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

Abstract

A production method according to one embodiment is a method in which a cis-form and / or a trans-form of 4-(aminomethyl)cyclohexane-1-carboxylic acid is produced by using a compound represented by general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from among a protein having an activity of converting a hydroxy group into an aldehyde group, a protein having an activity of converting an aldehyde group into a carboxy group and a protein having an activity of reversibly converting between an aldehyde group and an amino group. [In the formula, R1 and R2 each independently represent CH2OH, CHO, COOH, or CH2NH2 (excluding the case where one of R1 and R2 is COOH and the other is CH2NH2, the case where each of R1 and R2 is COOH, the case where each of R1 and R2 is CH2NH2, and the case where the compound represented by general formula (1) is a compound wherein one of R1 and R2 is CH2NH2 and the other is CHO and is an intermediate compound produced from a compound wherein each of R1 and R2 is CH2NH2).]

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-stage chemical synthesis reaction using 4-(chloromethyl)benzoic acid as a starting material, which involves an amination reaction and a hydrogenation reaction to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid, followed by 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 JP 63-152991 Publication CN114231507ACN115960854A

[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, 101-107Bioresources and Bioprocessing (2022) 9, 80

[0008] An object of the present invention is to provide a method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid based on a novel enzymatic reaction pathway, and a protein that can be used in the enzymatic reaction.

[0009] The present inventors assumed that an enzymatic reaction pathway involving aminotransferase and aldehyde dehydrogenase could be used to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid from 1,4-bis(aminomethyl)cyclohexane, and selected proteins that potentially possessed the desired activity. They also found that expressing and purifying these proteins in an Escherichia coli strain and performing an enzymatic reaction could produce 4-(aminomethyl)cyclohexane-1-carboxylic acid from 1,4-bis(aminomethyl)cyclohexane. As a result of further research, the present inventors assumed that an enzymatic reaction pathway involving choline oxidase, aminotransferase, and aldehyde dehydrogenase could be used to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid from 1,4-cyclohexanedimethanol, and selected proteins that potentially possessed the desired activity. They also found that expressing these proteins in an Escherichia coli strain and performing a resting cell reaction could produce 4-(aminomethyl)cyclohexane-1-carboxylic acid from 1,4-cyclohexanedimethanol, leading to the completion of the present invention.

[0010] The present invention relates to, for example, the following inventions: [A1] A method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２and R are both COOH)] Specifically, the compound represented by the general formula (1) is any one of Compounds 1, 2, 3, 4, 5, 6, 7, and 8. [A2] The method according to [A1], wherein the compound is produced in the presence of a protein having the activity of converting the hydroxy group to an aldehyde group, a protein having the activity of converting the aldehyde group to a carboxy group, and a protein having the activity of reversibly converting the aldehyde group and the amino group. [A3] R １ and R ２ One of them is CH ２ OH and the other is CH ２ OH, CHO, or CH ２ NH ２ When a compound represented by the general formula (1) is used as a substrate compound or an intermediate compound, the compound is produced in the presence of a protein having the activity of converting the hydroxy group to an aldehyde group, a protein having the activity of converting the aldehyde group to a carboxy group, and a protein having the activity of reversibly converting the aldehyde group and the amino group. [A4] The method according to [A2], １ and R ２ One of them is CH ２ NH ２ and the other is CHO, the method according to [A1], wherein the compound represented by general formula (1) is used as a substrate compound or an intermediate compound, and the compound is produced in the presence of a protein having the activity of converting the aldehyde group to a carboxy group. [A5] R １ and R ２ [A6] The method according to [A1], wherein a compound represented by general formula (1), in which one of R is CHO and the other is COOH, is used as a substrate compound or an intermediate compound, in the presence of a protein having an activity of reversibly converting the aldehyde group and the amino group. １ and R ２ One of them is CHO and the other is CH ２ NH ２ or CHO, or R １ and R ２ Both are CH ２ NH ２[A7] The method according to [A1], wherein R is a compound represented by general formula (1) and R is a carboxyl group, and R is a carboxyl group. １ and R ２ One of them is CH ２ OH and the other is CH ２ NH ２ [A8] The method according to [A1], wherein when a compound represented by general formula (1) is used as a substrate compound or an intermediate compound, the compound is produced in the presence of a protein having the activity of converting the hydroxy group to an aldehyde group and a protein having the activity of converting the aldehyde group to a carboxy group. １ and R ２ One of them is CH ２The method according to [A1], wherein the substrate compound or intermediate compound is a compound represented by general formula (1), in which the other is OH and the other is COOH, and the production is carried out in the presence of a protein having the activity of converting the hydroxy group to an aldehyde group and the protein having the activity of reversibly converting an aldehyde group and an amino group. [A9] The method according to any of [A1] to [A8], wherein the protein having the activity of converting a hydroxy group to an aldehyde group is at least one selected from proteins having oxidase activity, proteins having dehydrogenase activity, and proteins having aldehyde reductase activity, the protein having the activity of converting an aldehyde group to a carboxy group is a protein having aldehyde dehydrogenase activity and / or a protein having aldehyde oxidase activity, and the protein having the activity of reversibly converting an aldehyde group and an amino group is at least one selected from proteins having aminotransferase activity, proteins having amine dehydrogenase activity, and proteins having amine oxidase activity. [A10] The method according to any one of [A1] to [A9], wherein the protein having the activity of converting a hydroxy group to an aldehyde group is a protein having choline oxidase activity, the protein having the activity of converting an aldehyde group to a carboxy group is a protein having aldehyde dehydrogenase activity, and the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein having aminotransferase activity. [A11] The method according to [A10], wherein the protein having choline oxidase activity is a protein consisting of an amino acid sequence having 60% or more identity to the amino acid sequence represented by SEQ ID NO: 201, the protein having aldehyde dehydrogenase activity is a protein 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, and the protein having aminotransferase activity is a protein 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.[A12] In the above general formula (1), R. １ and R ２ The method according to [A5], wherein one of R is COOH and the other is CHO (i.e., compound 5) is a compound generated from any one of the substrate compounds or intermediate compounds (i) and (ii) below. １ and R ２ (ii) a compound in which R １ and R ２ One of them is COOH and the other is CH ２ OH (i.e., Compound 4) [A13] In the above general formula (1), R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, the compound (i.e., compound 7) is a compound generated from any one of the substrate compounds or intermediate compounds shown below in (i) and (ii). １ and R ２ (ii) a compound in which R １ and R ２ One of them is CH ２ NH ２ and the other is CH ２ OH (i.e., Compound 6) [A14] In the above general formula (1), R １ and R ２ and R are both CHO (i.e., Compound 3), １ and R ２ One of them is COOH and the other is CH ２ OH (i.e., Compound 4) or R １ and R ２ One of them is CH ２ NH ２ and the other is CH ２The method according to [A1], wherein cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid is produced using, as a substrate compound or intermediate compound, any one of compounds having a hydroxy group as a hydroxy group (i.e., compound 6) in the presence of at least one enzyme selected from proteins having the activity of converting a hydroxy group to an aldehyde group, proteins having the activity of converting an aldehyde group to a carboxy group, and proteins having the activity of reversibly converting an aldehyde group and an amino group. [A15] The method according to [A1], wherein, in the above general formula (1), R １ and R ２ In the case where a compound (i.e., Compound 3) in which R are both CHO is used as a substrate compound or an intermediate compound, in the presence of a protein having the activity of converting an aldehyde group to a carboxy group and a protein having the activity of reversibly converting an aldehyde group and an amino group, １ and R ２ One of them is COOH and the other is CH ２ When a compound having a hydroxy group as a substituent (i.e., compound 4) is used as a substrate compound or an intermediate compound, the compound can be produced in the presence of a protein having an activity of converting a hydroxy group to an aldehyde group and a protein having an activity of reversibly converting an aldehyde group and an amino group, or １ and R ２ One of them is CH ２ NH ２ and the other is CH ２The method according to [A14], wherein cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid is produced in the presence of a protein having the activity of converting a hydroxy group to an aldehyde group and a protein having the activity of converting an aldehyde group to a carboxy group, when a compound having a hydroxy group (i.e., compound 6) is used as a substrate compound or an intermediate compound. [A16] A method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound, in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 127 to 139, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to any one of these amino acid sequences: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２and R are both COOH.] Specifically, the compound represented by the general formula (1) is any one of Compounds 1, 2, 3, 4, 5, 6, 7, and 8. [A17] The method according to [A16], wherein the compound is produced in the presence of a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group. [A18] A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting an aldehyde group to a carboxy group, wherein, in the general formula (1), R １ and R ２ One of them is CH ２ NH ２ and the other is CHO (i.e., compound 7) as a substrate compound or intermediate compound, to produce cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid. [A19] A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting an aldehyde group to a carboxy group and a protein having the activity of reversibly converting an aldehyde group and an amino group, comprising: １ and R ２ and R １ and R ２ Both are CH ２ NH ２ [A20] A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting an aldehyde group to a carboxy group and a protein having the activity of converting a hydroxy group to an aldehyde group, comprising:１ and R ２ One of them is CH ２ NH ２ and the other is CH ２ The method according to [A16], in which cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid is produced using a compound in which R is OH (i.e., compound 6) as a substrate compound or intermediate compound. [A21] A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein １ and R ２ One of them is CH ２ OH and the other is CH ２The method according to [A16], which produces cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound which is OH or CHO (i.e., compound 1 or compound 2) as a substrate compound or intermediate compound. [A22] The method according to any one of [A16] to [A21], wherein the protein having the activity of converting an aldehyde group to a carboxy group is at least one of the following 1) to 6): 1) A protein consisting of an amino acid sequence that has 71% 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 set forth in SEQ ID NO: 19. 2) A protein consisting of an amino acid sequence that has 68% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence set forth in SEQ ID NO: 20. 3) A protein that is an aldehyde dehydrogenase derived from the genus Psudomonas and has an amino acid sequence that has 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence set forth in SEQ ID NO: 20. 4) A protein consisting of an amino acid sequence that has 63%, 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 SEQ ID NO: 21. 5) A protein consisting of an amino acid sequence that has 56.5% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO: 40. 6) A protein consisting of an amino acid sequence that has 63.9% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO: 41. [A23] The method according to [A22], wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 127 to 131, 133 to 135, 138, and 139.[A24] The method according to [A22], wherein the protein having the activity of converting an aldehyde group to a carboxy group has aldehyde dehydrogenase activity of oxidizing the aldehyde group of a compound having an aldehyde group, and is an aldehyde dehydrogenase derived from a bacterium of the genus Pseudomonas. [A25] A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1), wherein the compound (i) below is used as a substrate compound or an intermediate compound, in the presence of at least one enzyme selected from proteins having the activity of converting a hydroxy group to an aldehyde group, proteins having the activity of converting an aldehyde group to a carboxy group, and proteins having the activity of reversibly converting an aldehyde group and an amino group: (i) In general formula (1), R: １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ However, in the general formula (1), R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ are both COOH, and R １ and R ２ One of them is CH ２ NH ２ and the other is CHO (i.e., the compound represented by general formula (1) is compound 7). That is, the compound represented by general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, and 8. [A26] A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid, using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound, in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of the amino acid sequence represented by SEQ ID NO:207, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２and (A27) are at least one selected from the group consisting of a protein having oxidase activity, a protein having dehydrogenase activity, and a protein having aldehyde reductase activity, and the protein having the activity of reversibly converting an aldehyde group and an amino group is at least one selected from the group consisting of a protein having aminotransferase activity, a protein having amine dehydrogenase activity, and a protein having amine oxidase activity. [A28] The method according to [A16], wherein the protein having the activity of converting a hydroxy group to an aldehyde group is a protein having choline oxidase activity, and the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein having aminotransferase activity. [A29] The method according to [A28], wherein the protein having choline oxidase activity is a protein consisting of an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 201, and the protein having aminotransferase activity is a protein consisting of an amino acid sequence having 60% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and 103 to 105. [A30] In the general formula (1), R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, wherein the compound (i.e., compound 7) is a compound generated from any one of the substrate compounds or intermediate compounds shown below in (i) and (ii). １ and R ２ (ii) a compound in which R １ and R ２ One of them is CH ２ OH and the other is CH ２ NH ２ [A31] A compound (i.e., compound 6) in which R１ and R ２ and R are both CHO (i.e., Compound 3), １ and R ２ One of them is COOH and the other is CH ２ OH (i.e., Compound 4) or R １ and R ２ One of them is CH ２ NH ２ and the other is CH ２ The method according to [A16], in which cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid is produced using, as a substrate compound or intermediate compound, any one of compounds having a hydroxy group as a hydroxy group (i.e., compound 6) in the presence of at least one enzyme selected from proteins having the activity of converting a hydroxy group to an aldehyde group, proteins having the activity of converting an aldehyde group to a carboxy group, and proteins having the activity of reversibly converting an aldehyde group and an amino group. [A32] The method according to [A16], in the above general formula (1), R １ and R ２ In the case where a compound (i.e., Compound 3) in which R are both CHO is used as a substrate compound or an intermediate compound, in the presence of a protein having the activity of converting an aldehyde group to a carboxy group and a protein having the activity of reversibly converting an aldehyde group and an amino group, １ and R ２ One of them is COOH and the other is CH ２ When a compound having a hydroxy group as a substituent (i.e., compound 4) is used as a substrate compound or an intermediate compound, the compound can be produced in the presence of a protein having an activity of converting a hydroxy group to an aldehyde group and a protein having an activity of reversibly converting an aldehyde group and an amino group, or １ and R ２ One of them is CH ２ NH ２ and the other is CH ２The method according to [A31], wherein cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid is produced in the presence of a protein having the activity of converting a hydroxy group to an aldehyde group and a protein having the activity of converting an aldehyde group to a carboxy group, when a compound having a hydroxy group (i.e., compound 6) is used as a substrate compound or an intermediate compound. [A33] A method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound, in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, and 277, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, and 277: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ When both are COOH, R １ and R ２ Both are CH ２ NH ２ and when the compound represented by general formula (1) is R １ and R ２ One of them is CH ２ NH ２and the other is CHO, and compound 8 (in the above general formula (1), R １ and R ２ Both are CH ２ NH ２ Specifically, the compound represented by general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, and 7, excluding the case where compound 7 is an intermediate compound generated from compound 8. [A34] A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid, using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound, in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein consisting of the amino acid sequence represented by SEQ ID NO:277, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO:277: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２ [A35] In the above general formula (1), R １ and R ２ One of them is CH ２ OH and the other is CH ２OH, CHO, or COOH (i.e., compounds 1, 2, and 4), or R １ and R ２ [A36] The method according to [A33], which produces cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound in which one of R is CHO and the other is CHO or COOH (i.e., compounds 3 and 5) as a substrate compound or an intermediate compound. １ and R ２ Both are CH ２ NH ２ The method according to [A34], wherein when a compound (i.e., compound 8) represented by the formula (I) is used as a substrate compound or an intermediate compound, the compound is produced in the presence of a protein having an activity of reversibly converting an aldehyde group and an amino group. [A37] A method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound, in the presence of at least one enzyme selected from the group consisting of a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of converting a hydroxy group to an aldehyde group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 201, 213, 221 to 230, 241, and 289 to 294, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by any one of these amino acid sequences: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R２ When both are COOH, R １ and R ２ Both are CH ２ NH ２ and when the compound represented by general formula (1) is R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, and compound 8 (in the above general formula (1), R １ and R ２ Both are CH ２ NH ２ Specifically, the compound represented by the general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, and 7, excluding the case where compound 7 is an intermediate compound generated from compound 8. [A38] In the general formula (1), R １ and R ２ One of them is CH ２ The method according to [A1], wherein the cis-isomer of 4-(aminomethyl)cyclohexane-1-carboxylic acid is produced using a compound in which the other is OH and the other is COOH (i.e., compound 4) as a substrate compound or an intermediate compound. [A39] The protein having the activity of converting a hydroxy group to an aldehyde group is a protein consisting of an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO: 201 or 213, or an amino acid sequence represented by any one of these, wherein, in the general formula (1), R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２ When both are COOH, R １ and R ２ Both are CH２ NH ２ and R １ and R ２ One of them is COOH and the other is CH ２ [A40] The method according to [A37], wherein the protein having the activity of converting a hydroxy group to an aldehyde group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 289 and 290, or an amino acid sequence having 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by any one of these SEQ ID NOs: 289 and 290, wherein in the general formula (1), R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２ When both are COOH, R １ and R ２ One of them is CH ２ OH and the other is CH ２ NH ２ If R １ and R ２ Both are CH ２ NH ２ [A41] The method according to [A37], which produces cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound (any one of compounds 1, 2, 3, 4, 5, or 7) as a substrate compound or intermediate compound, except when R １ and R ２is CHO and the other is COOH, the method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of reversibly converting an aldehyde group and an amino group, when a compound represented by general formula (1) (i.e., compound 5) in which one of R is CHO and the other is COOH is used as a substrate compound or an intermediate compound, wherein the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277. [A42] The method according to [A1], １ and R ２ One of them is CH ２ OH and the other is CH ２ NH ２a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting a hydroxy group to an aldehyde group, and a protein having the activity of converting the aldehyde group to a carboxy group, when a compound represented by general formula (1) (i.e., compound 6) is used as a substrate compound or an intermediate compound, wherein the protein having the activity of converting a hydroxy group to an aldehyde group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 201, 213, 221 to 230, 241, and 291 to 294, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by any one of these, The method according to [A1], wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequences represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, and 207. [A43] In the above general formula (1), R １ and R ２ One of them is CH ２a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having an activity of converting a hydroxy group to an aldehyde group and a protein having an activity of reversibly converting an aldehyde group and an amino group, when a compound represented by general formula (1) (i.e., compound 4) in which one hydroxy group is OH and the other is COOH is used as a substrate compound or an intermediate compound, the method comprising the steps of: (a) converting a hydroxy group to an aldehyde group into an aldehyde group; (b) converting an aldehyde group to an amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277; or (c) converting an aldehyde group to an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277; The method according to [A1], wherein the protein having the activity of converting a hydroxy group to an aldehyde group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 213, 221 to 230, 241, and 289 to 294, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by any one of these. １ and R ２and (iii) are both CHO (i.e., Compound 3) as a substrate compound or an intermediate compound, a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting an aldehyde group to a carboxy group and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277, The method according to [A1], wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequences represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, and 207. [A45] The method according to [A1], wherein, in the general formula (1), R １ and R ２ One of them is CH ２ OH and the other is CH ２A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, when a compound which is OH or CHO (i.e., Compound 1 or Compound 2) is used as a substrate compound or an intermediate compound, wherein the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277; The method according to [A1], wherein the protein having the activity of converting a hydroxy group to an aldehyde group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 201, 213, 221 to 230, 241, and 289 to 294, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to an amino acid sequence represented by any one of these, and the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, or 207, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to an amino acid sequence represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, or 207.[A46] A method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound, in the presence of at least one enzyme selected from proteins having the activity of converting a hydroxy group to an aldehyde group, proteins having the activity of converting an aldehyde group to a carboxy group, and proteins having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, or 207, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, or 207: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ When both are COOH, R １ and R ２ Both are CH ２ NH ２ and when the compound represented by general formula (1) is R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, and compound 8 (in the above general formula (1), R １ and R ２ Both are CH ２ NH ２(a compound represented by the formula (1)) is any one of compounds 1, 2, 3, 4, 5, 6, and 7, excluding the case where compound 7 is an intermediate compound formed from compound 8. [A47] The method according to [A46], wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 19 to 22, 35 to 46, or 207, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 19 to 22, 35 to 46, or 207. [A48] A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid, using a compound represented by the following general formula (1) as an intermediate compound, in the presence of at least one enzyme selected from proteins having the activity of converting a hydroxy group to an aldehyde group, proteins having the activity of converting an aldehyde group to a carboxy group, and proteins having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by any of SEQ ID NOs: 127 to 139, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to an amino acid sequence represented by any of SEQ ID NOs: 127 to 139: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ When both are COOH, R １ and R ２ Both are CH ２ NH２ and when the compound represented by general formula (1) is R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, and compound 8 (in the above general formula (1), R １ and R ２ Both are CH ２ NH ２ Specifically, the compound represented by general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, and 7, excluding the case where compound 7 is an intermediate compound generated from compound 8.

