JP2003088373A - Caffeine synthase and gene for cording the synthase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a polypeptide having enzymatic activity of a caffeine synthase, capable of effectively producing the caffeine synthase useful as an enzyme for industry, food or medical care, and to provide a method for producing secondary metabolites of plants, capable of effectively producing caffeine-based metabolite compounds by changing caffeine- biosynthesizing metabolism in caffeine-producing plants, plant tissues thereof or plant cells thereof, and capable of changing a generation ratio of a group of the compounds by changing the metabolism in the plants, the plant tissues or the plant cells. SOLUTION: This polypeptide has a specific amino acid sequence derived from Camellia irrawodiensis (refer to the specification) and the enzymatic activity of the caffeine synthase. A DNA and a RNA which code the polypeptide are provided in the specification, respectively. The caffeine synthase is produced by using microorganisms, plant bodies or cultured cells which are transformed by a vector containing the whole or a part of the DNA or the RNA. The caffeine synthase is controlled in its representation by an antisense RNA. Thus, caffeine contents in the plants are changed, and the caffeine synthase is produced.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to caffeine synthase, DNA or RNA encoding the enzyme, and DN.
A method for producing caffeine or a precursor thereof using the microorganism and plant transformed with A, and the plant or a cultured cell or tissue thereof, and determining the amount of caffeine or a precursor thereof by a nucleic acid structure containing the RNA. Regarding how to change.

[0002]

2. Description of the Related Art Caffeine is used in tea ( Camellia sinensi
s ) and other camellia plants, such as coffee ( Coffea ar
abica ) is a purine alkaloid contained in Rubiaceae coffee genus plants and the like, and is used as a pharmaceutical raw material and a food additive. At present, caffeine is produced by extraction from caffeine-producing plants such as the above plant species or by organic synthesis. In addition, in the case of favorite products such as tea and coffee, it has been attempted to reduce or increase the content of caffeine and its intermediates by using classical breeding methods or the like in order to reduce or enhance their irritancy. ing.

Caffeine is a three-stage N from xanthosine.
Biosynthesis by -methylation has been demonstrated by ¹⁴ C-tracer experiments. [Phytochemistry, 3
1, 2575- (1992)]. The enzyme activity that catalyzes this methylation was first reported in 1975 in a study using crude extracts of tea leaves [Biochem. J., 146, 87- (1975)]. Attempts have been made to purify methyltransferase in coffee [Phytochemistry, 37, 1577- (1994)], but the purification rate was extremely low. In Cha, there is a report of partial purification of methyltransferase [Physiol.Plant., 98, 629-
(1996)], the enzyme protein was not isolated. In such a situation, caffeine synthase, that is, the amino acid sequence of N-methyltransferase that catalyzes the two-step methylation reaction of 7-methylxanthine-theobromine-caffeine, which is the final reaction of caffeine biosynthesis, and its amino acid sequence encoding respect to DNA, tea (Ca
mellia sinensis ), Kato et al. were the first in the world to isolate and purify and report (Japanese Patent Application No. 11-146358, Japanese Patent Application No. 12-1517).
18, Japanese Patent Application No. 12-275063).

On the other hand, in the tea, Camellia irrawadiensis
There is also a type known to accumulate theobromine instead of caffeine, but what kind of diversity the enzymes of the biosynthetic system of caffeine in these species show However, it was unknown at all.

[0005]

The present invention achieves the following objects by incorporating all or part of DNA or RNA encoding caffeine synthase into microorganisms or plant cells in the form of sense or antisense. It is what (1) Efficiently produce caffeine synthase which can be used as an industrial, food or medical enzyme. (2) By modifying the caffeine biosynthetic metabolism of a caffeine-producing plant, plant tissue or plant cell, a caffeine metabolism system compound is efficiently produced. (3) Modifying the caffeine biosynthetic metabolism of caffeine producing plants, plant tissues or plant cells to modify the production ratio of the compounds in the caffeine metabolism system.

[0006]

As a result of earnest research, the present inventors have prepared a DNA probe based on the DNA sequence of caffeine synthase of tea ( Camellia sinensis ) isolated earlier, and prepared this probe. It was successfully used to isolate the target DNA from different teas ( Camellia irrawadiensis ) known to accumulate theobromine by the 3'RACE method and the 5'RACE method. Next this DN
After incorporating A into a vector, it was introduced into E. coli and the DN
A large amount of the protein derived from A was expressed. When the enzymatic properties of this protein were examined, it was confirmed that the same reaction as that of caffeine synthase isolated from the young leaves of tea ( Camellia sinensis ) previously reported, that is, the production of caffeine from 7-methylxanthine was observed. Recognized, DN
It was confirmed that A is a gene encoding caffeine synthase. The present inventors have completed the present invention based on the above findings.

