Method for producing methyl compound

Abstract

The purpose of the present invention is to provide a method for producing a methyl compound. Provided is a method for producing a methyl compound using formic acid.

Description

Technical Field

[0001] The present invention relates to a method for producing a methyl compound using formic acid and a method for regenerating S-adenosylmethionine. Background Art

[0002] A variety of methyltransferases are known in nature, performing position- and stereo-selective methylation on various compounds. Most of these methyltransferases use S-adenosylmethionine (SAM) as a methyl donor to transfer a methyl group to the target compound.

[0003] On the other hand, the supply of SAM, which serves as a methyl group donor, is limited within cells and is easily depleted.

[0004] In this regard, a method for recycling SAM by adding methanol to the culture medium of a microorganism that co-expresses a methylase and a methanol dehydrogenase is known (Patent Document 1: International Publication No. 2019 / 160059). This document describes modifying a microorganism to increase the amount of methionine or SAM. Specifically, as methods for reducing formaldehyde decomposition activity, methods are described for reducing the activity of formaldehyde dehydrogenase, which directly converts formaldehyde to formic acid, and for reducing the activity of an enzyme that converts formaldehyde to formic acid by binding it to glutathione, that is, a method for inhibiting the conversion of formaldehyde to formic acid.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2019 / 160059 Summary of the Invention

[0008] Problems to be solved by the invention

[0009] The method described in Patent Document 1 establishes a recycling pathway for S-adenosylmethionine, which supplies methyl carbons through the added methanol, but the effect of promoting the methylation reaction is insufficient. There is a need for a new method that can more efficiently regenerate SAM and promote the methylation reaction of the compound. Furthermore, there is a need for a new method for producing a methyl compound.

[0010] Means for solving problems

[0011] The present inventors succeeded in efficiently regenerating SAM and producing a methyl compound by using formic acid or a salt thereof (hereinafter, sometimes simply referred to as "formic acid"), thereby completing the present invention.

[0012] That is, the present invention is as follows.

[0013] [1-1]

[0014] A method for producing a methyl compound, comprising:

[0015] In the presence of formic acid or its salt,

[0016] (a) a process for producing 5-methyltetrahydrofolate from formic acid or a salt thereof and tetrahydrofolate;

[0017] (b) a step of converting S-adenosylhomocysteine generated by demethylating S-adenosylmethionine into homocysteine;

[0018] (c) a step of generating methionine by transferring a methyl group of 5-methyltetrahydrofolate to homocysteine, and converting the methionine into S-adenosylmethionine; and

[0019] (d) A step of performing a methylation reaction using S-adenosylmethionine as a methyl donor to produce a methyl compound.

[0020] [1-2]

[0021] The method according to the above-mentioned [1-1], wherein in step (a), the production of 5-methyltetrahydrofolate from formate and tetrahydrofolate is carried out in the presence of formate-tetrahydrofolate ligase (FtfL), methenyl-tetrahydrofolate cyclohydrolase (Fch), methylene-tetrahydrofolate dehydrogenase (MtdA) and 5,10-methylene-tetrahydrofolate reductase (MetF).

[0022] [1-3]

[0023] The method according to the above-mentioned [1-1] or [1-2], wherein, in the above-mentioned step (b), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine nucleosidase (Mtn) and S-ribosylhomocysteine lyase (LuxS).

[0024] [1-4]

[0025] The method according to any one of [1-1] to [1-3] above, wherein in the step (b), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine hydrolase (SahH).

[0026] [1-5]

[0027] The method according to any one of [1-1] to [1-4] above, wherein in the step (b), demethylation of S-adenosylhomocysteine is performed in the presence of an S-adenosylmethionine-dependent methyltransferase.

[0028] [1-6]

[0029] The method according to any one of [1-1] to [1-5] above, wherein, in the aforementioned step (c), the production of methionine from homocysteine is carried out in the presence of methionine synthetase (MetH or MetE), and the conversion of the methionine to S-adenosylmethionine is carried out in the presence of methionine adenosyltransferase (MetK).

[0030] [1-7]

[0031] The method according to any one of [1-1] to [1-6], further comprising: (e) converting methanol into formaldehyde, and converting the formaldehyde into formic acid.

[0032] [1-8]

[0033] The method according to the above-mentioned [1-7], wherein in the step (e), the conversion of methanol to formaldehyde is carried out in the presence of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox).

[0034] [1-9]

[0035] The method according to [1-7] or [1-8] above, wherein in the step (e), the conversion of formaldehyde to formic acid is carried out in the presence of formaldehyde dehydrogenase (FdhA).

[0036] [1-10]

[0037] The method according to any one of [1-7] to [1-9] above, wherein in the step (e), the conversion of formaldehyde to formic acid is carried out in the presence of a spontaneous reaction between formaldehyde and glutathione, S-hydroxymethylglutathione dehydrogenase (FrmA) and S-formylglutathione hydrolase (FrmB).

[0038] [1-11]

[0039] The method according to any one of [1-1] to [1-10] above, wherein cells modified so as to enhance the activity or expression of S-adenosylmethionine-dependent methyltransferase are used.

[0040] [1-12]

[0041] The method according to any one of [1-1] to [1-11] above, wherein the cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0042] [1-13]

[0043] The method according to any one of [1-1] to [1-12] above, wherein the methyl compound is a derivative of an aromatic amino acid.

[0044] [1-14]

[0045] A method for producing an organic compound, comprising the step of using a methyl compound produced by the method described in any one of [1-1] to [1-13] as an intermediate.

[0046] [1-15]

[0047] A cell modified in such a way that the activity or expression of formate-tetrahydrofolate ligase (FtfL) and S-adenosylmethionine-dependent methyltransferase is enhanced.

[0048] [1-16]

[0049] The cell according to [1-15] above, further modified so that the activity or expression of methenyltetrahydrofolate cyclohydrolase (Fch) and / or methylenetetrahydrofolate dehydrogenase (MtdA) is enhanced.

[0050] [1-17]

[0051] The cell according to the above-mentioned [1-15] or [1-16] is further modified in a manner such that the activity or expression of at least one enzyme selected from the group consisting of S-adenosylhomocysteine nucleosidase (Mtn), S-ribosylhomocysteine lyase (LuxS) and S-adenosylhomocysteine hydrolase (SahH) is enhanced.

[0052] [1-18]

[0053] The cell according to any one of [1-15] to [1-17] above, further modified so as to enhance the activity or expression of at least one enzyme selected from the group consisting of ribose diphosphokinase (Prs), adenine phosphoribosyltransferase (Apt) and adenosine kinase (ADK).

[0054] [1-19]

[0055] The cell according to any one of [1-15] to [1-18] above, further modified so as to enhance the activity or expression of at least one enzyme selected from 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthetase (MetH or MetE), and methionine adenosyltransferase (MetK).

[0056] [1-20]

[0057] The cell according to any one of [1-15] to [1-19] above, further modified so that the activity or expression of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox) is enhanced.

[0058] [1-21]

[0059] The cell according to the above-mentioned [1-20], which is further modified so as to enhance the activity or expression of at least one enzyme selected from the group consisting of formaldehyde dehydrogenase (FdhA), S-hydroxymethylglutathione dehydrogenase (FrmA) and S-formylglutathione hydrolase (FrmB).

[0060] [1-22]

[0061] The cell according to any one of [1-15] to [1-21] above, which is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0062] [1-23]

[0063] A method for regenerating S-adenosylmethionine, comprising:

[0064] (a) a process for producing 5-methyltetrahydrofolate from formic acid or a salt thereof and tetrahydrofolate;

[0065] (b) a step of converting S-adenosylhomocysteine produced by demethylating S-adenosylmethionine into homocysteine; and

[0066] (c) A step of transferring the methyl group of the 5-methyltetrahydrofolate to the homocysteine to generate methionine, and converting the methionine into S-adenosylmethionine.

[0067] [2-1]

[0068] A method for regenerating S-adenosylmethionine, comprising:

[0069] (a) a process for producing 5-methyltetrahydrofolate from formic acid or a salt thereof and tetrahydrofolate;

[0070] (b) a step of converting S-adenosylhomocysteine produced by demethylating S-adenosylmethionine into homocysteine; and

[0071] (c) A step of generating methionine by transferring the methyl group of 5-methyltetrahydrofolate to the homocysteine, and converting the methionine into S-adenosylmethionine.

[0072] [2-2]

[0073] The method for regenerating S-adenosylmethionine according to the above-mentioned [2-1], wherein, in the above-mentioned step (a), the production of 5-methyltetrahydrofolate from formate and tetrahydrofolate is carried out in the presence of formate-tetrahydrofolate ligase (FtfL), methenyl-tetrahydrofolate cyclohydrolase (Fch), methylene-tetrahydrofolate dehydrogenase (MtdA) and 5,10-methylene-tetrahydrofolate reductase (MetF).

[0074] [2-3]

[0075] The method for regenerating S-adenosylmethionine according to the above-mentioned [2-1] or [2-2], wherein, in the above-mentioned step (b), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine nucleosidase (Mtn) and S-ribosylhomocysteine lyase (LuxS).

[0076] [2-4]

[0077] The method for regenerating S-adenosylmethionine according to any one of [2-1] to [2-3] above, wherein in the step (b), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine hydrolase (SahH).

[0078] [2-5]

[0079] The method for regenerating S-adenosylmethionine according to any one of [2-1] to [2-4] above, wherein in the step (b), demethylation of S-adenosylhomocysteine is performed in the presence of an S-adenosylmethionine-dependent methyltransferase.

[0080] [2-6]

[0081] The method for regenerating S-adenosylmethionine according to any one of [2-1] to [2-5] above, wherein, in the step (c), the production of methionine from homocysteine is carried out in the presence of methionine synthetase (MetH or MetE), and the conversion of the methionine into S-adenosylmethionine is carried out in the presence of methionine adenosyltransferase (MetK).

[0082] [2-7]

[0083] The method for regenerating S-adenosylmethionine according to any one of [2-1] to [2-6] above, wherein cells modified so as to enhance the activity or expression of S-adenosylmethionine-dependent methyltransferase are used.

[0084] [2-8]

[0085] The method for regenerating S-adenosylmethionine according to any one of [2-1] to [2-7] above, wherein the cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0086] [2-9]

[0087] A method for producing a methyl compound, comprising:

[0088] (a) a process for producing 5-methyltetrahydrofolate from formic acid or a salt thereof and tetrahydrofolate;

[0089] (b) a step of converting S-adenosylhomocysteine generated by demethylation of S-adenosylmethionine into homocysteine;

[0090] (c) a step of generating methionine by transferring a methyl group of 5-methyltetrahydrofolate to homocysteine, and converting the methionine into S-adenosylmethionine; and

[0091] (d) A step of performing a methylation reaction using S-adenosylmethionine as a methyl donor to produce a methyl compound.

[0092] [2-10]

[0093] The method for producing a methyl compound according to the above-mentioned [2-9], wherein, in the above-mentioned step (a), the production of 5-methyltetrahydrofolate from formate and tetrahydrofolate is carried out in the presence of formate-tetrahydrofolate ligase (FtfL), methenyl-tetrahydrofolate cyclohydrolase (Fch), methylene-tetrahydrofolate dehydrogenase (MtdA) and 5,10-methylene-tetrahydrofolate reductase (MetF).

[0094] [2-11]

[0095] The method for producing a methyl compound according to the above-mentioned [2-9] or [2-10], wherein, in the above-mentioned step (b), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine nucleosidase (Mtn) and S-ribosylhomocysteine lyase (LuxS).

[0096] [2-12]

[0097] The method for producing a methyl compound according to any one of [2-9] to [2-11] above, wherein in the step (b), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine hydrolase (SahH).

[0098] [2-13]

[0099] The method for producing a methyl compound according to any one of [2-9] to [2-12] above, wherein in the step (b), demethylation of S-adenosylhomocysteine is performed in the presence of an S-adenosylmethionine-dependent methyltransferase.

[0100] [2-14]

[0101] The method for producing a methyl compound according to any one of [2-9] to [2-13] above, wherein, in the aforementioned step (c), the production of methionine from homocysteine is carried out in the presence of methionine synthetase (MetH or MetE), and the conversion of the methionine to S-adenosylmethionine is carried out in the presence of methionine adenosyltransferase (MetK).

[0102] [2-15]

[0103] The method for producing a methyl compound according to any one of [2-9] to [2-14] above, wherein cells modified so as to enhance the activity or expression of S-adenosylmethionine-dependent methyltransferase are used.

[0104] [2-16]

[0105] The method for producing a methyl compound according to any one of [2-9] to [2-15] above, wherein the cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0106] [2-17]

[0107] A method for producing an organic compound, comprising the step of using a methyl compound produced by the method described in any one of [2-9] to [2-16] as an intermediate.

[0108] [2-18]

[0109] A cell modified in such a way that the activity or expression of formate-tetrahydrofolate ligase (FtfL) and S-adenosylmethionine-dependent methyltransferase is enhanced.

[0110] [2-19]

[0111] The cell according to [2-18] above, further modified so that the activity or expression of methenyltetrahydrofolate cyclohydrolase (Fch) and / or methylenetetrahydrofolate dehydrogenase (MtdA) is enhanced.

[0112] [2-20]

[0113] The cell according to the above-mentioned [2-18] or [2-19] is further modified in a manner such that the activity or expression of at least one enzyme selected from the group consisting of S-adenosylhomocysteine nucleosidase (Mtn), S-ribosylhomocysteine lyase (LuxS) and S-adenosylhomocysteine hydrolase (SahH) is enhanced.

[0114] [2-21]

[0115] The cell according to any one of [2-18] to [2-20] above, further modified so as to enhance the activity or expression of at least one enzyme selected from 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthetase (MetH or MetE), and methionine adenosyltransferase (MetK).

[0116] [2-22]

[0117] The cell according to any one of [2-18] to [2-21] above, which is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0118] [2-23]

[0119] A method for producing a methyl compound using the cell according to any one of [2-18] to [2-22] above.

[0120] [2-24]

[0121] A method for producing an organic compound, comprising the step of producing the organic compound using a methyl compound produced by the cell according to any one of [2-18] to [2-22] as an intermediate.

[0122] [3-1]

[0123] A method for regenerating S-adenosylmethionine, comprising:

[0124] (a) a process of converting methanol into formaldehyde, and then converting the formaldehyde into formic acid;

[0125] (b) a step of producing 5-methyltetrahydrofolate from the formic acid and tetrahydrofolate;

[0126] (c) a step of converting S-adenosylhomocysteine produced by demethylating S-adenosylmethionine into homocysteine; and

[0127] (d) A step of transferring the methyl group of the 5-methyltetrahydrofolate to the homocysteine to generate methionine, and converting the methionine into S-adenosylmethionine.

[0128] [3-2]

[0129] The method for regenerating S-adenosylmethionine according to the above [3-1], wherein in the step (a), the conversion of methanol to formaldehyde is carried out in the presence of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox).

[0130] [3-3]

[0131] The method for regenerating S-adenosylmethionine according to [3-1] or [3-2] above, wherein in the step (a), the conversion of formaldehyde to formic acid is carried out in the presence of formaldehyde dehydrogenase (FdhA).

[0132] [3-4]

[0133] The method for regenerating S-adenosylmethionine according to any one of [3-1] to [3-3] above, wherein, in the step (a), the conversion of formaldehyde to formic acid is carried out in the presence of a spontaneous reaction between formaldehyde and glutathione, S-hydroxymethylglutathione dehydrogenase (FrmA) and S-formylglutathione hydrolase (FrmB).

[0134] [3-5]

[0135] The method for regenerating S-adenosylmethionine according to any one of [3-1] to [3-4] above, wherein, in the step (b), the production of 5-methyltetrahydrofolate from formate and tetrahydrofolate is carried out in the presence of formate-tetrahydrofolate ligase (FtfL), methenyl-tetrahydrofolate cyclohydrolase (Fch), methylene-tetrahydrofolate dehydrogenase (MtdA), and 5,10-methylene-tetrahydrofolate reductase (MetF).

[0136] [3-6]

[0137] The method for regenerating S-adenosylmethionine according to any one of [3-1] to [3-5] above, wherein, in the aforementioned step (c), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine nucleosidase (Mtn) and S-ribosylhomocysteine lyase (LuxS).

[0138] [3-7]

[0139] The method for regenerating S-adenosylmethionine according to any one of [3-1] to [3-6] above, wherein in the step (c), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine hydrolase (SahH).

[0140] [3-8]

[0141] The method for regenerating S-adenosylmethionine according to any one of [3-1] to [3-7] above, wherein in the step (c), demethylation of S-adenosylhomocysteine is performed in the presence of an S-adenosylmethionine-dependent methyltransferase.

[0142] [3-9]

[0143] The method for regenerating S-adenosylmethionine according to any one of [3-1] to [3-8] above, wherein, in the step (d), the production of methionine from homocysteine is carried out in the presence of methionine synthetase (MetH or MetE), and the conversion of the methionine into S-adenosylmethionine is carried out in the presence of methionine adenosyltransferase (MetK).

[0144] [3-10]

[0145] The method for regenerating S-adenosylmethionine according to any one of [3-1] to [3-9] above, wherein cells modified so as to enhance the activity or expression of S-adenosylmethionine-dependent methyltransferase are used.

[0146] [3-11]

[0147] The method for regenerating S-adenosylmethionine according to any one of [3-1] to [3-10] above, wherein the cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0148] [3-12]

[0149] A method for producing a methyl compound, comprising:

[0150] (a) a process of converting methanol into formaldehyde, and then converting the formaldehyde into formic acid;

[0151] (b) a step of producing 5-methyltetrahydrofolate from the formic acid and tetrahydrofolate;

[0152] (c) a step of converting S-adenosylhomocysteine generated by demethylating S-adenosylmethionine into homocysteine;

[0153] (d) a step of generating methionine by transferring a methyl group of 5-methyltetrahydrofolate to the homocysteine, and converting the methionine into S-adenosylmethionine;

[0154] (e) A step of performing a methylation reaction using S-adenosylmethionine as a methyl donor to produce a methyl compound.

[0155] [3-13]

[0156] The method for producing a methyl compound according to [3-12] above, wherein in the step (a), the conversion of methanol to formaldehyde is carried out in the presence of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox).

[0157] [3-14]

[0158] The method for producing a methyl compound according to [3-12] or [3-13] above, wherein in the step (a), the conversion of formaldehyde to formic acid is carried out in the presence of formaldehyde dehydrogenase (FdhA).

