Novel gene for apiin production and method for producing apiin using the same

Abstract

To provide a method for identifying a gene encoding an apiose transferase for converting apigenin into apiin and a gene encoding a glucose transferase, and consistently producing apiin from apigenin by using the apiose transferase and the glucose transferase encoded by the gene.SOLUTION: Provided is an apiose transferase including a specific amino acid sequence. Further, provided is a method for producing apiin from apigenin 7-O glucoside which includes a step of bringing the apiose transferase, apigenin 7-O glucoside and UDP-apiose in contact each other.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel glucosyltransferase having the activity of producing apigenin 7-O glucoside from apigenin and UDP-glucose, a novel apiosetransferase having the activity of producing apiin from apigenin 7-O glucoside and UDP-apiose, and a method and a production kit for producing apiin from apigenin using them. [Background technology]

[0002] Apigenin, a type of flavone produced by plants, has anti-anxiety and cancer cell proliferation inhibitory effects, and has been reported to be useful for treating diabetes, amnesia, depression, etc. Although there have been reports of methods for producing apigenin in Escherichia coli (Non-Patent Document 1) and naringenin (Non-Patent Document 2), which is a precursor of apigenin, it was necessary to bind sugar to apigenin to make it soluble in order to administer it effectively to humans.

[0003] Apiin is known as a glycoside of apigenin, and is known to be abundant in parsley and celery, which are members of the Umbelliferae family. Apiin is thought to be produced by apigenin 7-O glucoside, which is biosynthesized by a glucose transferase using UDP-glucose as a donor substrate, and then apiose is transferred to apigenin 7-O glucoside by apiose transferase using UDP-apiose as a donor substrate. However, the gene encoding the apiose transferase required to produce apiin from apigenin 7-O glucoside remains unknown, so the technology to produce apiin has not been established. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Miyahisa et al., Appl Microbiol Biotehcnol, 71, 53-58 (2006) [Non-Patent Document 2] Zhou et al., J Microbiol Biotechnol, 30, 1574-1582 (2020) Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is to identify a gene encoding an apiosetransferase and a gene encoding a glucosyltransferase for converting apigenin to apiin, and to establish a method for consistently producing apiin from apigenin using the apiosetransferase and glucosyltransferase encoded by the genes. [Means for solving the problem]

[0006] The inventors have succeeded in identifying a novel gene encoding a glucosyltransferase and a gene encoding an apiose transferase by selecting and cloning a candidate apiose transferase gene and a candidate glucosyltransferase gene by RNA-Seq analysis of parsley or celery, and confirming their activity. The inventors have also succeeded in producing apigenin 7-O-glucoside and the like by introducing a gene encoding the identified glucosyltransferase into E. coli, and allowing the glucosyltransferase purified from the resulting transformant to act in vitro on apigenin as an acceptor substrate and UDP-glucose as a donor substrate. Similarly, the inventors have succeeded in producing apiin and the like by introducing a gene encoding the identified apiose transferase into E. coli, and allowing the apiose transferase purified from the resulting transformant to act in vitro on apigenin 7-O-glucoside as an acceptor substrate and UDP-apiose as a donor substrate. Furthermore, the inventors confirmed that apigenin 7-O-glucoside and apiin were produced by introducing a gene encoding an apiose transferase and a gene encoding a glucose transferase into Escherichia coli and culturing the resulting transformant in the presence of apigenin. Based on these findings, the inventors conducted further investigations and have now completed the present invention.

[0007] That is, the present invention provides the following. [1] An apiose transferase comprising an amino acid sequence selected from the group consisting of the following (i) to (iv): (i) an amino acid sequence represented by SEQ ID NO:2; (ii) an amino acid sequence represented by SEQ ID NO:4; (iii) an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:2; (iv) An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:4. [2] The apiosyltransferase according to [1], which comprises the following amino acid sequence (i) or (ii): (i) an amino acid sequence represented by SEQ ID NO:2; (ii) The amino acid sequence represented by SEQ ID NO:4. [3] A glucose transferase comprising an amino acid sequence selected from the group consisting of the following (i) to (iv): (i) the amino acid sequence represented by SEQ ID NO:6; (ii) the amino acid sequence represented by SEQ ID NO:8; (iii) an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:6; (iv) An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:8. [4] The glucosyltransferase according to [3], which comprises the following amino acid sequence (i) or (ii): (i) the amino acid sequence represented by SEQ ID NO:6; (ii) The amino acid sequence represented by SEQ ID NO:8. [5] A method for producing apiin from apigenin 7-O glucoside, comprising the step of contacting the apiosyltransferase according to [1] or [2] with apigenin 7-O glucoside and UDP-apiose. [6] The method according to [5], characterized in that the step is carried out in a host cell which contains a nucleic acid having a base sequence encoding the apiose transferase according to [1] or [2] and produces UDP-apiose. [7] A method for producing apigenin 7-O glucoside from apigenin, comprising a step of contacting the glucosyltransferase according to [3] or [4] with apigenin and UDP-glucose. [8] The method according to [7], wherein the step is carried out in a host cell that contains a nucleic acid having a base sequence encoding the glucose transferase according to [3] or [4] and produces UDP-glucose. [9] A method for producing apiin from apigenin, comprising the following steps (i) and (ii): (i) contacting the glucosyltransferase according to [3] or [4] with apigenin and UDP-glucose to produce apigenin 7-O-glucoside; (ii) contacting the apiosyltransferase according to [1] or [2] with the apigenin 7-O-glucoside and UDP-apiose to produce apiin.

[10] The method according to [9], characterized in that steps (i) and (ii) are carried out in the same host cell which contains a nucleic acid having a base sequence encoding the glucose transferase described in [3] or [4] and a nucleic acid having a base sequence encoding the apiose transferase described in [1] or [2] and which produces UDP-glucose and UDP-apiose.

[11] A kit for producing apiin from apigenin 7-O glucoside, comprising the following (i) to (iii): (i) an apiosyltransferase according to [1] or [2]; (ii) apigenin 7-O-glucoside, (iii) UDP-apiose.

[12] A kit for producing apiin from apigenin 7-O glucoside, comprising the following (i) and (ii): (i) a host cell that contains a nucleic acid having a nucleotide sequence encoding the apiosyltransferase according to [1] or [2] and produces UDP-apiose; (ii) Apigenin 7-O-glucoside.

[13] A kit for producing apigenin 7-O glucoside from apigenin, comprising the following (i) to (iii): (i) The glucosyltransferase according to [3] or [4], (ii) apigenin, (iii) UDP-glucose.

[14] A kit for producing apigenin 7-O glucoside from apigenin, comprising the following (i) and (ii): (i) a host cell that contains a nucleic acid having a nucleotide sequence encoding the glucosyltransferase according to [3] or [4] and produces UDP-glucose; (ii) Apigenin.

[15] A kit for producing apiin from apigenin, comprising the following (i) to (v): (i) The glucosyltransferase according to [3] or [4], (ii) an apiosyltransferase according to [1] or [2]; (iii) apigenin, (iv) UDP-glucose, (v) UDP-apiose.

[16] A kit for producing apiin from apigenin, comprising the following (i) and (ii): (i) a host cell which contains a nucleic acid having a nucleotide sequence encoding the glucose transferase according to [3] or [4] and a nucleic acid having a nucleotide sequence encoding the apiose transferase according to [1] or [2], and which produces UDP-glucose and UDP-apiose; (ii) Apigenin. Effect of the Invention

[0008] By introducing the gene encoding the apiosetransferase and the gene encoding the glucosyltransferase of the present invention into a host cell that produces UDP-glucose and UDP-apiose and culturing the cell in the presence of apigenin, it becomes possible to easily produce apiin from apigenin. [Brief description of the drawings]

[0009] [Figure 1] (A) The amino acid sequence of AgApiT. The 44 amino acids on the C-terminal side are the PSPG motif characteristic of UGTs. (B) The alignment of the partial amino acid sequence of AgApiT, the partial amino acid sequence of UGT94-like proteins from the Apiaceae family (UGT94P1, UGT89A2), the partial amino acid sequence of Gl16345 from Glehnia littoralis (littoral seaweed), and the partial amino acid sequences of Fv4536, Fv22755.1, and Fv18654.1 from Foeniculum vulgare (fennel). The open Ile in AgApiT is predicted to be the pentose recognition site. The open Ile in UGT94P1 and UGT89A2 is the amino acid in the pentose recognition site. The open Val in Gl16345, Fv4536, Fv22755.1 and Fv18654.1 is an amino acid in the pentose recognition site. [Diagram 2] 1 shows the AgApiT expression construct, which has a His tag and a ProS2 tag at the N-terminus of AgApiT. [Diagram 3] This is a polyacrylamide gel electrophoresis image of recombinant AgApiT expressed in E. coli. (-): 500 μL of E. coli culture before expression induction was centrifuged, and the cells were dissolved in 50 μL of 2x sample buffer. After incubation at 100°C for 3 minutes, 5 μL was applied. (+): 0.8 mM IPTG was added, and the E. coli culture after expression induction at 15°C for 24 hours was treated in the same manner as above. (AgApiT) Calculated molecular weight is 49 kDa. (AgApiT + tag protein) Calculated molecular weight is 73 kDa. (Tag protein) Calculated molecular weight is 24 kDa. [Figure 4] This is a diagram showing the detection of AgApiT activity. The upper chromatogram shows negative control 1 (tag protein), the middle chromatogram shows negative control 2 (without donor substrate UDP-Api), and the lower chromatogram shows the results after 2 hours of reaction. ▼ indicates apiin, and ▽ indicates apigenin 7-O-β-D-glucoside. [Diagram 5] FIG. 1 shows the enzyme activity of AgApiT. [Figure 6] (A) Time-dependence of the apiine synthesis reaction by AgApiT (B) Enzyme amount-dependence of the apiine synthesis reaction by AgApiT. [Figure 7] (A) Reaction rate for apigenin 7-O-β-D-glucoside (B) Reaction rate for UDP-Api. [Figure 8] Donor substrate dependence of AgApiT. 50 μM apigenin 7-O-β-D-glucoside, 0.5 mM sugar nucleotide, and 20 μg AgApiT were reacted at pH 7.0 and 23 °C for 2 h. Technical replicates were performed three times, and error bars represent the standard deviation of the results. The detection limit is 20 pU / g. [Figure 9] Figure 1 shows the acceptor substrate dependence of AgApiT. 50 μM acceptor substrate, 0.5 mM UDP-Api, and 20 μg AgApiT were reacted at pH 7.0 and 23°C for 2 h. Technical replicates were performed three times, and error bars represent the standard deviation of the results. The detection limit is 20 pU / g. [Figure 10] This figure shows the enzymatic activity of AgApiT towards various acceptor substrates. 50 μM acceptor substrate, 0.5 mM UDP-Api, and 20 μg AgApiT were reacted at pH 7.0 and 23°C for 2 hours. The upper chromatogram shows the negative control (no donor substrate), and the lower chromatogram shows the results after 2 hours of reaction. (A) Apigenin 7-O-β-D-glucoside (B) Quercetin 7-O-β-D-glucoside (C) Luteolin 7-O-β-D-glucoside (D) Naringenin 7-O-β-D-glucoside [Figure 11] Figure 1 shows the enzymatic activity of point mutants of AgApiT towards UDP-Api. 50 μM apigenin 7-O-β-D-glucoside, 0.5 mM UDP-Api, and 20 μg of point mutants of AgApiT were reacted at pH 7.0 and 23 °C for 2 h. Technical replicates were performed three times, and the error bars represent the standard deviation of the results. The detection limit is 20 pU / g. [Figure 12]FIG. 1 shows the expression level of the AgApiT gene at each developmental stage of celery. [Figure 13] Phylogenetic tree of AgGlcT candidate genes and UGTs. Celery RNA-seq read data (SRR1023730) was used to search for homologs of highly expressed UGT71, 72, 73, 78, and 88. Clusters were divided based on the sugar binding position. Circles represent nine candidate genes (UGT71, 73, and 88 encoded in the celery genome), squares represent seven candidate genes (genes whose expression was observed in celery leaf RNA-seq), and stars represent three candidate genes (the apiin biosynthetic enzyme gene AgFNS I and genes whose expression behavior was relatively similar to AgApiT). [Figure 14] Semi-quantitative PCR of AgGlcT candidate genes at each developmental stage of celery. (A) The gene expression levels of apiin biosynthesis-related enzyme genes (FNS I, ApiT) and seven candidate genes at each developmental stage were quantified by semi-quantitative PCR. GAPDH was used as an internal standard. (B) Graph showing the results of semi-quantitative PCR quantification of gene expression levels of apiin biosynthesis-related enzyme genes (FNS I, ApiT) and seven candidate genes at each developmental stage. [Figure 15] This is a diagram showing the expression levels of apiin biosynthesis-related enzyme genes (AgGlcT, AgApiT, AgFNS I) at each developmental stage of celery true leaves. (A) Expression level of AgGlcT at each developmental stage of celery true leaves. (B) Expression level of AgApiT at each developmental stage of celery true leaves. (C) Expression level of AgFNS I at each developmental stage of celery true leaves. The expression level of each gene at 0.5 cm was set to 1. [Figure 16] FIG. 1 shows expression of AgGlcT candidate proteins. [Figure 17] This shows the enzyme activity of the AgGlcT candidate protein. (A) The product was quantified using HPLC <Condition 1>. From the top, the peaks of DN23862, DN16008, and DN9954 are shown. ▼ indicates the peak of apigenin 7-O-glucoside, and ▽ indicates the peak of apigenin. (B) The enzyme activity of the product is shown. [Figure 18]This shows the enzyme amount and time dependence of AgGlcT. (A) 1 mM apigenin, 0.5 mM UDP-Glc, and 1 μg of parsley enzyme solution were reacted at pH 7.0, 23°C, and 0 to 4 hours. The amount of the product was quantified by HPLC <Condition 1>. (B) 1 mM apigenin, 0.5 mM UDP-Glc, and 10 to 1.8 μg of celery enzyme solution were reacted at pH 7.0, 23°C, and 2 hours. The amount of the product was quantified by HPLC <Condition 1>. [Figure 19] Figure 1 shows an amino acid sequence alignment of AgGlcT, in which underlines indicate catalytic residues, italics indicate PSPG motifs, and squares indicate GSS motifs. [Figure 20] FIG. 1 shows a predicted three-dimensional structure of AgGlcT. [Figure 21] FIG. 1 shows the pH dependence of AgGlcT. [Figure 22] This figure shows the donor substrate specificity of AgGlcT. (A) The product was quantified using HPLC <Condition 1>. (B) The structures of the top two substrates for which enzyme activity was detected are shown. AgGlcT recognizes the circled portion of UDP-Glc. [Figure 23] This shows the acceptor substrate dependency of AgGlcT. (A) The product was quantified using HPLC <Condition 1>. (B) The structures of the six substrates for which enzyme activity was detected are shown. AgGlcT recognizes the circled portions of apigenin, quercetin, luteolin, and chrysoeriol. [Figure 24] Figure showing the reaction kinetics of AgGlcT. (A) The reaction rate for UDP-Glc was determined. UDP-Glc was used at multiple concentrations from 0 to 500 μM. Km = 192.2 μM, Vmax = 36.2 pmol / min / μg. (B) The reaction rate for apigenin was determined. Apigenin was used at multiple concentrations from 0 to 1,000 μM. Km = 190.0 μM, Vmax = 37.6 pmol / min / μg. [Diagram 25] FIG. 1 shows amino acid sequence alignments between PcGlcT and AgGlcT, and between PcApiT and AgApiT. [Figure 26](A) Expression construct for recombinant PcGlcT protein, (B) SDS-PAGE of purified recombinant PcGlcT protein, (C) Expression construct for recombinant PcApiT protein, (D) SDS-PAGE of purified recombinant ProS2-PcApiT fusion protein and tag-cleaved PcApiT protein. [Figure 27] (A) A diagram showing apiin biosynthesis catalyzed by PcGlcT and PcApiT, (B) HPLC analysis of the enzymatic reaction of PcGlcT, and (C) HPLC analysis of the enzymatic reaction of PcApiT. [Figure 28] (A) Acceptor substrate specificity of PcGlcT. (B) Donor substrate specificity of PcGlcT. (C) Acceptor substrate specificity of PcApiT. (D) Donor substrate specificity of PcApiT. The average and standard deviation of triplicate measurements are shown. [Figure 29] (A) The optimum pH of PcGlcT (B) The optimum pH of PcApiT. [Diagram 30] (A) Expression patterns of PcFNSI in each organ and the expression levels at each stage of true leaf development measured by qRT-PCR. (B) Expression patterns of PcGlcT in each organ and the expression levels at each stage of true leaf development measured by qRT-PCR. (C) Expression patterns of PcApiT in each organ and the expression levels at each stage of true leaf development measured by qRT-PCR. [Diagram 31] FIG. 1 shows the gene base sequences (coding regions only) and protein amino acid sequences of PcGlcT and PcApiT. [Diagram 32] Figure 1 shows the results of SDS-PAGE and Western blot analysis of the soluble fractions of API001 (left) and API002 (right). Top: CBB staining, middle: Western blotting using Anti-His tag, bottom: Western blotting using Anti-S tag. [Diagram 33]1 shows the results of reverse-phase HPLC for API001 and API002. NC: E. coli strain into which only pColdProS2, pCDF-1b, and pACYCDuet were introduced. Apiin: Peak position of an apiin preparation. A7G: Peak position of an A7G preparation. Apigenin: Peak position of an apigenin preparation. [Diagram 34] FIG. 1 shows the production amounts of A7G and apiin in API001 and API002. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 1. Apiosyltransferase and glucosyltransferase The present invention provides an apiosetransferase (hereinafter, the apiosetransferase of the present invention). The present invention also provides a glucosyltransferase (hereinafter, the glucosyltransferase of the present invention).

