Method for evaluating enzyme activity and kit for enzyme activity evaluation

Abstract

To provide a simple method for evaluating the activity of an enzyme with S-adenosylmethionine as a substrate, and a simple kit for evaluating the activity of an enzyme in S-adenosylmethionine-consuming reactions.SOLUTION: A method for evaluating the activity of a first enzyme comprises: a step of inducing competition between consumption of S-adenosylmethionine by a reaction catalyzed by the first enzyme and consumption of S-adenosylmethionine by a reaction catalyzed by a second enzyme; and a step of evaluating fluorescence and / or coloration resulting from the reaction catalyzed by the second enzyme.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for evaluating enzyme activity and a kit for evaluating enzyme activity. [Background technology]

[0002] S-Adenosylmethionine is used as a substrate for various enzyme reactions in the body. For example, S-adenosylmethionine-dependent methyltransferase is an enzyme responsible for the methylation reaction using S-adenosylmethionine as a methyl donor, and its substrates are diverse, including low molecular weight compounds, DNA, RNA, and proteins. Natural products biosynthesized through methylation reactions by S-adenosylmethionine-dependent methyltransferase include many industrially important substances that exhibit physiological activity and health functionality. Examples of such natural products include vanillin, which is widely used as a fragrance, and morphine, which is widely used as a painkiller. In addition, S-adenosylmethionine decarboxylase decarboxylates S-adenosylmethionine to produce S-adenosyl 3-(methylsulfanyl)propylamine, which is important as the initial reaction in the biosynthesis of long-chain polyamines. In addition, S-adenosylmethionine-8-amino-7-oxononanoate aminotransferase exists in the biotin biosynthesis pathway and transfers an amino group from S-adenosylmethionine to 8-amino-7-oxononanoate. Nicotianamine synthase synthesizes nicotianamine from three molecules of S-adenosylmethionine. Nicotianamine is a synthetic intermediate of mugineic acid, an iron chelating substance secreted by plants, and is also a compound that has angiotensin-converting enzyme inhibitory activity. Therefore, the discovery or modification of enzymes that use S-adenosylmethionine as a substrate is an important technical issue due to its importance in cell physiology and high industrial usefulness.

[0003] As a method for evaluating enzyme activity for searching or modifying an enzyme that uses S-adenosylmethionine as a substrate, for example, a transcription factor-type biosensor that responds to vanillic acid has been developed to search for an S-adenosylmethionine-dependent methyltransferase suitable for high production of vanillic acid (Non-Patent Document 1), and a substrate specificity profile of an S-adenosylmethionine-dependent methyltransferase synthesized in a cell-free transcription / translation system has been performed by quantifying the amount of S-adenosylmethionine in the evaluation system (Non-Patent Document 2), etc. In addition, a methyltransferase activity detection system (Patent Document 1) has been reported that utilizes the fact that L-cysteine ​​requirement is complemented in a methylation activity-dependent manner via S-adenosyl-L-homocysteine, a by-product generated by a methyl group transfer reaction in a microorganism to which an L-cysteine ​​requirement has been imparted. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Pat. No. 1,147,9758 [Non-patent literature]

[0005] [Non-Patent Document 1] Kunjapur et al., “Development of a Vanillate Biosensor for the Vanillin Biosynthesis Pathway in E. coli”, ACS Synthetic Biology, vol.8. 1958-1967 (2019) [Non-Patent Document 2] Haslinger et al., “Rapid in vitro prototyping of O-methyltransferases for pathway applications in Escherichia coli”, Cell Chemical Biology, vol.28. 876-886.e4 (2021) Summary of the Invention [Problem to be solved by the invention]

[0006] However, these methods have issues in terms of versatility and simplicity, since the combinations of enzymes that use the target S-adenosylmethionine as a substrate and the substrates of those enzymes are limited, the enzyme preparation methods and kits used for measuring S-adenosylmethionine are expensive, and quantitative analysis is difficult because these are assays that complement auxotrophy.

[0007] The present invention has been made in consideration of the above circumstances, and aims to provide a method for simply evaluating the activity of an enzyme that uses S-adenosylmethionine as a substrate, or a kit for simply evaluating the activity of an enzyme in a reaction that consumes S-adenosylmethionine. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors have found that when the consumption of S-adenosylmethionine by an enzyme that consumes S-adenosylmethionine to produce a compound that exhibits fluorescence and / or color, and the consumption of S-adenosylmethionine by a reaction catalyzed by a target enzyme, are made to compete with each other, the fluorescence and / or color are weakened in accordance with the consumption of S-adenosylmethionine by the reaction catalyzed by the target enzyme, and have completed the present invention.

[0009] The present invention provides, for example, the following inventions. [1] Competing consumption of S-adenosylmethionine by a reaction catalyzed by a first enzyme and consumption of S-adenosylmethionine by a reaction catalyzed by a second enzyme; evaluating the fluorescence and / or color produced by the reaction catalyzed by the second enzyme; A method for assessing the activity of the first enzyme, comprising: [2] introducing a first expression vector containing a gene encoding the first enzyme into a cell; The method further comprises the step of introducing into the cell a second expression vector comprising a gene encoding the second enzyme, Competition for consumption of S-adenosylmethionine occurs within the cell. The method described in [1]. [3] The method according to [2], wherein the cell is a bacterium belonging to the genus Escherichia. [4] The method according to any of [1] to [3], wherein the second enzyme comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 27 and has S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase activity. [5] The method according to any one of [1] to [4], wherein the fluorescence and / or coloration is fluorescence of trimethylpyrocorpin. [6] The method according to any one of [1] to [5], wherein the second enzyme is a fusion protein containing a fluorescent protein. [7] The method according to any one of [1] to [6], wherein the first enzyme is an enzyme selected from the group consisting of methyltransferase, radical S-adenosylmethionine enzyme, S-adenosylmethionine decarboxylase, N-acylhomoserine lactone synthase, opine metallophore synthase, 5'-deoxy-5'-fluoroadenosine synthase, 5'-deoxy-5'-chloroadenosine synthase, and azetidine-2-carboxylic acid synthase. [8] An enzyme having S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase activity, S-adenosylmethionine, and Uroporphyrinogen III A kit for evaluating the activity of an enzyme different from the enzyme in a reaction consuming S-adenosylmethionine, comprising: [9] An enzyme having S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase activity, S-adenosylmethionine, and 5-aminolevulinic acid, porphobilinogen synthase, porphobilinogen deaminase, and uroporphyrinogen III synthase A kit for evaluating the activity of an enzyme different from the enzyme in a reaction consuming S-adenosylmethionine, comprising: Effect of the Invention

[0010] According to the present invention, it is possible to provide a method for simply evaluating the activity of an enzyme which uses S-adenosylmethionine as a substrate, or a kit for simply evaluating the activity of an enzyme in a reaction which consumes S-adenosylmethionine. [Brief description of the drawings]

[0011] [Figure 1] 1 is a plasmid map of plasmid p15A-Ptrc-cysG(A). [Diagram 2] 1 shows photographs of colonies of MG1655 / p15A-Ptrc-cysG(A) taken in Example 1 under a 365 nm ultraviolet lamp in the presence or absence of IPTG. [Diagram 3] 1 is a plasmid map of plasmid pQE80L-CCS1. [Figure 4] Photographs of colonies of MG1655 / pTrc99a+p15A-Ptrc-cysG(A) and MG1655 / pQE80L-CCS1+p15A-Ptrc-cysG(A) taken in Example 2 under a 365 nm ultraviolet lamp in the presence or absence of various concentrations of theobromine. [Diagram 5] 1 is a plasmid map of plasmid pQE80L-EgtD. [Figure 6] Photographs of colonies of MG1655 / pTrc99a+p15A-Ptrc-cysG(A) and MG1655 / pQE80L-EgtD+p15A-Ptrc-cysG(A) taken in Example 3 under a 365 nm ultraviolet lamp in the presence or absence of L-histidine at various concentrations. [Figure 7] 1 is a plasmid map of plasmid pQE80L-EgtD(AA). [Figure 8] Photographs of colonies of MG1655 / pQE80L-EgtD+p15A-Ptrc-cysG(A) and MG1655 / pQE80L-EgtD(AA)+p15A-Ptrc-cysG(A) taken in Example 4 under a 365 nm ultraviolet lamp in the presence of various concentrations of L-histidine or L-tryptophan, or in the absence of substrate. [Figure 9] 1 is a plasmid map of plasmid p15A-Ptrc-sfGFP-cysG(A). [Figure 10] Photographs of colonies of MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG(A) and MG1655 / pQE80L-CCS1+p15A-Ptrc-sfGFP-cysG(A) taken in Example 5 under a 365 nm ultraviolet lamp in the presence or absence of various concentrations of theobromine. [Figure 11] Photographs of colonies of MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG(A) and MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG(A) taken in Example 6 in the presence or absence of L-histidine at various concentrations under a 365 nm ultraviolet lamp. [Figure 12] Photographs of colonies of MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG(A) and MG1655 / pQE80L-EgtD(AA)+p15A-Ptrc-sfGFP-cysG(A) taken in Example 7 under a 365 nm ultraviolet lamp in the presence of various concentrations of L-histidine or L-tryptophan, or in the absence of substrate. [Figure 13] 1 is a plasmid map of plasmid pQE80L-NpN4OMT1. [Figure 14]Photographs of colonies of MG1655 / pTrc99a+p15A-Ptrc-cysG(A) and MG1655 / pQE80L-NpN4OMT1+p15A-Ptrc-cysG(A) taken in Example 8 under a 365 nm ultraviolet lamp in the presence or absence of dopamine hydrochloride. [Figure 15] Photographs of colonies of MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG(A) and MG1655 / pQE80L-NpN4OMT1+p15A-Ptrc-sfGFP-cysG(A) taken in Example 9 under a 365 nm ultraviolet lamp in the presence or absence of dopamine hydrochloride. [Figure 16] 1 is a plasmid map of plasmid pQE80L-SpeD. [Figure 17] Photographs of colonies of MG1655 / pTrc99a+p15A-Ptrc-cysG(A) and MG1655 / pQE80L-speD+p15A-Ptrc-cysG(A) taken in Example 10 under a 365 nm ultraviolet lamp in the presence or absence of IPTG. [Figure 18] Photographs of colonies of MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG(A) and MG1655 / pQE80L-speD+p15A-Ptrc-sfGFP-cysG(A) taken in Example 11 under a 365 nm ultraviolet lamp in the presence or absence of IPTG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The method for evaluating the activity of a first enzyme according to this embodiment includes a step of competing the consumption of S-adenosylmethionine by a reaction catalyzed by the first enzyme with the consumption of S-adenosylmethionine by a reaction catalyzed by the second enzyme (competition step), and a step of evaluating the fluorescence and / or color produced by the reaction catalyzed by the second enzyme (evaluation step).