That is, the present invention is as follows. [1] A polypeptide which is defined by the amino acid sequences 1 to 365 of SEQ ID NO: 1 in the sequence listing and has an enzymatic activity of caffeine synthase. [2] A DNA encoding the polypeptide according to [1] above. [3] The DNA according to [2] above, which is defined by the base sequence from 1 to 1432 of SEQ ID NO: 1 in the sequence listing. [4] RNA encoding the polypeptide according to [1] above. [5] The RNA according to the above [4], which is defined by the base sequence from No. 1 to No. 1432 of SEQ ID NO: 2 in the sequence listing. [6] A vector containing the DNA according to [2] or [3] above. [7] The vector according to [6] above, which is capable of expressing a polypeptide having the enzymatic activity of caffeine synthase in the cells of a microorganism and / or plant. [8] The vector according to [6] above, which is capable of expressing an antisense RNA having an effect of suppressing the expression of the enzyme of caffeine synthase in the cells of a microorganism and / or a plant. [9] A vector containing the RNA according to [4] or [5] above. [10] The vector according to [9] above, which can express an antisense RNA having an effect of suppressing the expression of the enzyme of caffeine synthase in the cells of a microorganism and / or a plant. [11] A microorganism transformed with the vector according to any one of [6] to [10] above. [12] A method for producing a polypeptide having an enzymatic activity of caffeine synthase, using the transformed microorganism according to the above [11]. [13] A plant cell, plant tissue or plant transformed with the vector according to any one of the above [6] to [10]. [14] A method for producing a plant secondary metabolite using the transformed plant according to [13] above, a cultured cell of the plant, or a plant. [15] The method described in [14] above, wherein the plant secondary metabolite is at least one compound selected from xanthosine, 7-methylxanthosine, 7-methylxanthine, theobromine, theophylline, and caffeine. [16] Transformed plants are Camellia genus plants, Coffee ( Coffea ) genus plants, Cola ( Cola ) genus plants, Coca ( Erythroxylum ) genus plants, Molex ( Ilex ) genus plants, Neea ( Neea ) genus plants, Aogiri ( Firmiana ) genus plant, Paulinia genus plant or cacao tree ( T
The method according to the above [15], which is a plant of the genus heobroma ).

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, as an embodiment of the present invention, a method for constructing an expression vector containing caffeine synthase, a method for producing a transformed plant and a method for suppressing the expression of caffeine synthase enzyme by the RNAi method will be described.

In order to express the caffeine synthase gene or its antisense RNA in this report in plant cells, (i) a promoter transcribable in plant cells, (ii) a sense direction or an antisense gene downstream of the promoter. Caffeine synthase gene D linked in the sense direction
An expression cassette containing all or part of NA, and (iii) a terminator sequence containing a polyadenylation site necessary for stabilization of a transcription product linked to the downstream of the gene DNA, is introduced into plant cells. .

The expression cassette may contain a promoter for expressing the inserted DNA constitutively or inducibly. As a promoter for constitutively expressing, for example, 35 of cauliflower mosaic virus is used.
The S promoter, the rice actin promoter and the like can be mentioned. In addition, as a promoter for inducible expression, it is known to be expressed by external factors such as infection and invasion of filamentous fungi, bacteria, viruses, low temperature, high temperature, dryness, anaerobic conditions, and application of specific compounds. Promoters and the like. Examples of such a promoter include a rice chitinase gene promoter expressed by filamentous fungi, bacteria, and virus infection or invasion, a tobacco PR protein gene promoter, and a low-temperature-induced rice "lip19" gene promoter. , "H of Arabidopsis thaliana induced by high temperature
Examples include the promoter of the SP18.2 "gene, the promoter of the rice" rab "gene induced by drought, the promoter of the maize alcohol dehydrogenase gene induced by anaerobic conditions, and the like. The rice chitinase gene promoter and the tobacco PR protein gene promoter are also induced by a specific compound such as salicylic acid, and the rice “rab” gene promoter is also induced by spraying the plant hormone abscisic acid.

Alternatively, as a promoter for expressing the DNA inserted in the expression cassette, a method of isolating and utilizing the promoter of the caffeine synthase gene can also be mentioned. One example of a specific method for isolating a promoter is to select a genomic DNA fragment by using a hybridization technique using a caffeine synthase gene, for example, all or part of DNA of the caffeine synthase gene as a probe, Upstream DN
A method of identifying A can be mentioned.

To prepare for the introduction of recombinant DNA molecules into plants, many cloning vectors are available which contain an E. coli replication signal and a marker gene for selecting transformed bacterial cells. Examples of such vectors include pBR322, pUC system, M13mp system and the like. The desired sequence can be introduced into the vector at an appropriate restriction enzyme cleavage site. The obtained plasmid DN
To clarify the characteristics of A, restriction enzyme cleavage site analysis,
Gel electrophoresis and other biochemical-molecular biology methods are commonly used. After each operation, the plasmid DNA can be cleaved and ligated to another DNA. The sequence of each plasmid DNA can be cloned in the same plasmid or another plasmid.