[0011] The apiosyltransferase of the present invention comprises an amino acid sequence selected from the group consisting of the following (i) to (iv): (i) The amino acid sequence represented by SEQ ID NO:2. (ii) The amino acid sequence represented by SEQ ID NO:4. (iii) An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:2. (iv) An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:4. The apiosyltransferase of the present invention preferably comprises the following amino acid sequence (i) or (ii): (i) The amino acid sequence represented by SEQ ID NO:2. (ii) The amino acid sequence represented by SEQ ID NO:4.

[0012] The glucosyltransferase of the present invention comprises an amino acid sequence selected from the group consisting of the following (i) to (iv): (i) The amino acid sequence represented by SEQ ID NO:6. (ii) The amino acid sequence represented by SEQ ID NO:8. (iii) An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:6. (iv) An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:8. The glucosyltransferase of the present invention preferably comprises the following amino acid sequence (i) or (ii): (i) The amino acid sequence represented by SEQ ID NO:6. (ii) The amino acid sequence represented by SEQ ID NO:8.

[0013] The apiosetransferase containing the amino acid sequence represented by SEQ ID NO:2 and the glucosyltransferase containing the amino acid sequence represented by SEQ ID NO:6 may be proteins isolated and purified from celery. They may also be proteins chemically synthesized or biochemically synthesized in a cell-free translation system, or they may be recombinant proteins produced from a transformant into which a nucleic acid having a base sequence encoding the above amino acid sequence has been introduced. Similarly, the apiosetransferase containing the amino acid sequence represented by SEQ ID NO:4 and the glucosyltransferase containing the amino acid sequence represented by SEQ ID NO:8 may be proteins isolated and purified from parsley. They may also be proteins chemically synthesized or biochemically synthesized in a cell-free translation system, or they may be recombinant proteins produced from a transformant into which a nucleic acid having a base sequence encoding the above amino acid sequence has been introduced.

[0014] Examples of amino acid sequences substantially identical to the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8 include amino acid sequences having a homology of about 90% or more, preferably about 93% or more, more preferably about 95% or more, and particularly preferably about 98% or more, to the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8. Here, "homology" refers to the ratio (%) of identical and similar amino acid residues to the total overlapping amino acid residues in optimal alignment (preferably an algorithm that can take into account the introduction of gaps into one or both of the sequences for optimal alignment) when two amino acid sequences are aligned using a mathematical algorithm known in the art. "Similar amino acids" refer to amino acids that are similar in physicochemical properties, and examples of such amino acids include aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), and amino acids with small side chains (Gly, Ala, Ser, Thr, Met) that are classified into the same group. It is predicted that substitution with such similar amino acids will not cause a change in the phenotype of the protein (i.e., it is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various documents (see, for example, Bowie et al., Science, 247:1306-1310 (1990)).

[0015] The amino acid sequence homology in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [this algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], the algorithm described in Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [this algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [this algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package], the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [this algorithm is incorporated into the FASTA program in the GCG software package], and the like, which can also be preferably used.

[0016] More preferably, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8 is an amino acid sequence that has an identity of about 90% or more, preferably about 93% or more, even more preferably about 95% or more, and particularly preferably about 98% or more to the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8.

[0017] Preferred examples of the apiosetransferase comprising substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or 4 include proteins that comprise substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or 4 and have substantially the same activity as the apiosetransferase comprising the amino acid sequence represented by SEQ ID NO: 2 or 4. Preferred examples of the apiosetransferase comprising substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 6 or 8 include proteins that comprise substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 6 or 8 and have substantially the same activity as the glucose transferase comprising the amino acid sequence represented by SEQ ID NO: 6 or 8.

[0018] Examples of activities substantially equivalent to those of an apiosetransferase comprising the amino acid sequence represented by SEQ ID NO:2 include the activity of producing apiin, quercetin 7-O-glucoside, luteolin 7-O-glucoside, or naringenin 7-O-glucoside using apigenin 7-O-glucoside, quercetin 7-O-glucoside, luteolin 7-O-glucoside, or naringenin 7-(2-O-apiosylglucoside) using UDP-apiose as a donor substrate, preferably the activity of producing apiin from apigenin 7-O-glucoside and UDP-apiose. Furthermore, examples of activities substantially equivalent to those of an apiosetransferase containing the amino acid sequence represented by SEQ ID NO:4 include the activity of producing apiin, quercetin 7-(2-O-apiosylglucoside), or chrysoeriol 7-(2-O-apiosylglucoside) using apigenin 7-O-glucoside, quercetin 7-O-glucoside, or chrysoeriol 7-O-glucoside as an acceptor substrate and UDP-apiose as a donor substrate, preferably the activity of producing apiin from apigenin 7-O-glucoside and UDP-apiose. As an example of an activity substantially equivalent to that of a glucose transferase containing the amino acid sequence represented by SEQ ID NO:6, apigenin 7-O-glucoside, quercetin 7-O-glucoside, luteolin 7-O-glucoside, chrysoeriol 7-O-glucoside, naringenin, or genistein is used as an acceptor substrate, and UDP-glucose or UDP-xylose is used as a donor substrate. Examples of the activity that can produce apigenin 7-O-glucoside, naringenin 7-O-glucoside, genistein 7-O-glucoside, apigenin 7-O-xyloside, quercetin 7-O-xyloside, luteolin 7-O-xyloside, chrysoeriol 7-O-xyloside, naringenin 7-O-xyloside or genistein 7-O-xyloside, preferably, the activity that can produce apigenin 7-O-glucoside from apigenin and UDP-glucose, and the like.Furthermore, examples of the activity substantially equivalent to that of the glucosyltransferase containing the amino acid sequence represented by SEQ ID NO: 8 include, for example, the activity of producing apigenin 7-O-glucoside, quercetin 7-O-glucoside, luteolin 7-O-glucoside, chrysoeriol 7-O-glucoside, naringenin 7-O-glucoside, or genistein 7-O-glucoside using apigenin, quercetin, luteolin, chrysoeriol, naringenin, or genistein as an acceptor substrate and UDP-glucose as a donor substrate, and preferably the activity of producing apigenin 7-O-glucoside from apigenin and UDP-glucose. "Substantially equivalent" means that the activities are qualitatively (e.g., enzymatically or biochemically) the same. Therefore, it is preferable that the above activities are equivalent (e.g., about 0.5 to about 2 times). The above activity can be measured according to a method known per se, for example, the activity can be measured by quantifying apiin, etc. using HPLC.

[0019] Examples of amino acid sequences that are substantially identical to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8 include: (1) an amino acid sequence having one or more (preferably about 1 to 14, more preferably about 1 to 10, and even more preferably one or several (2, 3, 4, or 5)) amino acid substitutions in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8; (2) an amino acid sequence having one or more (preferably about 1 to 14, more preferably about 1 to 10, and even more preferably one or several (2, 3, 4, or 5)) amino acid deletions in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8; (3) an amino acid sequence having one or more (preferably about 1 to 14, more preferably about 1 to 10, and even more preferably one or several (2, 3, 4, or 5)) amino acid insertions in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8; and (4) Also included are amino acid sequences in which one or more (preferably about 1 to 14, more preferably about 1 to 10, and even more preferably one to several (2, 3, 4 or 5)) amino acids have been added to the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8, or (5) amino acid sequences that are a combination thereof. When an amino acid sequence is substituted, deleted, inserted or added as described above, the position is not particularly limited as long as the apiosyltransferase or glucosyltransferase activity is maintained.

[0020] The apiosetransferase of the present invention is preferably a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. More preferably, the apiosetransferase of the present invention is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4. Furthermore, the glucosyltransferase of the present invention is preferably a protein comprising the amino acid sequence shown in SEQ ID NO: 6 or 8. More preferably, the glucosyltransferase of the present invention is a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 or 8.

[0021] The apiosetransferase or glucosyltransferase of the present invention can be produced by a publicly known method for purifying proteins from the above-mentioned celery, parsley, etc. Specifically, a method in which celery or parsley is crushed and then a crude extract of soluble proteins is obtained by centrifugation or filtration is appropriately used. The apiosetransferase or glucosyltransferase contained in the soluble fraction thus obtained can be isolated and purified by a method known per se. Examples of such methods include methods that utilize solubility, such as salting out or solvent precipitation; methods that mainly utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reversed-phase high performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing. These methods can also be combined as appropriate.

[0022] The apiosyltransferase or glucosyltransferase of the present invention can also be produced according to known peptide synthesis methods. The peptide synthesis method may be, for example, either solid phase synthesis or liquid phase synthesis. The target protein can be produced by condensing an amino acid derivative having a protecting group on the carboxyl group and the functional group of the side chain with an amino acid derivative having a protected amino group and the functional group of the side chain, and then removing the protecting group. Here, the condensation and removal of the protecting group are carried out according to a method known per se, for example, the methods described in (1) to (5) below. (1) M. Bodanszky and M.A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966) (2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965) (3) Nobuo Izumiya et al., Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975) (4) Haruaki Yajima and Shumpei Sakakibara, Biochemistry Experiment Course 1, Chemistry of Proteins IV 205 (1977) (5) Haruaki Yajima, editor, Pharmaceutical Development, Vol. 14, Peptide Synthesis, Hirokawa Shoten

[0023] The apiosetransferase or glucosyltransferase thus obtained can be purified and isolated by known purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, or a combination thereof.

[0024] The apiosetransferase or glucosyltransferase of the present invention may be a recombinant protein produced from a host cell (host cell of the present invention) into which a nucleic acid having a nucleotide sequence encoding the enzyme has been introduced. The nucleic acid having a nucleotide sequence encoding the apiosetransferase or glucosyltransferase of the present invention may be DNA or RNA, or may be a DNA / RNA chimera. DNA is preferred. The nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. If single-stranded, it may be the sense strand (i.e., the coding strand) or the antisense strand (i.e., the non-coding strand).

