JPWO2019160059A1 - How to recycle S-adenosylmethionine - Google Patents

Abstract

A method for recycling S-adenosylmethionine, which comprises demethylating S-adenosylmethionine and then converting it from methionine obtained by transferring a methyl group derived from methanol to regenerate it. According to the present invention, by adding methanol, S-adenosylmethionine (SAM) can be regenerated, and the application to various methylation reactions can be expanded. Therefore, pharmaceutical manufacturing and chemistry It is extremely useful in fields related to product manufacturing, functional compound manufacturing, and the like.

Description

The present invention relates to a method for recycling S-adenosylmethionine. More specifically, it relates to a method for regenerating and recycling S-adenosylmethionine and its use.

S-Adenosylmethionine (hereinafter referred to as SAM) has a role as a methyl group donor in the living body, and methyl of various compounds such as nucleic acids, proteins, phospholipids, hormones, and neurotransmitters. Involved in the chemical reaction. The methylation reaction using SAM as a methyl group donor is catalyzed by a group of enzymes called methylase, but according to the metabolic reaction database KEGG (Kyoto Encyclopedia of Genes and Genomes), there are more than 200 types of methylase. Exists. Among these are enzymes that work in the biosynthetic pathway of antibiotics such as gentamicin (see Non-Patent Document 1) and enzymes that catalyze the methylation of polycyclic aromatics such as hydroxycoumarins and flavonoids (non-patent). Many of them are involved in biosynthesis and modification of functional molecules having a complex skeleton, such as (see Reference 2). Therefore, it is expected that various functional molecules using these methylase groups and SAM will be created, but there are no cases in which SAM-dependent methylases have been industrially applied. The reason is the complexity of SAM regeneration in vivo.

SAM is decomposed into homocysteine, adenine, and ribose via S-Adenosylhomosystein (hereinafter referred to as SAH) after passing a methyl group to the substrate by a methylase reaction (Fig. 1). .. Regeneration of SAM requires remethylation of the homocysteine produced here to methionine, which involves other methyl group donors such as S-methylmethionine, betaine, and 5-methyltetrahydropteroyltriglutamic acid. Is required. However, the synthesis of these methyl group donors requires SAM, resulting in a "dignified cycle" of metabolic reactions that uses SAM to regenerate SAM. On the other hand, 5-methyltetrahydrofolic acid (5-methylTHF), which can be used as a methyl group donor, does not utilize SAM for its synthesis, but uses the amino acids serine and glycine for its synthesis. Since amino acids play an important role in the biosynthesis of proteins and the like, it is practically impossible to utilize these amino acids only for the regeneration of SAM.

Due to such difficulty in supplying SAM, there is no research focusing on SAM recycling, and it is used for techniques related to the addition of precursor methionine and inhibition of metabolism (see Patent Documents 1 and 2), and for the synthesis and decomposition of SAM. Studies have been conducted only from the viewpoint of high SAM production, such as high expression of related genes and introduction of mutations (see Patent Documents 3 to 4).

Japanese Patent Application Laid-Open No. 2009-17849 JP 2011-177125 Japanese Patent Application Laid-Open No. 2008-17756 JP-A-2009-34043

Kim HJ, et al., J Am Chem Soc, 2013, 135, 8093-8096 Dhar K, et al., Appl Environ Microbiol, 2000, 66, 4877-4882

However, conventional methods do not provide a fundamental solution to the problem of supplying methyl groups for methylation of homocysteine, which is essential for SAM regeneration, and the amount of SAM that can be produced is limited. Moreover, since it is expensive to use SAM as a reagent, further development of technology has been required.

The present invention relates to a method for regenerating and recycling S-adenosylmethionine (SAM) and its use.

As a result of diligent studies to solve the above problems, the present inventors supplied methanol as an initial methyl group donor and operated a pathway for promoting regeneration from homocysteine to SAM, thereby entering the methyl group of SAM. For the first time, we succeeded in creating a SAM recycling route in which the (supply side) and outlet (demand side) are unified.

That is, the present invention relates to the following [1] to [15].
[1] Recycling of S-adenosylmethionine, which is characterized in that S-adenosylmethionine is demethylated and then converted from methionine obtained by transferring a methyl group derived from methanol to be regenerated. Method.
[2] The method according to [1] above, wherein S-adenosylmethionine is demethylated and converted to S-adenosyl homocysteine and then to homocysteine.
[3] The method according to [1] or [2] above, wherein demethylation of S-adenosylmethionine is performed via S-adenosylmethionine-dependent methylase.
[4] The method according to any one of [1] to [3] above, wherein the methyl group derived from methanol is obtained by reacting methanol with methanol dehydrogenase or methanol oxidase.
[5] The method according to [3] or [4] above, wherein a host organism co-expressing methylase and methanol dehydrogenase is used.
[6] The method according to [3] or [4] above, wherein a host organism co-expressing methylase and methanol oxidase is used.
[7] The method according to [5] or [6] above, wherein the host organism is selected from animal cells, insect cells, plant cells and microorganisms.
[8] The method according to [7] above, wherein the host organism is modified to increase the amount of methionine or S-adenosylmethionine.
[9] The method according to any one of [1] to [8] above, wherein further, conversion from methionine via adenosine triphosphate obtained by adding an energy source for adenosine triphosphate regeneration.
[10] Methylation characterized in that S-adenosylmethionine regenerated by the method according to any one of [1] to [9] above is used as a methyl group donor to carry out a methylation reaction. How to make a body.
[11] A host organism co-expressing methylase and methanol dehydrogenase or methanol oxidase for use in the method according to any one of [1] to [10] above.
[12] The host organism according to [11] above, wherein methylase and methanol dehydrogenase or methanol oxidase have been introduced.
[13] The host organism according to [12] above, further modified to increase the amount of methionine or S-adenosylmethionine.
[14] Modifications overexpression of methionine or S-adenosylmethionine synthase, mutations that enhance the function of methionine or S-adenosylmethionine synthase, expression of methionine or S-adenosylmethionine synthase gene A mutation that suppresses the function of an enzyme or protein that suppresses the function of an enzyme, a mutation that improves the function of an enzyme that suffers feedback inhibition in the synthesis of methionine or S-adenosylmethionine, and a mutation that improves the function of a degrading enzyme of S-adenosylhomocysteine. The host organism according to [13] above, which is selected from, and a mutation that suppresses the function of a formaldehyde-degrading enzyme.
[15] An expression vector into which methylase and methanol dehydrogenase or methanol oxidase have been introduced.

The present invention also includes the following [16] to [18].
[16] A method for promoting methylation, which comprises using S-adenosylmethionine regenerated by the method according to any one of [1] to [9] above.
[17] A method for producing a pharmaceutical product, a chemical product or a functional compound, which comprises using S-adenosylmethionine regenerated by the method according to any one of the above [1] to [9].
[18] A pharmaceutical product, chemical product or functional compound obtained by the method according to the above [17].

According to the present invention, the addition of methanol makes it possible to regenerate S-adenosylmethionine (SAM), which has an excellent effect of expanding its application to various methylation reactions. To.

FIG. 1 is a diagram showing a schematic example of a SAM regeneration path. FIG. 2 is a diagram showing one aspect of the SAM recycling method of the present invention. FIG. 3 is a diagram showing one aspect of the SAM recycling method of the present invention. FIG. 4 is a diagram showing the results of measuring the activity of the introduced enzyme. FIG. 5 is a diagram showing the results of confirming the enzyme activity endogenous to the microorganism. FIG. 6 is a diagram showing the results of esculetin methylation reaction using the SAM recycling route by a transformant of Escherichia coli BW25113 strain. FIG. 7 is a diagram showing the results of the methylation reaction of esculetin using the SAM recycling route when glucose was separately added by the transformant of Escherichia coli BW25113 strain. FIG. 8 is a diagram showing the results of esculetin methylation reaction using the SAM recycling route by a transformant of Escherichia coli BL21 (DE3) strain. Figure (A) shows the results of the SaOMT single expression strain, and Figure (B) shows the results of the SaOMT and CnMDH2 co-expressing strain. FIG. 9 is a diagram showing a synthetic pathway of methionine and SAM in Escherichia coli and a gene whose expression is suppressed by its master regulator protein, MetJ. FIG. 10 is a diagram showing the results of enhancing the methylation reaction of esculetin by disrupting the metJ gene and frmA gene of Escherichia coli BL21 (DE3) strain. FIG. 11 is a diagram showing the results of enhancing the methylation reaction of esculetin when the feedback inhibitory resistance enzyme genes of CysE and MetA were overexpressed in a derivative strain of Escherichia coli BL21 (DE3). FIG. (A) shows the results of the ΔmetJ strain, and FIG. (B) shows the results of the ΔmetJ ΔfrmA strain. FIG. 12 is a diagram showing the results of esculetin methylation reaction when various enzymes that convert methanol to formaldehyde were introduced into a metaA (fdr) overexpressing strain of Escherichia coli BL21 (DE3) ΔmetJ ΔfrmA. FIG. 13 is a diagram showing the results of methylation reactions of various compounds using the SAM recycling pathway by transformants of Escherichia coli BW25113 strain or BL21 (DE3) ΔmetJ ΔfrmA metaA (fdr) overexpressing strain.

The basic principle of the method for recycling S-adenosylmethionine (SAM) of the present invention is shown in FIG. Hereinafter, a recycling route in which the inlet (supply side) and the outlet (demand side) of the methyl group of SAM are unified may be simply referred to as a SAM recycling route. Here, SAM is first demethylated to S-adenosyl homocysteine (SAH) by a SAM-dependent methylase and then degraded to homocysteine, adenine, and ribose. On the other hand, methionine was synthesized by transferring the methyl group of the added methanol to the obtained homocysteine via the methyl group donating precursor by methanol dehydrogenase, and adenosine triphosphate (ATP) was added to the methionine. ) Acts and it is thought that SAM is finally regenerated. In the above pathway, any enzyme capable of converting methanol into formaldehyde to transfer a methyl group can be used. Therefore, for example, when methanol oxidase is used instead of methanol dehydrogenase, FIG. 2 A route similar to that will be established. In the recycling route of the present invention, various methylation reactions are possible depending on the type of methylase, and by extension, methylation techniques for various substances can be provided.

