JP2010022323A - Prenyltransferase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for isolating DNA of a membrane-associated prenyltransferase, DNA obtained thereby and a membrane-associated prenyltransferase. <P>SOLUTION: Disclosed is a membrane-associated prenyltransferase wherein the prenyltransferase has at least one conserved motif selected from the group consisting of NDxxDxxxD, NQxxDxxxD, NQxxExxxD, DDxxDxxxD, DQxxDxxxD, DQxxExxxD, NExxDxxxD, NExxExxxD and DExxExxxD, and exhibits enzymatic activity which induces a prenyl group using a flavonoid such as naringenin, an isoflavonoid such as genistein and a chalcone such as isoliquiritigenin as a substrate. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a membrane-bound prenyltransferase, a method for producing the same, a DNA encoding the transferase, and a method for isolating the DNA.

  The number of secondary metabolites produced by plants is said to exceed 50,000, and this is a great resource for natural organic compounds. Although it is classified into several groups for convenience from the structural features, there are actually a large number of compounds having features between different groups. A prenylated aromatic compound is a good example, and the prenyl group contributes greatly to the diversity of structure and physiological activity of plant secondary metabolites. Biosynthetically, these compounds biosynthesized through a pathway called the complex pathway play a role necessary for plant life maintenance by bearing insect resistance, disease resistance, etc. Some of them contribute to their pharmacological action (Non-patent Document 1). For example, many prenylflavonoids having various physiological activities such as antitumor activity and antibacterial activity have been reported (Non-patent Document 2), which is a very important compound group in the pharmaceutical and food industries. They also play an important role in plant damage and defense.

In particular, the importance of the prenyl group for the physiological activity of prenyl aromatic compounds such as prenyl flavonoids has been pointed out by many researchers, and in fact, there are many cases where the mother nucleus compound having no prenyl group does not exhibit the physiological activity. . Therefore, enzymes that catalyze the prenylation of these aromatic compounds are recognized as very important industrially. However, many prenyltransferases with aromatic substrates are membrane-bound, and due to the difficulty of biochemical research, molecular biological analysis has been delayed so far. Plant prenyltransferases with flavonoids as substrates are As a result, the cloning of the gene has not been reported yet.
S. Mesia-Vela et al., Phytomedicine, 8 (2001), p.481-488 HY Sohn et al., Phytomedicine, 11 (2004), p.666-672

  Therefore, an object of the present invention is to provide a method for isolating a membrane-bound prenyltransferase DNA using an aromatic as a substrate, a DNA obtained by the isolation method, and a membrane-bound prenyltransferase.

  The inventors of the present invention have made extensive studies to solve the above-mentioned problems. Conventionally, it has been difficult to express a membrane protein in an expression system using Escherichia coli. By combining screening methods, we succeeded in isolating plant-derived membrane-bound prenyltransferase cDNA and completed the present invention with further improvements.

That is, the present invention provides the following.
Item 1.
A membrane-bound prenyltransferase comprising:
It has at least one conserved motif selected from the group consisting of NDxxDxxxD, NQxxDxxxD, NQxxExxxD, DDxxDxxxD, DQxxDxxxD, DQxxExxxD, NExxDxxxD, NExxExxxD and DExxExxxD and has the chemical formula (I):

(Wherein R ₁ and R ₂ are the same or different and each represents a hydrogen group, a hydroxyl group, a phenyl group, a phenol group or an aryl group;
R ₃ and R ₄ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group),
Chemical formula (II):

(Wherein R ₅ and R ₆ are the same or different and each represents a hydrogen group, a hydroxyl group, a phenyl group, a phenol group or an aryl group;
R ₇ and R ₈ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group),
Chemical formula (III):

(Wherein R ₉ represents a phenyl group, a phenol group or an aryl group,
R ₁₀ , R ₁₁ and R ₁₂ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group), or chemical formula (IV):

(Wherein R ₁₃ represents a phenyl group, a phenol group or an aryl group;
R ₁₄ , R ₁₅ and R ₁₆ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group)
A membrane-bound prenyltransferase exhibiting an enzyme activity for introducing a prenyl group into a compound represented by the formula:
Item 2.
At least selected from the group consisting of dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and phytyl diphosphate (PDP) as a prenyl donor Item 2. The membrane-bound prenyltransferase according to Item 1, wherein one kind is used as a substrate.
Item 3.
The following polypeptide (i) or (ii):
(I) a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11 or 13;
(Ii) A polypeptide having the amino acid sequence in which one or more amino acid residues are deleted, added or substituted in the polypeptide of (i), and having prenyltransferase activity.
Item 4.
Item 4. The membrane-bound prenyltransferase according to any one of Items 1 to 3, which is derived from a plant.
Item 5.
Item 5. The membrane-bound prenyltransferase according to Item 4, wherein the plant is selected from leguminous, mulberry, hypericum, citrus, sericaceae, chrysanthemum, peas, or hops.
Item 6.
A gene consisting of DNA shown in any of (iii) to (v) below:
(Iii) DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12 or 14;
(Iv) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence of (iii) and that encodes a membrane-bound prenyltransferase,
(V) DNA comprising a base sequence having at least 70% homology with DNA comprising the base sequence of (iii).
Item 7.
Item 7. A vector comprising the gene according to item 6.
Item 8.
Item 8. A transformant into which the vector according to Item 7 has been introduced.
Item 9.
Item 9. A method for preparing a membrane-bound prenyltransferase comprising culturing the transformant according to Item 8 and collecting a membrane-bound prenyltransferase from the transformant and / or the culture.
Item 10.
A membrane-bound prenyltransferase encoded by the gene of Item 6.
Item 11.
Item 9. A method for producing an aromatic prenylated compound, comprising culturing the transformant according to Item 8, and collecting the aromatic prenylated compound from the transformant and / or the culture.
Item 12.
Item 12. The method according to Item 9 or 11, wherein the transformant is yeast, a plant, or a plant cell.