[0025] Examples of nucleic acids having a nucleotide sequence encoding the apiosetransferase of the present invention include nucleic acids containing the nucleotide sequence shown in SEQ ID NO: 2 or 4, or nucleic acids containing a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 2 or 4 and that encode a protein having substantially the same activity as the apiosetransferase of the present invention. Examples of nucleic acids having a nucleotide sequence encoding the glucosyltransferase of the present invention include nucleic acids containing the nucleotide sequence shown in SEQ ID NO: 6 or 8, or nucleic acids containing a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 6 or 8 and that encode a protein having substantially the same activity as the glucosyltransferase of the present invention. Examples of base sequences that can hybridize under stringent conditions to the base sequence represented by SEQ ID NO: 2, 4, 6 or 8 include base sequences that have a homology of about 80% or more, preferably about 85% or more, more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more to the base sequence represented by SEQ ID NO: 2, 4, 6 or 8. The homology of base sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3). As other algorithms for determining the homology of base sequences, the above-mentioned amino acid sequence homology calculation algorithms are also preferably exemplified.

[0026] Hybridization can be performed according to a method known per se or a method similar thereto, for example, the method described in Molecular Cloning, 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When using a commercially available library, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed under stringent conditions. Highly stringent conditions include, for example, a hybridization reaction in 6×SSC (sodium chloride / sodium citrate) at 45° C., followed by one or more washes in 0.2×SSC / 0.1% SDS at 65° C. Those skilled in the art can easily adjust the stringency to a desired level by appropriately changing the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the probe length, the number of mismatches, the hybridization reaction time, the salt concentration of the washing solution, the washing temperature, etc.

[0027] The nucleic acid having a nucleotide sequence encoding the apiosetransferase of the present invention is preferably a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 2 or 4, more preferably a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 2 or 4. The nucleic acid having a nucleotide sequence encoding the glucosyltransferase of the present invention is preferably a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 6 or 8, more preferably a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 6 or 8.

[0028] The nucleic acid having a base sequence encoding the apiosetransferase or glucosyltransferase of the present invention can be amplified by PCR using a synthetic DNA primer having a portion of the base sequence encoding the apiosetransferase or glucosyltransferase of the present invention, using a cDNA derived from celery or parsley as a template, or can be cloned by hybridizing a cDNA derived from celery or parsley incorporated into an appropriate expression vector with a labeled portion of a nucleic acid having a base sequence encoding the apiosetransferase or glucosyltransferase of the present invention. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd Edition (mentioned above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.

[0029] The host cell of the present invention can be obtained by known means, for example, by ligating a nucleic acid having a nucleotide sequence encoding the apiosetransferase or glucosyltransferase of the present invention cloned as described above to an expression vector in such an expressible manner that the nucleic acid can be expressed, and then transforming a cell with the expression vector.

[0030] An expression vector containing a nucleic acid having a base sequence encoding the apiosetransferase or glucosyltransferase of the present invention can be produced, for example, by excising a nucleic acid having a base sequence encoding the apiosetransferase or glucosyltransferase of the present invention and ligating the nucleic acid fragment downstream of a promoter in an appropriate expression vector. As an expression vector, a plasmid capable of expression in Escherichia bacteria (e.g., pCold, pCDF-1b, pBR322, pBR325, pUC12, pUC13) can be used. The promoter may be any promoter suitable for the host used to express the gene. For example, when the host cell is an Escherichia bacterium, the trp promoter, the lac promoter, the recA promoter, the λP promoter, L promoter, lpp promoter, T7 promoter, etc. are preferred.

[0031] In addition to the above, the expression vector may contain an enhancer, a selection marker, etc., if desired. The selection marker may, for example, be an ampicillin resistance gene (hereinafter, referred to as amp r (sometimes abbreviated as " If necessary, a base sequence (signal codon) encoding a signal sequence suitable for the host cell may be added to the 5'-end of the DNA encoding YjbI (or may replace the native signal codon). For example, when the host cell is a bacterium of the genus YjbI, the PhoA signal sequence, OmpA signal sequence, etc., may be used.

[0032] As the host cell, for example, a bacterium belonging to the genus Escherichia is used. Examples of Escherichia bacteria include Escherichia coli K12 DH1 (Proceedings of the National Academy of Sciences of the USA, Vol. 60, p. 160 (1968)), Escherichia coli JM103 (Nucleic Acids Research, Vol. 9, p. 309 (1981)), Escherichia coli JA221 (Journal of Molecular Biology, Vol. 120, p. 517 (1978)), Escherichia coli HB101 (Journal of Molecular Biology, Vol. 41, p. 459 (1969)), Escherichia coli E. coli C600 (Genetics, Vol. 39, 440 (1954)) is used.

[0033] Transformation can be carried out according to known methods depending on the type of host cell. Bacteria of the genus Escherichia can be transformed, for example, according to the methods described in Proc. Natl. Acad. Sci. USA, vol. 69, pp. 2110 (1972) or Gene, vol. 17, pp. 107 (1982).

[0034] The host cell of the present invention is also preferably a cell that produces UDP-apiose or UDP-glucose, and most preferably a cell that produces UDP-apiose and UDP-glucose. When the host cell of the present invention cannot produce UDP-apiose and / or UDP-glucose, genes necessary for producing UDP-apiose and / or UDP-glucose can be introduced into the host cell. For example, when the host cell of the present invention is a bacterium of the genus Escherichia, since Escherichia can produce UDP-glucose but cannot produce UDP-apiose, the AXS1 gene (AtAXS1) derived from Arabidopsis thaliana can be introduced into Escherichia to enable the production of UDP-apiose.

[0035] As described above, the host cell of the present invention can be obtained. The apiosyltransferase or glucosyltransferase of the present invention can also be produced by culturing the above-mentioned cell.

[0036] 2. Method for producing apiin from apigenin 7-O-glucoside and production kit thereof The present invention provides a method for producing apiin from apigenin 7-O glucoside (hereinafter, apiin production method 1 of the present invention).

[0037] The apiin producing method 1 of the present invention comprises a step of contacting the apiosetransferase of the present invention, apigenin 7-O-glucoside, and UDP-apiose. In one embodiment, the apiin producing method 1 of the present invention may be carried out in an aqueous medium. Examples of the aqueous medium include deionized water, distilled water, and buffer solutions, and buffer solutions are preferred. Examples of the buffer solution include Tris-HCl buffer solution, phosphate buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution, Good's buffer solution, and the like.

[0038] In the apiin production method 1 of the present invention, the enzyme activity of the apiosyltransferase of the present invention in an aqueous medium may be, for example, 1.0 to 5.0 μU / μg. The concentration of apigenin 7-O-glucoside in the aqueous medium may be, for example, 10 to 100 μM. The concentration of UDP-apiose in the aqueous medium may be, for example, 0.1 to 10 mM.

[0039] In the apiin production method 1 of the present invention, when the apiosyltransferase of the present invention, apigenin 7-O-glucoside, and UDP-apiose are contacted in an aqueous medium, the reaction conditions may be appropriately determined by those skilled in the art, and examples thereof include a pH of 6.0 to 8.0, a reaction temperature of 20 to 30°C, and a reaction time of 0.5 to 5 hours.

[0040] In another embodiment, the apiin producing method 1 of the present invention may also be carried out in a host cell. When the apiin producing method 1 of the present invention is carried out in a host cell, the host cell may be the host cell of the present invention, preferably a host cell that contains a nucleic acid having a base sequence encoding the apiose transferase of the present invention and produces UDP-apiose.

[0041] In the apiin production method 1 of the present invention, when the apiosyltransferase of the present invention, apigenin 7-O-glucoside, and UDP-apiose are contacted in a host cell, a medium for culturing the host cell can be appropriately determined by a person skilled in the art depending on the host cell. For example, when the host is a bacterium of the genus Escherichia, preferred media include LB medium and M9 medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory, p. 431, 1972). In such cases, the culture can be carried out usually at 14 to 43°C for about 3 to 24 hours with aeration and stirring as necessary.

[0042] As described above, apiin can be produced from apigenin 7-O glucoside by apiin production method 1 of the present invention.

[0043] The present invention also provides a kit for producing apiin from apigenin 7-O glucoside (hereinafter, apiin production kit 1 of the present invention).

[0044] In one embodiment, the apiin production kit 1 of the present invention comprises, for example, the following (i) to (iii). (i) The apiosyltransferase of the present invention. (ii) Apigenin 7-O-glucoside. (iii) UDP-apiose.

[0045] The apiin production kit 1 of the present invention may further comprise other components necessary for producing apiin from apigenin 7-O-glucoside. For example, the apiin production kit 1 of the present invention may further comprise, as an aqueous medium, deionized water, distilled water, or a buffer solution (Tris-HCl buffer, phosphate buffer, citrate buffer, borate buffer, carbonate buffer, Good's buffer, etc.).

[0046] In another embodiment, the apiin production kit 1 of the present invention includes, for example, the following (i) and (ii). (i) A host cell which contains a nucleic acid having a nucleotide sequence encoding the apiosyltransferase of the present invention and produces UDP-apiose. (ii) Apigenin 7-O-glucoside.

[0047] The apiin production kit 1 of the present invention may further comprise other components necessary for producing apiin from apigenin 7-O-glucoside. For example, the apiin production kit 1 of the present invention may further comprise a medium for culturing a host cell. For example, when the host is a bacterium of the genus Escherichia, preferred media include LB medium, M9 medium, and the like.

[0048] 3. Method for producing apigenin 7-O glucoside from apigenin and production kit thereof The present invention provides a method for producing apigenin 7-O glucoside from apigenin (hereinafter, referred to as the present invention's method for producing apigenin 7-O glucoside).

[0049] The method for producing apigenin 7-O glucoside of the present invention comprises a step of contacting the glucosyltransferase of the present invention, apigenin and UDP-glucose. In one embodiment, the method for producing apigenin 7-O glucoside of the present invention may be carried out in an aqueous medium. Examples of the aqueous medium include deionized water, distilled water, and buffer solutions, and buffer solutions are preferred. Examples of the buffer solution include Tris-HCl buffer solution, phosphate buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution, Good's buffer solution, and the like.

[0050] In the method for producing apigenin 7-O-glucoside of the present invention, the enzyme activity of the glucosyltransferase of the present invention in an aqueous medium may be, for example, 1.0 to 5.0 μU / μg. The concentration of apigenin in the aqueous medium may be, for example, 10 to 100 μM. The concentration of UDP-glucose in the aqueous medium may be, for example, 0.1 to 10 mM.

[0051] In the method for producing apigenin 7-O-glucoside of the present invention, when the glucosyltransferase of the present invention, apigenin, and UDP-glucose are contacted in an aqueous medium, the reaction conditions may be appropriately determined by those skilled in the art, and examples thereof include a pH of 6.0 to 8.0, a reaction temperature of 20 to 30° C., and a reaction time of 0.5 to 5 hours.

[0052] In another embodiment, the method for producing apigenin 7-O glucoside of the present invention may also be carried out in a host cell. When the method for producing apigenin 7-O glucoside of the present invention is carried out in a host cell, the host cell may be the host cell of the present invention, preferably a host cell that contains a nucleic acid having a base sequence encoding the glucose transferase of the present invention and produces UDP-glucose.

[0053] In the method for producing apigenin 7-O-glucoside of the present invention, when the glucosyltransferase of the present invention, apigenin, and UDP-glucose are contacted in a host cell, a medium for culturing the host cell can be appropriately determined by a person skilled in the art according to the host cell. For example, when the host is a bacterium of the genus Escherichia, preferred media include LB medium and M9 medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory, p. 431, 1972). In such cases, the culture can be carried out usually at 14 to 43°C for about 3 to 24 hours with aeration and stirring as necessary.

[0054] As described above, apigenin 7-O glucoside can be produced from apigenin by the method for producing apigenin 7-O glucoside of the present invention.

[0055] The present invention also provides a kit for producing apigenin 7-O glucoside from apigenin (hereinafter, the apigenin 7-O glucoside production kit of the present invention).

[0056] The apigenin 7-O glucoside production kit of the present invention includes the following (i) to (iii): (i) a glucosyltransferase of the present invention, (ii) apigenin, (iii) UDP-glucose.

[0057] The apigenin 7-O glucoside production kit of the present invention may further comprise other components necessary for producing apigenin 7-O glucoside from apigenin. For example, the apiin production kit 1 of the present invention may further comprise, as an aqueous medium, deionized water, distilled water, or a buffer solution (Tris-HCl buffer, phosphate buffer, citrate buffer, borate buffer, carbonate buffer, Good's buffer, etc.).

[0058] In another embodiment, the apigenin 7-O glucoside production kit of the present invention includes, for example, the following (i) and (ii). (i) A host cell which contains a nucleic acid having a nucleotide sequence encoding the glucosyltransferase of the present invention and produces UDP-glucose. (ii) Apigenin.

[0059] The apigenin 7-O glucoside production kit of the present invention may further comprise other components necessary for producing apiin from apigenin 7-O glucoside. For example, the apigenin 7-O glucoside production kit of the present invention may further comprise a medium for culturing a host cell. For example, when the host is a bacterium of the genus Escherichia, preferred media include LB medium, M9 medium, and the like.

[0060] 4. Method for Producing Apiin from Apigenin and Production Kit Thereof The present invention provides a method for producing apiin from apigenin (hereinafter, apiin production method 2 of the present invention).