Further, as shown in FIG. 2, ATP is used when methionine is converted to SAM, and this ATP is derived from adenine and ribose generated when SAM is decomposed into homocysteine, such as glucose. It may be possible to resynthesize adenosine diphosphate (ADP) if there is a suitable energy source to convert it to ATP. Therefore, as another aspect of the present invention, as a system for separately adding an energy source for ATP regeneration (for example, glucose or the like), for example, the recycling route shown in FIG. 3 can be mentioned. Here, SAM-dependent methylase demethylates SAM to S-adenosyl homocysteine (SAH) and then degrades it to homocysteine, adenine, and ribose. At that time, ATP is resynthesized from the obtained adenine and ribose via an adenosine salvage synthesis pathway driven by using the energy obtained by the metabolism of added glucose. On the other hand, for the obtained homocysteine, methionine is synthesized by transferring the methyl group of the added methanol by methanol dehydrogenase via the methyl group donating precursor in the same manner as described above, and the methionine is converted to the methionine. It is considered that the separately resynthesized ATP acts to finally regenerate SAM. It can be seen that the same pathway as in FIG. 3 is established when, for example, methanol oxidase is used instead of methanol dehydrogenase in the system. As a result, it is not necessary to input expensive ATP from the outside, so that a more efficient recycling method can be provided. When adenine, ribose, AMP, and ADP are added, ATP is similarly regenerated, which can be an efficient recycling method.

The recycling pathways described above may be driven in series, and are included in the present invention regardless of whether they are driven in the same system or in different systems by enzymes. However, these speculations do not limit the present invention.

In the method for recycling S-adenosylmethionine (SAM) of the present invention, S-adenosylmethionine (SAM) is demethylated and then converted from methionine obtained by transferring a methyl group derived from methanol. It is characterized by being regenerated. Here, the methyl group derived from methanol means a methyl group obtained by a reaction of methanol and eliminated from methanol.

The recycling method of the present invention can be carried out without particular limitation as long as it is a system in which SAM exists. Examples of the system in which SAM exists include animal cells, insect cells, plant cells, microorganisms (including yeast, Escherichia coli, Bacillus subtilis, coryneform bacteria, lactic acid bacteria, etc.). In addition, the substance production system is not limited to living cells, and there are so-called in vitro substance production systems such as a substance production system using only an enzyme and a substance production system using a cell-free protein synthesis system. An embodiment in a cell in which SAM is present will be described as an example.

First, demethylation is performed from SAM, which is performed via SAM-dependent methylase (SAM-dependent methyltransferase). Hereinafter, "SAM-dependent methyltransferase" and "SAM-dependent methyltransferase" may be collectively referred to as "methyltransferase".

As the methylase, as long as it is SAM-dependent, it may be one that is originally endogenous to the cell or one that is introduced from a foreign substance. The foreign substance is not particularly limited as long as it is known, and can be appropriately selected and used according to a known technique according to the object to be methylated. Specifically, known DNA methylases may be used to methylate DNA, eg Lyko, 2018, Nature Review, 19: 81-92 (Human DNA Methylation and Epigenetics (DNA). A review of the relationship between gene expression and cell phenotype changes without changes in base sequence) and Vasu and Nagaraja, 2013, Microbiol. Mol. Biol. Rev., 77: 53-72. Those described in the system (a review of the mechanism of methylating DNA, recognizing its own DNA by its methylation pattern, and destroying foreign DNA) can be used. Known protein methylases can be used to methylate proteins, eg, those described in Boriack-Sjodin and Swinger, 2016, Biochemistry, 55: 1557-1569 (Review of Protein Methylation and Epigenetics). Can be used. Known methylases can be used to methylate low molecular weight compounds and polycyclic aromatics, such as Struck et al., 2012, ChemBioChem, 13: 2642-2655 (compounds with a benzene ring, such as catechols). Or, those described in (Review of application and utilization of various methylases) such as production of polycyclic aromatic compounds such as flavonoids and alkaloids can be used. You may also search from the database and use it. For example, if you use BRENDA (https://www.brenda-enzyme), which is one of the enzyme information databases, and search using methylase or methyltransferase as keywords, 400 Since more than one enzyme species is registered, SAM-dependent methylase may be referred to from these enzyme species. Similarly, KEGG's compound search (http://www.genome.jp/kegg/compound/) was used to search for SAM as a keyword (compound registration number: C00019), and from more than 400 reactions involving SAM, It can be selected by referring to the Reaction item. In addition, these foreign methylases may be mutated to improve their activity and function as long as they can methylate the object to be methylated.

Specific examples of methylase are shown below. For example, as an enzyme that methylates a low molecular weight compound or a polycyclic aromatic compound, O-, N-, C-, S-, Se-, depending on the type of atom that methylates the object to be methylated, , As-methylase, etc. Examples of O-methylases include caffeic acid O-methyltransferase, catechol O-methyltransferase, acetylserotonin O-methyltransferase, apigenin 4'-O-methyltransferase, cafferoyl CoA O-methyltransferase, chlorophenol O-methyltransferase. , Corumbamine O-Methyltransferase, Demethylmacrosin O-Methyltransferase, 3'-Demethylstaurosporin O-Methyltransferase, 12-Hydroxydihydrokerylbin 12-O-Methyltransferase, 6-Hydroxymelene O-methyl Transtransferase, 3'-Hydroxy-N-Methyl- (S) -Cochlaurin 4'-O-Methyltransferase, 8-Hydroxykelcetin 8-O-Methyltransferase, Iodophenol O-Methyltransferase, Isobutylaldoxime O-Methyltransferase, (Iso) Eugenol O-methyltransferase, isoflavone 4'-O-methyltransferase, isoflavone 7-O-methyltransferase, isoliquilithigenin 2'-O-methyltransferase, isoorientin 3'-O-methyltransferase, Jasmonate O-Methyltransferase, Kemperol 4'-O-Methyltransferase, Lidokine 2'-O-Methyltransferase, Loganic Acid O-Methyltransferase, Luteolin O-Methyltransferase, Macrosin O-Methyltransferase, Methylkelcetagenin 6- O-Methyltransferase, 3-Methylkelcetin 7-O-Methyltransferase, Myricetin O-methyltransferase, N-benzoyl-4-hydroxyanthranyl acid 4-O-methyltransferase, O-demethylpuromycin O-methyltransferase, 6 -O-methylnorlaudanosoline 5'-O-methyltransferase, phenol O-methyltransferase, quercetin 3-O-methyltransferase, (RS) -norcochlorin 6-O-methyltransferase, (S) -scourelin 9 -O-methyltransferase, steligmatocystin 8-O-methyltransferase, Taberso Examples thereof include nin 16-O-methyltransferase, tetrahydrocolumbamine 2-O-methyltransferase, tocopherol O-methyltransferase, and xanthotoxol O-methyltransferase. Examples of N-methylases that methylate nitrogen atoms include histidine-N-α-methyltransferase, (RS) -1-benzyl-1,2,3,4-tetrahydroisoquinolin N-methyltransferase, 3-hydroxy-16. -Methoxy-2,3-dihydrotabersonine N-methyltransferase, amine N-methyltransferase, anthranylate N-methyltransferase, carnosin N-methyltransferase, (S) -cochlaurin N-methyltransferase, dimethylhistidine N-methyltransferase , Glycin N-methyltransferase, guanidinoacetic acid N-methyltransferase, histamine N-methyltransferase, methylamine-glutamate N-methyltransferase, nicotine amide N-methyltransferase, nicotinic acid N-methyltransferase, phenylethanolamine N-methyltransferase , Phosphatidylethanolamine N-methyltransferase, phosphatidyl-N-methylethanolamine N-methyltransferase, phosphoethanolamine N-methyltransferase, putresin N-methyltransferase, pyridine N-methyltransferase, (S) -tetrahydroprotoberberin N- Examples thereof include methyltransferase, trimityl sulfonium-tetrahydrofolate N-methyltransferase, tryptamine N-methyltransferase, and tyramine N-methyltransferase. Examples of C-methylases that methylate carbon atoms include phenylpyruvate 3C-methyltransferase, 3-hydroxyanthranylic acid 4C-methyltransferase, 24-methylenesterol C-methyltransferase, sterol 24C-methyltransferase, and tryptophan 2C-methyl. Examples include transferase, glutamic acid 3C-methyltransferase, 2-methyl-6-phytyl-1,4-hydroquinone methyltransferase and the like. In addition, although no name is given to the compound that acts, CouO (GenBank accession number AAG29793.1) derived from Streptomyces rishiriensis, NovO (GenBank accession number AAF67508.1) derived from Streptomyces spheroids, and SgvM derived from Streptomyces griseoviridis. (GenBank accession number JX508597.1), AcmI (GenBank accession number ADG27364.1) derived from Streptomyces chrysomallus, AcmL (GenBank accession number ADG27351.1), etc. can also be mentioned. Although most of these methylases are given names for the substrates on which they act, methylases generally have a wide range of substrate properties and act on substrates other than the substrates described in the enzyme names. It is possible.

When a foreign substance is used as the above-mentioned methylase, the combination of the derived cell and the cell to be introduced is not particularly limited as long as it can be introduced by a known gene transfer method or the like.