According to the present invention,
The following chemical formula (I):

(Wherein R ₁ and R ₂ are the same or different and each represents a hydrogen group, a hydroxyl group, a phenyl group, a phenol group or an aryl group;
R ₃ and R ₄ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group),
Chemical formula (II):

(Wherein R ₅ and R ₆ are the same or different and each represents a hydrogen group, a hydroxyl group, a phenyl group, a phenol group or an aryl group;
R ₇ and R ₈ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group),
Chemical formula (III):

(Wherein R ₉ represents a phenyl group, a phenol group or an aryl group,
R ₁₀ , R ₁₁ and R ₁₂ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group), or chemical formula (IV):

(Wherein R ₁₃ represents a phenyl group, a phenol group or an aryl group;
R ₁₄ , R ₁₅ and R ₁₆ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group)
A prenyl transferase and a gene encoding the enzyme exhibiting an enzyme activity for introducing a prenyl group into the compound represented by the formula:

  In addition, prenyl aromatic compounds exhibiting various useful physiological activities can be produced in large quantities and at low cost by the enzyme of the present invention. Furthermore, using the gene sequence of the present invention, it is possible to isolate a gene of an enzyme that prenylates an aromatic compound from a plant containing a large amount of prenylated secondary metabolites.

  The membrane-bound prenyltransferase gene of the present invention can also be used as a molecular breeding tool in the agricultural field.

(Membrane-bound prenyltransferase)
The membrane-bound prenyltransferase of the present invention has at least one conserved motif in the membrane-bound prenyltransferase and has the following chemical formula (I):

(Wherein R ₁ and R ₂ are the same or different and each represents a hydrogen group, a hydroxyl group, a phenyl group, a phenol group or an aryl group;
R ₃ and R ₄ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group),
Chemical formula (II):

(Wherein R ₅ and R ₆ are the same or different and each represents a hydrogen group, a hydroxyl group, a phenyl group, a phenol group or an aryl group;
R ₇ and R ₈ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group),
Chemical formula (III):

(Wherein R ₉ represents a phenyl group, a phenol group or an aryl group,
R ₁₀ , R ₁₁ and R ₁₂ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group), or chemical formula (IV):

(Wherein R ₁₃ represents a phenyl group, a phenol group or an aryl group;
R ₁₄ , R ₁₅ and R ₁₆ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group)
Is a membrane-bound prenyltransferase exhibiting an enzyme activity for introducing a prenyl group into a compound represented by the formula:

  Examples of the conserved motif possessed by the membrane-bound prenyltransferase of the present invention include at least one conserved motif selected from the group consisting of NDxxDxxxD, NQxxDxxxD, NQxxExxxD, DDxxDxxxD, DQxxDxxxD, DQxxExxxD, NExxDxxxD, NExxExxxD, and DExxExxxD. In these conserved motifs, the position represented by x may be any amino acid. The same applies to the following.

  Among these, those having a conserved motif of N (Q / E) LxDx (D / E) xD are preferable. In these conserved motifs, the part represented as (A / B) represents an A or B amino acid. The same applies to the following.

  Furthermore, a membrane-bound prenyltransferase having an amino acid sequence of K (V / I) NK immediately after any one of the above conserved motifs is more preferable.

  The membrane-bound prenyltransferase of the present invention preferably further has a VIAxxKD amino acid sequence motif. Further, those having an amino acid sequence motif of VIAxxKDxxxxxGD are more preferable, and those having VIAxxKDIPDxxGD are particularly preferable.

  The membrane-bound prenyltransferase of the present invention is also at least one selected from the group consisting of YxxxxxxxG * AT, LxFxIG (W / L) LQ, (S / A) Gxx (S / T) FR, TxPxxxxFCxxI and AEYxxxPLF. It may have an amino acid sequence motif. The part represented by x is the same as described above. In addition, these motifs are preferably located at positions spaced apart from each other by the degree of multi-alignment shown in FIG. 3 described later, but are not limited thereto.

  Examples of the substrate on which the membrane-bound prenyltransferase of the present invention exhibits activity include flavonoids, isoflavonoids, pterocarpan, coumarin, chalcone and / or phloroglucinol.

  Preferred flavonoids include naringenin, hesperetin, liquiritigenin, galangin, chrysin, sakuranetin, isosakuranetin, taxifolin, apigenin, kaempferol, quercetin and the like.

  Preferred isoflavonoids include genistein, daidzein, biochanin A, formononetin and the like.

  Preferred chalcones include isoliquiritigenin, 2 ', 4', 4-trihydroxy-6'-methoxy chalcone, naringenin chalcone, methyl naringenin chalcone and the like.

  Preferred pterocarpans include glycinol (also known as (6aS, 11aS) -3,9,6a-trihydroxypterocarpan) maackiain and the like.

  The membrane-bound prenyltransferase of the present invention has dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranyl geranyl diphosphate (GGPP), phytyl diphosphate (PGP) as prenyl donors. PDP) is used as a substrate.

