Method for producing methyl compound

Abstract

The purpose of the present invention is to provide a novel method for producing a methyl compound by regenerating SAM and promoting a methylation reaction of a compound. Provided is a method for producing a methyl compound using glycine, serine, or an organic starting material.

Description

Technical Field

[0001] The present invention relates to a method for producing a methyl compound. Background Art

[0002] In nature, a variety of methyltransferases are known, and these enzymes perform position-selective and stereoselective methylation on various compounds as substrates. Most of these methyltransferases use S-adenosyl-L-methionine (SAM) as a methyl donor to transfer a methyl group to a target compound.

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

[0004] In response to this, a method of recycling SAM by adding methanol to the culture medium of microorganisms that co-express a methylase and methanol dehydrogenase is known (Patent Document 1: International Publication No. 2019 / 160059).

[0005] Prior Art Documents

[0006] Patent Documents

[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 constructs a recycling pathway of S-adenosylmethionine that supplies methyl carbon through 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, promote the methylation reaction of a compound, and produce a methyl compound. In addition, since SAM plays an important role in the methylation of a compound, there is a need for a cell useful for the efficient regeneration of SAM.

[0010] Means for Solving the Problems

[0011] The present inventors have completed the present invention by successfully and efficiently regenerating SAM and promoting the methylation reaction to produce a methyl compound by using glycine or serine. In addition, the present inventors have completed the present invention by successfully regenerating SAM from an organic raw material efficiently by using a cell modified in such a way as to enhance the activity or expression of serine hydroxymethyltransferase (GlyA) and S-adenosylmethionine (SAM)-dependent methyltransferase.

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

[0013] [1-1]

[0014] A method for producing a methyl compound, comprising the following steps: using cells modified in a manner that enhances the activity or expression of S-adenosylmethionine-dependent methyltransferase, and performing a methylation reaction in the presence of glycine or serine.

[0015] [1-2]

[0016] The method according to the above [1-1], wherein the aforementioned cells are further modified in a manner that enhances the activity or expression of serine hydroxymethyltransferase (GlyA).

[0017] [1-3]

[0018] A method for producing a methyl compound, comprising the following steps: using cells modified in a manner that enhances the activity or expression of serine hydroxymethyltransferase (GlyA) and S-adenosylmethionine-dependent methyltransferase, and performing a methylation reaction.

[0019] [1-4]

[0020] The method according to the above [1-3], wherein the aforementioned cells are further modified in a manner that increases the activity or expression of at least one enzyme selected from the group consisting of D-3-phosphoglycerate dehydrogenase (SerA), phosphoserine aminotransferase (SerC), and phosphoserine phosphatase (SerB).

[0021] [1-5]

[0022] The method according to any one of the above [1-1] to [1-4], wherein the aforementioned cells are further modified in a manner that enhances 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).

[0023] [1-6]

[0024] The method according to any one of the above [1-1] to [1-5], wherein the aforementioned cells are further modified in a manner that enhances the activity or expression of at least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK).

[0025] [1-7]

[0026] According to the method described in any one of [1-1] to [1-6] above, wherein the aforementioned cell is a cell that has been modified in such a way as to enhance the activity or expression of at least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthase (MetH or MetE), and methionine adenosyltransferase (MetK).

[0027] [1-8]

[0028] According to the method described in any one of [1-1] to [1-7] above, wherein the aforementioned cell is a cell that has been modified in such a way as to enhance the activity or expression of at least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl-[GcvH]: protein N-octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP).

[0029] [1-9]

[0030] According to the method described in any one of [1-1] to [1-8] above, wherein the aforementioned cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0031] [1-10]

[0032] According to the method described in [1-9] above, wherein the aforementioned cell is a microorganism.

[0033] [1-11]

[0034] According to the method described in [1-10] above, wherein the aforementioned microorganism is at least one selected from the group consisting of Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, and yeast.

[0035] [1-12]

[0036] According to the method described in any one of [1-1] to [1-11] above, it includes a step of allowing an organic raw material to act on the aforementioned cell.

[0037] [1-13]

[0038] According to the method described in [1-12] above, wherein the aforementioned organic raw material is an organic compound having 3 or more carbon atoms.

[0039] [1-14]

[0040] According to the method described in [1-12] above, wherein the organic raw material comprises one selected from the group consisting of saccharides, alcohols, and organic acids.

[0041] [1-15]

[0042] According to the method described in [1-1] above, wherein the cells are cultured in the presence of an organic raw material containing glycine or serine.

[0043] [1-16]

[0044] According to the method described in any one of [1-1] to [1-15] above, it further includes a step of recovering the methyl compound generated by the aforementioned methylation reaction.

[0045] [1-17]

[0046] According to the method described in any one of [1-1] to [1-16] above, it further includes a step of purifying the methyl compound generated by the aforementioned methylation reaction.

[0047] [1-18]

[0048] According to the method described in any one of [1-12] to [1-14] above, it further includes a step of recovering the methyl compound generated by the action of the aforementioned organic raw material.

[0049] [1-19]

[0050] According to the method described in any one of [1-12] to [1-14] and [1-18] above, it further includes a step of purifying the methyl compound generated by the action of the aforementioned organic raw material.

[0051] [1-20]

[0052] According to the method described in any one of [1-1] to [1-19] above, wherein the methyl compound is a derivative of an aromatic amino acid.

[0053] [1-21]

[0054] A cell modified in such a way that the activities or expressions of serine hydroxymethyltransferase (GlyA) and S-adenosylmethionine-dependent methyltransferase are enhanced.

[0055] [1-22]

[0056] The cell according to [1-21] above is further modified in such a way 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.

[0057] [1-23]

[0058] The cell according to [1-21] or [1-22] above is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of ribose phosphate diphosphokinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) is enhanced.

[0059] [1-24]

[0060] The cell according to any one of [1-21] to [1-23] above is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthase (MetH or MetE), and methionine adenosyltransferase (metK) is enhanced.

[0061] [1-25]

[0062] The cell according to any one of [1-21] to [1-24] above is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl-[GcvH]:protein N-octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP) is increased.

[0063] [1-26]

[0064] The cell according to any one of [1-21] to [1-25] above is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of D-3-phosphoglycerate dehydrogenase (SerA), phosphoserine transaminase (SerC), and phosphoserine phosphatase (SerB) is increased.

[0065] [1-27]

[0066] The cell according to any one of [1-21] to [1-26] above is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

[0067] [1-28]

[0068] The cell according to the above [1-27] is a microorganism.

[0069] [1-29]

[0070] The cell according to the above [1-28], wherein the microorganism is at least one selected from the group consisting of Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, and yeasts.

[0071] [1-30]

[0072] A method for producing an organic compound, which includes a step of producing an organic compound using, as an intermediate, a methyl compound obtained by the method according to any one of the above [1-1] to [1-20].

[0073] [2-1]

[0074] A method for producing a methyl compound, which includes the following step: a methylation reaction is carried out using a microorganism modified in such a way that the activity or expression of an S-adenosylmethionine-dependent methyltransferase is enhanced, in the presence of glycine.

[0075] [2-2]

[0076] The method for producing a methyl compound according to the above [2-1], wherein the microorganism is a microorganism further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of S-adenosylhomocysteinase (Mtn), S-ribosylhomocysteine lyase (LuxS), and S-adenosylhomocysteine hydrolase (SahH) is enhanced.

[0077] [2-3]

[0078] The method for producing a methyl compound according to the above [2-1] or [2-2], wherein the microorganism is a microorganism further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthase (MetH or MetE), and methionine adenosyltransferase (MetK) is enhanced.

[0079] [2-4]

[0080] The method for producing a methyl compound according to any one of [2-1] to [2-3] above, wherein the microorganism is a microorganism modified in such a manner as to enhance the activity or expression of at least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl-[GcvH]: protein N-octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP).

[0081] [2-5]

[0082] The method for producing a methyl compound according to any one of [2-1] to [2-4] above, wherein the microorganism is at least one selected from the group consisting of Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, and yeasts.

[0083] [2-6]

[0084] The method for producing a methyl compound according to any one of [2-1] to [2-5] above, wherein the microorganism is a microorganism cultured in the presence of an organic raw material containing glycine.

[0085] [2-7]

[0086] The method for producing a methyl compound according to any one of [2-1] to [2-6] above, further comprising a step of recovering the methyl compound produced by the methylation reaction.

[0087] [2-8]

[0088] The method for producing a methyl compound according to any one of [2-1] to [2-7] above, further comprising a step of purifying the methyl compound produced by the methylation reaction.

[0089] [2-9]

[0090] A method for producing an organic compound, comprising a step of producing an organic compound using the methyl compound obtained by the method according to any one of [2-1] to [2-8] above as an intermediate.

[0091] [3-1]

[0092] A method for producing a methyl compound, comprising: a step of performing a methylation reaction using a microorganism modified in such a way as to enhance the activity or expression of an S-adenosylmethionine-dependent methyltransferase in the presence of serine.

[0093] [3-2]

[0094] The method for producing a methyl compound according to [3-1] above, wherein the microorganism is a microorganism further modified in such a way as to enhance the activity or expression of at least one enzyme selected from the group consisting of S-adenosylhomocysteinase (Mtn), S-ribosylhomocysteine lyase (LuxS), and S-adenosylhomocysteine hydrolase (SahH).

[0095] [3-3]

[0096] The method for producing a methyl compound according to [3-1] or [3-2] above, wherein the microorganism is a microorganism further modified in such a way as to enhance the activity or expression of at least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthase (MetH or MetE), and methionine adenosyltransferase (MetK).

[0097] [3-4]

[0098] The method for producing a methyl compound according to any one of [3-1] to [3-3] above, wherein the microorganism is a microorganism further modified in such a way as to enhance the activity or expression of serine hydroxymethyltransferase (GlyA).

[0099] [3-5]

[0100] The method for producing a methyl compound according to any one of [3-1] to [3-4] above, wherein the microorganism is a microorganism further modified in such a way as to enhance the activity or expression of at least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl-[GcvH]: protein N-octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP).

[0101] [3-6]

[0102] The method for producing a methyl compound according to any one of [3-1] to [3-5] above, wherein the microorganism is at least one selected from the group consisting of Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, and yeasts.

[0103] [3-7]

[0104] The method for producing a methyl compound according to any one of [3-1] to [3-6] above, wherein the microorganism is a microorganism cultured in the presence of an organic raw material containing serine.

[0105] [3-8]

[0106] The method for producing a methyl compound according to any one of [3-1] to [3-7] above, further comprising a step of recovering the methyl compound generated by the methylation reaction.

[0107] [3-9]

[0108] The method for producing a methyl compound according to any one of [3-1] to [3-8] above, further comprising a step of purifying the methyl compound generated by the methylation reaction.

[0109] [3-10]

[0110] A method for producing an organic compound, comprising a step of producing an organic compound using the methyl compound obtained by the method according to any one of [3-1] to [3-9] above as an intermediate.

[0111] [4-1]

[0112] A microorganism modified in such a way that the activities or expressions of serine hydroxymethyltransferase (GlyA) and S-adenosylmethionine-dependent methyltransferase are enhanced.

[0113] [4-2]

[0114] The microorganism according to [4-1] above, further modified in such a way that the activities or expressions of at least one enzyme selected from the group consisting of S-adenosylhomocysteine nucleosidase (Mtn), S-ribosylhomocysteine lyase (LuxS), and S-adenosylhomocysteine hydrolase (SahH) are enhanced.

[0115] [4-3]

[0116] The microorganism according to [4-1] or [4-2] above is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthase (MetH or MetE), and methionine adenosyltransferase (metK) is enhanced.

[0117] [4-4]

[0118] The microorganism according to any one of [4-1] to [4-3] above is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl-[GcvH]: protein N-octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP) is increased.

[0119] [4-5]

[0120] The microorganism according to any one of [4-1] to [4-4] above is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of D-3-phosphoglycerate dehydrogenase (SerA), phosphoserine aminotransferase (SerC), and phosphoserine phosphatase (SerB) is increased.

[0121] [4-6]

[0122] The microorganism according to any one of [4-1] to [4-5] above is at least one selected from the group consisting of Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, and yeasts.

[0123] [4-7]

[0124] A method for producing a methyl compound, which comprises a step of allowing an organic raw material to act on the microorganism according to any one of [4-1] to [4-6] above.

[0125] [4-8]

[0126] In the method for producing a methyl compound according to [4-7] above, the organic raw material contains one or more selected from the group consisting of saccharides, alcohols, and organic acids.

[0127] [4-9]

[0128] The method for producing a methyl compound according to the above [4-7] or [4-8] further includes a step of recovering the methyl compound produced by reacting the aforementioned organic raw material.

[0129] [4-10]

[0130] The method for producing a methyl compound according to any one of the above [4-7] to [4-9] further includes a step of purifying the methyl compound produced by reacting the aforementioned organic raw material.

[0131] [4-11]

[0132] A method for producing an organic compound includes a step of producing an organic compound using the methyl compound obtained by the method according to any one of the above [4-7] to [4-10] as an intermediate.

[0133] Advantages of the Invention

[0134] According to the present invention, SAM can be efficiently regenerated, and the methylation reaction of substances can be promoted. In addition, according to the present invention, cells useful for the regeneration of SAM can be provided. Further, according to the present invention, a methyl compound can be produced. Further, the amount of the methyl compound produced can be increased as compared with the case where the reaction system of the present invention is not used. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 is a diagram showing an example of the reaction system used in the method of the present invention.

[0136] Figure 2 is a diagram showing an example of the reaction system used in the method of the present invention.

[0137] Figure 3 is a diagram showing an example of the reaction system in the cells of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0138] Hereinafter, the present invention will be described in detail. The following embodiments are illustrative of the present invention and are not intended to limit the present invention only to these embodiments. The present invention can be implemented in various ways without departing from its gist.

[0139] In addition, this specification includes the contents described in the specifications and drawings of Japanese Patent Applications filed on March 31, 2023 (Japanese Patent Application Nos. 2023-056933, 2023-056935, and 2023-056939), which are the basis for claiming the priority of this application.

[0140] 1. Abstract

[0141] ​​​SAM is useful for the methylation reaction of compounds. On the other hand, its supply in cells is limited and it is easily depleted.

[0142] Previously, a method of recycling SAM by adding methanol to the culture medium of microorganisms co-expressing methylase and methanol dehydrogenase has been known.

[0143] However, a method of promoting the methylation reaction and efficiently producing methyl compounds using glycine or serine is unknown.

[0144] In view of this, the present inventors have successfully and efficiently regenerated SAM by using glycine or serine, thereby promoting the methylation reaction, and thus completed the present invention.

[0145] In addition, since SAM plays an important role in the methylation of compounds, cells useful for the efficient regeneration of SAM are required. The present inventors have successfully and efficiently regenerated SAM from organic raw materials by using cells modified in such a way as to enhance the activity or expression of serine hydroxymethyltransferase (GlyA) and S-adenosylmethionine (SAM)-dependent methyltransferase. In addition, the present inventors have successfully promoted the methylation reaction to produce methyl compounds, and thus completed the present invention.

[0146] 2-1. Method for producing methyl compounds using glycine

[0147] (1) Glycine

[0148] Glycine is one of the amino acids and is cleaved by the glycine decarboxylase complex composed of aminomethyltransferase (GcvT), octanoyl-[GcvH]: protein N-octanoyltransferase (GcvH), glycine dehydrogenase (GcvP) and dihydrolipoamide dehydrogenase (LpdA). Along with this reaction, tetrahydrofolic acid is converted to 5,10-methylenetetrahydrofolic acid (glycine cleavage system).

[0149] (2) S-adenosylmethionine

[0150] S-adenosylmethionine (SAM; S-adenosyl-L-methionine, S-adenosyl-L-methionine) is a substance that functions as a methyl donor when a methyltransferase transfers a methyl group to a methylation target substance (substance to be methylated). When the methyl group of SAM is transferred to the methylation target substance and it loses the methyl group (demethylation), it becomes S-adenosyl-L-homocysteine (SAH). SAH is decomposed into homocysteine (Hcy) via S-ribosylhomocysteine (SRH). Since the supply of SAM in cells is limited, the supply of SAM is easily depleted unless there is new methionine.

[0151] (3) S-adenosylmethionine-dependent methyltransferase

[0152] S-adenosylmethionine (SAM)-dependent methyltransferase is an enzyme that transfers the methyl group of SAM to a methylation target substance (substance to be methylated). Examples of the methylation target substance include DNA, protein, and low-molecular-weight compounds that are the targets of methylation, with low-molecular-weight compounds being preferred. SAM is converted to SAH by being demethylated by SAM-dependent methyltransferase. On the other hand, the methylation target substance is methylated to generate a methyl compound. In this specification, SAM-dependent methyltransferase is also abbreviated as "methyltransferase".

[0153] In the present invention, the methyltransferase is not limited as long as it is a substance that transfers the methyl group of SAM to a methylation target substance. Examples of such methyltransferases include methyltransferases that methylate DNA, methyltransferases that methylate proteins, and transferases that methylate low-molecular-weight compounds, but are not limited to these. In addition, as methyltransferases, for example, in BRENDA (https: / / www.brenda-enzyme), which is a well-known database, "methyltransferase" can be used as a keyword for retrieval, and any enzyme recorded in the "Synonyms" column as "S-adenosyl-L-methionine" can be selected. Examples of such methyltransferases include, but are not limited to, L-tyrosine C3-methyltransferase, L-histidine N-alpha-methyltransferase, perillic acid O-methyltransferase, and catechol O-methyltransferase.

[0154] (4) Cell

[0155] In the present invention, cells modified in such a manner that the activity or expression of an S-adenosylmethionine (SAM)-dependent methyltransferase is enhanced can be used. As the cells used in the present invention, there is no limitation as long as they are cells in which SAM is present, and examples thereof include microorganisms, animal (mammalian) cells, insect cells, plant cells (including plant cultured cells), and the like. As the microorganisms, there is no limitation, and examples thereof include Escherichia coli (E. coli), coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, yeasts, and the like. As the animal cells, there is no limitation, and examples thereof include mouse cells, rat cells, rabbit cells, dog cells, monkey cells, human-derived cultured cells, and the like. As the insect cells, there is no limitation as long as they can be used for protein expression using baculoviruses or the like. As the plant cells, there is no limitation, and examples thereof include cells of plants of the genus Nicotiana (for example, Nicotiana benthamiana, Nicotiana tabacum, Nicotiana excelsior, etc.), Solanaceae plants, Gramineae plants, Brassicaceae plants, Asteraceae plants, bryophytes, and the like, but are not limited thereto. The cells used in the present invention are preferably cells capable of expressing foreign genes.