The reaction conditions of the methylase can be appropriately selected and set according to a known technique according to the type of methylase used with the cells of the embodiment. For example, when Escherichia coli, Bacillus subtilis, or lactic acid bacterium is used as a host organism, the condition is not particularly limited depending on the type of methylase, and is preferably 30 to 45 ° C, more preferably 35 to 40 ° C, and further preferably 36 to 38 ° C. It may be cultured in. When a coryneform bacterium or yeast is used as a host organism, it is not particularly limited depending on the type of methylase, and is preferably cultured under the conditions of 20 to 40 ° C, more preferably 25 to 35 ° C, and further preferably 29 to 31 ° C. Just do it. It should be noted that the subsequent reactions can also be set with reference to the above conditions.

This demethylates SAM to produce S-adenosyl homocysteine (SAH).

SAH is degraded by hydrolases present in the cells and converted to homocysteine. At that time, SAH may be directly converted to homocysteine and adenosine, or SAH may be converted to S-ribosyl homocysteine and adenine and then to homocysteine and ribose. The hydrolase is not particularly limited as long as it can hydrolyze SAH, and examples thereof include adenosyl homocystinase when directly converting to homocysteine. In addition, when converting to S-ribosylhomocysteine, S-adenosylhomocysteine nucleosidase can be mentioned. Here, the obtained S-ribosyl homocysteine is decomposed by a hydrolase also present in the cell and converted into homocysteine and ribose. The hydrolase is not particularly limited as long as it can hydrolyze S-ribosylhomocysteine, and examples thereof include S-ribosylhomocysteine lyase.

This consumes SAM and accumulates homocysteine. In the present invention, this homocysteine is converted to methionine to regenerate SAM.

The conversion from homocysteine to methionine may be carried out by introducing a methyl group into homocysteine, but in the present invention, it is carried out by utilizing the oxidation reaction from methanol to formaldehyde. Specifically, formaldehyde can be obtained by reacting methanol with, for example, methanol dehydrogenase or methanol oxidase as an enzyme that oxidizes the methanol to formaldehyde. The methyl group associated with formaldehyde thus obtained is transferred to homocysteine via a methyl group donating precursor.

The oxidation reaction from methanol to formaldehyde is carried out in a system in which a methyl group donating precursor that transfers a methyl group associated with formaldehyde is present. The methyl group donor precursor is not particularly limited as long as it functions as a methyl group donor by receiving a methyl group, and a system that functions in the same system as the above-mentioned system in which SAM exists may be a different system. It may function as a function. For example, if it functions in the same system, the received methyl group can be transferred to homocysteine as it is, and if it functions in a different system, the received methyl group can be transferred to the system in which SAM exists. Can be transferred to homocysteine. Specifically, for example, tetrahydrofolic acid (THF), which receives a methyl group, is converted to a methyl group donor of 5,10-methylenetetrahydrofolic acid (5,10-methyleneTHF).

Among the enzymes that oxidize methanol to formaldehyde, the methanol dehydrogenase may be any alcohol dehydrogenase that oxidizes methanol to formaldehyde. The existing one is not particularly limited, and for example, an exotic one may be introduced even if it is originally endogenous to the cell. Specifically, for example, ^{an enzyme having oxidative activity against methanol among NAD (P) +} -dependent alcohol dehydrogenase, or a mutant enzyme having improved methanol utilization; pyrroloquinoline quinone (PQQ) -dependent methanol dehydrogenase Can be mentioned. Specifically, as NAD ⁺ -dependent enzymes, Bacillus methanolicus (Krog et al., 2013, PLOS one, 8: e59188), Bacillus stearothermophilus (Sheehan et al., 1988, Biochem. J., 252: 661-666), an enzyme derived from Cupriavidus necator (Wu et al., 2016, Appl. Microbiol. Biotechnol., 100: 4969-4983) can be used. In addition, Acidomonas methnolica, Bacillus coagulans, Beggiatoa alba, Desulfitobacterium hafniense, Desulfotomaculum kuznetsovii, Lysinbacillus fusiformis, Lysinibacillus sphaericus, Methylobacterium lusitanum, Methylobacterium oryzae, Methylobacterium salsugi, etc. As the NADP ⁺ -dependent enzyme, an enzyme derived from Thermococcus hydrothermalis (Antonie et al., 1988, Eur. J. Biochem., 264: 880-889) can be used. In the present invention, a NAD ⁺ -dependent enzyme is preferable, and an enzyme called an activator protein, which does not require a partner protein necessary for improving the enzyme activity and exhibits the oxidizing activity of methanol by itself, is more preferable.

Specifically, as the oxidation reaction of methanol using the enzyme, for example, when NAD ^{+ -dependent methanol dehydrogenase is used, NAD +} is reduced to NADH by the oxidation reaction of methanol to formaldehyde. So, in order to maintain the oxidative-reduction balance of NADH and NAD ^{+ in the cell, it is necessary to oxidize the generated NADH to NAD +} , and 5,10-methylene THF is 5-methyltetrahydrofolic acid (5-methylTHF). NADH can be oxidized ^{to NAD +} by the reaction converted to. When other enzymes are used, it is preferable to separately design a pathway for reducing ^{NAD + to NADH.}

In addition, NADP ⁺ -dependent enzymes generate NADPH with the oxidation of methanol, so it is necessary to design a pathway for oxidizing ^{NADPH to NADP +.} Alternatively, when using PQQ-dependent methanol dehydrogenase, it is preferable to use a strain in which the host strain can produce (or have been genetically modified to produce PQQ).

Among the enzymes that oxidize methanol to formaldehyde, the methanol oxidase may be any alcohol oxidase that oxidizes methanol to formaldehyde. The existing one is not particularly limited, and for example, an exotic one may be introduced even if it is originally endogenous to the cell. Specifically, Aspergillus ochraceus, Aspergillus terreus, Byssochlamys spectabilis, Candida boidinii, Candida methanosorbosa, Candida sithepensis, Komagataella pastoris (Pichia pastoris), Ogataea angusta (Hansenula polymorpha), Ogataea angusta (Hansenula polymorpha), Ogataea Examples include enzymes derived from microorganisms such as chrysogenum, Penicillium purpurascens, Phanerochaete chrysosporium, Phlebiopsis gigantea, Pichia putida, Poria contigua, Thermoascus aurantiacus. When methanol oxidase is used, oxygen is required as an electron receptor, and a strain that can convert hydrogen peroxide generated in the reaction into water, or a strain that has been metabolically modified so that it can be converted. Is preferable.

As the enzyme that oxidizes methanol to formaldehyde, an enzyme having a high activity of oxidizing methanol to formaldehyde is preferable, and for example, an enzyme selected by evaluating the activity in advance according to a known technique may be used.

When a foreign substance is used as the methanol dehydrogenase or methanol oxidase described above, the combination of the derived cell and the cell to be introduced is not particularly limited as long as it can be introduced by a known gene transfer method or the like.

The reaction conditions for methanol dehydrogenase and methanol oxidase can be appropriately selected and set according to known techniques according to the type of cell and enzyme used in the embodiment. For example, the cells may be cultured at preferably 30 to 45 ° C, more preferably 35 to 40 ° C, and even more preferably 36 to 38 ° C.

The transfer of a methyl group from a methyl group donor that has received a methyl group to homocysteine, for example, is endogenous to the organism if the methyl group donor that received the methyl group from formaldehyde is 5,10-methylene THF. This can be done by converting to 5-methyl THF with an enzyme such as 5,10-methylene THF reductase introduced from the outside, and then acting with an enzyme such as methionine synthase.

As a result, methionine is produced by transferring the methyl group from the added methanol to homocysteine via the methyl group donating precursor.

The conversion of produced methionine to SAM is carried out by the action of SAM synthase in the presence of ATP. Here, ATP may be added and used separately, and as described above, an appropriate energy source such as glucose is added to the reaction system based on adenine and ribose generated when SAM is decomposed into homocysteine. It may be resynthesized and used. As a method of resynthesizing ATP based on adenine and ribose, for example, ribose is converted to ribose 5-phosphate by the action of ribokinase endogenously or externally introduced into an organism, and phosphoribosylpyrrophosphate (PRPP). After conversion to PRPP by the action of a synthase, it is condensed with adenine by the action of adenine phosphoribosyl transferase and converted to adenosine monophosphate (AMP). Then, after converting AMP to adenosine diphosphate (ADP) by the action of adenylate kinase, various ATP synthesis reactions during glucose metabolism (eg, 1,3-bisphosphoglycerate by phosphoglycerate kinase) 3-Phosphoglycerate conversion reaction and phosphoenolpyruvate conversion reaction to pyruvic acid) can be used.

Thus, in the present invention, SAM can be regenerated by the functioning of methylase (methyltransferase) and methanol dehydrogenase or methanol oxidase, but any system (host) in which these enzymes are co-expressed can be regenerated. For example, SAM can be regenerated more preferably. It should be noted that these enzymes may be co-expressed by the endogenous ones, or may be co-expressed by introducing either one or both of them.

The specific host may be, for example, prokaryotic cells or isolated eukaryotic cells, preferably industrial microorganisms (preferably Escherichia coli, Corine, Bacillus, cyanobacteria, lactic acid bacteria, yeast, etc. Filamentous fungi, actinomycetes) cells, plant cells, insect cells or animal cells. Specific examples of industrial microorganisms include Escherichia, Cupriavidus, Pseudomonas, Thermos, Corynebacterium, Bacillus, Geobacillus, Synechocucus, Synechoccystis, Lactobacillus, Lactococcus, Bifidobacterium, Saccharomyces. , Pichia, Yarrowia, Kluyveromyces, Ogataea, Hansenula, Aspergillus, Rhizopus, Streptomyces and the like. Specifically, for example, Escherichia coli, Cupriavidus necator, Pseudomonas putida, Pseudomonas fluorescens, Thermophilus, Corynebacterium glutamicum, Bacillus subtilis, Geobacillus stearothermophilus, Synechococcus sp. , Bifidobacterium longum, Saccharomyces cerevisiae, Shizosaccharomyces pombe, Komagataella pastoris (Pichia pastoris), Yarrowia lipolytica, Kluyveromyce lactis, Ogataea angusta (Hansenula polymorpha), Ogataea angusta (Hansenula polymorpha), Ogataea methanolica, Ogataea minuta, Aspergill However, Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, Bacillus subtilis, Lactobacillus plantarum, and Lactococcus lactis are preferred.