[0061] The apiin production method 2 of the present invention comprises the following steps (i) and (ii). (i) A step of contacting the glucosyltransferase of the present invention, apigenin and UDP-glucose to produce apigenin 7-O glucoside. (ii) a step of producing apiin by contacting the apiosyltransferase of the present invention with the apigenin 7-O-glucoside obtained in step (i) and UDP-apiose.

[0062] In one embodiment, the apiin producing method 2 of the present invention may be carried out in an aqueous medium. Examples of the aqueous medium include deionized water, distilled water, and buffer solutions, and buffer solutions are preferred. Examples of the buffer solution include Tris-HCl buffer solution, phosphate buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution, Good's buffer solution, and the like.

[0063] In the apiin production method 2 of the present invention, the enzyme activity of the glucosyltransferase of the present invention in the aqueous medium may be, for example, 1.0 to 5.0 μU / μg. The concentration of apigenin in the aqueous medium may be, for example, 10 to 100 μM. The concentration of UDP-glucose in the aqueous medium may be, for example, 0.1 to 10 mM. The enzyme activity of the apiosetransferase of the present invention in the aqueous medium may be, for example, 1.0 to 5.0 μU / μg. The concentration of apigenin 7-O-glucoside in the aqueous medium may be, for example, 10 to 100 μM. The concentration of UDP-apiose in the aqueous medium may be, for example, 0.1 to 10 mM.

[0064] In the apiin production method 2 of the present invention, when the apiosetransferase of the present invention, apigenin 7-O-glucoside, and UDP-apiose are contacted in an aqueous medium, the reaction conditions may be appropriately determined by those skilled in the art, and examples of such conditions include a pH of 6.0 to 8.0, a reaction temperature of 20 to 30° C., and a reaction time of 0.5 to 5 hours. When the glucosyltransferase of the present invention, apigenin, and UDP-glucose are contacted in an aqueous medium, the reaction conditions may be appropriately determined by those skilled in the art, and examples of such conditions include a pH of 6.0 to 8.0, a reaction temperature of 20 to 30° C., and a reaction time of 0.5 to 5 hours.

[0065] In another embodiment, steps (i) and (ii) of the apiin producing method 2 of the present invention may be carried out in the same host cell. When the apiin producing method 2 of the present invention is carried out in the same host cell, the host cell may be the host cell of the present invention, preferably a host cell that contains a nucleic acid having a nucleotide sequence encoding the glucose transferase of the present invention and a nucleic acid having a nucleotide sequence encoding the apiose transferase of the present invention, and produces UDP-glucose and UDP-apiose.

[0066] In the apiin producing method 2 of the present invention, when steps (i) and (ii) are carried out in the same host cell, a medium for culturing the host cell can be appropriately determined by a person skilled in the art depending on the host cell. For example, when the host is a bacterium of the genus Escherichia, preferred media include LB medium and M9 medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory, p. 431, 1972). In such cases, the culture can be carried out usually at 14 to 43°C for about 3 to 24 hours with aeration and stirring as necessary.

[0067] As described above, apiin can be consistently produced from apigenin by apiin production method 2 of the present invention.

[0068] The present invention also provides a kit for producing apiin from apigenin (hereinafter, apiin production kit 2 of the present invention).

[0069] In one embodiment, the apiin production kit 2 of the present invention includes, for example, the following (i) to (v). (i) The glucosyltransferase of the present invention. (ii) The apiosyltransferase of the present invention. (iii) Apigenin. (iv) UDP-glucose. (v) UDP-apiose.

[0070] The apiin production kit 2 of the present invention may further comprise other components necessary for producing apiin from apigenin. For example, the apiin production kit 2 of the present invention may further comprise, as an aqueous medium, deionized water, distilled water, or a buffer solution (Tris-HCl buffer, phosphate buffer, citrate buffer, borate buffer, carbonate buffer, Good's buffer, etc.).

[0071] As another embodiment, the apiin production kit 2 of the present invention also includes, for example, the following (i) and (ii). (i) A host cell which contains a nucleic acid having a nucleotide sequence encoding the glucosyltransferase of the present invention and a nucleic acid having a nucleotide sequence encoding the apiosetransferase of the present invention and which produces UDP-glucose and UDP-apiose. (ii) Apigenin 7-O-glucoside.

[0072] The apiin production kit 2 of the present invention may further comprise other components necessary for producing apiin from apigenin. For example, the apiin production kit 2 of the present invention may further comprise a medium for culturing a host cell, and when the host is a bacterium of the genus Escherichia, preferred media include LB medium, M9 medium, and the like. EXAMPLES

[0073] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.

[0074] Example 1: Search for candidate genes for ceroliapiin biosynthesis apiose transferase (AgApiT) 1-1. Experimental materials and methods This example was carried out with the cooperation of Associate Professor Kobayashi Masaru of the Laboratory of Plant Nutrition at Kyoto University. The RNAseq data for celery was obtained from accession number SRR1023730 (https: / / trace.ddbj.nig.acjp / DRASearch / run?acc=SRR1023730). This data was de novo assembled using Trinity to create a reference sequence. This was then mapped using HISAT2 and read counted using StringTie. From the results, it was determined that UGT79, UGT91, and UGT94 homologs (among UGTs, enzymes that specifically transfer sugar to the sugar moiety of the glycoside rather than to the aglycone are sugar-sugar UGTs or glycoside-specific glycosyltransferases (GGTs: G lycoside specific G Lycosyl T We searched for UGT79, UGT91, and UGT94, which are known as transgenes. We also estimated the expression level of each gene from the read counts using RNA-Seq data of celery that was independently analyzed by the Laboratory of Plant Nutrition, Kyoto University. 1-2.Results Since celery contains a relatively large amount of flavonoid glycosides with many apioses bound thereto, we hypothesized that the highly expressed GGT genes were likely to be UGTs that transfer apiose. GGT genes were selected from the RNA-seq data (SRR1023730) and ranked in order of increasing expression (Table 1).

[0075] [Table 1]

[0076] The expression levels of these genes were examined by correlation of read counts of two types of RNA-Seq data (SRR1023730). It was found that these data were correlated, and that the read counts correlated with the expression levels. Among them, DN15087, which had the highest read count, was selected as a candidate gene for AgApiT. The molecular weight of the protein encoded by DN15087 is 49 kDa and the isoelectric point is 5.7. The molecular weight and isoelectric point of the protein encoded by DN15087 are approximately equal to those of the protein estimated by partial purification of the parsley enzyme previously reported, which is 50 kDa and 4.8 (Ortmann, R., et al., (1972) Biochim. Biophys. Acta 289, 293-302.). The C-terminal 44 amino acids of the amino acid sequence of DN15087 contained a PSPG motif conserved in UGTs (Figure 1A).

[0077] Example 2: Expression of AgApiT in Escherichia coli, and activity, kinetic and biochemical analysis of AgApiT 2-1. Experimental materials and methods (1) Expression of AgApiT in E. coli The vector used to construct the E. coli expression construct was pColdProS2, the template was an artificially synthesized gene for expression in E. coli (DN15087), the primers were AgApiT F and AgApiT R (Table 2), and the restriction enzymes were Nde I and Xba I.

[0078] [Table 2]

[0079] PCR cloning was performed using the synthetic gene as a template and AgApiT F and AgApiT R as primers using KOD-Plus Neo to amplify the full length (1314 bp). PCR was performed with denaturation at 95°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 1.5 minutes, with 35 cycles. The PCR product and pColdProS2 were digested with Nde I and Xba I, respectively. This was ligated with Ligation Kit Mighty mix, and E. coli DH5α was transformed and cultured on an LB medium plate containing 100 μg / mL ampicillin. Colony PCR was performed using Go-taq Green Master mix to obtain E. coli transformed with the pColdProS2-AgApiT expression construct (Figure 2). The construct was confirmed to be constructed correctly by base sequence analysis. The constructed expression construct was transformed into E. coli BL21(DE3) strain. The transformed E. coli BL21 was suspended in 3 mL of LB medium and precultured overnight at 37°C. 3 mL of the culture was inoculated into 100 mL of LB medium, and the OD 600 = 0.6 to 0.8, and then cultured at 37°C. After cooling the culture on ice, IPTG (final concentration 0.8 mM) was added and expression induction was carried out at 15°C and 100 to 110 rpm for 24 hours. After expression induction, the E. coli solution was centrifuged (5,000×g, 4°C, 5 min) to harvest the bacteria, and the supernatant was removed. The harvested bacteria were suspended in 5 mL of E. coli solubilization solution (BugBuster Protein Extraction Reagent 5 mL, 10 mM sodium phosphate buffer (pH 7.4) 4.5 mL, benzonase nuclease 25 U, lysozyme (Novagen: 5 KU or Wako Pure Chemical Industries: 3.75 mg) per 1 g of bacteria). After standing for 20 minutes, the mixture was centrifuged (20,000×g, 4°C, 20 min), and the supernatant was used as a crude enzyme solution. The crude enzyme solution was purified using a Ni ion chelating column (HisTrap HP 1 mL). The flow rate was a maximum of 1 mL / min. First, the column was washed with 3 mL of ultrapure water. After washing, the column was equilibrated with 3 mL of equilibration buffer 1 (50 mM imidazole hydrochloride, 50 mM sodium phosphate pH 7.4, 500 mM sodium chloride). The crude enzyme solution was then added. Equilibration buffer 1 was poured in an amount 5 times the amount of the bacterial solution, and 5 mL of equilibration buffer 2 (100 mM imidazole hydrochloride, 50 mM sodium phosphate pH 7.4, 500 mM sodium chloride) was poured to wash away non-adsorbed components. The target protein was eluted with 3 mL of elution buffer (200 mM imidazole hydrochloride, 50 mM sodium phosphate pH 7.4, 500 mM sodium chloride). The expressed protein was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was 13% polyacrylamide, the molecular weight marker was XL-Ladder Broad, and CBB R-250 was used for CBB staining. During elution, the column was fractionated into Eppendorf tubes at approximately 300 μL each. 10 μL of each was dispensed, and 150 μL of Pierce 660 nm Protein Assay solution was added, then the mixture was left to stand for 5 minutes and the absorbance at 660 nm was measured. <When cutting the tag> Five fractions with high protein concentration were collected in 2 mL tubes. The mixture was reacted with HRV3C protease for 2 hours at 4°C (total volume 1.6 mL) to cleave the tag (full length 24 kDa). The solution was passed through a Ni ion chelating column equilibrated with 1× HRV 3C Cleavage Buffer, and the flow-through was collected. The tagged protein was collected with 3 mL of elution buffer (200 mM imidazole hydrochloride, 50 mM sodium phosphate pH 7.4, 500 mM sodium chloride). The expressed protein was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was 14% polyacrylamide, XL-Ladder Broad was used as a molecular weight marker, and CBB R-250 was used for CBB staining. Each collected solution was centrifuged at 14,000×g for 15 minutes at 4°C using an ultrafiltration filter (Amicon Ultra-0.5 mL, Ultracel-10K). The filter was then placed upside down in a new tube and centrifuged at 1,000 x g for 2 minutes at 4°C to recover the concentrated AgApiT and tagged proteins. 10 μL of the solution containing the appropriate diluted AgApiT and tagged proteins was combined with 150 μL of Pierce 660nm Protein Assay solution, left to stand for 5 minutes, and the absorbance at 660 nm was measured. The amount of protein was calculated from a standard curve using BSA. <If you do not want to cut the tag> The solutions of the five fractions with high protein concentrations were centrifuged at 14,000×g at 4℃ for 15 minutes using ultrafiltration filters (Amicon Ultra-0.5 mL, Ultracel-10K). The filter was then placed upside down in a new tube and centrifuged at 1,000×g at 4℃ for 2 minutes to recover concentrated AgApiT. Concentrated AgApiT was diluted to an appropriate concentration, and 10 μL of the solution was combined with 150 μL of Pierce 660nm Protein Assay solution, left to stand for 5 minutes, and the absorbance at 660 nm was measured. The protein amount was calculated from the standard curve using BSA. (2) Activity, kinetics, and biochemical analysis of AgApiT Activity detection was carried out under the following conditions. 50 μM apigenin 7-O-β-D-glucoside 1 mM UDP-Api Purified AgApiT protein Reaction buffer (100 mM Tris-HCl buffer, pH 7.0, 50 mM NaCl) These were reacted at 23° C. for 2 hours. The reaction was stopped by inactivating the enzyme by incubation at 100° C. for 3 minutes. The reaction products were quantified by high performance liquid chromatography (HPLC) analysis under the following conditions. Quantitative determination of apiin Equipment: Shimadzu LC2010C HT Column: GL Sciences Inertsil ODS-3 (4.6×250 mm) 5 μm Flow rate: 1.0 mL / min Mobile phase: A) 0.1% TFA B) Acetonitrile Gradient conditions B) 20-40 T) 5-25 Detection: 330 nm <Kinetic analysis> AgApiT K m , V max , and k cat Values ​​were determined by assaying with various concentrations of apigenin 7-O-β-D-glucoside (50–200 μM) and UDP-Api (125–1000 μM). Mean values ​​of triplicate experiments with standard deviations were plotted to the Michaelis-Menten equation, and kinetic parameters were calculated by nonlinear regression analysis using Prism version 8. <Biochemical analysis> The donor substrates used were UDP-Api, UDP-Araf, UDP-Rha, UDP-Glc, UDP-GlcUA, UDP-Gal, UDP-GalUA, UDP-Xyl, UDP-GlcNAc, and GDP-Fuc. The acceptor substrates used were apigenin 7-O-β-D-glucoside, quercetin 7-O-β-D-glucoside, luteolin 7-O-β-D-glucoside, naringenin 7-O-β-D-glucoside, apigenin, apiin, and quercetin 3-O-β-D-glucoside. 2-2.Results Expression of the recombinant AgApiT protein was induced with 0.8 mM IPTG. It was purified on a Ni column to obtain AgApiT with a molecular mass of 73 kDa (calculated value 73 kDa) tagged with His and ProS2 tags (Fig. 3). It was treated with HRV3C protease to obtain AgApiT with a molecular mass of 49 kDa (calculated value 49 kDa) from which the tags were cleaved (Fig. 3). A tag protein (His tag and ProS2 tag) with a molecular mass of 24 kDa (calculated value 24 kDa) was also detected (Fig. 3). We created an expression construct using the pET15b vector in addition to the pColdProS2 vector, transformed it into BL21, and cultured and induced expression, but we could not obtain much of the target protein. The pColdProS2 vector contains a ProS2 tag, which is a solubility tag, and it is believed that the increased expression level was due to the increased solubility. In a reaction buffer of pH 7.0, 50 μM apigenin 7-O-β-D-glucoside was used as the acceptor substrate, and the prepared 0.5 mM UDP-Api was used as the donor substrate. The reaction was carried out at 23°C for 2 hours, and an enzyme reaction product was detected at the same position as apiin (Figure 4). Therefore, it was concluded that DN15087 is the gene encoding the apiose transferase AgApiT involved in apiin biosynthesis. Furthermore, since apiose transferase activity was not detected from the tag protein, it can be said that this product is an enzyme reaction product by AgApiT (Figure 4). The enzymatic activity of the tag-cleaved AgApiT (2.8 μU / μg) was 1.6 times that of tagged AgApiT (4.6 μU / μg) (Figure 5). Only about 300 μg of tag-cleaved AgApiT could be recovered from 100 mL of E. coli culture, whereas more than 1.5 mg of tagged AgApiT could be recovered. Unless the enzyme concentration is high, the amount of product is small, making quantitative analysis difficult, so we used tagged AgApiT, which allows the recovery of more protein, for analysis. The amount of the AgApiT reaction product increased in an enzyme- and time-dependent manner (FIG. 6). K of AgApiT for apigenin 7-O-β-D-glucoside mand V max The values ​​were 119 ± 8 μM and 0.539 ± 0.019 nmol min -1 mg -1 (Table 3, Figure 7). cat and k cat / K m The values ​​are 1.585 s -1 and 0.0083 s -1 μM -1 The K for UDP-Api was 1.0. m and V max The values ​​were 190 ± 12 μM and 0.183 ± 0.004 nmol min -1 mg -1 (Table 3, Figure 7). cat and k cat / K m The values ​​are 0.538 s -1 and 0.0028 s -1· μM -1 These values ​​were similar to those of identified UGTs (Ono, E., et al. (2020) Plant J. doi: 10.1111).