[0156] As the cells used in the present invention, cells (for example, microorganisms) modified in such a manner that the activity or expression of an S-adenosylmethionine (SAM)-dependent methyltransferase is enhanced can be used. In the present invention, "modified cells" include "modified microorganisms".

[0157] In addition, in the present invention, in addition to SAM-dependent methyltransferases, cells modified in such a manner that the activity or expression of at least one enzyme selected from the group consisting of S-adenosylhomocysteinase (Mtn), S-ribosylhomocysteine lyase (LuxS), and S-adenosylhomocysteine hydrolase (SahH) (for example, only SahH; Mtn and LuxS) is enhanced can also be used.

[0158] Mtn is an enzyme for converting S-adenosylhomocysteine (SAH) into S-ribosylhomocysteine (SRH), LuxS is an enzyme for converting SRH into homocysteine (Hcy), and SahH is an enzyme for directly (without passing through the conversion to SRH) converting SAH into Hcy ( Figure 1 ).

[0159] Further, in the present invention, in addition to the SAM-dependent methyltransferase, cells modified in such a way as to enhance the activity or expression of the enzymes in the following (i) and / or (ii) can also be used:

[0160] (i) At least one enzyme selected from the group consisting of Mtn, LuxS, and SahH (e.g., only SahH; Mtn and LuxS),

[0161] (ii) At least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (e.g., Prs and Apt; only ADK, etc.).

[0162] Further, in the present invention, in addition to the SAM-dependent methyltransferase, cells modified in such a way as to enhance the activity or expression of the enzymes in the following (i) to (iii) can also be used:

[0163] (i) At least one enzyme selected from the group consisting of Mtn, LuxS, and SahH (e.g., only SahH; Mtn and LuxS),

[0164] (ii) At least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (e.g., Prs and Apt; only ADK, etc.), and

[0165] (iii) At least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthase (MetH or MetE), and methionine adenosyltransferase (MetK).

[0166] The methionine synthase can be either cobalamin-dependent (MetH) or cobalamin-independent (MetE).

[0167] MetF is an enzyme for generating 5-methyltetrahydrofolate (5-CH3-THF) from 5,10-methylenetetrahydrofolate (5,10-CH2-THF). Methionine synthase (MetH or MetE) is an enzyme for the reaction of transferring the methyl group of 5-methyltetrahydrofolate (5-CH3-THF) to Hcy and converting Hcy into methionine. MetK is an enzyme for the conversion from methionine to SAM ( Figure 1 ).

[0168] The conversion from methionine to SAM requires adenosine triphosphate (ATP), which can be synthesized by the reaction of at least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK).

[0169] Prs can synthesize phosphoribosyl pyrophosphate (PRPP) from ribose-5-phosphate (R5P), which is a metabolite of the pentose phosphate pathway. Apt can synthesize adenosine monophosphate (AMP) from PRPP and adenine. In addition, ADK can synthesize AMP. AMP can be converted to ATP by further phosphorylation.

[0170] Adenine is a metabolite produced by the reaction of Mtn, and adenosine is a metabolite produced by the reaction of SahH. By using these metabolites as substrates to synthesize ATP, the ATP required for the synthesis of SAM can be efficiently supplied.

[0171] Furthermore, in the present invention, in addition to SAM-dependent methyltransferase, cells modified in such a way as to enhance the activity or expression of at least one enzyme selected from the group consisting of the following (i) to (iv) can also be used:

[0172] (i) At least one enzyme selected from the group consisting of Mtn, LuxS, and SahH (for example, only SahH; Mtn and LuxS),

[0173] (ii) At least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (for example, Prs and Apt; only ADK, etc.),

[0174] (iii) At least one enzyme selected from the group consisting of MetF, methionine synthase (MetH or MetE), and MetK, and

[0175] (iv) At least one enzyme selected from the group consisting of glycine amide ribonucleotide transferase (GcvT), octanoyl-[GcvH]: protein N-octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP).

[0176] GcvT, GcvH, and GcvP are enzymes that constitute the glycine decarboxylase complex. Using this complex, glycine is cleaved. Along with this reaction, tetrahydrofolic acid (THF) is converted to 5,10-methylene-tetrahydrofolic acid (5,10-CH2-THF) (glycine cleavage system) ( Figure 1 ).

[0177] Cells modified in such a manner as to enhance the activity or expression of SAM-dependent methyltransferases include, for example, cells containing DNA encoding a SAM-dependent methyltransferase. As described above, SAM-dependent methyltransferases can be appropriately selected based on well-known databases such as BRENDA (https: / / www.brenda-enzyme).

[0178] Cells modified in such a manner as to enhance the activity or expression of Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, MetK, GcvT, GcvH, and GcvP respectively include, for example, cells containing DNA encoding Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, MetK, GcvT, GcvH, and GcvP respectively.

[0179] In the present invention, "modified cells" and cells containing DNA encoding each enzyme can also be referred to as "transformants".

[0180] In the present invention, "modified microorganisms" and microorganisms containing DNA encoding each enzyme can also be referred to as "transformants".

[0181] DNA encoding a SAM-dependent methyltransferase, Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, MetK, GcvT, GcvH, and GcvP respectively can obtain information on their base sequences from well-known databases such as NCBI (https: / / www.ncbi.nlm.nih.gov / nucleotide / ) etc. and can be prepared using well-known genetic engineering methods.

[0182] In addition, in the present invention, in order to introduce DNA encoding each enzyme into cells, a vector containing the DNA can be used. Examples of such vectors include plasmid vectors, viral vectors, etc. The type of vector can be appropriately selected by those skilled in the art according to the type of host cell (e.g., host microorganism) and the purpose. In the vector of the present invention, in addition to DNA encoding the above enzymes, cis-elements such as promoters and enhancers, splicing signals, poly-A addition signals, ribosome-binding sequences (SD sequences), selectable marker genes, reporter genes, etc. can also be included, and they can also be modified.

[0183] As the vector of the present invention, there is no limitation as long as it can express the enzymes used in the present invention. For example, a vector capable of expressing two or more enzyme genes can be used. As such a vector, in the case of a plasmid vector, for example, pET Duet-1, pCOLADuet-1, pACYC Duet-1, pCDF Duet-1 (Novagen), etc. can be cited, but it is not limited to these.

[0184] In the present invention, the vector may contain DNA encoding one or more enzymes. For example, a single vector may also contain: DNA encoding Mtn and LuxS; DNA encoding GcvT, GcvH, and GcvP; DNA encoding MetF, MetH, and MetK; DNA encoding MetF, MetH, MetK, Mtn, and LuxS; DNA encoding Prs and Apt; DNA encoding Prs, Apt, and ADK; or DNA encoding ADK.

[0185] In the present invention, for example, by introducing DNA encoding GcvT, GcvH, and GcvP into a cell, a glycine cleavage reaction system can be introduced into the cell. In addition, in the present invention, for example, in addition to GcvT, GcvH, and GcvP, by introducing DNA encoding LpdA into a cell, a glycine cleavage reaction system can also be introduced into the cell. That is, in the present invention, cells having a glycine cleavage reaction system can be used.

[0186] As the microorganism used in the present invention, there is no limitation as long as it can express SAM-dependent methyltransferase. For example, Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, yeasts, etc. can be cited.

[0187] The culture conditions (temperature, time, composition of the medium, etc.) of the cells used in the present invention and the reaction conditions (temperature, reaction time, pH, etc.) of the enzymes used in the present invention can be appropriately set by those skilled in the art according to the type of cells.

[0188] The cells used in the present invention are preferably cells cultured in the presence of an organic raw material containing glycine, that is, cells cultured in the presence of an organic raw material containing glycine before the start of the methylation reaction (before the addition of the methylation target substance). The inventors of the present invention found that by culturing the modified cells of the present invention in the presence of glycine, the methylation reaction can be promoted.

[0189] In the present invention, the "organic raw material" refers to a carbon source or a nitrogen source that cells (such as microorganisms) can assimilate and proliferate. As the carbon source, for example, organic compounds having 3 or more carbon atoms can be cited, such as sugars, alcohols, and organic acids. As the nitrogen source, for example, organic nitrogen compounds can be cited, but are not limited thereto. In the present invention, as the "sugar", for example, glucose, fructose, sucrose, xylose, arabinose, cellulose, etc. can be cited, but are not limited to these. In the present invention, as the "alcohol", for example, but not limited to, methanol, ethanol, propanol, butanol, glycerol, mannitol, inositol, etc. can be cited. In the present invention, as the "organic acid", for example, formic acid, acetic acid, benzoic acid, citric acid, lactic acid, oxalic acid, succinic acid, etc. can be cited, but are not limited to these. In the present invention, as the "organic nitrogen compound", for example, urea, soybean hydrolyzate, casein hydrolyzate, peptone, yeast extract, meat extract, corn steep liquor, etc. can be cited, but are not limited to these.

[0190] (5) Methyl compound

[0191] In the present invention, the "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 derived from SAM (transferred from SAM). In the present invention, the methyl compound is generated, for example, by the action of SAM-dependent methyltransferase to transfer the methyl group of SAM to the methylation target substance. That is, the methyl compound in the present invention is not limited as long as it is a substance that can be generated or can be generated in the presence of SAM. As the methyl compound in the present invention, for example, derivatives of aromatic amino acids can be cited. As the aromatic amino acid, for example, tyrosine, histidine, tryptophan, phenylalanine, etc. can be cited. As the methyl compound that is a derivative of the aromatic amino acid, for example, 3-methyltyrosine, histidine betaine, ergothioneine, melatonin, vanillic acid, vanillin, isovanillic acid, ferulic acid, isoferulic acid, pinosylvin, pterostilbene, anonaine, thebaine, codeine, magnoflorine, berberine, pinitol, pinositol, etc. can be cited, but are not limited to these.

[0192] In one embodiment of the present invention, the methyl compound can be, for example, at least one selected from the group consisting of 3-methyltyrosine, histidine betaine, ergothioneine, melatonin, anonaine, vanillic acid, vanillin, isovanillic acid, ferulic acid, isoferulic acid, pinosylvin, pterostilbene, thebaine, codeine, magnoflorine, berberine, pinitol, and pinositol.

[0193] In another embodiment of the present invention, the methyl compound can be, for example, at least one selected from the group consisting of 3-methyltyrosine, histidine betaine, ergothioneine, melatonin, and anonaine.

[0194] In another aspect 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, and melatonin.

[0195] (6) Method for producing methyl compounds

[0196] In the present invention, the method for producing a methyl compound includes a step of performing a methylation reaction in the presence of glycine using a cell modified in such a way that the activity or expression of an S-adenosylmethionine-dependent methyltransferase is enhanced.

[0197] In addition, in the present invention, as described above, as the cell, in addition to the SAM-dependent methyltransferase, a cell modified in such a way that the activity or expression of at least one or more enzymes selected from the group consisting of the following (i) to (iv) is enhanced can also be used:

[0198] (i) At least one or more enzymes selected from the group consisting of Mtn, LuxS, and SahH (for example, only SahH; Mtn and LuxS),

[0199] (ii) At least one or more enzymes selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (for example, Prs and Apt; only ADK, etc.),

[0200] (iii) At least one or more enzymes selected from the group consisting of MetF, methionine synthase (MetH or MetE), and MetK, and

[0201] (iv) At least one or more enzymes selected from the group consisting of aminomethyltransferase (GcvT), octanoyl-[GcvH]: protein N-octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP).

[0202] In addition, the method for producing a methyl compound of the present invention may include, for example, the following steps.

[0203] (a) A step of preparing and culturing a cell (e.g., a microorganism) containing at least one DNA selected from (i), (ii), (iii), and (iv) below: (i) a DNA encoding a SAM-dependent methyltransferase; (ii) a DNA encoding at least one enzyme selected from the group consisting of Mtn, LuxS, and SahH; (ii) a DNA encoding at least one enzyme selected from the group consisting of ribose phosphate diphosphokinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK); (iii) a DNA encoding at least one enzyme selected from the group consisting of MetF, methionine synthase (MetH or MetE), and MetK; and (iv) a DNA encoding at least one enzyme selected from the group consisting of GcvT, GcvH, and GcvP;

[0204] (b) A step of adding glycine to the culture solution of the cells obtained in step (a) and further culturing.

[0205] In step (a), the culturing can be carried out in the presence of glycine.

[0206] In addition, the production method of the present invention may further include a step of recovering the methyl compound generated by the methylation reaction.

[0207] Furthermore, the production method of the present invention may further include a step of purifying the methyl compound generated by the methylation reaction.

[0208] For the recovery and purification of the generated methyl compound, those skilled in the art can perform it by known methods suitable for the physical properties of the methyl compound, such as distillation, membrane dehydration, desalting using ion exchange resins, crystallization, column chromatography, etc.

[0209] In the present invention, when Escherichia coli is used as the microorganism, the production method of the methyl compound can be carried out as follows, for example, but is not limited thereto.

[0210] First, DNA encoding each enzyme is introduced into Escherichia coli as a host to prepare a transformant. The transformant is cultured overnight at about 37 °C in a known medium (e.g., LB medium). Then, the proliferated transformant is suspended in a minimal medium (e.g., M9 medium) and cultured at about 30 °C for an appropriate time (e.g., 6 hours), and then gene expression is induced using IPTG, etc., and further cultured for an appropriate time (e.g., 16 hours). Thus, a transformant with enhanced activity or expression of each enzyme can be prepared. The aforementioned minimal medium may contain glycine.

[0211] Next, a transformant with enhanced activity or expression of each enzyme is contacted with a solution containing glycine (reaction buffer) (for example, the transformant is suspended in a solution containing glycine), a methylation target substance is added thereto, and further cultured at an appropriate temperature (for example, about 30°C) and time (for example, about 6 to 24 hours), whereby a methylation reaction occurs to produce a methyl compound.

[0212] In addition to glycine, the solution containing glycine may, for example, also contain a buffer and glucose as an ATP supply source, and may also contain a divalent metal salt (for example, magnesium sulfate).

[0213] 2-2. Method for producing methyl compounds using serine

[0214] (1) Serine

[0215] Serine is an amino acid and is converted to glycine by serine hydroxymethyltransferase (GlyA). Along with this reaction, tetrahydrofolic acid is converted to 5,10-methylene tetrahydrofolic acid.

[0216] (2) S-adenosylmethionine

[0217] Regarding S-adenosylmethionine (SAM; S-adenosyl-L-methionine), as described in the above "2-1(2)".

[0218] (3) S-adenosylmethionine-dependent methyltransferase

[0219] Regarding S-adenosylmethionine (SAM)-dependent methyltransferase, as described in the above "2-1(3)".

[0220] (4) Cell

[0221] Regarding the types of "cells" that can be used in the present invention, as described in the above "2-1(4)". Specifically, as the "cells" in the present invention, for example, microorganisms, animal cells, insect cells, plant cells (including plant cultured cells), etc. can be mentioned, but are not limited thereto.

[0222] In addition, as the cells used in the present invention, cells (for example, microorganisms) modified in such a way that the activity or expression of S-adenosylmethionine (SAM)-dependent methyltransferase is enhanced can be used.

[0223] In addition, in the present invention, in addition to the SAM-dependent methyltransferase, cells modified in such a way as to enhance the activity or expression of 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) (for example, only SahH; Mtn and LuxS) can also be used.

[0224] Furthermore, in the present invention, in addition to the SAM-dependent methyltransferase, cells modified in such a way as to enhance the activity or expression of the enzymes in the following (i) and / or (ii) can also be used:

[0225] (i) At least one or more enzymes selected from the group consisting of Mtn, LuxS, and SahH (for example, only SahH; Mtn and LuxS),

[0226] (ii) At least one or more enzymes selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (for example, Prs and Apt; only ADK, etc.).

[0227] Furthermore, in the present invention, in addition to the SAM-dependent methyltransferase, cells modified in such a way as to enhance the activity or expression of the enzymes in the following (i) to (iii) can also be used:

[0228] (i) At least one or more enzymes selected from the group consisting of Mtn, LuxS, and SahH (for example, only SahH; Mtn and LuxS),

[0229] (ii) At least one or more enzymes selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (for example, Prs and Apt; only ADK, etc.), and

[0230] (iii) At least one or more enzymes selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthase (MetH or MetE), and methionine adenosyltransferase (MetK).

[0231] The methionine synthase can be either cobalamin-dependent (MetH) or cobalamin-independent (MetE).

[0232] Furthermore, in the present invention, in addition to the SAM-dependent methyltransferase, cells modified in such a way as to enhance the activity or expression of at least one or more enzymes selected from the group consisting of the following (i) to (iv) can also be used:

[0233] (i) At least one enzyme selected from the group consisting of Mtn, LuxS, and SahH (for example, only SahH; Mtn and LuxS),

[0234] (ii) At least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (for example, Prs and Apt; only ADK, etc.),

[0235] (iii) At least one enzyme selected from the group consisting of MetF, methionine synthase (MetH or MetE), and MetK, and

[0236] (iv) At least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl - [GcvH]: protein N - octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP).

[0237] Furthermore, in the present invention, in addition to SAM - dependent methyltransferase, cells modified by a method of enhancing the activity or expression of at least one enzyme selected from the group consisting of the following (i) to (v) can also be used:

[0238] (i) At least one enzyme selected from the group consisting of Mtn, LuxS, and SahH (for example, only SahH; Mtn and LuxS),

[0239] (ii) At least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (for example, only Prs and Apt; only ADK, etc.),

[0240] (iii) At least one enzyme selected from the group consisting of MetF, methionine synthase (MetH or MetE), and MetK,

[0241] (iv) At least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl - [GcvH]: protein N - octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP), and

[0242] (v) Serine hydroxymethyltransferase (GlyA).

[0243] Regarding Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, methionine synthase (MetH or MetE), and MetK, GcvT, GcvH, GcvP, as described in the above "2 - 1(4)".

[0244] Serine hydroxymethyltransferase (GlyA) is an enzyme used to convert serine to glycine, and with this reaction, tetrahydrofolate (THF) is converted to 5,10-methylenetetrahydrofolate (5-CH3-THF)( Figure 2 ).