[0013] Furthermore, the method for evaluating the activity of a first enzyme in this embodiment may further include, prior to the competitive step, a step of introducing a first expression vector containing a gene encoding the first enzyme into a cell (first introduction step) and a step of introducing a second expression vector containing a gene encoding the second enzyme into the cell (second introduction step) (hereinafter, the "first introduction step" and the "second introduction step" are collectively referred to as the "introduction step").

[0014] In this specification, the term "consumption" used for S-adenosylmethionine means that an enzyme acts on S-adenosylmethionine to cause a chemical reaction, thereby reducing the amount of S-adenosylmethionine in the reaction system.The concept of "consumption" also includes the case where the first enzyme does not reduce the amount of S-adenosylmethionine due to at least one reason selected from the group consisting of the first enzyme not having the activity required to act on S-adenosylmethionine to cause a chemical reaction, the first enzyme acting on S-adenosylmethionine to cause a chemical reaction, the substrate required for the first enzyme to act on S-adenosylmethionine to cause a chemical reaction not being present in the reaction system, and the first enzyme being inhibited by an inhibitor of the first enzyme.

[0015] In this specification, the term "competition" used in relation to the consumption of S-adenosylmethionine means that two enzymes consume S-adenosylmethionine with each other, and thus the amount of S-adenosylmethionine consumed by each enzyme is reduced compared to when the other enzyme does not exist. Note that the concept of "competition" also includes the case where the amount of S-adenosylmethionine consumed by the second enzyme is not reduced compared to when the first enzyme does not exist, because the first enzyme does not reduce the amount of S-adenosylmethionine in the reaction system due to at least one reason selected from the group consisting of the first enzyme not having the activity required to act on S-adenosylmethionine to cause a chemical reaction, the first enzyme not having a substrate required to act on S-adenosylmethionine to cause a chemical reaction in the reaction system, and the first enzyme being inhibited by an inhibitor of the first enzyme.

[0016] As used herein, "fluorescence and / or color" resulting from a reaction catalyzed by a second enzyme means fluorescence and / or color exhibited by a fluorescent compound and / or a compound that induces a color reaction, which are contained in the product of the reaction catalyzed by the second enzyme.

[0017] The method for measuring the activity of a first enzyme of the present invention utilizes the fact that fluorescence and / or coloration is produced by a reaction catalyzed by the second enzyme consuming S-adenosylmethionine, and that such fluorescence and / or coloration is attenuated in response to the consumption of S-adenosylmethionine by the reaction catalyzed by the first enzyme during competition between the consumption of S-adenosylmethionine by the reaction catalyzed by the first enzyme and the consumption of S-adenosylmethionine by the reaction catalyzed by the second enzyme.

[0018] The application of the method for evaluating the activity of the first enzyme according to this embodiment is not particularly limited, and may be used, for example, to evaluate the presence or absence of enzyme activity for a target reaction, to screen for an enzyme that exhibits high activity, to screen for a substrate for which the enzyme exhibits high activity, to screen for a combination of an enzyme and a substrate for which the enzyme exhibits high activity, to search for an inhibitor for the enzyme, or to screen for an enzyme with high substrate selectivity.

[0019] (First enzyme) The first enzyme according to the present embodiment is not particularly limited and can be appropriately selected according to the purpose, and may be an enzyme that catalyzes a reaction consuming S-adenosylmethionine, or may be an enzyme that is unclear whether it catalyzes a reaction consuming S-adenosylmethionine. Examples of the first enzyme according to the present embodiment include methyltransferases such as CCS1 (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 28), EgtD (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 29), dopamine-O-methyltransferase, radical S-adenosylmethionine enzyme, S-adenosylmethionine decarboxylase, N-acylhomoserine lactone (AHL) synthase, opine metallophore synthases such as nicotianamine synthase, 5'-deoxy-5'-fluoroadenosine synthase, 5'-deoxy-5'-chloroadenosine synthase, and azetidine-2-carboxylic acid synthase. Examples of the dopamine-O-methyltransferase include dopamine-O-methyltransferase NpN4OMT1 derived from Narcissus plants (a protein consisting of the amino acid sequence represented by SEQ ID NO: 37; Kilgore, Matthew B., et al. "Cloning and characterization of anorbelladine 4'-O-methyltransferase involved in the biosynthesis of theAlzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus." PloS one 9.7 (2014): e103223.). Examples of the S-adenosylmethionine decarboxylase include S-adenosylmethionine decarboxylase SpeD derived from Escherichia coli (a protein consisting of the amino acid sequence represented by SEQ ID NO: 39). These enzymes may be known enzymes or may be enzymes modified by amino acid substitution or the like.

[0020] (Second enzyme) The second enzyme according to the present embodiment is not particularly limited as long as it is an enzyme that catalyzes a reaction that consumes S-adenosylmethionine and produces fluorescence and / or color, and examples of the second enzyme include an enzyme having S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase (SUMT) activity (including SUMT), an enzyme having esculetin methylation activity, etc. The enzyme having SUMT activity may be a protein containing an amino acid sequence that has a high identity to the amino acid sequence represented by SEQ ID NO: 27, and examples of the enzyme having SUMT activity include Escherichia coli CysG, CysG (A)(i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 27) and the like. In this specification, high identity refers to a sequence identity of 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) in the entire amino acid sequence or the entire nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined by the BLAST algorithm by Karlin and Altschul (S. Karlin et al., "Applications and statistics for multiple high-scoring segments in molecular sequences." Proceedings of the National Academy of Sciences, 90(12), 5873-5877 (1993)). Based on this algorithm, programs called BLASTN and BLASTX have been developed (SF Altschul et al., "Basic local alignment search tool." Journal of molecular biology, 215(3), 403-410 (1990)). When analyzing a base sequence by BLASTN based on BLAST, the parameters are, for example, score=100 and wordlength=12. When analyzing an amino acid sequence by BLASTX based on BLAST, the parameters are, for example, score=50 and wordlength=3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known (information can be found on the BLAST (Basic Local Alignment Search Tool) website of NCBI (National Center for Biotechnology Information)).

[0021] An enzyme with SUMT activity is an enzyme that uses S-adenosylmethionine as a methyl donor and catalyzes the methylation reaction of uroporphyrinogen III one or more times. When uroporphyrinogen III is methylated three times by an enzyme with SUMT activity, fluorescent trimethylpyrocorpin is produced. (J. Feliciano et al., “Novel reporter gene in a fluorescent-based whole cell sensing system”, Biotechnology and Bioengineering, vol.93. 989-997 (2006)).

[0022] In addition, Escherichia coli CysG is a multifunctional enzyme that has SUMT activity, precorrin-2 dehydrogenase activity, and scyllohydrochlorin ferrochelatase activity. (A) is a protein obtained by excising only the C-terminal domain, which has SUMT activity, from Escherichia coli CysG, and is known to have SUMT activity (MJ Warren et al., "Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein", Biochemical Journal, vol.302, 837-844 (1994)).

[0023] The second enzyme according to the present embodiment may be a fusion protein containing a fluorescent protein and an enzyme that catalyzes a reaction that consumes S-adenosylmethionine and produces fluorescence and / or color. The fluorescent protein may be any protein that emits fluorescence when excited by excitation light, and examples of such a protein include avGFP, EGFP, GFPuv, superfolder GFP (sfGFP), CFP, BFP, DsRED, mCherry, and EYFP. When the second enzyme is a fusion protein containing a fluorescent protein, the fluorescence and / or color produced by the reaction that consumes S-adenosylmethionine catalyzed by the second enzyme can be evaluated by evaluating the change in hue. In this case, the activity of the first enzyme according to the present embodiment can be more easily evaluated. When the second enzyme is a fusion protein containing a fluorescent protein, the second enzyme may be, for example, CysG (A) and superfolder GFP fusion proteins.

[0024] <Introduction process> In the introduction step, a first expression vector containing a gene encoding a first enzyme is introduced into a cell, and a second expression vector containing a gene encoding a second enzyme is introduced into the cell. The introduction of the first expression vector containing the gene encoding the first enzyme into the cell and the introduction of the second expression vector containing the gene encoding the second enzyme into the cell may be performed simultaneously, or the introduction of the first expression vector containing the gene encoding the first enzyme into the cell may be performed first, or the introduction of the second expression vector containing the gene encoding the second enzyme into the cell may be performed first.

[0025] Furthermore, when the method for evaluating the activity of a first enzyme according to this embodiment includes an introduction step, the competition between the consumption of S-adenosylmethionine by the reaction catalyzed by the first enzyme and the consumption of S-adenosylmethionine by the reaction catalyzed by the second enzyme takes place in a cell into which the first expression vector and the second expression vector have been introduced.

[0026] (First Expression Vector and Second Expression Vector) The first expression vector according to the present embodiment is not particularly limited as long as it contains a gene encoding a first enzyme and has a function of producing the first enzyme in either a prokaryotic cell and / or a eukaryotic cell. The second expression vector according to the present embodiment is not particularly limited as long as it contains a gene encoding a second enzyme and has a function of producing the second enzyme in either a prokaryotic cell and / or a eukaryotic cell. The first expression vector according to the present embodiment and the second expression vector according to the present embodiment can be independently selected (hereinafter, the first expression vector according to the present embodiment and the second expression vector according to the present embodiment are collectively referred to as "the expression vector according to the present embodiment"). The expression vector according to the present embodiment may contain a promoter-operator region, a start codon, a stop codon, and a terminator region when bacteria belonging to the genus Escherichia are used as the host cell. These can be those commonly used by those skilled in the art, and examples of the promoter include the T5 promoter and the trc promoter, and examples of the operator include the Lac operator.

[0027] In addition to these, the host cell may contain a marker gene (antibiotic resistance gene, etc.) that enables the selection of transformed host cells, a gene encoding a transcriptional regulator that promotes or suppresses the transcription of the gene encoding the first enzyme, etc. Examples of antibiotic resistance genes include an ampicillin resistance gene and a chloramphenicol resistance gene. Examples of genes encoding transcriptional regulators include, for example, the lacIq gene that overexpresses the Lac repressor when the first expression vector contains the Lac operator as an operator.

