JP2015047142A - Specific protein, gene encoding the protein, transformant of isoprenoid producing plant with the gene introduced, and method of improving amount of biosynthesis or productivity of specific compound by introducing the gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide analysis of an amino acid sequence and a base sequence of a specific enzyme related to a biosynthetic pathway of polyisoprenoid in Solidago altissima which can be a stable supply source of natural rubber other than a mature tree of Hevea brasiliensis.SOLUTION: Protein of the present invention is any of A1 to A3: (A1) protein composed of a specific amino acid sequence; (A2) protein which is composed of the specific amino acid sequence in which one or plural amino acids are substituted, deleted, inserted, and/or added, and has enzyme activity catalyzing a reaction in which isopentenyl diphosphate and dimethylallyl diphosphate are used as substrates, or a reaction in which isopentenyl diphosphate and geranyl diphosphate are used as substrates; (A3) protein which is composed of an amino acid sequence having a sequence identity of 80% or more with the specific amino acid sequence, and has enzyme activity catalyzing a reaction in which isopentenyl diphosphate and dimethylallyl diphosphate are used as substrates, or a reaction in which isopentenyl diphosphate and geranyl diphosphate are used as substrates.

Description

The present invention improves a biosynthesis amount or productivity of a specific compound by introducing a specific protein, a gene encoding the protein, a transformant of an isoprenoid-producing plant into which the gene is introduced, and the gene. Regarding the method.

Natural rubber (a type of polyisoprenoid) currently used in industrial rubber products is collected from isoprenoid-producing plants such as Hevea brasiliensis and Mulberry plant Ficus elastica. Is obtained.

At present, the natural rubber used in industrial rubber products is almost exclusively collected from para rubber tree. Para rubber tree is a plant that can grow only in limited areas such as Southeast Asia and South America. Furthermore, it takes about 7 years for para rubber trees to become an adult tree from which rubber can be collected, and the period during which natural rubber can be collected is limited to 20-30 years. In the future, demand for natural rubber is expected to increase mainly in developing countries. However, for the reasons described above, it is difficult to significantly increase the production of natural rubber using para rubber tree.

Examples of a method for improving the productivity of natural rubber in Para rubber tree include a method of collecting a larger amount of latex in order to obtain a larger amount of natural rubber. Specifically, for example, a method of adding stimulation to the trunk of rubber tree with ethylene or Etherephone (2-chloroethylphosphonic acid), lanolin containing jasmonic acid or linolenic acid of jasmonic acid precursor, etc. There is a method of promoting (for example, Non-Patent Document 1).

However, the method of increasing latex production by ethylene stimulation may cause cracking of the bark if it is used for a long period of time on the trunk. In addition, ethylene stimulation is a method that makes the latex outflow from the milk duct more smooth, and does not improve the latex production capacity of the tree itself. .

It is also possible to increase the number of milk ducts by using jasmonic acid or the like to increase the number of milk ducts, but when collecting latex by tapping, the latex flowing out from the milk duct coagulates at the wound and is produced. There is a problem that the collected latex cannot be recovered sufficiently.

Thus, there is concern about the depletion of natural rubber resources, and development of a stable natural rubber supply source other than the adult tree of Para rubber tree and the development of technology for improving the productivity of isoprenoids are desired.

Hao, et al., Anals of Botany, 2000, 85, 37-43.

The present invention solves the above-mentioned problems and analyzes the amino acid sequence and base sequence of a specific enzyme involved in the biosynthetic pathway of polyisoprenoids in Solidago japonica, which can be a source of stable natural rubber other than adult rubber trees The purpose is to provide. Another object of the present invention is to provide a transformant in which the isoprenoid-producing plant is improved by introducing the gene of the base sequence into an isoprenoid-producing plant, and a method for improving the isoprenoid productivity.

