEQ ID NO: 187.

[0258] A PCR reaction was carried out using a mixture of the BsAlaDH gene fragment and the NOX gene fragment obtained above as a template and oligonucleotides having the base sequences represented by SEQ ID NOs: 185 and 86 as a primer set to obtain a DNA fragment in which the two fragments were ligated (hereinafter referred to as BsAlaDH-BsNOX).

[0259] The 5' end of the base sequence represented by SEQ ID NO: 185 contains a sequence complementary to the 5' end of the base sequence represented by SEQ ID NO: 184, and the 5' end of the base sequence represented by SEQ ID NO: 86 contains a sequence complementary to the 5' end of the base sequence represented by SEQ ID NO: 182.

[0260] The pQE80L-PatA fragment and the BsAlaDH-BsNOX fragment obtained above were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain the plasmid pQE80L-PatA-BsAlaDH-BsNOX.

[0261] Using the plasmid obtained above as a template, PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs: 188 and 189 as a primer set to obtain a DNA fragment (hereinafter referred to as pQE80L-PatA-BsAlaDH-BsNOX).

[0262] Using the base sequence represented by SEQ ID NO: 24 as a template, a PCR reaction was carried out using oligonucleotides having the base sequences represented by SEQ ID NOs: 190 and 94 as a primer set to obtain the XylC fragment.

[0263] The 5' end of the base sequence represented by SEQ ID NO: 188 contains a sequence complementary to the 5' end of the base sequence represented by SEQ ID NO: 190, and the 5' end of the base sequence represented by SEQ ID NO: 189 contains a sequence complementary to the 5' end of the base sequence represented by SEQ ID NO: 94.

[0264] The pQE80L-PatA-BsAlaDH-BsNOX fragment and the XylC fragment obtained above were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pQE80L-PatA-BsAlaDH-XylC-BsNOX.

[0265] Escherichia coli MG1655 was transformed with the expression plasmid obtained above to obtain the MG1655 / pQE80L-PatA-BsAlaDH-XylC-BsNOX strain.

[0266] c) Construction of MG1655ΔaceE / pQE80L-patA-xylC-ald-nox strain <Obtaining DNA fragment used as a marker for gene deletion> PCR was performed using DNA consisting of the base sequences represented by 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-resistant cat gene and the sucrose-sensitive sacB gene.

[0267] <Construction of Escherichia coli lacking pyruvate dehydrogenase activity> Escherichia coli lacking the DNA encoding pyruvate dehydrogenase (hereinafter referred to as the aceE gene) was constructed by the following method. Using genomic DNA of Escherichia coli MG1655 strain prepared by a conventional method as a template, PCR was performed using DNA primer sets consisting of the base sequences shown in "Primer set" in Table 23 to amplify each DNA fragment.

[0268] aceE upstream 1 and aceE upstream 2 comprise the region from the initiation codon of the aceE gene to about 1000 bp upstream of the initiation codon. aceE downstream 1 and aceE downstream 2 comprise the region from about 50 bp to about 1000 bp downstream of the termination codon of the aceE gene.

[0269] PCR was performed using a mixture of aceE upstream 1, aceE downstream 1, and cat-sacB fragments in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 193 and 196 as a primer set to obtain a DNA fragment consisting of a sequence in which the cat-sacB fragment was inserted into the sequence of the region surrounding the aceE gene (hereinafter referred to as aceE::cat-sacB).

[0270] PCR was performed using a mixture of aceE upstream 2 and aceE downstream 2 in an equimolar ratio as a template and DNA consisting of the base sequences represented by SEQ ID NOs: 193 and 196 as a primer set to obtain a DNA fragment (hereinafter referred to as ΔaceE) that does not contain aceE and consists of a sequence in which the upstream and downstream regions of aceE are directly linked.

[0271] The aceE::cat-sacB fragment was introduced by electroporation into the MG1655 strain harboring the plasmid pKD46 [Datsenko, K. A., Warner, B. L., Proc. Natl. Acad. Sci., USA, Vol. 97, 6640-6645 (2000)] containing a gene encoding λ recombinase, and a transformant exhibiting chloramphenicol resistance and sucrose sensitivity (a transformant in which the aceE gene had been replaced with aceE::cat-sacB) was obtained.

[0272] The ΔaceE fragment was introduced into the transformant by electroporation to obtain transformants that were sensitive to chloramphenicol and resistant to sucrose (transformants in which aceE::cat-sacB had been replaced with ΔaceE). From these, a transformant that was sensitive to ampicillin (transformant from which pKD46 had been lost) was further obtained. This transformant was designated MG1655ΔaceE.