[0080] [Table 3]

[0081] We performed biochemical analysis of AgApiT. The donor substrate dependency of AgApiT was examined using UDP-Api, UDP-Araf, UDP-Rha, UDP-Glc, UDP-GlcUA, UDP-Gal, UDP-GalUA, UDP-Xyl, UDP-GlcNAc, and GDP-Fuc (Fig. 8). AgApiT did not react with sugar nucleotides other than UDP-Api, indicating high donor substrate specificity. In addition, the acceptor substrate dependency of AgApiT was examined using apigenin 7-O-β-D-glucoside, quercetin 7-O-β-D-glucoside, luteolin 7-O-β-D-glucoside, naringenin 7-O-β-D-glucoside, apigenin, apiin, and quercetin 3-O-β-D-glucoside (Figs. 9 and 10). Apigenin 7-O-β-D-glucoside showed the highest activity, and activity was also detected in quercetin 7-O-β-D-glucoside, luteolin 7-O-β-D-glucoside, and naringenin 7-O-β-D-glucoside. No activity was detected in apigenin, apiin, or quercetin 3-O-β-D-glucoside. Because the proportion of aglycone in celery is higher in apigenin than in luteolin, it was expected that the substrate specificity for apigenin 7-O-β-D-glucoside would be higher than that of luteolin 7-O-β-D-glucoside. The characteristic amino acids found in UGTs that transfer pentoses have been predicted by homology modeling, and it has been reported that changing Ile at around position 140 to Ser results in recognition of UDP-Glc rather than UDP-pentose (Ohgami, S., et al., (2015) Plant Physiol. 168, 464-477; Chen, HY, Li, X. (2017) Plant J. 89, 195-203). In AgApiT, Ile139 corresponds to the characteristic amino acid and is predicted to be involved in pentose recognition (Fig. 1B). Substrate specificity was examined by substituting Ile139 with Val or Ser (Fig. 11). I139V showed the same substrate specificity and activity as the wild type, but I139S lost activity against UDP-Api. Neither UDP-Xyl nor UDP-Glc had any activity (data not shown). In the future, it is expected that crystal structure analysis of AgApiT will enable elucidation of the enzyme's substrate recognition mechanism.

[0082] Example 3: Analysis of the expression level of AgApiT gene at each developmental stage 3-1. Experimental materials and methods Following the method of Yan et al. (Yan, J., et al., (2014) Sci. Hortic. (Amsterdam) 165, 218-224), the developmental stages were divided according to the length of the plant body as follows: 1: 0.5 cm or less, 2: 0.5-1 cm, 3: 1-1.5 cm, 4: 1.5 cm-2 cm, 5: 2-2.5 cm, 6: 2.5-3 cm, 7: 3 cm or more. Samples were collected at each developmental stage, and RNA was extracted using the RNeasy Plus Mini Kit. PrimeScript TM cDNA was synthesized from 0.5 μg of RNA using the II 1st strand cDNA Synthesis Kit. PCR was performed using each cDNA as a template and GAPDH F, GAPDH R, FNS IF, FNS IR, DN15087 F, and DN15087 R (Table 1) as primers using Go-taq Green Master mix, and semi-quantitative analysis was performed. 3-2.Results The expression levels of two genes (FNS I, AgApiT (DN15087)) were analyzed for each celery developmental stage (1: 0.5 cm or less, 2: 0.5-1 cm, 3: 1-1.5 cm, 4: 1.5 cm-2 cm, 5: 2-2.5 cm, 6: 2.5-3 cm, 7: 3 cm or more). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal standard. FNS I (flavone synthase I) is an enzyme that synthesizes apigenin from naringenin, and its gene was identified by Yan et al. In the results of Yan et al., the expression level of the FNS I gene increased from stage 1 to 2 and decreased from stage 5. In our results, the expression level of the FNS I gene decreased at stage 5, similar to the results of Yan et al. (Figure 12). The expression level of the FNS I gene corresponds to the increase in apigenin. The expression level of the AgApiT gene, like that of the FNS I gene, was high until stage 4 and decreased from stage 5 (Fig. 12). We expect that apiin will also increase at the same time as apigenin.

[0083] Example 4: Screening of candidate genes for ceroliapiin biosynthesis glucosyltransferase 4-1. Experimental materials and methods (1) Experimental materials Celery (New Cornell 619) seeds were purchased from Tohoku. Seeds were grown on MS agar medium at 22°C with a 16-h light / 8-h dark cycle. After 10 days, seedlings were transferred to a soil mixture of Metro-Mix 360 (Sun Gro Horticulture, Agawam, OH) and vermiculite in a 4:1 ratio and grown under the same conditions. Restriction enzymes Nde I, Xba I, pCold ProS2 vector, and HRV3C Protease were purchased from Takara Bio. Go-taq Green Master mix was purchased from Promega. BugBuster Protein Extraction Reagent and Benzonase Nuclease were purchased from Novagen. HisTrap HP 1 mL was purchased from GE Healthcare. Pierce 660nm Protein Assay was purchased from Thermo Fisher Scientific. Inertsil ODS-3 (4.6 × 250 mm) was purchased from GL Sciences. Amicon Ultra-0.5 mL was purchased from Merck Millipore. Apigenin was purchased from Cayman Chemical (Ann Arbor, MI). Naringenin, luteolin, quercetin, and genistein were purchased from Tokyo Kasei. Apigenin 7-O-β-D-glucoside was purchased from Ark Pharm (Arlington Heights, IL). Naringenin 7-O-β-D-glucoside, luteolin 7-O-β-D-glucoside, and quercetin 7-O-β-D-glucoside were purchased from Extrasynthese (Lyon, France). UDP-Glc, UDP-galactose (Gal), and lysozyme were purchased from Fujifilm Wako Chemical. UDP-glucuronic acid (GlcA) was purchased from Sigma-Aldrich. (2) Selection of celery-derived GlcT (AgGlcT) gene The celery genome sequence was obtained from cleleryDB. The celery RNA-seq data (SRR102370) was obtained from NCBI's Sequence Read Archive (SRA) (http: / ww.ncbi.nlm.nih.gov / sra). The data was de novo assembled using Trinity on the Galaxy server. In addition, mapping was performed using HISAT2, and TPM values ​​were calculated for each predicted gene using StringTie. The celery RNA-seq read data was searched for homologs of UGT71, UGT73, and UGT88, which belong to flavone glucosyltransferases. The amino acid sequences of UGT71, UGT73, and UGT88 were assembled using ClustalW, and a phylogenetic tree was created using the neighbor-joining method. Sequences other than celery were obtained from the UGT Nomenclature Committee Website (https: / / prime.vetmed.wsu.edu) or public databases by BLAST with the transcriptome shotgun assemblies. (3) Glucosyltransferase gene expression analysis Celery leaves were divided into seven developmental stages according to their vertical length: stage 1: less than 0.5 cm, stage 2: 0.5-1.0 cm, stage 3: 1.0-1.5 cm, stage 4: 1.5-2.0 cm, stage 5: 2.0-2.5 cm, stage 6: 2.5-3.0 cm, and stage 7: 3.0 cm or more. Celery leaves were immediately frozen in liquid nitrogen after harvest and stored at -80°C. RNA was extracted using RNeasy plus mini kit (Qiagen, Hilden, Germany) and reverse transcribed to cDNA using PrimeScript II 1st strand cDNA synthesis kit (Takara Bio). Reverse transcription PCR was performed using cDNA from each developmental stage of celery as a template, and the following sequences were obtained: AgGAPDH_F, AgGAPDH_R, AgFNS I_F, AgFNS I_R, AgApiT_F, AgApiT_R, AgGlcT_DN9954_F, AgGlcT_DN9954_R, AgGlcT_DN15140_F, AgGlcT_DN15140_R, AgGlcT_DN15830_F, AgGlcT_DN15830_R, AgGlcT_DN15918_F, AgGlcT_DN15918_R, AgGlcT_DN16008_F, The PCR was performed using AgGlcT_DN16008_R, AgGlcT_DN23862_F, AgGlcT_DN23862_R, AgGlcT_DN41599_F, and AgGlcT_DN41599_R (Table 4) as primers and Go-taq Green Master mix. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a control. A TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio) was used. The PCR reaction was thermally denatured at 95°C for 1 minute, followed by 30 cycles of heating at 55°C for 30 seconds and 72°C for 1 minute. Loading Buffer (Takara Bio) was added to the PCR reaction product, and electrophoresis was performed using a 1% agarose gel (Funa Submarine, Funakoshi). Real-time PCR was performed using cDNA from each celery developmental stage as a template and GAPDH_F, GAPDH_R, FNSI_F, FNSI_R, AgApiT_F, AgApiT_R, AgGlcT_F, and AgGlcT_R (Table 4) as primers. Step One Plus (Applied Biosystems) was used. The reaction solution was prepared with 50 ng / μL cDNA, 0.4 μM primers, TB Green Premix Ex Taq II, ROX reference dye, and sterile purified water. The PCR reaction was performed by thermal denaturation at 95°C for 30 seconds, followed by 40 cycles of heating at 95°C for 5 seconds and 60°C for 30 seconds. Loading Buffer (Takara Bio) was added to the PCR reaction product, and electrophoresis was performed using a 3% agarose gel (Funa Submarine, Funakoshi).

[0084] [Table 4]

[0085] 4-2.Results Flavonoid glycosyltransferases are classified into 3-O-glycosyltransferases, 5-O-glycosyltransferases, and 7-O-glycosyltransferases. The celeriapiin biosynthetic glycosyltransferase was thought to be a homolog of flavonoid 7-O-glycosyltransferases UGT71, 73, and 88, which are similar to the previously identified apigenin:glucosyltransferases. The celery genome encodes many UGTs. Since the amount of apiin synthesized in the young leaves of celery is very high (2.28 g / g weight), we speculated that the gene with the highest expression level in the young leaves, where apiin is synthesized in large amounts, among the many UGT genes, is the glycosyltransferase (AgGlcT) involved in the biosynthesis of apiin derived from celery. When we searched for homologs of UGT71, 73, and 88 in the genome sequence to select candidate genes, 23 UGT genes were found. We created a molecular phylogenetic tree by combining these with known glycosyltransferases (Figure 13). Nine celery genes (DN9954, DN15450, DN15830, DN15918, DN16008, DN19752, DN23862, and DN41599) belonging to UGT71, 73, and 88 were selected as candidates for apiin biosynthetic glucosyltransferases. When the expression of the nine genes classified into UGT71, 73, and 88 was analyzed using RNA-seq data, expression of seven genes was observed (Table 5). Among them, the read counts (TPM values) of the DN9954 gene belonging to UGT71 and the DN16008 and DN23862 genes belonging to UGT88 were relatively high (Table 5).