[0028] The expression vector according to this embodiment is not limited to a plasmid, but may be, for example, an artificial chromosome, a vector using a transposon, or a cosmid. When a bacterium belonging to the genus Escherichia is used as a host cell into which an expression vector according to this embodiment is introduced, examples of the vector include pTrc-luxR (Y. Kimura et al., “Directed evolution of Vibrio fik-aphscheri LuxR signal sensitivity”, Journal of Bioscience and Bioengineering, Vol.122. 533-538 (2016)), Plux-sfgfp-HSVtk-aph (Y. Kimura et al., “Directed Evolution of the Stringency of the LuxR Vibrio fischeri Quorum Sensor without OFF-State Selection”, ACS Synthetic Biology, Vol.9. 567-575 (2016)), and the like. (2020)), pColdI, pSTV28, pSTV29, pUC118 (all from Takara Bio), pMW118, pMW119, pMW218 (all from Nippon Gene), pET21a, pET28a, pCDF-1b, pRSF-1b (all from Merck Millipore), pMAL-c5x (New England Biolabs), pGEX-4T-1, pTrc99A (all from GE Healthcare Biosciences), pTrcHis, pSE280 (all from Thermo Fisher Scientific), pGEMEX-1 (Promega), pQE-30, pQE-60, pQE80L (all from Qiagen), pET-3, pBluescriptII SK(+), pBluescriptII KS(-) (all manufactured by Agilent Technologies), pKYP10 (JP Patent Publication No. 110600 / 1983), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, p6378-6385], pPE167 (Appl. Environ. Microbiol. 2007, 73: 6378-6385), pPAC31 (WO98 / 12343), pUC19 [Gene, 33, 103 (1985)], pPA1 (JP Patent Publication No. 63-233798), etc. When the above vectors are used, any promoter may be used as long as it functions in the cells of bacteria belonging to the genus Escherichia, and examples of promoters that can be used include promoters derived from Escherichia coli, phages, etc., such as the trp promoter, gapA promoter, lac promoter, PL promoter, PR promoter, PSE promoter, and T5 promoter. Artificially designed and modified promoters such as a promoter in which two trp promoters are connected in series, the tac promoter, the trc promoter, the lacT5 promoter, the lacT7 promoter, and the let I promoter can also be used.

[0029] (cell) The cells according to this embodiment are not particularly limited as long as they are compatible with the first and second expression vectors and can be transformed, and may be prokaryotic cells such as bacteria, animal cells, eukaryotic cells such as fungi, etc. that are commonly used by those skilled in the art. These cells may be natural cells or recombinant cells. The cell according to the present embodiment is preferably, for example, a prokaryote or a yeast, and more preferably a bacterium belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, or a yeast belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, or Candida, and examples thereof include Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli BL21(DE3) (manufactured by Novagen), Escherichia coli BL21(DE3)pLysS (manufactured by Merck Millipore), Escherichia coli DH5α, Escherichia coli HST08Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm―, Escherichia coli JM109, Escherichia coli HB101, Escherichia coliCJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio), Escherichia coli W, Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli coli NM522 or Escherichia coliBacteria belonging to the genus Escherichia such as ATCC9637, bacteria belonging to the genus Serratia such as Serratia ficaria, Serratia fonticola, Serratia liquefaciens, or Serratia marcescens, bacteria belonging to the genus Bacillus such as Bacillus subtilis or Bacillus amyloliquefaciens, bacteria belonging to the genus Brevibacterium such as Brevibacterium immariophilum ATCC14068 or Brevibacterium saccharolyticum ATCC14066, bacteria belonging to the genus Brevibacterium such as Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, or Corynebacterium acetoacidophilum More preferred are bacteria belonging to the genus Corynebacterium, such as ATCC13870, bacteria belonging to the genus Microbacterium, such as Microbacterium ammoniaphilum ATCC15354, bacteria belonging to the genus Pseudomonas, such as Pseudomonas sp. D-0110, yeasts belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae, yeasts belonging to the genus Schizosaccharomyces, such as Schizosaccharomyces pombe, yeasts belonging to the genus Kluyveromyces lactis, yeasts belonging to the genus Trichosporon, such as Trichosporon pullulans, yeasts belonging to the genus Schwanniomyces, such as Schwanniomyces alluvius, yeasts belonging to the genus Pichia, such as Pichia pastoris, and yeasts belonging to the genus Candida, such as Candida utilis.

[0030] The introduction (transformation) of the first expression vector into a cell and the introduction (transformation) of the second expression vector into a cell according to this embodiment are not particularly limited and can be performed by a known method, for example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], a protoplast method (Japanese Patent Publication No. 63-248394), an electroporation method [Nucleic Acids Res., 16, 6127 (1988)], a particle gun method [TM Klein et al.: Nature, 327, 70 (1987)], a TSS method [Chung et al., PNAS, Vol. 86, 2172-2175 (1989)], a conjugal transfer method [Thomas CM,et al., Nat Rev Microbiol. 2005 Sep;3(9):711-21.], the spheroplast method [Proc. Natl. Acad. Sci., USA, 81, 4889(1984)], and the lithium acetate method [J. Bacteriol., 153, 163(1983)].

[0031] After the first expression vector and / or the second expression vector are introduced into the cells, cell culture, selection of the introduced (transformed) cells, induction of expression of the first enzyme, induction of expression of the second enzyme, etc. may be appropriately performed by methods commonly performed by those skilled in the art depending on the expression vector, the type of cell, etc. Examples of methods for inducing expression of the first enzyme and / or the second enzyme include inhibiting the Lac repressor with isopropyl-β-thiogalactopyranoside (IPTG) when the expression of the first enzyme and / or the second enzyme is suppressed by the Lac repressor.

[0032] <Competitive process> In the competition step, the consumption of S-adenosylmethionine by the reaction catalyzed by the first enzyme and the consumption of S-adenosylmethionine by the reaction catalyzed by the second enzyme are made to compete with each other. The competition in the competition step is carried out in the presence of the first enzyme, the second enzyme, S-adenosylmethionine, and a substrate of the second enzyme (excluding S-adenosylmethionine), and may be carried out in the presence of a substrate of the first enzyme (excluding S-adenosylmethionine) if necessary.

[0033] In the competition step, the first enzyme, the second enzyme, and S-adenosylmethionine may be present in sufficient amounts in advance, or may be supplied as needed, as long as the competition is carried out appropriately.

[0034] The substrate of the first enzyme (excluding S-adenosylmethionine) is not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, a substrate (excluding S-adenosylmethionine) in a reaction catalyzed by the first enzyme consuming S-adenosylmethionine, or a substance (excluding S-adenosylmethionine) that is unclear whether it is a substrate or not in the reaction. In the competitive step, the substrate of the first enzyme (excluding S-adenosylmethionine) may be present in a sufficient amount in advance or may be supplied appropriately so that a sufficient amount is present for the first enzyme or S-adenosylmethionine in the above reaction.

[0035] The substrate of the second enzyme (excluding S-adenosylmethionine) refers to a substrate (excluding S-adenosylmethionine) in a reaction catalyzed by the second enzyme, consuming S-adenosylmethionine, and producing fluorescence and / or color. The substrate of the second enzyme (excluding S-adenosylmethionine) may be appropriately determined depending on the type of the second enzyme, and may be, for example, uroporphyrinogen III when the second enzyme is an enzyme having SUMT activity. In the competitive step, the substrate of the second enzyme (excluding S-adenosylmethionine) may be present in sufficient amount in advance or may be appropriately supplied so that there is a sufficient amount for the second enzyme or S-adenosylmethionine in the above reaction.

[0036] The competitive step may be carried out in the presence of an inhibitor of the first enzyme, if necessary. The inhibitor of the first enzyme is not particularly limited as long as it does not inhibit the reaction catalyzed by the second enzyme, which consumes S-adenosylmethionine and produces fluorescence and / or coloration, and can be appropriately selected depending on the purpose. For example, it may be a substance that inhibits the reaction catalyzed by the first enzyme, which consumes S-adenosylmethionine, or it may be a substance that is unclear whether it inhibits the reaction or not. In the competitive step, the inhibitor of the first enzyme may be present in a sufficient amount in advance, or may be supplied as appropriate.

[0037] The competitive step is not particularly limited, and may be performed, for example, in a cell or a cell-free system, or may be performed in a cell capable of biosynthesizing S-adenosylmethionine and / or a substrate for the second enzyme. When the competitive step is performed in a cell capable of biosynthesizing S-adenosylmethionine and / or a substrate for the second enzyme, S-adenosylmethionine and / or a substrate for the second enzyme may not be supplied from the outside before or during the competitive step. For example, when the second enzyme is an enzyme having SUMT activity, the substrate for the second enzyme is uroporphyrinogen III, and the competitive step is performed in a cell capable of biosynthesizing S-adenosylmethionine and uroporphyrinogen III, such as a bacterium belonging to the genus Escherichia, S-adenosylmethionine and uroporphyrinogen III may not be supplied from the outside before or during the competitive step.

[0038] Furthermore, conditions of the competitive step, such as reaction time and reaction temperature, may be appropriately set so that competition between consumption of S-adenosylmethionine by the reaction catalyzed by the first enzyme and consumption of S-adenosylmethionine by the reaction catalyzed by the second enzyme occurs appropriately.

[0039] <Evaluation process> In the evaluation step, the fluorescence and / or color generated by the reaction consuming S-adenosylmethionine catalyzed by the second enzyme in the competitive step is evaluated. The evaluation of the fluorescence and / or color is not particularly limited, and may be by measuring the fluorescence and / or color, or by detecting the fluorescence and / or color.

[0040] The fluorescent compound and / or the compound that induces a color reaction contained in the product of the reaction catalyzed by the second enzyme is not particularly limited, and may be, for example, trimethylpyrocorpin when the second enzyme is an enzyme having SUMT activity.

[0041] The measurement of fluorescence in the evaluation step is not particularly limited, and the excitation wavelength, fluorescence wavelength, measuring device, etc. may be appropriately set according to the compound that emits fluorescence, and the fluorescence intensity, etc. may be measured. In addition, the detection of fluorescence in the evaluation step is not particularly limited, and the excitation wavelength, etc. may be appropriately set according to the compound that emits fluorescence, and the fluorescence may be observed visually. For example, when trimethylpyrocorpin is included in the product of the reaction consuming S-adenosylmethionine catalyzed by the second enzyme, the fluorescence intensity at wavelengths of 600 nm to 640 nm (maximum at 620 nm) may be measured with a plate reader or a spectrofluorophotometer by irradiating with excitation light of wavelengths of 340 nm to 380 nm (maximum at 357 nm), or the pink fluorescence may be observed visually by irradiating with excitation light of wavelengths of 340 nm to 380 nm (maximum at 357 nm).

[0042] The measurement of color in the evaluation step is not particularly limited, and the color reaction conditions, measurement method, measurement device, etc. may be appropriately set depending on the compound that induces the color reaction. Furthermore, the detection of color in the evaluation step is not particularly limited, and the color reaction conditions, etc. may be appropriately set depending on the compound that induces the color reaction, and the color may be observed visually.