For example, examples of the membrane-bound prenyltransferase of the present invention include the following polypeptides (a) to (j):
(A) a polypeptide having the amino acid sequence of SEQ ID NO: 1,
(B) a polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in SEQ ID NO: 1 and having prenyltransferase activity;
(C) a polypeptide having the amino acid sequence of SEQ ID NO: 3,
(D) a polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in SEQ ID NO: 3 and having prenyl transferase activity;
(E) a polypeptide having the amino acid sequence of SEQ ID NO: 5,
(F) a polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in SEQ ID NO: 5 and having prenyltransferase activity;
(G) a polypeptide having the amino acid sequence of SEQ ID NO: 7,
(H) a polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in SEQ ID NO: 7 and having prenyltransferase activity;
(I) a polypeptide having the amino acid sequence of SEQ ID NO: 9,
(J) A polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in SEQ ID NO: 9 and having prenyltransferase activity.
(K) a polypeptide having the amino acid sequence of SEQ ID NO: 11,
(L) A polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in SEQ ID NO: 11, and having prenyltransferase activity.
(M) a polypeptide having the amino acid sequence of SEQ ID NO: 13,
(N) A polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in SEQ ID NO: 13, and having prenyltransferase activity.

  The polypeptide (b) is a polypeptide in which one or more, for example 1 to 50, preferably 1 to 30 amino acids are substituted, added, deleted or inserted in the polypeptide (a). May be. The polypeptide (b) may be a polypeptide having 70% or more homology, preferably 80% or more homology with the polypeptide (a).

  The polypeptide (d) is a polypeptide in which one or more, for example, 1 to 50, preferably 1 to 30, amino acids are substituted, added, deleted or inserted in the polypeptide (c). May be. The polypeptide (d) may be a polypeptide having 70% or more homology, preferably 80% or more homology with the polypeptide (c).

  The polypeptide of (f) is a polypeptide in which one or more, for example, 1 to 50, preferably 1 to 30 amino acids are substituted, added, deleted or inserted in the polypeptide of (e). May be. The polypeptide (f) may be a polypeptide having 70% or more homology, preferably 80% or more homology with the polypeptide (e).

  The polypeptide (h) is a polypeptide in which one or more, for example, 1 to 50, preferably 1 to 30, amino acids are substituted, added, deleted or inserted in the polypeptide (g). May be. The polypeptide (h) may be a polypeptide having 70% or more homology, preferably 80% or more homology with the polypeptide (g).

  The polypeptide (j) is a polypeptide in which one or more, for example, 1 to 50, preferably 1 to 30, amino acids are substituted, added, deleted or inserted in the polypeptide (i). May be. The polypeptide (j) may be a polypeptide having 70% or more homology, preferably 80% or more homology with the polypeptide (i).

  The polypeptide (l) is a polypeptide in which one or more, for example, 1 to 50, preferably 1 to 30, amino acids are substituted, added, deleted or inserted in the polypeptide of (k). May be. The polypeptide (1) may be a polypeptide having 70% or more homology, preferably 80% or more homology with the polypeptide (k).

  The polypeptide (n) is a polypeptide in which one or more, for example 1 to 50, preferably 1 to 30 amino acids are substituted, added, deleted or inserted in the polypeptide (m). May be. The polypeptide (n) may be a polypeptide having 70% or more homology, preferably 80% or more homology with the polypeptide (m).

  Although it is not limited, specifically, substitution is possible as follows: For example, in the case of amino acid substitution, from the viewpoint of maintaining the structure of the protein, it is polar, charged, soluble, hydrophilic / hydrophobic. In this respect, the amino acid can be substituted with an amino acid having properties similar to those of the amino acid before substitution. For example, glycine, alanine, valine, leucine, isoleucine, and proline are classified as nonpolar amino acids; serine, threonine, cysteine, methionine, asparagine, and glutamine are classified as polar amino acids; phenylalanine, tyrosine, and tryptophan are classified as aromatic amino acids. Aspartic acid and glutamic acid are classified as acidic amino acids. Accordingly, substitution can be made by selecting from the same group of amino acids.

  Specific examples of preferable substitution include substitution of 126th methionine with alanine and substitution of 158th valine with methionine in the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing.

  In the present invention, the homology of amino acid sequences is a numerical value when analyzed according to the default setting of NCBI's BLAST program. Preferably, (Database: nr, Matrix: BLOSUM62, GapCosts: Existence = 11, Extension = 1) can be used.

Further, the membrane-bound prenyltransferase of the present invention is preferably derived from a plant, and particularly preferably derived from a legume, mulberry, hypericaceae, citrus, asteraceae, chrysanthemum, or genus, hops. .
(Membrane-bound prenyltransferase gene)
The present invention includes a membrane-bound prenyltransferase gene comprising any of the following DNAs (o) to (z) and (α) (β):
(O) DNA consisting of the base sequence of SEQ ID NO: 2,
(P) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 2 and encodes a membrane-bound prenyltransferase;
(Q) DNA consisting of the base sequence of SEQ ID NO: 4,
(R) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 4 and encodes a membrane-bound prenyltransferase;
(S) DNA consisting of the base sequence of SEQ ID NO: 6,
(T) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 6 and which encodes a membrane-bound prenyltransferase;
(U) DNA consisting of the base sequence of SEQ ID NO: 8,
(V) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 8, and which encodes a membrane-bound prenyltransferase;
(W) hybridizes under stringent conditions with DNA comprising the nucleotide sequence of SEQ ID NO: 10 and (x) DNA comprising the nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 10 and membrane-bound DNA encoding sex prenyltransferase.
(Y) hybridizes under stringent conditions with DNA consisting of the base sequence of SEQ ID NO: 12 and (z) DNA consisting of the base sequence complementary to the base sequence shown in SEQ ID NO: 12 and membrane-bound DNA encoding sex prenyltransferase.
Hybridizes under stringent conditions with (α) DNA consisting of the base sequence of SEQ ID NO: 14 and (β) DNA consisting of the base sequence complementary to the base sequence shown in SEQ ID NO: 14 and membrane-bound DNA encoding sex prenyltransferase.