[0245] Cells modified in such a way as to enhance the activity or expression of SAM-dependent methyltransferases include, for example, cells containing DNA encoding a SAM-dependent methyltransferase. As described above, SAM-dependent methyltransferases can be appropriately selected based on known databases such as BRENDA (https: / / www.brenda-enzyme).

[0246] Cells modified in such a way as to enhance the activity or expression of Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, MetK, GcvT, GcvH, GcvP, and GlyA respectively include, for example, cells containing DNA encoding Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, MetK, GcvT, GcvH, GcvP, and GlyA respectively.

[0247] In the present invention, "modified cells" and cells containing DNA encoding each enzyme can also be referred to as "transformants".

[0248] In the present invention, "modified microorganisms" and microorganisms containing DNA encoding each enzyme can also be referred to as "transformants".

[0249] DNA encoding SAM-dependent methyltransferase, Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, MetK, GcvT, GcvH, GcvP, and GlyA respectively can obtain information on their base sequences from known databases such as NCBI (https: / / www.ncbi.nlm.nih.gov / nucleotide / ) etc., and can be prepared using known genetic engineering methods.

[0250] In addition, in the present invention, in order to introduce DNA encoding each enzyme into cells, a vector containing the DNA can be used. The types of vectors that can be used in the present invention are as described in "2-1(4)" above.

[0251] In the present invention, the vector may contain DNA encoding one or more enzymes. For example, a vector may also contain: DNA encoding MetF, MetH or MetE, MetK, Mtn and LuxS; DNA encoding MetF, MetH or MetE and MetK; DNA encoding Mtn and LuxS; DNA encoding GcvT, GcvH and GcvP; DNA encoding Prs and Apt; DNA encoding Prs, Apt and ADK; or DNA encoding ADK.

[0252] Regarding the cells used in the present invention and their culture conditions, as described in the above “2-1(4)”.

[0253] The cells used in the present invention are preferably cells cultured in the presence of an organic raw material containing serine, that is, cells cultured in the presence of an organic raw material containing serine before the start of the methylation reaction (before the addition of the methylation target substance). The inventors of the present invention found that by culturing the modified cells of the present invention in the presence of serine, the methylation reaction can be promoted.

[0254] Regarding the “organic raw material” used in the present invention, as described in the above “2-1(4)”.

[0255] (5) Methyl compound

[0256] Regarding the “methyl compound” in the present invention, as described in the above “2-1(5)”.

[0257] (6) Method for producing methyl compounds

[0258] In the present invention, the method for producing a methyl compound includes a step of performing a methylation reaction using cells modified in such a way as to enhance the activity or expression of S-adenosylmethionine-dependent methyltransferase in the presence of serine.

[0259] In addition, in the present invention, as described above, as the cells, in addition to SAM-dependent methyltransferase, cells modified in such a way as to enhance the activity or expression of at least one or more enzymes selected from the group consisting of the following (i) to (v) can also be used:

[0260] (i) At least one or more enzymes selected from the group consisting of Mtn, LuxS and SahH (for example, only SahH; Mtn and LuxS),

[0261] (ii) At least one or more enzymes selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt) and adenosine kinase (ADK) (for example, only Prs and Apt; only ADK, etc.),

[0262] (iii) at least one enzyme selected from the group consisting of MetF, methionine synthase (MetH or MetE), and MetK,

[0263] (iv) at least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl - [GcvH]: protein N - octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP), and

[0264] (v) serine hydroxymethyltransferase (GlyA).

[0265] In addition, the method for producing a methyl compound of the present invention may include, for example, the following steps.

[0266] (a) A step of preparing a cell containing at least one DNA selected from the following (i), (ii), (iii), (iv), and (v) and culturing it. (i) DNA encoding a SAM - dependent methyltransferase, (ii) DNA encoding at least one enzyme selected from the group consisting of Mtn, LuxS, and SahH, (ii) DNA encoding at least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK), (iii) DNA encoding at least one enzyme selected from the group consisting of MetF, methionine synthase (MetH or MetE), and MetK, (iv) DNA encoding at least one enzyme selected from the group consisting of GcvT, GcvH, and GcvP, and (v) DNA encoding serine hydroxymethyltransferase (GlyA), and

[0267] (b) A step of adding serine to the culture solution of the cells obtained in step (a) and further culturing.

[0268] In step (a), the culture can be carried out in the presence of serine.

[0269] In addition, regarding the recovery and purification of the methyl compound in the production method of the present invention, it is as described in the above "2 - 1(6)".

[0270] In the present invention, when Escherichia coli is used as the microorganism, the method for producing a methyl compound using serine is the same as the method using glycine described in the above "2 - 1(6)".

[0271] 2 - 3. Having S-adenosylmethionine regeneration ability of the cells

[0272] (1) S-adenosylmethionine

[0273] Regarding S-adenosylmethionine (SAM; S-adenosyl-L-methionine), as described in "2-1(2)" above.

[0274] (2) Regeneration of S-adenosylmethionine

[0275] In the present invention, the "regeneration" of SAM means generating SAM without adding methionine or SAM from outside the system. Regarding whether SAM is generated, those skilled in the art can evaluate it using well-known methods, such as methods for measuring the methyltransferase activity in a test sample. For example, by measuring the methyltransferase activity in a test sample, when the activity in the test sample is higher than that of the control, it can be evaluated that SAM is generated. As the control, a sample with a different condition from the test sample can be used, such as a sample without the substance added to the test sample. The methyltransferase activity can be evaluated, for example, by introducing a foreign methyltransferase gene into cells (such as microorganisms), inducing the expression of the enzyme, adding a methylation target substance to the cell culture medium to react with the enzyme, and measuring the production amount of methyl compounds using a mass spectrometry method such as LC-MS. That is, in a test sample, without adding methionine or SAM from outside the reaction system and when the methyltransferase activity is higher than that of the control, it can be said that SAM is regenerated in the test sample. It should be noted that in the present invention, the method for measuring the methyltransferase activity can be appropriately adopted by those skilled in the art using well-known methods, and is not limited to the above method.

[0276] In addition, the above "higher than the control" means 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 higher than the methyltransferase activity in the control.

[0277] (3) Cell

[0278] Regarding the types of "cells" that can be used in the present invention, as described in "2-1(4)" above. Specifically, as the "cells" in the present invention, for example, microorganisms, animal cells, insect cells, plant cells (including plant cultured cells), etc. can be mentioned, but are not limited to these.

[0279] In addition, in the present invention, cells having the ability to regenerate S-adenosylmethionine refer to cells (such as microorganisms) that have been modified for efficient regeneration of SAM.

[0280] In the present invention, as such cells, for example, cells modified in such a manner that the activities or expressions of serine hydroxymethyltransferase (GlyA) and S-adenosylmethionine (SAM)-dependent methyltransferase are enhanced can be used.

[0281] In the present invention, serine hydroxymethyltransferase (GlyA) is an enzyme that converts serine into glycine and converts tetrahydrofolic acid into 5,10-methylenetetrahydrofolic acid.

[0282] In the present invention, the S-adenosylmethionine (SAM)-dependent methyltransferase is an enzyme that transfers the methyl group of SAM to a methylation target substance (substance to be methylated). Regarding the S-adenosylmethionine (SAM)-dependent methyltransferase, as described in the above “2-1(3)”.

[0283] In the present invention, in addition to GlyA and SAM-dependent methyltransferase, cells modified in such a manner that the activities or expressions of 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) (for example, only SahH; Mtn and LuxS) are enhanced can also be used.

[0284] Furthermore, in the present invention, in addition to GlyA and SAM-dependent methyltransferase, cells modified in such a manner that the activities or expressions of the enzymes in the following (i) and / or (ii) are enhanced can also be used:

[0285] (i) At least one or more enzymes selected from the group consisting of Mtn, LuxS, and SahH (for example, only SahH; Mtn and LuxS),

[0286] (ii) At least one or more enzymes selected from the group consisting of ribose phosphate diphosphokinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (for example, Prs and Apt; only ADK, etc.).

[0287] Furthermore, in the present invention, in addition to GlyA and SAM-dependent methyltransferase, cells modified in such a manner that the activities or expressions of the enzymes in the following (i) to (iii) are enhanced can also be used:

[0288] (i) At least one or more enzymes selected from the group consisting of Mtn, LuxS, and SahH (for example, only SahH; Mtn and LuxS),

[0289] (ii) at least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (e.g., Prs and Apt), and

[0290] (iii) at least one enzyme selected from the group consisting of 5,10 - methylene - tetrahydrofolate reductase (MetF), methionine synthase (MetH or MetE), and methionine adenosyltransferase (metK).

[0291] The methionine synthase can be either cobalamin - dependent (MetH) or cobalamin - independent (MetE).

[0292] Furthermore, in addition to GlyA and SAM - dependent methyltransferase, the present invention can also use cells modified in such a way as to enhance the activity or expression of the enzymes in the following (i) - (iv):

[0293] (i) at least one enzyme selected from the group consisting of Mtn, LuxS, and SahH (e.g., only SahH; Mtn and LuxS),

[0294] (ii) at least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (e.g., Prs and Apt; only ADK, etc.),

[0295] (iii) at least one enzyme selected from the group consisting of MetF, methionine synthase (MetH or MetE), and MetK, and

[0296] (iv) at least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl - [GcvH]: protein N - octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP).

[0297] Furthermore, in the present invention, in addition to GlyA and SAM - dependent methyltransferase, cells modified in such a way as to enhance the activity or expression of the enzymes in the following (i) - (v) can also be used:

[0298] (i) at least one enzyme selected from the group consisting of Mtn, LuxS, and SahH (e.g., only SahH; Mtn and LuxS),

[0299] (ii) at least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) (e.g., Prs and Apt; only ADK, etc.),

[0300] (iii) at least one enzyme selected from the group consisting of MetF, methionine synthase (MetH or MetE), and MetK,

[0301] (iv) at least one enzyme selected from the group consisting of GcvT, GcvH, and GcvP, and

[0302] (v) at least one enzyme selected from the group consisting of D-3-phosphoglycerate dehydrogenase (SerA), phosphoserine aminotransferase (SerC), and phosphoserine phosphatase (SerB).

[0303] Regarding Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, methionine synthase (MetH or MetE), and MetK, GcvT, GcvH, GcvP, as described in "2-1(4)" above.

[0304] Regarding GlyA, as described in "2-2(4)" above.

[0305] SerA is an enzyme for converting glyceraldehyde-3-phosphate (3-PG) to 3-phosphonooxypyruvate (3-PHP), SerC is an enzyme for converting 3-PHP to 3-phosphoserine (3-PS), and SerB is an enzyme for converting 3-PS to serine ( Figure 3 ).

[0306] Examples of cells modified in such a way as to enhance the activity or expression of GlyA and SAM-dependent methyltransferase include cells containing DNA encoding GlyA and DNA encoding SAM-dependent methyltransferase. As described above, SAM-dependent methyltransferase can be appropriately selected based on known databases such as BRENDA (https: / / www.brenda-enzyme).

[0307] Examples of cells modified in such a way as to enhance the activity or expression of Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, MetK, GcvT, GcvH, GcvP, SerA, SerC, and SerB respectively include cells containing DNA encoding Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, MetK, GcvT, GcvH, GcvP, SerA, SerC, and SerB.

[0308] In the present invention, "modified cells" and cells containing DNA encoding each enzyme may also be referred to as "transformants".

[0309] In the present invention, the "modified microorganism" and the microorganism containing the DNA encoding each enzyme may also be referred to as "transformant".

[0310] The DNAs encoding SAM-dependent methyltransferase, Mtn, LuxS, SahH, Prs, Apt, ADK, MetF, MetH, MetE, MetK, GcvT, GcvH, GcvP, SerA, SerC, and SerB can obtain the information of their base sequences from publicly known databases such as NCBI (https: / / / www.ncbi.nlm.nih.gov / nucleotide / ) respectively, and can be prepared using publicly known genetic engineering methods.

[0311] In addition, in the present invention, in order to introduce the DNA encoding each enzyme into cells, a vector containing the DNA can be used. The types of vectors that can be used in the present invention are as described in the above "2-1(4)".

[0312] In the present invention, the vector may contain the DNA encoding one or more enzymes. For example, a vector may contain: the DNA encoding Mtn and LuxS, the DNA encoding MetF, MetH or MetE, MetK, Mtn and LuxS, the DNA encoding MetF, MetH or MetE and MetK, the DNA encoding Mtn and LuxS, or the DNA encoding GcvT, GcvH and GcvP, the DNA encoding Prs and Apt, the DNA encoding Prs, Apt and ADK, or the DNA encoding ADK.

[0313] In the present invention, for example, by introducing the DNA encoding GlyA into cells, a reaction system that can convert serine produced from organic raw materials into glycine and convert tetrahydrofolic acid into 5,10-methylene tetrahydrofolic acid can be introduced into the cells.

[0314] In addition, in the present invention, for example, by introducing the DNA encoding GcvT, GcvH and GcvP into cells, a glycine cleavage reaction system can be introduced into the cells. Further, in the present invention, for example, in addition to GcvT, GcvH and GcvP, the DNA encoding LpdA can also be introduced into cells to introduce the glycine cleavage reaction system into the cells.

[0315] The culture conditions of the cells used in the present invention are as described in the above "2-1(4)".

[0316] (4) Methyl compound

[0317] Regarding the "methyl compound" in the present invention, it is as described in the above "2-1(5)".

[0318] (5) Method for producing methyl compounds

[0319] In the present invention, a method for producing a methyl compound includes the following steps: performing a methylation reaction using cells modified in such a way as to enhance the activity or expression of serine hydroxymethyltransferase (GlyA) and S-adenosylmethionine-dependent methyltransferase. Regarding the term "cells", as described in the above "2-1(4)".

[0320] In the method for producing a methyl compound of the present invention, the cells described in the above "2-3(3)" can be used.

[0321] In the present invention, the method for producing a methyl compound includes a step of allowing an organic raw material to act on the cells described in the above "2.(3) cells". In the present invention, "allowing an organic raw material to act on cells" means culturing the cells in the presence of the organic raw material.

[0322] Regarding the "organic raw material" used in the present invention, as described in the above "2-1(4)".

[0323] In addition, the method for producing a methyl compound of the present invention may, for example, include the following steps.

[0324] (a) A step of preparing cells containing at least one DNA selected from the following (i), (ii), (iii), (iv), (v), (vi), and (vii) and culturing them: (i) DNA encoding GlyA; (ii) DNA encoding SAM-dependent methyltransferase; (iii) DNA encoding at least one enzyme selected from the group consisting of Mtn, LuxS, and SahH (e.g., only SahH; Mtn and LuxS); (iv) DNA encoding at least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK); (v) DNA encoding at least one enzyme selected from the group consisting of MetF, methionine synthase (MetH or MetE), and MetK; (vi) DNA encoding at least one enzyme selected from the group consisting of GcvT, GcvH, and GcvP; (vii) DNA encoding at least one enzyme selected from the group consisting of SerA, SerC, and SerB; and

[0325] (b) A step of adding an organic raw material to the culture solution of the cells obtained in step (a) and further culturing them.

[0326] In addition, the production method of the present invention may further include a step of recovering the generated methyl compound.

[0327] Furthermore, the manufacturing method of the present invention may further include a step of purifying the generated methyl compound.

[0328] For the recovery and purification of the generated methyl compound, those skilled in the art can perform it by known methods suitable for the physical properties of the methyl compound, such as distillation, membrane dehydration, desalting using ion exchange resins, crystallization, column chromatography, and other treatments.

[0329] In the present invention, when Escherichia coli is used as the microorganism, the manufacturing method of the methyl compound can be carried out as follows, for example, but is not limited thereto.

[0330] First, DNA encoding each enzyme is introduced into Escherichia coli as the host to produce a transformant. The transformant is cultured overnight at about 37 °C in a known medium (such as LB medium). Then, the proliferated transformant is suspended in a minimal medium (such as M9 medium) and cultured at about 30 °C for an appropriate time (such as 6 hours), and then gene expression is induced using IPTG or the like, and further cultured for an appropriate time (such as 16 hours). Thus, a transformant with enhanced activity or expression of each enzyme can be produced.

[0331] Next, the transformant with enhanced activity or expression of each enzyme is contacted with a solution (reaction buffer) containing an organic raw material (such as glucose) (for example, the transformant is suspended in the solution containing the organic raw material), a methylation target substance is added thereto, and further cultured at an appropriate temperature (such as about 30 °C) and time (such as about 6 to 24 hours), whereby a methylation reaction occurs to generate a methyl compound.

[0332] In addition to the organic raw material, the solution containing the organic raw material may, for example, also contain a buffer and glucose as an ATP supply source, and may further contain a divalent metal salt (such as magnesium sulfate).

[0333] 3. Method for producing organic compounds

[0334] In the present invention, the methyl compound generated by the manufacturing method of the methyl compound of the present invention can be used as an intermediate to manufacture an organic compound. That is, the present invention provides a method for manufacturing an organic compound, which includes a step of manufacturing an organic compound by using the methyl compound generated by the SAM regeneration method or the methyl compound manufacturing method of the present invention as an intermediate.

[0335] In the present invention, an organic compound refers to a compound generated by using a methyl compound as an intermediate. As the methyl compound used as an intermediate, examples include substances generated in the SAM regeneration method or the methyl compound manufacturing method of the present invention.

[0336] The production of an organic compound using a methyl compound as an intermediate can be carried out by a person skilled in the art based on a known method. In addition, the production conditions can be appropriately set by a person skilled in the art.

[0337] 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, etc. can be cited. Tyrosine is methylated by the regeneration method of the SAM of the present invention or the method for producing a methyl compound to generate 3-methyltyrosine and 3,5-dimethyltyrosine, and then, these methyl compounds are respectively recognized as substrates by using a lyase, so that they can 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 method for producing a 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. 2018 Feb; 7 (2): 201-203.). Further, by the method for producing a methyl compound of the present invention, L-histidine can be methylated, and ergothioneine as an organic compound can be generated by using histidine betaine as an intermediate.

[0338] Hereinafter, the present invention will be described in detail by way of 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.

[0339] Example

[0340] [Example 1]

[0341] For the Escherichia coli HMS174 (DE3) strain, expression of the sfmM2 gene was enhanced as follows.