Various techniques can be used to introduce the expression cassette into plant host cells. These techniques include Agrobacterium tumefaciens as a transforming factor.
Or, Agrobacterium rhizogenes ( Agrobacteriu
transformation of plant cells with T-DNA using m rhizogenes ), direct introduction into protoplasts (injection method, electroporation method, etc.), particle gun method, and other possibilities.

For direct introduction into protoplasts, no special vector is required. For example, a simple plasmid such as pUC derivative can be used. Depending on the method of introducing the gene of interest into plant cells, other DN
The A sequence may be required. For example Ti or Ri
When the plasmid is used to transform plant cells, T
At least the rightmost sequences, and usually the sequences at both ends, of the T-DNA regions of the i and Ri plasmids must be joined so that they are adjacent to the gene to be introduced.

When Agrobacterium is used for transformation, it is necessary to clone the expression cassette to be introduced into a special plasmid, that is, an intermediate vector or a binary vector. The intermediate vector does not replicate in Agrobacterium. The intermediate vector is transferred into Agrobacterium by helper plasmid or electroporation. Since the intermediate vector has a region homologous to the T-DNA sequence, it is incorporated into the Ti or Ri plasmid of Agrobacterium by homologous recombination. Agrobacterium used as a host needs to contain a vir region. Usually, the Ti or Ri plasmid contains a vir region, and by its function,
T-DNA can be transferred to plant cells.

On the other hand, since the binary vector can be replicated and maintained in the bacterium of the genus Agrobacterium, when incorporated into the bacterium of the genus Agrobacterium by the helper plasmid or electroporation method,
T-D on a binary vector by the function of the r region
NA can be transferred to plant cells.

The intermediate vector or binary vector containing the thus obtained expression cassette, and microorganisms such as Escherichia coli and Agrobacterium belong to the present invention.

The transformed plant cells can be transformed into plants by undergoing a regeneration process. The method of regeneration depends on the type of plant cell, but in rice, for example, Fu
jimura et al. [Plant Tissue Culture Lett., 2, 74- (199
5)] method, in corn, Shillito et al [Bio / Techn
ology, 7, 581- (1989)], A in Arabidopsis
The method of Kama et al. [Plant Cell Rep., 12, 7- (1992)] can be mentioned.

Plants produced by these methods are
The expression level of the caffeine synthase protein of the present invention changes as compared to a wild-type plant that produces caffeine or its precursor, and changes in the production amount of the caffeine metabolism system compound due to the modification of the metabolism of the host plant and , The production ratio of caffeine metabolite compounds changes due to the alteration of metabolism of host plants. The transgenic plants thus obtained are the subject of the present invention. In the present invention, the “plant” refers to all or part of organs (eg, leaves, stems, roots, flowers, fruits, seeds) or cultured cells of organisms classified as plants.

The above-mentioned plants that produce caffeine by using SAM as a methyl group donor include Camellia plants belonging to the Camellia family such as tea, plants belonging to the Coffea family such as coffee, and the genus Caucas ( Theobroma ). Caffeine-producing plants such as plants and plants belonging to the genus Cola of the family Aquiliaceae such as Coranoki can be exemplified.

Examples of microorganisms for introducing a DNA encoding caffeine synthase according to the present invention to express a large amount of caffeine synthase protein include bacteria such as Escherichia coli and Bacillus subtilis and viruses such as baculovirus. You can

For the purpose of modifying the metabolism of the host cell to improve the productivity of a specific compound or modify the production ratio of a specific compound group, the DNA encoding the caffeine synthase according to the present invention is sensed or antisense. As a plant to be incorporated in the form of sense, any plant that produces caffeine or a precursor thereof can be used, but among them, a camellia ( Camellia ) genus plant such as tea, and a Rubiaceae coffee ( Coffea ) genus such as coffee. Plant, cacao ( Th
Examples thereof include plants of the genus eobroma ) and plants of the genus Cola of the family Aequiliceae .

The plants produced by these methods are
The expression level of the N-methyltransferase of the present invention changes as compared to a wild-type plant that produces caffeine or a precursor thereof, and changes in the production amount of the caffeine metabolism system compound due to the modification of the metabolism of the host plant, , The production ratio of caffeine metabolite compounds changes due to the alteration of metabolism of host plants. The transgenic plants thus obtained are the subject of the present invention.