In addition, these hosts are modified so that the amount of methionine or SAM (also referred to as the abundance or production amount of methionine or SAM) is increased from the viewpoint of increasing the amount of SAM itself or the raw material used for the recycling reaction. It may be. Specifically, for example, it may be modified to improve the intracellular methionine or SAM synthesis activity or the SAH decomposition activity to homocysteine, or it may be modified to reduce the formaldehyde decomposition activity. It may be, for example, a modification that overexpresses a methionine or SAM synthase, a mutation that improves the function of the methionine or SAM synthase, or an enzyme or protein that suppresses the expression of the methionine or SAM synthase gene. Mutations that suppress the function, mutations that improve the function of enzymes that suffer feedback inhibition in the synthesis of methionine or SAM, mutations that improve the function of SAH degrading enzymes, mutations that suppress the function of formaldehyde degrading enzymes, etc. were introduced. It may be a thing. In addition, as a method for improving the synthetic activity of methionine or SAM, a method of introducing a mutation that deletes the function of a master regulator protein (for example, MetJ) of the synthetic pathway of methionine or SAM, or a production reaction in the synthetic pathway is used. A method of overexpressing the gene of the enzyme to be promoted, a method of replacing the gene of an enzyme (for example, MetA, CysE) that suffers from feedback inhibition among the enzymes related to the synthetic pathway with a feedback inhibitory resistance enzyme gene, a method of replacing the gene of the feedback inhibitory enzyme gene. Examples include, but are not limited to, methods of overexpressing. Examples of the method for improving the activity of decomposing SAH into homocysteine include the above-mentioned method for improving the activity of the enzyme group that hydrolyzes SAH (for example, LuxS, Mtn). In addition, as a method for reducing the decomposition activity of formaldehyde, a method for reducing the activity of formaldehyde dehydrogenase (for example, FdhA) which directly converts formaldehyde into formic acid, and a method for combining formaldehyde with glutathione and converting it into formic acid. Decreases the activity of any of S-hydroxymethylglutathione synthase (eg Gfa), S-hydroxymethylglutathione dehydrogenase (eg FrmA, ADH5, AdhC), S-formylglutathione hydrolase (eg FrmB, ESD, FghA) There is a method of making it.

When introducing a foreign substance as the enzyme into these hosts, genetic engineering techniques known per se can be applied. For example, a method of introducing an expression vector containing the desired nucleic acid can be mentioned. The desired nucleic acid is treated with an appropriate restriction enzyme, cleaved at a specific site, mixed with the similarly treated plasmid DNA, and recombined with ligase. The method of introducing the plasmid DNA thus obtained is used. Alternatively, the desired plasmid DNA obtained by ligating an appropriate linker to the target gene and inserting it into the multicloning site of the plasmid suitable for the target may be used. In addition, a method such as Giboson assembly, in-fusion, or Gene-art that seamlessly clones using a homologous sequence of linearized plasmid DNA and a desired nucleic acid may be used. A known promoter region or repressor region can be incorporated into this plasmid, if desired, as long as it can be expressed in cells. For example, in the present invention, an expression vector in which a methylase (methyltransferase) and a gene for methanol dehydrogenase or methanol oxidase is inserted can be used. In addition to the above genes, an expression vector incorporating a gene into which a mutation that improves intracellular methionine or SAM synthesis activity or SAH degradation activity into homocysteine can be further used can also be used. For example, in addition to methylase (methyltransferase) and methanol dehydrogenase or methanol oxidase, MetA or CysE feedback-inhibiting resistant enzymes, plasmid DNA incorporating some or all of these genes can be mentioned. In addition, regulatory factors such as MetJ that suppresses the expression of enzyme genes in the synthetic pathway of methionine and SAM, plasmids for disrupting formaldehyde-degrading enzyme genes, and endogenous genes such as MetA and CysE are feedback-inhibited resistant enzymes. A plasmid for replacing with a functionally modified gene such as a gene can also be introduced separately.

The method for introducing the obtained plasmid DNA into the host is not particularly limited as long as it is an introduction method capable of introducing the plasmid DNA into the host cell and expressing the target gene in the host cell, and is appropriately selected depending on the species of the host cell. Any known method may be used. For example, the heat shock method, the electroporation method, the lithium acetate method, the calcium chloride method, the lipofection method and the like can be exemplified.

The host cells thus obtained are cultured under conditions suitable for the species of the host cells, and the SAM recycling method of the present invention can be carried out if at least methanol is present at that time. Specifically, for example, when Escherichia coli is used as a host cell, methanol 1 mM to 10 M, ATP 1 to 100 mM or glucose 1 mM to 1 M, Tris-HCl 1 to _{100 mM, DDL 4} 0.1 to 100 mM are used in a known cell culture medium. May be added and cultured. When SAM is used as a methyl group donor for the methylation reaction of a substance, the host cell itself, cell extract, culture solution or supernatant thereof, or a mixture thereof is used as the methyl of the object to be methylated. It can be mixed and used in a chemical reaction system.

Therefore, the present invention also provides a method for producing a methylated product, which comprises using the SAM regenerated by the recycling method of the present invention as a methyl group donor to carry out a methylation reaction. The object to be methylated is not particularly limited, and examples thereof include nucleic acids, proteins, phospholipids, hormones, neurotransmitters, catechols, polyphenols such as flavonoids and coumarins, and alkaloids. These methylation reactions can be carried out according to known techniques.

In this way, in a system in which both methylase (methyltransferase) and an enzyme that oxidizes methanol to methanol, such as methanol dehydrogenase or methanol oxidase, function, SAM is repeated by using inexpensive methanol as a source of methyl groups. It was first discovered by the inventors of the present application that it can be used, methylation of various substances can be efficiently carried out, and it is also useful in industry. Such techniques can be suitably carried out in fields such as pharmaceutical production, chemical production, and functional compound production. For example, polyphenols are known to have various useful effects on the human body, but unmethylated substances have the property of being difficult to be absorbed by the living body and easily excreted from the body. On the other hand, it is known that methylation promotes absorption into a living body and improves its effectiveness in the living body. In addition, many cases have been found in which new functionality is imparted by methylation. For example, resveratrol, which has an anti-aging effect, has been found to have new functions such as an effect of suppressing an increase in blood glucose level by methylation. By applying the present invention, it becomes possible to efficiently carry out the methylation reaction of various polyphenols, so that it is possible to produce pharmaceuticals and functional foods having improved absorbability, efficacy and functionality in vivo. It will be possible and further market expansion can be expected. In addition to the above-mentioned applications for methylating existing substances to improve their functionality, this technology can also be used as a technology for fermenting and producing existing methylated compounds. For example, vanillin, the main component of vanilla scent, is used as a fragrance and perfume and has a market of hundreds of millions of dollars. Fermentation production using sugar as a raw material, which enables stable supply, requires a methylation reaction using SAM, and the application of this technology enables efficient fermentation production. It can also be used for fermentative production of opiates (narcotic analgesics), which are very important as pharmaceuticals. Morphine and codeine are opioids obtained by extraction from poppy seeds, but since they are made from natural products, stable production and distribution are regarded as a problem. On the other hand, in recent years, opioids have been fermented and produced using Escherichia coli (see A Nakagawa et al., Nat Commun, 2016, 7, 10390) and yeast (see S. Galanie et al., Science, 2015, 349, 1095-1100). Attempts have been made. The opioid synthesis pathway requires multiple SAM-based methylation reactions. At present, only a few mg / L of opioids can be obtained in fermentative production, and it has not been put into practical use. If fermented opioid production can be put to practical use by applying this technology, it will be possible to enter the huge opioid market of tens of billions of dollars and to supply stable opioids that are not affected by climate change. On the other hand, in the production of substances using such SAM-dependent methylase, it is not realistic to directly add extremely expensive SAM. In addition, the method of adding methionine, which is a SAM precursor, as in the existing technology, can promote the methylation reaction, but its effect is limited because the ATP required for the conversion of methionine to SAM is deficient. Is. Like SAM, ATP is also expensive and its addition is still impractical. Therefore, this technology, which can be used for the methylation reaction by recycling SAM only by adding inexpensive methanol and further adding an energy source such as glucose, is more cost effective than the existing technology. It can be said that it has an advantage.

Hereinafter, the present invention will be described in detail with reference to Examples, but these Examples are examples of the present invention, and the present invention is not limited thereto.

Preparation Example 1
(1) plasmid construction Table 1 shows a list of primer sequences used in this study (all obtained from IDT). Escherichia coli TG1 strain manufactured by Zymo Research was used as an Escherichia coli host for plasmid construction. Prior to constructing a co-expression plasmid for O-methyltransferase (OMT) and methanol dehydrogenase (MDH), pRCI (Ninh PH, et al) enables heat-induced gene expression using the Lambda PR promoter and CI857 repressor. , Biotechnol Bioeng., 2015, 112, 189-196)). Using pRCI as template DNA, PCR was performed using primers pRCI-MCS_F and pRCI-MCS_R, digested with SpeI, and then self-ligated to construct pRCI-MCS. Using the constructed pRCI-MCS multi-cloning site, OMT and MDH genes were introduced. OMT (SaOMT5) gene (NCBI accession number NC_003155) (Yoon Y, et a., J Microbiol Biotechnol, 2010,) derived from Streptomyces avermitilis MA-4680, which was codon-optimized for Escherichia coli by IDT's gblocks artificial gene synthesis contract. 20, 1359-136) was amplified by PCR using primers SaOMT5_F and SaOMT5_R. The SaOMT5 gene is a SAM-dependent OMT. Then, after digestion with SpeI and SacI, it was inserted into the same site of pRCI-MCS to obtain pRCI-SaOMT5. Similarly, the triple mutant enzyme genes of A26V, A31V, and A169V of MDH (CnMDH2) (GenBank accession number CP002878) derived from Cupriavidus necator N-1 (Wu TY, et al., Appl Microbiol Biotechnol, 2016, 100, 4969) -4983) was codon-optimized and artificially synthesized. This enzyme is a mutant enzyme that improves the availability of methanol for ^{NAD + -dependent alcohol dehydrogenase.} The gene was amplified by PCR using primers CnMDH2_F and CnMDH2_R, digested with SacI and XhoI, and then inserted into the same site of pRCI-SaOMT5 to obtain pRCI-SaOMT5-CnMDH2.