As used herein, “polynucleotide hybridizing under stringent conditions” refers to a polynucleotide obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Meaning, for example, a probe is allowed to act on a support on which a polynucleotide to be detected is immobilized, and after prehybridization at 42 ° C. for 2 hours in the presence of 0.7 to 1.0 M NaCl, Hybridization was performed at 42 ° C. for 12 to 16 hours in the presence of 0.7 to 1.0 M NaCl, and then about 0.1 to 2 times the SSC solution (
Examples of the composition of the SSC solution having a 1-fold concentration include DNA that can be identified by washing the filter at 42 ° C. using 150 mM sodium chloride, 15 mM sodium citrate). Hybridization can be performed according to the method described in Molecular Cloning: A laboratory Mannual, 2nd Ed., (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989).

  In the present invention, any one of (o) to (z) and (α) (β) and, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and further preferably 93% or more. Particularly preferred is DNA comprising a base sequence having a homology of 95% or more, most preferably 98% or more.

In the present invention, the homology of the base sequence is a numerical value when analyzed according to the default setting of the NCBI BLAST program. Preferably, (Database: nr, Match, Mismatch: 1, -2, GapCosts: Linear) can be used.
(vector)
The vector of the present invention is any one of the above (o) to (z) and (α) (β), or two or more DNAs, or the above (o) to (z) and (α) (β). This is a recombinant vector into which a DNA having a base sequence having homology with the DNA is inserted. Known vectors for yeast, plant cells, etc. can be widely used. Known vectors include pDR196, pYES-DEST 52, Yip5, Yrp17, and Yep24 for yeast vectors, and pGWB vectors for plant cells (pGWB2, pGWB5, pGWB80, etc .; from Shimane University, Tsuyoshi Nakagawa PBiEl2-GUS, pIG121-Hm, pBI121, pBiHyg-HSE, pB119, pBI101, pGV3850, pABH-Hm1, and the like. The vector used in the present invention contains a selectable marker (for example, drug resistance gene, antibiotic resistance gene, reporter gene) as necessary.
(Transformant)
The transformant of the present invention is a transformant carrying the recombinant vector of the present invention. What is necessary is just to use a host suitable for a vector. For example, yeast, plants, plant cells, insect cells (Sf9 etc.) are preferred. Particularly preferred transformants include yeast, plants or plant cells. Transformation methods are well known to those skilled in the art.
(Method for preparing membrane-bound prenyltransferase)
By recovering the membrane-bound prenyltransferase from the transformant and / or the culture solution thereof, a recombinant membrane-bound prenyltransferase retaining the activity can be obtained.

  Specifically, for example, the method described in Yazaki et al (JBC, 2002, Vol. 277, p. 6240-6246) can be used.

  In addition, the membrane-bound prenyltransferase of the present invention can be obtained, for example, by mass production using a yeast expression system or by expression in an edible plant such as soybean.

  More specifically, since the membrane-bound prenyltransferase of the present invention can be expressed as a protein having high activity in yeast, flavonoids such as naringenin, isoflavonoids such as genistein, or isoliquiritigenin in the culture solution of transformed yeast It is possible to efficiently mass-produce prenylated flavonoids by administering a chalcone as a substrate. Naringenin, genistein, isoliquiritigenin and the like have moderate water solubility and hydrophobicity, and thus can pass through biological membranes and contact with prenyltransferase in yeast cells. Since yeast has a pathway (mevalonate pathway) for biosynthesizing DMAPP as a prenyl donor in the cytosol, the prenyl substrate is supplied in vivo. The produced prenylated flavonoids, prenylated isoflavonoids, prenylated chalcones and the like are expected to be efficiently produced by recovering them from yeast cells or culture media.

  Alternatively, prenylated flavonoids, prenylated isoflavonoids, prenylated chalcones, and the like can be produced in plants by expressing the membrane-bound prenyltransferase of the present invention in plants such as soybeans or plant cells. In the case of plant cells, there are two pathways for biosynthesis of DMAPP, one is the cytosolic mevalonate pathway and the other is the non-mevalonate pathway localized in plastids. In order to produce prenylated flavonoids, prenylated isoflavonoids, prenylated chalcones, etc. using the former DMAPP, a modified gene excluding the plastid localization signal is expressed. In addition, in order to produce prenylated flavonoids, prenylated isoflavonoids, prenylated chalcones, etc. using the latter DMAPP, use endogenous plastid localization signals or plastid stations of other genes such as RuBisCo small subunits. The localization signal is ligated and expressed, and the prenyltransferase is localized to the plastid. Since endogenous substances such as flavonoids, isoflavonoids, and chalcones are prenylated, plants are grown and the prenylated substances are recovered from the tissues. Alternatively, it is also effective for efficient production that flavonoids, isoflavonoids, chalcones and the like are absorbed to be prenylated and recovered from the plant tissue. Which tissue in the plant should produce prenylated flavonoids can be controlled by using a tissue-specific promoter.

  Plants producing prenylated flavonoids, prenylated isoflavonoids, prenylated chalcones, etc. can be used for the purpose of compound production as described above, but many of these compounds are produced as phytoalexins, Since it has high antibacterial activity, it can be considered to show high resistance against pathogenic bacteria and pests. Therefore, the membrane-bound prenyltransferase gene of the present invention can also be used as a molecular breeding tool in the agricultural field.