[0342] (A) Construction of SfmM2 enhanced plasmid

[0343] 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 restriction enzyme Nde I is assigned to the 5' end, and a recognition sequence for restriction enzyme Bgl II is assigned to the 3' end). It should be noted that a sequence with optimized codons for efficient expression in Escherichia coli was designed as the base sequence of the sfmM2 gene. The synthesized DNA was 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 was named pET_P T7-sfmM2.

[0344] (B) Production of SfmM2 enhanced strain

[0345] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -sfmM2, pCDF Duet-1 (Novagen), and pACYC Duet-1 (Novagen) constructed in Example 1(A), and the resulting strain was designated HMS174(DE3) / pET_P T7 -sfmM2.

[0346] [Example 2]

[0347] For the Escherichia coli HMS174(DE3) strain, the expression of the sfmM2 gene, gcvT gene, gcvH gene, and gcvP gene was enhanced as described below.

[0348] (A) Construction of GcvTHP enhanced plasmid

[0349] A DNA containing the base sequences of the gcvT gene, gcvH gene, and gcvP gene from Escherichia coli (also collectively referred to as "GcvTHP" for GcvT, GcvH, and GcvP) (a recognition sequence for the restriction enzyme Nco I was given to the 5'-terminal side, and a recognition sequence for the restriction enzyme BamHI was given to the 3'-terminal side) was synthesized. The synthesized DNA was inserted into the restriction enzyme sites Nco I and BamHI of the Escherichia coli expression vector pACYC Duet-1 (Novagen) and ligated downstream of the T7 promoter. The constructed plasmid was named pACYC_P T7 -gcvTHP.

[0350] (B) Production of SfmM2, GcvTHP enhanced strains

[0351] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -sfmM2 constructed in Example 1(A), the plasmid pACYC_P T7 -gcvTHP constructed in Example 2(A), and pCDF Duet-1 (Novagen), and the resulting strain was designated HMS174(DE3) / pET_P T7 -sfmM2 / pACYC_P T7 -gcvTHP.

[0352] [Example 3]

[0353] For the Escherichia coli HMS174(DE3) strain, the expression of the sfmM2 gene and the luxS gene was enhanced as described below.

[0354] (A) Construction of Mtn, LuxS enhanced plasmid

[0355] Synthesize DNA (synthetic DNA base sequence: SEQ ID NO: 3) containing the base sequences of the mtn gene and the luxS gene from Escherichia coli (with a recognition sequence for restriction enzyme Nde I assigned to the 5'-terminal side and a recognition sequence for restriction enzyme Bgl II assigned to the 3'-terminal side). Insert the synthesized DNA into the restriction enzyme sites Nde I and Bgl II of the Escherichia coli expression vector pCDF Duet-1 (Novagen) and ligate it downstream of the T7 promoter. The constructed plasmid was named pCDF_P T7 -mtn-luxS.

[0356] (B) Construction of LuxS enhanced plasmid

[0357] Treat the plasmid pCDF_P T7 -mtn-luxS constructed in Example 2(A) with the restriction enzyme NdeI, separate it by agarose gel electrophoresis, and then recover and purify it from the gel to obtain a DNA fragment with the mtn gene region removed. Ligate the two ends of the obtained DNA fragment. The constructed plasmid was named pCDF_P T7 -luxS.

[0358] (C) Production of SfmM2, LuxS enhanced strains

[0359] Transform the Escherichia coli HMS174(DE3) strain (Novagen) with the plasmid pET_P T7 -sfmM2 constructed in Example 1(A), the plasmid pCDF_P T7 -luxS constructed in Example 3(B), and pACYC Duet-1 (Novagen). The resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -luxS.

[0360] [Example 4]

[0361] For the Escherichia coli HMS174(DE3) strain, the expression of the sfmM2 gene, the luxS gene, the gcvT gene, the gcvH gene, and the gcvP gene was enhanced as described below.

[0362] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -sfmM2 constructed in Example 1(A), the plasmid pCDF_P T7 -luxS constructed in Example 3(B), and the plasmid pACYC_P T7 -gcvTHP constructed in Example 2(A). The resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -luxS / pACYC_P T7 -gcvTHP.

[0363] [Example 5]

[0364] For the Escherichia coli HMS174(DE3) strain, the expression of the sfmM2 gene, mtn gene, and luxS gene was enhanced as described below.

[0365] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -sfmM2 constructed in Example 1(A), the plasmid pCDF_P T7 -mtn-luxS constructed in Example 3(A), and pACYC Duet-1 (Novagen). The resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -mtn-luxS.

[0366] [Example 6]

[0367] For the Escherichia coli HMS174(DE3) strain, the expression of the sfmM2 gene, mtn gene, luxS gene, gcvT gene, gcvH gene, and gcvP gene was enhanced as described below.

[0368] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -sfmM2 constructed in Example 1(A), the plasmid pCDF_P T7 -mtn-luxS constructed in Example 3(A), and the plasmid pACYC_P T7 -gcvTHP constructed in Example 2(A). The resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -mtn-luxS / pACYC_P T7 -gcvTHP.

[0369] [Example 7]

[0370] For Escherichia coli HMS174(DE3) strain, as described below, the expression of sfmM2 gene and sahH gene was enhanced.

[0371] (A) Construction of SahH enhanced plasmid

[0372] Synthesize DNA (synthetic DNA base sequence: SEQ ID NO: 4) containing the base sequence of sahH gene derived from Pseudomonas aeruginosa (giving the recognition sequence of restriction enzyme Nde I to the 5'-terminal side and the recognition sequence of restriction enzyme Bgl II to the 3'-terminal side). It should be noted that for the sahH gene, a sequence with codons optimized for efficient expression in Escherichia coli was designed. Insert the synthesized DNA into the restriction enzyme sites Nde I and Bgl II of the Escherichia coli expression vector pCDFDuet-1 (Novagen) and ligate it downstream of the T7 promoter. The constructed plasmid was named pCDF_P T7 -sahH.

[0373] (B) Production of SfmM2, SahH enhanced strains

[0374] Transform Escherichia coli HMS174(DE3) strain (Novagen) with the plasmid pET_P T7 -sfmM2 constructed in Example 1(A), the plasmid pCDF_P T7 -sahH constructed in Example 7(A), and pACYC Duet-1 (Novagen), and the resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -sahH.

[0375] [Example 8]

[0376] For Escherichia coli HMS174(DE3) strain, as described below, the expression of sfmM2 gene, sahH gene, gcvT gene, gcvH gene and gcvP gene was enhanced.

[0377] Transform Escherichia coli HMS174(DE3) strain (Novagen) with the plasmid pET_P T7 -sfmM2 constructed in Example 1(A), the plasmid pCDF_P T7 -sahH constructed in Example 7(A), and the plasmid pACYC_P T7-Transform with gcvTHP, and designate the resulting strain as HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -sahH / pACYC_P T7 -gcvTHP.

[0378] [Example 9]

[0379] For the SfmM2 enhanced strain prepared in Example 1 (B), the production evaluation of 3-methyltyrosine was carried out as follows.

[0380] The HMS174(DE3) / pET_P prepared in Example 1 (B) T7 -sfmM2 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 34 μg / mL chloramphenicol. 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 34 μg / mL chloramphenicol was added to a 50 mL test tube, and the culture solution in the above LB medium was inoculated in such a way that it was diluted 1 / 50, and shaken at 200 rpm and 30 °C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and further shaken for 16 hours.

[0381] The resulting culture solution was pelleted by centrifugation at 3000×g for 5 minutes, and suspended in a reaction buffer (100 mM Tris-HCl pH 8.0, 20 mM MgSO4, 20 mM glucose) containing 20 mM glycine, 100 μg / mL carbenicillin, 100 μg / mL spectinomycin, 68 μg / mL chloramphenicol and 1 mM IPTG at OD 600 =10. 1 mL of the 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 start the methylation reaction, and shaken at 200 rpm and 30 °C for 24 hours. In addition, in order to clarify the promotion of the methylation reaction by the efficient regeneration of the methyl carbon of SAM, an experiment was also carried out under the same conditions without adding glycine as a supply source of methyl carbon, and the results were compared with the results of the experiment carried out under the condition of adding glycine.

[0382] Mix 20 μL of the resulting reaction solution with 20 μL of pure water, 80 μL of 1N hydrochloric acid, and 80 μL of acetonitrile, and collect the supernatant after centrifugation at 3000×g for 5 minutes for LC-MS analysis. The conditions for LC-MS analysis are shown in Table 1.

[0383] As a result of the measurement, the concentration of 3-methyltyrosine in the reaction solution increased by 0.212 mM compared to the condition without glycine addition, and was 0.340 mM under the condition with glycine addition.

[0384] [Table 1]

[0385]

[0386] [Example 10]

[0387] For the SfmM2 enhanced strain prepared in Example 1 (B), the production evaluation of 3-methyltyrosine was carried out under the condition of adding glycine during cultivation as described below.

[0388] Regarding the production evaluation of 3-methyltyrosine in this example, after culturing in LB medium, use 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol. Otherwise, it was carried out in the same manner as in Example 9.

[0389] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.267 mM compared to the condition without glycine addition, and was 0.442 mM under the condition with glycine addition.

[0390] [Example 11]

[0391] For the SfmM2, GcvTHP enhanced strain prepared in Example 2 (B), the production evaluation of 3-methyltyrosine was carried out as follows.

[0392] Regarding the production evaluation of 3-methyltyrosine in this example, use HMS174(DE3) / pET_P T7 -sfmM2 / pACYC_P T7 -gcvTHP prepared in Example 2 (B). Otherwise, it was carried out in the same manner as in Example 9.

[0393] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.099 mM compared to the condition without glycine addition, and was 0.196 mM under the condition with glycine addition.

[0394] [Example 12]

[0395] For the SfmM2 and GcvTHP enhanced strains prepared in Example 2 (B), the production evaluation of 3-methyltyrosine was carried out under the condition of adding glycine during cultivation as described below.

[0396] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 2 (B) was used T7 -sfmM2 / pACYC_P T7 -gcvTHP. After culturing in LB medium, 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin and 34 μg / mL chloramphenicol was used, and except for this, it was carried out in the same manner as in Example 9.

[0397] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.310 mM compared to the condition without glycine addition, and was 0.470 mM under the condition with glycine addition.

[0398] [Example 13]

[0399] For the SfmM2 and LuxS enhanced strains prepared in Example 3 (C), the production evaluation of 3-methyltyrosine was carried out under the condition of adding glycine during cultivation as described below.

[0400] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 3 (C) was used T7 -sfmM2 / pCDF_P T7 -luxS. After culturing in LB medium, 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin and 34 μg / mL chloramphenicol was used, and except for this, it was carried out in the same manner as in Example 9.

[0401] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.215 mM compared to the condition without glycine addition, and was 0.536 mM under the condition with glycine addition.

[0402] [Example 14]

[0403] For the SfmM2, LuxS, and GcvTHP enhanced strains prepared in Example 4, as described below, the evaluation of 3-methyltyrosine production was carried out under the condition of adding glycine during cultivation.

[0404] Regarding the evaluation of 3-methyltyrosine production in this example, HMS174(DE3) / pET_P prepared in Example 4 was used T7 -sfmM2 / pCDF_P T7 -luxS / pACYC_P T7 -gcvTHP. After culturing in LB medium, 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used. Except for this, it was carried out in the same manner as in Example 9.

[0405] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.142 mM compared to the condition without glycine addition, and was 0.576 mM under the condition with glycine addition.

[0406] [Example 15]

[0407] For the SfmM2, Mtn, and LuxS enhanced strains prepared in Example 5, the evaluation of 3-methyltyrosine production was carried out as follows.

[0408] Regarding the evaluation of 3-methyltyrosine production in this example, HMS174(DE3) / pET_P prepared in Example 5 was used T7 -sfmM2 / pCDF_P T7 -mtn-luxS. Except for this, it was carried out in the same manner as in Example 9.

[0409] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.372 mM compared to the condition without glycine addition, and was 0.558 mM under the condition with glycine addition.

[0410] [Example 16]

[0411] For the SfmM2, Mtn, and LuxS enhanced strains prepared in Example 5, as described below, the production evaluation of 3-methyltyrosine was carried out under the condition that glycine was added during cultivation.

[0412] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 5 was used. T7 -sfmM2 / pCDF_P T7 -mtn-luxS. After culturing in LB medium, 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used. Except for this, it was carried out in the same manner as in Example 9.

[0413] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.331 mM compared with the condition without adding glycine, and was 0.787 mM under the condition of adding glycine.

[0414] [Example 17]

[0415] For the SfmM2, Mtn, LuxS, and GcvTHP enhanced strains prepared in Example 6, the production evaluation of 3-methyltyrosine was carried out as follows.

[0416] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 6 was used. T7 -sfmM2 / pCDF_P T7 -mtn-luxS / pACYC_P T7 -gcvTHP. Except for this, it was carried out in the same manner as in Example 9.

[0417] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.150 mM compared with the condition without adding glycine, and was 0.258 mM under the condition of adding glycine.

[0418] [Example 18]

[0419] For the SfmM2, Mtn, LuxS, and GcvTHP enhanced strains prepared in Example 6, as described below, the production evaluation of 3-methyltyrosine was carried out under the condition that glycine was added during cultivation.

[0420] Regarding the evaluation of 3-methyltyrosine production in this example, HMS174(DE3) / pET_P prepared in Example 6 was used. T7 -sfmM2 / pCDF_P T7 -mtn-luxS / pACYC_P T7 -gcvTHP. After culturing in LB medium, 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used. Otherwise, the procedure was the same as in Example 9.

[0421] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.567 mM compared to the condition without glycine addition, and was 0.829 mM under the condition with glycine addition.

[0422] [Example 19]

[0423] For the SfmM2 and SahH enhanced strains prepared in Example 7(B), the evaluation of 3-methyltyrosine production was carried out under the condition of adding glycine during culturing as described below.

[0424] Regarding the evaluation of 3-methyltyrosine production in this example, HMS174(DE3) / pET_P prepared in Example 7(B) was used. T7 -sfmM2 / pCDF_P T7 -sahH. After culturing in LB medium, 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used. Otherwise, the procedure was the same as in Example 9.

[0425] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.196 mM compared to the condition without glycine addition, and was 0.284 mM under the condition with glycine addition.

[0426] [Example 20]

[0427] For the SfmM2, SahH, and GcvTHP enhanced strains prepared in Example 8, as described below, the production evaluation of 3-methyltyrosine was carried out under the condition of adding glycine during cultivation.

[0428] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 8 was used. T7 -sfmM2 / pCDF_P T7 -sahH / pACYC_P T7 -gcvTHP. After culturing in LB medium, 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used. Otherwise, it was carried out in the same manner as in Example 9.

[0429] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.219 mM compared with the condition without adding glycine, and was 0.313 mM under the condition of adding glycine.

[0430] The results of Examples 9 to 12 and Examples 15 to 18 are summarized in Table 2. In these examples, the accumulation concentrations of 3-methyltyrosine under the condition of adding glycine during the methylation reaction were increased by 2.7 times, 2.5 times, 2.0 times, 2.9 times, 3.0 times, 1.7 times, 2.4 times, and 3.2 times, respectively, compared with the condition without adding glycine. From the above results, it can be seen that by carrying out the methylation reaction in the presence of glycine, efficient regeneration of SAM can be achieved, the methylation reaction can be promoted, and the production amount of any methyl compound can be increased.

[0431] In addition, in Examples 10, 12, 16, and 18, the accumulation concentrations of 3-methyltyrosine under the condition of adding glycine during the methylation reaction were 1.3 times, 2.4 times, 1.4 times, and 3.2 times, respectively, compared with Examples 9, 11, 15, and 17. From the above results, it can be seen that by preparing the cells by adding glycine during cultivation (before the start of the methylation reaction), the efficiency of SAM regeneration can be improved, the methylation reaction can be further promoted, and the production amount of any methyl compound can be increased.

[0432] That is, the present invention shows that by culturing cells in the presence of an organic raw material containing glycine (adding glycine before the start of the methylation reaction, i.e., before adding the methylation target substance), the methylation reaction can be significantly promoted.

[0433] [Table 2]

[0434]

[0435] The results of Examples 10, 12 to 14, 16, 18 to 20 were summarized in Table 3. In Examples 13, 14, 19, and 20, the accumulation concentrations of 3-methyltyrosine under the conditions where glycine was added during the methylation reaction were increased by 1.7-fold, 1.3-fold, 3.2-fold, and 3.3-fold, respectively, compared with the conditions where glycine was not added. From the above results, it can be seen that in these strains, efficient regeneration of SAM can also be achieved by performing the methylation reaction in the presence of glycine, and the methylation reaction can be promoted.

[0436] In Examples 12, 14, 18, and 20, the accumulation concentrations of 3-methyltyrosine under the conditions where glycine was added during the methylation reaction were increased by 6.3%, 7.4%, 5.3%, and 10%, respectively, compared with Examples 10, 13, 16, and 19. From the above results, it can be seen that by enhancing GcvTHP, the efficiency of SAM regeneration is improved, the methylation reaction can be further promoted, and the production amount of any methyl compound is increased.

[0437] In addition, in Examples 13 and 16, the accumulation concentrations of 3-methyltyrosine under the conditions where glycine was added during the methylation reaction were increased by 21% and 78%, respectively, compared with Example 10. In Examples 14 and 18, the accumulation concentrations of 3-methyltyrosine under the conditions where glycine was added during the methylation reaction were increased by 23% and 76%, respectively, compared with Example 12. From the above results, it can be seen that by enhancing LuxS, Mtn, and LuxS, the efficiency of SAM regeneration is improved, the methylation reaction can be further promoted, and the production amount of any methyl compound is increased.

[0438] That is, the method of the present invention shows that by using glycine, SAM can be efficiently regenerated, the methylation reaction can be promoted, and the production amount of any methyl compound is increased.

[0439] [Table 3]

[0440]

[0441] [Example 21]

[0442] For Escherichia coli HMS174(DE3) strain, the expression of the TCMT gene was enhanced as follows.