[0011] The present invention also relates to, for example, the following inventions: [1] A protein having aminotransferase 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 having 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 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 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 production method according to any one of

[25] to

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

[31] A recombinant DNA comprising the DNA according to [2] and [7] or

[22] , and / or DNA encoding a protein having the activity of converting a hydroxy group in the compound having a hydroxy group to an aldehyde group.

[32] A recombinant cell comprising the DNA according to [2] and [7] or

[22] , and / or DNA encoding a protein having the activity of converting a hydroxy group in the compound having a hydroxy group to an aldehyde group, or obtained by transforming a host cell with the recombinant DNA according to

[31] .

[33] The recombinant cell according to

[32] , further comprising DNA encoding an enzyme having the activity of converting the hydroxy group in the compound having a hydroxy group to an aldehyde group, or obtained by transforming a host cell with recombinant DNA further comprising DNA encoding an enzyme having the activity of converting the hydroxy group in the compound having a hydroxy group to an aldehyde group.

[0012] The proteins of the present invention having the activity of converting a hydroxy group of a compound having a hydroxy group to an aldehyde group (e.g., proteins having oxidase activity), the proteins of the present invention having the activity of reversibly converting an aldehyde group and an amino group (e.g., proteins having aminotransferase activity), and / or the proteins of the present invention having the activity of converting an aldehyde group to a carboxy group (e.g., proteins having aldehyde dehydrogenase activity) can be used to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid based on a novel enzymatic reaction pathway. Furthermore, by utilizing the substrate specificity of the proteins having aldehyde dehydrogenase activity depending on the stereostructure of the target 4-(aminomethyl)cyclohexane-1-carboxylic acid, a compound of the desired structure can be selectively produced in large amounts even when a mixture of cis and trans isomers is used as a substrate.

[0013] According to the production method of the present invention, 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced efficiently while reducing production costs without requiring high temperatures and high pressures. Furthermore, by introducing oxygen and / or a coenzyme into the reaction system when a protein having oxidase activity is used, a compound that accepts an amino group (e.g., a keto acid) or a compound that donates an amino group (e.g., an amino acid) when a protein having aminotransferase activity is used, or a coenzyme and / or a regeneration system thereof when a protein having dehydrogenase activity or a protein having aldehyde reductase activity is used, the efficiency of the enzymatic reaction can be improved, 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, a protein of the present invention having the activity of converting an aldehyde group to a carboxy group (e.g., a protein having aldehyde dehydrogenase activity) that selectively acts on trans 4-(aminomethyl)cyclohexane-1-carbaldehyde and the reaction is carried out under neutral or basic conditions promotes the isomerization of 4-(aminomethyl)cyclohexane-1-carbaldehyde, one of the intermediate compounds, 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. In addition, when 1,4-cyclohexanedimethanol is used as a substrate, 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced more inexpensively than when 1,4-bis(aminomethyl)cyclohexane is used as a substrate.

[0014] 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 a plasmid (pQE80L-PpAT8) expressing a protein having aminotransferase activity (AT) in Example 1.

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

[0036] FIG. 5A and 5B show the results of an alignment of the amino acid sequence of a protein having aldehyde dehydrogenase activity (ALDH) in Example 11. 5A and 5B are diagrams showing the results of alignment of the amino acid sequence of a protein having aldehyde dehydrogenase activity (ALDH) in Example 11.

[0033] Figure 5A shows a scheme for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid from a compound represented by general formula (1) in one embodiment.

[0034] Figure 5B shows a table showing the substituents of each compound represented by general formula (1), predicted synthetic routes to 4-(aminomethyl)cyclohexane-1-carboxylic acid when each compound is used as a substrate compound or intermediate compound, and the enzymes used therein.

[0035] Figure 5B shows a chromatogram of the enzyme reaction solution in Example 15 after derivatization with o-phthalaldehyde (OPA).

[0036] Figure 5C shows a chromatogram of the enzyme reaction solution in Example 15 after derivatization with dinitrophenylhydrazine (DNPH).

[0015] 1. Compounds and Enzyme Reactions The target compound of the present invention, 4-(aminomethyl)cyclohexane-1-carboxylic acid (hereinafter sometimes referred to as "AMCHA"), is an unnatural amino acid (hereinafter sometimes referred to as the target compound in the present invention). 4-(aminomethyl)cyclohexane-1-carboxylic acid exists in two forms: the cis-4-(aminomethyl)cyclohexane-1-carboxylic acid (cis-4-(aminomethyl)cyclohexane-1-carboxylic acid) and the trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (trans-4-(aminomethyl)cyclohexane-1-carboxylic 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 called cis-tranexamic acid or cis-TXA (cis-TXA).

[0016] In the present invention, a substrate compound (sometimes simply referred to as a "substrate") or intermediate compound (sometimes simply referred to as an "intermediate") for producing a target compound is a compound represented by the following general formula (1). In the present invention, a substrate compound refers to a compound that is catalyzed by an enzyme in the production of 4-(aminomethyl)cyclohexane-1-carboxylic acid. Furthermore, an intermediate compound refers to a substrate compound other than the target compound that is produced by catalysis of an enzyme in the production process of 4-(aminomethyl)cyclohexane-1-carboxylic acid and that is further catalyzed by the same or another enzyme. The substituents of each compound (Compounds 1 to 8) are shown in Figure 7, and the chemical name of each compound is shown in Figure 6. All compounds that can be in either cis or trans form include both cis and trans forms unless otherwise specified. [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２ and the like are COOH. Specifically, the compound represented by the general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, 7 and 8.

[0017] The names of compounds 1 to 8, which are substrate compounds or intermediate compounds in the present invention, are as follows. Among these compounds, compounds that are readily commercially available or can be easily synthesized (e.g., compound 1 and compound 8) are preferably used as starting substrate compounds. On the other hand, compounds that are not commercially available, are difficult to synthesize and purify, or are unstable are preferably used as intermediate compounds.

[0018] Compound 1: 1,4-cyclohexanedimethanol, a compound represented by general formula (1), １ and R ２ Both are CH ２ Compound 2: 4-(hydroxymethyl)cyclohexane-1-carbaldehyde, a compound represented by the general formula (1) where R １ and R ２ One of them is CH ２ Compound 3: 1,4-cyclohexanedicarbaldehyde (1,4-cyclohexanedicarboxylic acid), a compound represented by general formula (1) in which R １ and R ２and R are both CHO. Compound 4: 4-(hydroxymethyl)cyclohexane-1-carboxylic acid, a compound represented by general formula (1), １ and R ２ One of them is COOH and the other is CH ２ Compound 5: 4-Formylcyclohexane-1-carboxylic acid, a compound represented by general formula (1) where R １ and R ２ Compound 6: [4-(aminomethyl)cyclohexyl]methanol, a compound represented by general formula (1) in which R １ and R ２ One of them is CH ２ NH ２ and the other is CH ２ Compound 7: 4-(aminomethyl)cyclohexane-1-carbaldehyde, R １ and R ２ One of them is CH ２ NH ２ and the other is CHO. Compound 8: 1,4-bis(aminomethyl)cyclohexane, a compound represented by general formula (1) where R １ and R ２ Both are CH ２ NH ２ A compound that is

[0019] In the present invention, the enzymatic reaction for producing a target compound includes at least one of the following (1) to (3): (1) an enzymatic reaction that converts a hydroxy group of a compound having a hydroxy group into an aldehyde group; (2) an enzymatic reaction that reversibly converts an aldehyde group and an amino group, i.e., both an enzymatic reaction that converts an aldehyde group of a compound having an aldehyde group into an amino group and the reverse reaction, an enzymatic reaction that converts an amino group of a compound having an amino group into an aldehyde group; and (3) an enzymatic reaction that converts an aldehyde group of a compound having an aldehyde group into a carboxy group.

[0020] The enzymatic reaction of the present invention is carried out in the presence of an enzyme. The enzyme used in the enzymatic reaction (1) is a protein having the activity of converting a hydroxy group to an aldehyde group, and may be referred to herein as "enzyme A." Examples of enzyme A include proteins with oxidase activity (OX), proteins with dehydrogenase activity, and proteins with aldehyde reductase activity. The enzyme used in the enzymatic reaction (2) is a protein having the activity of reversibly converting an aldehyde group to an amino group, and may be referred to herein as "enzyme B." Examples of enzyme B include proteins with aminotransferase activity (AT), proteins with amine dehydrogenase activity, and proteins with amine oxidase activity. The enzyme used in the enzymatic reaction (3) is a protein having the activity of converting an aldehyde group to a carboxy group, and may be referred to herein as "enzyme C." Examples of enzyme C include proteins with aldehyde dehydrogenase activity (ALDH) and proteins with aldehyde oxidase activity. These enzymes are described in detail in "2. Proteins and DNA of the Present Invention" below.

[0021] In the enzymatic reaction of the present invention, when a protein having aminotransferase activity is used as enzyme B, a compound that donates an amino group or a compound that accepts an amino group may be used. In the enzymatic reaction of the present invention, when a protein having dehydrogenase activity or a protein having aldehyde reductase activity is used as enzyme A, when a protein having amine dehydrogenase activity is used as enzyme B, and / or when a protein having aldehyde dehydrogenase activity is used as enzyme C, a coenzyme may be used as an electron acceptor. In the enzymatic reaction of the present invention, when a protein having oxidase activity is used as enzyme A, when a protein having amine oxidase activity is used as enzyme B, and / or when a protein having aldehyde oxidase activity is used as enzyme C, oxygen and / or a coenzyme may be used as an electron acceptor. Examples of compounds that donate an amino group include amino acids (e.g., alanine, glutamic acid, α-aminobutyric acid, isopropylamine), and the like. Examples of compounds that accept an amino group include keto acids (e.g., pyruvic acid, α-ketoglutaric acid, α-ketobutyric acid), and the like. The coenzyme is NAD(P) ＋ , FAD, etc.

[0022] In one embodiment of the enzymatic reaction, compound 1 can be converted to compound 2 by converting the hydroxy group of compound 1 to an aldehyde group in the presence of a protein (enzyme A) having the activity of converting a hydroxy group to an aldehyde group.

[0023] In one embodiment of the enzymatic reaction, compound 2 can be converted to compound 4 by converting the aldehyde group in compound 2 to a carboxy group in the presence of a protein (enzyme C) having the activity of converting an aldehyde group to a carboxy group. Compound 2 can also be converted to compound 3 by converting the hydroxy group in compound 2 to an aldehyde group in the presence of enzyme A. Compound 2 can also be converted to compound 6 by converting the aldehyde group in compound 2 to an amino group in the presence of a protein (enzyme B) having the activity of reversibly converting an aldehyde group and an amino group.

[0024] In one embodiment of the enzymatic reaction, compound 3 can be converted to compound 5 by converting one of the aldehyde groups of compound 3 to a carboxy group in the presence of enzyme C. Compound 3 can also be converted to compound 7 by converting one of the aldehyde groups of compound 3 to an amino group in the presence of enzyme B.

[0025] In one embodiment of the enzymatic reaction, compound 4 can be converted to compound 5 in the presence of enzyme A by converting the hydroxy group of compound 4 to an aldehyde group.

[0026] In one embodiment of the enzymatic reaction, compound 5 can be converted to 4-(aminomethyl)cyclohexane-1-carboxylic acid (the target compound) in the presence of enzyme B by converting the aldehyde group of compound 5 to an amino group.

[0027] In one embodiment of the enzymatic reaction, compound 6 can be converted to compound 7 in the presence of enzyme A by converting the hydroxy group of compound 6 to an aldehyde group. Compound 6 can also be converted to compound 2 in the presence of enzyme B by converting the amino group of compound 6 to an aldehyde group.

[0028] In one embodiment of the enzymatic reaction, compound 7 can be converted to a target compound by converting the aldehyde group of compound 7 to a carboxy group in the presence of enzyme C. Compound 7 can also be converted to compound 3 by converting the amino group of compound 7 to an aldehyde group in the presence of enzyme B.

[0029] In one embodiment of the enzymatic reaction, compound 8 can be converted to compound 7 in the presence of enzyme B by converting the amino group of compound 8 to an aldehyde group.

[0030] In one embodiment of the enzymatic reaction, 1,4-bis(aminomethyl)cyclohexane (compound 8) is used as a substrate. This enzymatic reaction produces a target compound from compound 8 via compound 7, an intermediate. The enzymatic reaction includes two steps, as shown below. In the first step, the substrate compound 1,4-bis(aminomethyl)cyclohexane (compound 8) reacts with a keto acid (e.g., pyruvic acid), which is a compound capable of accepting an amino group, and is converted by a protein (AT) having aminotransferase activity as enzyme B into the intermediate 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7) and the compound that has accepted the amino group (alanine when pyruvic acid is used as the keto acid). In the second step, the intermediate 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7) is converted to NAD(P) by a protein with aldehyde dehydrogenase activity (ALDH) as enzyme C. ＋ The enzyme reacts with 4-(aminomethyl)cyclohexane-1-carboxylic acid and NAD(P)H as a coenzyme (Figures 1, 6, and 7). The enzymatic reaction in the first step and the enzymatic reaction in the second step may be carried out in separate reaction systems, but it is preferable to carry them out in a single reaction system.

[0031] In addition, when pyruvate is used as a compound to accept an amino 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

[0032] The substrate compound or intermediate compound 1,4-bis(aminomethyl)cyclohexane (compound 8) may be a mixture of cis and trans isomers, or either the trans or cis isomer. Similarly, 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7) may be a mixture of cis and trans isomers, or either the trans or cis isomer.

[0033] In another embodiment of the enzymatic reaction, 1,4-cyclohexanedimethanol (compound 1) is used as a substrate. As shown in FIGS. 6 and 7 , the enzymatic reaction includes multiple enzymatic reactions that produce a target compound from compound 1. The enzymatic reaction may include the enzymatic reactions shown below. First, the substrate compound 1,4-cyclohexanedimethanol (compound 1) is converted to 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2) by an enzyme (enzyme A) that has the activity of converting a hydroxy group in a compound having a hydroxy group to an aldehyde group. Then, through various enzymatic reactions, it is finally converted to the target compound, 4-(aminomethyl)cyclohexane-1-carboxylic acid. These enzymatic reactions may be carried out sequentially in multiple reaction systems, but are preferably carried out in a single reaction system.

[0034] Specifically, the above-mentioned 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2) can be converted into the target compound 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of an enzyme having the activity of converting a hydroxy group of a compound having a hydroxy group to an aldehyde group (enzyme A; for example, a protein having oxidase activity), an enzyme having the activity of reversibly converting an aldehyde group and an amino group (enzyme B; for example, a protein having aminotransferase activity), and an enzyme having the activity of converting an aldehyde group to a carboxy group (enzyme C; for example, a protein having aldehyde dehydrogenase activity). In the presence of these enzymes, the enzymatic reaction may convert 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2) to various compounds (compounds 3, 4, and 6), which may then be converted to 4-formylcyclohexane-1-carboxylic acid (compound 5) and / or 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7), followed by the final conversion to the target compound, 4-(aminomethyl)cyclohexane-1-carboxylic acid (Figure 6). Note that compounds 3 and 7, as well as compounds 2 and 6, can be mutually converted in the presence of an enzyme (enzyme B) that has the activity of reversibly converting an aldehyde group and an amino group.

[0035] In the above-mentioned enzymatic reaction, among proteins (enzyme B) having the activity of reversibly converting an aldehyde group and an amino group, a protein having aminotransferase activity donates an amino group from an amino acid (e.g., alanine or glutamic acid), which is a compound capable of donating an amino group, to a substrate and / or intermediate having an aldehyde group, thereby producing a product that has accepted the amino group and a keto acid (pyruvic acid when alanine is used as the amino acid, and α-ketoglutaric acid when glutamic acid is used). Similarly, in the above-mentioned reverse reaction, a product having an aldehyde group and an amino acid are produced from a substrate and / or intermediate having an amino group and a keto acid.

[0036] When alanine or glutamic acid is used as the compound to donate an amino group, the produced pyruvate or α-ketoglutaric acid can be converted (regenerated) to alanine or glutamic acid by alanine dehydrogenase or glutamate dehydrogenase, which will be described later. Regeneration is also possible in the reverse reaction.

[0037] In the above enzymatic reaction, when dehydrogenases (e.g., alcohol dehydrogenase as enzyme A, amine dehydrogenase as enzyme B, aldehyde dehydrogenase as enzyme C, etc.) are used as enzymes A, B, and / or C, the substrate and / or intermediate is the coenzyme NAD(P). ＋ Similarly, when aldehyde reductase is used as enzyme A, the substrate and / or intermediate is oxidized by the coenzyme NAD(P) to produce the product and NAD(P)H. ＋ to produce a product and NAD(P)H.