[0086] [Table 5]

[0087] For these seven candidate genes, the expression level of the AgGlcT candidate gene was analyzed by semi-quantitative PCR for each celery developmental stage (stage 1: 0.5 cm or less, stage 2: 0.5-1.0 cm, stage 3: 1.0-1.5 cm, stage 4: 1.5-2.0 cm, stage 5: 2.0-2.5 cm, stage 6: 2.5-3.0 cm, stage 7: 3.0 cm or more). Among these, the expression level of DN23862, which belongs to UGT88, increased in celery stages 1-3 and decreased in stages 4-6, similar to the apiin biosynthesis-related genes (AgFNS I, AgApiT) (Figure 14A, B). As for the other genes, DN9954 and DN16008 showed expression behavior similar to DN23862. Among these, the expression level of DN23862 was analyzed by RT-PCR. The expression level of AgGlcT increased most at stage 3, and decreased significantly from stage 6 (Figure 15A). The expression level of the apiin biosynthesis-related enzyme genes (AgFNS I, AgApiT) was highest at stage 2 and decreased significantly from stage 5 onwards, with the expression pattern being similar to that of AgGlcT (Fig. 15B, C). The apiin content was also highest at stage 1 and decreased as the plant grew (Fig. 15A). This is roughly consistent with the expression behavior of AgGlcT. Based on these results, DN9954 (UGT71), DN16008 (UGT88), and DN23862 (UGT88) were selected as candidate genes for the apiin biosynthetic glucosyltransferase from celery. Among them, DN23862 (UGT88), whose expression profile was most similar to that of the apiin biosynthetic enzyme genes (AgFNS I, AgApiT), was selected as the most promising candidate.

[0088] Example 5 Identification of the GlcT gene from celery 5-1. Experimental materials and methods (1) Expression and purification of candidate glucosyltransferase proteins Constructs were made to express three types of AgGlcT candidate proteins in E. coli. The template was celery cDNA, and the primers were AgGlcT_DN9954_F, AgGlcDN9954_R, AgGlcT_DN16008_F, AgGlcDN16008_R, AgGlcT_DN23862_F, and AgGlcDN23862_R (Table 4). 25 mM MgSO4, 2 mM dNTPs, 10 μM primers (forward, reverse), 100 ng / μL cDNA, PCR Buffer for KOD Plus Neo, 1.0 U / μL KOD Plus Neo, and sterile purified water were mixed. The PCR reaction was performed by heat denaturation at 95°C for 1 minute, followed by 30 cycles of heating at 55°C for 30 seconds and 72°C for 1 minute. Loading Buffer (Takara Bio) was added to the PCR reaction product, and electrophoresis was performed using 1% agarose gel (Funa Submarine, Funakoshi). Restriction enzymes used were Nde I and Xba I. The pColdProS2 vector was treated with restriction enzymes Nde I and Xba I at 37°C for 2 hours. Infusion cloning was performed at 50°C for 30 minutes using the In Fusion HD Cloning Kit. This was transformed into E. coli DH5α and cultured on LB medium containing 100 μg / mL ampicillin. Colony PCR was performed using Go-taq Green Master mix to obtain the desired construct. Nucleotide sequence analysis was performed to confirm that the correct construct had been created. The purified plasmid was transformed into E. coli BL21(DE3) and cultured in 3 mL of LB medium at 37°C for 16 hours with shaking. 3 mL of the culture was added to 100 mL of LB medium, and the OD 600It was cultured with shaking at 37 °C until it reached 0.6 - 0.8. The culture solution was cooled on ice for 10 minutes, and expression induction was carried out at 15 °C for 24 hours using 1 mM IPTG. Bacteria were collected by centrifugation at 5,000 × g, 4 °C for 15 minutes. After collecting the bacteria, 5 mL of Escherichia coli solubilization solution (20 mM sodium phosphate buffer, pH 7.4, 5 U / mL benzonase and 1 kU / mL lysozyme) was added per 1 g of the bacterial cells and suspended. It was left standing on ice for 20 minutes, and the supernatant was recovered by centrifugation at 20,000 × g, 4 °C for 20 minutes to obtain a crude enzyme solution. This crude enzyme solution was equilibrated with (50 mM sodium phosphate and 500 mM sodium chloride, pH 7.4) containing 50 mM imidazole, applied to a Ni ion chelate column, and the protein was eluted with the same buffer containing 200 mM imidazole. This protein was treated with HRV3C protease at 4 °C for 24 hours to remove the tag. The purified protein was analyzed by SDS-PAGE. This protein was concentrated by ultrafiltration using Amicon Ultra-0.5 mL (10 kDa cutoff, Merck Millipore) at 14,000 × g, 4 °C for 15 minutes. The protein concentration was measured using the Pierce 660 nm protein assay kit. (2) Detection of the activity of glucose transferase Using 1 mM apigenin as the acceptor substrate and 0.5 mM UDP-Glc as the donor substrate, the reaction solution was prepared using 200 mM Tris-HCl buffer (pH 7.0). The reaction conditions were 23 °C for 2 hours, and the reaction was stopped by incubation at 100 °C for 3 minutes. The quantification of the reaction product was carried out using high-performance liquid chromatography (HPLC) under the following conditions. <HPLC condition 1> Apparatus: Hitachi Chromaster 5110 pump, 5440 FL Detector Flow rate: 1.0 mL / min Detection: 330 nm Column: GL Sciences Inertsil ODS-3 (4.6×250 mm) 5 μm Mobile phase: A) 0.1% TFA B) 100% acetonitrile Gradient conditions: 5-25 min (25-55% B) 5-2.Results The full length of the candidate genes DN9954, DN16008, and DN23862 were recombined into the pColdProS2 vector and transformed into E. coli BL21 strain. The ProS2 tag fusion proteins were expressed and purified to obtain the target proteins. After protease treatment, the tags were removed to obtain 53, 53, and 54 kDa proteins (calculated values: 53, 53, and 54 kDa) (Figure 16). The yields were 0.51, 0.48, and 0.42 mg per 100 mL of culture, respectively. In a pH 7.0 reaction buffer, 0.5 mM UDP-Glc was used as the donor substrate, and 1 mM apigenin was used as the acceptor substrate. When 1 μg of the expressed protein was reacted at 23°C for 2 hours, a reaction product was observed at the same elution position as apigenin 7-O-glucoside by reversed-phase chromatography (Figure 17A). The enzyme activities were highest in the order of DN23862, DN9954, and DN16008, at 63.5, 4.2, and 3.1 μU / μg, respectively (Figure 17B). DN23862 had 15-20 times higher enzyme activity than the other candidate proteins. The amount of the reaction product increased with DN23862 in an enzyme amount-dependent (0-1.8 μg) and reaction time-dependent (0-4 hours) manner (Figures 18A and B). Since the expression pattern of the DN23862 gene was most similar to those of AgFNS I and AgApiT (Fig. 15) and the enzyme activity was the highest (Fig. 17), we concluded that DN23862 is AgGlcT, a celery-derived glucosyltransferase that produces apigenin 7-O-glucoside from apigenin and UDP-Glc. This is the first apiin biosynthetic glucosyltransferase identified in celery.

[0089] Example 6: Amino acid sequence alignment and three-dimensional structure prediction of AgGlcT 6-1. Experimental materials and methods The amino acids of AgGlcT, PcGlcT, UGT88D5, UGT88D3, UGT88A1, UGT88A7, UGT73A17, and PaUGT1 were aligned using Clustalw (https: / / www.genome.jp / tools-bin / clustalw). The three-dimensional structure of AgGlcT was analyzed using AlphaFold2 (https: / / colab.research.google.com / github / sokrypton / The predictions were made using ColabFold / blob / main / AlphaFold2.ipynb). 6-2.Results AgGlcT is composed of 469 amino acid residues and belongs to the 7-O-glycosyltransferase UGT88. It contains the PSPG motif found in UGTs at positions 344-387 and the GSS motif found specifically in GlcTs at positions 454-456 (Figure 19). It contains the catalytic residues His19 and Asp123. It showed 90% homology with parsley glucosyltransferase (PcGlcT), which is also a member of the Umbelliferae family and has a high apiin content, as described below. It showed 46%, 47%, 41%, and 44% homology with the GlcT genes from other plant species belonging to UGT88, UGT88A1 (Brassicaceae), UGT88D3 (Burdock family), UGT88D5 (Lamiaceae), and UGT88A7 (Lamiaceae), respectively. AgGlcT showed 26% and 22% homology with UGT73A17 (Theaceae) and PaUGT1 (UGT72) (Marchantialaceae), enzymes that transfer Glc to apigenin, respectively. AgGlcT contained the PSPG motif found in UGTs and the GSS motif found specifically in GlcTs. Thus, AgGlcT had an amino acid sequence characteristic of apigenin:GlcT. The three-dimensional structure of AgGlcT predicted by AlphaFold2 is shown (Figure 20). The three-dimensional structure is similar to that of UGT88D7, which belongs to the same UGT88 as AgGlcT, and the relative positions of the catalytic residues, PSPG motif and GSS motif, are also similar.

[0090] Example 7 Biochemical analysis of AgGlcT 7-1. Experimental materials and methods The reaction mixture was prepared using 1 mM apigenin as the acceptor substrate and 0.5 mM UDP-Glc as the donor substrate at pH 4.0, 5.0, 6.0, 6.5, 7.0, 7.5, and 8.0 (200 mM sodium acetate buffer, pH 4.0 and 5.0; 200 mM MES-potassium hydroxide buffer, pH 6.0 and 6.5; 200 mM HEPES-potassium hydroxide buffer, pH 7.0, 7.5, and 8.0). The reaction was carried out at 23°C for 2 hours and stopped by incubation at 100°C for 3 minutes. The reaction product was quantified under HPLC condition 1. The acceptor substrate specificity of AgGlcT was determined by analyzing the reaction mixtures of 1 mM apigenin, luteolin, quercetin, chrysoeriol, naringenin, genistein, apigenin 7-O-glucoside, and apiin as acceptor substrates and 0.5 mM UDP-Glc as donor substrates in the presence of 200 mM Tris-HCl buffer, pH 7.0. The donor substrate specificity was determined by analyzing the reaction mixtures of 1 mM apigenin as acceptor substrates and 0.5 mM UDP-Glc, UDP-Xyl, UDP-GlcA, UDP-Gal, UDP-GalA, UDP-Araf, and UDP-Api as donor substrates in the presence of 200 mM Tris-HCl buffer, pH 7.0. kAgGlcT against apigenin and UDP-Glc cat , K m The values ​​were 1 mM apigenin and several concentrations of UDP-Glc (15-500 μM), and several concentrations of apigenin (30-1000 μM) and 0.5 mM UDP-Glc. These two substances were used as substrates and reacted in the presence of 200 mM Tris-HCl buffer, pH 7.0. The enzyme kinetic constants were calculated from the Michaelis-Menten equation by nonlinear regression analysis using Prism version 9.0, which was used to analyze the average values ​​of three experiments. 7-2.Results pH Dependence: When the enzyme activity was measured at pH 4.0 to 8.0 using apigenin and UDP-Glc as substrates, the enzyme activity was highest at pH 7.0, with 51.1 μU / μg (Figure 21). This was similar to the optimum pH of other glucose transferases. This enzyme is said to be present on the endoplasmic reticulum membrane, which is a characteristic of glucose transferases. The pH at which the enzyme exhibited 50% of its maximum activity was 5.75 and 7.50, which are the pK values ​​of the side chains of Asp123 and His19, respectively. a was considered to be equivalent to Donor substrate specificity: Seven sugar nucleotides were used for donor substrate specificity analysis. When UDP-Glc was used as the donor substrate, the enzyme activity was the highest at 59.2 μU / μg. UDP-Xly also showed an enzyme activity of 6.3 μU / μg, but no enzyme activity was observed with other substrates (Figure 22A). UDP-Xly has a structure that lacks -CH2OH at the 6th position of the glucose of UDP-Glc, and it was thought that the common structure of these two substrates was recognized to some extent (Figure 22B). Acceptor substrate specificity: Eight flavonoids were used for the acceptor substrate specificity analysis. When apigenin was used as the acceptor substrate, the enzyme activity was the highest at 69.7 μU / μg (Figure 23A). The enzyme activity was then highest for quercetin, luteolin, chrysoeriol, naringenin, and genistein, in that order. No enzyme activity was observed for apigenin 7-O-glucoside, which is a sugar bonded to apigenin, and apiin. Based on these substrate specificities, AgGlcT was considered to be involved in the biosynthesis of all of apigenin 7-O-glucoside, luteolin 7-O-glucoside, and chrysoeriol 7-O-glucoside synthesized in celery. This enzyme was considered to recognize the flavone skeleton and the double bond between the 1-2 positions, which are common to the top four substrates with high activity (Figure 23B). Kinetic analysis: 1 mM apigenin and several concentrations of UDP-Glc (15-500 μM), or several concentrations of apigenin (30-1000 μM) and 0.5 mM UDP-Glc were reacted with AgGlcT to determine the enzyme kinetic constants of UDP-Glc or apigenin for AgGlcT. m , k catThe values ​​are 192 μM and 0.0031 s, respectively. -1 , k cat / K m The value is 0.17 s -1 mm -1 The K for apigenin was m , k cat The values ​​are 190 μM and 0.0032 s, respectively. -1 , k cat / K m The value is 0.16 s -1 mm -1 The k of AgGlcT was cat / K m The values ​​were 10 to 100 times lower than those of glucose transferases from other plants such as moss and perilla. cat / K m The values ​​are 0.076 and 0.058 s, respectively. -1 mm -1 And AgApiT is also k cat / K m The k value of AgGlcT is low. cat Although the value was relatively low, the expression level of AgGlcT was relatively high according to the RNA-seq analysis, suggesting that the biosynthesis of apiin is regulated by the expression level of AgGlcT.