[0443] (A) Construction of TCMT enhanced plasmid

[0444] Synthesize DNA (synthetic DNA base sequence: SEQ ID NO: 5) with a base sequence of the TCMT gene from Lonsdalea populi, which encodes tyrosine 3C-methyltransferase, and confers a recognition sequence for restriction enzyme Nde I on the 5'-terminal side and a recognition sequence for restriction enzyme Bgl II on the 3'-terminal side. Note that as the base sequence of this TCMT gene, a sequence with codons optimized for high expression in Escherichia coli was designed. Insert the synthesized DNA into the restriction enzyme sites Nde I and Bgl II of the E. coli expression vector pET Duet-1 (Novagen), and ligate it downstream of the T7 promoter. The constructed plasmid was named pET_P T7 -TCMT.

[0445] (B) Production of TCMT enhanced strain

[0446] Transform the E. coli HMS174(DE3) strain (Novagen) with the plasmid pET_P T7 -TCMT, pCDF Duet-1 (Novagen), and pACYC Duet-1 (Novagen) constructed in Example 21(A), and name the resulting strain HMS174(DE3) / pET_P T7 -TCMT.

[0447] [Example 22]

[0448] For the E. coli HMS174(DE3) strain, enhance the expression of the TCMT gene, mtn gene, and luxS gene as follows.

[0449] Transform the E. coli HMS174(DE3) strain (Novagen) with the plasmid pET_P T7 -TCMT constructed in Example 21(A), the plasmid pCDF_P T7 -mtn-luxS constructed in Example 3(A), and pACYC Duet-1 (Novagen), and name the resulting strain HMS174(DE3) / pET_P T7 -TCMT / pCDF_P T7 -mtn-luxS.

[0450] [Example 23]

[0451] For the E. coli HMS174(DE3) strain, enhance the expression of the TCMT gene, mtn gene, luxS gene, prs gene, and apt gene as follows.

[0452] (A) Construction of Mtn, LuxS, Prs, Apt enhanced plasmid

[0453] Synthesize a DNA (synthetic DNA base sequence: SEQ ID NO: 6) containing the base sequences of the prs gene and the apt gene from Escherichia coli, with a recognition sequence for restriction enzyme Bgl II assigned to the 5'-terminal side and a recognition sequence for restriction enzyme Fse I assigned to the 3'-terminal side. Insert the synthesized DNA into the plasmid pCDF_P constructed in Example 3(A) T7 -mtn-luxS at the restriction enzyme sites Bgl II and Fse I, and ligate it to the downstream of the luxS gene. The constructed plasmid was named pCDF_P T7 -mtn-luxS-prs-apt.

[0454] (B) Production of TCMT, Mtn, LuxS, Prs, Apt enhanced strains

[0455] Transform Escherichia coli HMS174(DE3) strain (Novagen) with the plasmid pET_P T7 -TCMT constructed in Example 21(A), the plasmid pCDF_P T7 -mtn-luxS-prs-apt constructed in Example 23(A), and pACYC Duet-1 (Novagen). The resulting strain was designated as HMS174(DE3) / pET_P T7 -TCMT / pCDF_P T7 -mtn-luxS-prs-apt.

[0456] [Example 24]

[0457] For Escherichia coli HMS174(DE3) strain, enhance the expression of the TCMT gene, mtn gene, luxS gene, prs gene, apt gene, gcvT gene, gcvH gene, and gcvP gene as follows.

[0458] Transform Escherichia coli HMS174(DE3) strain (Novagen) with the plasmid pET_P T7 -TCMT constructed in Example 21(A), the plasmid pCDF_P T7 -mtn-luxS-prs-apt constructed in Example 23(A), and the plasmid pACYC_P T7 -gcvTHP constructed in Example 2(A). The resulting strain was designated as HMS174(DE3) / pET_P T7 -TCMT / pCDF_P T7 -mtn-luxS-prs-apt / pACYC_P T7 -gcvTH P.

[0459] [Example 25]

[0460] For Escherichia coli HMS174(DE3) strain, the expression of TCMT gene, sahH gene, gcvT gene, gcvH gene and gcvP gene was enhanced as follows.

[0461] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -TCMT constructed in Example 21(A), the plasmid pCDF_P T7 -sahH constructed in Example 7(A), and the plasmid pACYC_P T7 -gcvTHP constructed in Example 2(A). The obtained strain was designated as HMS174(DE3) / pET_P T7 -TCMT / pCDF_P T7 -sahH / pACYC_P T7 -gcvTHP.

[0462] [Example 26]

[0463] For Escherichia coli HMS174(DE3) strain, the expression of TCMT gene, sahH gene, ADO1 gene, gcvT gene, gcvH gene and gcvP gene was enhanced as follows. The ADO1 gene is a gene encoding adenosine kinase (ADK).

[0464] (A) Construction of SahH-ADO1 enhanced plasmid

[0465] A DNA containing the base sequence of the ADO1 gene from Saccharomyces cerevisiae (a recognition sequence for restriction enzyme Bgl II was given to the 5'-terminal side and a recognition sequence for restriction enzyme Mfe I was given to the 3'-terminal side) was synthesized (base sequence of the synthesized DNA: SEQ ID NO: 7). It should be noted that as the base sequence of this ADO1 gene, a sequence with codons optimized for high expression in Escherichia coli was designed. The synthesized DNA was inserted into the restriction enzyme sites Bgl II and Mfe I of the plasmid pCDF_P T7 -sahH and ligated to the downstream of the sahH gene. The constructed plasmid was named pCDF_P T7 -sahH-ADO1.

[0466] (B) Production of TCMT, SahH, ADO1, GcvTHP enhanced strains

[0467] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -TCMT constructed in Example 21(A), the plasmid pCDF_P T7 -sahH-ADO1 constructed in Example 26(A), and the plasmid pACYC_P T7 -gcvTHP constructed in Example 2(A). The resulting strain was designated as HMS174(DE3) / pET_P T7 -TCMT / pCDF_P T7 -sahH-ADO1 / pACYC_P T7 -gcvTHP.

[0468] [Example 27]

[0469] For the TCMT enhanced strain prepared in Example 21(B), the production evaluation of 3-methyltyrosine was carried out as follows.

[0470] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P T7 -TCMT prepared in Example 21(B) was cultured in LB medium and then used in 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol. It was suspended in the reaction buffer to an OD 600 = 5, and otherwise, carried out in the same manner as in Example 9.

[0471] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.154 mM compared to the condition without added glycine and was 0.175 mM under the condition with added glycine.

[0472] [Example 28]

[0473] For the TCMT, Mtn, LuxS enhanced strain prepared in Example 22, the production evaluation of 3-methyltyrosine was carried out as follows.

[0474] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P T7 -TCMT / pCDF_P T7 -mtn-luxS was used, and otherwise, carried out in the same manner as in Example 27.

[0475] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.308 mM compared with the condition without glycine addition, and was 0.481 mM under the condition with glycine addition.

[0476] [Example 29]

[0477] For the TCMT, Mtn, LuxS, Prs, Apt enhanced strains prepared in Example 23(B), the production evaluation of 3-methyltyrosine was carried out as follows.

[0478] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 23(B) was used T7 -TCMT / pCDF_P T7 -mtn-luxS-prs-apt, and except for this, it was carried out in the same manner as in Example 27.

[0479] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.352 mM compared with the condition without glycine addition, and was 0.568 mM under the condition with glycine addition.

[0480] [Example 30]

[0481] For the TCMT, Mtn, LuxS, Prs, Apt, GcvTHP enhanced strains prepared in Example 24, the production evaluation of 3-methyltyrosine was carried out as follows.

[0482] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 24 was used T7 -TCMT / pCDF_P T7 -mtn-luxS-prs-apt / pACYC_P T7 -gcvTH P, and except for this, it was carried out in the same manner as in Example 27.

[0483] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.227 mM compared with the condition without glycine addition, and was 0.571 mM under the condition with glycine addition.

[0484] [Example 31]

[0485] For the TCMT, SahH, GcvTHP enhanced strains prepared in Example 25, the production evaluation of 3-methyltyrosine was carried out as follows.

[0486] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 25 was usedT7 -TCMT / pCDF_P T7 -sahH / pACYC_P T7 -gcvTHP, except for this, it was carried out in the same manner as in Example 27.

[0487] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.167 mM compared to the condition without glycine addition, and was 0.189 mM under the condition with glycine addition.

[0488] [Example 32]

[0489] For the TCMT, SahH, ADO1, and GcvTHP overexpressing strains prepared in Example 26(B), the production evaluation of 3-methyltyrosine was carried out as follows.

[0490] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 26(B) was used T7 -TCMT / pCDF_P T7 -sahH-ADO1 / pACYC_P T7 -gcvTHP, except for this, it was carried out in the same manner as in Example 27.

[0491] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.226 mM compared to the condition without glycine addition, and was 0.288 mM under the condition with glycine addition.

[0492] The results of Examples 27 to 32 are summarized in Table 4. In Examples 27, 28, 29, 30, 31, and 32, the accumulated concentrations of 3-methyltyrosine under the condition of adding glycine during the methylation reaction increased by 8.3 times, 2.8 times, 2.6 times, 1.7 times, 8.6 times, and 4.6 times, respectively, compared to the condition without glycine addition. From the above results, it can be seen that in these strains, efficient regeneration of SAM can also be achieved by carrying out the methylation reaction in the presence of glycine, and the methylation reaction can be promoted.

[0493] In Examples 28, 29, and 30, the accumulated concentrations of 3-methyltyrosine under the condition of adding glycine during the methylation reaction increased by 2.7 times, 3.2 times, and 3.3 times, respectively, compared to Example 27. From the above results, it can be seen that by enhancing Mtn, LuxS, Prs, Apt, and GcvTHP, the efficiency of SAM regeneration is improved, the methylation reaction can be further promoted, and the production amount of any methyl compound can be increased.

[0494] In addition, in Examples 31 and 32, the accumulation concentration of 3-methyltyrosine under the condition of adding glycine during the methylation reaction increased by 8% and 65%, respectively, compared with Example 27. From the above results, it can be seen that the efficiency of SAM regeneration is improved by enhancing SahH, ADO1 and GcvTHP, which can further promote the methylation reaction and increase the production amount of any methyl compound.

[0495] That is, the method of the present invention has shown that the use of glycine can efficiently regenerate SAM, promote the methylation reaction, and increase the amount of production of any methyl compound.

[0496] [Table 4]

[0497]

[0498] [Example 33]

[0499] For the Escherichia coli HMS174 (DE3) strain, the expression of the EgtB gene, EgtD gene, and EgtE gene was enhanced as follows.

[0500] (A) Construction of EgtBDE enhanced plasmid

[0501] DNA (base sequence of synthetic DNA: sequence number 8) containing the base sequences of 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 EgtE gene (a recognition sequence for the restriction enzyme Nco I is assigned to the 5' end side, and a recognition sequence for the restriction enzyme EcoR I is assigned to the 3' end side) was synthesized. It should be noted that as the base sequences of the EgtB gene, the EgtD gene, and the EgtE gene, sequences with optimized codons were designed for efficient expression in Escherichia coli. The synthesized DNA was inserted into the restriction enzyme sites Nco I and EcoR I 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 -EgtBDE.

[0502] (B) Production of EgtBDE enhanced strain

[0503] Escherichia coli HMS174 (DE3) strain (Novagen) was transformed with the plasmid pET_P constructed in Example 33 (A). T7-EgtBDE, pCDF Duet-1 (Novagen), and pACYC Duet-1 (Novagen) were used for transformation, and the resulting strain was designated as HMS174(DE3) / pET_P T7 -EgtBDE.

[0504] [Example 34]

[0505] For Escherichia coli HMS174(DE3) strain, the expression of EgtB gene, EgtD gene, EgtE gene, mtn gene, and luxS gene was enhanced as follows.

[0506] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -EgtBDE constructed in Example 33(A), the plasmid pCDF_P T7 -mtn-luxS constructed in Example 3(A), and pACYC Duet-1 (Novagen), and the resulting strain was designated as HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS.

[0507] [Example 35]

[0508] For Escherichia coli HMS174(DE3) strain, the expression of EgtB gene, EgtD gene, EgtE gene, mtn gene, luxS gene, prs gene, and apt gene was enhanced as follows.

[0509] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -EgtBDE constructed in Example 33(A), the plasmid pCDF_P T7 -mtn-luxS-prs-apt constructed in Example 23(A), and pACYC Duet-1 (Novagen), and the resulting strain was designated as HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt.

[0510] [Example 36]

[0511] For Escherichia coli HMS174(DE3) strain, the expression of EgtB gene, EgtD gene, EgtE gene, mtn gene, luxS gene, prs gene, apt gene, gcvT gene, gcvH gene, and gcvP gene was enhanced as follows.

[0512] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -EgtBDE constructed in Example 33(A), the plasmid pCDF_P T7 -mtn-luxS-prs-apt constructed in Example 23(A), and the plasmid pACYC_P T7 -gcvTHP constructed in Example 2(A). The resulting strain was designated as HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt / pACYC_P T7 -gcvT HP.

[0513] [Example 37]

[0514] For the Escherichia coli HMS174(DE3) strain, the expression of the EgtB gene, EgtD gene, EgtE gene, sahH gene, ADO1 gene, gcvT gene, gcvH gene, and gcvP gene was enhanced as follows.

[0515] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -EgtBDE constructed in Example 33(A), the plasmid pCDF_P T7 -sahH-ADO1 constructed in Example 26(A), and the plasmid pACYC_P T7 -gcvTHP. The resulting strain was designated as HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -sahH-ADO1 / pACYC_P T7 -gcvTHP.

[0516] [Example 38]

[0517] For the EgtBDE overexpressing strain prepared in Example 33(B), the production of histidine betaine and ergothioneine was evaluated as described below.

[0518] The HMS174(DE3) / pET_P prepared in Example 33(B) T7-EgtBDE 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 34 μg / mL chloramphenicol. 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 34 μg / mL chloramphenicol was added to a 50 mL test tube, and the culture solution in the above LB medium was inoculated into this M9 medium at a dilution of 1 / 50, followed by shaking culture at 200 rpm and 30 °C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and further shaking culture was carried out for 16 hours.

[0519] The obtained culture solution was collected by centrifugation at 3000×g for 5 minutes, and suspended in a reaction buffer (100 mM Tris-HCl pH 8.0, 20 mM MgSO4, 20 mM glucose) containing 20 mM glycine, 100 μg / mL carbenicillin, 100 μg / mL spectinomycin, 68 μg / mL chloramphenicol, and 1 mM IPTG to achieve an OD 600 = 4. 1 mL of the cell suspension was added to a 14 mL test tube and mixed with 1 mL of a substrate solution containing 10 mM L-histidine and 10 mM L-cysteine as substrates, thereby starting the synthesis reaction of methylated ergothioneine. The mixture was shaken at 200 rpm and 30 °C for 48 hours. In addition, in order to clarify the promotion of the methylation reaction by the efficient regeneration of the methyl carbon of SAM, a test was also carried out under the same conditions without adding glycine as the supply source of methyl carbon, and the results were compared with those of the test carried out under the condition of adding glycine.

[0520] Regarding the obtained reaction solution, the supernatant after centrifugation at 3000×g for 5 minutes was collected for LC-MS analysis. The conditions for LC-MS analysis are shown in Table 5.

[0521] The measurement results showed that compared with the condition without adding glycine, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.003 mM and 0.015 mM, respectively, and were 0.031 mM and 0.054 mM, respectively, under the condition of adding glycine. In addition, compared with the condition without adding glycine, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.017 mM, and was 0.084 mM under the condition of adding glycine.

[0522] [Table 5]

[0523]

[0524] [Example 39]

[0525] For the EgtBDE enhanced strain prepared in Example 33(B), the production evaluation of histidine betaine and ergothioneine was carried out under the condition of adding glycine during cultivation as described below.

[0526] Regarding the production evaluation of histidine betaine and ergothioneine in this example, after culturing in LB medium, 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin and 34 μg / mL chloramphenicol was used, and except for this, it was carried out in the same manner as in Example 38.

[0527] As a result, compared with the condition without adding glycine, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.004 mM and 0.058 mM respectively, and were 0.028 mM and 0.088 mM respectively under the condition of adding glycine. In addition, compared with the condition without adding glycine, the total of the accumulated concentration of histidine betaine and the accumulated concentration of ergothioneine increased by 0.062 mM, and was 0.116 mM under the condition of adding glycine.

[0528] [Example 40]

[0529] For the EgtBDE, Mtn, LuxS enhanced strain prepared in Example 34, the production evaluation of histidine betaine and ergothioneine was carried out as described below.

[0530] Regarding the production evaluation of histidine betaine and ergothioneine in this example, HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS prepared in Example 34 was used, and except for this, it was carried out in the same manner as in Example 38.

[0531] As a result, compared with the condition without glycine addition, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.003 mM and 0.034 mM respectively, and were 0.030 mM and 0.111 mM respectively under the condition with glycine addition. In addition, compared with the condition without glycine addition, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.037 mM, and was 0.141 mM under the condition with glycine addition.

[0532] [Example 41]

[0533] For the EgtBDE, Mtn, LuxS enhanced strains prepared in Example 34, the production evaluation of histidine betaine and ergothioneine was carried out as described below under the condition of adding glycine during cultivation.

[0534] Regarding the production evaluation of histidine betaine and ergothioneine in this example, use HMS174(DE3) / pET_P prepared in Example 34 T7 -EgtBDE / pCDF_P T7 -mtn-luxS. After culturing in LB medium, use 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin and 34 μg / mL chloramphenicol, and except for this, carry out in the same manner as in Example 38.

[0535] As a result, compared with the condition without glycine addition, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.051 mM and 0.093 mM respectively, and were 0.083 mM and 0.164 mM respectively under the condition with glycine addition. In addition, compared with the condition without glycine addition, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.144 mM, and was 0.247 mM under the condition with glycine addition.

[0536] [Example 42]

[0537] For the EgtBDE, Mtn, LuxS, Prs, Apt enhanced strains prepared in Example 35, the production evaluation of histidine betaine and ergothioneine was carried out as described below.

[0538] Regarding the production evaluation of histidine betaine and ergothioneine in this example, use HMS174(DE3) / pET_P prepared in Example 35 T7-EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt, except for this, it was carried out in the same manner as in Example 38.

[0539] As a result, compared with the condition without glycine addition, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.013 mM and 0.017 mM respectively, and were 0.045 mM and 0.180 mM respectively under the condition with glycine addition. In addition, compared with the condition without glycine addition, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.029 mM, and was 0.224 mM under the condition with glycine addition.