(2) Induction of enzyme expression The various plasmids constructed were introduced into the E. coli BW25113 strain (provided by the National Institute of Genetics). The obtained recombinant Escherichia coli was aerobically cultured at 30 ° C. using Luria-Bertani medium containing 100 μg / mL ampicillin, and then 10% (v / v) was added to the M9 minimum medium. After aerobically culturing at 30 ° C., which is a non-inducible condition, for 2 hours, the expression of SaOMT5 and CnMDH2 was induced by shifting to 42 ° C. After culturing at 42 ° C. for 4 hours, the cells were collected by centrifugation, washed twice with 50 mM Tris-HCl buffer (pH 8.0), and then used for enzyme activity measurement and methylation reaction experiment.

(3) Measurement of activity of foreign enzyme The in vivo activity of OMT and MDH was measured using the prepared Escherichia coli cells. The activity of SaOMT5 is measured in a reaction solution consisting of 0.2 mM escretin or protocatechuic acid, 1 mM SAM, 50 mM Tris-HCl buffer (pH 8.0), 10 mM DDL ₄ and an appropriate amount of Escherichia coli cells. The methylated form of protocatechuic acid was quantified by HPLC. The separation column is a Nacalai Tesque 5C ₁₈ AR-II column (4.6 ID x 250 mm), and the eluate is a 20% (v / v) acetonitrile solution containing 5% (v / v) formic acid. Using. The flow rate was maintained at 1 mL / min, the column temperature was maintained at 40 ° C., and the eluate was monitored at 254 nm. The activity of CnMDH2 was measured in a ^{reaction solution consisting of 1M methanol, 1mM NAD +} , 50mM Tris-HCl buffer (pH 8.0), 10mM DDL ₄ and an appropriate amount of E. coli cells, and at 340 nm associated with the formation of NADH. The increase in absorbance was monitored. The molar extinction coefficient of NADH at the same wavelength of 6.2 mM ^-1 cm ^-1 was used to calculate the reaction rate. The reaction was carried out at 37 ° C., and the activity of each enzyme was defined as 1 unit (U) in an amount that catalyzes the production of 1 μmol of product per minute under the present measurement conditions.

(4) Confirmation of endogenous enzyme activity Using the prepared Escherichia coli cells, methionine synthase, 5-methylTHF homocysteine transmethylase, methylene THF reductase, SAH nucleosidase, S-ribosylhomo, which are Escherichia coli endogenous enzymes. The activity of cysteine nucleosidase was confirmed, and the presence or absence of spontaneous condensation reaction between formaldehyde and THF was confirmed. As a basic composition, use a reaction solution consisting of 0.2 mM escretin, 50 mM Tris-HCl buffer (pH 8.0), 1 mM ATP, 10 mM DDL ₄ and an appropriate amount of Escherichia coli cells, and use 1 mM methionine to confirm the activity of methionine synthase. To confirm the activity of 5-methylTHF homocysteine transmethylase and methylene THF reductase, add 1 mM 5,10-methyleneTHF, 1 mM homocysteine, 1 mM NADH, to confirm the spontaneous condensation reaction of formaldehyde and THF. Add 1 mM formaldehyde, 1 mM THF, 1 mM homocysteine, 1 mM NADH, and add 1 mM formaldehyde, 1 mM THF, 1 mM SAH, 1 mM NADH to confirm the activity of SAH nucleosidase and S-ribosyl homocysteine lyase. The methylated product was quantified by HPLC according to the method described in the previous section (3).

(5) Methylation reaction using the SAM recycling route An esculetin methylation reaction was carried out using the prepared Escherichia coli cells. The methylation reaction consists of 1 mM escretin as a substrate, 1 M methanol as a source of methyl groups, 50 mM Tris-HCl buffer (pH 8.0), 1 mM ATP, 10 mM DDL ₄ , and 20 mg-wet cell / mL E. coli cells. It was carried out in the reaction solution. The reaction was carried out at 37 ° C. for 18 hours, and the produced scopoletin and isoscopoletin were quantified by HPLC according to the method described in (3) above. In addition, since this methylation reaction utilizes viable cells of Escherichia coli, ATP is self-sustaining by the action of endogenous enzymes such as glycolytic enzymes and adenosine salvage synthesis system without adding ATP from the outside. There is a possibility that it can be produced in. Therefore, we also conducted an experiment in which 10 mM glucose was added instead of ATP.

Example 1 Evaluation of activity of foreign enzyme
The activities of OMT and MDH were measured using pRCI-SaOMT5-CnMDH2 or E. coli BW25113 introduced with the empty vector pRCI (hereinafter referred to as pRCI-SaOMT5-CnMDH2 / BW25113 and pRCI / BW25113). The enzyme activity was evaluated as the activity per cell, assuming that the methylation reaction using the SAM recycling route was finally carried out using live cells. In the methylation reaction by OMT, scopoletin, which is a methylated form of the hydroxyl group at the 6-position, and isoscopoletin, which is a methylated form of the hydroxyl group at the 7-position, are produced from escretin, and vanillic acid and isovanillic acid are produced from protocatechuic acid. Uru (shown below).

As a result of performing a methylation reaction using pRCI-SaOMT5-CnMDH2 / BW25113 using esculetin as a substrate and SAM as a co-substrate, scopoletin, which is a methylated form of the hydroxyl group at the 6-position of esculetin, and a methylated form of the hydroxyl group at the 7-position. The production of isoscopoletin was confirmed by HPLC, and the activity value of OMT was 5.9 mU / mg-wet cell (Fig. 4). On the other hand, the negative control pRCI / BW25113 did not produce scopoletin or isoscopoletin. When protocatechuic acid was used as the substrate, the production of vanillic acid and isovanillic acid was confirmed in pRCI-SaOMT5-CnMDH2 / BW25113.

Subsequently, MDH activity was measured using methanol as a substrate and NAD ^{+ as a co-substrate.} In pRCI-SaOMT5-CnMDH2 / BW25113, an increase in NADH was observed with the oxidation of methanol, and the MDH activity value was 4.7 mU / mg-wet cell (Fig. 4). On the other hand, in pRCI / BW25113, NADH production hardly occurred, and the MDH activity value was 0.08 mU / mg-wet cell. From the above results, it can be said that the functional expression of SaOMT5 and CnMDH2 was successful.

Example 2 Confirmation of endogenous enzyme activity
Using pRCI-SaOMT5-CnMDH2 / BW25113, the activity of Escherichia coli endogenous enzyme and the spontaneous condensation reaction of formaldehyde and THF were confirmed. The results are shown in FIG. The production of scopoletin and isoscopoletin was also observed when methionine was used as a starting material, confirming that methionine synthase has activity. In addition, 5-methylTHF homocysteine transmethylase and methylene THF reductase are active because the production of methylated substances was confirmed even when 5,10-methyleneTHF, homocysteine, and NADH were used as starting substances. I was able to confirm that. Furthermore, since the production of methylated products was confirmed using formaldehyde, THF, homocysteine, and NADH as starting materials, it was confirmed that the spontaneous condensation reaction between formaldehyde and THF was proceeding. Regarding SAH nucleosidase and S-ribosyl homocysteine lyase, the production of methylated compounds was confirmed using formaldehyde, THF, SAH, and NADH as starting materials, and the activity was confirmed.

Example 3 Methylation reaction using the SAM recycling route Subsequently, a methylation reaction of esculetin accompanied by recycling of SAM was carried out using methanol as a methyl group donor. The results are shown in FIG. Since pRCI / BW25113 does not have OMT activity, no production of scopoletin or isoscopoletin was observed. In pRCI-SaOMT5 / BW25113, which expresses only OMT, the production of 0.055 mM methylated compound was observed in 18 hours. From this result, it was suggested that the methylation reaction of esculetin was carried out by using SAM in which SaOMT5 is present in a trace amount in the cell. On the other hand, in pRCI-SaOMT5-CnMDH2 / BW25113, production of 0.17 mM methylated compound was observed in 18 hours, and a larger amount of methylated compound could be produced than the single expression strain of SaOMT5. From this result, it was shown that the co-expression of SaOMT5 and CnMDH2 functions the SAM recycling pathway using methanol as a methyl group source.

Subsequently, it was examined whether esculetin methylation reaction accompanied by SAM recycling is possible by adding glucose instead of ATP. As shown in FIG. 7, although the amount of scopoletin and isoscopoletin produced was lower than that when ATP was added, it was confirmed that the reaction proceeded even when glucose was used. The amount of scopoletin and isoscopoletin produced was 0.085 mM in 18 hours.

Preparation Example 2
In addition to the temperature-induced expression system described above, the E. coli BL21 (DE3) strain was used to confirm whether the IPTG-induced expression system commonly used in Escherichia coli can enhance the methylation reaction using the SAM recycling pathway. Escherichia coli methylation reaction was carried out using.