That is, by expressing the membrane-bound prenyltransferase of the present invention in crop plants such as cereals, vegetables, potatoes and fruits, high resistance to pathogenic bacteria and pests can be obtained.
(Gene isolation method)
Further, if the homology with the base sequence described in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14 in the sequence listing or a base sequence comprising a part thereof is utilized, membrane binding The prenyl transferase gene can be isolated.

  Specific methods include, for example, a method of screening a gene library expected to contain a membrane-bound prenyltransferase gene using the base sequence as a probe, or preparing a primer based on the base sequence information. And a method of performing PCR using a sample expected to contain a membrane-bound prenyltransferase gene as a template.

  Alternatively, a method of searching for a gene having a DNA sequence having a high homology with the base sequence by searching a base sequence database with an appropriate homology search program (for example, BLAST), etc. may be mentioned.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated in more detail, this invention is not limited to these.

1. Construction of expression library
In order to induce flavonoid prenylation enzyme, 20 μl of 0.1 M methyl jasmonate DMSO solution was added to S. flavescens cultured cells and cultured for 36 hours (final concentration 0.1 mM). Poly (A) ⁺ RNA was isolated from the cultured cells using Oligotex-MAG mRNA Purification Kit (Takara Bio Inc.). A cDNA plasmid library was constructed using the cDNA Synthesis Kit (STRATAGENE) and the yeast expression vector pDR196 for membrane-bound proteins. The first strand of cDNA was synthesized using 7 μg poly (A) + RNA, an oligo (dT) ₁₈ anchor primer containing a XhoI restriction site. After synthesizing the second strand of the cDNA, a blunt end adapter containing an EcoRI restriction site was ligated to the double stranded DNA, and the fragment was then incorporated into a plasmid with the constitutive promoter PMA1, pDR196.

2. Cloning and functional analysis of SfN8DT cDNA The cDNA library constructed above was introduced into Escherichia coli DH10B, about 10,000 clones were randomly selected and sequenced from the 5 'end using primers that anneal to the PMA1 promoter region. Among them, 200 clones having any one of the six conserved motifs (NDxxDxxxD, NQxxDxxxD, NQxxExxxD, DDxxDxxxD, DQxxDxxxD, DQxxExxxD) in the known membrane-bound prenyltransferase were selected. The cDNA clone was analyzed using the SOSUI program (http://bp.nuap.nagoya-u.ac.jp/sosui/), and 20 clones having a transmembrane region in at least the sequenced region were detected. 200 clones from three subcellular localization prediction programs: ChloroP, PSORT, and WoLF_PSORT (ChloroP; http://www.cbs.dtu.dk/services/ChloroP/, PSORT; http: //psort.ims.u- tokyo.ac.jp/form.html, WoLF_PSORT; http://wolfpsort.seq.cbrc.jp/), 30 clones were found to have a plastid translocation signal.

  These clones were introduced into the yeast strain W303-1A-Δcoq2 using the lithium acetate method. Recombinant proteins are expressed in yeast transformants by culturing in SD-Ura liquid medium (180 ml) until reaching the logarithmic growth phase, and then described in Yazaki et al. (JBC, 2002, 277, 6240-6246) The microsomal fraction was prepared using the method described above. The membrane fraction of each transformant was resuspended in 500 μl of 0.1 mM Tris-HCl buffer (pH 8.5), and then screened for enzyme activity using naringenin and DMAPP as substrates.

Enzyme products were screened using HPLC analysis. HPLC analysis was performed under the following conditions:
Shimadzu LC-10A system (Shimadzu, Kyoto, Japan): column YMC-Pack Pro C18 RS (YMC, Kyoto, Japan) 4.6 x 250 mm; solvent system, methanol: H ₂ O: acetic acid (70:30: 0.3) ; Flow rate, 1 ml min ^-1 ; Detection, 230-320 nm with SPD6A photodiode array detector.

  As a result, it was found that one of 200 yeast transformants had the same retention time as that of 8-dimethylallylnaringenin (FIG. 1). Furthermore, the product showed a UV spectrum equivalent to that of a standard sample in the detection of a photodiode array (FIG. 1).

  From this, it was found that the prenyl transferase was encoded in the clone used for transformation of the one yeast transformant.

  Thus, a cDNA (total length 1,495 bp, ORF 410 a.a.) of naringenin 8-dimethylallyltransferase (hereinafter referred to as SfN8DT), which is the first flavonoid prenyltransferase in biosynthesis of sophoraflavanone G (SFG), was obtained.

  The base sequence of SfN8DT is shown in SEQ ID NO: 2 in the sequence listing. The amino acid sequence of SfN8DT is shown in SEQ ID NO: 1. An example of the mechanism of the prenylation reaction performed by SfN8DT is shown in FIG.

  SfN8DT is also referred to as SfN8DT-1.

3. Cloning of HPT-like clones other than SfN8DT From the Clara cDNA library sequence information, 6 clones (Sf1c12f, Sf1C12c, SfL17a, SfL17b, SfG6DT, SfN8DT-2, respectively) having high homology with homogentisic acid prenyltransferase (HPT) as well as SfN8DT Sf1C12f is also referred to as SfN8DT-3, and Sf1C12c is also referred to as SfChal1DT.), And cDNA was isolated by the RACE method.

  Specifically, a 5'-end or 3'-end of each cDNA was specifically amplified by PCR using a cDNA mixture prepared using a GeneRacer kit manufactured by Invitrogen as a RACE template. The RNA sample used here is the same total RNA derived from Clara cultured cells treated with methyl jasmonate as the library was prepared. In this PCR, the 5'-RACE forward primer was the adapter sequence of the kit, and the reverse primer was the internal sequence of each gene. In the case of 3'-RACE, the forward primer was used as the internal sequence of each gene, and oligo-dT (20 mer) was used as the reverse primer. The amplified DNA fragment was confirmed to be the cDNA end of each gene by sequencing. In order to obtain full-length cDNA, PCR was performed again using a mixture of GeneRacer with 5'-end and oligo-dT (20 mer) as primers.