[0043] In the evaluation step, the first enzyme may be determined to have activity when the fluorescence and / or color produced by the reaction catalyzed by the second enzyme consuming S-adenosylmethionine is attenuated to a certain level or more compared to the fluorescence and / or color produced by the reaction catalyzed by the second enzyme under the same conditions except for the absence of the first enzyme. Also, the first enzyme may be determined to have activity on the substrate of the first enzyme when the fluorescence and / or color produced by the reaction catalyzed by the second enzyme consuming S-adenosylmethionine is attenuated to a certain level or more compared to the fluorescence and / or color produced by the reaction catalyzed by the second enzyme under the same conditions except for the absence of the substrate of the first enzyme. In addition, when two or more types of first enzymes are evaluated for fluorescence and / or coloration caused by a reaction catalyzed by a second enzyme consuming S-adenosylmethionine under the same conditions except that the first enzymes are different, the activity of the first enzyme showing strong fluorescence and / or coloration may be evaluated as relatively low, and the activity of the first enzyme showing weak fluorescence and / or coloration may be evaluated as relatively high. In addition, when two or more types of substrates for the first enzymes are evaluated for fluorescence and / or coloration caused by a reaction catalyzed by a second enzyme consuming S-adenosylmethionine under the same conditions except that the substrates for the first enzymes are different, the activity of the first enzyme on the substrate for the first enzyme showing strong fluorescence and / or coloration may be evaluated as relatively low, and the activity of the first enzyme on the substrate for the first enzyme showing low fluorescence intensity and / or coloration may be evaluated as relatively high. Furthermore, when two or more types of first enzymes and two or more types of substrates for the first enzymes are used, and the fluorescence and / or color produced by the reaction catalyzed by the second enzyme to consume S-adenosylmethionine is evaluated under the same conditions except that the first enzymes and the substrates for the first enzymes are different, the activity of the first enzyme in a combination of the first enzyme and the substrate for the first enzyme that shows strong fluorescence and / or color is relatively low, and the activity of the first enzyme in a combination of the first enzyme and the substrate for the first enzyme that shows low fluorescence intensity and / or color intensity may be evaluated as being relatively high.In addition, when the fluorescence and / or color caused by the reaction catalyzed by the second enzyme consuming S-adenosylmethionine in the presence of a first enzyme inhibitor is enhanced by a certain level or more compared to the fluorescence and / or color caused by the reaction catalyzed by the second enzyme under the same conditions except for the absence of the first enzyme inhibitor, the first enzyme inhibitor may be judged to have an inhibitory effect. In addition, when the fluorescence and / or color caused by the reaction catalyzed by the second enzyme consuming S-adenosylmethionine is evaluated for two or more first enzyme inhibitors under the same conditions except for the first enzyme inhibitors being different, the inhibitory effect of the first enzyme inhibitor showing strong fluorescence and / or color may be evaluated as being relatively high, and the inhibitory effect of the first enzyme inhibitor showing low fluorescence intensity and / or color intensity may be evaluated as being relatively low.

[0044] In addition, when the second enzyme is a fusion protein containing a fluorescent protein, the fluorescence emitted by the fluorescent protein may be further measured in the evaluation step. The measurement of this fluorescence is not particularly limited, and the excitation wavelength, fluorescence wavelength, measurement device, etc. may be appropriately set according to the fluorescent protein, and the fluorescence intensity may be measured. In this case, the ratio of the fluorescence intensity and / or color intensity generated by the reaction catalyzed by the second enzyme to the fluorescence intensity derived from the fluorescent protein in the evaluation step may be calculated.

[0045] In the evaluation step, when the second enzyme is a fusion protein containing a fluorescent protein, the first enzyme may be determined to have activity when the ratio is smaller than a certain level compared to the ratio under the same conditions except that the first enzyme is absent. The first enzyme may be determined to have activity on the substrate of the first enzyme when the ratio is smaller than a certain level compared to the ratio under the same conditions except that the substrate of the first enzyme is absent. When the ratio is calculated for two or more types of first enzymes under the same conditions except that the first enzymes are different, the activity of the first enzyme showing a large ratio may be evaluated as being relatively low, and the activity of the first enzyme showing a small ratio may be evaluated as being relatively high. When the ratio is calculated for two or more types of substrates of the first enzymes under the same conditions except that the substrates of the first enzymes are different, the activity of the first enzyme showing a large ratio on the substrate of the first enzyme showing a small ratio may be evaluated as being relatively low, and the activity of the first enzyme showing a small ratio on the substrate of the first enzyme showing a small ratio may be evaluated as being relatively high. In addition, when the above ratio is calculated for two or more types of first enzymes and two or more types of substrates for the first enzymes under the same conditions except that the first enzymes and the substrates for the first enzymes are different, the activity of the first enzyme in the combination of the first enzyme and the substrate for the first enzyme that shows a large ratio may be evaluated as being relatively low, and the activity of the first enzyme in the combination of the first enzyme and the substrate for the first enzyme that shows a small ratio may be evaluated as being relatively high. In addition, when the above ratio in the presence of a first enzyme inhibitor is greater than the above ratio under the same conditions except that the first enzyme inhibitor is not present, it may be determined that the first enzyme inhibitor has an inhibitory effect. In addition, when the above ratio is calculated for two or more types of first enzyme inhibitors under the same conditions except that the first enzyme inhibitors are different, the inhibitory effect of the first enzyme inhibitor that shows a large ratio may be evaluated as being relatively high, and the inhibitory effect of the first enzyme inhibitor that shows a small ratio may be evaluated as being relatively low.

[0046] In addition, when the second enzyme is a fusion protein containing a fluorescent protein, the fluorescence emitted by the fluorescent protein may be further detected in the evaluation step. The detection of this fluorescence is not particularly limited, and the excitation wavelength, etc. may be appropriately set according to the fluorescent protein, and the fluorescence may be observed visually. In this case, the hue of the fluorescence derived from the fluorescent protein and the fluorescence and / or color produced by the reaction catalyzed by the second enzyme may be detected in the evaluation step.

[0047] In the evaluation step, when the second enzyme is a fusion protein containing a fluorescent protein, the first enzyme may be determined to have activity when the above hue is closer to the hue of the fluorescence derived from the fluorescent protein compared to the hue (initial hue) under the same conditions except for the absence of the first enzyme. Also, when the above hue is closer to the hue of the fluorescence derived from the fluorescent protein compared to the hue (initial hue) under the same conditions except for the absence of the substrate of the first enzyme, the first enzyme may be determined to have activity on the substrate of the first enzyme. Also, when the above hues are detected under the same conditions except for the different first enzymes, the activity of the first enzyme that shows a hue close to the initial hue may be evaluated as relatively low, and the activity of the first enzyme that shows a hue close to the hue of the fluorescence derived from the fluorescent protein may be evaluated as relatively high. In addition, when the above-mentioned hue is detected under the same conditions except that the substrates of the first enzyme are different for two or more types of substrates of the first enzyme, the activity of the first enzyme for the substrate of the first enzyme that shows a hue close to the initial hue may be evaluated as being relatively low, and the activity of the first enzyme for the substrate of the first enzyme that shows a hue close to the hue of the fluorescence derived from the fluorescent protein may be evaluated as being relatively high. In addition, when the above-mentioned hue is detected under the same conditions except that the substrates of the first enzyme and the substrates of the first enzyme are different for two or more types of substrates of the first enzyme, the activity of the first enzyme in the combination of the first enzyme and the substrate of the first enzyme that shows a hue close to the initial hue may be evaluated as being relatively low, and the activity of the first enzyme in the combination of the first enzyme and the substrate of the first enzyme that shows a hue close to the hue of the fluorescence derived from the fluorescent protein may be evaluated as being relatively high. In addition, when the above-mentioned hue in the presence of a first enzyme inhibitor is closer to the initial hue than the above-mentioned hue under the same conditions except that the inhibitor of the first enzyme is not present, it may be determined that the inhibitor of the first enzyme has an inhibitory effect. Furthermore, when the above-mentioned hues are detected under the same conditions for two or more types of first enzyme inhibitors, except that the first enzyme inhibitors are different, it may be evaluated that the inhibitory effect of a first enzyme inhibitor that exhibits a hue close to the initial hue is relatively high, and that the inhibitory effect of a first enzyme inhibitor that exhibits a hue close to the hue of the fluorescence derived from the fluorescent protein is relatively low.

[0048] <Kit> According to one embodiment, a kit for evaluating the activity of an enzyme in a reaction consuming S-adenosylmethionine includes an enzyme having SUMT activity, S-adenosylmethionine, and uroporphyrinogen III. The kit may further include a buffer, a thiol reagent (dithiothreitol, etc.), an enzyme stabilizer, a metal ion, a metal ion chelating agent, a protease inhibitor, and a salt (sodium chloride, etc.). The use of the kit may be the same as the use of the method for evaluating the activity of the first enzyme according to the present embodiment described above. In addition, the evaluation of the activity of an enzyme in a reaction consuming S-adenosylmethionine using the kit may include the competitive step and the evaluation step, with the enzyme as the first enzyme and the enzyme having SUMT activity as the second enzyme. In the above kit, the enzyme having SUMT activity, S-adenosylmethionine, and uroporphyrinogen III may all be present in a cell into which an expression vector containing a gene encoding the enzyme has been introduced, the cell having the ability to biosynthesize S-adenosylmethionine and uroporphyrinogen III. In this case, the above kit may contain the above cell. When the above kit contains the above cell, evaluation of the activity of the enzyme in a reaction consuming S-adenosylmethionine using the above kit may further include the above first introduction step, with the enzyme as the first enzyme, the enzyme having SUMT activity as the second enzyme, and an expression vector containing a gene encoding the enzyme as the second expression vector.

[0049] In another embodiment, a kit for evaluating the activity of an enzyme in a reaction consuming S-adenosylmethionine includes an enzyme having SUMT activity, S-adenosylmethionine, and 5-aminolevulinic acid, porphobilinogen synthase (E. coli HemB, etc.), porphobilinogen deaminase (E. coli HemC, etc.), and uroporphyrinogen III synthase (E. coli HemD, etc.). In the kit, uroporphyrinogen III is synthesized from 5-aminolevulinic acid through reactions caused by porphobilinogen synthase (E. coli HemB, etc.), porphobilinogen deaminase (E. coli HemC, etc.), and uroporphyrinogen III synthase in this order. The kit may further include a buffer, a thiol reagent (dithiothreitol, etc.), an enzyme stabilizer, a metal ion, a metal ion chelating agent, a protease inhibitor, and a salt (sodium chloride, etc.). The use of the kit may be the same as the use of the method for evaluating the activity of the first enzyme according to the present embodiment described above. Furthermore, evaluation of the activity of an enzyme in a reaction consuming S-adenosylmethionine using the kit may include the competitive step and the evaluation step, with the enzyme as the first enzyme and an enzyme having SUMT activity as the second enzyme. EXAMPLES

[0050] The present invention will be specifically described below based on test examples, but the present invention is not limited to these.