[0038] In the above reaction, the produced NAD(P)H is converted to NAD(P) by NAD(P)H oxidase, which will be described later. ＋ can be converted (reproduced) into

[0039] In the above enzyme reaction, the substrate compound 1,4-cyclohexanedimethanol (Compound 1) may be a mixture of cis and trans isomers, or either the trans or cis isomer. 4-(aminomethyl)cyclohexane-1-carbaldehyde (Compound 7) and 4-formylcyclohexane-1-carboxylic acid (Compound 5) may also be a mixture of cis and trans isomers, or either the trans or cis isomer.

[0040] Patent Document 3 and Non-Patent Document 5 disclose a choline oxidase mutant derived from Arthrobacter chlorophenolicus that produces 1,4-cyclohexanedicarboxaldehyde from 1,4-cyclohexanedimethanol, and Patent Document 4 discloses a putrescine aminotransferase (PatA) mutant derived from Escherichia coli that produces 1,4-bis(aminomethyl)cyclohexane from 1,4-cyclohexanedicarboxaldehyde. However, it is not known that 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced from 1,4-cyclohexanedimethanol based on the enzymatic reaction pathway of the present invention.

[0041] 2. Proteins and DNA of the Present Invention (1) Protein (Enzyme B) Having the Activity of Reversibly Converting an Aldehyde Group to an Amino Group of the Present Invention and DNA Encoding the Same As used herein, "the activity of reversibly converting an aldehyde group to an amino group" refers to the activity of catalyzing both an enzymatic reaction that converts an aldehyde group in a compound having an aldehyde group to an amino group, and the reverse reaction, that is, an enzymatic reaction that converts an amino group in a compound having an amino group to an aldehyde group. As used herein, these proteins are collectively referred to as "Enzyme B." A protein having the activity of reversibly converting an aldehyde group to an amino group is also referred to as an "enzyme that reversibly converts an aldehyde group to an amino group."

[0042] Specifically, the protein (enzyme B) of the present invention having the activity of reversibly converting an aldehyde group and an amino group is a protein having the activity of converting the amino group of [4-(aminomethyl)cyclohexyl]methanol (compound 6), 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7), or 1,4-bis(aminomethyl)cyclohexane (compound 8), which are compounds having an amino group shown in Figures 6 and 7, to an aldehyde group, or the activity of converting the aldehyde group of 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2), 1,4-cyclohexanedicarboxaldehyde (compound 3), or 4-formylcyclohexane-1-carboxylic acid (compound 5), which are compounds having an aldehyde group shown in Figures 6 and 7, to an amino group.

[0043] Examples of proteins (enzyme B) having the activity of reversibly converting an aldehyde group and an amino group include proteins having aminotransferase activity, proteins having amine dehydrogenase activity, and proteins having amine oxidase activity, and preferred are proteins having aminotransferase activity or amine oxidase activity.

[0044] Aminotransferase (also referred to as aminotransferase; AT, or transaminase) is a general term for enzymes that catalyze a reaction between an amino acid and an α-keto acid in biochemistry. As used herein, having aminotransferase activity refers to the activity of catalyzing a reversible reaction in which an amino group is transferred from a compound having an amino group to another compound, converting it into a carbonyl group. As used herein, a protein having aminotransferase activity refers to a protein that has the activity of catalyzing a reversible reaction in which an amino group is transferred from a compound having an amino group to another compound, converting it into a carbonyl group, and is sometimes referred to as "AT."

[0045] In one embodiment, the AT is a protein consisting of an amino acid sequence having 60% or more (preferably, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more) identity to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, 103 to 105, and 217, and having aminotransferase activity.

[0046] The AT of this embodiment may be a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, 103 to 105, and 217. 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. The protein having the amino acid sequence shown in SEQ ID NO: 217 is the aminotransferase SmAT derived from Shimia marina. These proteins have not been known to have the activity of transferring an amino group from a compound having an amino group shown in Figure 6 above to another compound, or the activity of transferring an amino group to a compound having an aldehyde group shown in Figure 6 above.

[0047] In another embodiment, the AT may be a mutant protein or a homologous protein of the above-mentioned protein having amino acid transferase activity, for example, a mutant protein or a homologous protein of the protein consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, 103 to 105, and 217, which consists of an amino acid sequence having 60% or more (preferably 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more) identity to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, 103 to 105, and 217, and which has amino acid transferase activity.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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

[0052] The amino acid sequence of 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, 103 to 105, and 217 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, 103 to 105, and 217.

[0053] 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.

[0054] Amine dehydrogenase is a enzyme that converts NAD(P) into NAD(P) in biochemistry. ＋Amine dehydrogenase is a general term for a group of enzymes that catalyze an oxidation-reduction reaction between a compound having an amino group and a compound having an aldehyde group, using amine dehydrogenase as a coenzyme. As used herein, "having amine dehydrogenase activity" refers to the activity of catalyzing two reversible reactions: a reaction of converting an amino group in a compound having an amino group to an aldehyde group via an oxidation reaction, and a reaction of converting an aldehyde group in a compound having an aldehyde group to an amino group via a reduction reaction. As used herein, a protein having amine dehydrogenase activity refers to a protein having the above-mentioned amine dehydrogenase activity.

[0055] In one embodiment, the protein having amine dehydrogenase activity is not particularly limited as long as it has the activity of reversibly converting the amino group and aldehyde group of the compound shown in Figure 6, and may be a known amine dehydrogenase. Examples of known amine dehydrogenases include amine dehydrogenases derived from bacteria of the genus Bacillus (e.g., amine dehydrogenase derived from Bacillus badius, amine dehydrogenase derived from Bacillus stearothermophilus, amine dehydrogenase derived from Streptomyces virginiae, amine dehydrogenase derived from Pseudomonas putida, amine dehydrogenase derived from Escherichia coli, and amine dehydrogenase derived from Salmonella enterica).

[0056] In one embodiment, the protein having amine dehydrogenase activity may be a mutant protein or a homologous protein of amine dehydrogenase, which has the activity of reversibly converting the amino group and aldehyde group of the compounds shown in Figure 6 (e.g., Compound 2, Compound 3, Compound 5, Compound 6, Compound 7, and Compound 8). Such a protein may be, for example, a protein consisting of an amino acid sequence that shares 60% or more (preferably, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more) identity with the amino acid sequence of the above-mentioned known amine dehydrogenase, and which has amine dehydrogenase activity.

[0057] In biochemistry, amine oxidase is a general term for enzymes that catalyze an oxidation-reduction reaction between a compound having an amino group and a compound having an aldehyde group, using oxygen as an electron acceptor. As used herein, having amine oxidase activity refers to the activity of catalyzing a reversible reaction that converts an amino group in a compound having an amino group to an aldehyde group via a reaction that oxidizes the amino group. As used herein, a protein having amine oxidase activity refers to a protein that has the activity of catalyzing a reversible reaction that converts an amino group in a compound having an amino group to an aldehyde group via a reaction that oxidizes the amino group.

[0058] In one embodiment, the protein having amine oxidase activity is not particularly limited as long as it has the activity of reversibly converting an amino group and an aldehyde group in the compound shown in Figure 6, and may be a known amine oxidase. The known amine oxidase may be a primary amine oxidase, a diamine oxidase, or a putrescine oxidase.

[0059] In one embodiment, the putrescine oxidase is a putrescine oxidase derived from a fungus of the genus Aspergillus (e.g., putrescine oxidase derived from Aspergillus nidulans, putrescine oxidase derived from Aspergillus luchuensis, etc.), a putrescine oxidase derived from Bos taurus, a putrescine oxidase derived from a fungus of the genus Candida (e.g., putrescine oxidase derived from Candida albicans, putrescine oxidase derived from Candida parapsilosis, putrescine oxidase derived from Candida tropicalis, etc.), a putrescine oxidase derived from a bacterium of the genus Kocuria (e.g., putrescine oxidase derived from Kocuria rosea, etc.), a putrescine oxidase derived from a bacterium of the genus Meyerozyma (e.g., putrescine oxidase derived from Meyerozyma nidulans ... putrescine oxidase derived from P. guilliermondii, putrescine oxidase derived from Micrococcus luteus, putrescine oxidase derived from Paenarthrobacter bacteria (e.g., putrescine oxidase derived from Paenarthrobacter aurescens), putrescine oxidase derived from Pichia kudriavzevii, putrescine oxidase derived from Pisum sativum, putrescine oxidase derived from Pseudomonas bacteria (e.g., putrescine oxidase derived from Pseudomonas putida), putrescine oxidase derived from Rhodococcus bacteria (e.g., Rhodococcus putrescine oxidase derived from Bacillus erythropolis, putrescine oxidase derived from Rhodococcus opacus, etc.

[0060] The putrescine oxidase of this embodiment may be a protein consisting of the amino acid sequence shown in SEQ ID NO: 277. The protein having the amino acid sequence shown in SEQ ID NO: 277 is putrescine oxidase RePO derived from Rhodococcus erythropolis, and these proteins have not been known to have the activity of transferring an amino group from a compound having an amino group shown in Figure 6 to another compound or the activity of transferring an amino group to a compound having an aldehyde group shown in Figure 6.

[0061] The amino acid sequence of a mutant protein or a homologous protein of the protein consisting of the amino acid sequence represented by SEQ ID NO:277 has an identity of 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 to the amino acid sequence represented by SEQ ID NO:277.

[0062] In one embodiment, the primary amine oxidase is selected from the group consisting of a primary amine oxidase derived from Paenarthrobacter aurescens, a primary amine oxidase derived from Trifolium pratense, a primary amine oxidase derived from Huperzia serrata, a primary amine oxidase derived from Homo sapiens, a primary amine oxidase derived from Malus domestica, a primary amine oxidase derived from Pisum sativum, a primary amine oxidase derived from Nicotiana tabacum, a primary amine oxidase derived from Arabidopsis thaliana, a primary amine oxidase derived from Euphorbia characias, a primary amine oxidase derived from Huperzia serrata, a primary amine oxidase derived from Lens primary amine oxidase derived from Aspergillus culinaris, primary amine oxidase derived from Trifolium pratense, primary amine oxidase derived from Arabidopsis thaliana, primary amine oxidase derived from Arthrobacter globiformis, primary amine oxidase derived from Aspergillus fungi (e.g., primary amine oxidase derived from Aspergillus carbonarius, primary amine oxidase derived from Aspergillus nidulans, primary amine oxidase derived from Aspergillus niger, primary amine oxidase derived from Aspergillus luchuensis, etc.), primary amine oxidase derived from Bos taurus, primary amine oxidase derived from Escherichia coli, primary amine oxidase from E. coli, primary amine oxidase from Lathyrus sativus, primary amine oxidase from Mus musculus, primary amine oxidase from Mycobacterium sp., primary amine oxidase from Ogataea angusta, primary amine oxidase from Onobrychis viciifolia, primary amine oxidase from Rattus norvegicus, primary amine oxidase from Rhodococcus opacus, primary amine oxidase from Schizosaccharomyces pombe, primary amine oxidase from Sus scrofa, primary amine oxidase from Trifolium pratense,It is a primary amine oxidase derived from Vicia faba.

[0063] In one embodiment, the diamine oxidase is selected from the group consisting of diamine oxidase derived from Brugmansia xcandida, diamine oxidase derived from Glycine max, diamine oxidase derived from Homo sapiens, diamine oxidase derived from Hyoscyamus niger, diamine oxidase derived from plants of the genus Lathyrus (e.g., diamine oxidase derived from Lathyrus sativus, diamine oxidase derived from Lathyrus cicera, etc.), diamine oxidase derived from Nicotiana tabacum, diamine oxidase derived from plants of the genus Pisum (e.g., diamine oxidase derived from Pisum sativum, diamine oxidase derived from Pinus sylvestris, etc.), and diamine oxidase derived from Sus diamine oxidase derived from A. scrofa, diamine oxidase derived from Trifolium subterraneum, diamine oxidase derived from Yarrowia lipolytica, diamine oxidase derived from bacteria of the genus Arthrobacter (for example, diamine oxidase derived from Arthrobacter sp., diamine oxidase derived from Arthrobacter globiformis, etc.), diamine oxidase derived from Papaver somniferum, diamine oxidase derived from Arachis hypogaea, diamine oxidase derived from Euphorbia characias, diamine oxidase derived from Lens culinaris, diamine oxidase derived from Vicia faba, diamine oxidase derived from Rattus norvegicus-derived diamine oxidase, and Aspergillus fungal diamine oxidase (e.g., Aspergillus niger-derived diamine oxidase, Aspergillus luchuensis-derived diamine oxidase, etc.).

[0064] In one embodiment, the protein having amine oxidase activity may be a mutant or homologous protein of amine oxidase, which has the activity of reversibly converting an amino group of an amino group-containing compound into an aldehyde group. Such a protein may be, for example, a protein 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) with the amino acid sequence of the above-mentioned amine oxidase, and which has amine oxidase activity.

[0065] The activity of reversibly converting an aldehyde group and an amino group can be confirmed, for example, by the following method. First, a recombinant DNA containing a DNA encoding a 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. In the medium, a compound having an amino group shown in Figure 6 (e.g., Compound 6, Compound 7, or Compound 8), a compound having an aldehyde group shown in Figure 6 (e.g., Compound 2, Compound 3, Compound 5, or Compound 7), a keto acid such as pyruvic acid, an amino acid such as alanine, or NAD(P) is added. ＋ A coenzyme such as α- or β-amino acid is added to generate a compound in which the corresponding amino group has been converted to an aldehyde group, or a compound in which an aldehyde group has been converted to an amino group. Finally, by detecting the generated compound in the bacterial cells or in the culture supernatant using a general analytical method such as HPLC, it can be confirmed that the target protein has the activity of reversibly converting an aldehyde group to an amino group.

[0066] Enzyme B of this embodiment includes proteins consisting of amino acid sequences represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, and 277, or amino acid sequences having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequences represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, and 277. Preferably, the proteins consist of amino acid sequences represented by SEQ ID NOs: 1, 4, 217, and 277, or amino acid sequences having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequences represented by SEQ ID NOs: 1, 4, 217, and 277.

[0067] In one embodiment, the DNA encoding Enzyme B is a DNA that consists of an amino acid sequence that has 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, 103 to 105, 217, and 277, and encodes a protein having aminotransferase activity. In this specification, the DNA may be a gene, a part of a gene, or artificially synthesized DNA (e.g., DNA encoding a mutant protein or codon-optimized DNA).

[0068] The DNA encoding the enzyme B of this embodiment is, for example, a base sequence encoding a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, 103 to 105, 217, and 277, and examples thereof include DNA consisting of a base sequence represented by any one of SEQ ID NOs: 5 to 8, 108 to 110, 218, and 280.

[0069] In another embodiment, the DNA encoding Enzyme B is, for example, 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, 103 to 105, 217, and 277. 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, 108 to 110, 218, and 280, 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 having 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.

[0070] DNA consisting of any one of the base sequences represented by SEQ ID NOs: 5-8, 108-110, 218, and 280, 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 DNA sequences for cloning into an expression vector, such as restriction enzyme sites, at their ends. 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 base sequence.

[0071] 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.

[0072] In one embodiment, the DNA encoding Enzyme B may also be a DNA that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to any one of the nucleotide sequences represented by SEQ ID NOs: 5 to 8, 108 to 110, 218, and 280. Hybridization refers to the process of hybridizing a DNA to a DNA having a specific nucleotide sequence or a part of the DNA. Therefore, the nucleotide sequence of the DNA having the specific nucleotide sequence or the DNA that hybridizes to a part of the DNA may be a DNA of a length that is useful as a probe for Northern or Southern blot analysis, or that can be used as an oligonucleotide primer for PCR analysis.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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, 108 to 110, 218, and 280, when calculated using a program such as BLAST or FASTA.

[0079] In one embodiment, the DNA encoding Enzyme B may also be a DNA encoding the above-mentioned protein having aminotransferase activity, protein having amine dehydrogenase activity, or protein having amine oxidase activity.

[0080] (2) Protein (Enzyme C) Having the Activity of Converting an Aldehyde Group to a Carboxy Group of the Present Invention and DNA Encoding the Same As used herein, "the activity of converting an aldehyde group to a carboxy group" refers to the activity of catalyzing an enzymatic reaction that converts the aldehyde group of a compound having an aldehyde group to a carboxy group. As used herein, these proteins are collectively referred to as "Enzyme C." A protein having the activity of converting an aldehyde group to a carboxy group is also referred to as an "enzyme that converts an aldehyde group to a carboxy group."

[0081] Specifically, the protein (enzyme C) of the present invention having the activity of converting an aldehyde group to a carboxy group is a protein having the activity of catalyzing a reaction of converting 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2), 1,4-cyclohexanedicarboxaldehyde (compound 3), or 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7), which are compounds having an aldehyde group as shown in Figures 6 and 7, into 4-(hydroxymethyl)cyclohexane-1-carboxylic acid (compound 4), 4-formylcyclohexane-1-carboxylic acid (compound 5), or 4-(aminomethyl)cyclohexane-1-carboxylic acid (target compound), which are carboxylic acids as shown in Figures 6 and 7, respectively.

[0082] Examples of proteins having the activity of converting an aldehyde group to a carboxy group include proteins having aldehyde dehydrogenase activity and proteins having aldehyde oxidase activity, with proteins having aldehyde dehydrogenase activity being preferred.

[0083] Aldehyde dehydrogenases (ALDH) are enzymes that convert NAD(P) into ＋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 a reaction that oxidizes the aldehyde group of a compound having an aldehyde group and converts it to a carboxy group. As used herein, a protein having aldehyde dehydrogenase activity refers to a protein that has the activity of catalyzing a reaction that oxidizes the aldehyde group of a compound having an aldehyde group and converts it to a carboxy group, and may be referred to as "ALDH."

[0084] 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, 127 to 139, and 207, for example, and having aldehyde dehydrogenase activity.

[0085] 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, 127 to 139, and 207. 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 represented by SEQ ID NOs: 35 to 46 and 127 to 139 are homologous proteins of the proteins (PatD, XylC, or StyD) consisting of the amino acid sequences represented by SEQ ID NOs: 19, 20, or 21, which are derived from the microorganisms listed in Table 13 and Table 20. The protein consisting of the amino acid sequence represented by SEQ ID NO: 207 is a benzaldehyde dehydrogenase derived from Xanthomonas campestris. These proteins have not been known to have the activity of converting the aldehyde group of the aldehyde-containing compound shown in Figure 6 above to a carboxy group.