[0159] [3-15]

[0160] The method for producing a methyl compound according to any one of [3-12] to [3-14] above, wherein, in the step (a), the conversion of formaldehyde to formic acid is carried out in the presence of a spontaneous reaction between formaldehyde and glutathione, S-hydroxymethylglutathione dehydrogenase (FrmA) and S-formylglutathione hydrolase (FrmB).

[0161] [3-16]

[0162] The method for producing a methyl compound according to any one of [3-12] to [3-15] above, wherein, in the step (b), the production of 5-methyltetrahydrofolate from formate and tetrahydrofolate is carried out in the presence of formate-tetrahydrofolate ligase (FtfL), methenyl-tetrahydrofolate cyclohydrolase (Fch), methylene-tetrahydrofolate dehydrogenase (MtdA) and 5,10-methylene-tetrahydrofolate reductase (MetF).

[0163] [3-17]

[0164] The method for producing a methyl compound according to any one of [3-12] to [3-16] above, wherein, in the aforementioned step (c), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine nucleosidase (Mtn) and S-ribosylhomocysteine lyase (LuxS).

[0165] [3-18]

[0166] The method for producing a methyl compound according to any one of [3-12] to [3-17] above, wherein in the step (c), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine hydrolase (SahH).

[0167] [3-19]

[0168] The method for producing a methyl compound according to any one of [3-12] to [3-18] above, wherein in the step (c), demethylation of S-adenosylhomocysteine is performed in the presence of an S-adenosylmethionine-dependent methyltransferase.

[0169] [3-20]

[0170] The method for producing a methyl compound according to any one of [3-12] to [3-19] above, wherein, in the aforementioned step (d), the production of methionine from homocysteine is carried out in the presence of methionine synthetase (MetH or MetE), and the conversion of the methionine to S-adenosylmethionine is carried out in the presence of methionine adenosyltransferase (MetK).

[0171] [3-21]

[0172] The method for producing a methyl compound according to any one of [3-12] to [3-20] above, wherein cells modified so as to enhance the activity or expression of S-adenosylmethionine-dependent methyltransferase are used.

[0173] [3-22]

[0174] The method for producing a methyl compound according to any one of [3-12] to [3-21] above, wherein the cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0175] [3-23]

[0176] A method for producing an organic compound, comprising the step of using a methyl compound produced by the method described in any one of [3-12] to [3-22] as an intermediate.

[0177] [3-24]

[0178] A cell modified in such a way that the activity or expression of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox), formate-tetrahydrofolate ligase (FtfL) and S-adenosylmethionine-dependent methyltransferase is enhanced.

[0179] [3-25]

[0180] The cell according to [3-24] above, further modified so as to enhance the activity or expression of at least one enzyme selected from the group consisting of formaldehyde dehydrogenase (FdhA), S-hydroxymethylglutathione dehydrogenase (FrmA) and S-formylglutathione hydrolase (FrmB).

[0181] [3-26]

[0182] The cell according to [3-24] or [3-25], further modified so that methylenetetrahydrofolate cyclohydrolase (Fch) and / or methylenetetrahydrofolate dehydrogenase (MtdA) is enhanced.

[0183] [3-27]

[0184] The cell according to any one of [3-24] to [3-26] above, which is further modified in a manner such that the activity or expression of at least one enzyme selected from the group consisting of S-adenosylhomocysteine nucleosidase (Mtn), S-ribosylhomocysteine lyase (LuxS) and S-adenosylhomocysteine hydrolase (SahH) is enhanced.

[0185] [3-28]

[0186] The cell according to any one of [3-24] to [3-27] above, further modified so as to increase the activity or expression of at least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthetase (MetH or MetE), and methionine adenosyltransferase (MetK).

[0187] [3-29]

[0188] The cell according to any one of [3-24] to [3-28] above, wherein the cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0189] [3-30]

[0190] A method for producing a methyl compound, characterized by using the cell according to any one of [3-24] to [3-29] above.

[0191] [3-31]

[0192] A method for producing an organic compound, comprising the step of using a methyl compound produced by the cell according to any one of [3-24] to [3-29] above as an intermediate.

[0193] [3-32]

[0194] A method for regenerating S-adenosylmethionine, comprising:

[0195] (a) a process for converting methanol into formaldehyde;

[0196] (b) a step of producing 5-methyltetrahydrofolate from the formaldehyde and tetrahydrofolate;

[0197] (c) a step of converting S-adenosylhomocysteine produced by demethylating S-adenosylmethionine into homocysteine; and

[0198] (d) A step of transferring the methyl group of the 5-methyltetrahydrofolate to the homocysteine to generate methionine, and converting the methionine into S-adenosylmethionine.

[0199] [3-33]

[0200] The method for regenerating S-adenosylmethionine according to [3-32] above, wherein in the step (a), the conversion of methanol to formaldehyde is carried out in the presence of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox).

[0201] [3-34]

[0202] The method for regenerating S-adenosylmethionine according to [3-32] or [3-33], wherein in the step (b), the production of 5-methyltetrahydrofolate from formaldehyde and tetrahydrofolate is carried out in the presence of 5,10-methylenetetrahydrofolate reductase (MetF).

[0203] [3-35]

[0204] The method for regenerating S-adenosylmethionine according to any one of [3-32] to [3-34] above, wherein, in the aforementioned step (c), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine nucleosidase (Mtn) and S-ribosylhomocysteine lyase (LuxS).

[0205] [3-36]

[0206] The method for regenerating S-adenosylmethionine according to any one of [3-32] to [3-35] above, wherein in the step (c), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine hydrolase (SahH).

[0207] [3-37]

[0208] The method for regenerating S-adenosylmethionine according to any one of [3-32] to [3-36] above, wherein in the step (c), demethylation of S-adenosylhomocysteine is performed in the presence of an S-adenosylmethionine-dependent methyltransferase.

[0209] [3-38]

[0210] The method for regenerating S-adenosylmethionine according to any one of [3-32] to [3-37] above, wherein, in the aforementioned step (d), the production of methionine from homocysteine is carried out in the presence of methionine synthetase (MetH or MetE), and the conversion of the methionine into S-adenosylmethionine is carried out in the presence of methionine adenosyltransferase (MetK).

[0211] [3-39]

[0212] The method for regenerating S-adenosylmethionine according to any one of [3-32] to [3-38] above, wherein cells modified so as to enhance the activity or expression of S-adenosylmethionine-dependent methyltransferase are used.

[0213] [3-40]

[0214] The method for regenerating S-adenosylmethionine according to any one of [3-32] to [3-39] above, wherein the cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0215] [3-41]

[0216] A method for producing a methyl compound, comprising:

[0217] (a) a process for converting methanol into formaldehyde;

[0218] (b) a step of producing 5-methyltetrahydrofolate from the formaldehyde and tetrahydrofolate;

[0219] (c) a step of converting S-adenosylhomocysteine generated by demethylating S-adenosylmethionine into homocysteine;

[0220] (d) a step of generating methionine by transferring the methyl group of the 5-methyltetrahydrofolate to the homocysteine, and converting the methionine into S-adenosylmethionine;

[0221] (e) A step of performing a methylation reaction using the S-adenosylmethionine produced in the step (d) as a methyl donor to produce a methyl compound.

[0222] [3-42]

[0223] The method for producing a methyl compound according to the above-mentioned [3-41], wherein in the step (a), the conversion of methanol to formaldehyde is carried out in the presence of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox).

[0224] [3-43]

[0225] The method for producing a methyl compound according to [3-41] or [3-42], wherein in the step (b), the production of 5-methyltetrahydrofolate from formaldehyde and tetrahydrofolate is carried out in the presence of 5,10-methylenetetrahydrofolate reductase (MetF).

[0226] [3-44]

[0227] The method for producing a methyl compound according to any one of [3-41] to [3-43] above, wherein, in the aforementioned step (c), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine nucleosidase (Mtn) and S-ribosylhomocysteine lyase (LuxS).

[0228] [3-45]

[0229] The method for producing a methyl compound according to any one of [3-41] to [3-44] above, wherein in the step (c), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine hydrolase (SahH).

[0230] [3-46]

[0231] The method for producing a methyl compound according to any one of [3-41] to [3-45] above, wherein in the step (c), demethylation of S-adenosylhomocysteine is performed in the presence of an S-adenosylmethionine-dependent methyltransferase.

[0232] [3-47]

[0233] The method for producing a methyl compound according to any one of [3-41] to [3-46] above, wherein, in the aforementioned step (d), the production of methionine from homocysteine is carried out in the presence of methionine synthetase (MetH or MetE), and the conversion of the methionine to S-adenosylmethionine is carried out in the presence of methionine adenosyltransferase (MetK).

[0234] [3-48]

[0235] The method for producing a methyl compound according to any one of [3-41] to [3-47] above, wherein cells modified so as to enhance the activity or expression of S-adenosylmethionine-dependent methyltransferase are used.

[0236] [3-49]

[0237] The method for producing a methyl compound according to any one of [3-41] to [3-48] above, wherein the cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0238] [3-50]

[0239] A method for producing an organic compound, comprising the step of using a methyl compound produced by the method described in any one of [3-41] to [3-49] as an intermediate.

[0240] [3-51]

[0241] A cell modified in such a way that the activity or expression of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox) and S-adenosylmethionine-dependent methyltransferase is enhanced.

[0242] [3-52]

[0243] The cell according to the above-mentioned [3-51] is further modified in a manner such that the activity or expression of at least one enzyme selected from the group consisting of S-adenosylhomocysteine nucleosidase (Mtn), S-ribosylhomocysteine lyase (LuxS) and S-adenosylhomocysteine hydrolase (SahH) is enhanced.

[0244] [3-53]

[0245] The cell according to [3-51] or [3-52] above, further modified so as to increase the activity or expression of at least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthetase (MetH or MetE), and methionine adenosyltransferase (MetK).

[0246] [3-54]

[0247] The cell according to any one of [3-51] to [3-53] above, wherein the cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0248] [3-55]

[0249] A method for producing a methyl compound, characterized by using the cell according to any one of [3-51] to [3-54] above.

[0250] [3-56]

[0251] A method for producing an organic compound, comprising the step of using a methyl compound produced by the cell according to any one of [3-51] to [3-54] above as an intermediate.

[0252] Effects of the Invention

[0253] According to the present invention, SAM can be efficiently regenerated. BRIEF DESCRIPTION OF THE DRAWINGS

[0254] [ Figure 1 ] is a diagram showing an example of a reaction system used in the method of the present invention.

[0255] [ Figure 2 ] is a diagram showing an example of a reaction system used in the method of the present invention. DETAILED DESCRIPTION

[0256] The present invention will be described in detail below. The following embodiments are for illustrating the present invention and are not intended to limit the present invention to these embodiments. The present invention can be implemented in various ways without departing from the gist of the present invention.

[0257] In addition, this description incorporates the contents described in the description and drawings of Japanese patent applications (Japanese Patent Application Nos. 2023-056931 and 2023-056932) filed on March 31, 2023, on which the priority claim of this application is based.

[0258] 1. summary

[0259] SAM is useful for the methylation reaction of compounds. However, its supply is limited within cells and it is easily depleted.

[0260] Conventionally, a method for recycling SAM by adding methanol to the culture medium of a microorganism that co-expresses a methylase and a methanol dehydrogenase is known. This method requires a spontaneous condensation reaction between formaldehyde and tetrahydrofolate (THF). In a document describing this method (International Publication No. 2019 / 160059), methods for reducing the decomposition activity of formaldehyde include reducing the activity of formaldehyde dehydrogenase, which directly converts formaldehyde into formic acid, and reducing the activity of an enzyme that converts formaldehyde into formic acid by binding it to glutathione, i.e., inhibiting the conversion of formaldehyde to formic acid.

[0261] In contrast, the present inventors succeeded in efficiently regenerating SAM by intentionally using formic acid for SAM regeneration or intentionally using the reaction pathway from formaldehyde to formic acid for SAM regeneration, thereby completing the present invention.

[0262] 2-1. Method for regenerating S-adenosylmethionine using formic acid

[0263] (1) Formic acid

[0264] Formic acid is the carboxylic acid with the smallest molecular weight and is a compound represented by the chemical formula HCOOH. In the present invention, formic acid can be used in the form of formic acid or its salts. Examples of formate salts include, but are not limited to, potassium formate, sodium formate, lithium formate, cesium formate, thallium formate, and ammonium formate. Furthermore, in the present invention, hydrates of formic acid or its salts can also be used as formic acid.

[0265] (2) S-adenosylmethionine

[0266] S-adenosylmethionine (SAM) is a substance that functions as a methyl donor when methyltransferases transfer a methyl group to a target methylation substance (a substance to be methylated). When SAM loses its methyl group (demethylation) due to the transfer of its methyl group to the target methylation substance, it becomes S-adenosyl-L-homocysteine (SAH). SAH is broken down into homocysteine (Hcy) by S-ribosylhomocysteine (SRH). Because the supply of SAM in cells is limited, SAM is easily depleted without new methionine.

[0267] (3) Method for regenerating S-adenosylmethionine

[0268] In the present invention, "regeneration" of SAM refers to the generation of SAM without the addition of methionine or SAM from outside the system. Those skilled in the art can assess the generation of SAM using known methods, such as methods for measuring methyltransferase activity in a test sample. For example, if the methyltransferase activity in a test sample is measured and the activity is higher than that in a control, SAM can be assessed as being generated. As a control, a sample under different conditions from the test sample can be used, such as a sample without the addition of the substance added to the test sample. Methyltransferase activity can be assessed, for example, by genetically introducing an exogenous methyltransferase into cells to induce enzyme expression, then adding a methylation target substance to the cell culture medium to react with the enzyme, and measuring the amount of methyl compound generated using mass spectrometry such as LC-MS. That is, if the methyltransferase activity in the test sample is higher than that in the control without the addition of methionine or SAM from outside the reaction system, SAM can be considered to have been regenerated in the test sample. In the present invention, the method for measuring methyltransferase activity can be appropriately adopted by those skilled in the art using known methods and is not limited to the above-mentioned method.

[0269] In addition, the above-mentioned "higher than the control" means that the methyltransferase activity is higher than that in the control by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more.

[0270] The method for regenerating S-adenosylmethionine of the present invention may include, for example, the following steps.

[0271] (a) a process for producing 5-methyltetrahydrofolate from formic acid or a salt thereof and tetrahydrofolate;

[0272] (b) a step of converting S-adenosylhomocysteine produced by demethylating S-adenosylmethionine into homocysteine; and

[0273] (c) A step of transferring the methyl group of the 5-methyltetrahydrofolate to the homocysteine to generate methionine, and converting the methionine into S-adenosylmethionine.

[0274] Step (a) is a step of producing 5-methyltetrahydrofolate (5-CH3-THF) from formic acid or a salt thereof and tetrahydrofolate (THF). Figure 1 ).

[0275] Step (a) may include, for example: (i) a step of generating 10-formyltetrahydrofolate (10-CHO-THF) from formic acid or a salt thereof and tetrahydrofolate (THF); (ii) a step of generating 5,10-methylenetetrahydrofolate (5,10-CH═THF) from 10-CHO-THF; (iii) a step of generating 5,10-methylenetetrahydrofolate (5,10-CH2-THF) from 5,10-CH═THF; and (iv) a step of generating 5-methyltetrahydrofolate (5-CH3-THF) from 5,10-CH2-THF.

[0276] In step (i), the production of 10-CHO-THF from formic acid or a salt thereof and tetrahydrofolate (THF) can be carried out, for example, in the presence of formate-tetrahydrofolate ligase (FtfL) or by using this enzyme. In step (ii), the production of 5,10-CH═THF from 10-CHO-THF can be carried out, for example, in the presence of methenyl-tetrahydrofolate cyclohydrolase (Fch) or by using this enzyme. In step (iii), the production of 5,10-CH₂-THF from 5,10-CH₂-THF can be carried out, for example, in the presence of methylene-tetrahydrofolate dehydrogenase (MtdA) or by using this enzyme. In step (iv), the production of 5-CH₃-THF from 5,10-CH₂-THF can be carried out, for example, in the presence of 5,10-methylene-tetrahydrofolate reductase (MetF) or by using this enzyme.

[0277] That is, in step (a), the production of 5-methyltetrahydrofolate from formate and tetrahydrofolate can be carried out in the presence of, for example, formate-tetrahydrofolate ligase (FtfL), methenyl-tetrahydrofolate cyclohydrolase (Fch), methylene-tetrahydrofolate dehydrogenase (MtdA), and 5,10-methylene-tetrahydrofolate reductase (MetF), or by these enzymes.

[0278] Step (b) is a step of converting S-adenosylhomocysteine (SAH) generated by demethylation of S-adenosylmethionine (SAM) into homocysteine (Hcy). Figure 1 ).

[0279] Step (b) may include, for example, (i) a step of converting S-adenosylhomocysteine (SAH) into S-ribosylhomocysteine (SRH) and (ii) a step of converting SRH into homocysteine (Hcy).

[0280] In the above step (i), the conversion of S-adenosylhomocysteine (SAH) to S-ribosylhomocysteine (SRH) can be carried out, for example, in the presence of S-adenosylhomocysteine nucleosidase (Mtn) or by the enzyme, and in the above step (ii), the conversion of SRH to homocysteine (Hcy) can be carried out, for example, in the presence of S-ribosylhomocysteine lyase (LuxS) or by the enzyme.

[0281] That is, in step (b), the conversion of S-adenosylhomocysteine to homocysteine can be performed in the presence of S-adenosylhomocysteine nucleosidase (Mtn) and S-ribosylhomocysteine lyase (LuxS) or by these enzymes.

[0282] In another embodiment, step (b) may include, for example, (iii) a step of converting S-adenosylhomocysteine (SAH) directly (without conversion to S-ribosylhomocysteine (SRH)) to homocysteine (Hcy).

[0283] In the above step (iii), the conversion of S-adenosylhomocysteine to homocysteine can be performed in the presence of S-adenosylhomocysteine hydrolase (SahH) or by the enzyme.

[0284] In step (b), demethylation of S-adenosylmethionine (SAM) can be performed, for example, in the presence of an S-adenosylmethionine-dependent methyltransferase or by the enzyme.