[0051] Example 1. CysG (A) Fluorescence caused by (Construction of plasmids) CysG from Escherichia coli MG1655 (A) PCR was performed using a plasmid containing the sequence (SEQ ID NO: 23) as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 1 and 2 as a primer set to obtain CysG (A)A DNA fragment containing a gene encoding the gene was amplified. Next, PCR was performed using pTrc-luxR (Y. Kimura et al., “Directed evolution of Vibrio fischeri LuxR signal sensitivity”, Journal of Bioscience and Bioengineering, Vol.122. 533-538 (2016)) as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 3 and 4 as a primer set to amplify a DNA fragment corresponding to the expression vector portion. The two DNA fragments obtained above were ligated using In-Fusion Snap Assembly Master Mix (Takara Bio Inc.) to obtain the plasmid pTrc-cysG (A) was obtained. pTrc-cysG (A) PCR was performed using the above as a template and oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 5 and 6 as a primer set to clone the lacIq gene, the trc promoter, and the cysG (A) A DNA fragment containing a gene encoding the LuxR Vibrio fischeri Quorum Sensor was amplified. Next, PCR was performed using Plux-sfgfp-HSVtk-aph (Y. Kimura et al., “Directed Evolution of the Stringency of the LuxR Vibrio fischeri Quorum Sensor without OFF-State Selection”, ACS Synthetic Biology, Vol.9. 567-575 (2020)) as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs:7 and 8 as a primer set to amplify a DNA fragment corresponding to the expression vector portion. The two DNA fragments obtained above were ligated using In-Fusion Snap Assembly Master Mix to obtain the plasmid p15A-Ptrc-cysG (A) Figure 1 shows the p15A-Ptrc-cysG (A) 1 is a plasmid map of

[0052] (Introduction of plasmids into E. coli, etc.) p15A-Ptrc-cysG (A) The E. coli MG1655 strain was transformed by electroporation to obtain MG1655 / p15A-Ptrc-cysG. (A) The obtained MG1655 / p15A-Ptrc-cysG (A) was cultured in LB liquid medium containing 30 mg / L chloramphenicol (Cm) at 37° C. for 6 hours with shaking. The resulting culture solution was diluted and inoculated onto LB solid medium containing 30 mg / L Cm and LB solid medium containing 30 mg / L Cm and 0.1 mM IPTG, and cultured at 30° C. for 16 hours.

[0053] (CysG (A) Detection of fluorescence caused by After the incubation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (Figure 2).

[0054] As shown in Figure 2, IPTG stimulated the production of CysG (A) When expression of CysG was induced, (A) The colonies showed stronger pink fluorescence than those without induction of p15A-Ptrc-cysG expression. (A) By introducing the above gene into E. coli MG1655, the CysG (A) It was shown that the SUMT activity of was enhanced and the fluorescence of trimethylpyrocorpin was also enhanced.

[0055] (CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped off from the LB solid medium into physiological saline, and the fluorescence intensity at a wavelength of 620 nm when excited at OD595 and 359 nm was measured using Cytation5 (BioTek). The values ​​of the fluorescence intensity divided by OD595 (fluorescence intensity / OD) are shown in Table 1 (rounded off to the nearest whole number).

[0056] [Table 1]

[0057] As shown in Table 1, similar to the above detection, IPTG increased CysG (A) When expression of CysG was induced, (A) The fluorescence intensity / OD was higher when the expression of was not induced. This indicates that the fluorescent compounds produced can be measured by the fluorescence intensity / OD. It also suggests that the quantitative analysis of the fluorescent compounds produced can be performed by the fluorescence intensity / OD.

[0058] Example 2: Evaluation of caffeine synthase activity (Construction of plasmids) PCR was performed using a plasmid containing an artificially synthesized sequence (SEQ ID NO: 24) encoding caffeine synthase (CCS1) derived from Coffee arabica as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 9 and 10 as a primer set to amplify a DNA fragment containing a gene encoding CCS1. Next, PCR was performed using pQE80L (Qiagen) as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 11 and 12 as a primer set to amplify the expression vector sequence and obtain a linearized DNA fragment. The two DNA fragments obtained above were ligated using In-Fusion Snap Assembly Master Mix to obtain the plasmid pQE80L(pre)-CCS1. Using pQE80L(pre)-CCS1 as a template, PCR was performed with a primer set consisting of oligonucleotides with the base sequences shown in SEQ ID NOs: 13 and 14, and oligonucleotides with the base sequences shown in SEQ ID NOs: 15 and 16, to amplify two corresponding DNA fragments. The two DNA fragments obtained above were ligated using In-Fusion Snap Assembly Master Mix to obtain plasmid pQE80L-CCS1. Figure 3 shows the plasmid map of pQE80L-CCS1. In addition, pTrc99a, which does not contain the gene encoding CCS1, was prepared for a control experiment (Void). pTrc99a has the same replication origin and antibiotic resistance gene as pQE80L, and also has the lacIq gene like pQE80L.

[0059] (Introduction of plasmids into E. coli, etc.) pTrc99a, and p15A-Ptrc-cysG (A) , and pQE80L-CCS1, and p15A-Ptrc-cysG (A) Then, E. coli MG1655 strain was transformed by electroporation to obtain MG1655 / pTrc99a+p15A-Ptrc-cysG. (A) and MG1655 / pQE80L-CCS1+p15A-Ptrc-cysG (A) The resulting MG1655 / pTrc99a+p15A-Ptrc-cysG (A) and MG1655 / pQE80L-CCS1+p15A-Ptrc-cysG (A) Each was cultured with shaking in LB liquid medium containing 100 mg / L sodium ampicillin (Amp) and 30 mg / L Cm at 37° C. for 6 hours. The obtained culture solution was diluted and inoculated onto LB solid medium containing 0.25 mM, 0.5 mM or no theobromine, which is a substrate of CCS1, and containing 100 mg / L Amp, 30 mg / L Cm, and 0.1 mM IPTG, and cultured at 30° C. for 21 hours.

[0060] (CysG (A) Detection of fluorescence caused by After the incubation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (Figure 4).

[0061] As shown in Figure 4, IPTG stimulated the production of CysG (A) and MG1655 / pQE80L-CCS1+p15A-Ptrc-cysG, which induced the expression of CCS1. (A)In the presence of theobromine, the colony fluorescence was attenuated in a theobromine concentration-dependent manner. The attenuation of the colony fluorescence was due to the consumption of S-adenosylmethionine by the methylation reaction catalyzed by CCS1 and the (A) This is thought to be due to the competition between the consumption of S-adenosylmethionine by the SUMT activity of CCS1, resulting in a decrease in the production of trimethylpyrocorpin, a fluorescent product of SUMT activity. Therefore, CCS1 activity is visualized and can be detected.

[0062] (CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped off from the LB solid medium into physiological saline, and the fluorescence intensity at a wavelength of 620 nm when excited at OD595 and 359 nm was measured using Cytation5 (BioTek). The values ​​of the fluorescence intensity divided by OD595 (fluorescence intensity / OD) are shown in Table 2 (rounded off to the nearest whole number).

[0063] [Table 2]

[0064] As shown in Table 2, similar to the above detection, IPTG inhibited CysG (A) and MG1655 / pQE80L-CCS1+p15A-Ptrc-cysG, which induced the expression of CCS1. (A) In the presence of theobromine, the colony fluorescence intensity / OD decreased in a theobromine concentration-dependent manner. This indicates that CCS1 activity can be measured by fluorescence intensity / OD. It also suggests that quantitative analysis of CCS1 activity is possible by fluorescence intensity / OD.

[0065] Example 3: Evaluation of L-histidine N-α-methyltransferase activity (Construction of plasmids) Using a plasmid containing a sequence (SEQ ID NO: 25) encoding L-histidine N-α-methyltransferase (EgtD) derived from Mycobacterium smegmatis as a template, PCR was performed using oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 17 and 18 as a primer set to amplify a DNA fragment containing a gene encoding EgtD. Next, PCR was performed using pQE80L (Qiagen) as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 11 and 12 as a primer set to amplify the expression vector sequence and obtain a linearized DNA fragment. The two DNA fragments obtained above were ligated using In-Fusion Snap Assembly Master Mix to obtain the plasmid pQE80L(pre)-EgtD. Using pQE80L(pre)-EgtD as a template, PCR was performed using a primer set consisting of oligonucleotides with the base sequences shown in SEQ ID NOs: 13 and 14, and oligonucleotides with the base sequences shown in SEQ ID NOs: 15 and 16, to amplify two corresponding DNA fragments. The two DNA fragments obtained above were ligated using In-Fusion Snap Assembly Master Mix to obtain plasmid pQE80L-EgtD. Figure 5 shows the plasmid map of pQE80L-EgtD. In addition, pTrc99a not containing the gene encoding EgtD was prepared for a control experiment (Void).

[0066] (Introduction of plasmids into E. coli, etc.) pTrc99a, and p15A-Ptrc-cysG (A) , and pQE80L-EgtD, and p15A-Ptrc-cysG (A) Then, E. coli MG1655 strain was transformed by electroporation to obtain MG1655 / pTrc99a+p15A-Ptrc-cysG. (A) and MG1655 / pQE80L-EgtD+p15A-Ptrc-cysG (A) The resulting MG1655 / pTrc99a+p15A-Ptrc-cysG(A) and MG1655 / pQE80L-EgtD+p15A-Ptrc-cysG (A) Each was cultured with shaking in LB liquid medium containing 100 mg / L Amp and 30 mg / L Cm at 37° C. for 6 hours. The obtained culture solution was diluted and inoculated onto LB solid medium containing 2 g / L, 4 g / L, or no L-histidine, which is a substrate for EgtD, and containing 100 mg / L Amp, 30 mg / L Cm, and 0.1 mM IPTG, and cultured at 30° C. for 21 hours.

[0067] (CysG (A) Detection of fluorescence caused by After the incubation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (Figure 6).

[0068] As shown in Figure 6, IPTG stimulated the production of CysG (A) and MG1655 / pQE80L-EgtD+p15A-Ptrc-cysG, which induced the expression of EgtD. (A) In the presence of L-histidine, the fluorescence of the colonies was attenuated in a concentration-dependent manner. The attenuation of the fluorescence of the colonies was due to the consumption of S-adenosylmethionine by the methylation reaction catalyzed by EgtD and the (A) The consumption of S-adenosylmethionine by the SUMT activity of CysG (A) This is thought to be due to a decrease in the amount of trimethylpyrocorphin produced, which is a fluorescent product of SUMT activity in EgtD. Therefore, EgtD activity is visualized, and it can be said that EgtD activity can be detected.