Further, in recent years, based on the research on post-translational gene silencing of plants, it is possible to suppress the expression of a target gene by using the defense mechanism originally possessed by plants against foreign nucleic acids such as viruses. (Cell, 95, 177-187 (1998), Chemistry and Biology, 3
7, 532- (1999), Protein Nucleic Acid Enzyme, 44, 1396- (1999)).
According to this, when a DNA virus, an RNA virus, or the like enters a plant, the plant transcribes averant RNA from these templates, and produces a double-stranded RNA sequence-specifically with the transcript of the sequence originally possessed by the plant. Form. This double-stranded R
When NA is degraded by RNase, it becomes possible to suppress the expression of the target gene (Cell, 96, 3
03- (1999)). One of the important features of this method is that it is not always necessary to transform the target plant with the sequence whose expression is to be suppressed. Further, a further feature of this method is that, when a target nucleic acid is introduced into a part of a plant (lower leaf etc.) by infection or the like, its effect is spread to the whole plant body. A specific method for suppressing expression is a double-stranded RN containing all or part of a sequence of a target gene or a sequence having high homology with it.
The lower leaves of the plant are infected with A or Agrobacterium having a double-stranded DNA. Here, high homology means 60% or more homology in each nucleotide sequence comparison, preferably 75% or more homology, more preferably 90% or more homology, most preferably 95% or more homology. Point to.

The plants subjected to these methods have a different expression level of the N-methyltransferase protein of the present invention as compared with the wild-type plants producing caffeine or its precursor, and the host plants Changes in the amount of caffeine-metabolizing compounds produced due to alteration of metabolism and changes in the production ratio of caffeine-metabolising compounds due to alteration of metabolism of host plants. The plants thus obtained are the subject of the present invention. The plant as referred to in the present invention also includes specific tissues or cells of plants such as leaves, flowers, fruits and seeds.

The plant cells or plant tissues transformed or infected with the vector having the above-mentioned constitution are cultured, or similarly transformed plant is cultivated, and the caffeine synthesis system is used. The production of secondary metabolites and the composition of secondary metabolites produced by these transformants can be modified. As this secondary metabolite,
Examples thereof include at least one compound selected from the group consisting of 7-methylxanthine, paraxanthine, theobromine and caffeine.

As a source of plant cells, plant tissues or plants used for transformation, for example, transformed plants are Camellia genus plants, Coffee (Coffea) genus plants, Cola genus plants, Plants of the genus Ilex, Neea, Firmiana, Paulinia or cacao (T)
Heobroma) plants can be mentioned. Among them, preferable examples are Camellia plants such as tea, Coffea plants such as coffee, and Cola plants such as Coranaceae.

Furthermore, since the N-methyltransferase according to the present invention can methylate structural similar compounds such as 7-methylxanthine in addition to theobromine, the structures of these xanthines can be obtained even in other plant species. The method of the present invention can be applied to any plant containing a similar compound.

[0029]

EXAMPLES The present invention will be described in more detail below with reference to examples and comparative examples, but the scope of the present invention is not limited to these examples.

[Example 1] Cloning of caffeine synthase (1) Isolation of total RNA 5 g of young tea leaves ( Camellia irrawadiensis ) obtained from the Ministry of Agriculture, Forestry and Fisheries Experiment Station were pestle in the presence of liquid nitrogen.
It was crushed using a mortar. After sublimation of liquid nitrogen, 5 ml of 2%
CTAB, 0.1M Tris (pH9.5), 20mM EDTA, 1.4M NaCl, 5%
Dissolve in β-mercaptoethanol and leave at 65 ℃ for 10 minutes. Add an equal volume of chloroform solution and stir to bring the total volume to 10
It was set to about ml. After centrifugation at 15,000 rpm for 10 minutes, the aqueous layer is collected, 2.5 ml of 10M lithium chloride solution is added, and the mixture is left at -20 ° C for at least 2 hours. 4 ° C, 1
Centrifugation was performed at 5,000 rpm for 10 minutes, the supernatant was removed, the precipitate was dissolved in 1 ml of TE, and centrifugation was performed again. The supernatant was recovered, 1 ml of a TE-saturated phenol solution was added, mixed by inverting, and centrifuged at 15,000 rpm for 10 minutes. Further, phenol / chloroform was added to the supernatant, and the mixture was stirred and centrifuged at 15,000 rpm for 10 minutes. The aqueous layer is collected, an equal volume of chloroform solution is added, and the mixture is stirred. Centrifuge at 15,000 rpm for 10 minutes, collect the aqueous layer, add 1/4 volume of 10M lithium chloride solution, and leave at -20 ° C for at least 2 hours. 1
Centrifugation was performed at 5,000 rpm for 10 minutes, 70% ethanol was added to the obtained precipitate, and centrifugation was performed again. After drying up the precipitate with a vacuum pump, 20
It was dissolved in 0 μl of water to give a fraction of total RNA.