(1) plasmid construction A list of primer sequences used in Table 2 (all obtained from IDT) is shown. The SaOMT5 gene used in Preparation Example 1 was amplified by PCR using primers SaOMT5_F2 and SaOMT5_R2. Then, after digestion with NcoI and HindIII, it was inserted into the same site of pETDuet1 (Novagen) to obtain pETDuet-SaOMT5. Next, the triple mutant enzyme genes of A26V, A31V, and A169V of CnMDH2 used in Preparation Example 1 were amplified by PCR using primers CnMDH2_F2 and CnMDH2_R2. Then, after digestion with NdeI and XhoI, it was inserted into the same site of pETDuet-SaOMT5 to obtain pETDuet-SaOMT5-CnMDH2.

(2) Induction of enzyme expression The various plasmids constructed were introduced into the E. coli BL21 (DE3) strain (Merck Millipore). The obtained recombinant Escherichia coli was aerobically cultured at 37 ° C. using Luria-Bertani medium containing 100 μg / mL ampicillin, and then 5% (v / v) was added to the M9 minimum medium. After culturing aerobically for 2 hours, the expression of SaOMT5 and CnMDH2 was induced by adding IPTG so that the final concentration was 0.2 mM. After culturing for 3 hours, the cells were collected by centrifugation, washed twice with 50 mM Tris-HCl buffer (pH 8.0), and then used for a methylation reaction experiment.

(3) Methylation reaction using the SAM recycling route An esculetin methylation reaction was carried out using the prepared Escherichia coli cells. The methylation reaction was carried out in a reaction solution consisting of 0.5 mM esculetin as a substrate, 50 mM Tris-HCl buffer (pH 8.0), 1 mM ATP, 10 mM DDL ₄ , and 10 mg-wet cell / mL E. coli cells. For methanol, which is a source of methyl groups, the reaction was carried out by varying the concentration between 0 and 200 mM. The reaction was carried out at 37 ° C. for 24 hours, and the produced scopoletin and isoscopoletin were quantified by HPLC according to the method described in Preparation Example 1.

Example 4 Methylation reaction using the SAM recycling route Using methanol as a methyl group donor, an esculetin methylation reaction accompanied by SAM recycling was carried out. The results are shown in FIG. Since pETDuet / BL21 (DE3) does not have OMT activity, no production of scopoletin or isoscopoletin was observed. In pETDuet-SaOMT5 / BL21 (DE3), which expresses only OMT, the formation of 0.084 mM methylated compound was observed in 24 hours under the condition of 100 mM methanol, but the difference in product concentration due to the change in methanol concentration was observed. I couldn't. On the other hand, in pETDuet-SaOMT5-CnMDH2 / BL21 (DE3), the production of methylated compounds increased as the methanol concentration increased up to a methanol concentration of 100 mM, and the methanol-dependent SAM recycling system was functioning. Shown. Under the condition of 100 mM methanol, 0.22 mM methylated compound was produced in 24 hours, and a larger amount of methylated compound could be produced than the single expression strain of SaOMT5. From this result, it was shown that the SAM recycling pathway using methanol as a methyl group source functions by co-expression of SaOMT5 and CnMDH2 even in the induced expression system using IPTG.

Preparation Example 3
As described above, it was found that the co-expression of SaOMT5 and CnMDH2 can drive the SAM recycling pathway using methanol as a methyl group source, and the methylation reaction can be carried out efficiently. Therefore, by strengthening the SAM synthesis pathway of the host strain, further improvement in its efficiency can be expected. FIG. 9 shows, by way of example, the synthetic pathway of methionine and SAM in E. coli. SAM is produced using aspartic acid and serine as starting substances, but many enzyme genes in the pathway are suppressed by the MetJ protein, which is a master regulator. Therefore, it is considered that the disruption of the metaJ gene releases the suppression of intracellular SAM synthesis gene expression and increases the amount of SAM synthesis. As a result, it is expected that the amount of SAM used for recycling and methylation reactions will increase, so we decided to disrupt the metJ gene. In addition, since formaldehyde converted from methanol by methanol dehydrogenase is toxic to cells, microorganisms originally have a decomposition route thereof. Therefore, by suppressing the decomposition of formaldehyde, it is expected that the amount of formaldehyde used for recycling SAM shown in FIG. 2 will increase. Therefore, we decided to verify the effect of disrupting the frmA gene, which encodes S-hydroxymethylglutathione dehydrogenase, which is one of the enzymes of the formaldehyde decomposition pathway.

(1) Constructing a plasmid for disrupting the metJ and frmA genes
The CRISPR-Cas9 system was used to disrupt the metJ and frmA genes (see Jiang Y, et al., Appl. Environ. Microbiol., 2015, 81, 2506-2514). The plasmid pCas expressing Cas9 nuclease and λ recombinant enzyme and the plasmid pTargetF for expressing single-guide RNA (sgRNA) and inserting the template sequence for homologous recombination were purchased from addgene (Plasmid # 62225 and Plasmad # 62226). .. Table 3 shows a list of primer sequences used (all obtained from IDT). Using pTargetF as template DNA, PCR was performed using primers sgRNA (metJ) _F and pTargetF_R, digested with SpeI, and then self-ligated to insert sgRNA for metaJ gene disruption into pTargetF, pTargetF-metJ. (sgRNA) was constructed. Next, from the genome of the Escherichia coli BL21 (DE3) strain, the upstream 448 bp of the metJ gene was amplified using the primers metJ up_F and metJ up_R, and the downstream 549 bp was amplified using the primers metJ down_F and metJ down_R. Then, by overlapping PCR using primers metJ up_F and metJ down_R, a fragment in which the upstream and downstream regions of the metJ gene were linked was prepared, digested with HindIII and XhoI, and then transferred to the same site of pTargetF-metJ (sgRNA). It was inserted to obtain pTargetF-ΔmetJ. Similarly, pTargetF-metJ (sgRNA) was obtained by using primers sgRNA (frmA) _F and pTargetF_R, and pTargetF-ΔfrmA was obtained by using primers frmA up_F and frmA up_R, and frmA down_F and frmA down_R.

(2) Disruption of metJ and frmA genes First, pCas expressing Cas9 nuclease and λ recombinant enzyme was introduced into the E. coli BL21 (DE3) strain. The obtained recombinant Escherichia coli was cultured in an arabinose-added medium, and pTargetF-ΔmetJ or pTargetF-ΔfrmA was introduced in a state where the expression of λ-recombinant enzyme was induced. By this step, only strains lacking the metJ or frmA gene can form colonies. The obtained gene-disrupted strain was cultured in a medium supplemented with IPTG to induce the expression of sgRNA that recognizes the sequence on TargetF encoded on pCas, and pTargetF was shed. Subsequently, using the temperature sensitivity of pCas, Escherichia coli was cultured at 37 ° C. at which pCas could not be replicated, thereby causing pCas to be shed. A double-disrupted strain of metaJ frmA of E. coli BL21 (DE3) strain was also constructed by the same method. Then, pETDuet-SaOMT5-CnMDH2 prepared in Preparation Example 2 was introduced into the E. coli BL21 (DE3) strain and its ΔmetJ strain, ΔfrmA strain, and ΔmetJ ΔfrmA strain, and Luria-Bertani medium containing 100 μg / mL ampicillin was introduced. After aerobically culturing at 37 ° C., 5% (v / v) was added to the M9 minimum medium. After culturing aerobically for 2 hours, the expression of SaOMT5 and CnMDH2 was induced by adding IPTG so that the final concentration was 0.2 mM. After culturing for 3 hours, the cells were collected by centrifugation, washed twice with 50 mM Tris-HCl buffer (pH 8.0), and then used for a methylation reaction experiment.

(3) Methylation reaction using the SAM recycling route An esculetin methylation reaction was carried out using the prepared Escherichia coli cells. Methylation reaction is performed from 0.5 mM escretin as a substrate, 100 mM methanol as a methyl group source, 50 mM Tris-HCl buffer (pH 8.0), 1 mM ATP, 10 mM DDL ₄ , and 10 mg-wet cell / mL of Escherichia coli cells. It was carried out in the reaction solution. The reaction was carried out at 37 ° C. for 18 hours, and the produced scopoletin and isoscopoletin were quantified by HPLC according to the method described in Preparation Example 1.

Example 5 Methylation reaction using various gene-disrupted strains The effect of gene disruption on the host strain on the methylation reaction of esculetin was examined. The results are shown in FIG. While pETDuet-SaOMT5-CnMDH2 / BL21 (DE3) produced 0.21 mM methylated compound in 18 hours, pETDuet-SaOMT5-CnMDH2 / ΔmetJ showed an increase in product volume of 0.26 mM in 18 hours. This suggests that disruption of the metJ gene releases the suppression of gene expression in the methionine (SAM) synthetic pathway and increases the amount of intracellular SAM pooled, thereby improving the efficiency of the methylation reaction. It was. On the other hand, with pETDuet-SaOMT5-CnMDH2 / ΔfrmA, the amount of product decreased to 0.19 mM in 18 hours. It is considered that this is because the decomposition of formaldehyde is suppressed by the destruction of frmA, but the regeneration rate of SAM is not sufficient, and formaldehyde, which is toxic to cells, is accumulated. Therefore, it was considered that the effect of frmA destruction could be sufficiently obtained by destroying frmA in ΔmetJ where the reproduction speed of SAM was improved. As a result, pETDuet-SaOMT5-CnMDH2 / ΔmetJ ΔfrmA produced the largest amount of methylated product, 0.30 mM in 18 hours. From the above results, it was shown that the SAM recycling pathway using methanol as a methyl group source functions more efficiently by strengthening the methionine (SAM) synthesis pathway and blocking the formaldehyde decomposition pathway.