[0069] (CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped off from the LB solid medium into physiological saline, and the fluorescence intensity at a wavelength of 620 nm when excited at OD595 and 359 nm was measured using Cytation5 (BioTek). The values ​​of the fluorescence intensity divided by OD595 (fluorescence intensity / OD) are shown in Table 3 (rounded off to the nearest whole number).

[0070] [Table 3]

[0071] As shown in Table 3, similar to the above detection, IPTG inhibited CysG (A) and MG1655 / pQE80L-EgtD+p15A-Ptrc-cysG, which induced the expression of EgtD. (A) In the presence of L-histidine, the colony fluorescence intensity / OD decreased in a concentration-dependent manner. This indicates that EgtD activity can be measured by fluorescence intensity / OD. It also suggests that quantitative analysis of EgtD activity is possible by fluorescence intensity / OD.

[0072] Example 4: Evaluation of the activity of L-histidine N-α-methyltransferase mutants (Construction of plasmids) Using pQE80L-EgtD prepared in Example 3 as a template, PCR was performed using oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 19 and 20, and oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 21 and 22 as a primer set, and two corresponding DNA fragments were amplified. The two DNA fragments obtained above were linked by the Golden Gate Assembly method (C. Engler et al., "A one pot, one step, precision cloning method with high throughput capability", PLoS ONE, Vol.3 e3647 (2008)), and a plasmid pQE80L-EgtD(AA) containing a sequence (SEQ ID NO: 26) encoding EgtD(AA) (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 30) in which the 252nd methionine and the 282nd glutamic acid in the amino acid sequence of EgtD (SEQ ID NO: 29) were replaced with alanine was obtained. EgtD(AA) with this mutation has a reduced affinity for its original substrate, L-histidine, but is known to prefer L-tryptophan and L-phenylalanine as substrates (A. Vit et al., “Ergothioneine biosynthetic methyltransferase EgtD reveals the structural basis of aromatic amino acid betaine biosynthesis”, ChemBioChem Vol.16 119-125 (2015)). Figure 7 shows the plasmid map of pQE80L-EgtD(AA).

[0073] (Introduction of plasmids into E. coli, etc.) pQE80L-EgtD and p15A-Ptrc-cysG (A) , and pQE80L-EgtD(AA), and p15A-Ptrc-cysG (A) Then, E. coli MG1655 strain was transformed by electroporation to obtain MG1655 / pQE80L-EgtD+p15A-Ptrc-cysG. (A)and MG1655 / pQE80L-EgtD(AA)+p15A-Ptrc-cysG (A) The resulting MG1655 / pQE80L-EgtD+p15A-Ptrc-cysG (A) and MG1655 / pQE80L-EgtD(AA)+p15A-Ptrc-cysG (A) Each was cultured with shaking in LB liquid medium containing 100 mg / L Amp and 30 mg / L Cm at 37° C. for 3 hours. The obtained culture solution was diluted and inoculated onto LB solid medium containing 4 g / L L-histidine, which is a substrate for EgtD, 5 g / L L-tryptophan, which is a substrate for EgtD(AA), or neither, but containing 100 mg / L Amp, 30 mg / L Cm, and 0.1 mM IPTG, and cultured at 30° C. for 21 hours.

[0074] (CysG (A) Detection of fluorescence caused by After the incubation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (Figure 8).

[0075] As shown in Figure 8, MG1655 / pQE80L-EgtD+p15A-Ptrc-cysG (A) In the presence of L-histidine, the fluorescence of the colonies was significantly attenuated in MG1655 / pQE80L-EgtD(AA)+p15A-Ptrc-cysG. (A) In the presence of L-tryptophan, the fluorescence of the colonies was significantly attenuated. In both cases, the decrease in the fluorescence of the colonies was due to the consumption of S-adenosylmethionine by the methylation reaction catalyzed by EgtD(AA) and the increase in CysG (A) The consumption of S-adenosylmethionine by the SUMT activity of CysG (A) This is thought to be due to a decrease in the amount of trimethylpyrocorphin produced, which is a fluorescent product of the SUMT activity of EgtD. Therefore, the difference in substrate specificity between EgtD and EgtD(AA) is visualized by the fluorescence of trimethylpyrocorphin, and it can be said that the difference in substrate specificity between EgtD and EgtD(AA) is detectable.

[0076] (CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped off from the LB solid medium into physiological saline, and the fluorescence intensity at a wavelength of 620 nm when excited at OD595 and 359 nm was measured using Cytation5 (BioTek). The values ​​of the fluorescence intensity divided by OD595 (fluorescence intensity / OD) are shown in Table 4 (rounded off to the nearest whole number).

[0077] [Table 4]

[0078] As shown in Table 4, similar to the above detection, MG1655 / pQE80L-EgtD+p15A-Ptrc-cysG (A) In the presence of L-histidine, the (fluorescence intensity / OD) of the colonies was small, and in the case of MG1655 / pQE80L-EgtD(AA)+p15A-Ptrc-cysG (A) In the presence of L-tryptophan, the (fluorescence intensity / OD) of the colonies was reduced. This indicates that the difference in substrate specificity between EgtD and EgtD(AA) can be measured by (fluorescence intensity / OD). It is also suggested that the difference in substrate specificity between EgtD and EgtD(AA) can be quantitatively analyzed by (fluorescence intensity / OD).

[0079] <Example 5. Evaluation of caffeine synthase activity 2> (Construction of plasmids) PCR was performed using a plasmid containing a sequence encoding superfolder GFP (SEQ ID NO: 35) as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 31 and 32 as a primer set to amplify a DNA fragment containing a gene encoding superfolder GFP. (A)PCR was performed using the above as a template and a primer set of oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 33 and 34 to amplify the expression vector sequence and obtain a linearized DNA fragment. The two DNA fragments obtained above were ligated using In-Fusion Snap Assembly Master Mix to obtain the plasmid p15A-Ptrc-sfGFP-cysG (A) In this plasmid, superfolder GFP (SEQ ID NO: 36) and CysG (A) is the fusion protein sfGFP-CysG (A) Figure 9 shows the expression of p15A-Ptrc-sfGFP-cysG (A) 1 is a plasmid map of

[0080] (Introduction of plasmids into E. coli, etc.) pTrc99a, and p15A-Ptrc-sfGFP-cysG (A) , and pQE80L-CCS1, and p15A-Ptrc-sfGFP-cysG (A) The E. coli MG1655 strain was transformed by electroporation to obtain MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG. (A) and MG1655 / pQE80L-CCS1+p15A-Ptrc-sfGFP-cysG (A) pTrc99a, which does not contain the gene encoding CCS1, was prepared as a control (Void). (A) and MG1655 / pQE80L-CCS1+p15A-Ptrc-sfGFP-cysG (A) Each was cultured with shaking in LB liquid medium containing 100 mg / L Amp and 30 mg / L Cm at 37° C. for 6 hours. The obtained culture solution was diluted and inoculated into LB solid medium containing 0.25 mM, 0.5 mM or no theobromine, which is a substrate of CCS1, and containing 100 mg / L Amp, 30 mg / L Cm, and 0.1 mM IPTG, and cultured at 30° C. for 21 hours.

[0081] (Superfolder GFP and CysG (A) Detection of fluorescence caused by After the cultivation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (Figure 10).

[0082] As shown in Figure 10, sfGFP-CysG (A) MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG was induced to express only (A) In the sfGFP-CysG colonies, the colonies showed yellow fluorescence, whereas in the sfGFP-CysG (A) and MG1655 / pQE80L-CCS1+p15A-Ptrc-sfGFP-cysG, which induced the expression of CCS1. (A) In the presence of 0.5 mM theobromine, the fluorescence hue changed from yellow to green in a theobromine concentration-dependent manner, and the colonies showed green fluorescence. (A) The consumption of S-adenosylmethionine by the SUMT activity of CysG (A) The amount of trimethylpyrocorphin, a fluorescent product of SUMT activity in (A) It is believed that the hue of the fluorescence changed from yellow, which is the result of overlapping with the fluorescence of trimethylpyrocorpin, a fluorescent product of the SUMT activity of CCS1, to green, which is the result of the fluorescence of superfolder GFP. Therefore, the activity of CCS1 is visualized as a change in the hue of the fluorescence, and it can be said that the activity of CCS1 can be detected.

[0083] (Superfolder GFP and CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped from the LB solid medium into physiological saline, and the fluorescence intensity at a wavelength of 535 nm (fluorescence from superfolder GFP) when excited at 485 nm and the fluorescence intensity at a wavelength of 620 nm (fluorescence from CysG) when excited at 359 nm were measured using Cytation5 (BioTek). (A)The fluorescence intensity at a wavelength of 620 nm when excited at 359 nm was divided by the fluorescence intensity at a wavelength of 535 nm when excited at 485 nm (CysG (A) The fluorescence intensity / GFP fluorescence intensity is shown in Table 5 (rounded to the fifth decimal place).

[0084] [Table 5]

[0085] As shown in Table 5, sfGFP-CysG was expressed by IPTG. (A) and MG1655 / pQE80L-CCS1+p15A-Ptrc-sfGFP-cysG, which induced the expression of CCS1. (A) In the 1990s, theobromine concentration was dependent on the number of colonies (CysG (A) The fluorescence intensity / GFP fluorescence intensity) became smaller. (A) It was shown that the activity of CCS1 can be measured by the ratio of CysG (A) It was suggested that quantitative analysis of CCS1 activity is possible using the ratio (fluorescence intensity / GFP fluorescence intensity).

[0086] <Example 6. Evaluation of L-histidine N-α-methyltransferase activity 2> (Introduction of plasmids into E. coli, etc.) pTrc99a, and p15A-Ptrc-sfGFP-cysG (A) , and pQE80L-EgtD, and p15A-Ptrc-sfGFP-cysG (A) The E. coli MG1655 strain was transformed by electroporation to obtain MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG. (A) and MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG (A) pTrc99a, which does not contain the gene encoding EgtD, was prepared as a control (Void). (A)and MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG (A) Each was cultured with shaking in LB liquid medium containing 100 mg / L Amp and 30 mg / L Cm at 37° C. for 6 hours. The obtained culture solution was diluted and inoculated onto LB solid medium containing 2 g / L, 4 g / L, or no L-histidine, which is a substrate for EgtD, and containing 100 mg / L Amp, 30 mg / L Cm, and 0.1 mM IPTG, and cultured at 30° C. for 21 hours.

[0087] (Superfolder GFP and CysG (A) Detection of fluorescence caused by After the cultivation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (FIG. 11).