[0273] <Obtaining expression plasmid> Using the plasmid pQE80L-PatA-XylC obtained in (a) as a template, PCR was performed using DNA consisting of the base sequences shown in SEQ ID NOs: 199 and 182 as a primer set to obtain a DNA fragment (hereinafter referred to as pQE80L-PatA-XylC).

[0274] Using the BsAlaDH-BsNOX fragment obtained in (a) as a template, PCR was performed using DNA consisting of the base sequences shown in SEQ ID NOs: 200 and 86 as a primer set to obtain a DNA fragment (hereinafter referred to as BsAlaDH-BsNOX2).

[0275] The 5' end of the base sequence represented by SEQ ID NO: 200 contains a sequence complementary to the 5' end of the base sequence represented by SEQ ID NO: 199, and the 5' end of the base sequence represented by SEQ ID NO: 86 contains a sequence complementary to the 5' end of the base sequence represented by SEQ ID NO: 182.

[0276] The pQE80L-PatA-XylC fragment and the BsAlaDH-BsNOX2 fragment obtained above were ligated using In-Fusion™ HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid pQE80L-PatA-XylC-BsAlaDH-BsNOX. Escherichia coli MG1655ΔaceE was transformed with the expression plasmid obtained above to obtain the MG1655ΔaceE / pQE80L-PatA-XylC-BsAlaDH-BsNOX strain.

[0277] (2) Resting Cell Reaction Using Strains Introduced with a Coenzyme Regeneration System The productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid in a resting cell reaction was evaluated using the MG1655 / pQE80L-XylC-PatA strain, the MG1655 / pQE80L-PatA-BsAlaDH-XylC-BsNOX strain, and the MG1655ΔaceE / pQE80L-PatA-XylC-BsAlaDH-BsNOX strain constructed in (1) above.

[0278] Each strain was inoculated into a test tube containing 2 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking at 30°C for 16 hours. The culture was inoculated into a 250 mL Erlenmeyer flask containing 40 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking at 28°C for 2 hours, after which IPTG was added to a final concentration of 1 mM and cultured with shaking at 28°C for an additional 24 hours. However, for the MG1655ΔaceE / pQE80L-PatA-XylC-BsAlaDH-BsNOX strain, the culture time before addition of ITPG was 3.5 hours.

[0279] After the culture, the culture medium was centrifuged to obtain wet cells, to which xylene was added to give a final concentration of 10 mL / L, and the wet cells were subjected to membrane treatment at 30° C. and 850 rpm for 30 minutes.

[0280] The mixture contained 100 g / L of the above-mentioned membrane-treated wet cells, 140 mM glucose, 50 mM cis- and trans-isomer mixture of 1,4-bis(aminomethyl)cyclohexane (trans ratio 45.7%), 50 mM magnesium chloride, 10 mM pyruvic acid, and 10 mM NAD. ＋A 0.2 mL reaction solution containing the above was prepared, and the reaction was carried out at 30° C. and 850 rpm for 24 hours.

[0281] After the reaction was completed, the reaction products and the remaining substrate were analyzed in the same manner as in Example 1. The results are shown in Table 24.

[0282] From Table 24, pyruvate and NAD ＋ Introduction of the regeneration gene improved the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid and the consumption of the substrate, 1,4-bis(aminomethyl)cyclohexane. Furthermore, disruption of the pyruvate degradation gene in the host E. coli also improved target production and substrate consumption, with the improvement in substrate consumption being particularly significant. This suggests that disruption of the pyruvate degradation gene promoted the transamination reaction of the substrate.

[0283] These results demonstrate that in the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid by a resting cell reaction, it is effective to enhance the supply of keto acid and coenzyme by introducing genes encoding enzymes that regenerate keto acid and coenzyme or by blocking the keto acid degradation system in the host.

[0284] Example 11 Extraction of Amino Acid Residues Common to ALDHs with High Specificity for trans-4-(Aminomethyl)cyclohexane-1-carbaldehyde In Examples 3, 4, and 9, ALDHs that produced AMCHA with a high trans ratio in reactions using a cis-trans mixed substrate are useful in the production of AMCHA, particularly TXA. To identify amino acid residues characteristic of these ALDHs, a comparison of amino acid sequences was performed.

[0285] In Examples 3, 4, and 9, ALDHs (SEQ ID NOS: 20, 40, 41, 127, 128, 130, and 131) that produced AMCHA in a trans ratio of 70% or more by reaction using a cis-trans mixed substrate were extracted as ALDHs with particularly high specificity for the trans isomer of 4-(aminomethyl)cyclohexane-1-carbaldehyde. As a result, all of these enzymes were 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) that produced AMCHA in a trans ratio of less than 50% by reaction using a cis-trans mixed substrate were extracted as ALDHs that do not have specificity for the trans isomer of 4-(aminomethyl)cyclohexane-1-carbaldehyde.