(2) cDNA synthesis required for cloning of 3'end by 3'-RACE method 3'-RACE is 3'-Full RACE Core Se of TAKARA.
used t. The primers not included in the sequence listing are those attached to this set. Using 1 μg of total RNA heat-treated at 65 ° C for 5 minutes as a template, use the method described in the instruction manual for this set to use oligo dT-3-sites A
cDNA was synthesized by a reverse transcription reaction using daptor Primer. The composition of the reaction solution is as follows. This reaction solution is Ge
30 using ne Amp PCR System 2400 (Perkin Elmer)
A reaction product was obtained under the conditions of reacting at 10 ° C for 10 minutes, 50 ° C for 1 hour, and 95 ° C for 5 minutes. Template cDNA 3 μl 10 × buffer 2 μl 10 mM dNTP 2 μl 25 mM MgCl _{2 4} μl Oligo-dT 3 sites adaptor primer 1 μl RNase inhibitor 0.5 μl H ₂ O 7 μl AMV-Reverse transcriptase 0.5 μl

(3) Cloning of caffeine synthase gene by 3'-RACE method The following reaction solution was prepared using the single-stranded cDNA prepared by the above-mentioned method as a template. Primer TCS6
The sequence is shown in SEQ ID NO: 3 in the sequence listing. This reaction solution is Ge
Using ne Amp PCR System 2400 (Perkin Elmer), 94
After the reaction at ℃ / 30 seconds, 94 ℃ / 30 seconds, 59 ℃ / 30 seconds,
PCR under conditions of 30 cycles of 72 ° C / 2 minutes
Was carried out to obtain a reaction product. 20 μl 10 × buffer 10 μl 2.5 mM NTP 16 μl TCS6 1 μl (20 pmol) 3 sites Adapter Primer 1 μl (20 pmol) H ₂ O 51 μl ExTaq (TAKARA) 1 μl

(4) Subcloning into a plasmid vector The reaction product was electrophoresed in TAE using 0.8% agarose gel, the resulting band of the desired product was cut out, and GENE CLEAN (Funakoshi) was used. DNA was recovered from the gel. Recovered DNA is pT7bl
After ligation to the ue vector (Novagen), it was transformed into E. coli DH5α.
After performing color selection using X-gal,
Liquid culture was performed in LB medium containing ampicillin, and the plasmid was extracted by the alkali-SDS method. The presence or absence of the insert was confirmed by agarose electrophoresis.

(5) Determination of nucleotide sequence Using the isolated plasmid, primer extension was performed in the following termination mix reaction solution. Regarding the reaction conditions, after reacting at 98 ° C. for 5 minutes, 25 cycles of 98 ° C. for 1 minute, 50 ° C. for 45 seconds, 72 ° C. for 1.5 minutes were performed, and then 72 ° C. for 1 minute. After the reaction was completed, 8 μl of the reaction stop solution was added to each tube. Shimadzu DSQ-2000 after heat denaturing at 98 ℃ for 5 minutes and quenching the sample in ice
Analysis was performed using the L (S) system. Reagent is ThermoSequen
The one attached to the ase Cycle Sequencing Kit was used. Sample mix reaction solution Plasmid DNA (1pmol) 3 μl Labeled Primer (2pmol) 1 μl H ₂ O 22 μl Termination mix reaction solution (4 types per sample) A reaction solution 2 μl Sample mix 6 μl G reaction solution 2 μl Sample mix 6 μl C reaction solution 2 μl sample Mix 6 μl T reaction 2 μl Sample mix 6 μl

(6) Isolation of 5'upstream region of caffeine synthase mRNA by 5'RACE method For isolation of 5'upstream region, 5'-Full RACE Core Set
(TAKARA) was used. Using the primer of TCS11R (SEQ ID NO: 7 in the sequence listing), 1st stranddcDN
A is carried out, and after degrading the hybrid RNA and cyclizing the single-stranded cDNA by the ligation reaction, a PCR reaction is carried out using the primers TCS9 (SEQ ID NO: 4 in the sequence listing) and TCS8R (SEQ ID NO: 5 in the sequence listing). It was Reaction conditions are 9
After the reaction at 4 ° C / 3 minutes, 94 ° C / 30 seconds, 55 ° C / 30 seconds,
A reaction product was obtained by setting 72 ° C./1 minute for 30 cycles.
Further, using this as a template, TCS7R (SEQ ID NO: 8 in the sequence listing)
And TCS10 (SEQ ID NO: 6 in the Sequence Listing), after reacting at 94 ° C / 3 minutes, 94 ° C / 30 seconds, 60 ° C / 30
Seconds and 72 ° C./30 seconds for 30 cycles to obtain a reaction product. The band of the reaction product was separated by acrylamide gel electrophoresis, and the DNA was recovered from the gel and subcloned into the pT7blue vector.
After preparing a sample using the enase Cycle Sequencing Kit, the nucleotide sequence of DNA was determined using the Shimadzu DSQ-2000L (S) system. The results of 3'RACE and 5'RACE were combined and combined, and the resulting full-length sequence is shown in SEQ ID NO: 1 of the sequence listing. The sequence was named ICS1.