[0088] As shown in FIG. 11, sfGFP-CysG (A) MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG was induced to express only (A) In the sfGFP-CysG colonies, the colonies showed yellow fluorescence, whereas in the sfGFP-CysG (A) and MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG, which induced the expression of EgtD. (A) In the presence of 4 g / L L-histidine, the fluorescence hue changed from yellow to green depending on the L-histidine concentration, and the colonies showed green fluorescence. (A) The consumption of S-adenosylmethionine by the SUMT activity of CysG (A) The amount of trimethylpyrocorphin, a fluorescent product of SUMT activity in (A)It is believed that the hue of the fluorescence changed from yellow, which is the result of overlapping with the fluorescence of trimethylpyrocorpin, a fluorescent product of SUMT activity, to green, which is the result of the fluorescence of superfolder GFP. Therefore, EgtD activity is visualized as a change in the hue of the fluorescence, and it can be said that EgtD activity can be detected.

[0089] (Superfolder GFP and CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped from the LB solid medium into physiological saline, and the fluorescence intensity at a wavelength of 535 nm (fluorescence from superfolder GFP) when excited at 485 nm and the fluorescence intensity at a wavelength of 620 nm (fluorescence from CysG) when excited at 359 nm were measured using Cytation5 (BioTek). (A) The fluorescence intensity at a wavelength of 620 nm when excited at 359 nm was divided by the fluorescence intensity at a wavelength of 535 nm when excited at 485 nm (CysG (A) The fluorescence intensity / GFP fluorescence intensity is shown in Table 6 (rounded to the fifth decimal place).

[0090] [Table 6]

[0091] As shown in Table 6, sfGFP-CysG was expressed by IPTG. (A) and MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG, which induced the expression of EgtD. (A) In the present study, the (CysG (A) The fluorescence intensity / GFP fluorescence intensity) became smaller. (A) It was shown that the activity of EgtD can be measured by the ratio of CysG (A) It was suggested that quantitative analysis of EgtD activity is possible by using the ratio (fluorescence intensity / GFP fluorescence intensity).

[0092] <Example 7. Evaluation of the activity of L-histidine N-α-methyltransferase mutants 2> (Introduction of plasmids into E. coli, etc.) pQE80L-EgtD and p15A-Ptrc-sfGFP-cysG (A) , and pQE80L-EgtD(AA), and p15A-Ptrc-sfGFP-cysG (A) The E. coli MG1655 strain was transformed with each of the vectors by electroporation to obtain MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG. (A) and MG1655 / pQE80L-EgtD(AA)+p15A-Ptrc-sfGFP-cysG (A) The resulting MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG (A) and MG1655 / pQE80L-EgtD(AA)+p15A-Ptrc-sfGFP-cysG (A) Each was cultured with shaking in LB liquid medium containing 100 mg / L Amp and 30 mg / L Cm at 37° C. for 3 hours. The obtained culture solution was diluted and inoculated onto LB solid medium containing 4 g / L L-histidine, which is a substrate for EgtD, 5 g / L L-tryptophan, which is a substrate for EgtD(AA), or neither, but containing 100 mg / L Amp, 30 mg / L Cm, and 0.1 mM IPTG, and cultured at 30° C. for 21 hours.

[0093] (Superfolder GFP and CysG (A) Detection of fluorescence caused by After the cultivation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (FIG. 12).

[0094] As shown in FIG. 12, MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG (A) In the presence of L-histidine, the fluorescence of the colonies changed from yellow to green, whereas in the absence of L-histidine, the fluorescence of the colonies changed from yellow to green.(A) In the presence of L-tryptophan, the fluorescence of the colonies changed from orange in the absence of L-tryptophan to green. Therefore, the difference in substrate specificity between EgtD and EgtD(AA) was visualized as a change in the hue of the fluorescence, and it can be said that the difference in substrate specificity between EgtD and EgtD(AA) is detectable.

[0095] (Superfolder GFP and CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped from the LB solid medium into physiological saline, and the fluorescence intensity at a wavelength of 535 nm (fluorescence from superfolder GFP) when excited at 485 nm and the fluorescence intensity at a wavelength of 620 nm (fluorescence from CysG) when excited at 359 nm were measured using Cytation5 (BioTek). (A) The fluorescence intensity at a wavelength of 620 nm when excited at 359 nm was divided by the fluorescence intensity at a wavelength of 535 nm when excited at 485 nm (CysG (A) The fluorescence intensity / GFP fluorescence intensity is shown in Table 7 (rounded to the fifth decimal place).

[0096] [Table 7]

[0097] As shown in Table 7, similar to the above detection, MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG (A) In the presence of L-histidine, the (CysG (A) Fluorescence intensity / GFP fluorescence intensity) is smaller, and MG1655 / pQE80L-EgtD(AA)+p15A-Ptrc-sfGFP-cysG (A) In the presence of L-tryptophan, the (CysG (A) The fluorescence intensity / GFP fluorescence intensity) became smaller. (A) It was shown that the difference in substrate specificity between EgtD and EgtD(AA) can be measured by the ratio of CysG (A)It was suggested that quantitative analysis of the difference in substrate specificity between EgtD and EgtD(AA) is possible by using the ratio of fluorescence intensity to GFP fluorescence intensity.

[0098] Example 8: Evaluation of dopamine-O-methyltransferase activity (Construction of plasmids) PCR was performed using a plasmid containing an artificially synthesized sequence (SEQ ID NO: 38) encoding dopamine-O-methyltransferase NpN4OMT1 (amino acid sequence SEQ ID NO: 37) derived from Narcissus genus plants as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 41 and 42 as a primer set to amplify a DNA fragment containing a gene encoding NpN4OMT1. Next, PCR was performed using pQE80L-EgtD as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 11 and 12 as a primer set to obtain a DNA fragment in which the expression vector sequence was amplified and linearized. The two DNA fragments obtained above were ligated using In-Fusion Snap Assembly Master Mix (manufactured by Takara Bio Inc.) to obtain the plasmid pQE80L-NpN4OMT1. Figure 13 is a plasmid map of pQE80L-NpN4OMT1.

[0099] (Introduction of plasmids into E. coli, etc.) pTrc99a, and p15A-Ptrc-cysG (A) , and pQE80L-NpN4OMT1, and p15A-Ptrc-cysG (A) Then, E. coli MG1655 strain was transformed by electroporation to obtain MG1655 / pTrc99a+p15A-Ptrc-cysG. (A) and MG1655 / pQE80L-NpN4OMT1+p15A-Ptrc-cysG (A) The pTrc99a gene, which does not contain the gene encoding NpN4OMT1, was prepared as a control (Void). (A) and MG1655 / pQE80L-NpN4OMT1+p15A-Ptrc-cysG(A) Each was cultured with shaking in LB liquid medium containing 100 mg / L Amp and 30 mg / L Cm at 37° C. for 3.5 hours. The obtained culture solution was diluted and inoculated onto LB solid medium containing 100 mg / L Amp, 30 mg / L Cm, and 0.1 mM IPTG, with or without 1 mM dopamine hydrochloride, which is a substrate of NpN4OMT1, and cultured at 30° C. for 24 hours.

[0100] (CysG (A) Detection of fluorescence caused by After the cultivation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (FIG. 14).

[0101] As shown in FIG. 14, IPTG inhibited CysG (A) and MG1655 / pQE80L-NpN4OMT1+p15A-Ptrc-cysG, which induced the expression of NpN4OMT1. (A) In the presence of dopamine hydrochloride, the fluorescence of the colonies was attenuated in a dopamine hydrochloride-dependent manner. The attenuation of the fluorescence of the colonies was due to the consumption of S-adenosylmethionine by the methylation reaction catalyzed by NpN4OMT1 and the (A) The consumption of S-adenosylmethionine by the SUMT activity of CysG (A) This is thought to be due to a decrease in the amount of trimethylpyrocorphin produced, which is a fluorescent product of the SUMT activity of NpN4OMT1. Therefore, the activity of NpN4OMT1 is visualized, and it can be said that the activity of NpN4OMT1 can be detected.

[0102] (CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped off from the LB solid medium and placed in physiological saline, and the fluorescence intensity at 620 nm when excited at OD595 and 359 nm (CysG (A) The fluorescence intensity was divided by OD595 (fluorescence intensity / OD) and the values ​​are shown in Table 8 (rounded off to the nearest whole number).

[0103] [Table 8]

[0104] As shown in Table 8, similar to the above detection, IPTG inhibited CysG (A) and MG1655 / pQE80L-NpN4OMT1+p15A-Ptrc-cysG, which induced the expression of NpN4OMT1. (A) In the case of the void, a significant decrease in the (fluorescence intensity / OD) of the colonies was confirmed by the addition of dopamine hydrochloride compared to the void. Therefore, it was shown that the activity of NpN4OMT1 can be measured by (fluorescence intensity / OD). It was also suggested that the activity of NpN4OMT1 can be quantitatively analyzed by (fluorescence intensity / OD).

[0105] <Example 9. Evaluation of dopamine-O-methyltransferase activity 2> (Introduction of plasmids into E. coli, etc.) pTrc99a, and p15A-Ptrc-sfGFP-cysG (A) , and pQE80L-NpN4OMT1, and p15A-Ptrc-sfGFP-cysG (A) The E. coli MG1655 strain was transformed by electroporation to obtain MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG. (A) and MG1655 / pQE80L-NpN4OMT1+p15A-Ptrc-sfGFP-cysG (A) pTrc99a, which does not contain the gene encoding NpN4OMT1, was prepared as a control (Void). (A) and MG1655 / pQE80L-NpN4OMT1+p15A-Ptrc-sfGFP-cysG (A)Each was cultured with shaking in LB liquid medium containing 100 mg / L Amp and 30 mg / L Cm for 3.5 hours at 37° C. The resulting culture was diluted and inoculated onto LB solid medium containing 100 mg / L Amp, 30 mg / L Cm, and 0.1 mM IPTG, or without 1 mM dopamine hydrochloride, which is a substrate of NpN4OMT1, and cultured at 30° C. for 24 hours.

[0106] (Superfolder GFP and CysG (A) Detection of fluorescence caused by After the cultivation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (FIG. 15).

[0107] As shown in FIG. 15, sfGFP-CysG (A) MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG was induced to express only (A) In the sfGFP-CysG colonies, the fluorescence color was orange to pink, whereas in the sfGFP-CysG colonies, the fluorescence color was orange to pink. (A) and MG1655 / pQE80L-EgtD+p15A-Ptrc-sfGFP-cysG induced expression of NpN4OMT1. (A) In the presence of dopamine hydrochloride, a change in the fluorescence hue to green was observed. (A) The consumption of S-adenosylmethionine by the SUMT activity of CysG (A) The amount of trimethylpyrocorphin, a fluorescent product of SUMT activity in (A) It is considered that the hue of the fluorescence changed from orange to pink, which is overlapped with the fluorescence of trimethylpyrocorpin, a fluorescent product of SUMT activity, to green, which is dominated by the fluorescence of superfolder GFP. Therefore, the activity of NpN4OMT1 is visualized as a change in the hue of the fluorescence, and it can be said that the activity of NpN4OMT1 can be detected.