[0285] S-adenosylmethionine-dependent methyltransferase is an enzyme that transfers the methyl group of SAM to a methylation target substance (methylated substance). As the methylation target substance, DNA, proteins, low molecular weight compounds, etc. that are the objects of methylation can be cited, preferably low molecular weight compounds. SAM is converted into SAH by demethylation by the S-adenosylmethionine-dependent methyltransferase. On the other hand, the methylation target substance is methylated to generate a methyl compound. In this specification, S-adenosylmethionine-dependent methyltransferase is also referred to as "methyltransferase".

[0286] In the present invention, the term "methyltransferase" is not limited as long as it is a substance that transfers the methyl group of SAM to the target substance to be methylated. Examples of such methyltransferases include, but are not limited to, methyltransferases that methylate DNA, methyltransferases that methylate proteins, and transferases that methylate low molecular weight compounds. Furthermore, as a methyltransferase, for example, in the well-known database BRENDA (https: / / www.brenda-enzyme), "methyltransferase" can be searched as a keyword, and any enzyme with "S-adenosyl-L-methionine" listed in the "Synonyms" column can be selected. Examples of such methyltransferases include, but are not limited to, tyrosine 3C-methyltransferase, histidine N-α-methyltransferase, perillicacid O-methyltransferase, and catechol O-methyltransferase.

[0287] Step (c) is a step in which methionine is produced by transferring the methyl group of 5-methyltetrahydrofolate produced in step (a) to homocysteine (Hcy) converted from S-adenosylhomocysteine (SAH) in step (b), and the produced methionine is converted to S-adenosylmethionine (SAM). This step regenerates SAM.

[0288] Step (c) may include, for example: (i) a step of generating methionine by transferring the methyl group of 5-methyltetrahydrofolate generated in step (a) to homocysteine (Hcy) converted from S-adenosylhomocysteine (SAH) in step (b); and (ii) a step of converting the generated methionine into S-adenosylmethionine (SAM).

[0289] In the above step (i), the production of methionine from homocysteine can be carried out, for example, in the presence of methionine synthetase (MetH or MetE) or by the enzyme, and in the above step (ii), the conversion of methionine to S-adenosylmethionine can be carried out, for example, in the presence of methionine adenosyltransferase (MetK) or by the enzyme.

[0290] That is, the production of methionine from homocysteine can be carried out in the presence of or by methionine synthetase (MetH or MetE), and the conversion of methionine to S-adenosylmethionine can be carried out in the presence of or by methionine adenosyltransferase (MetK). Methionine synthetase can be either cobalamin-dependent (MetH) or cobalamin-independent (MetE).

[0291] In the above step (ii), the conversion of methionine to S-adenosylmethionine requires adenosine triphosphate (ATP), and the ATP can be synthesized by a step comprising a reaction of at least one enzyme selected from the group consisting of ribose diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt) and adenosine kinase (ADK).

[0292] Ribose-5-phosphate (R5P), a metabolite of the pentose phosphate pathway, can be synthesized into phosphoribosyl pyrophosphate (PRPP) by ribose-5-phosphate kinase (Prs), and adenine phosphoribosyltransferase (Apt) can be used to synthesize adenosine monophosphate (AMP) from PRPP and adenine. Furthermore, adenosine monophosphate (AMP) can be synthesized from adenosine by adenosine kinase (ADK). AMP can be converted into ATP through further phosphorylation.

[0293] Adenine is a metabolite produced by the Mtn reaction in step (b), and adenosine is a metabolite produced by the SahH reaction in step (b). By synthesizing ATP using these metabolites as substrates, the ATP required for the synthesis of SAM can be efficiently supplied.

[0294] When cells are used for regeneration of SAM, the enzyme used in the method of the present invention may be an enzyme existing in the cells, or may be an enzyme expressed by introducing foreign DNA encoding the enzyme into the cells.

[0295] The reaction conditions (temperature, reaction time, pH, etc.) of the enzyme in the method of the present invention can be appropriately set by those skilled in the art according to the type of enzyme, the type of cell used, etc. As temperature conditions, for example, about 25 to 40°C, for example, about 30 to 37°C, but are not limited thereto. As reaction time, for example, about 15 minutes to 36 hours, for example, about 30 minutes to 24 hours (for example, 1, 2, 4, 6, 8, 10, 12, 16, 18, 24 hours) can be mentioned, but are not limited thereto. pH can be appropriately set within the range of about 6 to 9.

[0296] The above steps can be carried out in a cell-free system by known methods by those skilled in the art (Microbial Cell Factories, Issue 11, No. 120, September 2012, Journal of Bioscience and Bioengineering, Issue 129, Article 3, March 2020, pp. 269-275).

[0297] (4) cell

[0298] The method for regenerating S-adenosylmethionine of the present invention can be performed using cells (e.g., microorganisms) (in cells (e.g., microorganisms)). The method of the present invention can be used with any cell that has SAM. Therefore, the cells used in the present invention are not limited as long as they have SAM, and examples include microorganisms, mammalian cells, insect cells, and plant cells (including cultured plant cells). Microorganisms are not limited, and examples include Escherichia coli (E. coli), coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, yeast, etc. Mammalian cells are not limited, and examples include mouse cells, rat cells, rabbit cells, dog cells, monkey cells, and cultured cells of human origin. Insect cells are not limited as long as they can express proteins using baculovirus or the like. Plant cells are not limited to these, and examples thereof include, but are not limited to, cells of plants of the genus Nicotiana (e.g., Nicotiana benthamiana, N. tabacum, N. excelsior), plants of the Solanaceae family, plants of the Gramineae family, plants of the Cruciferae family, plants of the Asteraceae family, and plants of the Bryophyte family. The cells used in the present invention are preferably cells capable of expressing foreign genes.

[0299] In the present invention, cells modified so that the activity or expression of S-adenosylmethionine-dependent methyltransferase is enhanced can be used. In the present invention, "modified cells" include "modified microorganisms."

[0300] Furthermore, in the present invention, cells modified so that the activity or expression of formate-tetrahydrofolate ligase (FtfL) and S-adenosylmethionine-dependent methyltransferase is enhanced can be used.

[0301] Furthermore, in the present invention, cells modified to enhance the activity or expression of methenyltetrahydrofolate cyclohydrolase (Fch) and / or methylenetetrahydrofolate dehydrogenase (MtdA) in addition to FtfL and SAM-dependent methyltransferase can also be used.

[0302] Furthermore, in the present invention, in addition to FtfL and SAM-dependent methyltransferase, cells can also be used that have been modified in a manner that enhances the activity or expression of at least one enzyme (for example, Mtn and LuxS) selected from the group consisting of S-adenosylhomocysteine nucleosidase (Mtn), S-ribosylhomocysteine lyase (LuxS) and S-adenosylhomocysteine hydrolase (SahH).

[0303] Furthermore, in the present invention, in addition to FtfL and SAM-dependent methyltransferases, cells modified in such a manner that the activity or expression of at least one or more enzymes (e.g., Prs and Apt) selected from the group consisting of ribose diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt) and adenosine kinase (ADK) are enhanced can also be used.

[0304] Furthermore, in the present invention, cells modified to enhance the activity or expression of at least one enzyme selected from the group consisting of the following (i) to (iv) can be used in addition to FtfL and SAM-dependent methyltransferase:

[0305] (i) Fch and / or MtdA,

[0306] (ii) at least one enzyme selected from the group consisting of Mtn, LuxS and SahH,

[0307] (iii) at least one enzyme selected from the group consisting of Prs, Apt, and ADK (e.g., Prs and Apt; ADK only, etc.), and

[0308] (iv) At least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthetase (MetH or MetE), and methionine adenosyltransferase (MetK).

[0309] In this specification, "modification so as to enhance the activity or expression" of a certain enzyme means modification so as to enhance the activity or expression of the enzyme by, for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more, compared to the activity or expression of the enzyme in non-modified cells (unmodified cells). Whether the activity or expression of the enzyme is enhanced can be evaluated by measuring the expression level of the enzyme in the modified cells and examining whether the expression level is increased compared to that in the non-modified cells.

[0310] Examples of cells modified to enhance the activity or expression of a SAM-dependent methyltransferase include cells containing DNA encoding a SAM-dependent methyltransferase. As described above, a SAM-dependent methyltransferase can be appropriately selected based on a known database, such as BRENDA (https: / / www.brenda-enzyme).

[0311] Examples of cells modified so that the activity or expression of each of FtfL, Fch, MtdA, Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, and MetK is enhanced include cells containing DNA encoding FtfL, Fch, MtdA, Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, and MetK, respectively.

[0312] In the present invention, "modified cells" and cells containing DNA encoding each enzyme may also be referred to as "transformants."

[0313] The base sequence information of DNA encoding SAM-dependent methyltransferase, FtfL, Fch, MtdA, Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE and MetK can be obtained from publicly known databases, such as NCBI (https: / / www.ncbi.nlm.nih.gov / nucleotide / ), and prepared using publicly known genetic engineering methods.

[0314] In addition, in the present invention, in order that the DNA encoding each enzyme is carried out gene import to cell, the vector containing this DNA can be used. As such a vector, for example, plasmid vectors, viral vectors etc. can be enumerated. Those skilled in the art can suitably select the kind of vector according to the kind and purpose of host cell. In the vector of the present invention, except the DNA encoding the above-mentioned enzyme, cis elements such as promoter, enhancer, splicing signal, poly A additional signal, ribosome binding sequence (SD sequence), selective marker gene, reporter gene etc. can also be included, in addition, they can also be modified.

[0315] The vector of the present invention is not limited as long as it can express the enzyme used in the present invention in the host cell. For example, a vector capable of expressing two or more enzyme genes can be used. Examples of such vectors, in the case of plasmid vectors, include, but are not limited to, pET Duet-1, pCOLA Duet-1, pACYC Duet-1, and pCDF Duet-1 (Novagen).

[0316] In the present invention, a vector may contain DNA encoding one or more enzymes. For example, a vector may contain DNA encoding FtfL, Fch, and MtdA, DNA encoding MetF, MetH, and MetK, DNA encoding Mtn and Lux S, or DNA encoding MetF, MetH, MetK, Mtn, and Lux S.

[0317] In the present invention, for example, by introducing DNA encoding FtfL, Fch, and MtdA into cells, a formate immobilization pathway for producing 5-methyltetrahydrofolate from formate can be introduced into the cells. That is, in the present invention, cells having a formate immobilization pathway can be used.

[0318] (5) Regeneration method of SAM using cells

[0319] The method for regenerating S-adenosylmethionine of the present invention can be carried out using (in cells) cells modified to enhance the activity or expression of each enzyme as described in (4) above. That is, the present invention provides a method for regenerating SAM using cells.

[0320] In the present invention, the method for regenerating SAM using cells may include, for example, the following steps.

[0321] (a) a step of preparing cells having a formate immobilization pathway and culturing them; and

[0322] (b) A step of adding formic acid or a salt thereof to the culture medium of the cells obtained in step (a) and further culturing the cells.

[0323] In step (a) above, "cells having a formate immobilization pathway" can be produced by, for example, introducing DNA encoding FtfL, Fch, and MtdA into cells using known gene transfer methods. Specifically, in the present invention, "cells having a formate immobilization pathway" include, for example, cells containing DNA encoding FtfL, Fch, and MtdA. Cell culture conditions (temperature, time, medium composition, etc.) can be appropriately determined by those skilled in the art depending on the cell type.

[0324] The "formic acid or its salt" in the step (b) is as described above in "(1) Formic acid." In the step (b), a methylation target substance (a substance to be methylated) may be further added.

[0325] The reaction conditions (temperature, reaction time, pH, etc.) of the enzyme used in the present invention are as described above.

[0326] In the present invention, the SAM regeneration method using cells can be performed, for example, as follows when Escherichia coli is used as the cells, but is not limited thereto.

[0327] First, DNA encoding each enzyme is introduced into Escherichia coli as a host cell to produce a transformant. The transformant is cultured overnight at about 37°C using a known culture medium (e.g., LB culture medium). Next, the proliferated transformant is suspended in a minimal culture medium (e.g., M9 culture medium), cultured at about 30°C for an appropriate time (e.g., 6 hours), and then induced to express the gene using IPTG or the like, and further cultured for an appropriate time (e.g., 16 hours). Thus, a transformant in which the activity or expression of each enzyme is enhanced can be produced.

[0328] Next, the transformant in which the activity or expression of each enzyme is enhanced is brought into contact with a formic acid-containing solution (reaction buffer) (for example, the transformant is suspended in a formic acid-containing solution), a methylation target substance is added thereto, and the culture is further incubated at an appropriate temperature (for example, about 30° C.) and time (for example, about 6 to 24 hours), thereby causing a methylation reaction to produce a methyl compound, and SAM can be regenerated from SAH produced by demethylation of SAM.

[0329] The formic acid-containing solution may contain, in addition to formic acid, for example, a buffer and glucose as an ATP supply source, and may further contain a divalent metal salt (eg, magnesium sulfate).

[0330] In the present invention, whether SAM is produced can be evaluated by those skilled in the art using known methods, such as a method of measuring methyltransferase activity in a test sample, as described in the above-mentioned "(3) Method for Regenerating S-adenosylmethionine".

[0331] 2-2. Method for regenerating S-adenosylmethionine using methanol

[0332] (1) Methanol

[0333] Methanol is a type of alcohol and is a compound represented by the chemical formula CH3OH. In the present invention, methanol is converted into formaldehyde.

[0334] (2)S -Adenosylmethionine

[0335] S-adenosylmethionine is as described in the above-mentioned "2-1. (2)".

[0336] (3) Method for regenerating S-adenosylmethionine

[0337] The "regeneration" of SAM, the evaluation of whether SAM is produced, the control, the evaluation of methyltransferase activity, and the term "higher than the control" in the present invention are as described in "2-1. (3)" above.

[0338] The method for regenerating S-adenosylmethionine of the present invention may include, for example, the following steps.

[0339] (a) a process of converting methanol into formaldehyde, and then converting the formaldehyde into formic acid;

[0340] (b) a step of producing 5-methyltetrahydrofolate from the formic acid and tetrahydrofolate;

[0341] (c) a step of converting S-adenosylhomocysteine produced by demethylating S-adenosylmethionine into homocysteine; and

[0342] (d) A step of transferring the methyl group of the 5-methyltetrahydrofolate to the homocysteine to generate methionine, and converting the methionine into S-adenosylmethionine.

[0343] Step (a) is a step of converting methanol into formaldehyde and producing formic acid from the formaldehyde. Figure 2 ).

[0344] Step (a) may include, for example, (i) a step of converting methanol into formaldehyde and (ii) a step of converting formaldehyde into formic acid.

[0345] In step (i), the conversion of methanol to formaldehyde can be carried out in the presence of, for example, methanol dehydrogenase (Medh) and / or methanol oxidase (Mox) or by these enzymes.

[0346] Examples of step (ii) include: (ii-1) a step of directly converting formaldehyde into formic acid; or (ii-2) a step of generating hydroxymethylglutathione (HM-GSH) by a spontaneous reaction between formaldehyde and glutathione (GSH), converting HM-GSH into S-formylglutathione, and converting S-formylglutathione into formic acid.

[0347] In step (ii-1), the conversion of formaldehyde to formic acid can be performed in the presence of formaldehyde dehydrogenase (FdhA) or by the enzyme, for example.

[0348] In step (ii-2), the production of hydroxymethylglutathione (HM-GSH) is carried out by a spontaneous reaction between formaldehyde and glutathione (GSH). The conversion of HM-GSH to S-formylglutathione can be carried out, for example, in the presence of S-hydroxymethylglutathione dehydrogenase (FrmA) or by this enzyme, and the conversion of S-formylglutathione to formic acid can be carried out, for example, in the presence of S-formylglutathione hydrolase (FrmB) or by this enzyme.

[0349] That is, in step (a), the conversion of formaldehyde into formic acid can be performed in the presence of formaldehyde dehydrogenase (FdhA) or by the enzyme, for example.

[0350] In another embodiment, in step (a), the conversion of formaldehyde to formic acid can be carried out, for example, after the spontaneous reaction of formaldehyde and glutathione, in the presence of S-hydroxymethylglutathione dehydrogenase (FrmA) and S-formylglutathione hydrolase (FrmB) or by these enzymes.

[0351] Step (b) is a step of producing 5-methyltetrahydrofolate (5-CH 3 -THF) from formic acid and tetrahydrofolate (THF).

[0352] Step (b) may include, for example: (i) a step of generating 10-formyltetrahydrofolate (10-CHO-THF) from formic acid and tetrahydrofolate (THF); (ii) a step of generating 5,10-methylenetetrahydrofolate (5,10-CH═THF) from 10-CHO-THF; (iii) a step of generating 5,10-methylenetetrahydrofolate (5,10-CH2-THF) from 5,10-CH═THF; and (iv) a step of generating 5-methyltetrahydrofolate (5-CH3-THF) from 5,10-CH2-THF.

[0353] In step (i), the production of 10-CHO-THF from formic acid and tetrahydrofolate (THF) can be carried out, for example, in the presence of or by formate-tetrahydrofolate ligase (FtfL).

[0354] In step (ii), the production of 5,10-CH=THF from 10-CHO-THF can be carried out, for example, in the presence of or by methylene-tetrahydrofolate cyclohydrolase (Fch).

[0355] In step (iii), the production of 5,10-CH2-THF from 5,10-CH=THF can be carried out, for example, in the presence of or by methylenetetrahydrofolate dehydrogenase (MtdA).

[0356] In step (iv), the production of 5-CH 3 -THF from 5,10-CH 2 -THF can be carried out, for example, in the presence of 5,10-methylenetetrahydrofolate reductase (MetF) or by the enzyme.

[0357] That is, in step (b), the production of 5-methyltetrahydrofolate from formate and tetrahydrofolate can be carried out in the presence of, for example, formate-tetrahydrofolate ligase (FtfL), methenyl-tetrahydrofolate cyclohydrolase (Fch), methylene-tetrahydrofolate dehydrogenase (MtdA), and 5,10-methylene-tetrahydrofolate reductase (MetF), or by these enzymes.

[0358] Step (c) is a step of converting S-adenosylhomocysteine (SAH) generated by demethylation of S-adenosylmethionine (SAM) into homocysteine (Hcy). Figure 2 ).

[0359] Step (c) may include, for example, (i) a step of converting S-adenosylhomocysteine (SAH) into S-ribosylhomocysteine (SRH) and (ii) a step of converting SRH into homocysteine (Hcy).

[0360] In the above step (i), the conversion of S-adenosylhomocysteine (SAH) to S-ribosylhomocysteine (SRH) can be carried out in the presence of or by S-adenosylhomocysteine nucleosidase (Mtn), for example.