[0086] In another embodiment, the ALDH may be a mutant protein or a homologous protein of a protein having aldehyde dehydrogenase activity, for example, 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, 127 to 139, and 207, which consists of an amino acid sequence having 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, 127 to 139, and 207, and which 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, 127 to 139, and 207.

[0087] The activity of converting an aldehyde group to a carboxyl group can be confirmed, for example, by the following method. First, a recombinant DNA containing a DNA encoding a 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. Compound 2, 3, or 7 and NAD(P) are added to the medium. ＋ Alternatively, a coenzyme such as FAD is added to generate the target carboxylic acid. Finally, the target carboxylic acid can be 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.

[0088] In yet another embodiment, the ALDH may be a protein that has aldehyde dehydrogenase activity that oxidizes the aldehyde group of a compound having an aldehyde group, and is classified into any one or more of the following categories 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.

[0089] Examples of such ALDH include proteins consisting of the 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.

[0090] In yet another embodiment, the protein having aldehyde dehydrogenase activity (ALDH) is preferably an aldehyde dehydrogenase derived from a bacterium of the genus Pseudomonas.

[0091] 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.

[0092] In another embodiment, ALDH may be a benzaldehyde dehydrogenase, which is a protein having aldehyde dehydrogenase activity that oxidizes the aldehyde group of a compound having an aldehyde group.

[0093] The benzaldehyde dehydrogenase of the present embodiment has aldehyde dehydrogenase activity that oxidizes the aldehyde group of a compound having an aldehyde group. 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.

[0094] 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.

[0095] 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, 139, and 207. 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, 139, and 207, which 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, 139, and 207, and which has aldehyde dehydrogenase activity.

[0096] In a further embodiment, the benzaldehyde dehydrogenase may be a protein that has aldehyde dehydrogenase activity of oxidizing an aldehyde group of a compound having an aldehyde group, and is classified into any one or more of the following categories 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.

[0097] In a further embodiment, the ALDH may be a mutant protein or a homologous protein of the above-mentioned benzaldehyde dehydrogenase, for example, a protein that has aldehyde dehydrogenase activity of oxidizing the aldehyde group of a compound having an aldehyde group, and has 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 SEQ ID NO: 20, and that, when aligned with the amino acid sequence represented by SEQ ID NO: 20, contains at least one amino acid residue selected from the following (1) to (26):(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).

[0098] 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.

[0099] 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 residues (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."

[0100] The amino acid sequence of the protein having aldehyde dehydrogenase activity of this embodiment may contain at least one of the amino acid residues (1) to (26) above, and may contain, 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.

[0101] 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.

[0102] The protein having aldehyde dehydrogenase activity 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.

[0103] In biochemistry, aldehyde oxidase is a general term for a group of enzymes that catalyze the oxidation reaction of aldehydes to carboxylic acids using oxygen and FAD as a coenzyme. As used herein, "having aldehyde oxidase activity" refers to the activity of catalyzing a reaction that oxidizes the aldehyde group of a compound having an aldehyde group and converts it to a carboxy group. As used herein, "a protein having aldehyde oxidase activity" refers to a protein that has the activity of catalyzing a reaction that oxidizes the aldehyde group of a compound having an aldehyde group and converts it to a carboxy group.

[0104] In one embodiment, the protein having aldehyde oxidase activity is an aldehyde oxidase derived from Escherichia coli, an aldehyde oxidase derived from a bacterium of the genus Pseudomonas (e.g., an aldehyde oxidase derived from Pseudomonas putida, an aldehyde oxidase derived from Pseudomonas stutzeri, an aldehyde oxidase derived from Pseudomonas sp., etc.), an aldehyde oxidase derived from Cavia porcellus, an aldehyde oxidase derived from Oryctolagus cuniculus, an aldehyde oxidase derived from Mus musculus, an aldehyde oxidase derived from Arabidopsis thaliana, an aldehyde oxidase derived from Solanum Examples of aldehyde oxidases that can be used include aldehyde oxidase derived from R. lycopersicum, aldehyde oxidase derived from Rattus norvegicus, aldehyde oxidase derived from Streptomyces moderatus, aldehyde oxidase derived from Homo sapiens, aldehyde oxidase derived from Culex quinquefasciatus, aldehyde oxidase derived from Burkholderia sp., aldehyde oxidase derived from Arabidopsis thaliana, and aldehyde oxidase derived from Gluconobacter thailandicus. The protein having aldehyde oxidase activity of this embodiment may be a mutant protein or a homologous protein of these aldehyde oxidases, and may also have aldehyde oxidase activity. Alternatively, the protein may be a protein consisting of 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 with the amino acid sequence of any of these aldehyde oxidases, and that has aldehyde oxidase activity.

[0105] In one embodiment, enzyme C is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, and 207, or 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, 127 to 139, and 207.

[0106] The DNA encoding the enzyme C of this embodiment is a DNA encoding a protein having the above-mentioned aldehyde dehydrogenase activity. The DNA encoding ALDH of one embodiment 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, 127 to 139, and 207, and examples thereof include DNA consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, 140 to 152, and 208.

[0107] In another embodiment, the DNA encoding the enzyme C may be a DNA encoding an aldehyde dehydrogenase, for example, a DNA encoding a mutant protein or a homologous protein of a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, and 207. 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, 140 to 152, and 208, 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, 140 to 152, and 208, and more preferably comprises a base sequence represented by any one of SEQ ID NOs: 23 to 26, 47 to 58, 140 to 152, and 208.

[0108] In one embodiment, the DNA encoding the enzyme C may be a DNA encoding a protein having aldehyde dehydrogenase activity, and may be, for example, a DNA that hybridizes under stringent conditions to a DNA consisting of a nucleotide sequence complementary to any one of the nucleotide sequences represented by SEQ ID NOs: 23 to 26, 47 to 58, 140 to 152, and 208. Examples of DNA that can hybridize under stringent conditions include DNA consisting of a nucleotide 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 nucleotide sequences represented by SEQ ID NOs: 23 to 26, 47 to 58, 140 to 152, and 208, when calculated using a program such as BLAST or FASTA.

[0109] The DNA encoding the enzyme C of this embodiment may be, for example, a DNA encoding the protein having the above-mentioned aldehyde oxidase activity.

[0110] (3) Protein (Enzyme A) Having the Activity of Converting a Hydroxy Group to an Aldehyde Group of the Present Invention and DNA Encoding the Same As used herein, "the activity of converting a hydroxy group to an aldehyde group" refers to the activity of oxidizing the hydroxy group of a compound having a hydroxy group and converting the hydroxy group to an aldehyde group. As used herein, these proteins are collectively referred to as "Enzyme A." A protein having the activity of converting a hydroxy group to an aldehyde group is also referred to as an "enzyme that converts a hydroxy group to an aldehyde group."

[0111] The protein of the present invention having the activity of converting a hydroxy group into an aldehyde group is a protein having the activity of catalyzing the reaction of 1,4-cyclohexanedimethanol (compound 1), 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2), [4-(aminomethyl)cyclohexyl]methanol (compound 6), and 4-(hydroxymethyl)cyclohexane-1-carboxylic acid (compound 4), which are compounds having a hydroxy group shown in Figures 6 and 7, to 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2), 1,4-cyclohexanedicarboxaldehyde (compound 3), 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7), or 4-formylcyclohexane-1-carboxylic acid (compound 5), which are compounds having an aldehyde group shown in Figure 6, respectively.

[0112] Examples of the protein (enzyme A) having the activity of converting a hydroxy group to an aldehyde group in the present invention include a protein having oxidase activity, a protein having dehydrogenase activity, and a protein having aldehyde reductase activity, and preferably a protein having oxidase activity.

[0113] Oxidase is a general term for enzymes that oxidize a substrate using molecular oxygen as an electron acceptor. As used herein, having oxidase activity as enzyme A refers to the activity of catalyzing a reaction that oxidizes a hydroxy group in a compound having a hydroxy group and converts it to an aldehyde group. As used herein, a protein having oxidase activity as enzyme A refers to a protein that has the activity of catalyzing a reaction that oxidizes a hydroxy group in a compound having a hydroxy group and converts it to an aldehyde group, and may be referred to as "OX" in the present specification.

[0114] In one embodiment, the OX may be, for example, a flavin adenine dinucleotide (FAD)-binding oxidase or a phenol oxidase, and is preferably an FAD-binding oxidase. Note that the FAD-binding oxidase is a general term for oxidases in which FAD binds to the active site of the enzyme and uses FAD as a coenzyme, and the phenol oxidase is a general term for oxidases that use phenols as substrates.

[0115] As used herein, FAD-linked oxidase refers to an enzyme that uses a compound having a hydroxy group as a substrate, molecular oxygen as an electron acceptor, and converts the hydroxy group of the substrate to an aldehyde group. As used herein, FAD-linked oxidase may also include, for example, enzymes belonging to the glucose-methanol-choline (GMC) oxidoreductase family (e.g., choline oxidase, glucose oxidase, methanol oxidase, alcohol oxidase, cholesterol oxidase), sarcosine oxidase, L-α-glycerophosphate oxidase, etc. Preferred FAD-linked oxidases are those belonging to the GMC oxidoreductase family, with choline oxidase being particularly preferred. Examples of phenol oxidases include laccase, etc.

[0116] In one embodiment, OX as the enzyme A may be an alcohol oxidase, such as an alcohol oxidase derived from a fungus of the genus Pichia (e.g., an alcohol oxidase derived from Pichia pastoris, an alcohol oxidase derived from Pichia sp., etc.), an alcohol oxidase derived from Komagataella pastoris, an alcohol oxidase derived from a fungus of the genus Ogataea (e.g., an alcohol oxidase derived from Ogataea angusta, an alcohol oxidase derived from Ogataea thermomethanolica, an alcohol oxidase derived from Ogataea methanolica, etc.), an alcohol oxidase derived from Phanerodontia chrysosporium, or an alcohol oxidase derived from a fungus of the genus Aspergillus (e.g., an alcohol oxidase derived from Aspergillus alcohol oxidase derived from Aspergillus terreus, alcohol oxidase derived from Aspergillus ochraceus), alcohol oxidase derived from Ochrobactrum sp., alcohol oxidase derived from Thermoascus aurantiacus, alcohol oxidase derived from Gloeophyllum trabeu m, alcohol oxidase derived from Colletotrichum graminicola, alcohol oxidase derived from Arthrobacter globiformis, alcohol oxidase derived from Comamonas sp. alcohol oxidase derived from Candida species, alcohol oxidase derived from Cornus aspersum, alcohol oxidase derived from fungi of the genus Candida (for example, alcohol oxidase derived from Candida methanosorbosa, alcohol oxidase derived from Candida sp.), or alcohol oxidase derived from Rhodococcus triatomae.

[0117] In one embodiment, OX as the enzyme A may be a choline oxidase, and examples thereof include choline oxidase derived from bacteria of the genus Arthrobacter (e.g., choline oxidase derived from Arthrobacter chlorophenolicus, choline oxidase derived from Arthrobacter globiformis, etc.), choline oxidase derived from Achromobacter cholinophagum, choline oxidase derived from Arthrobacter pascens, choline oxidase derived from Fusarium oxysporum, choline oxidase derived from Cylindrocarpon didymum, choline oxidase derived from Glutamicibacter nicotianae, choline oxidase derived from Alcaligenes sp. oxidase derived from Arthrobacter genus, more preferably Arthrobacter chlorophenolicus choline oxidase (AcCOx) or Arthrobacter globiformis choline oxidase (AgCOx).

[0118] In one embodiment, OX as the enzyme A may be a glucoxidase, and examples thereof include glucose oxidase derived from the genus Fusarium (e.g., glucose oxidase derived from Fusarium graminearum, glucose oxidase derived from Fusarium oxysporum, etc.), glucose oxidase derived from fungi of the genus Aspergillus (e.g., glucose oxidase derived from Aspergillus niger, glucose oxidase derived from Aspergillus sp., etc.), glucose oxidase derived from Cladosporium neopsychrotolerans, glucose oxidase derived from fungi of the genus Penicillium (e.g., glucose oxidase derived from Penicillium adametzii, glucose oxidase derived from Penicillium glucose oxidase derived from Bacillus amagasakiense, Penicillium janthinellum, Penicillium sp., etc.), glucose oxidase derived from Streptomyces coelicolor, or glucose oxidase derived from Talaromyces (e.g., glucose oxidase derived from Talaromyces funiculosus, glucose oxidase derived from Talaromyces purpureogenus, etc.).

[0119] In one embodiment, OX as the enzyme A may be a laccase, such as a laccase derived from a bacterium of the genus Trametes (e.g., a laccase derived from Trametes versicolor or a laccase derived from Trametes villosa), a laccase derived from a bacterium of the genus Bacillus (e.g., a laccase derived from Bacillus subtilis or a laccase derived from Bacillus pumilus), a laccase derived from Escherichia coli, or a laccase derived from Pseudomonas putida.

[0120] In one embodiment, OX as enzyme A is, for example, a protein having an activity of converting a hydroxy group to an aldehyde group, and consisting of an amino acid sequence represented by SEQ ID NOs: 201, 213, 221 to 230, 241, 289 to 294, or an amino acid sequence having 60% or more (preferably, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more) identity to the amino acid sequence represented by SEQ ID NOs: 201, 213, 221 to 230, 241, 289 to 294.

[0121] OX as enzyme A in this embodiment may be a protein consisting of the amino acid sequence represented by SEQ ID NOs: 201, 213, 221 to 230, 241, and 289 to 294.

[0122] The protein having the amino acid sequence represented by SEQ ID NO: 201 is choline oxidase derived from Arthrobacter chlorophenolicus.

[0123] The protein having the amino acid sequence represented by SEQ ID NO: 213 is a mutant (also referred to as 5mut) in which five mutations have been introduced into the Arthrobacter chlorophenolicus-derived choline oxidase: 101st serine to alanine, 351st histidine to valine, 355th valine to threonine, 357th phenylalanine to arginine, and 376th threonine to glutamine (S101A / H351V / V355T / F357R / T376Q).

[0124] The protein having the amino acid sequence represented by SEQ ID NO: 221 is a choline oxidase derived from Arthrobacter globiformis.

[0125] The protein having the amino acid sequence represented by SEQ ID NO: 222 is annotated as a glucose-methanol-choline (GMC) oxidoreductase derived from Corynebacterium variabile.

[0126] The protein having the amino acid sequence represented by SEQ ID NO: 223 is annotated as glucose-methanol-choline (GMC) oxidoreductase derived from Saccharopolyspora erythraea.

[0127] The protein having the amino acid sequence represented by SEQ ID NO: 224 is annotated as glucose-methanol-choline (GMC) oxidoreductase derived from Streptomyces aureocirculatus.

[0128] The protein having the amino acid sequence represented by SEQ ID NO: 225 is annotated as an enzyme belonging to the glucose-methanol-choline (GMC) oxidoreductase family derived from Streptomyces hygroscopicus.

[0129] The protein having the amino acid sequence represented by SEQ ID NO: 226 is annotated as glucose-methanol-choline (GMC) oxidoreductase derived from Streptomyces lincolnensis.

[0130] The protein having the amino acid sequence represented by SEQ ID NO: 227 is annotated as glucose-methanol-choline (GMC) oxidoreductase derived from Streptomyces orinocci.

[0131] The protein having the amino acid sequence represented by SEQ ID NO: 228 is annotated as glucose-methanol-choline (GMC) oxidoreductase derived from Streptomyces rishiriensis.

[0132] The protein having the amino acid sequence represented by SEQ ID NO: 229 is annotated as an enzyme belonging to the glucose-methanol-choline (GMC) oxidoreductase family derived from Streptomyces thioluteus.

[0133] The protein having the amino acid sequence represented by SEQ ID NO: 230 is annotated as a glucose-methanol-choline (GMC) oxidoreductase derived from Streptomyces viridochromogenes.

[0134] The protein having the amino acid sequence represented by SEQ ID NO: 241 is a mutant (also called 6mut) in which six mutations have been introduced into the choline oxidase derived from Arthrobacter chlorophenolicus: 101st serine to alanine, 250th aspartic acid to glycine, 253rd phenylalanine to arginine, 355th valine to threonine, 357th phenylalanine to arginine, and 359th methionine to arginine (S101A / D250G / F253R / V355T / F357R / M359R).

[0135] The protein having the amino acid sequence represented by SEQ ID NO: 289 is a choline oxidase derived from Arthrobacter chlorophenolicus, in which the 250th aspartic acid is mutated to glycine, the 253rd phenylalanine is mutated to arginine, the 355th valine is mutated to threonine, the 357th phenylalanine is mutated to arginine, and the 359th methionine is mutated to arginine (D250G / F253R / V355T / F357R / M359R). This is a mutant into which five mutations have been introduced.

[0136] The protein having the amino acid sequence represented by SEQ ID NO: 290 is a choline oxidase derived from Arthrobacter chlorophenolicus, in which the 101st serine is mutated to alanine, the 250th aspartic acid is mutated to glycine, the 253rd phenylalanine is mutated to arginine, the 357th phenylalanine is mutated to arginine, and the 359th methionine is mutated to arginine (S101A / D250G / F253R / F357R / M359R). This is a mutant into which five mutations have been introduced.

[0137] The protein having the amino acid sequence represented by SEQ ID NO: 291 is a choline oxidase derived from Arthrobacter chlorophenolicus, in which the 101st serine is mutated to alanine, the 250th aspartic acid is mutated to glycine, the 253rd phenylalanine is mutated to arginine, the 355th valine is mutated to threonine, and the 359th methionine is mutated to arginine (S101A / D250G / F253R / V355T / M359R). This is a mutant into which five mutations have been introduced.

[0138] The protein having the amino acid sequence represented by SEQ ID NO: 292 is a choline oxidase derived from Arthrobacter chlorophenolicus. The 101st serine is mutated to alanine, the 250th aspartic acid is mutated to glycine, the 253rd phenylalanine is mutated to arginine, the 355th valine is mutated to threonine, and the 357th phenylalanine is mutated to arginine (S101A / D250G / F253R / V355T / F357R). This is a mutant into which five mutations have been introduced.

[0139] The protein having the amino acid sequence represented by SEQ ID NO: 293 is a choline oxidase derived from Arthrobacter chlorophenolicus, in which the 101st serine is mutated to alanine, the 250th aspartic acid is mutated to glycine, the 355th valine is mutated to threonine, the 357th phenylalanine is mutated to arginine, and the 359th methionine is mutated to arginine (S101A / D250G / V355T / F357R / M359R). This is a mutant into which five mutations have been introduced.