[0091] [Table 6]

[0092] Example 8 Identification of the PcApiT and PcGlcT genes 8-1. Experimental materials and methods (1) Experimental materials Parsley seeds (variety Curly Paramount) were purchased from Takii Seeds. Seeds were sterilized with 70% ethanol for 1 min, washed three times with distilled water, and surface-sterilized with sodium hypochlorite for 20 min. After washing three times with distilled water, 2 μL of PPM (plant preservative mixture, Nacalai Tesque) was added and vernalized for 1 day in the dark at 4 °C. Seeds were sown in a soil mixture of Metromix and vermiculite (1:1). The growth conditions were photoperiod, 16 h light / 8 h dark, light intensity, 132 μmol m-2 s-1, temperature, 22 °C, distilled water every day, and Hyponex every 3 days. (2) Screening of candidate genes for apiosyltransferase and glucosyltransferase from parsley Parsley RNA-Seq dataset was obtained from NCBI's Sequence Read Archive (SRA) (https: / / www.ncbi.nlm.nih.gov / sra). Reads were trimmed with trimomatic version 0.39 and de novo assembled with Trinity version 2.8.5 with k-mer size 32. Based on the results of Trinity, amino acid sequences were predicted with TransDecoder version 5.5.0, and expression levels were counted with Salmon version 0.14.1. Parsley piose transferase (PcApiT) candidate gene was obtained by searching the transcript catalog created by de novo assembly for sequences similar to known UGT87, 91, and 94 (including cerolelia piose transferase) with BLAST 2.12.0+. Among them, those with high TPM values ​​were selected as candidate genes. For the parsley glucosyltransferase (PcGlcT) candidate gene, a gene with high expression level and similarity to the celery glucosyltransferase gene was selected as the candidate gene. 8-2.Results RNA was extracted from primary leaves of parsley at the early stage and RNA-Seq analysis was performed. The dataset was trimmed, and 112154 transcripts were de novo assembled with a k-mer size of 32, and their abundance was counted by Transcripts Per Million (TPM) (Table 7). Among UGT79, 91, and 94 classified as GGTs to which ApiT is thought to belong, the expression level of Ubk32_id_4398_c1_g5 was high. This gene has 90% amino acid sequence homology with celery apiose transferase AgApiT (Figure 25), and was selected as a candidate gene for PcApiT. For PcGlcT candidate genes, those with high homology (78%) with celery AgGlcT were selected as candidate genes (Figure 25).

[0093] [Table 7]

[0094] Example 9 Expression, enzyme activity, and biochemical properties of PcGlcT and PcApiT 9-1. Experimental materials and methods (1) Expression of parsleyia piose transferase and glucosyl transferase in E. coli Candidate gene sequences for PcGlcT and PcApiT were chemically synthesized as codon-optimized genes (Eurofins) for protein expression in E. coli cells. The codon-optimized gene for PcApiT was amplified with pColdProS2_PcApiT_F and pColdProS2_PcApiT_R primers and cloned into pColdProS2 vector between NdeI and XbaI sites. The codon-optimized gene for PcGlcT was amplified with pET28b_PcGlcT_F and pET28b_PcGlcT_R primers and cloned into pET28b vector between NdeI and XhoI sites. The pColdProS2_PcApiT and pET28b_PcGlcT clone products were transformed into E. coli DH5a cell clone products by heat shock, and the cells were plated on LB-ampicillin agar plates and grown at 37°C for 12–16 h. Several colonies were inoculated into 3 mL of LB-ampicillin medium, and the culture was grown at 37°C for 12 to 16 hours, and the plasmid was isolated using the FastGene Plasmid Mini Kit (Nippon Genetics). The nucleotide sequence of the plasmid product was confirmed by DNA sequencing. E. coli BL21(DE3) transformed with pColdProS2_PcApiT and pET28b_PcGlcT were cultured in LB medium containing 100 mg / mL ampicillin at 37°C until the OD600 reached 0.6. IPTG was added to a final concentration of 1 mM and incubated at 15°C for 24 hours. Cells were harvested by centrifugation at 5,000 × g for 5 minutes at 4°C and lysed for 20 minutes in BugBuster Protein Extraction Reagent (Millipore) supplemented with 10 mM sodium phosphate buffer, pH 7.4, 5 U / mL benzonase, and 1 kU / mL lysozyme. Cell debris was removed by centrifugation at 20,000 × g for 20 minutes at 4°C. The supernatant was collected and an appropriate amount of buffer (20 mM sodium phosphate buffer, 200 mM sodium chloride, 20 mM imidazole pH 7.4) was added to the supernatant so that the solution contained 20 mM imidazole. The filtered sample was then passed through a 0.45 mm filter and applied to a 1 mL HisTrap HP equilibrated with buffer 1 (50 mM sodium phosphate buffer and 500 mM sodium chloride, 20 mM imidazole pH 7.4). The column was washed with 10 column volumes of buffer 1, followed by elution with 5 column volumes of buffer 2 (50 mM sodium phosphate buffer, 500 mM sodium chloride, 50 mM imidazole pH 7.4) and buffer 3 (50 mM sodium phosphate buffer, 500 mM sodium chloride, 200 mM imidazole pH 7.4). The expressed ProS2_PcApiT fusion protein was digested with HRV3C protease at 4 °C for 12 h to remove the proS2 tag. The resulting PcApiT protein was purified as the flow-through fraction on a 1 mL-HisTrap HP column equilibrated with buffer 1. The resulting expressed proteins (PcGlcT and PcApiT) were concentrated by ultrafiltration several times using an Amicon Ultra-0.5 mL (10 kD cutoff) at 14,000 × g, 4 °C, for 15 min. The proteins were analyzed by SDS-PAGE. (2) Activity measurement of PcApiT and PcGlcT To analyze the substrate specificity of the purified recombinant protein PcGlcT, a 10 μL reaction mixture consisting of 1 mM glycosyl donor, 1 mM glycosyl acceptor, 0.2 μg of PcGlcT, and 100 mM Tris-HCl buffer (pH 9.0) containing 50 mM NaCl was reacted at 23°C for 30 min. The glycosyl acceptors used were apigenin (Cayman Chemical), apigenin 7-O-glucoside (Ark Pharm), apiin (Ark Pharm), naringenin (TCI), luteolin (TCI), quercetin (TCI), genistein (TCI), and chrysoeriol (Extrasynthase), and the glycosyl donors UDP-Glc (Fujifilm Wako Chemical), UDP-Gal (Fujifilm Wako Chemical), UDP-GlcA (Sigma-Aldrich), and UDP-Araf (Peptide Institute). UDP-Xyl, UDP-Api, and UDP-GalA were prepared by the inventors. To analyze the substrate specificity of the purified recombinant protein PcApiT, 5 μg / μL of the PcApiT enzyme was incubated with 100 μM of sugar acceptor, 1 mM of sugar donor, and 120 mM Tris-HCl buffer containing 50 mM NaCl (pH 7.0) at 23°C for 1 h. The glycosyl acceptors used were apigenin 7-O-glucoside, apiin, apigenin, chrysoeriol 7-O-glucoside (ChemFaces), naringenin 7-O-glucoside (Extrasynthese), luteolin 7-O-glucoside (Extrasynthese), quercetin 7-O-glucoside (Extrasynthese), and quercetin 3-O-glucoside (Extrasynthese). The glycosyl donors used were UDP-Araf, UDP-Glc, UDP-Gal, UDP-GalA, UDP-GlcNAc (Fujifilm Wako Chemical), UDP-GlcA, GDP-Fuc (Sigma-Aldrich), UDP-Api, UDP-Xyl, and UDP-GalA. The enzyme reaction was stopped by incubation at 100°C for 3 min, and the biochemical properties of the enzyme were determined by quantification of the peak areas of the substrate and product by HPLC analysis. The enzyme activity was defined as 1 unit, which is the amount of enzyme that produces 1 μmol of product under the above reaction conditions. To determine the enzyme kinetic parameters (Km and kcat values) of PcGlcT and PcApiT, the enzyme activities were analyzed at the following concentrations: apigenin (25-1500 μM), UDP-Glc (50-1500 μM) for PcGlcT, and apigenin 7-O-glucoside (12.5-2000 μM), and UDP-apiose (2.4-500 μM) for PcApiT. To determine the optimum pH and temperature for PcGlcT and PcApiT, 100 mM sodium acetate buffer (pH 4.0-5.0), 100 mM MES-KOH buffer (pH 6), 100 mM HEPES-KOH buffer (pH 7.0-8.0), 100 mM Tris-HCl buffer (pH 8.5-9.5), 100 mM glycine-NaOH buffer (pH 10.0-10.5), and 100 mM Britton-Robinson buffer (pH 11.0-12.0) were used. To determine the optimum temperature, each reaction mixture was incubated at a series of temperatures (5-50°C, 5°C increments) for 20 min, and the enzyme activity was analyzed. (3) HPLC conditions The activities of PcGlcT and PcApiT were measured by quantifying the peak areas corresponding to the substrate and product on an Inertsil ODS-3 reversed-phase column (4.6 × 250 mm, GL Sciences). 10 μL of the reaction mixture was applied to the column and analyzed at a flow rate of 1.0 mL / min. PcGlcT was analyzed in 25% acetonitrile containing 0.1% TFA for 5 min, followed by increasing the acetonitrile concentration to 55% in 20 min. PcApiT was analyzed in 20% acetonitrile containing 0.1% TFA for 5 min, followed by increasing the acetonitrile concentration to 40% in 20 min. Apigenin, chrysoeriol, luteolin, naringenin, and quercetin were detected and quantified by absorbance at 330, 330, 350, 280, and 254 nm, respectively. (4) Gene expression analysis The expression profiles of PcGlcT and PcApiT in each organ and each developmental stage of parsley were analyzed by qRT-PCR. RNA was prepared from parsley seeds, roots, stems, and leaves at each developmental stage (~0.5 cm, 0.5-1.0 cm, 1.0-1.5 cm, 1.5-2.0 cm, 2.0 cm+) using the RNeasy Mini Kit (Qiagen). cDNA was synthesized using the PrimScript II 1st strand cDNA synthesis Kit (Takara Bio). The qRT-PCR reaction mixture (20 μL) containing 2.0 μL of cDNA (50 ng / μL), 0.8 μL of primer (final concentration 0.4 μM), 10 μL of TB Green Premix Ex Taq II (Tli RNaseH Plus), 0.4 μL of ROX reference dye, and 6.0 μL of RNA-free water5 was reacted at 95°C for 30 s, 95°C for 5 s and 60°C for 30 s (40 cycles), 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The housekeeping gene EF-1α was selected as the reference gene. The relative expression level was calculated using the ΔΔCt method, and the expression level in leaves less than 0.5 cm was set to 1. The results were expressed as the average of three replicates. 9-2.Results The PcGlcT candidate gene, codon-optimized for expression in E. coli, was cloned into the pET28b vector and expressed in E. coli. A protein with a molecular mass of 53 kDa was purified by Ni-chelate affinity chromatography (Figure 26). When the purified PcGlcT protein was reacted with apigenin (sugar acceptor) and UDP-glucose (sugar donor), apigenin 7-O-glucoside was quantitatively produced (Figure 27). The acceptor substrate specificity of PcGlcT was examined. PcGlcT showed activity toward flavonoids such as apigenin, luteolin, quercetin, naringenin, genistein, and chrysoeriol (Figure 28). As a donor substrate, PcGlcT preferred only UDP-glucose (Figure 28). The codon-optimized PcApiT was cloned into the pColdProS2 vector and expressed in E. coli. The ProS2-ApiT fusion protein with a molecular mass of 73 kDa was purified by Ni-chelate affinity chromatography. The ProS2 tag was cleaved, and the tagless PcApiT protein (molecular mass 49 kDa) was purified (Figure 26). When this was reacted with apigenin 7-O-glucoside (sugar acceptor) and UDP-apiose (sugar donor), apiin was quantitatively produced (Figure 27). The acceptor substrate specificity of PcApiT was mainly specific for flavonoid 7-O-glucosides (apigenin 7-O-glucoside, quercetin 7-O-glucoside, and chrysoeriol 7-O-glucoside) (Figure 28). As a glycosyl donor, PcApiT showed activity only for UDP-apiose, with high specificity (Figure 28). The optimum pH for both PcGlcT and PcApiT was 9.0 (Figure 29). m The value is 610 μM, k cat The value is 0.62 s -1 The K of PcGlcT against apigenin was m The value is 430 μM, k cat The value is 0.60 s -1 The K of PcApiT against UDP-apiose was m The value is 81 μM, k cat The value is 1.98 s -1 The K of PcGlcT against apigenin 7-O-glucoside was m The value is 64 μM, V max The value is 3.20 s -1 It was.

[0095] [Table 8]

[0096] The expression of the PcGlcT and PcApiT genes was analyzed by qRT-PCR (Figure 30). Both enzyme genes were most expressed in the true leaves, and even in the true leaves, they were most expressed at the young stage (leaf length 0.5 to 1.0 cm). This coincides with the time and space of apiin biosynthesis, indicating that these enzymes are involved in the biosynthesis of apiin. The nucleotide sequences of the PcGlcT and PcApiT genes and the amino acid sequences of the proteins are shown (Figure 31).

[0097] Example 10: Apiin production 10-1. Experimental materials and methods (1) Media used 2xYT medium was used as the growth medium for E. coli. The composition is shown below. (2×YT medium (1 L)) Hypopolypeptone 16g 10 g dry yeast extract Sodium chloride 5 g (15 g of agar powder) (2) E. coli expression vector used Five types of E. coli expression vectors were used for apiin production (Table 9). pCDF-1b-AgGlcT, pCDF-1b-AgApiT, and pACYCDuet-AtAXS1 were constructed in this example. The construction methods are described below.