[0361] In the above step (ii), the conversion of SRH to homocysteine (Hcy) can be performed in the presence of, for example, S-ribosylhomocysteine lyase (LuxS) or by the enzyme.

[0362] That is, in step (c), the conversion of S-adenosylhomocysteine to homocysteine can be performed in the presence of S-adenosylhomocysteine nucleosidase (Mtn) and S-ribosylhomocysteine lyase (LuxS) or by these enzymes.

[0363] In another embodiment, step (c) may include, for example, (iii) a step of converting S-adenosylhomocysteine (SAH) directly (without conversion to S-ribosylhomocysteine (SRH)) to homocysteine (Hcy).

[0364] In the above step (iii), the conversion of S-adenosylhomocysteine to homocysteine can be performed in the presence of S-adenosylhomocysteine hydrolase (SahH) or by the enzyme.

[0365] In step (c), demethylation of S-adenosylmethionine (SAM) can be performed, for example, in the presence of an S-adenosylmethionine-dependent methyltransferase or by the enzyme.

[0366] S-adenosylmethionine-dependent methyltransferase is as described in the above-mentioned "2-1(3)".

[0367] Step (d) is a step in which the methyl group of 5-methyltetrahydrofolate produced in step (b) is transferred to homocysteine (Hcy) converted from S-adenosylhomocysteine (SAH) in step (c), thereby producing methionine, and converting the produced methionine into S-adenosylmethionine (SAM). This step regenerates SAM.

[0368] Step (d) may include, for example: (i) a step of generating methionine by transferring the methyl group of 5-methyltetrahydrofolate generated in step (b) to homocysteine (Hcy) converted from S-adenosylhomocysteine (SAH) in step (c); and (ii) a step of converting the generated methionine into S-adenosylmethionine (SAM).

[0369] In the above step (i), the production of methionine from homocysteine can be carried out, for example, in the presence of methionine synthetase (MetH or MetE) or by the enzyme.

[0370] In the above step (ii), the conversion of methionine to S-adenosylmethionine can be performed in the presence of, for example, methionine adenosyltransferase (MetK) or by the enzyme.

[0371] That is, the production of methionine from homocysteine can be carried out in the presence of or by methionine synthetase (MetH or MetE), and the conversion of methionine to S-adenosylmethionine can be carried out in the presence of or by methionine adenosyltransferase (MetK). Methionine synthetase can be either cobalamin-dependent (MetH) or cobalamin-independent (MetE).

[0372] In the above step (ii), the conversion of methionine to S-adenosylmethionine requires adenosine triphosphate (ATP), and the ATP can be synthesized by a step comprising a reaction of at least one enzyme selected from the group consisting of ribose diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt) and adenosine kinase (ADK).

[0373] Ribose-5-phosphate (R5P), a metabolite of the pentose phosphate pathway, can be synthesized into phosphoribosyl pyrophosphate (PRPP) by ribose-5-phosphate kinase (Prs), and adenosine monophosphate (AMP) can be synthesized from PRPP and adenine by adenine phosphoribosyltransferase (Apt). In addition, adenosine monophosphate (AMP) can be synthesized from adenosine by adenosine kinase (ADK). AMP can be converted into ATP through further phosphorylation.

[0374] Adenine is a metabolite produced by the Mtn reaction in step (b), and adenosine is a metabolite produced by the SahH reaction in step (b). By synthesizing ATP using these metabolites as substrates, the ATP required for the synthesis of SAM can be efficiently supplied.

[0375] When cells are used for regeneration of SAM, the enzyme used in the method of the present invention may be an enzyme existing in the cells, or may be an enzyme expressed by introducing foreign DNA encoding the enzyme into the cells.

[0376] The enzyme reaction conditions (temperature, reaction time, pH, etc.) in the method of the present invention are the same as those described in the above-mentioned "2-1(3)".

[0377] In another embodiment, the method for regenerating S-adenosylmethionine of the present invention may include the following steps, for example.

[0378] (a) a process for converting methanol into formaldehyde;

[0379] (b) a step of producing 5-methyltetrahydrofolate from the formaldehyde and tetrahydrofolate;

[0380] (c) a step of converting S-adenosylhomocysteine produced by demethylating S-adenosylmethionine into homocysteine; and

[0381] (d) A step of transferring the methyl group of the 5-methyltetrahydrofolate to the homocysteine to generate methionine, and converting the methionine into S-adenosylmethionine.

[0382] Step (a) is a step of converting methanol into formaldehyde.

[0383] In step (a), the conversion of methanol to formaldehyde can be carried out in the presence of, for example, methanol dehydrogenase (Medh) and / or methanol oxidase (Mox) or by these enzymes.

[0384] Step (b) is a step of producing 5-methyltetrahydrofolate (5-CH 3 -THF) from formaldehyde and tetrahydrofolic acid.

[0385] Step (b) may include, for example: (i) a step of generating 5,10-methylenetetrahydrofolate (5,10-CH2-THF) from formaldehyde and tetrahydrofolate; and (ii) a step of generating 5-methyltetrahydrofolate (5-CH3-THF) from 5,10-CH2-THF.

[0386] In step (i), the formation of 5,10-CH2-THF from formaldehyde and tetrahydrofolic acid can be carried out by a spontaneous reaction of formaldehyde and tetrahydrofolic acid.

[0387] In step (ii), the production of 5-CH 3 -THF from 5,10-CH 2 -THF can be carried out, for example, in the presence of or by 5,10-methylenetetrahydrofolate reductase (MetF).

[0388] That is, in step (b), the production of 5-CH 3 -THF from formaldehyde and tetrahydrofolate can be carried out, for example, by a spontaneous reaction of formaldehyde and tetrahydrofolate in the presence of 5,10-methylenetetrahydrofolate reductase (MetF) or by these enzymes.

[0389] Regarding each process of the present invention, any person skilled in the art can carry out it in a cell-free system by a known method (Microbial Cell Factories, Issue 11, No. 120, September 2012, Journal of Bioscience and Bioengineering, Issue 129, Article 3, March 2020, Pages 269-275).

[0390] Steps (c) and (d) are as described above.

[0391] (4) cell

[0392] The types of cells that can be used in the method for regenerating S-adenosylmethionine of the present invention are as described in "2-1(4)" above.

[0393] In the present invention, cells modified so that the activity or expression of S-adenosylmethionine-dependent methyltransferase is enhanced can be used.

[0394] Furthermore, in the present invention, cells modified so that the activity or expression of formate-tetrahydrofolate ligase (FtfL) and SAM-dependent methyltransferase is enhanced can be used.

[0395] In the present invention, cells modified so as to enhance the activity or expression of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox) in addition to FtfL and SAM-dependent methyltransferase can also be used.

[0396] In the present invention, cells modified so that the activity or expression of the following enzymes (i) and / or (ii) is enhanced in addition to FtfL and SAM-dependent methyltransferase can be used:

[0397] (i) Medh and / or Mox,

[0398] (ii) at least one or more enzymes selected from the group consisting of formaldehyde dehydrogenase (FdhA), S-hydroxymethylglutathione dehydrogenase (FrmA), and S-formylglutathione hydrolase (FrmB) (e.g., FdhA alone; FrmA and FrmB; FdhA, FrmA, and FrmB; etc.).

[0399] In the present invention, cells modified to enhance the activity or expression of at least one enzyme selected from the group consisting of the following (i) to (iii) in addition to FtfL and SAM-dependent methyltransferase can be used:

[0400] (i) Medh and / or Mox,

[0401] (ii) at least one or more enzymes selected from the group consisting of FdhA, FrmA, and FrmB (e.g., only FdhA; FrmA and FrmB; FdhA, FrmA, and FrmB; etc.), and

[0402] (iii) Methylenetetrahydrofolate cyclohydrolase (Fch) and / or methylenetetrahydrofolate dehydrogenase (MtdA).

[0403] In the present invention, cells modified to enhance the activity or expression of at least one enzyme selected from the group consisting of the following (i) to (iv) in addition to FtfL and SAM-dependent methyltransferase can be used:

[0404] (i) Medh and / or Mox,

[0405] (ii) at least one or more enzymes selected from the group consisting of FdhA, FrmA, and FrmB (e.g., only FdhA; FrmA and FrmB; FdhA, FrmA, and FrmB; etc.),

[0406] (iii) Fch and / or MtdA, and

[0407] (iv) at least one or more enzymes selected from the group consisting of S-adenosylhomocysteine nucleosidase (Mtn), S-ribosylhomocysteine lyase (LuxS), and S-adenosylhomocysteine hydrolase (SahH) (e.g., only SahH; Mtn and Lux; etc.).

[0408] In the present invention, cells modified to enhance the activity or expression of at least one enzyme selected from the group consisting of the following (i) to (v) in addition to FtfL and SAM-dependent methyltransferase can be used:

[0409] (i) Medh and / or Mox,

[0410] (ii) at least one or more enzymes selected from the group consisting of FdhA, FrmA, and FrmB (e.g., only FdhA; FrmA and FrmB; FdhA, FrmA, and FrmB; etc.),

[0411] (iii) Fch and / or MtdA,

[0412] (iv) at least one enzyme selected from the group consisting of Mtn, LuxS, and SahH (e.g., only SahH; Mtn and Lux; etc.), and

[0413] (v) At least one or more enzymes selected from the group consisting of Prs, Apt, and ADK (eg, Prs and Apt; ADK alone; etc.).

[0414] In the present invention, cells modified to enhance the activity or expression of at least one enzyme selected from the group consisting of the following (i) to (vi) in addition to FtfL and SAM-dependent methyltransferase can be used:

[0415] (i) Medh and / or Mox,

[0416] (ii) at least one or more enzymes selected from the group consisting of FdhA, FrmA, and FrmB (e.g., only FdhA; FrmA and FrmB; FdhA, FrmA, and FrmB; etc.),

[0417] (iii) Fch and / or MtdA,

[0418] (iv) at least one enzyme selected from the group consisting of Mtn, LuxS, and SahH (e.g., only SahH; Mtn and Lux; etc.),

[0419] (v) at least one enzyme selected from the group consisting of Prs, Apt, and ADK (e.g., Prs and Apt; ADK alone; etc.),

[0420] (vi) At least one or more enzymes selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthetase (MetH or MetE), and methionine adenosyltransferase (MetK).

[0421] In the present invention, cells modified to enhance the activity or expression of at least one enzyme selected from the group consisting of the following (i) to (iv) in addition to FtfL and SAM-dependent methyltransferase can be used:

[0422] (i) Medh and / or Mox,

[0423] (ii) at least one or more enzymes selected from the group consisting of FdhA, FrmA, and FrmB (e.g., only FdhA; FrmA and FrmB; FdhA, FrmA, and FrmB; etc.),

[0424] (iii) Fch and / or MtdA, and

[0425] (iv) At least one enzyme selected from the group consisting of MetF, methionine synthetase (MetH or MetE), and MetK.

[0426] In another embodiment, in the present invention, cells modified so as to enhance the activity or expression of Medh and / or Mox in addition to SAM-dependent methyltransferase can be used.

[0427] In the present invention, cells modified in such a manner that the activity or expression of (i) Medh and / or Mox, and / or (ii) at least one or more enzymes selected from the group consisting of Mtn, LuxS and SahH (e.g., only SahH; Mtn and Lux; etc.) are enhanced in addition to SAM-dependent methyltransferases can be used.

[0428] The present invention further includes the following (i) to (iii) in addition to the SAM-dependent methyltransferase:

[0429] (i) Medh and / or Mox, (ii) at least one or more enzymes selected from the group consisting of Mtn, LuxS and SahH (e.g., only SahH; Mtn and Lux; etc.), and (iii) at least one or more enzymes selected from the group consisting of Prs, Apt and ADK (e.g., Prs and Apt, only ADK; etc.).

[0430] In the present invention, cells modified to enhance the activity or expression of at least one enzyme selected from the group consisting of the following (i) to (iv) in addition to the SAM-dependent methyltransferase can be used:

[0431] (i) Medh and / or Mox, (ii) at least one or more enzymes selected from the group consisting of Mtn, LuxS and SahH (e.g., only SahH; Mtn and Lux; etc.), (iii) at least one or more enzymes selected from the group consisting of Prs, Apt and ADK (e.g., Prs and Apt; only ADK; etc.), and (iv) at least one or more enzymes selected from the group consisting of MetF, methionine synthase (MetH or MetE) and MetK.

[0432] In this specification, the term "modified so that the activity or expression of a certain enzyme is enhanced" and cells modified so that the activity or expression of a SAM-dependent methyltransferase is enhanced are as described in "2-1(4)" above.

[0433] Examples of cells modified so that the activity or expression of each of FtfL, Medh, Mox, FdhA, FrmA, FrmB, Fch, MtdA, Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, and MetK is enhanced include cells containing DNA encoding FtfL, Medh, Mox, FdhA, FrmA, FrmB, Fch, MtdA, Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, and MetK, respectively.

[0434] In the present invention, "modified cells" and cells containing DNA encoding each enzyme may also be referred to as "transformants."

[0435] Regarding the DNAs encoding SAM-dependent methyltransferase, FtfL, Medh, Mox, FdhA, FrmA, FrmB, Fch, MtdA, Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE and MetK, the base sequence information can be obtained from publicly known databases such as NCBI (https: / / www.ncbi.nlm.nih.gov / nucleotide / ) and prepared using publicly known genetic engineering methods.

[0436] In addition, the vector that can be used in the present invention is as described in the above-mentioned "2-1(4)".

[0437] In the present invention, a vector may contain DNA encoding one or more enzymes. For example, a vector may include DNA encoding FtfL, Fch, and MtdA, DNA encoding MetF, MetH, and MetK, DNA encoding Mtn and Lux S, DNA encoding MetF, MetH, MetK, Mtn, and Lux S, DNA encoding Prs and Apt, DNA encoding Prs, Apt, and ADK, or DNA encoding ADK.

[0438] In the present invention, for example, by introducing DNA encoding Medh and / or Mox into cells, a reaction system for converting methanol into formaldehyde can be introduced into the cells. As DNA encoding Medh, for example, the mdh2 gene from Cupriavidus necator can be cited. In addition, it is known that by introducing mutations into the mdh2 gene, Medh can be modified in a manner that improves the substrate specificity and activity of Medh for methanol (Appl Microbiol Biotechnol. 2016 Jun; 100(11): 4969-83.), and such mutant genes can also be used.

[0439] In addition, in the present invention, by introducing into cells, for example, (i) DNA encoding FdhA and / or (ii) DNA encoding FrmA and FrmB in addition to DNA encoding Medh and / or Mox, a reaction system that enhances the reaction of converting formaldehyde from methanol into formic acid can be introduced into the cells.

[0440] Furthermore, in the present invention, for example, by introducing DNA encoding (iii) FtfL, Fch, and MtdA into cells in addition to DNA encoding Medh and / or Mox, (i) FdhA, and / or (ii) FrmA and FrmB, a formate immobilization pathway for producing 5-methyltetrahydrofolate from formate converted from formaldehyde can be introduced into the cells. That is, cells having a formate immobilization pathway can be used in the present invention.

[0441] (5) SAM regeneration method using cells

[0442] The method for regenerating S-adenosylmethionine of the present invention can be carried out using (in cells) cells modified to enhance the activity or expression of each enzyme as described in (4) above. That is, the present invention provides a method for regenerating SAM using cells.

[0443] In the present invention, the method for regenerating SAM using cells may include, for example, the following steps.

[0444] (a) a step of preparing and culturing cells comprising the following DNAs: (i) DNA encoding Medh and / or Mox, (ii-1) DNA encoding FdhA, (ii-2) DNA encoding FrmA and FrmB, (iii) DNA encoding FtfL, Fch, and MtdA; and

[0445] (b) A step of adding methanol to the culture medium of the cells obtained in step (a) and further culturing the cells.

[0446] In the above method, the culture conditions of the cell (temperature, time, composition of the culture medium, etc.) can be appropriately set according to the type of the cell as long as those skilled in the art. In addition, the reaction conditions of the enzyme used in the present invention (temperature, reaction time, pH, etc.) are as described above.

[0447] In the present invention, the method for regenerating SAM using cells can be performed, for example, as follows when Escherichia coli is used as the cells, but is not limited thereto.

[0448] First, DNA encoding each enzyme is introduced into Escherichia coli as a host cell to produce a transformant. The transformant is cultured overnight at about 37°C using a known culture medium (e.g., LB culture medium). Next, the proliferated transformant is suspended in a minimal culture medium (e.g., M9 culture medium), cultured at about 30°C for an appropriate time (e.g., 6 hours), and then induced to express the gene using IPTG or the like, and further cultured for an appropriate time (e.g., 16 hours). Thus, a transformant in which the activity or expression of each enzyme is enhanced can be produced.

[0449] Next, the transformant in which the activity or expression of each enzyme is enhanced is brought into contact with a methanol-containing solution (reaction buffer) (for example, the transformant is suspended in a methanol-containing solution), a methylation target substance is added thereto, and the culture is further incubated at an appropriate temperature (for example, about 30°C) and time (for example, about 6 to 24 hours), thereby causing a methylation reaction to produce a methyl compound, and SAM can be regenerated from SAH produced by demethylation of SAM.

[0450] The methanol-containing solution may contain, in addition to methanol, for example, a buffer and glucose as an ATP supply source, and may further contain a divalent metal salt (eg, magnesium sulfate).

[0451] In the present invention, whether SAM is produced can be evaluated by those skilled in the art using known methods, such as a method of measuring methyltransferase activity in a test sample, as described in the above-mentioned "(3) Method for Regenerating S-adenosylmethionine".

[0452] 3. Method for producing methyl compound

[0453] (1) Methyl compounds

[0454] In the present invention, "methyl compound" refers to a compound having a methyl group. Specifically, the methyl compound in the present invention is, for example, a compound having a methyl group from SAM (transferred from SAM). In the present invention, the methyl compound is produced by, for example, transferring the methyl group of SAM to a methylated target substance through the action of a SAM-dependent methyltransferase. That is, the methyl compound in the present invention is not limited as long as it is a substance generated or capable of being generated in the presence of SAM. As a methyl compound generated or capable of being generated in the presence of SAM, for example, a derivative of an aromatic amino acid can be mentioned. As an aromatic amino acid, for example, tyrosine, histidine, tryptophan, phenylalanine, etc. can be mentioned. As a methyl compound of a derivative of an aromatic amino acid, for example, 3-methyltyrosine, histidine betaine, ergothioneine, melatonin, vanillic acid, vanillin, isovanillic acid, ferulic acid, isoferulic acid, pinostearyl, pterostilbene, annonine, thebaine, codeine, magnolamine, berberine, formononetol, pinitol, etc. can be mentioned, but it is not limited to these.