[0140] The protein having the amino acid sequence represented by SEQ ID NO: 294 is a choline oxidase derived from Arthrobacter chlorophenolicus, in which the 101st serine is mutated to alanine, the 253rd phenylalanine is mutated to arginine, the 355th valine is mutated to threonine, the 357th phenylalanine is mutated to arginine, and the 359th methionine is mutated to arginine (S101A / F253R / V355T / F357R / M359R). This is a mutant into which five mutations have been introduced.

[0141] As used herein, dehydrogenase refers to an enzyme that decomposes NAD(P). ＋ In this specification, the term "enzyme A having dehydrogenase activity" refers to an enzyme that oxidizes the above substrate using a compound having a hydroxyl group as a substrate, and oxidizes the substrate to NAD(P). ＋ In this specification, a protein having dehydrogenase activity as enzyme A refers to the activity of catalyzing a reaction in which a substrate is oxidized using a coenzyme such as NAD(P) as an electron acceptor. ＋ The term "protein" refers to a protein that has the activity of catalyzing the reaction of oxidizing the above substrate using a coenzyme such as an electron acceptor.

[0142] In one embodiment, the protein having dehydrogenase activity as enzyme A may be an alcohol dehydrogenase, such as Sulfolobus solfataricus-derived alcohol dehydrogenase, Brevibacterium sp.-derived alcohol dehydrogenase, horse liver-derived alcohol dehydrogenase, or Pseudomonas putida-derived alcohol dehydrogenase.

[0143] As used herein, the term "aldehyde reductase" refers to an enzyme that converts NAD(P) into aldehyde reductase in biochemistry. ＋ In this specification, having aldehyde reductase activity means an enzyme that oxidizes a hydroxy group of a substrate having a hydroxy group to an aldehyde group using a coenzyme such as NAD(P) as an electron acceptor. ＋ In this specification, a protein having aldehyde reductase activity refers to an activity that catalyzes the reaction of oxidizing a hydroxy group of a substrate having a hydroxy group to an aldehyde group using a coenzyme such as NAD(P) as an electron acceptor. ＋ It refers to a protein that has the activity of catalyzing the reaction of oxidizing the hydroxy group of a substrate having a hydroxy group to an aldehyde group, using a coenzyme such as an electron acceptor.

[0144] In one embodiment, the protein having aldehyde reductase activity is selected from the group consisting of aldehyde reductase derived from Saccharomyces cerevisiae, aldehyde reductase derived from Rhodococcus rubber, aldehyde reductase derived from Magnusiomyces capitatus, aldehyde reductase derived from Saccharolobus solfataricus, aldehyde reductase derived from Thermus thermophilus, aldehyde reductase derived from Corynebacterium glutamicum, aldehyde reductase derived from Escherichia coli, aldehyde reductase derived from Geobacillus bacteria, and aldehyde reductase derived from Pseudomonas putida-derived aldehyde reductase, etc.

[0145] The activity of converting a hydroxy group to an aldehyde group can be confirmed, for example, by the following method. First, a recombinant DNA containing a DNA encoding a 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. Compound 1, Compound 2, Compound 6, or Compound 4 is added to the medium to produce the target aldehyde. Finally, the target aldehyde can be detected in the bacterial cells or in the culture supernatant using a common analytical method such as HPLC, thereby confirming that the target protein has the activity of converting a hydroxy group to an aldehyde group.

[0146] In one embodiment, the DNA encoding Enzyme A may be a DNA encoding a protein having choline oxidase activity, for example, 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 SEQ ID NO: 201, and encoding a protein having choline oxidase activity. In this specification, the DNA may be a gene or a part of a gene.

[0147] The DNA encoding the enzyme A of this embodiment may be a DNA encoding a protein having choline oxidase activity, for example, a base sequence encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 201, and one example of such a DNA is a DNA consisting of the base sequence represented by SEQ ID NO: 202.

[0148] In another embodiment, the DNA encoding Enzyme A may be a DNA encoding a protein having choline oxidase activity, for example, a DNA encoding a mutant protein or a homologous protein of a protein consisting of the amino acid sequence represented by SEQ ID NO: 201. 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 SEQ ID NO: 202, 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 having at least 60% or more identity to the base sequence shown in SEQ ID NO:202, 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, and more preferably consists of the base sequence shown in SEQ ID NO:202.

[0149] DNA consisting of the base sequence represented by SEQ ID NO: 202, 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 of the primer. 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 based on the determined DNA base sequence using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd.

[0150] In one embodiment, the DNA encoding Enzyme A may also be DNA encoding a protein having choline oxidase activity, and may be, for example, DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 202. Examples of DNA that can hybridize under stringent conditions include DNA consisting of a nucleotide sequence that has an identity of at least 50% or 60% or more, 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, to the nucleotide sequence represented by SEQ ID NO: 202, when calculated using a program such as BLAST or FASTA.

[0151] In one embodiment, the DNA encoding the enzyme A may be, for example, a DNA encoding a protein having oxidase activity as the enzyme A, a protein having dehydrogenase activity, and a protein having aldehyde reductase activity.

[0152] In one embodiment, the DNA encoding Enzyme A may be a DNA encoding a protein having choline oxidase activity, for example, 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 SEQ ID NO: 201, and encoding a protein having choline oxidase activity. In this specification, the DNA may be a gene or a part of a gene.

[0153] In one embodiment, the DNA encoding Enzyme A 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: 201, 213, 221 to 230, 241, and 289 to 294, and encoding a protein having aminotransferase activity. As used herein, the DNA may be a gene, a part of a gene, or artificially synthesized DNA (e.g., DNA encoding a mutant protein or codon-optimized DNA).

[0154] The DNA encoding the enzyme A of this embodiment is, for example, a base sequence encoding a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 201, 213, 221 to 230, 241, and 289 to 294, and examples thereof include DNA consisting of a base sequence represented by any one of SEQ ID NOs: 202, 214, 231 to 240, 242, 276, and 295 to 299.

[0155] In another embodiment, the DNA encoding Enzyme A is, for example, 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: 201, 213, 221 to 230, 241, and 289 to 294. 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: 202, 214, 231 to 240, 242, 276, and 295 to 299, 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 having 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: 202, 214, 231 to 240, 242, 276, and 295 to 299, and more preferably consists of a base sequence represented by any one of SEQ ID NOs: 202, 214, 231 to 240, 242, 276, and 295 to 299.

[0156] In one embodiment, the DNA encoding the enzyme A may also be a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to any one of the base sequences represented by SEQ ID NOs: 202, 214, 231 to 240, 242, 276, and 295 to 299.

[0157] 3. Recombinant DNA of the Present Invention The recombinant DNA of the present invention includes DNA encoding a protein (enzyme A) of the present invention having the activity of converting a hydroxy group in a compound having a hydroxy group to an aldehyde group, DNA encoding a protein (enzyme B) of the present invention having the activity of reversibly converting an aldehyde group and an amino group, and / or DNA encoding a protein (enzyme C) of the present invention having the activity of converting an aldehyde group to a carboxy group. Of these enzymes, the recombinant DNA may include DNA encoding one type of enzyme having the same enzymatic activity, or DNA encoding multiple types of enzymes having different enzymatic activities. When DNA encoding multiple types of enzymes is included, it may include DNA encoding combinations such as enzyme A and enzyme B, enzyme A and enzyme C, enzyme B and enzyme C, or enzyme A, enzyme B and enzyme C. Here, enzymes having the same enzymatic activity (e.g., enzyme A) may include multiple different enzymes.

[0158] The recombinant DNA of the present invention may further comprise DNA encoding alanine dehydrogenase or glutamate dehydrogenase, as described below, and / or DNA encoding NADH oxidase. The recombinant DNA of the present invention may comprise DNA encoding only one type of enzyme, or may comprise 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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)].

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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 or glutamate dehydrogenase, as described below, and / or DNA encoding NADH oxidase. 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.

[0170] 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. ,

[0171] 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)].

[0172] Examples of methods for introducing the recombinant DNA of the present invention into the chromosome of a host cell include homologous recombination. Examples of homologous recombination methods include a method using a plasmid for homologous recombination, which can be prepared by ligating a 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 using the lambda phage homologous recombination system to introduce recombinant DNA [Proc. Natl. Acad. Sci. USA, 97, 5978-5983 (2000)].

[0173] 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 sensitive to sucrose 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 sensitive to streptomycin 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)].

[0174] 5. Production Method of the Present Invention The production method of the present invention is a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from the group consisting of a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２ and the like are COOH. Specifically, the compound represented by the general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, 7 and 8.

[0175] In the production method of the present invention, the protein having the activity of converting a hydroxy group to an aldehyde group, the protein having the activity of converting an aldehyde group to a carboxy group, and the protein having the activity of reversibly converting an aldehyde group and an amino group are enzyme A, enzyme C, and enzyme B, respectively. In the production method of the present invention, production may be performed in the presence of one of these enzymes (enzyme A alone, enzyme B alone, or enzyme C alone), two of them (enzyme A + enzyme B, enzyme A + enzyme C, or enzyme B + enzyme C), or all three of them (enzyme A + enzyme B + enzyme C). In the production method of the present invention, compounds 1 to 8 can be used as substrate compounds or intermediate compounds. In such cases, the enzymatic reactions (synthetic pathways) and the enzymes used in each enzymatic reaction are shown in Figures 6 and 7. Here, the compounds, enzymes, and enzymatic reactions in the production method of the present invention are as described above in "1. Compounds and Enzymatic Reactions" and "2. Proteins and DNA of the Present Invention."

[0176] When at least one of the compounds 1 to 8 is used as an intermediate compound, at least one of the compounds 1 to 8 may be produced in the presence of at least one of the enzymes A, B, and C, as well as any enzyme and any substrate compound necessary for producing the compounds 1 to 8.

[0177] In one embodiment, the production method is carried out in the presence of a protein (enzyme A) having the activity of converting a hydroxy group to an aldehyde group, a protein (enzyme C) having the activity of converting an aldehyde group to a carboxy group, and a protein (enzyme B) having the activity of reversibly converting an aldehyde group and an amino group. １ and R ２ One of them is CH ２ OH and the other is CH ２ OH, CHO, or CH２ NH ２ When a compound represented by general formula (1) (compound 1, 2, or 6) is used as a substrate compound or an intermediate compound, the reaction is preferably carried out in the presence of a protein having the activity of converting a hydroxy group to an aldehyde group (enzyme A), a protein having the activity of converting an aldehyde group to a carboxy group (enzyme C), and a protein having the activity of reversibly converting an aldehyde group and an amino group (enzyme B).

[0178] Also, R １ and R ２ One of them is CH ２ NH ２ and the other is CHO (compound 7) is used as a substrate compound or an intermediate compound, the reaction is preferably carried out in the presence of a protein (enzyme C) having the activity of converting an aldehyde group into a carboxy group.

[0179] In this case, in the general formula (1), R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, the compound (i.e., compound 7) is more preferably a compound generated from any one of the substrate compounds or intermediate compounds shown in (i) and (ii) below: １ and R ２ (ii) a compound in which R １ and R ２ One of them is CH ２ NH ２ and the other is CH ２ OH (i.e., Compound 6)

[0180] R １ and R ２ In the case where a compound represented by general formula (1) (compound 5), in which one of the groups is CHO and the other is COOH, is used as a substrate compound or an intermediate compound, the reaction is preferably carried out in the presence of a protein (enzyme B) having the activity of reversibly converting an aldehyde group and an amino group.

[0181] In this case, in the general formula (1), R １ and R ２It is more preferable that the compound in which one of R is COOH and the other is CHO (i.e., compound 5) is a compound generated from any one of the substrate compounds or intermediate compounds shown in (i) and (ii) below. １ and R ２ (ii) a compound in which R １ and R ２ One of them is COOH and the other is CH ２ OH (i.e., Compound 4)

[0182] R １ and R ２ One of them is CHO and the other is CH ２ NH ２ or CHO, or R １ and R ２ Both are CH ２ NH ２ When a compound represented by general formula (1) (compound 7, 3, or 8) is used as a substrate compound or an intermediate compound, the reaction is preferably carried out in the presence of a protein (enzyme C) having the activity of converting an aldehyde group to a carboxy group and a protein (enzyme B) having the activity of reversibly converting an aldehyde group and an amino group.

[0183] R １ and R ２ One of them is CH ２ OH and the other is CH ２ NH ２ When a compound represented by general formula (1) (compound 6), which is:

[0184] R １ and R ２ One of them is CH ２When a compound represented by general formula (1) (compound 4) in which one hydroxyl group is OH and the other is COOH is used as a substrate compound or an intermediate compound, the reaction is preferably carried out in the presence of a protein (enzyme A) having the activity of converting a hydroxyl group to an aldehyde group and a protein (enzyme B) having the activity of reversibly converting an aldehyde group and an amino group.

[0185] The protein having the activity of converting a hydroxy group to an aldehyde group (enzyme A) is preferably an oxidase, and the oxidase is preferably a protein consisting of an amino acid sequence having 60% or more identity to the amino acid sequences shown in SEQ ID NOs: 201, 213, 221 to 230, 241, and 289 to 294. The protein having the activity of converting an aldehyde group to a carboxy group (enzyme C) is preferably an aldehyde dehydrogenase, and the aldehyde dehydrogenase is preferably a protein consisting of an amino acid sequence having 50% or more identity to the amino acid sequence shown in any one of SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, and 207. The protein (enzyme B) having the activity of reversibly converting an aldehyde group and an amino group is preferably an aminotransferase or an amine oxidase. The aminotransferase is preferably a protein consisting of an amino acid sequence having 60% or more identity to any one of the amino acid sequences shown in SEQ ID NOs: 1 to 4, 103 to 105, and 217, and the amine oxidase is preferably a protein consisting of an amino acid sequence having 60% or more identity to the amino acid sequence shown in SEQ ID NO: 277.

[0186] As the substrate compound, Compound 1 or Compound 8 is preferably used, and Compound 1 is more preferably used.

[0187] "In the presence of enzyme A, enzyme B, and / or enzyme C" means that active enzyme A, enzyme B, and / or enzyme C are present in the reaction system so as to catalyze the reaction. For example, this includes adding enzyme A, enzyme B, and / or enzyme C of the present invention to the reaction system, or allowing cells containing DNA encoding enzyme A, enzyme B, and / or enzyme C of the present invention and capable of expressing the enzymes, or recombinant cells obtainable by transforming a host cell with recombinant DNA containing DNA encoding enzyme A, enzyme B, and / or enzyme C of the present invention and capable of expressing the enzymes, 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.

[0188] Furthermore, the reaction carried out in the presence of enzyme A, enzyme B, and / or enzyme C may be carried out in one system, or may be carried out in multiple systems in a stepwise manner. When the reaction carried out in the presence of enzyme A, enzyme B, and / or enzyme C is carried out in one system, it is desirable that these enzymes are expressed in one cell. When the reaction carried out in the presence of enzyme A, enzyme B, and / or enzyme C is carried out in multiple systems in a stepwise manner, it is desirable that enzyme A, enzyme B, and / or enzyme C are expressed in separate cells.

[0189] For example, 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced by a reaction using 1,4-cyclohexanedimethanol (compound 1) as a substrate and cells expressing enzymes A, B, and C. Alternatively, 1,4-cyclohexanedimethanol (compound 1) can be first reacted using cells expressing enzymes A and B to produce 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7), and then a reaction can be carried out using cells expressing enzyme C to produce 4-(aminomethyl)cyclohexane-1-carboxylic acid from 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7).

[0190] One embodiment of the production method uses 1,4-bis(aminomethyl)cyclohexane (compound 8) as a substrate compound, and includes the steps of: (i) producing 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7) from 1,4-bis(aminomethyl)cyclohexane (compound 8) in the presence of a protein (enzyme B) having the activity of reversibly converting an aldehyde group and an amino group; and (ii) producing 4-(aminomethyl)cyclohexane-1-carboxylic acid (target compound) from 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7) in the presence of a protein (enzyme C) having the activity of converting an aldehyde group to a carboxy group.

[0191] Another embodiment of the production method is a production method using 1,4-cyclohexanedimethanol (compound 1) as a substrate compound, and includes: (iii) a step (step (iii)) of converting a hydroxy group of 1,4-cyclohexanedimethanol (compound 1) into an aldehyde group in the presence of a protein (enzyme A) having the activity of converting a hydroxy group into an aldehyde group to produce 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2).

[0192] As shown in Figures 6 and 7, the method for producing the target compound, 4-(aminomethyl)cyclohexane-1-carboxylic acid, from 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2) as a substrate compound or intermediate compound can take multiple routes, and the substrate compound or intermediate compound and enzyme can be selected depending on the desired reaction route. The compounds that can be converted using each enzyme are as follows: Enzyme A: Compound 1 to Compound 2, Compound 2 to Compound 3, Compound 4 to Compound 5, Compound 6 to Compound 7 (can be performed in the same manner as in step (iii)); Enzyme B: Compound 8 to Compound 7, Compound 7 to Compound 3 (reversible), Compound 5 to the target compound (reversible), Compound 2 to Compound 6 (reversible) (can be performed in the same manner as in step (i)); Enzyme C: Compound 2 to Compound 4, Compound 3 to Compound 5, Compound 7 to the target compound (can be performed in the same manner as in step (ii));

[0193] Another embodiment of the production method is a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound in the presence of at least one enzyme selected from a protein having an activity of converting a hydroxy group to an aldehyde group, a protein having an activity of converting an aldehyde group to a carboxy group, and a protein having an activity of reversibly converting an aldehyde group and an amino group: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ are both COOH, and the compound represented by general formula (1) is R １ and R ２ One of them is CH ２ NH ２ and the other is CHO or CH ２ NH ２ Specifically, the compound represented by the general formula (1) is any one of compounds 1, 2, 3, 4, 5 and 6.

[0194] A production method of another embodiment (hereinafter also referred to as the "production method of embodiment B") is a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as an intermediate compound in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ When both are COOH, R １ and R ２ Both are CH ２ NH ２ and when the compound represented by general formula (1) is R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, and compound 8 (general (1) in R １ and R ２ Both are CH ２ NH ２ Specifically, the compound represented by general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, and 7, excluding the case where compound 7 is an intermediate compound generated from compound 8.