[0540] [Example 43]

[0541] For the EgtBDE, Mtn, LuxS, Prs, Apt enhanced strains prepared in Example 35, the production evaluation of histidine betaine and ergothioneine was carried out under the condition of adding glycine during cultivation as described below.

[0542] Regarding the production evaluation of histidine betaine and ergothioneine in this example, HMS174(DE3) / pET_P prepared in Example 35 was used T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt. After culturing in LB medium, 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin and 34 μg / mL chloramphenicol was used. Except for this, it was carried out in the same manner as in Example 38.

[0543] As a result, compared with the condition without glycine addition, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.073 mM and 0.089 mM respectively, and were 0.101 mM and 0.215 mM respectively under the condition with glycine addition. In addition, compared with the condition without glycine addition, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.162 mM, and was 0.316 mM under the condition with glycine addition.

[0544] [Example 44]

[0545] For the EgtBDE, Mtn, LuxS, Prs, Apt, and GcvTHP enhanced strains prepared in Example 36, the production evaluations of histidine betaine and ergothioneine were carried out as described below.

[0546] Regarding the production evaluations of histidine betaine and ergothioneine in this example, HMS174(DE3) / pET_P prepared in Example 36 was used. T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt / pACYC_P T7 -gcvT HP, and except for this, it was carried out in the same manner as in Example 38.

[0547] As a result, compared with the condition without glycine addition, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.048 mM and 0.086 mM respectively, and were 0.071 mM and 0.157 mM respectively under the condition with glycine addition. In addition, compared with the condition without glycine addition, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.134 mM, and was 0.228 mM under the condition with glycine addition.

[0548] [Example 45]

[0549] For the EgtBDE, Mtn, LuxS, Prs, Apt, and GcvTHP enhanced strains prepared in Example 36, the production evaluations of histidine betaine and ergothioneine were carried out as described below under the condition of adding glycine during cultivation.

[0550] Regarding the production evaluations of histidine betaine and ergothioneine in this example, HMS174(DE3) / pET_P prepared in Example 36 was used. T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt / pACYC_P T7 -gcvT HP. After culturing in LB medium, 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used, and except for this, it was carried out in the same manner as in Example 38.

[0551] As a result, compared with the condition without glycine addition, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.075 mM and 0.137 mM respectively, being 0.095 mM and 0.222 mM respectively under the condition with glycine addition. In addition, compared with the condition without glycine addition, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.212 mM, being 0.317 mM under the condition with glycine addition.

[0552] [Example 46]

[0553] For the EgtBDE, SahH, ADO1, and GcvTHP overexpressing strains prepared in Example 37, the production evaluation of histidine betaine and ergothioneine was carried out as described below.

[0554] Regarding the production evaluation of histidine betaine and ergothioneine in this example, HMS174(DE3) / pET_P prepared in Example 37 was used T7 -EgtBDE / pCDF_P T7 -sahH-ADO1 / pACYC_P T7 -gcvTHP, and in other respects, it was carried out in the same manner as in Example 38.

[0555] As a result, under the condition without glycine addition, the concentrations of histidine betaine and ergothioneine in the reaction solution were 0.015 mM and 0.018 mM respectively. In contrast, under the condition with glycine addition, they were 0.015 mM and 0.036 mM respectively. In addition, compared with the condition without glycine addition, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.019 mM, being 0.052 mM under the condition with glycine addition.

[0556] [Example 47]

[0557] For the EgtBDE, SahH, ADO1, and GcvTHP overexpressing strains prepared in Example 37, the production evaluation of histidine betaine and ergothioneine was carried out as described below under the condition with glycine added during cultivation.

[0558] Regarding the production evaluation of histidine betaine and ergothioneine in this example, HMS174(DE3) / pET_P prepared in Example 37 was used T7 -EgtBDE / pCDF_P T7 -sahH-ADO1 / pACYC_P T7-gcvTHP was cultured in LB medium, and then 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 10 mM glycine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used. Otherwise, the procedure was the same as in Example 38.

[0559] As a result, compared with the condition without glycine addition, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.018 mM and 0.059 mM, respectively, and were 0.033 mM and 0.132 mM under the condition with glycine addition. In addition, compared with the condition without glycine addition, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.077 mM, and was 0.165 mM under the condition with glycine addition.

[0560] The results of Examples 38 to 47 are summarized in Table 6. To evaluate the efficiency of the methylation reaction, the total of the accumulated concentrations of histidine betaine and ergothioneine was calculated and compared. In these examples, the total accumulated concentrations under the condition with glycine addition were increased to 1.3 times, 2.1 times, 1.4 times, 2.4 times, 1.1 times, 2.1 times, 2.4 times, 3.0 times, 1.6 times, and 1.9 times, respectively, compared with the condition without glycine addition. From the above results, it can be seen that by carrying out the methylation reaction in the presence of glycine, efficient regeneration of SAM can be achieved, the methylation reaction can be promoted, and the production amount of any methyl compound can be increased.

[0561] In addition, in Examples 39, 41, 43, 45, and 47, the total accumulated concentrations under the condition with glycine addition during the methylation reaction were 1.4 times, 1.8 times, 1.4 times, 1.4 times, and 3.2 times, respectively, compared with Examples 38, 40, 42, 44, and 46. From the above results, it can be seen that by preparing the cells by adding glycine during the culture (before the start of the methylation reaction), the efficiency of SAM regeneration is improved, the methylation reaction can be further promoted, and the production amount of any methyl compound can be increased.

[0562] That is, the present invention shows that by culturing cells in the presence of an organic raw material containing glycine (adding glycine before the start of the methylation reaction, i.e., before adding the methylation target substance), the methylation reaction can be significantly promoted.

[0563] In Examples 40 and 41, the total accumulation concentrations under the condition of adding glycine during the methylation reaction increased to 1.7 times and 2.1 times, respectively, compared with Examples 38 and 39. From the above results, it can be seen that by enhancing Mtn and LuxS, the efficiency of SAM regeneration is improved, the methylation reaction can be further promoted, and the production amount of any methyl compound is increased.

[0564] In Examples 42 and 43, the total accumulation concentrations under the condition of adding glycine during the methylation reaction increased to 1.6 times and 1.3 times, respectively, compared with Examples 40 and 41. According to the above results, it can be seen that by enhancing Prs and Apt, the efficiency of SAM regeneration is improved, the methylation reaction can be further promoted, and the production amount of any methyl compound is increased.

[0565] In addition, in Example 47, the total accumulation concentration under the condition of adding glycine during the methylation reaction increased to 1.4 times compared with Example 39. From the above results, it can be seen that by enhancing Prs, Apt and GcvTHP, the efficiency of SAM regeneration is improved, the methylation reaction can be further promoted, and the production amount of any methyl compound is increased.

[0566] That is, the method of the present invention shows that by using glycine, SAM can be efficiently regenerated, the methylation reaction can be promoted, and the production amount of any methyl compound is increased.

[0567] [Table 6]

[0568]

[0569] [Example 48]

[0570] For Escherichia coli HMS174(DE3) strain, the expression of sfmM2 gene and glyA gene was enhanced as described below.

[0571] (A) Construction of GlyA enhanced plasmid

[0572] Synthesize DNA (base sequence of the synthesized DNA: SEQ ID NO: 9) containing the base sequence of the glyA gene from Escherichia coli (recognizing sequence of restriction enzyme Nde I is given to the 5'-terminal side, and recognizing sequence of restriction enzyme Mfe I is given to the 3'-terminal side). Insert the synthesized DNA into the restriction enzyme sites Nde I and Mfe I of the Escherichia coli expression vector pCDF Duet-1 (Novagen) and ligate it downstream of the T7 promoter. The constructed plasmid was named pCDF_P T7 -glyA.

[0573] (B) Production of SfmM2, GlyA enhanced strains

[0574] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -sfmM2 constructed in Example 1(A), the plasmid pCDF_P T7 -glyA constructed in Example 48(A), and pACYC Duet-1 (Novagen). The resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -glyA.

[0575] [Example 49]

[0576] For the Escherichia coli HMS174(DE3) strain, the expression of the sfmM2 gene, metF gene, metH gene, metK gene, mtn gene, and luxS gene was enhanced as described below.

[0577] (A) Construction of MetFHK enhanced plasmid

[0578] DNA (synthetic DNA base sequence: SEQ ID NO: 10) containing the base sequences of the metF gene, metH gene, and metK gene from Escherichia coli (collectively referred to as "MetFHK" for MetF, MetH, and MetK) with a recognition sequence for the restriction enzyme BspHI assigned to the 5'-terminal side and a recognition sequence for the restriction enzyme EcoRI assigned to the 3'-terminal side was synthesized. The synthesized DNA was inserted into the restriction enzyme sites Nco I and EcoRI of the Escherichia coli expression vector pACYC Duet-1 (Novagen) and ligated downstream of the T7 promoter. The constructed plasmid was named pACYC_P T7 -metFHK.

[0579] (B) Construction of MetFHK, Mtn, LuxS enhanced plasmid

[0580] The plasmid pCDF_P T7 -mtn-luxS constructed in Example 3(A) was treated with the restriction enzymes EcoRI and XhoI, separated by agarose gel electrophoresis, and then recovered and purified from the gel to obtain a DNA fragment of the T7 promoter, mtn gene, and luxS gene region. The obtained DNA fragment was inserted into the same restriction enzyme sites of the plasmid pACYC_P T7 -metFHK constructed in Example 49(A). The constructed plasmid was named pACYC_P T7 -metFHK_P T7 -mtn-luxS.

[0581] (C) Production of SfmM2, MetFHK, Mtn, LuxS enhanced strains

[0582] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -sfmM2 constructed in Example 1(A), the plasmid pACYC_P T7 -metFHK_P T7 -mtn-luxS, and pCDF Duet-1 (Novagen), and the resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pACYC_P T7 -metFHK_P T7 -mtn-luxS.

[0583] [Example 50]

[0584] For the Escherichia coli HMS174(DE3) strain, the expression of the sfmM2 gene, metF gene, metH gene, metK gene, mtn gene, luxS gene, and glyA gene was enhanced as described below.

[0585] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -sfmM2 constructed in Example 1(A), the plasmid pACYC_P T7 -metFHK_P T7 -mtn-luxS, and the plasmid pCDF_P T7 -glyA constructed in Example 48(A), and the resulting strain was designated as HMS174(DE3) / pET_P T7 -sfmM2 / pACYC_P T7 -metFHK_P T7 -mtn-luxS / pCDF_P T7 -g lyA.

[0586] [Example 51]

[0587] For the SfmM2-enhanced strain prepared in Example 1(B), the production evaluation of 3-methyltyrosine was carried out as follows.

[0588] The HMS174(DE3) / pET_P prepared in Example 1(B) T7-sfmM2 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 34 μg / mL chloramphenicol. 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 34 μg / mL chloramphenicol was added to a 50 mL test tube, and the culture solution in the above LB medium was inoculated in such a way that it was diluted 1 / 50, and shake-cultured at 200 rpm and 30 °C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and further shake-cultured for 16 hours.

[0589] The obtained culture solution was collected by centrifugation at 3000×g for 5 minutes and suspended in a reaction buffer (100 mM Tris-HCl pH 8.0, 20 mM MgSO4, 20 mM glucose) containing 20 mM L-serine, 100 μg / mL carbenicillin, 100 μg / mL spectinomycin, 68 μg / mL chloramphenicol, and 1 mM IPTG to a concentration of OD 600 = 10. 1 mL of the 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 start the methylation reaction, and shake-cultured at 200 rpm and 30 °C for 24 hours. In addition, in order to clarify the promotion of the methylation reaction by the efficient regeneration of the methyl carbon of SAM, an experiment was also carried out under the same conditions without adding L-serine as a supply source of methyl carbon, and the results were compared with the results of the experiment carried out under the condition of adding L-serine.

[0590] The LC-MS analysis of the obtained reaction solution was carried out in the same manner as in Example 9. As a result of the measurement, the concentration of 3-methyltyrosine in the reaction solution increased by 0.093 mM compared with the condition without adding L-serine, and was 0.221 mM under the condition of adding L-serine.

[0591] [Example 52]

[0592] For the SfmM2 enhanced strain prepared in Example 1 (B), the production evaluation of 3-methyltyrosine was carried out under the condition of adding L-serine during cultivation as described below.

[0593] Regarding the evaluation of 3-methyltyrosine production in this example, after culturing in LB medium, 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 10 mM L-serine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used, and otherwise, it was carried out in the same manner as in Example 51.

[0594] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.142 mM compared with the condition without adding L-serine, and was 0.255 mM under the condition of adding L-serine.

[0595] [Example 53]

[0596] For the SfmM2 and GlyA overexpressing strains prepared in Example 48(B), the evaluation of 3-methyltyrosine production was carried out as follows.

[0597] Regarding the evaluation of 3-methyltyrosine production in this example, HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7 -glyA prepared in Example 48(B) was used, and otherwise, it was carried out in the same manner as in Example 51.

[0598] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.027 mM compared with the condition without adding L-serine, and was 0.165 mM under the condition of adding L-serine.

[0599] [Example 54]

[0600] For the SfmM2 and GlyA overexpressing strains prepared in Example 48(B), the evaluation of 3-methyltyrosine production was carried out as described below under the condition of adding L-serine during culturing.

[0601] Regarding the evaluation of 3-methyltyrosine production in this example, HMS174(DE3) / pET_P T7 -sfmM2 / pCDF_P T7-glyA, after culturing in LB medium, 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 10 mM L-serine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin and 34 μg / mL chloramphenicol was used, and otherwise, it was carried out in the same manner as in Example 51.

[0602] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.183 mM compared with the condition without adding L-serine, and was 0.319 mM under the condition of adding L-serine.

[0603] [Example 55]

[0604] For the SfmM2, MetFHK, Mtn, LuxS enhanced strains prepared in Example 49(C), the production evaluation of 3-methyltyrosine was carried out as follows.

[0605] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 49(C) was used T7 -sfmM2 / pACYC_P T7 -metFHK_P T7 -mtn-luxS, and otherwise, it was carried out in the same manner as in Example 51.

[0606] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.131 mM compared with the condition without adding L-serine, and was 0.243 mM under the condition of adding L-serine.

[0607] [Example 56]

[0608] For the SfmM2, MetFHK, Mtn, LuxS enhanced strains prepared in Example 49(C), the production evaluation of 3-methyltyrosine was carried out under the condition of adding L-serine during culturing as described below.

[0609] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P prepared in Example 49(C) was used T7 -sfmM2 / pACYC_P T7 -metFHK_P T7-mtn-luxS. After culturing in LB medium, 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 10 mM L-serine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used, and otherwise, the procedure was the same as in Example 51.

[0610] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.324 mM compared to the condition without addition of L-serine, and was 0.543 mM under the condition with addition of L-serine.

[0611] [Example 57]

[0612] For the SfmM2, MetFHK, Mtn, LuxS, GlyA overexpressing strains prepared in Example 50, the evaluation of 3-methyltyrosine production was carried out as follows.

[0613] Regarding the evaluation of 3-methyltyrosine production in this example, HMS174(DE3) / pET_P prepared in Example 50 was used T7 -sfmM2 / pACYC_P T7 -metFHK_P T7 -mtn-luxS / pCDF_P T7 -glyA, and otherwise, the procedure was the same as in Example 51.

[0614] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.047 mM compared to the condition without addition of L-serine, and was 0.240 mM under the condition with addition of L-serine.

[0615] [Example 58]

[0616] For the SfmM2, MetFHK, Mtn, LuxS, GlyA overexpressing strains prepared in Example 50, the evaluation of 3-methyltyrosine production was carried out as described below under the condition of adding L-serine during cultivation.

[0617] Regarding the evaluation of 3-methyltyrosine production in this example, HMS174(DE3) / pET_P prepared in Example 50 was used T7 -sfmM2 / pACYC_P T7 -metFHK_P T7 -mtn-luxS / pCDF_P T7-glyA was cultured in LB medium, and then 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 10 mM L-serine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used. Otherwise, it was carried out in the same manner as in Example 51.

[0618] As a result, the concentration of 3-methyltyrosine in the reaction solution increased by 0.309 mM compared to the condition without the addition of L-serine, and was 0.551 mM under the condition of adding L-serine.

[0619] The results of Examples 51 to 58 were summarized in Table 7. In these examples, the accumulation concentrations of 3-methyltyrosine under the conditions of adding L-serine during the methylation reaction were increased by 1.7 times, 2.3 times, 1.2 times, 2.2 times, 2.2 times, 2.5 times, 1.2 times, and 2.3 times, respectively, compared to the conditions without the addition of L-serine. From the above results, it can be seen that by carrying out the methylation reaction in the presence of L-serine, efficient regeneration of SAM can be achieved, and the methylation reaction can be promoted.

[0620] In addition, in Examples 52, 54, 56, and 58, the accumulation concentrations of 3-methyltyrosine under the conditions of adding L-serine during the methylation reaction were 1.2 times, 1.9 times, 2.2 times, and 2.3 times, respectively, compared to Examples 51, 53, 55, and 57. From the above results, it can be seen that by preparing the cells by adding L-serine during the culture (before the start of the methylation reaction), the efficiency of SAM regeneration can be improved, the methylation reaction can be further promoted, and the production amount of any methyl compound can be increased.

[0621] That is, the present invention shows that by culturing cells in the presence of an organic raw material containing serine (adding serine before the start of the methylation reaction, i.e., before adding the methylation target substance), the methylation reaction can be significantly promoted.

[0622] In Examples 54 and 58, the accumulation concentrations of 3-methyltyrosine under the conditions of adding L-serine during the methylation reaction increased by 25% and 1.5%, respectively, compared to Examples 52 and 56. From the above results, it can be seen that by enhancing GlyA, the efficiency of SAM regeneration is improved, the methylation reaction can be further promoted, and the production amount of any methyl compound can be increased.

[0623] In addition, in Examples 55 and 57, the accumulation concentrations of 3-methyltyrosine under the conditions where L-serine was added during the methylation reaction increased by 10% and 45% respectively compared to Examples 51 and 53. In Examples 56 and 58, the accumulation concentrations of 3-methyltyrosine under the conditions where L-serine was added during the methylation reaction increased by 113% and 73% respectively compared to Examples 52 and 54. From the above results, it can be seen that by enhancing MetFHK, Mtn, and LuxS, the efficiency of SAM regeneration can be improved, the methylation reaction can be further promoted, and the production amount of any methyl compound can be increased.