[0455] In one embodiment of the present invention, the methyl compound may be, for example, at least one selected from the group consisting of 3-methyltyrosine, histidine betaine, ergothioneine, melatonin, annonine, vanillic acid, vanillin, isovanillic acid, ferulic acid, isoferulic acid, pinostearyl, pterostilbene, thebaine, codeine, magnolamine, berberine, formononetol, and pinitol.

[0456] In another embodiment of the present invention, the methyl compound may be at least one selected from the group consisting of, for example, 3-methyltyrosine, histidine betaine, ergothioneine, melatonin, and annonine.

[0457] In another embodiment of the present invention, the methyl compound may be at least one selected from the group consisting of, for example, 3-methyltyrosine, histidine betaine, ergothioneine, and melatonin.

[0458] (2-1) Method for producing methyl compounds-1

[0459] The method for producing the methyl compound of the present invention may include, for example, the following steps.

[0460] In the presence of formic acid or a salt thereof,

[0461] (a) a step of producing 5-methyltetrahydrofolate from formic acid or a salt thereof and tetrahydrofolate,

[0462] (b) a step of converting S-adenosylhomocysteine produced by demethylation of S-adenosylmethionine into homocysteine,

[0463] (c) a step of transferring a methyl group of 5-methyltetrahydrofolate to homocysteine to generate methionine, and converting the methionine into S-adenosylmethionine, and

[0464] (d) A step of performing a methylation reaction using S-adenosylmethionine as a methyl donor to produce a methyl compound.

[0465] Steps (a) to (c) are as described in the above-mentioned "(3) Method for regenerating S-adenosylmethionine".

[0466] Step (d) is a step of performing a methylation reaction using S-adenosylmethionine (SAM) as a methyl donor to produce a methyl compound. Here, the "S-adenosylmethionine" in step (d) may be produced in step (c) or by other methods.

[0467] Step (d) may include, for example, a step of generating a methyl compound from the methylated target substance. The generation of a methyl compound from the methylated target substance can be performed, for example, by transferring a methyl group from S-adenosylmethionine (SAM) to the methylated target substance in the presence of or by a SAM-dependent methyltransferase.

[0468] The method for producing the methyl compound of the present invention may further include the step (e): a step of converting methanol into formaldehyde and then converting the formaldehyde into formic acid.

[0469] In the step (e), the conversion of methanol to formaldehyde can be carried out in the presence of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox).

[0470] In the step (e), the conversion of formaldehyde to formic acid can be carried out in the presence of formaldehyde dehydrogenase (FdhA).

[0471] In the step (e), the conversion of formaldehyde to formic acid can be carried out by a spontaneous reaction between formaldehyde and glutathione in the presence of S-hydroxymethylglutathione dehydrogenase (FrmA) and S-formylglutathione hydrolase (FrmB).

[0472] The method for producing the methyl compound of the present invention can be carried out in a cell-free system by a known method using the enzymes described above.

[0473] The method for producing a methyl compound of the present invention can be carried out using (in cells) cells modified to enhance the activity or expression of each enzyme as described in "2. (4)" above. That is, the present invention provides a method for producing a methyl compound using cells.

[0474] In the present invention, the method for producing a methyl compound using cells may include the following steps, for example.

[0475] (a) a step of preparing cells having a formate immobilization pathway and culturing them; and

[0476] (b) A step of adding formic acid or a salt thereof and the methylation target substance to the culture medium of the cells obtained in step (a), further culturing the cells, and recovering the methyl compound.

[0477] In the above step (a), the "cells having a formic acid immobilization pathway" are as described in the above "2. (5) SAM regeneration method using cells".

[0478] The "formic acid or its salt" in the step (b) is as described in the above "2. (1) Formic acid". In addition, the step (b) may further include a step of purifying the generated methyl compound.

[0479] The recovery and purification of the methyl compound produced in step (b) can be carried out by those skilled in the art by known methods suitable for the physical properties of the methyl compound, such as distillation, membrane dehydration, desalination using an ion exchange resin, crystallization, column chromatography, and the like.

[0480] (2-2) Method for producing methyl compounds-2

[0481] In another embodiment, the method for producing the methyl compound of the present invention may include the following steps, for example.

[0482] (a) a process of converting methanol into formaldehyde, and then converting the formaldehyde into formic acid;

[0483] (b) a step of producing 5-methyltetrahydrofolate from the formic acid and tetrahydrofolate;

[0484] (c) a step of converting S-adenosylhomocysteine generated by demethylating S-adenosylmethionine into homocysteine;

[0485] (d) a step of transferring the methyl group of the 5-methyltetrahydrofolate to the homocysteine to generate methionine, and converting the methionine into S-adenosylmethionine;

[0486] (e) A step of performing a methylation reaction using the S-adenosylmethionine produced in the step (d) as a methyl donor to produce a methyl compound.

[0487] Steps (a) to (d) are as described in the above-mentioned "(3) Method for regenerating S-adenosylmethionine".

[0488] Step (e) is a step of performing a methylation reaction using the generated S-adenosylmethionine (SAM) as a methyl donor to generate a methyl compound.

[0489] Step (e) may include, for example, a step of generating a methyl compound from the methylated target substance. The generation of a methyl compound from the methylated target substance can be performed, for example, by transferring a methyl group from S-adenosylmethionine (SAM) to the methylated target substance in the presence of or by a SAM-dependent methyltransferase.

[0490] The method for producing a methyl compound of the present invention can be carried out using (in cells) cells modified to enhance the activity or expression of each enzyme described in "2. (4)" above. That is, the present invention provides a method for producing a methyl compound using cells.

[0491] In the present invention, the method for producing a methyl compound using cells may include the following steps, for example.

[0492] (a) a step of preparing and culturing cells comprising the following DNAs: (i) DNA encoding Medh and / or Mox, (ii-1) DNA encoding FdhA, (ii-2) DNA encoding FrmA and FrmB, (iii) DNA encoding FtfL, Fch, and MtdA,

[0493] (b) A step of adding methanol and the methylation target substance to the culture medium of the cells obtained in step (a), further culturing the cells, and recovering the methyl compound.

[0494] The step (b) may further include a step of purifying the generated methyl compound.

[0495] The recovery and purification of the methyl compound produced in step (b) can be carried out by those skilled in the art by known methods suitable for the physical properties of the methyl compound, such as distillation, membrane dehydration, desalination using an ion exchange resin, crystallization, column chromatography, and the like.

[0496] In the present invention, the method for producing a methyl compound using cells can be carried out, for example, as follows when Escherichia coli is used as the cell, but the method is not limited thereto.

[0497] First, DNA encoding each enzyme is introduced into Escherichia coli as a host cell to produce a transformant. The transformant is cultured overnight at about 37°C using a known culture medium (e.g., LB culture medium). Next, the proliferated transformant is suspended in a minimal culture medium (e.g., M9 culture medium), cultured at about 30°C for an appropriate time (e.g., 6 hours), and then induced to express the gene using IPTG or the like, and further cultured for an appropriate time (e.g., 16 hours). Thus, a transformant in which the activity or expression of each enzyme is enhanced can be produced.

[0498] Next, the transformant in which the activity or expression of each enzyme is enhanced is brought into contact with a formic acid solution or a methanol solution (reaction buffer) (for example, the transformant is suspended in a formic acid solution or a methanol solution), to which the methylation target substance is added, and the culture is further incubated at an appropriate temperature (for example, about 30°C) and for an appropriate time (for example, about 6 to 24 hours), thereby causing a methylation reaction to produce a methyl compound.

[0499] The formic acid-containing solution and the methanol-containing solution may contain, for example, a buffer and glucose as an ATP supply source, and may further contain a divalent metal salt (eg, magnesium sulfate), in addition to formic acid or methanol, respectively.

[0500] 4. Method for producing organic compounds

[0501] In the present invention, an organic compound can be produced using a methyl compound produced by the SAM regeneration method or the methyl compound production method of the present invention as an intermediate. Specifically, the present invention provides a method for producing an organic compound, comprising the step of producing the organic compound using a methyl compound produced by the SAM regeneration method or the methyl compound production method of the present invention as an intermediate.

[0502] In the present invention, an organic compound refers to a compound produced by using a methyl compound as an intermediate. Examples of the methyl compound used as an intermediate include those produced in the SAM regeneration method or the methyl compound production method of the present invention.

[0503] The production of an organic compound using a methyl compound as an intermediate can be carried out by those skilled in the art based on a known method, and the production conditions can also be appropriately set by those skilled in the art.

[0504] As an organic compound that can be generated by using a methyl compound as an intermediate, for example, o-cresol, 2,6-xylenol, cumic acid, terephthalic acid, ergothioneine, etc. can be mentioned. Tyrosine is methylated by the regeneration method of the SAM of the present invention or the manufacturing method of the methyl compound to generate 3-methyltyrosine and 3,5-dimethyltyrosine, and then, these methyl compounds are respectively recognized as substrates using a lyase, thereby being able to be converted into o-cresol and 2,6-xylenol. In addition, perillic acid can be methylated by the regeneration method of the SAM of the present invention or the manufacturing method of the methyl compound to generate perillic acid methyl ester, and then, it is converted into cumic acid and further converted into terephthalic acid by a known chemical synthesis reaction (Chemistry Open.2018Feb;7(2):201-203.). Furthermore, L-histidine can be methylated by the regeneration method of the SAM of the present invention or the manufacturing method of the methyl compound to generate ergothioneine as an organic compound by using histidine betaine as an intermediate.

[0505] Hereinafter, the present invention will be described in detail with reference to the Examples, but the present invention is not limited to these Examples. In the following Examples, "enhancement" means that the activity or expression of an enzyme is enhanced.

[0506] Example

[0507] [Example 1]

[0508] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene, ftfL gene, fch gene, and mtdA gene was enhanced as follows.

[0509] (A) Construction of SfmM2-enhanced plasmid

[0510] A DNA (base sequence of synthetic DNA: sequence number 1) containing the base sequence of the sfmM2 gene from Streptomyces lavendulae encoding tyrosine 3C-methyltransferase (a recognition sequence for the restriction enzyme Nde I is assigned to the 5' end side, and a recognition sequence for the restriction enzyme Bgl II is assigned to the 3' end side) is synthesized. It should be noted that a sequence with optimized codons is designed for efficient expression in Escherichia coli as the base sequence of the sfmM2 gene. The synthesized DNA is inserted into the restriction enzyme sites Nde I and Bgl II of the expression vector pET Duet-1 (Novagen) for Escherichia coli and connected to the downstream of the T7 promoter. The constructed plasmid is named pET_P T7 -sfmM2.

[0511] (B) Construction of FtfL, Fch, and MtdA Enhanced Plasmids

[0512] A DNA containing the base sequence of the ftfL gene, fch gene and mtdA gene from Methylobacterium extorquens (the recognition sequence of the restriction enzyme Nde I is assigned to the 5' end side, and the recognition sequence of the restriction enzyme Bgl II is assigned to the 3' end side) was synthesized (base sequence of the synthetic DNA: sequence number 2). It should be noted that for the base sequence containing the ftfL gene, fch gene and mtdA gene, a sequence with optimized codons was designed for efficient expression in Escherichia coli. The synthesized DNA was inserted into the restriction enzyme sites Nde I and Bgl II of the expression vector pCOLA Duet-1 (Novagen) for Escherichia coli and connected to the downstream of the T7 promoter. The constructed plasmid was named pCOLA_P T7 -ftfL-fch-mtdA.

[0513] (C) Production of SfmM2, FtfL, Fch, and MtdA-enhanced strains

[0514] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA and pCDFDuet-1 (Novagen) were used for transformation, and the resulting strain was designated HMS174 (DE3) / pET_P T7 -sfmM2 / pCOLA_P T7 -ftfL-fch-mtdA.

[0515] [Example 2]

[0516] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene, luxS gene, ftfL gene, fch gene, and mtdA gene was enhanced as follows.

[0517] (A) Construction of Mtn and LuxS enhanced plasmids

[0518] A DNA containing the base sequences of the mtn and luxS genes from Escherichia coli (with the recognition sequence for the restriction enzyme Nde I at the 5' end and the recognition sequence for the restriction enzyme Bgl II at the 3' end) was synthesized (base sequence of the synthetic DNA: SEQ ID NO: 3). The synthesized DNA was inserted into the restriction enzyme sites Nde I and Bgl II of the E. coli expression vector pCDF Duet-1 (Novagen) and ligated downstream of the T7 promoter. The constructed plasmid was named pCDF_P T7-mtn-luxS.

[0519] (B) Construction of LuxS-enhanced plasmid

[0520] The plasmid pCDF_P constructed in Example 2(A) was T7 -mtn-luxS was treated with the restriction enzyme Nde I, separated by agarose gel electrophoresis, and recovered from the gel and purified to obtain a DNA fragment in which the mtn gene region had been removed. The two ends of the obtained DNA fragment were connected by self-ligation. The constructed plasmid was named pCDF_P T7 -luxS.

[0521] (C) Production of SfmM2, LuxS, FtfL, Fch, and MtdA-enhanced strains

[0522] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 2(B) T7 -luxS was used for transformation, and the resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -luxS / pCOLA_P T7 -ftfL-fch-mtdA.

[0523] [Example 3]

[0524] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene, mtn gene, luxS gene, ftfL gene, fch gene, and mtdA gene was enhanced as follows.

[0525] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA and the plasmid pCDF_P constructed in Example 2(A) T7 -mtn-luxS transformation, and the resulting strain was designated HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -mtn-luxS / pCOLA_P T7 -ftfL-fch-mtdA.

[0526] [Example 4]

[0527] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene, sahH gene, ftfL gene, fch gene, and mtdA gene was enhanced as described below.

[0528] (A) Construction of SahH-enhanced plasmid

[0529] A DNA containing the base sequence of the sahH gene from Pseudomonas aeruginosa (a recognition sequence for the restriction enzyme Nde I is assigned to the 5' end side, and a recognition sequence for the restriction enzyme Bgl II is assigned to the 3' end side) was synthesized (base sequence of the synthetic DNA: sequence number 4). It should be noted that a sequence with optimized codons was designed as the base sequence of the sahH gene for efficient expression in Escherichia coli. The synthesized DNA was inserted into the restriction enzyme sites Nde I and Bgl II of the Escherichia coli expression vector pCDFDuet-1 (Novagen) and connected to the downstream of the T7 promoter. The constructed plasmid was named pCDF_P T7 -sahH.

[0530] (B) Production of SfmM2, SahH, FtfL, Fch, and MtdA-enhanced strains

[0531] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 4(A) T7 -sahH was used for transformation, and the resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -sahH / pCOLA_P T7 -ftfL-fch-mtdA.

[0532] [Example 5]

[0533] The SfmM2, FtfL, Fch, and MtdA-enhanced strains prepared in Example 1(C) were evaluated for 3-methyltyrosine production as follows.

[0534] The HMS174(DE3) / pET_P prepared in Example 1(C) T7 -sfmM2 / pCOLA_P T7-ftfL-fch-mtdA was cultured overnight in LB medium (16 g / L Bacto-trypton, 10 g / L yeast extract, 5 g / L NaCl) containing 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin. To a 50 mL test tube, 5 mL of M9 medium (6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 mM MgSO4·7H2O, 0.1 mM CaCl2·2H2O, 10 mg / L thiamine hydrochloride) containing 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin was added. The culture solution in the LB medium was inoculated into this M9 medium at a 1 / 50 dilution, and cultured with shaking at 200 rpm and 30°C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and shaking culture was further performed for 16 hours.

[0535] The obtained culture solution was centrifuged at 3000×g for 5 minutes to collect the bacteria to an OD of 600 =10 was suspended in a reaction buffer (100 mM Tris-HCl pH 8.0, 20 mM MgSO4, 20 mM glucose) containing 20 mM sodium formate, 100 μg / mL carbenicillin, 100 μg / mL spectinomycin, 100 μg / mL kanamycin, and 1 mM IPTG. 1 mL of the bacterial cell suspension was added to a 14 mL test tube and mixed with 1 mL of a 10 mM L-tyrosine solution (dissolved in 0.1 N hydrochloric acid) as a substrate to initiate the methylation reaction. The mixture was shaken at 200 rpm and 30°C for 6 hours.

[0536] In addition, in order to clarify that the methylation reaction is promoted by the efficient regeneration of methyl carbon in SAM, an experiment was also carried out under the same conditions except that sodium formate was not added as a methyl carbon supply source, and the results were compared with the results of the experiment carried out under the conditions of adding sodium formate.

[0537] 20 μL of the resulting reaction solution was mixed with 20 μL of pure water, 80 μL of 1N hydrochloric acid, and 80 μL of acetonitrile, and the supernatant was collected after centrifugation at 3000×g for 5 minutes and subjected to LC-MS analysis. The conditions for the LC-MS analysis are shown in Table 1.

[0538] The measurement results showed that the concentration of 3-methyltyrosine in the reaction solution increased by 0.030 mM compared to the condition without the addition of sodium formate, and was 0.092 mM under the condition with the addition of sodium formate.

[0539] [Table 1]

[0540]

[0541] [Example 6]

[0542] The SfmM2, LuxS, FtfL, Fch, and MtdA-enhanced strains prepared in Example 2(C) were evaluated for 3-methyltyrosine production as follows.

[0543] Regarding the evaluation of 3-methyltyrosine production in this example, the HMS174(DE3) / pET_P prepared in Example 2(C) was used. T7 -sfmM2 / pCDF_P T7 -luxS / pCOLA_P T7 The same procedure as in Example 5 was carried out except for the addition of -ftfL-fch-mtdA.

[0544] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.136 mM compared to the condition without the addition of sodium formate, and became 0.185 mM under the condition with the addition of sodium formate.

[0545] [Example 7]

[0546] The SfmM2, Mtn, LuxS, FtfL, Fch, and MtdA enhanced strains prepared in Example 3 were evaluated for 3-methyltyrosine production as follows.

[0547] Regarding the evaluation of 3-methyltyrosine production in this example, the HMS174 (DE3) / pET_P prepared in Example 3 was used. T7 -sfmM2 / pCDF_P T7 -Mtn-luxS / pCOLA_P T7 The same procedure as in Example 5 was carried out except for the addition of -ftfL-fch-mtdA.