[0286] In order to exclude the influence of the strength of the enzyme activity, ALDH, which produced less than 0.2 mM AMCHA in the reaction using the cis-trans mixed substrate in Examples 3, 4 and 9, was excluded.

[0287] The amino acid sequences of ALDH extracted by the above procedure were aligned using Clustal Omega (Figures 5A and 5B). Furthermore, amino acid residues that are conserved in ALDHs with high specificity for trans-4-(aminomethyl)cyclohexane-1-carbaldehyde but are not conserved in ALDHs lacking specificity for trans-4-(aminomethyl)cyclohexane-1-carbaldehyde 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).

[0288] The amino acid residues shown in Table 25 are suggested to be important for the particularly high specificity of ALDH for trans-4-(aminomethyl)cyclohexane-1-carbaldehyde.

Claims

1. A process for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid, comprising the steps of: A method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid from cis- and / or trans-4-(aminomethyl)cyclohexane-1-carbaldehyde in the presence of aldehyde dehydrogenase.

2. The method according to claim 1, further comprising the step of producing 4-(aminomethyl)cyclohexane-1-carbaldehyde from 1,4-bis(aminomethyl)cyclohexane in the presence of an aminotransferase.

3. The method according to claim 2, wherein the aminotransferase has an amino group transfection activity of transferring the amino group of 1,4-bis(aminomethyl)cyclohexane to another compound to produce 4-(aminomethyl)cyclohexane-1-carbaldehyde, and is a protein consisting of an amino acid sequence having 60% or more identity to an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105.

4. The production method described in any one of claims 1 to 3, wherein the aldehyde dehydrogenase is at least one protein selected from the group consisting of the following i) to vi): i) a protein having an aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, and having an amino acid sequence that has 50% or more identity with any one of the amino acid sequences represented by SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139. ii) a protein having an aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, and having an amino acid sequence having 60% or more identity with any one of the amino acid sequences represented by SEQ ID NOs: 19 to 22 and 35 to 46. iii) a benzaldehyde dehydrogenase, which has an aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid; iv) A protein having an aldehyde dehydrogenase activity derived from a Pseudomonas bacterium, which oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid. v) a protein having an aldehyde dehydrogenase activity that oxidizes the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, and having an amino acid sequence that has 50% or more identity with any one of the amino acid sequences represented by SEQ ID NOs: 19 to 22, 35 to 46, and 127 to 139. vi) A protein having an aldehyde dehydrogenase activity that produces 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carbaldehyde, an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 20, and comprising at least one amino acid residue selected from the following [1] to [26] when aligned with the amino acid sequence represented by SEQ ID NO: 20: [1] The amino acid residue at position 35 is a glycine residue (G). [2] The amino acid residue at the position corresponding to 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 at position 104 is a glycine residue (G). [5] The amino acid residue at position 109 is a glycine residue (G). [6] The amino acid residue at position 110 is an isoleucine residue (I). [7] The amino acid residue at 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 at position 124 is a leucine residue (L). [10] The amino acid residue at position 134 is a valine residue (V). [11] The amino acid residue at position 144 is a valine residue (V). [12] The amino acid residue at position 160 is a phenylalanine residue (F). [13] The amino acid residue at 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 at position 319 is an aspartic acid residue (D). [16] The amino acid residue at position 333 is an aspartic acid residue (D). [17] The amino acid residue at position 365 is a glutamine residue (Q). [18] The amino acid residue at 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 at position 423 is a leucine residue (L). [21] The amino acid residue at position 434 is a valine residue (V). [22] The amino acid residue at position 440 is a proline residue (P). [23] The amino acid residue at position 443 is a cysteine ​​residue (C). [24] The amino acid residue at position 445 is a proline residue (P). [25] The amino acid residue at position 466 is a serine residue (S). [26] The amino acid residue at position 467 is an isoleucine residue (I).

5. The method according to any one of claims 1 to 3, wherein alanine dehydrogenase and / or NADH oxidase is / are present in part or all of the method.

6. The process according to claim 2 or 3, wherein trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is produced from cis- and / or trans-1,4-bis(aminomethyl)cyclohexane.

7. The process according to claim 2 or 3, wherein cis-4-(aminomethyl)cyclohexane-1-carboxylic acid is produced from cis- and / or trans-1,4-bis(aminomethyl)cyclohexane.

8. The process according to any one of claims 1 to 3, wherein part or all of the process is carried out under neutral or basic conditions.

9. The process according to claim 8, wherein part or all of the process is carried out in the presence of a secondary amine.