[0624] That is, the method of the present invention shows that by using serine, SAM can be efficiently regenerated, the methylation reaction can be promoted, and the production amount of any methyl compound can be increased.

[0625] [Table 7]

[0626]

[0627] [Example 59]

[0628] For Escherichia coli HMS174(DE3) strain, the expression of EgtB gene, EgtD gene, and EgtE gene was enhanced as follows.

[0629] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -EgtBDE constructed in Example 33(A) and pCDF Duet-1 (Novagen), and the resulting strain was designated as HMS174(DE3) / pET_P T7 -EgtBDE.

[0630] [Example 60]

[0631] For Escherichia coli HMS174(DE3) strain, the expression of EgtB gene, EgtD gene, EgtE gene, mtn gene, and luxS gene was enhanced as follows.

[0632] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -EgtBDE constructed in Example 33(A) and the plasmid pCDF_P T7 -mtn-luxS constructed in Example 3(A), and the resulting strain was designated as HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS.

[0633] [Example 61]

[0634] For Escherichia coli strain HMS174(DE3), the expression of the EgtB gene, EgtD gene, EgtE gene, mtn gene, luxS gene, prs gene, and apt gene was enhanced as follows.

[0635] The Escherichia coli strain HMS174(DE3) (Novagen) was transformed with the plasmid pET_P T7 -EgtBDE constructed in Example 33(A), and the plasmid pCDF_P T7 -mtn-luxS-prs-apt constructed in Example 23(A). The resulting strain was designated as HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt.

[0636] [Example 62]

[0637] For the EgtBDE overexpressing strain prepared in Example 59, the production of histidine betaine and ergothioneine was evaluated as described below.

[0638] The HMS174(DE3) / pET_P T7 -EgtBDE prepared in Example 59 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 and 50 μg / mL spectinomycin. 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, and 50 μg / mL spectinomycin was added to a 50 mL test tube. The culture solution in the above LB medium was inoculated into this M9 medium at a dilution of 1 / 50, 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 further cultured with shaking for 16 hours.

[0639] The resulting culture solution was collected by centrifugation at 3000×g for 5 minutes to obtain an OD 600was suspended in a reaction buffer (100 mM Tris-HCl pH 8.0, 20 mM MgSO4, 20 mM glucose) containing 20 mM L-serine, 100 μg / mL carbenicillin, 100 μg / mL spectinomycin and 1 mM IPTG at a ratio of = 4. 1 mL of the cell suspension was added to a 14 mL test tube and mixed with 1 mL of a substrate solution containing 10 mM L-histidine and 10 mM L-cysteine as substrates, thereby starting the synthesis reaction of methylated ergothioneine. The mixture was shaken at 200 rpm and 30 °C for 48 hours. In addition, in order to clarify the promotion of the methylation reaction by the efficient regeneration of the methyl carbon of SAM, a test was also carried out under the same conditions without adding L-serine as the supply source of methyl carbon, and the results were compared with those of the test carried out under the condition of adding L-serine.

[0640] The LC-MS analysis of the obtained reaction solution was carried out in the same manner as in Example 38. The measurement results showed that compared with the condition without adding L-serine, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.008 mM and 0.005 mM, respectively, and were 0.019 mM and 0.016 mM, respectively, under the condition of adding L-serine. In addition, compared with the condition without adding L-serine, the total of the accumulated concentration of histidine betaine and the accumulated concentration of ergothioneine increased by 0.012 mM, and was 0.035 mM under the condition of adding L-serine.

[0641] [Example 63]

[0642] For the EgtBDE enhanced strain prepared in Example 59, the production evaluation of histidine betaine and ergothioneine was carried out under the condition of adding L-serine during cultivation as described below.

[0643] Regarding the production evaluation of histidine betaine and ergothioneine in this example, after culturing in LB medium, 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 10 mM L-serine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin and 34 μg / mL chloramphenicol was used, and otherwise, it was carried out in the same manner as in Example 62.

[0644] As a result, compared with the condition without the addition of L-serine, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.007 mM and 0.013 mM respectively, being 0.020 mM and 0.028 mM respectively under the condition with the addition of L-serine. In addition, compared with the condition without the addition of L-serine, the total of the accumulated concentration of histidine betaine and the accumulated concentration of ergothioneine increased by 0.020 mM, being 0.049 mM under the condition with the addition of L-serine.

[0645] [Example 64]

[0646] For the EgtBDE, Mtn, LuxS enhanced strains prepared in Example 60, the production evaluation of histidine betaine and ergothioneine was carried out as described below.

[0647] Regarding the production evaluation of histidine betaine and ergothioneine in this example, HMS174(DE3) / pET_P prepared in Example 60 was used T7 -EgtBDE / pCDF_P T7 -mtn-luxS, and except for this, it was carried out in the same manner as in Example 62.

[0648] As a result, compared with the condition without the addition of L-serine, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.206 mM and 0.110 mM respectively, being 0.230 mM and 0.164 mM respectively under the condition with the addition of L-serine. In addition, compared with the condition without the addition of L-serine, the total of the accumulated concentration of histidine betaine and the accumulated concentration of ergothioneine increased by 0.020 mM, being 0.049 mM under the condition with the addition of L-serine.

[0649] [Example 65]

[0650] For the EgtBDE, Mtn, LuxS enhanced strains prepared in Example 60, the production evaluation of histidine betaine and ergothioneine was carried out as described below under the condition of adding L-serine during cultivation.

[0651] Regarding the production evaluation of histidine betaine and ergothioneine in this example, HMS174(DE3) / pET_P prepared in Example 34 was used T7 -EgtBDE / pCDF_P T7-mtn-luxS was cultured in LB medium, and then 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 10 mM L-serine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin, and 34 μg / mL chloramphenicol was used. Otherwise, it was carried out in the same manner as in Example 62.

[0652] As a result, compared with the condition without adding L-serine, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.235 mM and 0.092 mM, respectively, and were 0.259 mM and 0.166 mM, respectively, under the condition of adding L-serine. In addition, compared with the condition without adding L-serine, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.327 mM, and was 0.425 mM under the condition of adding L-serine.

[0653] [Example 66]

[0654] For the EgtBDE, Mtn, LuxS, Prs, Apt overexpressing strains prepared in Example 61, the production evaluation of histidine betaine and ergothioneine was carried out as follows.

[0655] Regarding the production evaluation of histidine betaine and ergothioneine in this example, HMS174(DE3) / pET_P T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt prepared in Example 61 was used. Otherwise, it was carried out in the same manner as in Example 62.

[0656] As a result, compared with the condition without adding L-serine, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.358 mM and 0.091 mM, respectively, and were 0.371 mM and 0.168 mM, respectively, under the condition of adding L-serine. In addition, compared with the condition without adding L-serine, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.448 mM, and was 0.539 mM under the condition of adding L-serine.

[0657] [Example 67]

[0658] For the EgtBDE, Mtn, LuxS, Prs, Apt overexpressing strains prepared in Example 61, the production evaluation of histidine betaine and ergothioneine was carried out as follows under the condition of adding glycine during cultivation.

[0659] Regarding the production evaluation of histidine betaine and ergothioneine in this example, use HMS174(DE3) / pET_P prepared in Example 61 T7 -EgtBDE / pCDF_P T7 -mtn-luxS-prs-apt. After culturing in LB medium, use 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 10 mM L-serine, 0.5% glucose, 50 μg / mL carbenicillin, 50 μg / mL spectinomycin and 34 μg / mL chloramphenicol. Except for this, perform in the same manner as in Example 62.

[0660] As a result, compared with the condition without adding L-serine, the concentrations of histidine betaine and ergothioneine in the reaction solution increased by 0.413 mM and 0.082 mM respectively, and were 0.423 mM and 0.138 mM respectively under the condition of adding L-serine. In addition, compared with the condition without adding L-serine, the total of the accumulated concentrations of histidine betaine and ergothioneine increased by 0.495 mM, and was 0.561 mM under the condition of adding L-serine.

[0661] The results of Examples 62 to 67 are summarized in Table 8. To evaluate the efficiency of the methylation reaction, calculate and compare the total of the accumulated concentrations of histidine betaine and ergothioneine. In these examples, the total of the accumulated concentrations under the condition of adding glycine increased by 1.5 times, 1.7 times, 5.1 times, 4.3 times, 5.9 times, and 8.5 times respectively compared with the condition without adding L-serine. From the above results, it can be seen that by performing the methylation reaction in the presence of L-serine, efficient regeneration of SAM can be achieved, the methylation reaction can be promoted, and the production amount of any methyl compound can be increased.

[0662] In addition, in Examples 63, 65 and 67, the total of the accumulated concentrations under the condition of adding L-serine during the methylation reaction increased by 40%, 8% and 4% respectively compared with Examples 62, 64 and 66. From the above results, it can be seen that by preparing the cells by adding L-serine during the culture (before the start of the methylation reaction), the efficiency of SAM regeneration can be improved, the methylation reaction can be further promoted, and the production amount of any methyl compound can be increased.

[0663] That is, the present invention shows that by culturing cells in the presence of an organic raw material containing serine (adding serine before the start of the methylation reaction, that is, before adding the methylation target substance), the methylation reaction can be significantly promoted.

[0664] In Examples 64 and 65, the total accumulation concentration under the condition of adding L-serine during the methylation reaction increased to 11.3 times and 8.7 times respectively compared with Examples 62 and 63. From the above results, it can be seen that by enhancing Mtn and LuxS, the efficiency of SAM regeneration is improved, the methylation reaction can be further promoted, and the production amount of any methyl compound can be increased.

[0665] In Examples 66 and 67, the total accumulation concentration under the condition of adding L-serine during the methylation reaction increased to 1.4 times and 1.3 times respectively compared with Examples 64 and 65. From the above results, it can be seen that by enhancing Prs and Apt, the efficiency of SAM regeneration is improved, the methylation reaction can be further promoted, and the production amount of any methyl compound can be increased.

[0666] That is, the method of the present invention has shown that the use of L-serine can efficiently regenerate SAM, promote the methylation reaction, and increase the amount of production of any methyl compound.

[0667] [Table 8]

[0668]

[0669] [Example 68]

[0670] For the Escherichia coli HMS174 (DE3) strain, expression of the ASMT gene was enhanced as follows.

[0671] (A) Construction of ASMT enhanced plasmid

[0672] A DNA containing the base sequence of the ASMT gene from Homo sapiens encoding acetyl-serotonin O-methyltransferase (a recognition sequence for the restriction enzyme Nco I is assigned to the 5' end, and a recognition sequence for the restriction enzyme Bgl II is assigned to the 3' end) is synthesized (base sequence of the synthetic DNA: sequence number 11). 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 alanine at position 258 was further mutated to glutamic acid. The synthesized DNA was inserted into the restriction enzyme sites Nco I and Bgl II of the Escherichia coli expression vector pET Duet-1 (Novagen), and connected to the downstream of the T7 promoter. The constructed plasmid was named pET_P T7 -ASMT.

[0673] (B) Production of ASMT enhanced strain

[0674] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -ASMT constructed in Example 68(A), and pCDFDuet-1 (Novagen), and the resulting strain was designated as HMS174(DE3) / pET_P T7 -ASMT.

[0675] [Example 69]

[0676] For the Escherichia coli HMS174(DE3) strain, the expression of the ASMT gene, mtn gene, and luxS gene was enhanced as follows.

[0677] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -ASMT constructed in Example 68(A), and the plasmid pCDF_P T7 -mtn-luxS constructed in Example 3(A), and the resulting strain was designated as HMS174(DE3) / pET_P T7 -ASMT / pCDF_P T7 -mtn-luxS.

[0678] [Example 70]

[0679] For the Escherichia coli HMS174(DE3) strain, the expression of the ASMT gene, mtn gene, luxS gene, Prs gene, and Apt gene was enhanced as follows.

[0680] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -ASMT constructed in Example 68(A), and the plasmid pCDF_P T7 -mtn-luxS-prs-apt constructed in Example 23(A), and the resulting strain was designated as HMS174(DE3) / pET_P T7 -ASMT / pCDF_P T7 -mtn-luxS-prs-apt.

[0681] [Example 71]

[0682] For the Escherichia coli HMS174(DE3) strain, the expression of the ASMT gene, glyA gene, mtn gene, and luxS gene was enhanced as follows.

[0683] (A) Construction of GlyA, Mtn, LuxS enhanced plasmid

[0684] Synthesize DNA (synthetic DNA base sequence: SEQ ID NO: 12) containing the base sequences of the mtn gene and the luxS gene from Escherichia coli, with a recognition sequence for the restriction enzyme Mfe I assigned to the 5'-terminal side and a recognition sequence for the restriction enzyme Kpn I assigned to the 3'-terminal side. Insert the synthesized DNA into the plasmid pCDF_P constructed in Example 48(A). T7 The restriction enzyme sites Mfe I and Kpn I of -glyA are ligated downstream of the glyA gene. The constructed plasmid was named pCDF_P T7 -glyA-mtn-luxS.

[0685] (B) Production of ASMT, GlyA, Mtn, LuxS enhanced strains

[0686] Transform Escherichia coli HMS174(DE3) strain (Novagen) with the plasmid pET_P constructed in Example 68(A). T7 -ASMT, and the plasmid pCDF_P constructed in Example 71(A). T7 -glyA-mtn-luxS, and the resulting strain was designated HMS174(DE3) / pET_P T7 -ASMT / pCDF_P T7 -glyA-mtn-luxS.

[0687] [Example 72]

[0688] For the Escherichia coli HMS174(DE3) strain, enhance the expression of the ASMT gene, glyA gene, mtn gene, luxS gene, Prs gene, and Apt gene as follows.

[0689] (A) Construction of GlyA, Mtn, LuxS, Prs, Apt enhanced plasmid

[0690] Synthesize DNA (synthetic DNA base sequence: SEQ ID NO: 13) containing the base sequences of the prs gene and the apt gene from Escherichia coli, with a recognition sequence for the restriction enzyme Kpn I assigned to the 5'-terminal side and a recognition sequence for the restriction enzyme Avr II assigned to the 3'-terminal side. Insert the synthesized DNA into the restriction enzyme sites Kpn I and Avr II of the plasmid pCDF_P T7 -glyA-mtn-luxS, and ligate it downstream of the glyA gene. The constructed plasmid was named pCDF_P T7 -glyA-mtn-luxS-prs-apt.

[0691] (B) Production of ASMT, GlyA, Mtn, LuxS, Prs, Apt enhanced strains

[0692] The Escherichia coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pET_P T7 -ASMT constructed in Example 68(A), and the plasmid pCDF_P T7 -glyA-mtn-luxS-prs-apt constructed in Example 72(A). The resulting strain was designated as HMS174(DE3) / pET_P T7 -ASMT / pCDF_P T7 -glyA-mtn-luxS-prs-apt.

[0693] [Example 73]

[0694] For the ASMT enhanced strain prepared in Example 68(B), melatonin production was evaluated as follows.

[0695] The HMS174(DE3) / pET_P T7 -ASMT prepared in Example 68(B) 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 and 50 μg / mL spectinomycin. 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 10 mM L-serine, 0.5% glucose, 50 μg / mL carbenicillin and 50 μg / mL spectinomycin was added to a 50 mL test tube, and the culture solution in the above LB medium was inoculated into this M9 medium in a 1 / 50 dilution, and shake-cultured at 200 rpm and 30 °C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and further shake-cultured for 16 hours.

[0696] The resulting culture solution was collected by centrifugation at 3000×g for 5 minutes, and suspended in a reaction buffer (100 mM TriS-HCl pH 8.0, 20 mM MgSO4, 20 mM glucose) containing 20 mM L-serine, 100 μg / mL carbenicillin, 100 μg / mL spectinomycin and 1 mM IPTG to a concentration of OD 600 = 4. 1 mL of the 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, thereby starting the melatonin synthesis reaction including methylation, and shake-cultured at 200 rpm and 30 °C for 48 hours.

[0697] In addition, in order to promote the methylation reaction by efficiently regenerating the methyl carbon through SAM, an experiment was also conducted under the same conditions without adding L-serine as a supply source of methyl carbon, and the results were compared with those of the experiment conducted under the condition of adding L-serine.

[0698] 100 μL of the obtained reaction solution was mixed with 100 μL of methanol, and the supernatant after centrifugation at 3000×g for 5 minutes was collected for LC-MS analysis. The conditions for LC-MS analysis are shown in Table 9.

[0699] As a result of the measurement, the melatonin concentration in the reaction solution increased by 0.003 mM compared with the condition without adding L-serine, and was 0.094 mM under the condition of adding L-serine.

[0700] [Table 9]

[0701]

[0702] [Example 74]

[0703] For the ASMT, Mtn, LuxS enhanced strains prepared in Example 69, the evaluation of melatonin production was carried out as follows.

[0704] Regarding the evaluation of melatonin production in this example, HMS174(DE3) / pET_P prepared in Example 69 was used T7 -ASMT / pCDF_P T7 -mtn-luxS, and except for this, it was carried out in the same manner as in Example 73.

[0705] As a result, the melatonin concentration in the reaction solution increased by 0.012 mM compared with the condition without adding L-serine, and was 0.220 mM under the condition of adding L-serine.

[0706] [Example 75]

[0707] For the ASMT, Mtn, LuxS, Prs, Apt enhanced strains prepared in Example 70, the evaluation of melatonin production was carried out as follows.

[0708] Regarding the evaluation of melatonin production in this example, HMS174(DE3) / pET_P prepared in Example 70 was used T7 -ASMT / pCDF_P T7 -mtn-luxS-prs-apt, and except for this, it was carried out in the same manner as in Example 73.

[0709] As a result, the melatonin concentration in the reaction solution increased by 0.016 mM compared to the condition without the addition of L-serine, and was 0.248 mM under the condition with the addition of L-serine.

[0710] [Example 76]

[0711] For the ASMT, GlyA, Mtn, and LuxS enhanced strains prepared in Example 71(B), the evaluation of melatonin production was performed as follows.

[0712] Regarding the evaluation of melatonin production in this example, HMS174(DE3) / pET_P prepared in Example 71(B) was used T7 -ASMT / pCDF_P T7 -glyA-mtn-luxS, and except for this, it was carried out in the same manner as in Example 73.