[0548] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.128 mM compared to the condition without the addition of sodium formate, and became 0.236 mM under the condition with the addition of sodium formate.

[0549] [Example 8]

[0550] The SfmM2, SahH, FtfL, Fch, and MtdA-enhanced strains prepared in Example 4(B) were evaluated for 3-methyltyrosine production as follows.

[0551] Regarding the evaluation of 3-methyltyrosine production in this example, the HMS174(DE3) / pET_P prepared in Example 4(B) was used. T7 -sfmM2 / pCDF_PT7 -sahH / pCOLA_P T7 The same procedure as in Example 5 was carried out except for the addition of -ftfL-fch-mtdA.

[0552] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.024 mM compared to the condition without the addition of sodium formate, and became 0.091 mM under the condition with the addition of sodium formate.

[0553] [Comparative Example 1]

[0554] For Escherichia coli HMS174 (DE3) strain, expression of the sfmM2 gene was enhanced as follows.

[0555] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, pCDFDuet-1 (Novagen) and pCOLADuet-1 (Novagen), and the resulting strain was designated HMS174 (DE3) / pET_P T7 -sfmM2.

[0556] [Comparative Example 2]

[0557] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene and the luxS gene was enhanced as follows.

[0558] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pCDF_P constructed in Example 2(B) T7 -luxS and pCOLADuet-1 (Novagen), and the resulting strain was designated HMS174 (DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -luxS.

[0559] [Comparative Example 3]

[0560] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene, mtn gene, and luxS gene was enhanced as follows.

[0561] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pCDF_P constructed in Example 2(A) T7-mtn-luxS and pCOLADuet-1 (Novagen) were used to transform the obtained strain, and the resulting strain was designated as HMS174 (DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -mtn-luxS.

[0562] [Comparative Example 4]

[0563] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene and the sahH gene was enhanced as follows.

[0564] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pCDF_P constructed in Example 4(A) T7 -sahH and pCOLA Duet-1 (Novagen), and the resulting strain was designated HMS174 (DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -sahH.

[0565] [Comparative Example 5]

[0566] The SfmM2-enhanced strain prepared in Comparative Example 1 was evaluated for 3-methyltyrosine production as follows.

[0567] Regarding the evaluation of 3-methyltyrosine production in this comparative example, the HMS174 (DE3) / pET_P prepared in Comparative Example 1 was used. T7 The same procedure as in Example 5 was carried out except for replacing -sfmM2.

[0568] As a result, the 3-methyltyrosine concentration in the reaction solution did not increase compared to the condition without the addition of sodium formate.

[0569] [Comparative Example 6]

[0570] The SfmM2 and LuxS enhanced strains prepared in Comparative Example 2 were evaluated for 3-methyltyrosine production as follows.

[0571] Regarding the evaluation of 3-methyltyrosine production in this comparative example, the HMS174(DE3) / pET_P prepared in Comparative Example 2 was used. T7 -sfmM2 / pCDF_P T7 The same procedure as in Example 5 was carried out except that -luxS was used.

[0572] As a result, the 3-methyltyrosine concentration in the reaction solution did not increase compared to the condition without the addition of sodium formate.

[0573] [Comparative Example 7]

[0574] The enhanced strains SfmM2, Mtn, and LuxS prepared in Comparative Example 3 were evaluated for 3-methyltyrosine production as follows.

[0575] Regarding the evaluation of 3-methyltyrosine production in this comparative example, the HMS174 (DE3) / pET_P prepared in Comparative Example 3 was used. T7 -sfmM2 / pCDF_P T7 The same procedure as in Example 5 was carried out except that -mtn-luxS was used.

[0576] As a result, the 3-methyltyrosine concentration in the reaction solution did not increase compared to the condition without the addition of sodium formate.

[0577] [Comparative Example 8]

[0578] The SfmM2 and SahH-enhanced strains prepared in Comparative Example 4 were evaluated for 3-methyltyrosine production as follows.

[0579] Regarding the evaluation of 3-methyltyrosine production in this comparative example, the HMS174(DE3) / pET_P prepared in Comparative Example 4 was used. T7 -sfmM2 / pCDF_P T7 The same procedure as in Example 5 was carried out except for the addition of -sahH.

[0580] As a result, the 3-methyltyrosine concentration in the reaction solution did not increase compared to the condition without the addition of sodium formate.

[0581] The results of Examples 5 to 8 are summarized in Table 2. The accumulation concentrations of 3-methyltyrosine under the conditions of sodium formate addition in Examples 5, 6, 7, and 8 significantly increased by 1.5 times, 3.8 times, 2.2 times, and 1.4 times, respectively, compared to the conditions without sodium formate addition. On the other hand, no increase in 3-methyltyrosine accumulation was observed under the conditions of sodium formate addition in Comparative Examples 5, 6, 7, and 8.

[0582] From the above results, it can be seen that by using formic acid under conditions where the expression of FtfL, Fch, and MtdA is enhanced, SAM can be regenerated more efficiently, the methylation reaction can be promoted, and the production amount of any methyl compound can be increased compared to the case where no formic acid is used, or compared to the case where formic acid is used under conditions where the expression of the above enzymes is not enhanced.

[0583] In Examples 6 and 7, the accumulated concentrations of 3-methyltyrosine under the conditions of addition of sodium formate increased by 2.0 times and 2.6 times, respectively, compared to the result of Example 5.

[0584] The above results indicate that enhanced expression of LuxS and enhanced expression of Mtn and LuxS improves the efficiency of SAM regeneration and further promotes the methylation reaction.

[0585] That is, the method of the present invention has shown that the use of formic acid enables efficient regeneration of SAM, promotes the methylation reaction, and increases the production amount of any methyl compound.

[0586] [Table 2]

[0587]

[0588] [Example 9]

[0589] For the Escherichia coli HMS174 (DE3) strain, the expression of the TCMT gene, mtn gene, luxS gene, ftfL gene, fch gene, and mtdA gene was enhanced as follows.

[0590] (A) Construction of TCMT-enhanced plasmid

[0591] A DNA (base sequence of synthetic DNA: sequence number 5) containing the base sequence of the TCMT gene from Lonsdale apopuli encoding tyrosine 3C-methyltransferase (a recognition sequence for the restriction enzyme Nde I is assigned to the 5' end side, and a recognition sequence for the restriction enzyme Bgl II is assigned to the 3' end side) is synthesized. It should be noted that a sequence with optimized codons is designed for efficient expression in Escherichia coli. The synthesized DNA is inserted into the restriction enzyme sites Nde I and Bgl II of the expression vector pET Duet-1 (Novagen) for Escherichia coli and connected to the downstream of the T7 promoter. The constructed plasmid is named pET_P T7 -TCMT.

[0592] (B) Production of TCMT, Mtn, LuxS, FtfL, Fch, and MtdA-enhanced strains

[0593] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 9 (A) T7 -TCMT, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 2(A) T7 -mtn-luxS transformation, and the resulting strain was designated HMS174(DE3) / pET_P T7 -TCMT / pCDF_P T7 -mtn-luxS / pCOLA_PT7 -ftfL-fch-mtdA.

[0594] [Example 10]

[0595] For the Escherichia coli HMS174 (DE3) strain, expression enhancement of the TCMT gene, mtn gene, luxS gene, prs gene, apt gene, ftfL gene, fch gene, and mtdA gene was performed as follows.

[0596] (A) Construction of Mtn, LuxS, Prs, and Apt Enhanced Plasmids

[0597] A DNA containing the base sequences of the prs gene and apt gene from Escherichia coli (with a recognition sequence for the restriction enzyme Bgl II at the 5' end and a recognition sequence for the restriction enzyme Fse I at the 3' end) was synthesized (base sequence of the synthetic DNA: SEQ ID NO: 6). The synthesized DNA was inserted into the plasmid pCDF_P constructed in Example 2(A). T7 The restriction enzyme sites Bgl II and Fse I of -mtn-luxS were connected to the downstream of luxS gene. The constructed plasmid was named pCDF_P T7 -mtn-luxS-prs-apt.

[0598] (B) Production of TCMT, Mtn, LuxS, Prs, Apt, FtfL, Fch, and MtdA-enhanced strains

[0599] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 9 (A) T7 -TCMT, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 10(A) T7 -mtn-luxS-prs-apt was transformed and the resulting strain was designated as HMS174(DE3) / pET_P T7 -TCMT / pCDF_P T7 -mtn-luxS-prs-apt / pCOLA_P T7 -ftfL-fch-mtdA.

[0600] [Example 11]

[0601] For the Escherichia coli HMS174 (DE3) strain, the expression of the TCMT gene, sahH gene, ftfL gene, fch gene, and mtdA gene was enhanced as follows.

[0602] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 9 (A) T7 -TCMT, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 4(A) T7 -sahH was used for transformation, and the resulting strain was designated as HMS174(DE3) / pET_P T7 -TCMT / pCDF_P T7 -sahH / pCOLA_P T7 -ftfL-fch-mtdA.

[0603] [Example 12]

[0604] For Escherichia coli HMS174 (DE3) strain, expression enhancement of the TCMT gene, sahH gene, ADO1 gene, ftfL gene, fch gene, and mtdA gene was performed as follows: The ADO1 gene is a gene encoding adenosine kinase (ADK).

[0605] (A) Construction of SahH-ADO1 enhancing plasmid

[0606] A DNA containing the base sequence of the ADO1 gene from Saccharomyces cerevisiae (with a recognition sequence for the restriction enzyme Bgl II at the 5' end and a recognition sequence for the restriction enzyme Mfe I at the 3' end) was synthesized (base sequence of the synthetic DNA: SEQ ID NO: 7). It should be noted that the base sequence of this ADO1 gene was designed with codons optimized for efficient expression in Escherichia coli. The synthesized DNA was inserted into the plasmid pCDF_P constructed in Example 4(A). T7 -sahH restriction enzyme sites Bgl II and Mfe I were connected to the downstream of sahH gene. The constructed plasmid was named pCDF_P T7 -sahH-ADO1.

[0607] (B) Production of TCMT, SahH, ADO1, FtfL, Fch, and MtdA-enhanced strains

[0608] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 9 (A) T7 -TCMT, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 12(A) T7-sahH-ADO1 was transformed and the resulting strain was designated HMS174(DE3) / pET_P T7 -TCMT / pCDF_P T7 -sahH-ADO1 / pCOLA_P T7 -ftfL-fch-mtd A.

[0609] [Example 13]

[0610] The TCMT, Mtn, LuxS, FtfL, Fch, and MtdA-enhanced strains prepared in Example 9(B) were evaluated for 3-methyltyrosine production as follows.

[0611] Regarding the evaluation of 3-methyltyrosine production in this example, the HMS174(DE3) / pET_P prepared in Example 9(B) was used. T7 -TCMT / pCDF_P T7 -mtn-luxS / pCOLA_P T7 -ftfL-fch-mtdA to become OD 600 =4 in the reaction buffer, and the methylation reaction was carried out in the same manner as in Example 5, except that the reaction mixture was suspended in the reaction buffer at 200 rpm and 30°C for 24 hours.

[0612] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.093 mM compared to the condition without the addition of sodium formate, and became 0.162 mM under the condition with the addition of sodium formate.

[0613] [Example 14]

[0614] The TCMT, Mtn, LuxS, Prs, Apt, FtfL, Fch, and MtdA-enhanced strains prepared in Example 10(B) were evaluated for 3-methyltyrosine production as follows.

[0615] Regarding the evaluation of 3-methyltyrosine production in this example, the HMS174(DE3) / pET_P prepared in Example 10(B) was used. T7 -TCMT / pCDF_P T7 -mtn-luxS-prs-apt / pCOLA_P T7 The same procedure as in Example 13 was carried out except for the addition of -ftfL-fch-mtdA.

[0616] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.141 mM compared to the condition without the addition of sodium formate, and became 0.187 mM under the condition with the addition of sodium formate.

[0617] [Example 15]

[0618] The TCMT, SahH, FtfL, Fch, and MtdA-enhanced strains prepared in Example 11 were evaluated for 3-methyltyrosine production as follows.

[0619] Regarding the evaluation of 3-methyltyrosine production in this example, the HMS174 (DE3) / pET_P prepared in Example 11 was used. T7 -TCMT / pCDF_P T7 -sahH / pCOLA_P T7 The same procedure as in Example 13 was carried out except for the addition of -ftfL-fch-mtdA.

[0620] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.046 mM compared to the condition without the addition of sodium formate, and became 0.072 mM under the condition with the addition of sodium formate.

[0621] [Example 16]

[0622] The TCMT, SahH, FtfL, Fch, and MtdA-enhanced strains prepared in Example 12(B) were evaluated for 3-methyltyrosine production as follows.

[0623] Regarding the evaluation of 3-methyltyrosine production in this example, the HMS174(DE3) / pET_P prepared in Example 12(B) was used. T7 -TCMT / pCDF_P T7 -sahH-ADO1 / pCOLA_P T7 The same procedure as in Example 13 was carried out except for the addition of -ftfL-fch-mtd A.

[0624] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.036 mM compared to the condition without the addition of sodium formate, and became 0.078 mM under the condition with the addition of sodium formate.

[0625] [Comparative Example 9]

[0626] For Escherichia coli HMS174 (DE3) strain, expression of the TCMT gene was enhanced as follows.

[0627] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 9 (A) T7 -TCMT, pCDFDuet-1 (Novagen) and pCOLA Duet-1 (Novagen), and the resulting strain was designated HMS174 (DE3) / pET_P T7 -TCMT.

[0628] [Comparative Example 10]

[0629] The TCMT-enhanced strain prepared in Comparative Example 9 was evaluated for 3-methyltyrosine production as follows.

[0630] Regarding the evaluation of 3-methyltyrosine production in this comparative example, the HMS174 (DE3) / pET_P prepared in Comparative Example 9 was used. T7 The same procedure as in Example 13 was carried out except for the addition of TCMT.

[0631] As a result, the concentration of 3-methyltyrosine in the reaction solution was 0.058 mM under the condition of addition of sodium formate.

[0632] The results of Examples 13 to 16 are summarized in Table 3. The accumulation concentrations of 3-methyltyrosine under the conditions of sodium formate addition in Examples 13, 14, 15, and 16 significantly increased by 2.3 times, 4.1 times, 2.8 times, and 1.9 times, respectively, compared to the conditions without sodium formate addition. On the other hand, in Comparative Example 10, the accumulation concentration of 3-methyltyrosine under the conditions of sodium formate addition was the lowest.

[0633] In Examples 14 and 16, the accumulated concentrations of 3-methyltyrosine under the conditions of addition of sodium formate increased by 15% and 8%, respectively, compared to the results of Examples 13 and 15.

[0634] The above results indicate that by enhancing the expression of Prs and Apt in addition to enhancing the expression of Mtn and LuxS, and enhancing the expression of ADO1 in addition to enhancing the expression of SahH, the efficiency of SAM regeneration is improved, and the methylation reaction can be further promoted.

[0635] That is, the method of the present invention has shown that the use of formic acid enables efficient regeneration of SAM, promotes the methylation reaction, and increases the production amount of any methyl compound.

[0636] [Table 3]

[0637]

[0638] [Example 17]

[0639] For the Escherichia coli HMS174 (DE3) strain, expression enhancement of the EgtB gene, EgtD gene, EgtE gene, mtn gene, luxS gene, ftfL gene, fch gene, and mtdA gene was performed as follows.

[0640] (A) Construction of EgtBDE enhanced plasmid

[0641] DNA (synthesized DNA base sequence: SEQ ID NO: 8) containing the EgtB gene from Methylobacterium pseudosasicola encoding histidine betaine cysteine S-oxide synthase, the EgtD gene from Mycolicibacterium smegmatis encoding L-histidine Nα-methyltransferase and histidine betaine cysteine S-oxide lyase, and the base sequence of the EgtE gene (with a recognition sequence for the restriction enzyme Nco I conferred on the 5' end and a recognition sequence for the restriction enzyme EcoR I conferred on the 3' end). It should be noted that the base sequences of the EgtB, EgtD, and EgtE genes were designed with codons optimized for efficient expression in Escherichia coli. The synthesized DNA was inserted into the restriction enzyme sites Nco I and EcoR I of the Escherichia coli expression vector pET Duet-1 (Novagen) and connected downstream to the T7 promoter. The constructed plasmid was named pET_P T7 -EgtBDE.

[0642] (B) Production of EgtBDE, Mtn, LuxS, FtfL, Fch, and MtdA-enhanced strains

[0643] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 17 (A) T7 -EgtBDE, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 2(A) T7 -mtn-luxS transformation, and the resulting strain was designated HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS / pCOLA_P T7 -ftfL-fch-mtdA.

[0644] [Example 18]

[0645] For Escherichia coli HMS174 (DE3) strain, expression enhancement of the EgtB gene, EgtD gene, EgtE gene, mtn gene, luxS gene, Prs gene, Apt gene, ftfL gene, fch gene, and mtdA gene was performed as follows.

[0646] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 17 (A) T7-EgtBDE, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 10(A) T7 -mtn-luxS-prs-apt transformation, and the resulting strain was designated HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt / pCOLA_P T7 -ftfL-fc h-mtdA.

[0647] [Example 19]

[0648] The EgtBDE, Mtn, LuxS, FtfL, Fch, and MtdA-enhanced strains prepared in Example 17(B) were evaluated for the production of histidine betaine and ergothioneine as follows.

[0649] The HMS174(DE3) / pET_P prepared in Example 17(B) T7 -EgtBDE / pCDF_P T7 -mtn-luxS / pCOLA_P T7 -ftfL-fch-mtdA was cultured overnight in LB medium (16 g / L Bacto-trypton, 10 g / L yeast extract, 5 g / L NaCl) containing 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin. To a 50 mL test tube, 5 mL of M9 medium (6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 mM MgSO4·7H2O, 0.1 mM CaCl2·2H2O, 10 mg / L thiamine hydrochloride) containing 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin was added. The culture solution from the LB medium was inoculated into this M9 medium at a 1 / 50 dilution, and cultured with shaking at 200 rpm and 30°C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and shaking culture was further performed for 16 hours.