  Of these, the homology of the amino acid sequences excluding the signal peptide between SfL17b, Sf1C12f, Sf1C12c, SfL17a, SfL17b and SfN8DT and homogentisic prenyltransferase was compared (FIG. 3). As a result, Sf1C12f, Sf1C12c, SfL17a, and SfL17b had high homology with SfN8DT. Bases of Sf1C12f (full length 1684bp, ORF 410a.a.), Sf1C12c (full length 1527bp, ORF 391a.a.), SfL17a (full length 1325bp, ORF 407a.a.), and SfL17b (full length 1373bp, ORF 379a.a.) The sequences are shown in SEQ ID NOs: 4, 6, 8, and 10, respectively. These amino acid sequences are shown in 3, 5, 7 and 9 of the sequence listing, respectively.

  In addition, FIG. 4 shows a multiple alignment prepared using each partial sequence of SfN8DT-1 (SfN8DT), SfN8DT-2, SfG6DT, SfChal1DT (Sf1C12c) and a partial sequence of homogentisic prenyl transferase. As a result, SfN8DT-1 (SfN8DT), SfN8DT-2, SfG6DT, and SfChal1DT (Sf1C12c) had high homology. The nucleotide sequences of SfG6DT (ORF 407a.a.) and SfN8DT-2 (ORF 407a.a.) are shown in SEQ ID NOs: 12 and 14, respectively. In addition, these amino acid sequences are shown in SEQ ID NOS: 11 and 13, respectively.

4). Functional analysis of clones having high homology with SfN8DT Functional analysis of SfN8DT-2, SfN8DT-3 (Sf1C12f), and SfG6DT was performed in the same manner as in “2. Cloning and functional analysis of SfN8DT cDNA” above. It was.

  That is, as in the functional analysis of SfN8DT, each of these cDNA clones was introduced into yeast and cultured to express a recombinant protein, from which a microsomal fraction was prepared. Then, the fraction (membrane fraction) is resuspended in 500 μl of 0.1 mM Tris-HCl buffer (pH 8.5), and then enzyme activity when naringenin or genistein and DMAPP are used as substrates. Was measured.

  The enzyme activity was measured by measuring the enzyme product under the same HPLC analysis conditions as in the functional analysis of SfN8DT.

  As a result of measuring enzyme activity, it was found that the fraction prepared by expressing SfN8DT-2 and SfN8DT-3 (Sf1C12f) and using naringenin as a substrate contains a substance having the same retention time as 8-dimethylallylnaringenin. It was. In addition, the product showed a UV spectrum equivalent to 8-dimethylallylnaringenin preparation in photodiode array detection.

  This indicates that SfN8DT-2 and SfN8DT-3 (Sf1C12f) are also naringenin 8-dimethylallyltransferases, which are flavonoid prenyltransferases.

  Furthermore, as a result of measuring enzyme activity, the fraction prepared by expressing SfG6DT and using genistein and DMAPP as substrates contained a substance having the same retention time as 6-dimethylallylgenistine (FIG. 5). Separately, the enzyme reaction product was identified as 6-dimethylallylgenstein by direct comparison with a chemically synthesized sample.

  From this, it was found that SfG6DT is a prenyltransferase and at least works as genistein 6-dimethylallyltransferase.

  An example of the mechanism of the prenylation reaction performed by SfG6DT (an example that works as genistein 6-dimethylallyltransferase) is shown in FIG.

5. Analysis of substrate specificity of recombinant SfG6DT using yeast expression system The substrate specificity analysis of recombinant SfG6DT described above was performed by a radioactive assay using thin layer chromatography.
That is, first, a yeast microsome fraction expressing SfG6DT was prepared in the same manner as the SfG6DT functional analysis method described above.

Next, the fraction (membrane fraction) is resuspended in 500 μl of 0.1 mM Tris-HCl buffer (pH 8.5), and then the group consisting of ¹⁴ C-labeled genistein and DMAPP, GPP, FPP, GGPP. The reaction was carried out with one kind selected from as a substrate.

Separately, the fraction (membrane fraction) is resuspended in 500 μl of 0.1 mM Tris-HCl buffer (pH 8.5), and then from the group consisting of genistein, daidzein, and biochanin A (Biochanin A). One selected species was reacted with ¹⁴ C-labeled DMAPP as a substrate.

  After the reaction, each sample is separated using a thin film chromatography plate (Silica Gel 60 F254; Merck) (developing solvent is Toluene: EtOAc: AcOH = 70: 30: 0.3) and detected by BAS1800 (Fuji Film). went.

  As a result, recombinant SfG6DT was found to use biochanin A having the same isoflavanone skeleton as the substrate in addition to genistein. Furthermore, as a prenyl donor, it was found that DMAPP, GPP, and FPP can be used as substrates (FIG. 7).

6). Enzymatic analysis of recombinant SfN8DT and SfG6DT Using the yeast expression system described above, Km, optimum temperature, optimum pH, and divalent cation requirement of recombinant SfN8DT and SfG6DT were examined.

  Specifically, a recombinant yeast microsome fraction was prepared by the same method as described above, and the amount of the product was detected by HPLC.