The present invention relates to the protein described in any of [A1] to [A3] below.
[A1] a protein comprising the amino acid sequence of amino acid numbers 1-333 represented by SEQ ID NO: 1 [A2] substitution of one or more amino acids in the amino acid sequence of amino acid numbers 1-333 represented by SEQ ID NO: 1, A reaction comprising deletion, insertion, and / or addition, and using isopentenyl diphosphate and dimethylallyl diphosphate as substrates, or isopentenyl diphosphate and geranyl diphosphate as substrates Protein [A3] having an enzymatic activity that catalyzes the reaction. The protein comprises an amino acid sequence of 80% or more of the amino acid sequence of amino acid numbers 1-333 represented by SEQ ID NO: 1, and isopentenyl diphosphate and dimethyl Enzyme activity that catalyzes reactions using allyl diphosphate as a substrate or reactions using isopentenyl diphosphate and geranyl diphosphate as substrates Protein that

The present invention also relates to a gene encoding the above protein.
The present invention also relates to the gene described in [A4] or [A5] below.
[A4] DNA consisting of the base sequence of base numbers 1-999 represented by SEQ ID NO: 2
[A5] Hybridizes under stringent conditions with DNA comprising a nucleotide sequence complementary to the nucleotide sequence of nucleotide numbers 1-999 represented by SEQ ID NO: 2, and isopentenyl diphosphate and dimethylallyl diphosphate Which encodes a protein having an enzyme activity that catalyzes a reaction in which the enzyme is used as a substrate or a reaction in which isopentenyl diphosphate and geranyl diphosphate are used as substrates.
The present invention also relates to a transformant in which the biosynthesis amount of farnesyl diphosphate in the plant is improved by introducing the gene into an isoprenoid-producing plant.
The present invention also relates to a method for improving the amount of farnesyl diphosphate biosynthesis in the plant by introducing the gene into an isoprenoid-producing plant.

The present invention relates to the protein according to any one of [B1] to [B3] below.
[B1] a protein consisting of the amino acid sequence of amino acid numbers 1-360 represented by SEQ ID NO: 3 [B2] substitution of one or more amino acids in the amino acid sequence of amino acid numbers 1-360 represented by SEQ ID NO: 3, Reactions consisting of sequences containing deletions, insertions, and / or additions, and reactions using isopentenyl diphosphate and dimethylallyl diphosphate as substrates, reactions using isopentenyl diphosphate and geranyl diphosphate as substrates Or a protein having an enzymatic activity that catalyzes a reaction using isopentenyl diphosphate and farnesyl diphosphate as substrates [B3] and an amino acid sequence of amino acid numbers 1-360 represented by SEQ ID NO: 3 and a sequence of 80% or more A reaction comprising isopentenyl diphosphate and dimethylallyl diphosphate, each having an amino acid sequence having the same identity, and isopentenyl diphosphate Reaction of the acid with geranyl diphosphate as a substrate, or a protein having an enzyme activity that catalyzes the reaction using a isopentenyl diphosphate and farnesyl diphosphate as a substrate

The present invention also relates to a gene encoding the above protein.
The present invention also relates to the gene described in [B4] or [B5] below.
[B4] DNA consisting of the nucleotide sequence of nucleotide numbers 1-180 represented by SEQ ID NO: 4
[B5] It hybridizes under stringent conditions with DNA comprising a base sequence complementary to the base sequence 1-180 represented by SEQ ID NO: 4, and isopentenyl diphosphate and dimethylallyl diphosphate A protein having an enzyme activity that catalyzes a reaction in which isopentenyl diphosphate and geranyl diphosphate are used as substrates, or a reaction in which isopentenyl diphosphate and farnesyl diphosphate are used as substrates. DNA encoding
The present invention also relates to a transformant in which the biosynthesis amount of geranylgeranyl diphosphate in the plant is improved by introducing the gene into an isoprenoid-producing plant.
The present invention also relates to a method for improving the biosynthesis amount of geranylgeranyl diphosphate in the plant by introducing the gene into an isoprenoid-producing plant.