[0650] The obtained culture solution was centrifuged at 3000×g for 5 minutes to collect the bacteria to an OD of 600=4 mode is suspended in the reaction buffer (100mM Tris-HCl pH8.0, 20mM MgSO , 20mM glucose) containing 20mM sodium formate, 100 μg / mL carbenicillin, 100 μg / mL spectinomycin, 100 μg / mL kanamycin and 1mMIPTG.In 14mL test tube, add 1mL bacterial cell suspension, mix with the substrate solution 1mL containing 10mM L-histidine, 10mM L-cysteine as substrate, thus start to comprise methylated thioneine building-up reaction, at 200rpm, 30 ℃ of lower vibrations 48 hours.

[0651] In addition, in order to clarify that the methylation reaction is promoted by the efficient regeneration of methyl carbon in SAM, an experiment was also carried out under the same conditions except that sodium formate was not added as a methyl carbon supply source, and the results were compared with the results of the experiment carried out under the conditions of adding sodium formate.

[0652] The reaction solution was centrifuged at 3000×g for 5 minutes, and the supernatant was collected and subjected to LC-MS analysis. The conditions for LC-MS analysis are shown in Table 4.

[0653] The result of measuring is that, compared with the condition without adding sodium formate, the histidine betaine concentration and thioneine concentration in the reaction solution increase by 0.033mM, 0.039mM respectively, and are 0.047mM, 0.078mM respectively under the condition with the addition of sodium formate.As a result, compared with the condition without adding sodium formate, the total of the accumulation concentration of histidine betaine and thioneine increases by 0.073mM, and is 0.125mM under the condition with the addition of sodium formate.

[0654] [Table 4]

[0655]

[0656] [Example 20]

[0657] The EgtBDE, Mtn, LuxS, Prs, Apt, FtfL, Fch, and MtdA-enhanced strains prepared in Example 18 were evaluated for the production of histidine betaine and ergothioneine as follows.

[0658] Regarding the production evaluation of histidine betaine and ergothioneine in this embodiment, the HMS174 (DE3) / pET_P prepared in Example 18 was used. T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt / pCOLA_P T7 The same procedure as in Example 19 was carried out except for the addition of -ftfL-fch-mtdA.

[0659] As a result, the total accumulated concentration of histidine betaine and ergothioneine in the reaction solution increased by 0.014 mM compared to the condition without the addition of sodium formate, and became 0.228 mM under the condition with the addition of sodium formate.

[0660] [Comparative Example 11]

[0661] For the Escherichia coli HMS174 (DE3) strain, the expression of the EgtB gene, EgtD gene, and EgtE gene was enhanced as follows.

[0662] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 17 (A) T7 -EgtBDE, pCDFDuet-1 (Novagen) and pCOLA Duet-1 (Novagen), and the resulting strain was designated HMS174 (DE3) / pET_P T7 -EgtBDE.

[0663] [Comparative Example 12]

[0664] For the Escherichia coli HMS174 (DE3) strain, expression enhancement of the EgtB gene, EgtD gene, EgtE gene, mtn gene, and luxS gene was performed as follows.

[0665] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 17 (A) T7 -EgtBDE, plasmid pCDF_P constructed in Example 2(A) T7 -mtn-luxS and pCOLA Duet-1 (Novagen) were used to transform the obtained strain, and the resulting strain was designated as HMS174 (DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS.

[0666] [Comparative Example 13]

[0667] For the Escherichia coli HMS174 (DE3) strain, the expression of the EgtB gene, EgtD gene, EgtE gene, mtn gene, luxS gene, Prs gene, and Apt gene was enhanced as follows.

[0668] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 17 (A) T7 -EgtBDE, plasmid pCDF_P constructed in Example 10(A)T7 -mtn-luxS-prs-apt, and pCOLADuet-1 (Novagen), and the resulting strain was designated HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt.

[0669] [Comparative Example 14]

[0670] The EgtBDE-enhanced strain prepared in Comparative Example 11 was evaluated for the production of histidine betaine and ergothioneine as follows.

[0671] Regarding the production evaluation of histidine betaine and ergothioneine in this comparative example, the HMS174 (DE3) / pET_P prepared in Comparative Example 11 was used. T7 The same procedure as in Example 19 was carried out except for the addition of -EgtBDE.

[0672] As a result, compared with the condition without the addition of sodium formate, neither the histidine betaine concentration nor the ergothioneine concentration in the reaction solution increased, and the total of the accumulated histidine betaine concentration and the accumulated ergothioneine concentration did increase.

[0673] [Comparative Example 15]

[0674] The enhanced strains of EgtBDE, Mtn, and LuxS prepared in Comparative Example 12 were evaluated for the production of histidine betaine and ergothioneine as follows.

[0675] Regarding the production evaluation of histidine betaine and ergothioneine in this comparative example, the HMS174 (DE3) / pET_P prepared in Comparative Example 12 was used. T7 -EgtBDE / pCDF_P T7 The same procedure as in Example 19 was carried out except for replacing -mtn-luxS.

[0676] As a result, compared with the condition without the addition of sodium formate, the histidine betaine concentration and the ergothioneine concentration in the reaction solution were the same or did not increase, and the total of the accumulated histidine betaine concentration and the accumulated ergothioneine concentration did not increase.

[0677] [Comparative Example 16]

[0678] The enhanced strains of EgtBDE, Mtn, LuxS, Prs, and Apt prepared in Comparative Example 13 were evaluated for the production of histidine betaine and ergothioneine as follows.

[0679] Regarding the production evaluation of histidine betaine and ergothioneine in this comparative example, the HMS174 (DE3) / pET_P prepared in Comparative Example 13 was used. T7 -EgtBDE / pCDF_P T7 The same procedure as in Example 19 was carried out except for the substitution of -mtn-luxS-prs-apt.

[0680] As a result, under the condition of addition of sodium formate, the total accumulated concentration of histidine betaine and ergothioneine in the reaction solution was 0.123 mM.

[0681] The results of Examples 19 and 20 are summarized in Table 5. In order to evaluate the efficiency of the methylation reaction, the total cumulative concentration of histidine betaine and ergothioneine was calculated and compared. The total cumulative concentration under the conditions of adding sodium formate in Example 19 increased significantly to 2.4 times compared to the conditions without adding sodium formate. On the other hand, in Comparative Examples 14 and 15, no increase in the total cumulative concentration was confirmed under the conditions of adding sodium formate.

[0682] The total accumulated concentration under the condition of adding sodium formate in Example 20 increased to 1.1 times compared with the condition without adding sodium formate, and was 1.9 times the total accumulated concentration under the condition of adding sodium formate in Comparative Example 16.

[0683] From the above results, it can be seen that by using formic acid under conditions where the expression of FtfL, Fch, and MtdA is enhanced, SAM can be regenerated more efficiently, the methylation reaction can be promoted, and the production amount of any methyl compound can be increased compared to the case where no formic acid is used, or compared to the case where formic acid is used under conditions where the expression of the above enzymes is not enhanced.

[0684] In Example 20, the total accumulated concentration under the condition of adding sodium formate was 1.8 times the result of Example 19.

[0685] These results indicate that enhanced expression of Mtn, LuxS, Prs, and Apt improves the efficiency of SAM regeneration and further promotes methylation reactions.

[0686] That is, the method of the present invention has shown that the use of formic acid enables efficient regeneration of SAM, promotes the methylation reaction, and increases the production amount of any methyl compound.

[0687] [Table 5]

[0688]

[0689] [Example 21]

[0690] For Escherichia coli HMS174 (DE3) strain, expression enhancement of the ASMT gene, mtn gene, luxS gene, ftfL gene, fch gene, and mtdA gene was performed as follows.

[0691] (A) Construction of ASMT-enhancing plasmid

[0692] A DNA (base sequence of synthetic DNA: sequence number 9) containing the base sequence of the ASMT gene from Homo sapiens encoding acetylserotonin O-methyltransferase (a recognition sequence for the restriction enzyme Nco I was assigned to the 5' end side, and a recognition sequence for the restriction enzyme Bgl II was assigned to the 3' end side) was synthesized. It should be noted that as the base sequence of the ASMT gene, a sequence was designed in which codons were optimized for efficient expression in Escherichia coli and the alanine at position 258 was mutated to glutamic acid. The synthesized DNA was inserted into the restriction enzyme sites Nco I and Bgl II of the expression vector pET Duet-1 (Novagen) for Escherichia coli and connected to the downstream of the T7 promoter. The constructed plasmid was named pET_P T7 -ASMT.

[0693] (B) Preparation of ASMT, Mtn, LuxS, FtfL, Fch, and MtdA-enhanced strains

[0694] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 21 (A) T7 -ASMT, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 2(A) T7 -mtn-luxS transformation, and the resulting strain was designated HMS174(DE3) / pET_P T7 -ASMT / pCDF_P T7 -mtn-luxS / pCOLA_P T7 -ftfL-fch-mtdA.

[0695] [Example 22]

[0696] For Escherichia coli HMS174 (DE3) strain, expression enhancement of the ASMT gene, mtn gene, luxS gene, Prs gene, Apt gene, ftfL gene, fch gene, and mtdA gene was performed as follows.

[0697] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 21 (A) T7-ASMT, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 10(A) T7 -mtn-luxS-prs-apt transformation, and the resulting strain was designated HMS174(DE3) / pET_P T7 -ASMT / pCDF_P T7 -mtn-luxS-prs-apt / pCOLA_P T7 -ftfL-fch-mtdA.

[0698] [Example 23]

[0699] The ASMT, Mtn, LuxS, FtfL, Fch, and MtdA-enhanced strains prepared in Example 21(B) were evaluated for melatonin production as follows.

[0700] The HMS174(DE3) / pET_P prepared in Example 21(B) T7 -ASMT / pCDF_P T7 -mtn-luxS / pCOLA_P T7 -ftfL-fch-mtdA was cultured overnight in LB medium (16 g / L Bacto-trypton, 10 g / L yeast extract, 5 g / L NaCl) containing 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin. To a 50 mL test tube, 5 mL of M9 medium (6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 mM MgSO4·7H2O, 0.1 mM CaCl2·2H2O, 10 mg / L thiamine hydrochloride) containing 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin was added. The culture solution in the LB medium was inoculated into this M9 medium at a 1 / 50 dilution, and cultured with shaking at 200 rpm and 30°C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and shaking culture was further performed for 16 hours.

[0701] The obtained culture solution was centrifuged at 3000×g for 5 minutes to collect the bacteria to an OD of 600=4 was suspended in a reaction buffer (100 mM Tris-HCl pH 8.0, 20 mM MgSO4, 20 mM glucose) containing 20 mM sodium formate, 100 μg / mL carbenicillin, 100 μg / mL spectinomycin, 100 μg / mL kanamycin, and 1 mM IPTG. 1 mL of the bacterial cell suspension was added to a 14 mL test tube and mixed with 1 mL of a substrate solution containing 50 mM N-acetylserotonin as a substrate. This initiated the melatonin synthesis reaction involving methylation and was shaken at 200 rpm and 30°C for 48 hours.

[0702] In addition, in order to clarify that the methylation reaction is promoted by the efficient regeneration of methyl carbon in SAM, an experiment was also carried out under the same conditions except that sodium formate was not added as a methyl carbon supply source, and the results were compared with the results of the experiment carried out under the conditions of adding sodium formate.

[0703] 100 μL of the obtained reaction solution was mixed with 100 μL of methanol, and the supernatant was collected after centrifugation at 3000×g for 5 minutes and subjected to LC-MS analysis. The conditions for LC-MS analysis are shown in Table 6.

[0704] The results of the measurement showed that the melatonin concentration in the reaction solution increased by 0.036 mM compared to the condition without the addition of sodium formate, and was 0.144 mM under the condition with the addition of sodium formate.

[0705] [Table 6]

[0706]

[0707] [Example 24]

[0708] The ASMT, Mtn, LuxS, Prs, Apt, FtfL, Fch, and MtdA-enhanced strains prepared in Example 22 were evaluated for melatonin production as follows.

[0709] Regarding the melatonin production evaluation in this example, the HMS174 (DE3) / pET_P prepared in Example 22 was used. T7 -ASMT / pCDF_P T7 -mtn-luxS-prs-apt / pCOLA_P T7 The same procedure as in Example 23 was carried out except for the addition of -ftfL-fch-mtdA.

[0710] As a result, the melatonin concentration in the reaction solution increased by 0.031 mM compared to the condition without the addition of sodium formate, and became 0.153 mM under the condition with the addition of sodium formate.

[0711] [Comparative Example 17]

[0712] For Escherichia coli HMS174 (DE3) strain, expression of the ASMT gene was enhanced as follows.

[0713] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 21 (A) T7 -ASMT, pCDFDuet-1 (Novagen) and pCOLA Duet-1 (Novagen), and the resulting strain was designated HMS174 (DE3) / pET_P T7 -ASMT.

[0714] [Comparative Example 18]

[0715] The ASMT-enhanced strain prepared in Comparative Example 17 was evaluated for melatonin production as follows.

[0716] Regarding the melatonin production evaluation in this comparative example, the HMS174 (DE3) / pET_P prepared in Comparative Example 17 was used. T7 The same procedure as in Example 23 was carried out except for the addition of -ASMT.

[0717] As a result, the melatonin concentration in the reaction solution did not increase compared to the condition without the addition of sodium formate, but was 0.075 mM under the condition with the addition of sodium formate.

[0718] The results of Examples 23 and 24 are summarized in Table 7. The accumulated concentrations of melatonin in Examples 23 and 24, when sodium formate was added, increased by 33% and 25%, respectively, compared to the conditions without sodium formate. On the other hand, in Comparative Example 18, no increase in melatonin accumulation was observed when sodium formate was added.

[0719] From the above results, it can be seen that by using formic acid under conditions where the expression of FtfL, Fch, and MtdA is enhanced, SAM can be regenerated more efficiently, the methylation reaction can be promoted, and the production amount of any methyl compound can be increased compared to the case where no formic acid is used, or compared to the case where formic acid is used under conditions where the expression of the above enzymes is not enhanced.

[0720] In Examples 23 and 24, the accumulated concentrations of melatonin under the conditions of addition of sodium formate increased by 1.9 times and 2.0 times, respectively, compared to the result of Comparative Example 18.

[0721] These results indicate that enhanced expression of Mtn, LuxS, Prs, and Apt improves the efficiency of SAM regeneration and further promotes methylation reactions.

[0722] That is, the method of the present invention has shown that the use of formic acid enables efficient regeneration of SAM, promotes the methylation reaction, and increases the production amount of any methyl compound.

[0723] [Table 7]

[0724]

[0725] [Example 25]

[0726] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene, mdh2 gene, ftfL gene, fch gene, and mtdA gene was enhanced as described below.

[0727] (A) Construction of Mdh2-enhancing plasmid

[0728] A DNA (base sequence of synthetic DNA: sequence number 10) containing the base sequence of the mdh2 gene from Cupriavidus necator (a recognition sequence for the restriction enzyme Nde I is assigned to the 5' end side, and a recognition sequence for the restriction enzyme Aat II is assigned to the 3' end side) is synthesized. It should be noted that the base sequence of the mdh2 gene is designed to optimize the codons for efficient expression in Escherichia coli, and further mutates the 26th alanine to valine, the 31st alanine to valine, and the 169th alanine to valine. The synthesized DNA is inserted into the restriction enzyme sites Nde I and Aat II of the expression vector pACYCDuet-1 (Novagen) for Escherichia coli and connected to the downstream of the T7 promoter. The constructed plasmid is named pACYC_P T7 -mdh2.

[0729] (B) Production of SfmM2, Mdh2, FtfL, Fch, and MtdA-enhanced strains

[0730] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pACYC_P constructed in Example 25(A) T7 -mdh2, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and pCDF Duet-1 (Novagen), and the resulting strain was designated HMS174 (DE3) / pET_P T7 -sfmM2 / pACYC_P T7 -mdh2 / pCOLA_P T7 -ftfL-fch-mtdA.

[0731] [Example 26]

[0732] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene, mdh2 gene, luxS gene, ftfL gene, fch gene, and mtdA gene was enhanced as follows.

[0733] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pACYC_P constructed in Example 25(A) T7 -mdh2, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 2(B) T7 -luxS was used for transformation, and the resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pACYC_P T7 -mdh2 / pCDF_P T7 -luxS / pCOLA_P T7 -ftfL-fch-mtdA.

[0734] [Example 27]

[0735] For Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene, mdh2 gene, mtn gene, luxS gene, ftfL gene, fch gene, and mtdA gene was enhanced as follows.

[0736] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pACYC_P constructed in Example 25(A) T7 -mdh2, plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA, and the plasmid pCDF_P constructed in Example 2(A) T7 -mtn-luxS was transformed and the resulting strain was designated HMS174(DE3) / pET_P T7 -sfmM2 / pACYC_P T7 -mdh2 / pCDF_P T7 -mtn-luxS / pCOLA_P T7 -ftfL-fch-mtdA.

[0737] [Example 28]

[0738] The SfmM2, Mdh2, FtfL, Fch, and MtdA-enhanced strains prepared in Example 25(B) were evaluated for 3-methyltyrosine production as follows.

[0739] The HMS174(DE3) / pET_P prepared in Example 25(B) T7 -sfmM2 / pACYC_P T7 -mdh2 / pCOLA_P T7 -ftfL-fch-mtdA was cultured overnight in LB medium (16 g / L Bacto-trypton, 10 g / L yeast extract, 5 g / L NaCl) containing 50 μg / mL carbenicillin, 34 μg / mL chloramphenicol, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin. To a 50 mL test tube, 5 mL of M9 medium (6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 mM MgSO4·7H2O, 0.1 mM CaCl2·2H2O, 10 mg / L thiamine hydrochloride) containing 0.5% glucose, 50 μg / mL carbenicillin, 34 μg / mL chloramphenicol, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin was added. The culture solution in the LB medium was inoculated into this M9 medium at a 1 / 50 dilution, and cultured with shaking at 200 rpm and 30°C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and cultured with shaking for a further 16 hours.

[0740] The obtained culture solution was centrifuged at 3000×g for 5 minutes to collect the bacteria to an OD of 600 =10 was suspended in a reaction buffer (100mM Tris-HCl pH 8.0, 20mM MgSO4, 20mM glucose) containing 100mM methanol, 100μg / mL carbenicillin, 68μg / mL chloramphenicol, 100μg / mL spectinomycin, 100μg / mL kanamycin and 1mM IPTG. 1mL of the bacterial cell suspension was added to a 14mL test tube and mixed with 1mL of a 10mM L-tyrosine solution (dissolved in 0.1N hydrochloric acid) as a substrate to initiate the methylation reaction. The mixture was shaken at 200rpm and 30°C for 24 hours. In addition, in order to clarify whether the methylation reaction is promoted by the efficient regeneration of the methyl carbon of SAM, an experiment was also carried out under the same conditions except that methanol was not added as a source of methyl carbon, and the results were compared with the results of the experiment carried out under the conditions of adding methanol.