Preparation Example 4
Other methods for enhancing the methionine and SAM synthetic pathways include overexpressing inhibitory resistant enzymes with respect to enzymes in the pathway that undergo feedback inhibition. In the synthetic pathway for methionine and SAM in FIG. 9, serine-O-acetyltransferase (CysE), which converts serine to O-acetylserine, is converted to cysteine, and homoserine is converted to O-succinyl homoserine, which is homoserine-O-succinyl transferase (MetA). ) Is feedback-inhibited by SAM. Therefore, by overexpressing the genes encoding the feedback resistance inhibitory enzymes of CysE and MetA (denoted as cysE (fdr) and metaA (fdr)) in ΔmetJΔfrmA, the amount of SAM synthesis was further improved, and SAM recycling and methylation were performed. It is expected that the amount of SAM used for conversion will increase, and verification was conducted.

(1) Construction of plasmid for overexpression of cysE (fdr) and metaA (fdr) The list of primer sequences used in Table 4 (all obtained from IDT) is shown. MetA feedback inhibition resistance mutant (R27C, I296S, P298L) gene (see Usuda and Kurahashi, Appl. Environ. Microbiol., 2005, 71, 3228-3234) was artificially synthesized by IDT's gblocks artificial gene synthesis contract. It was amplified by PCR using the primers metaA-fdr_F and metaA-fdr_R. Then, after digestion with NcoI and HindIII, it was introduced into the same site of pACYCDuet-1 (Novagen) to obtain pACYCDuet-metA (fdr). Similarly, the feedback inhibition resistance mutant (V95R, D96P) gene of CysE (see Kai et al., Protein Eng. Des. Sel., 2006, 19, 163-167) was artificially synthesized and combined with the primer cysE-fdr_F. Amplified by PCR using cysE-fdr_R. Then, after digestion with NdeI and XhoI, it was introduced into the same site of pACYCDuet-1 and pACYCDuet-metA (fdr) to obtain pACYCDuet-cysE (fdr) and pACYCDuet-metA (fdr) -cysE (fdr).

(2) Induction of enzyme expression The various plasmids constructed were introduced into pETDuet-SaOMT5-CnMDH2 / ΔmetJ or pETDuet-SaOMT5-CnMDH2 / ΔmetJ ΔfrmA. The obtained recombinant Escherichia coli was aerobically cultured at 37 ° C. using Luria-Bertani medium containing 100 μg / mL ampicillin and 17 μg / mL chloramphenicol, and then 5% (v /) in M9 minimum medium. v) Added. After aerobic culturing for 2 hours, the expression of SaOMT5, CnMDH2, MetA (fdr), and CysE (fdr) was induced by adding IPTG to a final concentration of 0.2 mM. After culturing for 3 hours, the cells were collected by centrifugation, washed twice with 50 mM Tris-HCl buffer (pH 8.0), and then used for a methylation reaction experiment.

(3) Methylation reaction using the SAM recycling route An esculetin methylation reaction was carried out using the prepared Escherichia coli cells. Methylation reaction is performed from 0.5 mM escretin as a substrate, 100 mM methanol as a methyl group source, 50 mM Tris-HCl buffer (pH 8.0), 1 mM ATP, 10 mM DDL ₄ , and 10 mg-wet cell / mL of Escherichia coli cells. It was carried out in the reaction solution. The reaction was carried out at 37 ° C. for 18 hours, and the produced scopoletin and isoscopoletin were quantified by HPLC according to the method described in Preparation Example 1 described above.

Example 6 Methylation reaction using an overexpression strain of the feedback inhibitory resistance enzyme gene The effect of overexpression of the feedback inhibitory resistance enzyme gene on the methylation reaction of escretin in the host strain was investigated. The results are shown in FIG. The pETDuet-SaOMT5-CnMDH2 / ΔmetJ strain produced 0.26 mM methylated compound in 18 hours, whereas the strain overexpressing metA (fdr) showed an increase of 0.32 mM in 18 hours. In addition, the strain that overexpressed cysE (fdr) also showed an increase in the amount of methylated product produced at 0.31 mM in 18 hours. On the other hand, when both metA (fdr) and cysE (fdr) were overexpressed, a slight decrease was observed in the amount of methylated product produced. This strain is a strain that overexpresses four genes, SaOMT5, CnMDH2, metaA (fdr), and cysE (fdr), and the amount of each enzyme produced is reduced because the strain is overloaded. I thought it might be. Moreover, when the effect of overexpression of metA (fdr) was examined in the pETDuet-SaOMT5-CnMDH2 / ΔmetJ ΔfrmA strain, the concentration of the methylated compound increased from 0.30 mM to 0.33 mM. From the above results, it was shown that the expression of the feedback inhibition resistant enzyme causes the SAM recycling pathway using methanol as a methyl group source to function more efficiently.

Preparation Example 5
It is considered that the recycling of SAM in the present invention can be used in principle with any enzyme that oxidizes methanol to formaldehyde. Therefore, the verification was carried out using an enzyme derived from Bacillus methanolicus (BmMDH3) as a methanol dehydrogenase derived from other microorganisms and an enzyme derived from methanol-utilizing yeast Ogataea minuta (OmAOX1) as a methanol oxidase.

(1) plasmid construction A list of primer sequences used in Table 5 (all obtained from IDT) is shown. The methanol dehydrogenase 3 gene (Genbank accession number AIE60275.1) derived from Bacillus methanolicus was artificially synthesized by IDT's gblocks artificial gene synthesis contract, and amplified by PCR using primers BmMDH3_F and BmMDH3_R. Then, after digestion with NdeI and XhoI, it was introduced into the same site of pETDuet-SaOMT5 described in Preparation Example 2 to obtain pETDuet-SaOMT5-BmMDH3. Similarly, an alcohol dehydrogenase 1 gene (Genbank accession number AB242209.1) derived from Ogataea minuta was artificially synthesized and amplified by PCR using primers OmAOX1_F and OmMOX1_R. Then, after digestion with NdeI and XhoI, it was introduced into the same site of pETDuet-SaOMT5 described in Preparation Example 2 to obtain pETDuet-SaOMT5-OMAOX1.

(2) Induction of enzyme expression The various plasmids constructed in Preparation Example 4 and the pACYC Diet-metA (fdr) constructed in Preparation Example 4 were introduced into the Δmet J ΔfrmA strain constructed in Preparation Example 3. The obtained recombinant Escherichia coli was aerobically cultured at 37 ° C. using Luria-Bertani medium containing 100 μg / mL ampicillin and 17 μg / mL chloramphenicol, and then 5% (v /) in M9 minimum medium. v) Added. After culturing aerobically for 2 hours, the expression of SaOMT5, BmMDH3, OmAOX1, and CysE (fdr) was induced by adding IPTG to a final concentration of 0.2 mM. After culturing for 3 hours, the cells were collected by centrifugation, washed twice with 50 mM Tris-HCl buffer (pH 8.0), and then used for a methylation reaction experiment.

(3) Methylation reaction using the SAM recycling route An esculetin methylation reaction was carried out using the prepared Escherichia coli cells. Methylation reaction is performed from 0.5 mM escretin as a substrate, 100 mM methanol as a methyl group source, 50 mM Tris-HCl buffer (pH 8.0), 1 mM ATP, 10 mM DDL ₄ , and 10 mg-wet cell / mL of Escherichia coli cells. It was carried out in the reaction solution. The reaction was carried out at 37 ° C. for 18 hours, and the produced scopoletin and isoscopoletin were quantified by HPLC according to the method described in Preparation Example 1 described above.

Example 7 The results of the methylation reaction using various enzymes that oxidize methanol to formaldehyde are shown in FIG. The single expression strain of SaOMT5, pETDuet-SaOMT5 / pACYCDuet-metA (fdr) / ΔmetJ ΔfrmA, produced 0.077 mM methylated compound in 18 hours, whereas pETDuet-SaOMT5-BmMDH3 / pACYCDuet-metA (fdr) The product volume increased to 0.10 mM in 18 hours for the / ΔmetJ ΔfrmA strain and 0.17 mM in 18 hours for the pETDuet-SaOMT5-OmAOX1 / pACYCDuet-metA (fdr) / ΔmetJ ΔfrmA strain. From this result, it was shown that the recycling pathway of SAM in the present invention works by co-expressing methylase with an enzyme having an activity of oxidizing methanol to formaldehyde, regardless of the origin of the enzyme.

Preparation Example 6
It is considered that the enhancement of the methylation reaction by SAM recycling in the present invention can be used in principle with any enzyme as long as it is a SAM-dependent methylase. Therefore, it was verified whether it is possible to enhance the methylation reaction of compounds other than esculetin by co-expressing methanol dehydrogenase and various methylases.

(1) plasmid construction A list of primer sequences used in Table 6 (all obtained from IDT) is shown. IDT's gblocks artificial gene synthesis contracted catechol-O-methyltransferase (HsOMT; Genbank accession number NM_000754) derived from Homo sapiens, caffeate-O-methyltransferase (AtOMT; Genbank accession number AY062837.1) derived from Arabidopsis thaliana, Mycobacterium smegmatis-derived histidine-N-α-methyltransferase (EgtD; Genbank accession number ABK75457.1) was artificially synthesized and amplified by PCR using primers HsOMT_F and HsOMT_R, AtOMT_F and AtOMT_R, and EgtD_F and EgtD_R, respectively. Then, HsOMT and AtOMT were digested with SpeI and SacI, and then inserted into the same site of pRCI-MCS constructed in Preparation Example 1 to obtain pRCI-AtOMT and pRCI-HsOMT. Next, the CnMDH2 gene was excised by digesting pRCI-SaOMT5-CnMDH2 constructed in Preparation Example 1 with SacI and XhoI, inserted into the same site of pRCI-AtOMT and pRCI-HsOMT, and pRCI-AtOMT-CnMDH2 and pRCI. -HsOMT-CnMDH2 was obtained. EgtD was digested with NcoI and HindIII and then introduced to the same site of pETDuet to obtain pETDuet-EgtD. Next, by digesting pETDuet-SaOMT5-CnMDH2 constructed in Preparation Example 2 with NdeI and XhoI, the CnMDH2 gene was excised and inserted into the same site of pETDuet-EgtD to obtain pETDuet-EgtD-CnMDH2.