[0195] The production method of embodiment B is a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from a protein having an activity of converting a hydroxy group to an aldehyde group, a protein having an activity of converting an aldehyde group to a carboxy group, and a protein having an activity of reversibly converting an aldehyde group and an amino group: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ and the compound represented by the general formula (1) is １ and R ２ One of them is COOH and the other is CH ２(ii) a substrate compound or intermediate compound (i.e., Compound 4 or Compound 5), which is OH or CHO; １ and R ２ One of them is CH ２ NH ２ and the other is CH ２ a substrate compound or intermediate compound (i.e., compound 6), wherein R is OH; and １ and R ２ are both CHO, or R １ and R ２ One of them is CH ２ OH and the other is CH ２ NH ２ A compound derived from a compound represented by general formula (1) (i.e., compound 3 or 6), wherein R １ and R ２ One of them is CH ２ NH ２ and an intermediate compound in which the other is CHO (i.e., compound 7), or at least one of the following.] Specifically, the compound represented by the above general formula (1) is at least one of compounds 1, 2, 3, 4, 5, and 6 as substrate compounds, or at least one of compounds 1, 2, 3, 4, 5, and 6 as intermediate compounds, and compound 7 generated from compound 3 or 6.

[0196] In one embodiment, the manufacturing method comprises: １ and R ２ and R are both CHO (i.e., Compound 3), １ and R ２ One of them is COOH and the other is CH ２ OH (i.e., Compound 4) or R １ and R ２ One of them is CH ２ NH ２ and the other is CH ２Preferably, the method is a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using, as a substrate compound or an intermediate compound, any one of the compounds having a hydroxy group in a hydroxy group (i.e., compound 6) as a substrate compound or an intermediate compound, in the presence of at least one enzyme selected from a protein having an activity of converting a hydroxy group to an aldehyde group, a protein having an activity of converting an aldehyde group to a carboxy group, and a protein having an activity of reversibly converting an aldehyde group and an amino group.

[0197] In the manufacturing method of this embodiment, in the general formula (1), R １ and R ２ In the case where a compound (i.e., Compound 3) in which R are both CHO is used as a substrate compound or an intermediate compound, in the presence of a protein having the activity of converting an aldehyde group to a carboxy group and a protein having the activity of reversibly converting an aldehyde group and an amino group, １ and R ２ One of them is COOH and the other is CH ２ When a compound having a hydroxy group as a substituent (i.e., compound 4) is used as a substrate compound or an intermediate compound, the compound can be produced in the presence of a protein having an activity of converting a hydroxy group to an aldehyde group and a protein having an activity of reversibly converting an aldehyde group and an amino group, or １ and R ２ One of them is CH ２ NH ２ and the other is CH ２ When a compound having a hydroxy group as a substituent (i.e., compound 6) is used as a substrate compound or an intermediate compound, the method is more preferably a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting a hydroxy group to an aldehyde group and a protein having the activity of converting an aldehyde group to a carboxy group.

[0198] A production method of one embodiment (hereinafter also referred to as the "production method of embodiment A") is a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 127 to 139, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by any one of these amino acid sequences: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２ and the like are COOH. Specifically, the compound represented by the general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, 7 and 8.

[0199] The production method of embodiment A is preferably a production method in the presence of a protein having the activity of converting the hydroxy group to an aldehyde group, a protein having the activity of converting the aldehyde group to a carboxy group, and a protein having the activity of reversibly converting the aldehyde group and the amino group.

[0200] The production method of embodiment A is a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having an activity of converting an aldehyde group to a carboxy group, １ and R ２ One of them is CH ２ NH ２ and the other is CHO (i.e., Compound 7) as a substrate compound or an intermediate compound, to produce cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0201] In this case, in the general formula (1), R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, the compound (i.e., compound 7) is more preferably a compound generated from any one of the substrate compounds or intermediate compounds shown in (i) and (ii) below: １ and R ２ (ii) a compound in which R １ and R ２ One of them is CH ２ NH ２ and the other is CH ２ OH (i.e., Compound 6)

[0202] The production method of embodiment A is a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having an activity of converting an aldehyde group to a carboxy group and a protein having an activity of reversibly converting an aldehyde group and an amino group, １ and R ２ and R １ and R ２ Both are CH ２ NH ２The present invention is preferably a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the formula (i.e., compound 8) as a substrate compound or an intermediate compound.

[0203] The production method of embodiment A is a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having an activity of converting an aldehyde group to a carboxy group and a protein having an activity of converting a hydroxy group to an aldehyde group, １ and R ２ One of them is CH ２ NH ２ and the other is CH ２ Preferably, the method is a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound in which OH is OH (i.e., compound 6) as a substrate compound or intermediate compound.

[0204] The production method of embodiment A is a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having an activity of converting a hydroxy group to an aldehyde group, a protein having an activity of converting an aldehyde group to a carboxy group, and a protein having an activity of reversibly converting an aldehyde group and an amino group, １ and R ２ One of them is CH ２ OH and the other is CH ２ The method is preferably a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound which is OH or CHO (i.e., Compound 1 or Compound 2) as a substrate compound or intermediate compound.

[0205] In the production method of embodiment A, the protein having the activity of converting an aldehyde group to a carboxy group is preferably at least one of the following 1) to 6): 1) a protein consisting of an amino acid sequence having 71% 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 set forth in SEQ ID NO: 19; 2) a protein consisting of an amino acid sequence having 68% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence set forth in SEQ ID NO: 20; and 3) a protein that is an aldehyde dehydrogenase derived from the genus Psudomonas and has an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence set forth in SEQ ID NO: 20. 4) A protein consisting of an amino acid sequence having 63%, 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 SEQ ID NO:21. 5) A protein consisting of an amino acid sequence having 56.5% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO:40. 6) A protein consisting of an amino acid sequence having 63.9% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO:41.

[0206] In the production method of embodiment A, when the protein having the activity of converting an aldehyde group to a carboxy group is at least one of 1) to 6) above, it is preferable that the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 127 to 131, 133 to 135, 138, and 139.

[0207] In the production method of embodiment A, when the protein having the activity of converting an aldehyde group to a carboxy group is at least one of 1) to 6) above, it is preferable that the protein having the activity of converting an aldehyde group to a carboxy group has an aldehyde dehydrogenase activity of oxidizing the aldehyde group of a compound having an aldehyde group, and is an aldehyde dehydrogenase derived from a bacterium of the genus Pseudomonas.

[0208] In the production method of embodiment A, it is preferred that the protein having the activity of converting a hydroxy group to an aldehyde group is at least one selected from a protein having oxidase activity, a protein having dehydrogenase activity, and a protein having aldehyde reductase activity, and that the protein having the activity of reversibly converting an aldehyde group and an amino group is at least one selected from a protein having aminotransferase activity, a protein having amine dehydrogenase activity, and a protein having amine oxidase activity.

[0209] In the production method of embodiment A, it is preferred that the protein having the activity of converting a hydroxy group to an aldehyde group is a protein having choline oxidase activity, and the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein having aminotransferase activity.

[0210] In the production method of embodiment A, it is preferred that the protein having choline oxidase activity is a protein consisting of an amino acid sequence having 60% or more identity to the amino acid sequence represented by SEQ ID NO: 201, and the protein having aminotransferase activity is a protein 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.

[0211] The manufacturing method of embodiment A is to form a compound represented by the general formula (1) below, １ and R ２ and R are both CHO (i.e., Compound 3), １ and R ２One of them is COOH and the other is CH ２ OH (i.e., Compound 4) or R １ and R ２ One of them is CH ２ NH ２ and the other is CH ２ Preferably, the method is a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using, as a substrate compound or an intermediate compound, any one of the compounds having a hydroxy group in a hydroxy group (i.e., compound 6) as a substrate compound or an intermediate compound, in the presence of at least one enzyme selected from a protein having an activity of converting a hydroxy group to an aldehyde group, a protein having an activity of converting an aldehyde group to a carboxy group, and a protein having an activity of reversibly converting an aldehyde group and an amino group.

[0212] The manufacturing method of embodiment A is to form a compound represented by the general formula (1) below, １ and R ２ In the case where a compound (i.e., Compound 3) in which R are both CHO is used as a substrate compound or an intermediate compound, in the presence of a protein having the activity of converting an aldehyde group to a carboxy group and a protein having the activity of reversibly converting an aldehyde group and an amino group, １ and R ２ One of them is COOH and the other is CH ２ When a compound having a hydroxy group as a substituent (i.e., compound 4) is used as a substrate compound or an intermediate compound, the compound can be produced in the presence of a protein having an activity of converting a hydroxy group to an aldehyde group and a protein having an activity of reversibly converting an aldehyde group and an amino group, or １ and R ２ One of them is CH ２ NH ２ and the other is CH ２When a compound having a hydroxy group as a substituent (i.e., compound 6) is used as a substrate compound or an intermediate compound, the method is preferably a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having an activity of converting a hydroxy group to an aldehyde group and a protein having an activity of converting an aldehyde group to a carboxy group.

[0213] Another embodiment of the production method may be a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, which is a compound represented by the following general formula (1), using the following compound (i) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from a protein having an activity of converting a hydroxy group to an aldehyde group, a protein having an activity of converting an aldehyde group to a carboxy group, and a protein having an activity of reversibly converting an aldehyde group and an amino group: (i) In general formula (1), R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ However, in the general formula (1), R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ are both COOH, and R １ and R ２ One of them is CH ２ NH ２ and the other is CHO (i.e., the compound represented by general formula (1) is compound 7). That is, the compound represented by general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, and 8.

[0214] Another embodiment of the production method may be a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of the amino acid sequence represented by SEQ ID NO:207, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２ and the like are COOH. Specifically, the compound represented by the general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, 7 and 8.

[0215] In another embodiment, the production method is a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from a protein having an activity of converting a hydroxy group to an aldehyde group, a protein having an activity of converting an aldehyde group to a carboxy group, and a protein having an activity of reversibly converting an aldehyde group and an amino group: [In the formula, R １ and R ２ are each independently CH２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ If one of them is COOH, the other is CH ２ Specifically, the compound represented by the general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, 7, and 8.

[0216] The production method of embodiment A or B may be a method for producing cis- and / or trans-isomers of 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from a protein having an activity of converting a hydroxy group to an aldehyde group, a protein having an activity of converting an aldehyde group to a carboxy group, and a protein having an activity of reversibly converting an aldehyde group and an amino group, wherein the protein having an activity of reversibly converting an aldehyde group and an amino group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, and 277, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, and 277: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ When both are COOH, R １ and R ２ Both are CH ２ NH ２ and when the compound represented by general formula (1) is R １ and R２ One of them is CH ２ NH ２ and the other is CHO, and compound 8 (general (1) in R １ and R ２ Both are CH ２ NH ２ Specifically, the compound represented by general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, and 7, excluding the case where compound 7 is an intermediate compound generated from compound 8.

[0217] In this case, the manufacturing method of embodiment A or B is a method of manufacturing a compound having R １ and R ２ One of them is CH ２ OH and the other is CH ２ OH, CHO, or COOH (i.e., compounds 1, 2, and 4), or R １ and R ２ and the other is CHO or COOH (i.e., Compounds 3 and 5) as a substrate compound or an intermediate compound, to produce cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0218] The production method of embodiment A or B may be a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein consisting of the amino acid sequence represented by SEQ ID NO:277 or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO:277: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２ and the like are COOH. Specifically, the compound represented by the general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, 7 and 8.

[0219] The production method is a method for producing a compound represented by the general formula (1) in which R １ and R ２ Both are CH ２ NH ２ In the case where a compound represented by the formula (i.e., compound 8) is used as a substrate compound or an intermediate compound, the production may be carried out in the presence of a protein having the activity of reversibly converting an aldehyde group and an amino group.

[0220] The production method of embodiment A or B may be a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of converting a hydroxy group to an aldehyde group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 201, 213, 221 to 230, 241, and 289 to 294, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by any one of these. [In the formula, R １ and R ２are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ When both are COOH, R １ and R ２ Both are CH ２ NH ２ and when the compound represented by general formula (1) is R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, and compound 8 (in the above general formula (1), R １ and R ２ Both are CH ２ NH ２ Specifically, the compound represented by general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, and 7, excluding the case where compound 7 is an intermediate compound generated from compound 8.

[0221] In this case, the production method of embodiment A or B is a protein having the activity of converting a hydroxy group into an aldehyde group, the protein consisting of an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO: 201 or 213, or an amino acid sequence represented by any one of these, wherein in the general formula (1), R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２When both are COOH, R １ and R ２ Both are CH ２ NH ２ and R １ and R ２ One of them is COOH and the other is CH ２ The present invention may also be a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound (any one of compounds 1, 2, 3, 5, 6, or 7) (except when the compound is OH) as a substrate compound or an intermediate compound.

[0222] The manufacturing method of embodiment A or B is to form a compound represented by the general formula (1) below, １ and R ２ One of them is CH ２ The method may also be a method for producing cis-4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound in which one atom is OH and the other is COOH (i.e., compound 4) as a substrate compound or intermediate compound.

[0223] In this case, the production method of embodiment A or B is a method for producing a protein having the activity of converting a hydroxy group into an aldehyde group, the protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 289 and 290, or an amino acid sequence having 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by any one of these, wherein in the general formula (1), R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ and R １ and R ２ When both are COOH, R １ and R ２ One of them is CH ２ OH and the other is CH ２ NH ２ If R １and R ２ Both are CH ２ NH ２ The present invention may also be a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound (any one of Compounds 1, 2, 3, 4, 5, and 7) as a substrate compound or an intermediate compound (except when

[0224] The manufacturing method of embodiment A or B is to form a compound represented by the general formula (1) below, １ and R ２ and the other is COOH, the method may be a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of reversibly converting an aldehyde group and an amino group, when the compound represented by general formula (1) (i.e., compound 5) is used as a substrate compound or an intermediate compound, wherein one of the aldehyde group and the amino group is CHO and the other is COOH, and the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277.

[0225] The manufacturing method of embodiment A or B is to form a compound represented by the general formula (1) below, １ and R ２ One of them is CH ２ OH and the other is CH ２ NH ２a method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting a hydroxy group to an aldehyde group, and a protein having the activity of converting the aldehyde group to a carboxy group, when a compound represented by general formula (1) (i.e., compound 6) is used as a substrate compound or an intermediate compound, wherein the protein having the activity of converting a hydroxy group to an aldehyde group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 201, 213, 221 to 230, 241, and 291 to 294, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by any one of these, The method may be such that the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequences represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, and 207.

[0226] The manufacturing method of embodiment A or B is to form a compound represented by the general formula (1) below, １ and R ２ One of them is CH ２a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having an activity of converting a hydroxy group to an aldehyde group and a protein having an activity of reversibly converting an aldehyde group and an amino group, when a compound represented by general formula (1) (i.e., compound 4) in which one hydroxy group is OH and the other is COOH is used as a substrate compound or an intermediate compound, the method comprising the steps of: (a) converting a hydroxy group to an aldehyde group into an aldehyde group; (b) converting an aldehyde group to an amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277; or (c) converting an aldehyde group to an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277; The method may be such that the protein having the activity of converting a hydroxy group to an aldehyde group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 213, 221 to 230, 241, and 289 to 294, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by any one of these.

[0227] The manufacturing method of embodiment A or B is to form a compound represented by the general formula (1) below, １ and R ２and (iii) are both CHO (i.e., Compound 3) as a substrate compound or an intermediate compound, a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting an aldehyde group to a carboxy group and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277, The method may be such that the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequences represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, and 207.

[0228] The manufacturing method of embodiment A or B is to form a compound represented by the general formula (1) below, １ and R ２ One of them is CH ２ OH and the other is CH ２A method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, when a compound which is OH or CHO (i.e., Compound 1 or Compound 2) is used as a substrate compound or an intermediate compound, wherein the protein having the activity of reversibly converting an aldehyde group and an amino group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277; The method may be such that the protein having the activity of converting a hydroxy group to an aldehyde group is a protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 201, 213, 221 to 230, 241, and 289 to 294, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to an amino acid sequence represented by any one of these, and the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, or 207, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to an amino acid sequence represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, or 207.

[0229] In the production method of embodiment A or B, when 1,4-cyclohexanedimethanol (compound 1) is used as a substrate compound or an intermediate compound, the proteins having the activity of converting a hydroxy group to an aldehyde group are preferably those represented by SEQ ID NOs: 201, 213, 221 to 230, 241, and 289 to 294, more preferably those represented by SEQ ID NOs: 241, 290, and 293, and most preferably those represented by SEQ ID NO: 241.

[0230] In the production method of embodiment A or B, when 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2) is used as a substrate compound or an intermediate compound, the proteins having the activity of converting a hydroxy group to an aldehyde group are preferably those represented by SEQ ID NOs: 201, 213, 221 to 230, 241, and 289 to 294, more preferably those represented by SEQ ID NOs: 201, 213, and 241, and most preferably those represented by SEQ ID NO: 241.

[0231] In the production method of embodiment A or B, when 4-(hydroxymethyl)cyclohexane-1-carboxylic acid (compound 4) is used as a substrate compound or an intermediate compound, it is preferable to use SEQ ID NOs: 213, 221 to 230, 241, and 289 to 294 as proteins having the activity of converting a hydroxy group to an aldehyde group, with SEQ ID NO: 213 being more preferred. SEQ ID NO: 213 is particularly preferred when producing cis-4-(aminomethyl)cyclohexane-1-carboxylic acid.

[0232] In the production method of embodiment A or B, when [4-(aminomethyl)cyclohexyl]methanol (compound 6) is used as a substrate compound or an intermediate compound, it is preferable to use SEQ ID NOs: 201, 213, 221 to 230, 241, 291 to 294 as a protein having the activity of converting a hydroxy group to an aldehyde group, more preferably SEQ ID NOs: 201, 213, 221 to 225, 228, 230, 291 to 294, and most preferably SEQ ID NO: 201, 221, 222 or 291.

[0233] In the production method of embodiment A or B, when 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2) is used as a substrate compound or an intermediate compound, it is preferable to use SEQ ID NOs: 1 to 4, 103 to 105, and 217 as the protein having the activity of reversibly converting an aldehyde group and an amino group, and SEQ ID NO: 1, 4, or 217 is more preferable.

[0234] In the production method of embodiment A or B, when 1,4-cyclohexanedicarboxaldehyde (compound 3) is used as a substrate compound or an intermediate compound, it is preferable to use SEQ ID NOs: 1 to 4, 103 to 105, and 217 as the protein having the activity of reversibly converting an aldehyde group and an amino group, and SEQ ID NO: 1, 4, or 217 is more preferable.