[0741] The obtained reaction solution was analyzed by LC-MS in the same manner as in Example 5. The measurement results showed that the 3-methyltyrosine concentration in the reaction solution increased by 0.010 mM compared to the condition without adding methanol, and was 0.042 mM under the condition with adding methanol.

[0742] [Example 29]

[0743] The SfmM2, Mdh2, LuxS, FtfL, Fch, and MtdA enhanced strains prepared in Example 26 were evaluated for 3-methyltyrosine production as follows.

[0744] Regarding the evaluation of 3-methyltyrosine production in this example, the HMS174 (DE3) / pET_P prepared in Example 26 was used. T7 -sfmM2 / pACYC_P T7 -mdh2 / pCDF_P T7 -luxS / pCOLA_P T7 The same procedure as in Example 28 was carried out except for the addition of -ftfL-fch-mtdA.

[0745] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.014 mM compared to the condition without adding methanol, and became 0.080 mM under the condition with adding methanol.

[0746] [Example 30]

[0747] The SfmM2, Mdh2, Mtn, LuxS, FtfL, Fch, and MtdA enhanced strains prepared in Example 27 were evaluated for 3-methyltyrosine production as follows.

[0748] Regarding the evaluation of 3-methyltyrosine production in this example, the HMS174(DE3) / pET_P prepared in Example 27 was used. T7 -sfmM2 / pACYC_P T7 -mdh2 / pCDF_P T7 -mtn-luxS / pCOLA_P T7 The same procedure as in Example 28 was carried out except for the addition of -ftfL-fch-mtdA.

[0749] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.059 mM compared to the condition without adding methanol, and became 0.158 mM under the condition with adding methanol.

[0750] [Comparative Example 19]

[0751] For Escherichia coli HMS174 (DE3) strain, expression of the sfmM2 gene was enhanced as follows.

[0752] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, pACYC Duet-1 (Novagen), pCDF Duet-1 (Novagen), and pCOLA Duet-1 (Novagen) were used for transformation, and the resulting strain was designated HMS174 (DE3) / pET_P T7 -sfmM2.

[0753] [Comparative Example 20]

[0754] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene and the mdh2 gene was enhanced as follows.

[0755] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pACYC_P constructed in Example 25(A) T7 -mdh2, pCDF Duet-1 (Novagen) and pCOLA Duet-1 (Novagen), and the resulting strain was designated HMS174 (DE3) / pET_P T7 -sfmM2 / pACYC_P T7 -mdh2.

[0756] [Comparative Example 21]

[0757] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene, mtn gene, and luxS gene was enhanced as follows.

[0758] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pCDF_P constructed in Example 2(A) T7 -mtn-luxS, pACYC Duet-1 (Novagen), and pCOLA Duet-1 (Novagen) were used for transformation, and the resulting strain was designated HMS174 (DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -mtn-luxS.

[0759] [Comparative Example 22]

[0760] For the Escherichia coli HMS174 (DE3) strain, the expression of the sfmM2 gene, mdh2 gene, mtn gene, and luxS gene was enhanced as follows.

[0761] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 1 (A) T7 -sfmM2, plasmid pACYC_P constructed in Example 25(A) T7 -mdh2, plasmid pCDF_P constructed in Example 2(A) T7 -mtn-luxS and pCOLA Duet-1 (Novagen) were used to transform the obtained strain, and the resulting strain was designated as HMS174 (DE3) / pET_P T7 -sfmM2 / pACYC_P T7 -mdh2 / pCDF_P T7 -mtn-luxS.

[0762] [Comparative Example 23]

[0763] The SfmM2-enhanced strain prepared in Comparative Example 19 was evaluated for 3-methyltyrosine production as follows.

[0764] Regarding the evaluation of 3-methyltyrosine production in this comparative example, the HMS174(DE3) / pET_P prepared in Comparative Example 19 was used. T7 The same procedure as in Example 28 was carried out except for replacing -sfmM2.

[0765] As a result, the concentration of 3-methyltyrosine in the reaction solution did not increase compared with the condition without adding methanol.

[0766] [Comparative Example 24]

[0767] The SfmM2 and Mdh2 enhanced strains prepared in Comparative Example 20 were evaluated for 3-methyltyrosine production as follows.

[0768] Regarding the evaluation of 3-methyltyrosine production in this comparative example, the HMS174(DE3) / pET_P prepared in Comparative Example 20 was used. T7 -sfmM2 / pACYC_P T7 The same procedure as in Example 28 was carried out except that -mdh2 was used.

[0769] As a result, the concentration of 3-methyltyrosine in the reaction solution did not increase compared with the condition without adding methanol.

[0770] [Comparative Example 25]

[0771] The enhanced strains SfmM2, Mtn, and LuxS prepared in Comparative Example 21 were evaluated for 3-methyltyrosine production as follows.

[0772] Regarding the evaluation of 3-methyltyrosine production in this comparative example, the HMS174(DE3) / pET_P prepared in Comparative Example 21 was used. T7 -sfmM2 / pCDF_P T7 The same procedure as in Example 28 was carried out except for replacing -mtn-luxS.

[0773] As a result, the concentration of 3-methyltyrosine in the reaction solution did not increase compared with the condition without adding methanol.

[0774] [Comparative Example 26]

[0775] The enhanced strains SfmM2, Mdh2, Mtn, and LuxS prepared in Comparative Example 22 were evaluated for 3-methyltyrosine production as follows.

[0776] Regarding the evaluation of 3-methyltyrosine production in this comparative example, the HMS174(DE3) / pET_P prepared in Comparative Example 22 was used. T7 -sfmM2 / pACYC_P T7 -mdh2 / pCDF_P T7 The same procedure as in Example 28 was carried out except for replacing -mtn-luxS.

[0777] As a result, the concentration of 3-methyltyrosine in the reaction solution did not increase compared with the condition without adding methanol.

[0778] The results of Examples 28 to 30 are summarized in Table 8. The accumulated concentrations of 3-methyltyrosine under the conditions of methanol addition in Examples 28, 29, and 30 increased by 31%, 21%, and 60%, respectively, compared to the conditions without methanol addition. On the other hand, in Comparative Examples 23, 24, 25, and 26, no increase in 3-methyltyrosine accumulation was observed under the conditions of methanol addition.

[0779] From the above results, it can be seen that by using methanol under conditions where the expression of Mdh2, FtfL, Fch, and MtdA is enhanced, SAM can be regenerated more efficiently, the methylation reaction can be promoted, and the production of any methyl compound can be increased compared to the case where methanol is not used, or compared to the case where methanol is used under conditions where the expression of the above enzymes is not enhanced.

[0780] In Examples 29 and 30, the accumulated concentrations of 3-methyltyrosine under the conditions of adding methanol were 1.9 times and 3.8 times higher than those in Example 28, respectively.

[0781] The above results indicate that enhanced expression of LuxS and enhanced expression of Mtn and LuxS improves the efficiency of SAM regeneration, further promotes the methylation reaction, and increases the production amount of any methyl compound.

[0782] That is, the method of the present invention has shown that the use of methanol can efficiently regenerate SAM, promote the methylation reaction, and increase the production amount of any methyl compound.

[0783] [Table 8]

[0784]

[0785] [Example 31]

[0786] For Escherichia coli HMS174 (DE3) strain, expression enhancement of the EgtB gene, EgtD gene, EgtE gene, mdh2 gene, mtn gene, luxS gene, ftfL gene, fch gene, and mtdA gene was performed as follows.

[0787] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 17 (A) T7 -EgtBDE, plasmid pACYC_P constructed in Example 25(A) T7 -mdh2, plasmid pCDF_P constructed in Example 2(A) T7 -mtn-luxS, and the plasmid pCOLA_P constructed in Example 1(B) T7 -ftfL-fch-mtdA was transformed and the resulting strain was designated HMS174(DE3) / pET_P T7 -EgtBDE / pACYC_P T7 -mdh2 / pCDF_P T7 -mtn-luxS / pCOL A_P T7 -ftfL-fch-mtdA.

[0788] [Example 32]

[0789] The EgtBDE, Mdh2, Mtn, LuxS, FtfL, Fch, and MtdA-enhanced strains prepared in Example 31 were evaluated for the production of histidine betaine and ergothioneine as follows.

[0790] The HMS174(DE3) / pET_P prepared in Example 31 was T7 -EgtBDE / pACYC_P T7 -mdh2 / pCDF_P T7-mtn-luxS / pCOLA_P T7 -ftfL-fch-mtdA was cultured overnight in LB medium (16 g / LBacto-trypton, 10 g / L yeast extract, 5 g / L NaCl) containing 50 μg / mL carbenicillin, 34 μg / mL chloramphenicol, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin. To a 50 mL test tube, 5 mL of M9 medium (6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 mM MgSO4·7H2O, 0.1 mM CaCl2·2H2O, 10 mg / L thiamine hydrochloride) containing 0.5% glucose, 50 μg / mL carbenicillin, 34 μg / mL chloramphenicol, 50 μg / mL spectinomycin, and 50 μg / mL kanamycin was added. The culture solution in the LB medium was inoculated into this M9 medium at a 1 / 50 dilution, and cultured with shaking at 200 rpm and 30°C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and cultured with shaking for a further 16 hours.

[0791] The obtained culture solution was centrifuged at 3000×g for 5 minutes to collect the bacteria to an OD of 600 =4 mode is suspended in the reaction buffer (100mM Tris-HCl pH8.0, 20mM MgSO , 20mM glucose) containing 100mM methyl alcohol, 100 μ g / mL carbenicillin, 68 μ g / mL chloramphenicol, 100 μ g / mL spectinomycin, 100 μ g / mL kanamycin and 1mM IPTG.In 14mL test tube, add 1mL bacterial cell suspension, mix with the substrate solution 1mL containing 10mM L-histidine, 10mM L-cysteine as substrate, thus start to comprise methylated thioneine building-up reaction, at 200rpm, 30 ℃ of lower vibrations 48 hours.

[0792] In addition, in order to clarify that the methylation reaction is promoted by the efficient regeneration of methyl carbon in SAM, an experiment was also carried out under the same conditions except that methanol was not added as a methyl carbon supply source, and the results were compared with the results of the experiment carried out under the conditions of adding methanol.

[0793] The obtained reaction solution was analyzed by LC-MS in the same manner as in Example 19.

[0794] The result of measuring is that the histidine betaine concentration and thioneine concentration in the reaction solution are respectively 0.011mM and 0.023mM under the condition of not adding methanol, and relative to this, are respectively 0.011mM and 0.027mM under the condition of adding methanol.Compared with the condition without adding methanol, the total of the accumulation concentration of histidine betaine under the condition of adding methanol and the accumulation concentration of thioneine increased by 0.005mM, is 0.039mM.

[0795] [Comparative Example 27]

[0796] For the Escherichia coli HMS174 (DE3) strain, the expression of the EgtB gene, EgtD gene, and EgtE gene was enhanced as follows.

[0797] The plasmid pET_P constructed in Example 17(A) was cloned into Escherichia coli HMS174 (DE3) (Novagen). T7 -EgtBDE, pACYCDuet-1 (Novagen), pCDFDuet-1 (Novagen), and pCOLA Duet-1 (Novagen), and the resulting strain was designated HMS174 (DE3) / pET_P T7 -EgtBDE.

[0798] [Comparative Example 28]

[0799] The EgtBDE-enhanced strain prepared in Comparative Example 27 was evaluated for the production of histidine betaine and ergothioneine as follows.

[0800] Regarding the production evaluation of histidine betaine and ergothioneine in this comparative example, the HMS174 (DE3) / pET_P prepared in Comparative Example 27 was used. T7 The same procedure as in Example 32 was carried out except for the addition of -EgtBDE.

[0801] As a result, compared with the condition without adding methanol, the histidine betaine concentration and the thioneine concentration in the reaction solution did not increase, and the total of the histidine betaine accumulation concentration and the thioneine accumulation concentration did not increase. Under the condition with the addition of methanol, the total of the histidine betaine accumulation concentration and the thioneine accumulation concentration was 0.016mM.

[0802] The results of Example 32 are summarized in Table 9. To evaluate the efficiency of the methylation reaction, the total accumulation concentration of histidine betaine and ergothioneine was calculated and compared. The total accumulation concentration under the conditions of methanol addition in Example 32 increased by 15% compared to the conditions without methanol addition. On the other hand, in Comparative Example 28, no increase in the total accumulation concentration was observed under the conditions of methanol addition.

[0803] From the above results, it can be seen that by using methanol under conditions where the expression of Mdh2, FtfL, Fch and MtdA is enhanced, SAM can be regenerated more efficiently, the methylation reaction can be promoted, and the production amount of any methyl compound can be increased compared to the case where methanol is not used, or compared to the case where methanol is used under conditions where the expression of the above enzymes is not enhanced.

[0804] In Example 32, the total accumulated concentration under the condition of adding methanol was 2.4 times the result of Comparative Example 28.

[0805] These results indicate that enhanced expression of Mtn and LuxS improves the efficiency of SAM regeneration and further promotes the methylation reaction.

[0806] That is, the method of the present invention has shown that the use of methanol can efficiently regenerate SAM, promote the methylation reaction, and increase the production amount of any methyl compound.

[0807] [Table 9]

[0808]

[0809] Industrial Application Possibilities

[0810] The present invention can provide an industrially useful method for regenerating SAM and a method for producing a methyl compound.

[0811] Sequence Listing Free Text:

[0812] Sequence numbers 1 to 10: synthetic DNA.

Claims

1. A method for producing a methyl compound, comprising: In the presence of formic acid or a salt thereof, (a) a process for producing 5-methyltetrahydrofolate from formic acid or a salt thereof and tetrahydrofolate; (b) a step of converting S-adenosylhomocysteine generated by demethylating S-adenosylmethionine into homocysteine; (c) a step of generating methionine by transferring a methyl group of 5-methyltetrahydrofolate to homocysteine, and converting the methionine into S-adenosylmethionine; and (d) A step of performing a methylation reaction using S-adenosylmethionine as a methyl donor to produce a methyl compound.

2. The method according to claim 1, wherein In step (a), 5-methyltetrahydrofolate is produced from formate and tetrahydrofolate in the presence of formate-tetrahydrofolate ligase (FtfL), methenyl-tetrahydrofolate cyclohydrolase (Fch), methylene-tetrahydrofolate dehydrogenase (MtdA), and 5,10-methylene-tetrahydrofolate reductase (MetF).

3. The method according to claim 1, wherein In the step (b), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine nucleosidase (Mtn) and S-ribosylhomocysteine lyase (LuxS).

4. The method according to claim 1, wherein In the step (b), the conversion of S-adenosylhomocysteine to homocysteine is carried out in the presence of S-adenosylhomocysteine hydrolase, namely, SahH.

5. The method according to claim 1, wherein In the step (b), demethylation of S-adenosylhomocysteine is performed in the presence of an S-adenosylmethionine-dependent methyltransferase.

6. The method according to claim 1, wherein In the step (c), methionine is produced from homocysteine in the presence of methionine synthetase, MetH or MetE, and the conversion of methionine to S-adenosylmethionine is carried out in the presence of methionine adenosyltransferase, MetK.

7. The method according to claim 1, further comprising: (e) A step of converting methanol into formaldehyde and then converting the formaldehyde into formic acid.

8. The method according to claim 7, wherein: In the step (e), the conversion of methanol to formaldehyde is carried out in the presence of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox).

9. The method according to claim 7, wherein: In the step (e), the conversion of formaldehyde to formic acid is carried out in the presence of formaldehyde dehydrogenase, namely, FdhA.

10. The method according to claim 7, wherein: In the step (e), the conversion of formaldehyde to formic acid is carried out by a spontaneous reaction between formaldehyde and glutathione in the presence of S-hydroxymethylglutathione dehydrogenase (FrmA) and S-formylglutathione hydrolase (FrmB).

11. The method according to claim 1, wherein Cells modified so that the activity or expression of S-adenosylmethionine-dependent methyltransferase is enhanced are used.

12. The method according to claim 11, wherein The cell is at least one selected from the group consisting of animal cells, insect cells, plant cells and microorganisms.

13. The method according to claim 1, wherein Methyl compounds are derivatives of aromatic amino acids.

14. A method for producing an organic compound, comprising: A process for producing an organic compound using the methyl compound produced by the method according to claim 1 as an intermediate.

15. A cell modified in such a manner that the activity or expression of formate-tetrahydrofolate ligase (FtfL) and S-adenosylmethionine-dependent methyltransferase is enhanced. 16 . The cell according to claim 15 , which is further modified so that the activity or expression of methylenetetrahydrofolate cyclohydrolase (Fch) and / or methylenetetrahydrofolate dehydrogenase (MtdA) is enhanced.

17. The cell according to claim 15, which is further modified so as to enhance the activity or expression of at least one enzyme selected from the group consisting of S-adenosylhomocysteine nucleosidase (Mtn), S-ribosylhomocysteine lyase (LuxS), and S-adenosylhomocysteine hydrolase (SahH). The cell according to claim 15, further modified to enhance the activity or expression of at least one enzyme selected from the group consisting of ribose diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK). The cell according to claim 15, which is further modified so as to enhance the activity or expression of at least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthetase (MetH or MetE), and methionine adenosyltransferase (MetK).

20. The cell according to claim 15, which is further modified in such a way that the activity or expression of methanol dehydrogenase (Medh) and / or methanol oxidase (Mox) is enhanced.

21. The cell according to claim 20, which is further modified so as to enhance the activity or expression of at least one enzyme selected from the group consisting of formaldehyde dehydrogenase (FdhA), S-hydroxymethylglutathione dehydrogenase (FrmA), and S-formylglutathione hydrolase (FrmB). 22 . The cell according to claim 15 , which is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

23. A method for regenerating S-adenosylmethionine, comprising: (a) a process for producing 5-methyltetrahydrofolate from formic acid or a salt thereof and tetrahydrofolate; (b) a step of converting S-adenosylhomocysteine produced by demethylating S-adenosylmethionine into homocysteine; and (c) a step of transferring the methyl group of the 5-methyltetrahydrofolate to the homocysteine to generate methionine, and converting the methionine into S-adenosylmethionine.

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