(2) Induction of enzyme expression The constructed pRCI-AtOMT, pRCI-AtOMT-CnMDH2, pRCI-HsOMT, and pRCI-HsOMT-CnMDH2 were introduced into Escherichia coli BW25113 strain. pETDuet-EgtD and pETDuet-EgtD-CnMDH2 were introduced into the metA (fdr) overexpressing strain of Escherichia coli BL21 (DE3) ΔmetJ ΔfrmA constructed in Preparation Example 4. The obtained transformant of BW25113 strain was aerobically cultured at 30 ° C. using Luria-Bertani medium containing 100 μg / mL ampicillin, and then 5% (v / v) was added to the M9 minimum medium. After aerobically culturing at 30 ° C., which is a non-inducible condition, for 3 hours, the expression of methylase and CnMDH2 was induced by shifting to 42 ° C., and further culturing was performed for 4 hours. For transformants of BL21 (DE3) strain, use Luria-Bertani medium containing 100 μg / mL ampicillin and 17 μg / mL chloramphenicol, aerobically cultivate at 37 ° C, and then use M9 minimal medium. 5% (v / v) was added. After culturing aerobically for 2 hours, the expression of EgtD, CnMDH2, and MetA (fdr) was induced by adding IPTG to a final concentration of 0.2 mM. After culturing for 3 hours, the cells were collected by centrifugation, washed twice with 50 mM Tris-HCl buffer (pH 8.0), and then used for a methylation reaction experiment.

(3) Methylation reaction using the SAM recycling route Methylation reaction of various substrates was carried out using the prepared Escherichia coli cells. AtOMT, an O-methyltransferase that methylates oxygen atoms, can be used, for example, to methylate caffeic acid to ferulic acid or isoferulic acid, or to methylate resveratrol to pinostylben or pterostilben. In the reaction, HsOMT, for example, methylates protocatechuic acid to vanillic acid and isoferulic acid (shown below). As an example of methylation other than oxygen atom, EgtD, which is an N-methyltransferase that methylates nitrogen atom, methylates histidine in three steps to produce hercinin. Methylation reaction was performed with 0.5 mM substrate for each enzyme, 100 mM methanol as a source of methyl groups, 50 mM Tris-HCl buffer (pH 8.0), 1 mM ATP, 10 mM DDL ₄ , and 10 mg-wet cell / mL Escherichia coli cells. It was carried out in a reaction solution consisting of. The reaction was carried out at 37 ° C. for 18 hours, and the produced methylated product was quantified by HPLC. Isoferulic acid and ferulic acid were quantified according to the method described in Preparation Example 1. Isovanillic acid and vanillic acid were analyzed by setting the content of acetonitrile in the mobile phase composition of the method described in Preparation Example 1 to 10% (v / v). .. For pinostilbene and pterostilbene, the content of acetonitrile in the mobile phase composition of the method described in Preparation Example 1 was set to 50% (v / v), and analysis was performed. For hercinin, a Nacalai Tesque 5C ₁₈ AR-II column (4.6 ID x 250 mm) was _{used as the separation column, and 10 mM Na 2} HPO ₄ was used as the eluate. The flow rate was maintained at 1 mL / min, the column temperature was maintained at 40 ° C., and the eluate was monitored at 210 nm.

Example 8 The results of the methylation reaction using various methylases are shown in FIG. Regarding the methylation reaction of caffeic acid, the strain expressing AtOMT alone produced 0.061 mM methylated compound in 18 hours, whereas the strain co-expressing with CnMDH2 showed an increase in product amount of 0.12 mM in 18 hours. Was done. Regarding the methylation reaction of resveratrol, the strain expressing AtOMT alone produced 0.040 mM methylated compound in 18 hours, whereas the strain co-expressing with CnMDH2 showed an increase in product amount of 0.051 mM. .. Regarding the methylation reaction of protocatechuic acid, the strain expressing HsOMT alone produced a methylated compound of 0.059 mM in 18 hours, whereas the strain co-expressing with CnMDH2 showed an increase in product amount of 0.15 mM. Regarding the methylation reaction of histidine, the strain expressing EgtD alone produced a methylated compound of 0.18 mM in 18 hours, whereas the strain co-expressing with CnMDH2 showed an increase in the amount of product, 0.29 mM. From the above results, the methylation reaction using the SAM recycling system in the present invention is effective regardless of the type of methylation reaction, and is expected to be applied to various methylation reactions.

Preparation Example 7
The enhancement of the methylation reaction efficiency by SAM recycling in the present invention can be used not only in Escherichia coli but also in any host. As an example, we verified whether the enhancement of the methylation reaction using the SAM recycling pathway works in Bacillus subtilis B. subtilis.

(1) plasmid construction A list of primer sequences used in Table 7 (all obtained from IDT) is shown. The SaOMT5 gene used in Preparation Example 1 was amplified by PCR using primers SaOMT5_F3 and SaOMT5_R3. Then, after digestion with EcoRV and SalI, pCU (see Okano et al., Appl. Microbiol. Biotechnol, 2007, 75, 1007-1013), which is a vector that can be replicated in various hosts such as Escherichia coli, Bacillus subtilis, and lactic acid bacteria. I inserted it in the same site and got pCU-SaOMT5. In addition, the triple mutant enzyme genes of A26V, A31V, and A169V of CnMDH2 used in Preparation Example 1 were amplified by PCR using primers CnMDH2_F3 and CnMDH2_R. Then, after digestion with SalI and XhoI, it was inserted into the same site of pCU-SaOMT5 to obtain pCU-SaOMT5-CnMDH2.

(2) Preparation and culture of recombinant Bacillus subtilis The various plasmids constructed were introduced into Bacillus subtilis 168 strain by a two-step starvation method. The obtained recombinant B. subtilis was cultured aerobically for 6 hours at 37 ° C. using M9 medium containing 25 μg / mL erythromycin, 50 μg / mL tryptophan, leucine, arginine, and threonine, and then centrifuged. The body was collected, washed twice with 50 mM Tris-HCl buffer (pH 8.0), and then used in the methylation reaction experiment.

(3) Methylation reaction using the SAM recycling route An esculetin methylation reaction was carried out using the prepared B. subtilis cells. The methylation reaction was performed with 1 mM escretin as a substrate, 100 mM methanol as a source of methyl groups, 50 mM Tris-HCl buffer (pH 8.0), 1 mM ATP, 10 mM DDL ₄ , and 40 mg-wet cell / mL of B. subtilis cells. It was carried out in a reaction solution consisting of. The reaction was carried out at 37 ° C. for 18 hours, and the produced scopoletin and isoscopoletin were quantified by HPLC according to the method described in Preparation Example 1 described above.

Example 9 Methylation reaction using the SAM recycling route Using methanol as a methyl group donor, an esculetin methylation reaction was carried out with SAM recycling. Since the pCU / B. Subtilis strain does not have OMT activity, no production of scopoletin or isoscopoletin was observed. On the other hand, the SaOMT5-expressing strain showed the production of methylated compounds, and the strain co-expressing with CnMDH2 showed 1.4 times the production of methylated compounds as compared with the SaOMT5-single-expressing strain. From this result, it was shown that the SAM recycling pathway using methanol as a methyl group source functions by co-expression of SaOMT5 and CnMDH2 in microorganisms other than Escherichia coli.

According to the present invention, by adding methanol, S-adenosylmethionine (SAM) can be regenerated, and the application to various methylation reactions can be expanded. Therefore, pharmaceutical manufacturing and chemistry It is extremely useful in fields related to product manufacturing, functional compound manufacturing, and the like.

Claims (15)

A method for recycling S-adenosylmethionine, which comprises demethylating S-adenosylmethionine and then converting it from methionine obtained by transferring a methyl group derived from methanol to regenerate it. The method of claim 1, wherein S-adenosylmethionine is demethylated to S-adenosyl homocysteine and then to homocysteine. The method according to claim 1 or 2, wherein demethylation of S-adenosylmethionine is performed via S-adenosylmethionine-dependent methylase. The method according to any one of claims 1 to 3, wherein the methyl group derived from methanol is obtained by reacting methanol with methanol dehydrogenase or methanol oxidase. The method according to claim 3 or 4, wherein a host organism co-expressing methylase and methanol dehydrogenase is used. The method according to claim 3 or 4, wherein a host organism co-expressing methylase and methanol oxidase is used. The method according to claim 5 or 6, wherein the host organism is selected from animal cells, insect cells, plant cells and microorganisms. The method of claim 7, wherein the host organism is modified to increase the amount of methionine or S-adenosylmethionine. The method according to any one of claims 1 to 8, further comprising converting from methionine via adenosine triphosphate obtained by adding an energy source for adenosine triphosphate regeneration. A method for producing a methylated product, which comprises using S-adenosylmethionine regenerated by the method according to any one of claims 1 to 9 as a methyl group donor to carry out a methylation reaction. A host organism co-expressing methylase with methanol dehydrogenase or methanol oxidase for use in the method of any of claims 1-10. The host organism according to claim 11, wherein the methylase and methanol dehydrogenase or methanol oxidase have been introduced. The host organism of claim 12, further modified to increase the amount of methionine or S-adenosylmethionine. Modifications suppress overexpression of methionine or S-adenosylmethionine synthase, mutations that enhance the function of methionine or S-adenosylmethionine synthase, expression of methionine or S-adenosylmethionine synthase gene Mutations that suppress the function of enzymes or proteins, mutations that improve the function of enzymes that suffer feedback inhibition in the synthesis of methionine or S-adenosylmethionine, mutations that improve the function of degrading enzymes of S-adenosylhomocysteine, and The host organism according to claim 13, which is selected from mutations that suppress the function of formaldehyde-degrading enzymes. An expression vector into which methylase and methanol dehydrogenase or methanol oxidase have been introduced.