[0098] [Table 9]

[0099] First, pCDF-1b-AgApiT and pCDF-1b-AgGlcT were constructed. The AgApiT and AgGlcT expression cassettes (DNA fragments containing the cspA promoter to the T7 terminator) were PCR amplified using pColdProS2-AgApiT or pColdProS2-AgGlcT as a template, pColdProS2-F and pColdProS2-R as a primer pair, and KOD One DNA polymerase (TOYOBO) as a PCR enzyme. Next, the DNA fragment from the T7 promoter to the T7 terminator removed from pCDF-1b was amplified by inverse PCR using pCDF-1b-F and pCDF-1b-R with pCDF-1b as a template and KOD One DNA polymerase (TOYOBO). Each amplified fragment was then purified using a Gel / PCR Extraction kit (FastGene) and then ligated using NE-Builder HiFi DNA Assembly Master Mix (NEB). Next, pACYCDuet-AtAXS1 was constructed. The AtAXS1 gene fragment was amplified using pET28b-AtAXS1 as a template, AtAXS1-Bgl-F and AtAXS1-Kpn-R as a primer pair, and KOD-Plus-NEO DNA polymerase (TOYOBO) as a PCR enzyme. The PCR product and pACYCDuet-1 (Merck) were treated with restriction enzymes BglII and KpnI, and each fragment was purified using a Gel / PCR Extraction kit (FastGene), and then ligated using Ligation High Ver2 DNA ligase (TOYOBO). The primer sequences used for vector construction are shown in Table 10.

[0100] [Table 10]

[0101] The conditions for PCR and DNA ligation reaction are described below. (KOD One) 98℃ 10sec (T m -5)℃ 5 sec 68℃ 1~10sec / kbp (30 cycles) 68℃ 5 min 12℃∞ (KOD Plus Neo) 98℃ 10sec T m ℃ 30 seconds 68℃ 30sec / kbp (30 cycles) 68℃ 5 min 12℃∞ (NE-Builder HiFi DNA Assembly Master Mix) 2×NE builder HiFi DNA Assembly Master Mix: 1.2 μL Linearized vector + purified insert: 1.2 μL The above mixed solution was incubated at 50° C. for 15 minutes and then allowed to stand on ice. (Ligation High Ver2) Ligation High Ver2: 5.0 μL Linearized vector + purified insert: 5.0 μL The above mixed solution was incubated at 16° C. for 30 minutes and then allowed to stand on ice. (3) Colony PCR After obtaining E. coli transformants, colony PCR was performed using Go Taq Green Master Mix to confirm whether or not the transformants had the target gene. The reaction conditions are as follows: 95℃ 2 min 95℃ 30sec (Tm value -5) ℃ 30 sec (30 cycles) 72℃ 60sec / 1kbp 72℃ 5 min 12℃∞ (4) Construction of transformants for apiin production BL21(DE3) was used as the host for apiin production. The combination of the introduced plasmids and the strain names are shown in Table 11. The transformants were named API001 and API002, respectively.

[0102] [Table 11]

[0103] (5) Sample preparation for protein expression analysis From the frozen stocks of API001 and API002 strains, some of the cells were inoculated onto 2xYT+Amp+Cm+Sm agar medium and cultured overnight at 37°C. A part of the cells grown on the agar medium was scraped off and inoculated into 3 mL of 2xYT+Amp+Cm+Sm liquid medium and precultured at 37°C for 18 h. After that, the cells were inoculated into 20 mL of 2xYT+Amp+Cm+Sm liquid medium at OD 0. 600 Inoculate the preculture so that the OD = 0.2. 600 = 0.6-0.8, then apigenin was added to a final concentration of 50 μM, and protein expression was induced at 15 °C, 200 spm, and 24 h. (6) Sample preparation for protein expression analysis and reverse-phase HPLC analysis After induction of expression, the cells were harvested, suspended in a homogenizing buffer (1x PBS, 1 mM PMSF) and frozen in liquid nitrogen to form spheres of approximately 5 mm diameter. The spherical cells were then homogenized (2500 rpm, 20 sec) using a multi-beads shocker (YASUI KIKAI) until they were finely divided, and then thawed on ice. After thawing, the homogenized suspension was transferred to a new 1.5 mL tube and stirred at 1000 rpm, 4°C, for 5 min to separate the supernatant and precipitate. The supernatant was supplemented with an equal amount of 2x SDS Sample Buffer, and the precipitate was supplemented with twice the amount of 2x SDS Sample Buffer obtained by adding 1x SDS Sample Buffer to the supernatant, and the mixture was treated at 100°C for 5 min to prepare a sample for SDS-PAGE. The composition of the 2x SDS Sample Buffer is shown below. (2x SDS sample buffer (10 mL)) 0.5 M Tris-HCl (pH 6.8) 2.0 mL 10% SDS 4.0 mL Glycerol 2.0 mL β-Mercaptoethanol 1.2 mL 0.8 mL distilled water A few drops of 1% BPB In addition, to prepare a sample for reverse-phase HPLC analysis, the cell suspension after expression induction was centrifuged to separate the cells and the culture supernatant, and then an equal volume of methanol was added to the culture supernatant. (7) Protein detection by CBB staining and Western blotting The SDS-PAGE sample was applied to a 10% acrylamide gel and SDS-PAGE was performed. After electrophoresis, the separation gel was cut out and stained with CBB or transferred to a PVDF membrane. For electroblotting, filter paper (Absorbent Paper CB-09A (ATTO)) was soaked in Blotting Buffer A, Blotting Buffer B, and Blotting Buffer C for 30 min. The PVDF membrane was pretreated by soaking in methanol and then soaked in Blotting Buffer C. After SDS-PAGE, the membranes were stacked in the following order from the cathode side in the transfer device: Blotting Buffer C → electrophoresis gel → PVDF membrane → Blotting Buffer B → Blotting Buffer A, and then transferred to a 1 mM / 1 cm 2 The antibody was then transferred to a PVDF membrane at 10-15 V for 90 minutes. After transfer, the PVDF membrane was immersed in blocking buffer {5% skim milk, PBS + 0.05% Tween 20 (PBS-T)} and blocked by shaking at room temperature for 1 h. The PVDF membrane was then washed by shaking 3 times for 5 min with PBS-T at room temperature. After washing, the membrane was immersed in primary antibodies (1:10,000, Anti 6×Histidine, Monoclonal Antibody Lot.CAR2715; 1:5,000, Anti-S-tag Antibody, Rabbit) diluted in PBS-T for 1 h. The PVDF membrane was then washed again by shaking 3 times for 5 min with PBS-T at room temperature. Next, the sections were immersed in secondary antibodies (1:10,000, Anti-Mouse IgG (H+L) Lot.144601; 1:10,000, Anti-IgG (H+L chain) (Rabbit) pAb-HRP) diluted in PBS-T for 1 h. Finally, after washing for 5 min three times, the sections were immersed in 500 μL of Immobilon Forte Western HRP Substrate (Millipore), a chemiluminescent substrate, and the amount of chemiluminescence was measured using ChemiDocTM XRS+ with Image LabTM Software to detect the target protein. (8) Reverse-phase HPLC analysis After fermentation production, the prepared reverse phase HPLC analysis sample was analyzed under the following conditions. (Reverse-phase HPLC analysis conditions) Column: InterSustain AQ-C18 5 μm (4.6 × 150 mm) Solution: A) 20% CH3CN,0.1% TFA B) 40% CH3CN, 0.1% TFA Flow rate: 1.0 mL / min Detection: absorbance at 330 nm Column temperature: 30 °C Gradient: Time (min) 0→5→25→30→31 B(%)0→0→100→100→0 10-2.Results (1) Protein expression analysis The results of protein expression analysis using SDS-PAGE and Western blotting are shown in Figure 32. The left side shows the results when API001 was used, and the right side shows the results when API002 was used. A negative control strain in which the three types of AgGlcT, AgApiT, and AtAXS1 genes were not introduced, and a strain in which all three types of AgGlcT, AgApiT, and AtAXS1 genes were introduced (API001 or API002 strain), were cultured under conditions without and with the addition of apigenin, respectively. Soluble fractions were prepared from the cultured cells, and a total of four types of samples for SDS-PAGE were prepared. Specific bands that were thought to be AgGlcT, AgApiT, and AtAXS1 were confirmed. (2) Analysis of A7G and apiin production by reversed-phase HPLC Next, the results of reversed-phase HPLC analysis of the products are shown in Figure 33. Two specific peaks were confirmed in the reversed-phase HPLC samples prepared from API001 and API002, which were not observed in the negative control (NC). These peaks were co-eluted with the authentic samples of apigenin-7-O-Glc (A7G) and apiin, suggesting that A7G and apiin were produced in both API001 and API002. (3) Production and yield of A7G and apiin The production and yield of A7G and apiin quantified from the peak areas of the results of reversed-phase HPLC are shown in Figure 34. A7G was produced at 19 mg / L in API001 and 15 mg / L in API002. Apiin was produced at 1.4 mg / L in API001 and 0.38 mg / L in API002. The yields of A7G were 78% in API001 and 75% in API002, and the yields of apiin were 4.6% in API001 and 1.4% in API002. There was no significant difference in the production of A7G between the two strains, but API001 produced about 3.6 times more apiin than API002. The difference in the production of apiin between the two strains is thought to be related to the expression level of the AgApiT gene, which transfers apiose to A7G. In the two apiin-producing strains used in this study, API001 had AgApiT introduced into pCDF-1b, and API002 had AgApiT introduced into pColdProS2. The replication origin ColDF13 of pCDF-1b has a copy number of 20-40, and the replication origin ColE1 of ColdProS2 has a copy number of 15-20. These results suggest that the copy number of the vector into which AgApiT is introduced may have an effect, and the API001 strain, which had AgApiT introduced into pCDF-1b, which has a higher copy number, produced more apiin. [Industrial Applicability]

[0104] By introducing the gene encoding the apiosetransferase and the gene encoding the glucose transferase of the present invention into a host cell that produces UDP-glucose and UDP-apiose and culturing the cell in the presence of apigenin, it becomes possible to easily produce apiin from apigenin, facilitating its use as a medicine or food product.

Claims

1. An apiosyltransferase comprising an amino acid sequence selected from the group consisting of (i) to (iv) below: (i) an amino acid sequence represented by SEQ ID NO:2; (ii) an amino acid sequence represented by SEQ ID NO:4; (iii) an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:2; (iv) An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:

4.

2. The apiosyltransferase according to claim 1, comprising the following amino acid sequence (i) or (ii): (i) an amino acid sequence represented by SEQ ID NO:2; (ii) The amino acid sequence represented by SEQ ID NO:

4.

3. A glucosyltransferase comprising an amino acid sequence selected from the group consisting of (i) to (iv) below: (i) an amino acid sequence represented by SEQ ID NO:6; (ii) an amino acid sequence represented by SEQ ID NO:8; (iii) an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:6; (iv) An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO:

8.

4. The glucosyltransferase of claim 3, comprising the following amino acid sequence (i) or (ii): (i) an amino acid sequence represented by SEQ ID NO:6; (ii) The amino acid sequence represented by SEQ ID NO:

8.

5. A method for producing apiin from apigenin 7-O glucoside, comprising a step of contacting the apiosyltransferase according to claim 1 or 2, apigenin 7-O glucoside and UDP-apiose.

6. The method according to claim 5, characterized in that the process is carried out in a host cell which contains a nucleic acid having a base sequence encoding the apiosyltransferase according to claim 1 or 2 and which produces UDP-apiose.

7. A method for producing apigenin 7-O glucoside from apigenin, comprising a step of contacting the glucosyltransferase of claim 3 or 4, apigenin and UDP-glucose.

8. 8. The method according to claim 7, characterized in that the process is carried out in a host cell which contains a nucleic acid having a base sequence encoding the glucosyltransferase according to claim 3 or 4 and which produces UDP-glucose.

9. A method for producing apiin from apigenin, comprising the following steps (i) and (ii): (i) contacting the glucosyltransferase of claim 3 or 4 with apigenin and UDP-glucose to produce apigenin 7-O glucoside; (ii) a step of contacting the apiosyltransferase according to claim 1 or 2, the apigenin 7-O-glucoside and UDP-apiose to produce apiin.

10. The method according to claim 9, characterized in that steps (i) and (ii) are carried out in the same host cell, which contains a nucleic acid having a base sequence encoding the glucose transferase described in claim 3 or 4 and a nucleic acid having a base sequence encoding the apiose transferase described in claim 1 or 2, and produces UDP-glucose and UDP-apiose.

11. A kit for producing apiin from apigenin 7-O glucoside, comprising the following (i) to (iii): (i) the apiosyltransferase according to claim 1 or 2; (ii) apigenin 7-O-glucoside, (iii) UDP-apiose.

12. A kit for producing apiin from apigenin 7-O glucoside, comprising: (i) a host cell comprising a nucleic acid having a base sequence encoding the apiosyltransferase according to claim 1 or 2 and capable of producing UDP-apiose; (ii) Apigenin 7-O-glucoside.

13. A kit for producing apigenin 7-O glucoside from apigenin, comprising the following (i) to (iii): (i) a transglucosyltransferase according to claim 3 or 4, (ii) apigenin, (iii) UDP-glucose.

14. A kit for producing apigenin 7-O glucoside from apigenin, comprising: (i) a host cell comprising a nucleic acid having a base sequence encoding the glucosyltransferase according to claim 3 or 4 and capable of producing UDP-glucose; (ii) Apigenin.

15. A kit for producing apiin from apigenin, comprising the following (i) to (v): (i) a transglucosyltransferase according to claim 3 or 4, (ii) the apiosyltransferase according to claim 1 or 2, (iii) apigenin, (iv) UDP-glucose, (v) UDP-apiose.

16. A kit for producing apiin from apigenin, comprising the following (i) and (ii): (i) a host cell which contains a nucleic acid having a base sequence encoding the glucosyltransferase according to claim 3 or 4 and a nucleic acid having a base sequence encoding the apiosetransferase according to claim 1 or 2, and which produces UDP-glucose and UDP-apiose; (ii) Apigenin.

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