[0235] In the production method of embodiment A or B, when 4-formylcyclohexane-1-carboxylic acid (compound 5) is used as a substrate compound or an intermediate compound, it is preferable to use SEQ ID NOs: 1 to 4, 103 to 105, and 217 as the protein having the activity of reversibly converting an aldehyde group and an amino group, and SEQ ID NO: 1, 4, or 217 is more preferable.

[0236] In the production method of embodiment A, when 1,4-bis(aminomethyl)cyclohexane (compound 8) is used as a substrate compound or an intermediate compound, it is preferable to use SEQ ID NO: 1 to 4, 103 to 105, 217, or 277 as a protein having the activity of reversibly converting an aldehyde group and an amino group, more preferably SEQ ID NO: 217 or 277, and most preferably SEQ ID NO: 277.

[0237] In the production method of embodiment A or B, when 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2) is used as a substrate compound or an intermediate compound, the protein having the activity of converting an aldehyde group to a carboxy group is preferably SEQ ID NO: 19 to 22, 35 to 46, 127 to 139, or 207, more preferably SEQ ID NO: 19 to 22, 35 to 41, 44 to 46, 127 to 131, 133 to 136, 139, or 207, and most preferably SEQ ID NO: 19, 20, 40, 41, 130, or 131.

[0238] In the production method of embodiment A or B, when 1,4-cyclohexanedicarboxaldehyde (compound 3) is used as a substrate compound or an intermediate compound, the protein having the activity of converting an aldehyde group to a carboxy group is preferably one selected from the group consisting of SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, and 207, more preferably SEQ ID NOs: 19 to 22, 35 to 41, 44 to 46, 127 to 131, 133 to 136, 139, and 207, and most preferably SEQ ID NO: 19 or 20.

[0239] In the production method of embodiment A or B, when 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7) is used as a substrate compound or an intermediate compound, it is preferable to use SEQ ID NO: 19 to 22, 35 to 46, 127 to 139, or 207 as a protein having the activity of converting an aldehyde group to a carboxy group, and SEQ ID NO: 19 to 22, 35 to 41, 44 to 46, 127 to 131, 133 to 136, 139, or 207 is more preferable.

[0240] The production method of embodiment A or B may be a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, wherein the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, or 207, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, or 207: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ When both are COOH, R １ and R ２ Both are CH ２ NH ２ and when the compound represented by general formula (1) is R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, and compound 8 (general (1) in R １ and R ２ Both are CH ２ NH ２ Specifically, the compound represented by general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, and 7, excluding the case where compound 7 is an intermediate compound generated from compound 8.

[0241] In this case, the protein having the activity of converting an aldehyde group to a carboxy group may be a protein consisting of an amino acid sequence represented by SEQ ID NO: 19 to 22, 35 to 46, or 207, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO: 19 to 22, 35 to 46, or 207.

[0242] The production method of embodiment A or B may be a method for producing cis and / or trans 4-(aminomethyl)cyclohexane-1-carboxylic acid using a compound represented by the following general formula (1) as an intermediate compound in the presence of at least one enzyme selected from a protein having the activity of converting a hydroxy group to an aldehyde group, a protein having the activity of converting an aldehyde group to a carboxy group, and a protein having the activity of reversibly converting an aldehyde group and an amino group, except for cases where the protein having the activity of converting an aldehyde group to a carboxy group is a protein consisting of an amino acid sequence represented by any of SEQ ID NOs: 127 to 139, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to an amino acid sequence represented by any of SEQ ID NOs: 127 to 139: [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (where R １ and R ２ One of them is COOH and the other is CH ２ NH ２ If R １ and R ２ When both are COOH, R １ and R ２ Both are CH ２ NH ２ and when the compound represented by general formula (1) is R １ and R ２ One of them is CH ２ NH ２ and the other is CHO, and compound 8 (general (1) in R １ and R ２ Both are CH ２ NH ２ Specifically, the compound represented by general formula (1) is any one of compounds 1, 2, 3, 4, 5, 6, and 7, excluding the case where compound 7 is an intermediate compound generated from compound 8.

[0243] In each enzymatic reaction step in the production method of the present invention, the substrate compound or intermediate compound (e.g., 1,4-bis(aminomethyl)cyclohexane (Compound 8) in step (i) and 1,4-cyclohexanedimethanol (Compound 1) in step (iii)) may be in the trans or cis form, or a mixture thereof, and a mixture of the trans and cis forms is preferred in terms of availability of raw materials. Furthermore, the intermediate compound in each step (e.g., 4-(aminomethyl)cyclohexane-1-carbaldehyde (Compound 7) in step (i), 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (Compound 2) or 4-formylcyclohexane-1-carboxylic acid (Compound 5) in step (iii)) may be in the trans or cis form, or a mixture thereof. When the intermediate compound produced in each step is supplied to the production of tranexamic acid, it is preferably in the trans form. In the mixture of trans and cis forms, the ratio of the trans and cis forms is not important, but the ratio of the trans form is preferably greater than 50%.

[0244] The substrate compounds or intermediate compounds (e.g., 1,4-bis(aminomethyl)cyclohexane (compound 8) in step (i) and 1,4-cyclohexanedimethanol (compound 1) in step (iii)) used in each enzymatic reaction step in the production method of the present invention may be supplied from an ester by an enzymatic or non-enzymatic reaction. Examples of enzymatic reactions include reactions using esterases, and examples of non-enzymatic reactions include acid hydrolysis and saponification (hydrolysis with a base).

[0245] The substrate compound or intermediate compound of the present invention may be used in the form of a salt, such as an acid addition salt, an alkali metal salt, an alkaline earth metal salt, an ammonium salt, or an amine salt.

[0246] Acid addition salts include, for example, inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, nitrate, or organic acid salts such as acetate, lactate, tartrate, benzoate, citrate, methanesulfonate, ethanesulfonate, trifluoroacetate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate, or gluconate.

[0247] Examples of alkali metal salts include potassium salts and sodium salts.

[0248] Alkaline earth metal salts include, for example, calcium salts and magnesium salts.

[0249] Examples of ammonium salts include tetramethylammonium salts.

[0250] Examples of amine salts include triethylamine salts, methylamine salts, dimethylamine salts, cyclopentylamine salts, benzylamine salts, phenethylamine salts, piperidine salts, monoethanolamine salts, diethanolamine salts, tris(hydroxymethyl)aminomethane salts, lysine salts, arginine salts, and N-methyl-D-glucamine salts.

[0251] In the production method of the present invention, when the substrate compound in each enzymatic reaction step, for example, 1,4-bis(aminomethyl)cyclohexane (Compound 8) in step (i) or 4-(aminomethyl)cyclohexane-1-carbaldehyde (Compound 7) in step (ii) has a different stereostructure from, for example, 4-(aminomethyl)cyclohexane-1-carbaldehyde (Compound 7) in step (i) or 4-(aminomethyl)cyclohexane-1-carboxylic acid (target compound) in step (ii), it is preferable to carry out part or all of the production method of this embodiment under neutral or basic conditions. For example, it is preferable to carry out part or all of step (i) and / or part or all of step (ii) 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 (Compound 7), 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 (compound 7) reaches equilibrium at a trans ratio of approximately 60%. For example, when trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (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 for the production of tranexamic acid in high yield. On the other hand, when the trans isomer is used as the substrate, isomerization can be prevented by adjusting the temperature to an acidic range (e.g., pH 4 to 6), thereby enabling the production of a larger amount of the trans product.

[0252] 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.

[0253] When the substrate compound in each enzymatic reaction step of the production method of the present invention differs in conformation from the intermediate compound or target compound, 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, e.g., 60% or more, or a trans-to-cis isomerization rate of 20% or more, e.g., 30% or more or 35% or more.

[0254] In each enzymatic reaction step in the production method of the present invention, an enzyme that catalyzes the enzymatic reaction in that step (enzyme A, enzyme B, and / or enzyme C of the present invention) may be used as an enzyme source, and the enzyme source and substrate compound may be present in an aqueous medium, and an intermediate compound or target compound may be produced and accumulated in the aqueous medium. Alternatively, cells capable of producing the enzyme that catalyzes the enzymatic reaction in that step (enzyme A, enzyme B, and / or enzyme C of the present invention) may be cultured in a medium, and the intermediate compound or target compound may be produced and accumulated in the culture.

[0255] 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.

[0256] 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.

[0257] When a purified protein is used as the enzyme source in each step, the amount of the enzyme (enzyme A, enzyme B, and / or enzyme C of the present invention) catalyzing the enzymatic reaction in that step may be 0.01 to 100% by weight, preferably 0.1 to 50% by weight, relative to the substrate compound or intermediate compound. 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 the substrate compound or intermediate compound.

[0258] Examples of aqueous media include buffer solutions such as water, phosphate, carbonate, acetate, borate, citrate, Tris, 2-morpholinoethanesulfonic acid (hereinafter referred to as MES), 3-morpholinopropanesulfonic acid (hereinafter referred to as MOPS), N-cyclohexyl-2-aminoethanesulfonic acid (hereinafter referred to as CHES), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (hereinafter referred to as HEPES), alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, and amides such as acetamide. Furthermore, the culture medium of the microorganism used as the enzyme source can also be used as the aqueous medium.

[0259] In the enzymatic reaction step (e.g., step (i)) catalyzed by enzyme B, as described in 1. above, in the enzymatic reaction of converting a compound having an amino group to a compound having an aldehyde group in the presence of a protein having aminotransferase activity, which is one type of enzyme B, for example, the enzymatic reaction of converting a compound having an amino group such as 1,4-bis(aminomethyl)cyclohexane (compound 8) to a compound having an aldehyde group such as 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7), it is desirable to use a compound capable of accepting an amino group in the coexistence. Furthermore, as described in 1. above, in the enzymatic reaction of converting a compound having an aldehyde group to a compound having an amino group in the presence of enzyme B, for example, the reaction of converting compound 3 to compound 7, it is desirable to use a compound capable of donating an amino group in the coexistence.

[0260] Compounds capable of accepting an amino group include keto acids, preferably pyruvic acid, α-ketoglutaric acid, α-ketobutyric acid, etc. Compounds capable of donating an amino group include amino acids, preferably alanine, glutamic acid, α-aminobutyric acid, isopropylamine, etc.

[0261] The origin of the keto acid is not important as long as it can accept an amino group, but 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 the processed product of the culture. The origin of the amino acid is not important as long as it can donate an amino group, but a culture of a microorganism capable of producing an amino acid or a processed product of the culture may be used as is, or an amino acid recovered from the culture or the processed product of the culture.

[0262] Alternatively, the keto acid may be produced in the reaction system rather than added. For example, when culturing a microorganism in a medium to produce and accumulate an intermediate compound or a target compound (e.g., when producing and accumulating compound 7 from compound 8), a microorganism capable of producing a keto acid may be used. 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 or enzyme activity of a target gene (e.g., the gene for the pyruvate dehydrogenase complex).

[0263] Alternatively, amino acids may be produced in the reaction system rather than added. For example, when a microorganism is cultured in a medium to produce and accumulate an intermediate compound or a target compound (e.g., when producing and accumulating compound 6 from compound 2), a microorganism capable of producing an amino acid may be used. Examples of microorganisms capable of producing an amino acid include microorganisms in which the gene for glutamic acid decarboxylase involved in glutamic acid degradation in vivo has been deleted or weakened, and microorganisms in which a gene involved in glutamic acid biosynthesis has been enhanced. Here, "weakening" means reducing the expression level of a target gene or the activity of an enzyme, and "enhancing" means increasing the expression level of a target gene or the activity of an enzyme.

[0264] When a keto acid or amino acid is added in the enzymatic reaction step catalyzed by enzyme B, the amount of the keto acid or amino acid added may be, for example, a molar ratio of 0.01 to 100, preferably 0.01 to 60, relative to the substrate compound (e.g., compound 8 or compound 2).

[0265] The production method of this embodiment includes the coexistence of alanine dehydrogenase, glutamate dehydrogenase, and / or NAD(P)H oxidase in part or all of the method. In particular, when production is carried out in the presence of an aminotransferase as enzyme B (for example, in the first step (step (i)) or in the step of producing the target compound 4-aminomethylcyclohexane-1-carboxylic acid from 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2)), it is preferable to have alanine dehydrogenase, glutamate dehydrogenase, and / or NADH oxidase coexist. When production is carried out in the presence of an aldehyde dehydrogenase as enzyme C (for example, in the second step (step (ii)) or in the step of producing the target compound (4-aminomethylcyclohexane-1-carboxylic acid) from 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (compound 2)), it is preferable to have alanine dehydrogenase, glutamate dehydrogenase, and / or NADH oxidase coexist. In the case of producing the target compound (4-aminomethylcyclohexane-1-carboxylic acid) from 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (Compound 2) in the presence of amine dehydrogenase as Enzyme B (for example, the first step (Step (i)) or the step of producing the target compound (4-aminomethylcyclohexane-1-carboxylic acid) from 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (Compound 2)), and / or in the case of producing the target compound in the presence of dehydrogenase and / or aldehyde reductase as Enzyme A (for example, Step (iii) or the step of producing the target compound (4-aminomethylcyclohexane-1-carboxylic acid) from 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (Compound 2)), it is preferable to use NADH oxidase in the presence of amine dehydrogenase as Enzyme B (for example, the first step (Step (i)) or the step of producing the target compound (4-aminomethylcyclohexane-1-carboxylic acid) from 4-(hydroxymethyl)cyclohexane-1-carbaldehyde (Compound 2)).

[0266] In the enzymatic reaction step catalyzed by a protein having aminotransferase activity as enzyme B, when pyruvic acid is used as the keto acid or alanine is used as the amino acid, it is preferable to use alanine dehydrogenase (AlaDH) in the presence of the enzyme. ＋ AlaDH is an enzyme that can catalyze the conversion (regeneration) of the produced alanine to pyruvic acid or the conversion (regeneration) of the produced pyruvic acid to alanine, using AlaDH as a coenzyme. Therefore, by allowing AlaDH to coexist, pyruvic acid or alanine is reused, and the transamination reaction can proceed efficiently with a small amount of raw material. In addition, when AlaDH is coexisted, the NAD required for the reaction can be reduced. ＋ Similarly, when glutamic acid is used as the amino acid, it is preferable to add glutamate dehydrogenase (GDH). ＋ as a coenzyme and can catalyze the conversion (regeneration) of the produced α-ketoglutaric acid to glutamic acid or the conversion (regeneration) of the produced glutamic acid to α-ketoglutaric acid.

[0267] 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 method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid of the present invention, alanine produced when pyruvic acid is used as the keto acid can be converted to pyruvic acid. In addition, in the method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid of the present invention, pyruvic acid produced when alanine is used as the amino acid can be converted to alanine. Similarly, GDH is an oxidoreductase that catalyzes a reversible reaction between (glutamic acid + water + NAD ＋ ) and (α-ketoglutaric acid + NH ３ +NADH + H ＋) is an oxidoreductase that catalyzes a reversible reaction between

[0268] 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).

[0269] The GDH is not particularly limited, and examples thereof include glutamate dehydrogenase derived from Escherichia coli, glutamate dehydrogenase derived from bacteria of the genus Bacillus (e.g., glutamate dehydrogenase derived from Bacillus subtilis), glutamate dehydrogenase derived from bacteria of the genus Pseudomonas (e.g., glutamate dehydrogenase derived from Pseudomonas aeruginosa), glutamate dehydrogenase derived from bacteria of the genus Clostridium, glutamate dehydrogenase derived from fungi of the genus Aspergillus, glutamate dehydrogenase derived from Peptoniphilus asaccharolyticus, and glutamate dehydrogenase derived from Saccharomyces cerevisiae.

[0270] The coexistence of AlaDH or GDH may mean that in a reaction using an aminotransferase as enzyme B, a culture of cells expressing AlaDH or GDH or a processed product of the culture is added, or that cells expressing AlaDH or GDH are co-cultured, or that DNA encoding AlaDH or GDH is contained in the recombinant cells.

[0271] The amount of AlaDH or GDH may be 0.01 to 100% by weight, preferably 0.1 to 50% by weight, relative to the alanine, pyruvic acid, glutamic acid, or α-ketoglutaric acid produced in 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 5 to 1000% by weight, preferably 10 to 400% by weight, in terms of wet cell weight, relative to the substrate compound or intermediate compound (e.g., 1,4-bis(aminomethyl)cyclohexane (compound 8)). ＋ It is desirable to add NAD ＋ The amount of added may be in the range of 0.01 to 100, preferably 0.01 to 60, molar ratio relative to alanine, pyruvic acid, glutamic acid or α-ketoglutaric acid produced in the transamination reaction.

[0272] In the enzymatic reaction step catalyzed by enzyme C (for example, step (ii)), the enzymatic reaction step catalyzed by enzyme B (for example, step (i)), or the enzymatic reaction step catalyzed by enzyme A (for example, step (iii)), the above-mentioned 1. As described above, in the enzymatic reaction of converting a compound having an aldehyde group (e.g., 4-(aminomethyl)cyclohexane-1-carbaldehyde (compound 7)) to a compound having a carboxy group (e.g., 4-(aminomethyl)cyclohexane-1-carboxylic acid (target compound)) in the presence of a protein having aldehyde dehydrogenase activity, which is one of the enzymes C, the enzymatic reaction of converting a compound having a hydroxy group (e.g., compound 1) to a compound having an aldehyde group (e.g., compound 2) in the presence of a protein having dehydrogenase activity and / or a protein having aldehyde reductase activity, which is one of the enzymes A, and / or the enzymatic reaction of converting a compound having an amino group (e.g., compound 8) to a compound having an aldehyde group (e.g., compound 7) in the presence of a protein having amine dehydrogenase activity, which is one of the enzymes B, the coenzyme NAD(P) ＋The protein having aldehyde dehydrogenase activity among enzymes C, the protein having dehydrogenase activity and the protein having aldehyde reductase activity among enzymes A, and the protein having amine dehydrogenase activity among enzymes B require NAD as a coenzyme. ＋ NAD prefers ＋ Type, NADP ＋ NADP prefers ＋ type, NAD ＋ and NADP ＋ Therefore, when using any of the enzymes C, which are proteins having aldehyde dehydrogenase activity, the enzymes A, which are proteins having dehydrogenase activity and aldehyde reductase activity, and the enzymes B, which are proteins having amine dehydrogenase activity, in the production method of the present invention, it is preferable to use a coenzyme that is highly available to the protein in the presence of the coenzyme.