[Example 2] Expression of caffeine synthase in E. coli The isolated caffeine synthase gene was used as an expression vector p.
It was incorporated into ET23d (Novagen) and this plasmid was transformed into E. coli BL21 (DE3). After culturing the obtained Escherichia coli at 37 ° C. for 2 hours, IPTG was added to a final concentration of 0.3 mM, and the mixture was further cultured at 30 ° C. for 5 hours. After culturing, the cells were collected and 0.2 ml of 10 m was added to 3 ml of the culture solution.
M Tris-hydrochloric acid buffer (pH 7.5), 0.1M
Sonication was carried out intermittently in NaCl, 1 mM EDTA-Na2 for 1 minute. This is 14,000 rpm, 1
Centrifugation was performed for 0 minutes, and the obtained supernatant was used as an enzyme solution. The reaction solution for measuring caffeine synthase activity is
100 mM Tris-hydrochloric acid buffer (pH 8.5),
0.2 mM MgCl ₂ , 0.2 mM paraxanthine, 4 μM [methyl-14C] S-adenosylmethionine (0.9 kBq) to which 30 μl of enzyme solution was added,
The volume of the reaction solution was 100 μl. The reaction was carried out at 27 ° C for 15 minutes, the obtained 14C-caffeine was extracted by adding 1 ml of chloroform, and the radioactivity of the chloroform layer was measured. As a control, a reaction solution obtained by removing paraxanthine was used. As a result of activity measurement, 133.7 pmo
It was found that 1 l of caffeine was produced. The substrate specificity of the recombinant enzyme is shown in Table 1 (Table 1). It was shown as a relative activity (%) when the activity against 7-methylxanthine was set to 100. For comparison, tea ( Camellia sinensi
s ) The substrate specificity of leaf-derived caffeine synthase is also described.

[0037]

[Table 1]

[0038]

According to the present invention, it becomes possible to efficiently produce caffeine synthase which can be used as an industrial, food or medical enzyme. ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to modify the caffeine biosynthesis metabolism of a caffeine production plant, a plant tissue, or a plant cell, and to efficiently produce the compound of a caffeine metabolism system. According to the present invention, it becomes possible to modify the caffeine biosynthetic metabolism of caffeine-producing plants, plant tissues or plant cells to modify the production ratio of compounds in the caffeine metabolism system.

[0039]