  The results of Km values are shown in Table 1, the optimum temperature, the optimum pH of SfN8DT, and the results of divalent cation requirement are shown in FIGS. 8 to 10, and the results of SfG6DT are shown in FIGS. In addition, the optimal temperature of recombinant SfN8DT was 70-80 degreeC (FIG. 8), and the optimal temperature of recombinant SfG6DT was 25-35 degreeC (FIG. 11).

7). Expression analysis of mRNA of SfN8DT and SfG6DT Expression analysis of SfN8DT and SfG6DT in Clara plants and Clara cultured cells was performed by Northern blot and RT-PCR. (Probe: full length ORF) For the tissue-specific expression analysis, Clara plant bodies (plant height: about 170 cm, root diameter: ˜3 cm) sampled at Takeda Yakuzoen were used. As a result of Northern blot analysis, both SfN8DT and SfG6DT were found to be expressed only in the roots (FIG. 14). When RT-PCR analysis was further performed, SfN8DT was expressed specifically in the root skin part, while SfG6DT was rooted. It was found that the protein was specifically expressed inside the skin (FIG. 15).

  In addition, the expression responses to MJ (methyl jasmonate), SA (salicylic acid) and YE (yeast extract) were also examined in cultured cells. Three days after transplanting, MJ, SA, and YE were added to final concentrations of 100 μM, 100 μM, and 5 mg / ml, respectively, and collected 24 hours later. As a result, the expression of SfN8DT was remarkably increased by MJ, SA, and YE, and the expression of SfG6DT was remarkably increased by SA (FIG. 16).

8). Production of prenylflavonoids in plants using SfN8DT
Whether Sf8DT was expressed in plants and prenylflavonoids could be produced in plants was examined as follows.
<Introduction and expression of SfN8DT gene>
The full length of the SfN8DT gene was amplified by PCR using the clone cloned as described above as a template and using the SfN8DTfull_Fw primer (SEQ ID NO: 15) and the SfN8DTfull_Rv primer (SEQ ID NO: 16). The amplified product was subcloned into the pENTR1A vector and used as the GATEWAY entry vector. Further, this was incorporated into the pGWB2 vector to obtain a GATEWAY destination vector. The pGWB2-SfN8DTfull vector was introduced into Arabidopsis thaliana (Columbia wild strain) by the floral dip method using Agrobacterium tumefaciens GV3101. The T1 generation was grown in a medium containing 50 μg / mL of kanamycin and selected, and in the T2 generation, RT-PCR was performed using the SfN8DTfull_Fw primer (SEQ ID NO: 15) and the SfN8DTfull_Rv primer (SEQ ID NO: 16), and the SfN8DT gene was introduced. It was confirmed that it was expressed.
<Production of prenylflavonoids in SfN8DT gene expression strain>
Arabidopsis thaliana (T2 generation) expressing the above SfN8DT gene is cultivated on an agar medium supplemented with 100 μM naringenin for 2 weeks and then freeze-dried. This is about 100 mg (approximately 300 to 500 freeze-dried plant bodies). ) Collected. The freeze-dried Arabidopsis thaliana was used as a sample for 80% acetone extraction, and the extract was analyzed to obtain 8DN (8-dimethylallyl naringenin), 8DK (des-O-methylanhydroicaritin), DA (dimethylallylated apigenin), DQ (dimethylallylated quercetin) It was investigated whether it was produced. As a control, a similar experiment was performed using a wild Arabidopsis thaliana strain.
Enzyme products were screened using HPLC analysis. HPLC analysis was performed under the following conditions:
Shimadzu LC-10A system (Shimadzu, Kyoto, Japan): column YMC-Pack Pro C18 RS (YMC, Kyoto, Japan) 4.6 x 250 mm; solvent system, methanol: H ₂ O: acetic acid (70:30: 0.3) ; Flow rate, 1 ml min ^-1 ; Detection, 230-320 nm with SPD6A photodiode array detector.

  The results are shown in Table 2.

  As shown in Table 2, it was found that 8DN, 8DK, DA, and DQ were not produced in the wild strain, whereas all 8DN, 8DK, DA, and DQ were produced in the SfN8DT gene expression strain. .

  From the above, it was confirmed that prenylflavonoids can be produced by expressing the SfN8DT gene in the plant body.

9. Functional analysis of SfChal1DT
Similar to the analysis of the function of SfN8DT, a cDNA clone of SfChal1DT was introduced into yeast and cultured to express a recombinant protein, from which a microsomal fraction was prepared. Then, the fraction (membrane fraction) is resuspended in 500 μl of 0.1 mM Tris-HCl buffer (pH 8.5), and then the enzyme when the chalcone isoliquiritgenin and DMAPP are used as substrates. Activity was measured.

The enzyme activity was measured using LC / MS under the following conditions:
Shimadzu model 2010A system LC / MS (Shimadzu Corporation) (using LC-10AD Solvent Delivery system)
column: 4 x 250 mm LiChrospere 100RP-18 (Merck, Tokyo, Japan)
solvent system: ethanol: H ₂ O: formic acid (90: 10: 0.3)
flow rate: 0.2 ml min ^-1
The sample was measured in positive ion mode, and the measurement range of mass spectrometry was set to m / z 50 to 600.

  As a result, in the chromatogram traced with the molecular weight of 325 of prenylated isoliquiritigenin, it was confirmed that the dimethylallyl form of isoliquiritigenin was produced by the enzymatic reaction, as shown in FIG. This indicates that SfChal1DT is a prenyltransferase that can prenylate chalcones.