[0713] As a result, the melatonin concentration in the reaction solution increased by 0.004 mM compared to the condition without the addition of L-serine, and was 0.295 mM under the condition with the addition of L-serine.

[0714] [Example 77]

[0715] For the ASMT, GlyA, Mtn, LuxS, Prs, and Apt enhanced strains prepared in Example 72(B), the evaluation of melatonin production was performed as follows.

[0716] Regarding the evaluation of melatonin production in this example, HMS174(DE3) / pET_P prepared in Example 72(B) was used T7 -ASMT / pCDF_P T7 -glyA-mtn-luxS-prs-apt, and except for this, it was carried out in the same manner as in Example 73.

[0717] As a result, the melatonin concentration in the reaction solution increased by 0.029 mM compared to the condition without the addition of L-serine, and was 0.301 mM under the condition with the addition of L-serine.

[0718] The results of Examples 73 to 77 were summarized in Table 10. The accumulated concentrations of melatonin under the conditions with the addition of L-serine in these examples increased by 3%, 6%, 7%, 16%, and 11% respectively compared to the conditions without the addition of L-serine. From the above results, it can be seen that by performing the methylation reaction in the presence of L-serine, efficient regeneration of SAM can be achieved, the methylation reaction can be promoted, and the production amount of any methyl compound can be increased.

[0719] In Example 74, the accumulation concentration of melatonin under the condition of adding L-serine increased to 2.3 times compared with the result of Example 73. From the above results, it can be seen that by enhancing the expression of Mtn and LuxS, the efficiency of SAM regeneration is improved, and further the methylation reaction can be promoted.

[0720] In Examples 75 and 77, the accumulation concentrations of melatonin under the condition of adding L-serine increased by 13% and 2% respectively compared with the results of Examples 74 and 76. From the above results, it can be seen that in addition to enhancing the expression of Mtn and LuxS, by enhancing the expression of Prs and Apt, the efficiency of SAM regeneration is improved, and further the methylation reaction can be promoted.

[0721] In Examples 76 and 77, the accumulation concentrations of melatonin under the condition of adding L-serine increased by 34% and 21% respectively compared with the results of Examples 74 and 75. From the above results, it can be seen that in addition to enhancing the expression of Mtn, LuxS, Prs, and Apt, by enhancing the expression of GlyA, the efficiency of SAM regeneration is improved, and further the methylation reaction can be promoted.

[0722] That is, the method of the present invention shows that by using L-serine, SAM can be efficiently regenerated, the methylation reaction can be promoted, and the production amount of any methyl compound can be increased.

[0723] [Table 10]

[0724]

[0725] [Example 78]

[0726] For the SfmM2 and GlyA overexpressing strains prepared in Example 48(B), the production evaluation of 3-methyltyrosine was carried out as follows.

[0727] The HMS174(DE3) / pET_P prepared in Example 48(B) T7 -sfmM2 / pCDF_P T7-glyA 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 34 μg / mL chloramphenicol. 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 34 μg / mL chloramphenicol was added to a 50 mL test tube, and the culture solution in the above LB medium was inoculated in such a way that it was diluted 1 / 50, and shake-cultured at 200 rpm and 30 °C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and further shake-cultured for 16 hours.

[0728] The obtained culture solution was collected by centrifugation at 3000×g for 5 minutes and suspended in a reaction buffer (100 mM Tris-HCl pH 8.0, 20 mM MgSO4, 20 mM glucose) containing 100 μg / mL carbenicillin, 100 μg / mL spectinomycin, 68 μg / mL chloramphenicol, and 1 mM IPTG to an OD 600 = 10. 1 mL of the 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 start the methylation reaction, and shake-cultured at 200 rpm and 30 °C for 6 hours.

[0729] LC-MS analysis of the obtained reaction solution was carried out in the same manner as in Example 9. As a result of the measurement, the concentration of 3-methyltyrosine in the reaction solution was 0.075 mM.

[0730] [Example 79]

[0731] For the SfmM2, MetFHK, Mtn, LuxS, GlyA overexpressing strains prepared in Example 50, the production evaluation of 3-methyltyrosine was carried out as follows.

[0732] Regarding the production evaluation of 3-methyltyrosine in this example, HMS174(DE3) / pET_P T7 -sfmM2 / pACYC_P T7 -metFHK_P T7 -mtn-luxS / pCDF_P T7 -glyA prepared in Example 50 was used, and except for this, it was carried out in the same manner as in Example 78.

[0733] As a result, the concentration of 3-methyltyrosine in the reaction solution was 0.170 mM.

[0734] [Comparative Example 1]

[0735] For the SfmM2 enhanced strain prepared in Example 1, the production evaluation of 3-methyltyrosine was carried out as follows.

[0736] Regarding the production evaluation of 3-methyltyrosine in this comparative example, HMS174(DE3) / pET_P prepared in Example 1 was used. T7 -sfmM2, and except for this, it was carried out in the same manner as in Example 78.

[0737] As a result, the concentration of 3-methyltyrosine in the reaction solution was 0.036 mM.

[0738] [Comparative Example 2]

[0739] For the SfmM2, MetFHK, Mtn, LuxS enhanced strains prepared in Example 49(C), the production evaluation of 3-methyltyrosine was carried out as follows.

[0740] Regarding the production evaluation of 3-methyltyrosine in this comparative example, HMS174(DE3) / pET_P prepared in Example 49(C) was used. T7 -sfmM2 / pACYC_P T7 -metFHK_P T7 -mtn-luxS, and except for this, it was carried out in the same manner as in Example 78.

[0741] As a result, the concentration of 3-methyltyrosine in the reaction solution was 0.063 mM.

[0742] [Comparative Example 3]

[0743] For the SfmM2, GcvTHP enhanced strains prepared in Example 2(B), the production evaluation of 3-methyltyrosine was carried out as follows.

[0744] Regarding the production evaluation of 3-methyltyrosine in this comparative example, HMS174(DE3) / pET_P prepared in Example 2(B) was used. T7 -sfmM2 / pACYC_P T7 -gcvTHP, and except for this, it was carried out in the same manner as in Example 78.

[0745] As a result, the concentration of 3-methyltyrosine in the reaction solution was 0.021 mM.

[0746] [Comparative Example 4]

[0747] For the SfmM2, Mtn, and LuxS enhanced strains prepared in Example 5, the production evaluation of 3-methyltyrosine was carried out as follows.

[0748] Regarding the production evaluation of 3-methyltyrosine in this comparative example, HMS174(DE3) / pET_P prepared in Example 5 was used. T7 -sfmM2 / pCDF_P T7 -mtn-luxS, and except for this, it was carried out in the same manner as in Example 78.

[0749] As a result, the concentration of 3-methyltyrosine in the reaction solution was 0.029 mM.

[0750] [Comparative Example 5]

[0751] For the SfmM2, Mtn, LuxS, and GcvTHP enhanced strains prepared in Example 6, the production evaluation of 3-methyltyrosine was carried out as follows.

[0752] Regarding the production evaluation of 3-methyltyrosine in this comparative example, HMS174(DE3) / pET_P prepared in Example 6 was used. T7 -sfmM2 / pCDF_P T7 -mtn-luxS / pACYC_P T7 -gcvTHP, and except for this, it was carried out in the same manner as in Example 78.

[0753] As a result, the concentration of 3-methyltyrosine in the reaction solution was 0.020 mM.

[0754] The results of Example 78, 79, and Comparative Examples 1 to 5 were summarized in Table 11. The accumulated concentrations of 3-methyltyrosine in Example 78 and 79 were increased by 2.1 times and 2.7 times, respectively, compared with Comparative Examples 1 and 2. From the above results, it can be seen that by enhancing GlyA, efficient regeneration of SAM using glucose as a supply source of methyl carbon can be achieved, methylation reaction can be promoted, and the production amount of any methyl compound can be increased. It is considered that the methyl carbon of SAM was regenerated from glucose via L-serine.

[0755] In addition, the accumulated concentration of 3-methyltyrosine in Example 79 was increased by 2.3 times compared with Example 78. From this, it can be seen that by enhancing MetFHK, Mtn, and LuxS, the efficiency of SAM regeneration is improved, further methylation reaction can be promoted, and the production amount of any methyl compound can be increased.

[0756] That is, the present invention shows that by using the modified cells of the present invention, SAM can be efficiently regenerated from organic raw materials such as glucose, methylation reaction can be promoted, and the production amount of any methyl compound can be increased.

[0757] On the other hand, in Comparative Examples 3 and 5, no increase in the accumulation of 3-methyltyrosine was confirmed as compared with Comparative Examples 1 and 4, respectively. Although a metabolic pathway for regenerating the methyl carbon of SAM via glycine generated from glucose was considered, since the methylation reaction could not be promoted by enhancing GcvTHP, GlyA enhancement was considered important in terms of using general organic raw materials such as glucose as a supply source of methyl carbon.

[0758] [Table 11]

[0759]

[0760] [Example 80]

[0761] For the ASMT, GlyA, Mtn, and LuxS overexpressing strains prepared in Example 71(B), melatonin production was evaluated as follows.

[0762] The HMS174(DE3) / pET_P T7 -ASMT / pCDF_P T7 -glyA-mtn-luxS prepared in Example 71(B) 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 and 50 μg / mL spectinomycin. 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, and 50 μg / mL spectinomycin was added to a 50 mL test tube, and the culture solution in the above LB medium was inoculated into this M9 medium at a dilution of 1 / 50, and shake-cultured at 200 rpm and 30 °C for 6 hours. IPTG was added to a final concentration of 0.5 mM, and further shake-cultured for 16 hours.

[0763] The obtained culture solution was collected by centrifugation at 3000×g for 5 minutes to obtain an OD 600was suspended in a reaction buffer (100 mM Tris-HCl pH 8.0, 20 mM MgSO4, 20 mM glucose) containing 100 μg / mL carbenicillin, 100 μg / mL spectinomycin, and 1 mM IPTG at a ratio of 4. 1 mL of the 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, thereby starting the melatonin synthesis reaction involving methylation. The mixture was shaken at 200 rpm and 30 °C for 48 hours.

[0764] In addition, in order to clarify the promotion of the methylation reaction by the efficient regeneration of the methyl carbon of SAM, an experiment was also carried out under the same conditions without adding L-serine as a supply source of methyl carbon, and the results were compared with those of the experiment carried out under the condition of adding L-serine.

[0765] The LC-MS analysis of the obtained reaction solution was carried out in the same manner as in Example 73. As a result of the measurement, the melatonin concentration in the reaction solution was 0.248 mM.

[0766] [Example 81]

[0767] For the ASMT, GlyA, Mtn, LuxS, Prs, Apt enhanced strains prepared in Example 72(B), the melatonin production evaluation was carried out as follows.

[0768] Regarding the melatonin production evaluation in this example, HMS174(DE3) / pET_P prepared in Example 72(B) was used. T7 -ASMT / pCDF_P T7 -glyA-mtn-luxS-prs-apt, and except for this, the procedure was the same as in Example 80.

[0769] As a result, the melatonin concentration in the reaction solution was 0.281 mM.

[0770] [Comparative Example 6]

[0771] For the ASMT enhanced strain prepared in Example 68(B), the melatonin production evaluation was carried out as follows.

[0772] Regarding the melatonin production evaluation in this example, HMS174(DE3) / pET_P prepared in Example 68(B) was used. T7 -ASMT, and except for this, the procedure was the same as in Example 80.

[0773] As a result, the melatonin concentration in the reaction solution was 0.075 mM.

[0774] [Comparative Example 7]

[0775] For the ASMT, Mtn, and LuxS enhanced strains prepared in Example 69, melatonin production was evaluated as follows.

[0776] Regarding the melatonin production evaluation in this example, HMS174(DE3) / pET_P prepared in Example 69 was used T7 -ASMT / pCDF_P T7 -glyA-mtn-luxS, and other than this, it was carried out in the same manner as in Example 80.

[0777] As a result, the melatonin concentration in the reaction solution was 0.189 mM.

[0778] [Comparative Example 8]

[0779] For the ASMT, Mtn, LuxS, Prs, and Apt enhanced strains prepared in Example 70, melatonin production was evaluated as follows.

[0780] Regarding the melatonin production evaluation in this example, HMS174(DE3) / pET_P prepared in Example 70 was used T7 -ASMT / pCDF_P T7 -glyA-mtn-luxS-prs-apt, and other than this, it was carried out in the same manner as in Example 80.

[0781] As a result, the melatonin concentration in the reaction solution was 0.263 mM.

[0782] The results of Examples 80 and 81 and Comparative Examples 6 to 8 were summarized in Table 12. The accumulated concentrations of melatonin in Examples 80 and 81 increased by 31% and 7% respectively compared with Comparative Examples 7 and 8. From the above results, it was found that by enhancing GlyA, efficient regeneration of SAM using glucose as a supply source of methyl carbon could be achieved, methylation reaction could be promoted, and the production amount of any methyl compound could be increased. It was considered that the methyl carbon of SAM was regenerated from glucose via L-serine.

[0783] In addition, the accumulated concentration of melatonin in Example 81 increased by 13% compared with Example 80. From this, it was found that in addition to enhancing the expression of Mtn and LuxS, by enhancing the expression of Prs and Apt, the efficiency of SAM regeneration was improved, methylation reaction could be further promoted, and the production amount of any methyl compound could be increased.

[0784] That is, the present invention shows that by using the modified cells of the present invention, SAM can be efficiently regenerated from organic raw materials such as glucose, methylation reaction can be promoted, and the production amount of any methyl compound can be increased.

[0785] [Table 12A]

[0786]

[0787] [Table 12B]

[0788]

[0789] [Industrial Applicability]

[0790] The present invention can promote the methylation reaction of a compound to produce a methyl compound.

[0791] Sequence Listing Free Text:

[0792] SEQ ID NOs: 1 to 13: Synthetic DNA.

Claims

1. A method for producing a methyl compound, comprising the following steps: performing a methylation reaction in the presence of glycine or serine using a cell modified in such a way that the activity or expression of an S-adenosylmethionine-dependent methyltransferase is enhanced.

2. The method according to claim 1, wherein, The cell is further modified in such a way that the activity or expression of serine hydroxymethyltransferase (GlyA) is enhanced.

3. A method for producing a methyl compound, comprising the following steps: performing a methylation reaction using a cell modified in such a way that the activities or expressions of serine hydroxymethyltransferase (GlyA) and an S-adenosylmethionine-dependent methyltransferase are enhanced.

4. The method according to claim 3, wherein The cell is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of D-3-phosphoglycerate dehydrogenase (SerA), phosphoserine aminotransferase (SerC), and phosphoserine phosphatase (SerB) is increased.

5. The method according to claim 1 or 3, wherein, The cell is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of S-adenosylhomocysteinase (Mtn), S-ribosylhomocysteine lyase (LuxS), and S-adenosylhomocysteine hydrolase (SahH) is enhanced.

6. The method according to claim 1 or 3, wherein The cell is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of ribose phosphate diphosphokinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) is enhanced.

7. The method according to claim 1 or 3, wherein The cell is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthase (MetH or MetE), and methionine adenosyltransferase (MetK) is enhanced.

8. The method according to claim 1 or 3, wherein The cell is further modified in such a way that the activity or expression of at least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl-[GcvH]: protein N-octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP) is enhanced.

9. The method according to claim 1 or 3, wherein, The cell is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

10. The method according to claim 9, wherein The cell is a microorganism.

11. The method according to claim 10, wherein, The microorganism is at least one selected from the group consisting of Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, and yeasts.

12. The method according to claim 1 or 3, comprising a step of allowing an organic raw material to act on the cell.

13. The method according to claim 12, wherein, The organic raw material is an organic compound having 3 or more carbon atoms.

14. The method according to claim 12, wherein, The organic raw material contains one selected from the group consisting of saccharides, alcohols, and organic acids.

15. The method according to claim 1, wherein, The cells are cells cultured in the presence of an organic raw material containing glycine or serine.

16. The method according to claim 1 or 3, further comprising a step of recovering the methyl compound produced by the methylation reaction.

17. The method according to claim 1 or 3, further comprising a step of purifying the methyl compound produced by the methylation reaction.

18. The method according to claim 12, further comprising a step of recovering the methyl compound produced by the action of the organic raw material.

19. The method according to claim 12, further comprising a step of purifying the methyl compound produced by the action of the organic raw material.

20. The method according to claim 1 or 3, wherein The methyl compound is a derivative of an aromatic amino acid.

21. A cell modified in such a way that the activities or expressions of serine hydroxymethyltransferase (GlyA) and S-adenosylmethionine-dependent methyltransferase are enhanced.

22. The cell according to claim 21, further modified in such a way that the activities or expressions of at least one enzyme selected from the group consisting of S-adenosylhomocysteine nucleosidase (Mtn), S-ribosylhomocysteine lyase (LuxS), and S-adenosylhomocysteine hydrolase (SahH) are enhanced.

23. The cell according to claim 21, further modified in such a way that the activities or expressions of at least one enzyme selected from the group consisting of ribose phosphate diphosphate kinase (Prs), adenine phosphoribosyltransferase (Apt), and adenosine kinase (ADK) are enhanced.

24. The cell according to claim 21, further modified in such a way that the activities or expressions of at least one enzyme selected from the group consisting of 5,10-methylenetetrahydrofolate reductase (MetF), methionine synthase (MetH or MetE), and methionine adenosyltransferase (metK) are enhanced.

25. The cell according to claim 21, further modified in such a way that the activities or expressions of at least one enzyme selected from the group consisting of aminomethyltransferase (GcvT), octanoyl-[GcvH]: protein N-octanoyltransferase (GcvH), and glycine dehydrogenase (GcvP) are increased.

26. The cell according to any one of claims 21 to 25, further modified in such a way that the activities or expressions of at least one enzyme selected from the group consisting of D-3-phosphoglycerate dehydrogenase (SerA), phosphoserine aminotransferase (SerC), and phosphoserine phosphatase (SerB) are increased.

27. The cell according to claim 21 is at least one selected from the group consisting of animal cells, insect cells, plant cells, and microorganisms.

28. The cell according to claim 27 is a microorganism.

29. The cell according to claim 28, wherein, The microorganism is at least one selected from the group consisting of Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, and yeasts.

30. A method for producing an organic compound, comprising a step of producing an organic compound using the methyl compound obtained by the method according to claim 1 or 3 as an intermediate.

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