[Sequence list]                          SEQUENCE LISTING <110> MITSUI CHEMICALS, INC. <120> A Caffeine Synthase and A Gene thereof <130> P0000331 <160> 20 <210> 1 <211> 365 <212> PRT <213> Camellia irrawadiensis <400> 1 gtcactgctg tggcagctgg cctctttgcc ataaaaaatt acttttctga cgaggcgtgg 60 agctagctac t atg ggg aag gtg aac gaa gtg ttg ttc atg aac aga gga 110              Met Gly Lys Val Asn Glu Val Leu Phe Met Asn Arg Gly                1 5 10 gaa gga gaa att agt tat gca caa aac tct gct ttc aca caa aaa gtg 158 Glu Gly Glu Ile Ser Tyr Ala Gln Asn Ser Ala Phe Thr Gln Lys Val      15 20 25 gcc tca atg gca atg cca gcg cta gaa aat gca gtt gaa act ctc ttc 206 Ala Ser Met Ala Met Pro Ala Leu Glu Asn Ala Val Glu Thr Leu Phe  30 35 40 45 tcc aaa gat ttc cac ctt ctt caa gct ctt act gca gcg gac ttg ggt 254 Ser Lys Asp Phe His Leu Leu Gln Ala Leu Thr Ala Ala Asp Leu Gly                  50 55 60 tgt gca gcg ggt cca aac acg ttc gca gta att tct acg atc aag aga 302 Cys Ala Ala Gly Pro Asn Thr Phe Ala Val Ile Ser Thr Ile Lys Arg              65 70 75 atg atg gaa aag aaa tgc agg gaa ttg tat tgc caa aca ctg gaa ctt 350 Met Met Glu Lys Lys Cys Arg Glu Leu Tyr Cys Gln Thr Leu Glu Leu          80 85 90 cag gtt tac ttg aat gat ctt ttt gga aac gat ttc aat acc ctc ttc 398 Gln Val Tyr Leu Asn Asp Leu Phe Gly Asn Asp Phe Asn Thr Leu Phe      95 100 105 aaa ggc ctg tcg tct gag gtt gtt ggt aac aaa tgt gag gaa gtt tct 446 Lys Gly Leu Ser Ser Glu Val Val Gly Asn Lys Cys Glu Glu Val Ser 110 115 120 125 tgt tat gtg atg gga gta ccg ggg tct ttc cat ggc cgg ctt ttt cct 494 Cys Tyr Val Met Gly Val Pro Gly Ser Phe His Gly Arg Leu Phe Pro                 130 135 140 cgt aac agc ttg cat tta gtt cat tcc tct tac agt gtt cat tgg ctt 542 Arg Asn Ser Leu His Leu Val His Ser Ser Tyr Ser Val His Trp Leu             145 150 155 act cag gca cca aaa gga ctc aca agc aga gaa ggc ttg gca tta aac 590 Thr Gln Ala Pro Lys Gly Leu Thr Ser Arg Glu Gly Leu Ala Leu Asn         160 165 170 aag ggg aag att tac ata tca aag aca agc cct cct gtt gta aaa gaa 638 Lys Gly Lys Ile Tyr Ile Ser Lys Thr Ser Pro Pro Val Val Lys Glu     175 180 185 gcc tac tta tct caa ttt cat gaa gat ttc aca atg ttt ctc aac gct 686 Ala Tyr Leu Ser Gln Phe His Glu Asp Phe Thr Met Phe Leu Asn Ala 190 195 200 205 aga tcc caa gag gtg gtt cca aat ggt tgt atg gtg ttg ata ctt cat 734 Arg Ser Gln Glu Val Val Pro Asn Gly Cys Met Val Leu Ile Leu His                 210 215 220 ggt agg caa tct tct gat cct tca gag atg gag agc tgc ttt act tgg 782 Gly Arg Gln Ser Ser Asp Pro Ser Glu Met Glu Ser Cys Phe Thr Trp             225 230 235 gaa cta tta gct ata gcc att gct gaa ttg gtt tca cag gga ttg ata 830 Glu Leu Leu Ala Ile Ala Ile Ala Glu Leu Val Ser Gln Gly Leu Ile         240 245 250 gat gaa gat aaa tta gac acc ttc aat gta cct agc tat tgg cca tca 878 Asp Glu Asp Lys Leu Asp Thr Phe Asn Val Pro Ser Tyr Trp Pro Ser     255 260 265 ctt gag gaa gtg aaa gac ata gtg gag agg gac gga tca ttc aca att 926 Leu Glu Glu Val Lys Asp Ile Val Glu Arg Asp Gly Ser Phe Thr Ile 270 275 280 285 gat cat ttg gag ggg ttc gaa ctt gat agt cta gag atg caa gag aat 974 Asp His Leu Glu Gly Phe Glu Leu Asp Ser Leu Glu Met Gln Glu Asn                 290 295 300 gat aaa tgg gtt aga ggg gac aag ttt gcc aag atg gtc agg gcc ttc 1022 Asp Lys Trp Val Arg Gly Asp Lys Phe Ala Lys Met Val Arg Ala Phe             305 310 315 aca gag cct ata att tca aac cag ttt gga cat gaa atc atg gac aaa 1070 Thr Glu Pro Ile Ile Ser Asn Gln Phe Gly His Glu Ile Met Asp Lys         320 325 330 cta tat gac aaa ttc act cac att tta gtt tca gat ttg gaa gca gag 1118 Leu Tyr Asp Lys Phe Thr His Ile Leu Val Ser Asp Leu Glu Ala Glu     335 340 345 cta ccg aag acc aca agt atc atc cta gtg ctt tcc aag att gtt gga 1166 Leu Pro Lys Thr Thr Ser Ile Ile Leu Val Leu Ser Lys Ile Val Gly 350 355 360 365 tagtttttta gtgttctgaa ataaactgta gtccccatca catatatgct agtagagggt 1226 tgtgccaatg tattgcacaa gaagatttga gaagggtcaa ctatagaagc tttttgctct 1286 tgtgtggaga gagaatgttt tcttgattta aatctgtgat acccaaatcg taatggtggg 1346 aagaaatggg aagttgaaca tgaaatttta atttctttac cttatgtatg tatggaatta 1406 gtggttgttt gctaaaaaaa aaaaaa 1432 <210> 2 <211> 1432 <212> DNA <213> Camellia irrawadiensis <400> 2  gucacugcug uggcagcugg ccucuuugcc auaaaaaauu acuuuucuga cgaggcgugg 60  agcuagcuac uauggggaag gugaacgaag uguuguucau gaacagagga gaaggagaaa 120  uuaguuaugc acaaaacucu gcuuucacac aaaaaguggc cucaauggca augccagcgc 180  uagaaaaugc aguugaaacu cucuucucca aagauuucca ccuucuucaa gcucuuacug 240  cagcggacuu ggguugugca gcggguccaa acacguucgc aguaauuucu acgaucaaga 300  gaaugaugga aaagaaaugc agggaauugu auugccaaac acuggaacuu cagguuuacu 360  ugaaugaucu uuuuggaaac gauuucaaua cccucuucucaa aggccugucg ucugagguug 420  uugguaacaa augugaggaa guuucuuguu augugauggg aguaccgggg ucuuuccaug 480  gccggcuuuu uccucguaac agcuugcauu uaguucauuc cucuuacagu guucauuggc 540  uuacucaggc accaaaagga cucacaagca gagaaggcuu ggcauuaaac aaggggaaga 600  uuuacauauc aaagacaagc ccuccuguug uaaaagaagc cuacuuaucu caauuucaug 660  aagauuucac aauguuucuc aacgcuagau cccaagaggu gguuccaaau gguuguaugg 720  uguugauacu ucaugguagg caaucuucug auccuucaga gauggagagc ugcuuuacuu 780  gggaacuauu agcuauagcc auugcugaau ugguuucaca gggauugaua gaugaagaua 840  aauuagacac cuucaaugua ccuagcuauu ggccaucacu ugaggaagug aaagacauag 900  uggagaggga cggaucauuc acaauugauc auuuggaggg guucgaacuu gauagucuag 960  agaugcaaga gaaugauaaa uggguuagag gggacaaguu ugccaagaug gucagggccu 1020  ucacagagcc uauaauuuca aaccaguuug gacaugaaau cauggacaaa cuauaugaca 1080  aauucacuca cauuuuaguu ucagauuugg aagcagagcu accgaagacc acaaguauca 1140  uccuagugcu uuccaagauu guuggauagu uuuuuagugu ucugaaauaa acuguagucc 1200  ccaucacaua uaugcuagua gaggguugug ccaauguauu gcacaagaag auuugagaag 1260  ggucaacuau agaagcuuuu ugcucuugug uggagagaga auguuuucuu gauuuaaauc 1320  ugugauaccc aaaucguaau ggugggaaga aaugggaagu ugaacaugaa auuuuaauuu 1380  cuuuaccuua uguauguaug gaauuagugg uuguuugcua aaaaaaaaaa aa 1432 <210> 3 <211> 20 <212> DNA <400> 3 ggacttgggt tggtgcagcg 20 <210> 4 <211> 20 <212> DNA <400> 4 cggggtcttt ccatggccgg 20 <210> 5 <211> 18 <212> DNA <400> 5 ggcctttgaa gagggtat 18 <210> 6 <211> 23 <212> DNA <400> 6 ggcttactca ggcaccaaaa gga 23 <210> 7 <211> 21 <212> DNA <400> 7 ggagggcttg tctttgatat g 21 <210> 8 <211> 21 <212> DNA <400> 8 ccctgcattt cttttccatc t 21