The result of having analyzed the product when naringenin and DMAPP were added to the microsomal fraction of yeast transformed with SfN8DT by HPLC is shown. a is a chromatogram obtained by analyzing each of 8-DN, 3′DN and 6DN samples by HPLC, b is a chromatogram obtained by analyzing the product of yeast introduced with an empty vector by HPLC, and c is transformed by SfN8DT. In the chromatogram of the yeast product analyzed by HPLC, d shows the photodiode array detection result of the yeast product transformed with SfN8DT. An example of the mechanism of the prenylation reaction performed by SfN8DT is shown. A comparison of amino acid sequence homologies excluding signal peptides between SfL17b, SfN8DT, SfL17a, Sf1C12c and Sf1C12f and other homogentisic prenyltransferases is shown. GmVTE2-1, AtVTE2-1, TaVTE2-1, ZmVTE2-1, ApVTE2-1 and CpVTE2-1: Eva Collakova et al, Plant Phys., 2001, 127, 1113-1124 and Beth Savidge etal., Plant Phys., See 2002, 129, 321-332. HvHGGT, TaHGGT and OsHGGT: See Edgar B Cahoon et al., Nature Biotech, 2003, 21, 1082-1087. See GmVTE2-2 and AtVTE2-2: Venkatech et al., Planta, 2006, 223, 1134-1144. The multiple alignment produced using each partial sequence of SfN8DT-1 (SfN8DT), SfN8DT-2, SfG6DT, SfChal1DT (Sf1C12c) and a partial sequence of homogentisate prenyltransferase is shown. The result of having analyzed the product when genistein and DMAPP were added to the microsome fraction of yeast transformed with SfG6DT by HPLC is shown. An example of the mechanism of the prenylation reaction performed by SfG6DT is shown. The result of having examined the substrate specificity of SfG6DT by Radioactive assay is shown. The red and blue arrows indicate the detected 6-dimethylallylgenistein. It is a graph which shows the optimal temperature of recombinant SfN8DT. It is a graph which shows the optimal pH of recombinant SfN8DT. It is a graph which shows the bivalent cation requirement of recombinant SfN8DT. It is a graph which shows the optimal temperature of recombinant SfG6DT. It is a graph which shows the optimal pH of recombinant SfG6DT. It is a graph which shows the bivalent cation requirement of recombinant SfG6DT. The image of the northern blot analysis result which examined the expression of SfN8DT and SfG6DT in each organ of Clara is shown. The image of the RT-PCR analysis result which examined the expression site of SfN8DT and SfG6DT in the root of Clara is shown. Here, IFS represents isoflavone synthase, CHS represents chalcone synthase, and CHI represents chalcone isomerase. The image of the northern blot analysis result which examined the influence with respect to the expression of SfN8DT and SfG6DT of MJ, SA, and YE is shown. The results of LC / MS analysis of products obtained by adding isoliquiritigenin and DMAPP to the microsomal fraction of yeast transformed with SfChal1DT.

Claims (12)

A membrane-bound prenyltransferase comprising:
It has at least one conserved motif selected from the group consisting of NDxxDxxxD, NQxxDxxxD, NQxxExxxD, DDxxDxxxD, DQxxDxxxD, DQxxExxxD, NExxDxxxD, NExxExxxD and DExxExxxD and has the chemical formula (I):

(Wherein R ₁ and R ₂ are the same or different and each represents a hydrogen group, a hydroxyl group, a phenyl group, a phenol group or an aryl group;
R ₃ and R ₄ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group),
Chemical formula (II):

(Wherein R ₅ and R ₆ are the same or different and each represents a hydrogen group, a hydroxyl group, a phenyl group, a phenol group or an aryl group;
R ₇ and R ₈ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group),
Chemical formula (III):

(Wherein R ₉ represents a phenyl group, a phenol group or an aryl group,
R ₁₀ , R ₁₁ and R ₁₂ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group), or chemical formula (IV):

(Wherein R ₁₃ represents a phenyl group, a phenol group or an aryl group;
R ₁₄ , R ₁₅ and R ₁₆ are the same or different and each represents a hydrogen group, a hydroxyl group or a methoxyl group)
A membrane-bound prenyltransferase exhibiting an enzyme activity for introducing a prenyl group into a compound represented by the formula: At least selected from the group consisting of dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and phytyl diphosphate (PDP) as a prenyl donor The membrane-bound prenyltransferase according to claim 1, wherein one kind is used as a substrate. The following polypeptide (i) or (ii):
(I) a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11 or 13;
(Ii) A polypeptide having the amino acid sequence in which one or more amino acid residues are deleted, added or substituted in the polypeptide of (i), and having prenyltransferase activity. The membrane-bound prenyltransferase according to any one of claims 1 to 3, which is derived from a plant. The membrane-bound prenyltransferase according to claim 4, wherein the plant is selected from legume, mulberry, hypericum, citrus, sericaceae, chrysanthemum or aspens or hops. A gene consisting of DNA shown in any of (iii) to (v) below:
(Iii) DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12 or 14;
(Iv) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence of (iii) and that encodes a membrane-bound prenyltransferase,
(V) DNA comprising a base sequence having at least 70% homology with DNA comprising the base sequence of (iii). A vector comprising the gene according to claim 6. A transformant into which the vector according to claim 7 has been introduced. A method for preparing a membrane-bound prenyltransferase comprising culturing the transformant according to claim 8 and collecting a membrane-bound prenyltransferase from the transformant and / or the culture. A membrane-bound prenyltransferase encoded by the gene of claim 6. A method for producing an aromatic prenylated compound, comprising culturing the transformant according to claim 8 and collecting the aromatic prenylated compound from the transformant and / or the culture. The method according to claim 9 or 11, wherein the transformant is a yeast, a plant or a plant cell.

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