[0273] NAD(P) ＋ The amount of may be a molar ratio of 0.01 to 100, preferably 0.01 to 60, relative to the substrate compound or intermediate compound (for example, compound 1, 7 or 8).

[0274] Furthermore, in the production method of the present invention, when a protein having aldehyde dehydrogenase activity is used as enzyme C, a protein having dehydrogenase activity or a protein having aldehyde reductase activity is used as enzyme A, and / or a protein having amine dehydrogenase activity is used as enzyme B, a method for supplying a coenzyme can be used in which a microorganism capable of producing a coenzyme is used as a host for expressing the above protein. This is expected to further improve the production amount of the desired 4-(aminomethyl)cyclohexane-1-carboxylic acid. Examples of a microorganism capable of producing a coenzyme include, for example, a microorganism capable of producing NAD(P) in vivo. ＋ Microorganisms in which genes involved in degradation (e.g., mazG, nudC) are deleted, deleted, or weakened, ＋A microorganism in which a gene involved in the production (e.g., pncB, nadA, nadB, nadE, or nadD) has been enhanced or introduced, the above-mentioned NAD(P) ＋ Examples of such a microorganism include a microorganism in which a gene that suppresses the expression or function of a gene product involved in the production (for example, nadR) is deleted, eliminated, or weakened.

[0275] The amount of the enzyme C protein having aldehyde dehydrogenase activity, the enzyme A protein having dehydrogenase activity, the enzyme B protein having aldehyde reductase activity, and / or the enzyme B protein having amine dehydrogenase activity may be 0.01 to 100% by weight, preferably 0.1 to 50% by weight, relative to the substrate compound or intermediate compound (e.g., compound 1, 7, or 8). 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, 5 to 1000% by weight, preferably 10 to 400% by weight, in terms of wet cell weight, relative to the substrate compound or intermediate compound (e.g., compound 1, 7, or 8).

[0276] When a protein having dehydrogenase or aldehyde reductase activity, which is one of the enzymes A, a protein having amine dehydrogenase activity, which is one of the enzymes B, a protein having aldehyde dehydrogenase activity, which is one of the enzymes C, and / or a protein having aminotransferase activity is used as the enzyme B, it is preferable to use NAD(P)H oxidase (NOX) in the presence of alanine dehydrogenase or glutamate dehydrogenase in the step of carrying out the step.

[0277] 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 ２In the production method of the present invention, when a protein having dehydrogenase activity or a protein having aldehyde reductase activity, which is one of the enzymes A, a protein having amine dehydrogenase activity, which is one of the enzymes B, a protein having aldehyde dehydrogenase activity, which is one of the enzymes C, or / and a protein having aminotransferase activity is used as enzyme B, the NAD(P)H produced in the step carried out in the presence of alanine dehydrogenase or glutamate dehydrogenase is converted into NAD(P). ＋ Therefore, in this step, by allowing NAD(P)H oxidase to coexist, NAD(P) ＋ is recycled, and 4-(aminomethyl)cyclohexane-1-carboxylic acid can be produced efficiently using a small amount of raw materials.

[0278] 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).

[0279] 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.

[0280] The amount of NAD(P)H oxidase may be 0.001 to 100% by weight, and preferably 0.01 to 50% by weight, of the total amount of NAD(P)H produced when using a protein having alanine dehydrogenase activity, a protein having glutamate dehydrogenase activity, a protein having dehydrogenase activity among enzymes A, a protein having amine dehydrogenase activity among enzymes B, or a protein having aldehyde dehydrogenase activity among enzymes C. 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, and preferably 1 to 400% by weight, of the wet cell weight of the substrate compound or intermediate compound (e.g., compound 1 or 8).

[0281] Instead of using NAD(P)H oxidase, NAD(P)H is electrically oxidized to NAD(P). ＋ It can also be played back.

[0282] When the substrate compound and the intermediate compound or the target compound have different steric structures, it is preferable that part or all of the process is carried out in a neutral or basic environment, and further, it is preferable that part or all of the process is carried out in the presence of a secondary amine. When the substrate compound and the intermediate compound or the target compound have the same steric structure, it is desirable that part or all of the process is carried out in an acidic environment.

[0283] The target compound, 4-(aminomethyl)cyclohexane-1-carboxylic acid, may be in the cis form, the trans form, or a mixture thereof. When the target is the cis form (i.e., cis-tranexamic acid), it is preferable to use, as the protein having aldehyde dehydrogenase activity, an enzyme capable of accepting a cis substrate, as shown in one embodiment, any one 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 the trans form (i.e., tranexamic acid), it is preferable to use, as the protein having aldehyde dehydrogenase activity, an enzyme with high trans selectivity, as shown in one embodiment, any one of SEQ ID NOS: 20, 38, 40, 41, 127, 128, 129, 130, 131, 133, 139, and 207, 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, 133, and 207, 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.

[0284] As used herein, the term "highly trans-selective enzyme (ALDH)" refers to an enzyme that produces 4-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA; target compound) in a trans isomer ratio of greater than 50% (e.g., 51% or more, 55% or more, 60% or more, 65% or more, 70% or more) by a two-step reaction using a mixture of cis and trans isomers of 1,4-bis(aminomethyl)cyclohexane (compound 8), 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 in trans to the total amount of AMCHA produced in trans and cis by 100. These highly trans-specific enzymes (aldehyde dehydrogenases) can produce tranexamic acid in high yields and are therefore beneficial in the production of tranexamic acid.

[0285] Each step 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.

[0286] 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.

[0287] In the method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid by fermentation, it is desirable to add a substrate compound or an intermediate compound (e.g., 1,4-bis(aminomethyl)cyclohexane (compound 8) or 1,4-cyclohexanedimethanol (compound 1)) to the medium. The substrate compound or intermediate compound (e.g., 1,4-bis(aminomethyl)cyclohexane (compound 8) or 1,4-cyclohexanedimethanol (compound 1)) may be added to the medium before culture, or may be added to the culture solution during culture.

[0288] Alternatively, instead of adding a substrate compound or an intermediate compound (e.g., 1,4-bis(aminomethyl)cyclohexane (compound 8) or 1,4-cyclohexanedimethanol (compound 1)) to the medium, the substrate compound or intermediate compound (e.g., 1,4-bis(aminomethyl)cyclohexane (compound 8) or 1,4-cyclohexanedimethanol (compound 1)) may be supplied by co-culturing a microorganism capable of producing the substrate compound or intermediate compound (e.g., 1,4-bis(aminomethyl)cyclohexane (compound 8) or 1,4-cyclohexanedimethanol (compound 1)) with the above-mentioned cells.

[0289] 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.

[0290] 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.

[0291] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0292] In a method for producing 4-(aminomethyl)cyclohexane-1-carboxylic acid by fermentation, pyruvic acid, α-ketoglutaric acid, α-ketobutyric acid, alanine, glutamic acid, α-aminobutyric acid, isopropylamine, NAD, etc., which are substrates for a protein having dehydrogenase activity or a protein having aldehyde reductase activity, which is one of enzymes A, a protein having aminotransferase activity or a protein having amine dehydrogenase activity, which is one of enzymes B, or a protein having aldehyde dehydrogenase activity, which is one of enzymes C, are used. ＋ , 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.

[0293] 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.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] By the above-mentioned cultivation, an intermediate compound or a target compound (e.g., 4-(aminomethyl)cyclohexane-1-carboxylic acid) can be produced in the culture or in the cells, thereby producing an intermediate compound or a target compound (e.g., 4-(aminomethyl)cyclohexane-1-carboxylic acid), preferably tranexamic acid. Quantitation of 4-(aminomethyl)cyclohexane-1-carboxylic acid can be carried out using LCMS (e.g., an analytical apparatus LCMS-8040 manufactured by Shimadzu Corporation).

[0298] The intermediate compound or the target compound (e.g., 4-(aminomethyl)cyclohexane-1-carboxylic acid) can be collected from the culture by combining an ion exchange resin method, a precipitation method, or other known methods. When the intermediate compound or the target compound (e.g., 4-(aminomethyl)cyclohexane-1-carboxylic acid)) accumulates in the cells, the cells can be disrupted by, for example, ultrasonication, and centrifuged to remove the cells. The resulting supernatant can then be collected by an ion exchange resin method or the like.

[0299] [Analytical Examples] 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, and [4-(aminomethyl)cyclohexyl]methanol were carried out according to the procedures shown below.

[0300] 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, 4-(aminomethyl)cyclohexane-1-carboxylic acid, or [4-(aminomethyl)cyclohexyl]methanol 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.

[0301] [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 ([4-(aminomethyl)cyclohexyl]methanol) 144.25 > 67.05 m / z

[0302] The following examples are provided in detail, but the present invention is not limited to these examples.

[0303] Example 1 Search for proteins (AT) with aminotransferase activity for 1,4-bis(aminomethyl)cyclohexane (1) Selection of AT to be evaluated Until now, no enzymes with aminotransferase activity that transfer the amino group of 1,4-bis(aminomethyl)cyclohexane to produce 4-(aminomethyl)cyclohexane-1-carbaldehyde were known. To search for an enzyme with aminotransferase 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 aminotransferase 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 aminotransferase activity toward putrescine and cadaverine, which, like 1,4-bis(aminomethyl)cyclohexane, have two amino groups per molecule.

[0304] (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.

[0305] Using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 9 and 10 as a primer set and plasmid pQE80L (manufactured by QIAGEN) containing a T5 promoter and an N-terminal His tag sequence as a template, a vector fragment of approximately 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.

[0306] 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.

[0307] (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.

[0308] (4) Evaluation of aminotransferase 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.

[0309] 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.

[0310] 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 aminotransferase activity toward 1,4-bis(aminomethyl)cyclohexane.

[0311] <Evaluation of aminotransferase 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.

[0312] (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.

[0313] 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.

[0314] (7) Evaluation of aminotransferase 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).

[0315] 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.

[0316] After the reaction was completed, the reaction product was analyzed in the same manner as in (4). The results are shown in Table 5.

[0317] PcAT, PaAT, and HeAT1 were confirmed to have aminotransferase 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] 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.

[0323] 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.

[0324] (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 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.

[0325] 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.

[0326] 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%.

[0327] 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.

[0328] 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.

[0329] 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.

[0330] Example 3 Evaluation of the Activity of Proteins Having Aldehyde Dehydrogenase Activity (ALDH) (1) Selection of ALDH to be Evaluated Until now, no enzymes having aldehyde dehydrogenase activity that can oxidize the aldehyde group of 4-(aminomethyl)cyclohexane-1-carbaldehyde to produce 4-(aminomethyl)cyclohexanecarboxylic acid have been known.

[0331] 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).

[0332] (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.

[0333] 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.

[0334] 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.

[0335] 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.

[0336] 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.

[0337] 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.

[0338] (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.

[0339] (4) Evaluation of productivity of 4-(aminomethyl)cyclohexane-1-carboxylic acid by ALDH in combination with AT <NAD as a coenzyme for ALDH ＋ In the case of using 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] Next, Table 10 shows the results when only the trans isomer of 1,4-bis(aminomethyl)cyclohexane was used as the substrate.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] 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.

[0350] <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.

[0351] 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.

[0352] After the reaction was completed, the reaction product was analyzed in the same manner as described above. The results are shown in Table 12.

[0353] 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.

[0354] 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.

[0355] (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.

[0356] 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.

[0357] 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.

[0358] 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.

[0359] 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.

[0360] (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.

[0361] 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.

[0362] 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.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain BL21(DE3) / pQE80L-BsAlaDH.

[0367] (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.

[0368] 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.

[0369] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain BL21(DE3) / pQE80L-BsNOX.

[0370] (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.

[0371] (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.

[0372] 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.

[0373] 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.

[0374] 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.

[0375] 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.

[0376] 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.

[0377] 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.

[0378] 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).

[0379] 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.

[0380] 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).

[0381] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain the BL21(DE3) / pET28a-PatA-XylC strain.

[0382] 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.

[0383] 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.

[0384] 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.

[0385] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain the BL21(DE3) / pET28a-XylC-PatA strain.

[0386] 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.

[0387] 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).

[0388] 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.

[0389] 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.

[0390] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain the BL21(DE3) / pUC19-PatA-XylC strain.

[0391] 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.

[0392] 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.

[0393] 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.

[0394] Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain the BL21(DE3) / pUC19-XylC-PatA strain.

[0395] (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.

[0396] 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.

[0397] 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.

[0398] 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.

[0399] 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.

[0400] 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.

[0401] 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.

[0402] 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.

[0403] 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.

[0404] 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 lacking ampicillin. The culture was inoculated into a large test tube containing 4 mL of LB medium containing 30 mg / L kanamycin 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 mM final concentration of 1 g / L 1,4-bis(aminomethyl)cyclohexane cis- and trans-isomer mixture (trans ratio 45.7%) were added, and the culture was continued at 37°C for an additional 24 hours.

[0405] 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.

[0406] From Table 19, it was confirmed that TXA can be produced even by the supplemented culture method.

[0407] 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.

[0408] 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.

[0409] (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.

[0410] 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.

[0411] 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.

[0412] (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.

[0413] 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.

[0414] 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.

[0415] 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.

[0416] 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.

[0417] Using a mixture of the XylC fragment and the PatA fragment obtained above as a template, a PCR r...

Claims

1. A method for producing cis- and / or trans-4-(aminomethyl)cyclohexane-1-carboxylic acid in the presence of at least one enzyme selected from a protein having an activity of converting a hydroxy group into an aldehyde group, a protein having an activity of converting an aldehyde group into a carboxy group, and a protein having an activity of reversibly converting an aldehyde group and an amino group, using a compound represented by the following general formula (1) as a substrate compound or an intermediate compound. [In the formula, R １ and R ２ are each independently CH ２ OH, CHO, COOH, or CH ２ NH ２ (provided that when one of R １ and R ２ is COOH and the other is CH ２ NH ２ , when both R １ and R ２ are COOH, when both R １ and R ２ are CH ２ NH ２ , and when the compound represented by the general formula (1) is an intermediate compound generated from a compound in which one of R １ and R ２ is CH ２ NH ２ and the other is CHO, and both R １ and R ２ are CH ２ NH ２ are excluded).] 2. In the general formula (1), R １ and R ２ wherein one of them is CH ２ NH ２ and the other is CHO, the compound is a compound generated from any one of the following substrate compounds or intermediate compounds (i) and (ii). The method according to claim 1. (i) A compound in which both R １ and R ２ are CHO (ii) A compound in which one of R １ and R ２ is CH ２ NH ２ and the other is CH ２ OH 3. The method according to claim 1, which is produced in the presence of a protein having an activity of converting the hydroxy group into an aldehyde group, a protein having an activity of converting the aldehyde group into a carboxy group, and a protein having an activity of reversibly converting the aldehyde group and an amino group.

4. R １ and R ２ wherein one of them is CH ２ OH and the other is CH ２ OH, CHO, or CH ２ NH ２ When the compound represented by the general formula (1) is used as a substrate compound or an intermediate compound, the method according to claim 3, which is produced in the presence of a protein having an activity of converting the hydroxy group into an aldehyde group, a protein having an activity of converting the aldehyde group into a carboxy group, and a protein having an activity of reversibly converting the aldehyde group and an amino group.

5. R １ and R ２ wherein one of them is CH ２ NH ２ and the other is CHO, and when the compound represented by the general formula (1) is used as a substrate compound or an intermediate compound, the method according to claim 1, which is produced in the presence of a protein having an activity of converting the aldehyde group into a carboxy group.

6. R １ and R ２ When a compound represented by the general formula (1), in which one of them is CHO and the other is COOH, is used as a substrate compound or an intermediate compound, it is produced in the presence of a protein having an activity of reversibly converting an aldehyde group and an amino group. The method according to claim 1.

7. R １ and R ２ wherein one of them is CHO and the other is CH ２ NH ２ , or CHO, or R １ and R ２ are both CH ２ NH ２ When the compound represented by the general formula (1) is used as a substrate compound or an intermediate compound, the method according to claim 1, which is produced in the presence of a protein having an activity of converting the aldehyde group into a carboxy group and a protein having an activity of reversibly converting the aldehyde group and an amino group.

8. R １ and R ２ wherein one of them is CH ２ OH and the other is CH ２ NH ２ When the compound represented by the general formula (1) in which one of them is CH OH and the other is CH NH is used as a substrate compound or an intermediate compound, the method according to claim 1, which is produced in the presence of a protein having an activity of converting the hydroxy group into an aldehyde group and a protein having an activity of converting the aldehyde group into a carboxy group.

9. R １ and R ２ wherein one of them is CH ２ OH and the other is COOH, when the compound represented by the general formula (1) is used as a substrate compound or an intermediate compound, the method according to claim 1, which is produced in the presence of a protein having an activity of converting the hydroxy group into an aldehyde group and a protein having an activity of reversibly converting the aldehyde group and an amino group.

10. The protein having an activity of converting the hydroxy group into an aldehyde group is at least one selected from a protein having oxidase activity, a protein having dehydrogenase activity, and a protein having aldehyde reductase activity; the protein having an activity of converting the aldehyde group into a carboxy group is a protein having aldehyde dehydrogenase activity and / or a protein having aldehyde oxidase activity; and the protein having an activity of reversibly converting the aldehyde group and an amino group is at least one selected from a protein having aminotransferase activity, a protein having amine dehydrogenase activity, and a protein having amine oxidase activity. The method according to any one of claims 1 to 9.

11. The protein having an activity of converting the hydroxy group into an aldehyde group is a protein having choline oxidase activity; the protein having an activity of converting the aldehyde group into a carboxy group is a protein having aldehyde dehydrogenase activity; and the protein having an activity of reversibly converting the aldehyde group and an amino group is a protein having aminotransferase activity. The method according to any one of claims 1 to 9.

12. The protein having an activity of converting the hydroxy group into an aldehyde group is a protein consisting of an amino acid sequence having 60% or more identity with the amino acid sequences represented by SEQ ID NOs: 201, 213, 221 to 230, 241, 289 to 294, and the protein having an activity of converting the aldehyde group into a carboxy group is a protein consisting of an amino acid sequence having 50% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, 35 to 46, 127 to 139, and 207, and the protein having an activity of reversibly converting the aldehyde group and an amino group is a protein consisting of an amino acid sequence having 60% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, 103 to 105, 217, or 277. The method according to claim 10.

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