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 C12P 17/12 9/10 C12N 15/00 ZNAA C12P 17/12 5/00 C F term ( reference) 2B030 AA03 AB03 AD08 CA06 CA17 CA19 CB03 CD03 CD07 CD09 4B024 AA01 AA03 AA08 BA10 BA79 BA80 CA04 CA12 CA20 DA01 DA06 EA04 GA11 4B050 CC01 CC03 DD13 EE01 LL01 LL02 LL05 4B064 AE57 CA11 CA19 CC01 CC24 DA01 DA10 4B065 AA26X AA58X AA72X AA88X AA88Y AB01 AC14 BA02 CA18 CA29 CA41 CA53

Claims (16)

[Claims] 1. A sequence number 1 to 3 in SEQ ID NO: 1 of the sequence listing
A polypeptide defined by the 65th amino acid sequence and having caffeine synthase enzymatic activity. 2. A DNA encoding the polypeptide according to claim 1. 3. The DNA according to claim 2, which is defined by the nucleotide sequences from 1 to 1432 of SEQ ID NO: 1 in the sequence listing. 4. An RNA encoding the polypeptide according to claim 1. 5. The RNA according to claim 4, which is defined by the nucleotide sequences from 1 to 1432 of SEQ ID NO: 2 in the sequence listing. 6. A vector containing the DNA according to claim 2 or 3. 7. In a cell of a microorganism and / or plant,
The vector according to claim 6, which can express a polypeptide having an enzymatic activity of caffeine synthase. 8. An antisense RNA having an effect of suppressing the expression of the enzyme of caffeine synthase can be expressed in the cells of microorganisms and / or plants.
The described vector. 9. A vector containing the RNA according to claim 4 or 5. 10. In a cell of a microorganism and / or plant,
The vector according to claim 9, which can express an antisense RNA having an effect of suppressing the expression of the enzyme of caffeine synthase. 11. A microorganism transformed with the vector according to any one of claims 6 to 10. 12. A method for producing a polypeptide having an enzymatic activity of caffeine synthase, using the transformed microorganism according to claim 11. 13. A plant cell, plant tissue or plant transformed with the vector according to any one of claims 6 to 10. 14. A method for producing a secondary metabolite of a plant using the transformed plant according to claim 13, a cultured cell of the plant, or a plant. 15. The plant secondary metabolite is xanthosine,
The method according to claim 14, which is at least one compound selected from 7-methylxanthosine, 7-methylxanthine, theobromine, theophylline, and caffeine. 16. The transformed plant is a Camellia genus plant, a coffee ( Coffea ) genus plant, a Cola tree ( Cola ) genus plant, a coca ( Erythroxylum ) genus plant, and an ilex tree ( Il).
ex) plants of the genus, Neea (Neea) plants of the genus, Sterculiaceae (Firmia
The method according to claim 15, which is a plant belonging to the genus na ), a plant belonging to the genus Paullinia or a plant belonging to the cocoa tree ( Theobroma ).