[0108] (Superfolder GFP and CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped from the LB solid medium into physiological saline, and the fluorescence intensity at a wavelength of 535 nm (fluorescence from superfolder GFP) when excited at 485 nm and the fluorescence intensity at a wavelength of 620 nm (fluorescence from CysG) when excited at 359 nm were measured using an SH-9000Lab (Corona). (A) The fluorescence intensity at a wavelength of 620 nm when excited at 359 nm was divided by the fluorescence intensity at a wavelength of 535 nm when excited at 485 nm (CysG (A) The fluorescence intensity / GFP fluorescence intensity is shown in Table 9 (rounded to the fifth decimal place).

[0109] [Table 9]

[0110] As shown in Table 9, sfGFP-CysG was expressed by IPTG. (A) and MG1655 / pQE80L-NpN4OMT1+p15A-Ptrc-sfgfp-cysG, which induced the expression of NpN4OMT1. (A) In the case of β-lactamase, the (CysG (A) The fluorescence intensity / GFP fluorescence intensity) became smaller. (A) It was shown that the activity of NpN4OMT1 can be measured by the ratio of CysG (A) It was suggested that quantitative analysis of NpN4OMT1 activity is possible using the ratio (fluorescence intensity / GFP fluorescence intensity).

[0111] Example 10: Evaluation of S-adenosylmethionine decarboxylase activity (Construction of plasmids) PCR was performed using a sequence (SEQ ID NO: 40) encoding S-adenosylmethionine decarboxylase SpeD (amino acid sequence SEQ ID NO: 39, hereinafter also simply referred to as "SpeD") derived from Escherichia coli, with the genomic DNA of Escherichia coli MG1655 as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 43 and 44 as a primer set, to amplify a DNA fragment containing a gene encoding SpeD. Next, PCR was performed using pQE80L-EgtD as a template and oligonucleotides consisting of the base sequences represented by SEQ ID NOs: 11 and 12 as a primer set, to obtain a DNA fragment in which the expression vector sequence was amplified and linearized. The two DNA fragments obtained above were ligated using In-Fusion Snap Assembly Master Mix (manufactured by Takara Bio Inc.) to obtain the plasmid pQE80L-SpeD. Figure 16 is a plasmid map of pQE80L-SpeD.

[0112] (Introduction of plasmids into E. coli, etc.) pTrc99a, and p15A-Ptrc-cysG (A) , and pQE80L-speD, and p15A-Ptrc-cysG (A) Then, E. coli MG1655 strain was transformed by electroporation to obtain MG1655 / pTrc99a+p15A-Ptrc-cysG. (A) and MG1655 / pQE80L-speD+p15A-Ptrc-cysG (A) The pTrc99a gene, which does not contain the gene encoding speD, was prepared as a control (Void). (A) and MG1655 / pQE80L-speD+p15A-Ptrc-cysG (A) Each was cultured in LB liquid medium containing 100 mg / L Amp and 30 mg / L Cm with shaking at 37° C. for 3.5 hours. The resulting culture was diluted and inoculated onto LB solid medium containing 100 mg / L Amp, 30 mg / L Cm, and 0.1 mM IPTG, and cultured at 30° C. for 24 hours.

[0113] (CysG (A) Detection of fluorescence caused by After the cultivation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (FIG. 17).

[0114] As shown in FIG. 17, IPTG inhibited CysG (A) and MG1655 / pQE80L-speD+p15A-Ptrc-cysG, which induced the expression of SpeD. (A) In the case of CysG (A) MG1655 / pTrc99a+p15A-Ptrc-cysG was induced to express only (A) The fluorescence of the colonies was weakened compared to that of the control. The weakening of the fluorescence of the colonies was due to the consumption of S-adenosylmethionine by the SpeD-catalyzed S-adenosylmethionine decarboxylation reaction, which uses only S-adenosylmethionine present in the cells as a substrate, and the decarboxylation reaction of CysG. (A) The consumption of S-adenosylmethionine by the SUMT activity of CysG (A) This is thought to be due to a decrease in the amount of trimethylpyrocorphin produced, which is a fluorescent product of the SUMT activity of SpeD. Therefore, the activity of SpeD is visualized, and it can be said that the activity of SpeD can be detected.

[0115] (CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped off from the LB solid medium and placed in physiological saline, and the fluorescence intensity at 620 nm when excited at OD595 and 359 nm (CysG (A) The fluorescence intensity was divided by OD595 (fluorescence intensity / OD) and the values ​​are shown in Table 10 (rounded to the nearest whole number).

[0116] [Table 10]

[0117] As shown in Table 10, similar to the above detection, IPTG inhibited CysG (A)and MG1655 / pQE80L-speD+p15A-Ptrc-cysG, which induced the expression of SpeD. (A) In the case of the void, a significant decrease in the (fluorescence intensity / OD) of the colonies was confirmed compared to the void. Therefore, it was shown that the activity of SpeD can be measured by (fluorescence intensity / OD). It was also suggested that quantitative analysis of SpeD activity is possible by (fluorescence intensity / OD).

[0118] <Example 11. Evaluation of S-adenosylmethionine decarboxylase activity 2> (Introduction of plasmids into E. coli, etc.) pTrc99a, and p15A-Ptrc-sfGFP-cysG (A) , and pQE80L-speD, and p15A-Ptrc-sfGFP-cysG (A) The E. coli MG1655 strain was transformed by electroporation to obtain MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG. (A) and MG1655 / pQE80L-speD+p15A-Ptrc-sfGFP-cysG (A) pTrc99a, which does not contain the gene encoding SpeD, was prepared as a control (Void). (A) and MG1655 / pQE80L-speD+p15A-Ptrc-sfGFP-cysG (A) Each was cultured in LB liquid medium containing 100 mg / L Amp and 30 mg / L Cm with shaking at 37° C. for 3.5 hours. The resulting culture was diluted and inoculated onto LB solid medium containing 100 mg / L Amp, 30 mg / L Cm, and 0.1 mM IPTG, and cultured at 30° C. for 24 hours.

[0119] (Superfolder GFP and CysG (A) Detection of fluorescence caused by After the cultivation was completed, the grown colonies were placed under a 365 nm ultraviolet lamp and photographed (FIG. 18).

[0120] As shown in FIG. 18, sfGFP-CysG (A) MG1655 / pTrc99a+p15A-Ptrc-sfGFP-cysG was induced to express only (A) In the sfGFP-CysG colonies, the fluorescence color was orange to pink, whereas in the sfGFP-CysG colonies, the fluorescence color was orange to pink. (A) and MG1655 / pQE80L-speD+p15A-Ptrc-sfGFP-cysG, in which SpeD expression was induced. (A) In the case of β-adenosylmethionine, a hue change to green fluorescence was observed. The consumption of S-adenosylmethionine by the decarboxylation reaction of S-adenosylmethionine, which is catalyzed by SpeD and which uses only S-adenosylmethionine present in the cell as a substrate, and the consumption of CysG (A) The consumption of S-adenosylmethionine by the SUMT activity of CysG (A) The amount of trimethylpyrocorphin, a fluorescent product of SUMT activity in (A) This is thought to be due to a change in the hue of the fluorescence from the orange to pink color overlapping with the fluorescence of trimethylpyrocorpin, a fluorescent product of the SUMT activity of SpeD, to green, dominated by the fluorescence of superfolder GFP. Therefore, the activity of SpeD is visualized as a change in the hue of the fluorescence, and it can be said that the activity of SpeD can be detected.

[0121] (Superfolder GFP and CysG (A) Measurement of fluorescence produced by In addition, colonies were scraped from the LB solid medium into physiological saline, and the fluorescence intensity at a wavelength of 535 nm (fluorescence from superfolder GFP) when excited at 485 nm and the fluorescence intensity at a wavelength of 620 nm (fluorescence from CysG) when excited at 359 nm were measured using an SH-9000Lab (Corona). (A) The fluorescence intensity at a wavelength of 620 nm when excited at 359 nm was divided by the fluorescence intensity at a wavelength of 535 nm when excited at 485 nm (CysG (A) The fluorescence intensity / GFP fluorescence intensity is shown in Table 11 (rounded to the fifth decimal place).

[0122] [Table 11]

[0123] As shown in Table 11, similar to the above detection, IPTG stimulated the expression of sfGFP-CysG (A) and MG1655 / pQE80L-speD+p15A-Ptrc-cysG, which induced the expression of SpeD. (A) In the case of void, the colony size (CysG (A) A decrease in the fluorescence intensity / GFP fluorescence intensity was confirmed. (A) It was shown that the activity of SpeD can be measured by the ratio of CysG (A) It was suggested that quantitative analysis of SpeD activity is possible by using the ratio (fluorescence intensity / GFP fluorescence intensity).

Claims

1. Competing consumption of S-adenosylmethionine by a reaction catalyzed by a first enzyme and consumption of S-adenosylmethionine by a reaction catalyzed by a second enzyme; evaluating the fluorescence and / or color produced by the reaction catalyzed by the second enzyme; A method for assessing the activity of the first enzyme comprising:

2. introducing a first expression vector containing a gene encoding the first enzyme into a cell; introducing into the cell a second expression vector comprising a gene encoding the second enzyme, The competition for consumption of S-adenosylmethionine takes place within the cell. The method of claim 1.

3. The method of claim 2 , wherein the cell is a bacterium belonging to the genus Escherichia.

4. The method according to any one of claims 1 to 3, wherein the second enzyme comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 27 and has S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase activity.

5. The method according to claim 4 , wherein the fluorescence and / or color change is the fluorescence of trimethylpyrocorpin.

6. The method according to any one of claims 1 to 3, wherein the second enzyme is a fusion protein containing a fluorescent protein.

7. The method according to any one of claims 1 to 3, wherein the first enzyme is an enzyme selected from the group consisting of methyltransferase, radical S-adenosylmethionine enzyme, S-adenosylmethionine decarboxylase, N-acylhomoserine lactone synthase, opine metallophore synthase, 5'-deoxy-5'-fluoroadenosine synthase, 5'-deoxy-5'-chloroadenosine synthase, and azetidine-2-carboxylic acid synthase.

8. an enzyme having S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase activity; S-adenosylmethionine, and Uroporphyrinogen III and a kit for evaluating the activity of an enzyme different from the enzyme in a reaction consuming S-adenosylmethionine, comprising:

9. an enzyme having S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase activity; S-adenosylmethionine, and 5-aminolevulinic acid, porphobilinogen synthase, porphobilinogen deaminase, and uroporphyrinogen III synthase and a kit for evaluating the activity of an enzyme different from the enzyme in a reaction consuming S-adenosylmethionine, comprising:

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