The present invention relates to the protein according to any one of [C1] to [C3] below.
[C1] a protein comprising the amino acid sequence of amino acid number 1-302 represented by SEQ ID NO: 5 [C2] substitution of one or more amino acids in the amino acid sequence of amino acid number 1-302 represented by SEQ ID NO: 5, A protein [C3] consisting of a sequence containing a deletion, insertion, and / or addition, and having an enzyme activity that catalyzes a reaction to isomerize isopentenyl diphosphate or dimethylallyl diphosphate [C3] represented by SEQ ID NO: 5 A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of amino acid No. 1-302, and having an enzyme activity that catalyzes a reaction for isomerizing isopentenyl diphosphate or dimethylallyl diphosphate

The present invention also relates to a gene encoding the above protein.
The present invention also relates to the gene described in [C4] or [C5] below.
[C4] DNA consisting of the nucleotide sequence of nucleotide numbers 1-906 represented by SEQ ID NO: 6
[C5] It hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of nucleotide numbers 1-906 represented by SEQ ID NO: 6, and is isopentenyl diphosphate or dimethylallyl diphosphate DNA encoding a protein having an enzymatic activity that catalyzes a reaction to isomerize
The present invention also relates to a transformant in which the biosynthesis amount of dimethylallyl diphosphate or isopentenyl diphosphate in the plant is improved by introducing the gene into an isoprenoid-producing plant.
The present invention also relates to a method for improving the biosynthetic amount of dimethylallyl diphosphate or isopentenyl diphosphate in the plant by introducing the gene into an isoprenoid-producing plant.

The present invention relates to the protein according to any one of [D1] to [D3] below.
[D1] a protein comprising the amino acid sequence of amino acid number 1-263 represented by SEQ ID NO: 7 [D2] substitution of one or a plurality of amino acids in the amino acid sequence of amino acid number 1-263 represented by SEQ ID NO: 7; A protein consisting of a sequence containing a deletion, insertion, and / or addition, and having an enzyme activity that catalyzes a reaction for extending the chain length of an isoprenoid compound to cis type [D3] amino acid number 1- represented by SEQ ID NO: 7 A protein having an enzyme activity that catalyzes a reaction of extending the chain length of an isoprenoid compound to a cis type, comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of 263

The present invention also relates to a gene encoding the above protein.
The present invention also relates to the gene described in [D4] or [D5] below.
[D4] DNA consisting of the base sequence of base numbers 1 to 789 represented by SEQ ID NO: 8
[D5] Hybridizes with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of nucleotide numbers 1-789 represented by SEQ ID NO: 8 under stringent conditions, and extends the chain length of the isoprenoid compound to the cis type. DNA encoding a protein having enzyme activity that catalyzes the reaction
The present invention also relates to a transformant in which the amount of biosynthesis in the plant is improved by introducing the gene into an isoprenoid-producing plant.
The present invention also relates to a method for improving the amount of biosynthesis in the plant by introducing the gene into an isoprenoid-producing plant.

The present invention also relates to a transformant in which any of the above genes is introduced into an isoprenoid-producing plant to improve the isoprenoid productivity in the plant.
The present invention also relates to a method for improving isoprenoid productivity in a plant by introducing any of the above genes into the isoprenoid-producing plant.
The present invention also relates to a transformant in which the productivity of polyisoprenoid in the plant is improved by introducing any of the above genes into the isoprenoid-producing plant.
The present invention also relates to a method for improving the productivity of polyisoprenoid in a plant by introducing any of the above genes into the isoprenoid-producing plant.

The gene of the present invention is a gene encoding a specific enzyme involved in the biosynthetic pathway of polyisoprenoid. Therefore, by introducing the gene into an isoprenoid-producing plant, the biosynthetic amount of the product of the reaction catalyzed by the protein encoded by the gene, and hence the productivity of isoprenoids and polyisoprenoids, can be improved. A transformant with improved biosynthesis of the product of the reaction catalyzed by the protein and a transformant with improved productivity of isoprenoids and polyisoprenoids can be obtained.

It is a schematic diagram which shows a part of biosynthetic pathway of a polyisoprenoid. It is a schematic diagram which shows the reaction which each enzyme of the prenyl transferase group involved in the biosynthetic pathway of polyisoprenoid catalyzes.

In the isoprenoid-producing plant, various isoprenoids are biosynthesized by the isoprenoid biosynthesis pathway as shown in FIG. In the isoprenoid biosynthetic pathway, a reaction in which isopentenyl diphosphate (IPP) is sequentially linked to an allylic substrate proceeds. Enzymes that catalyze this reaction are generically called prenyltransferases in the sense that they sequentially connect isoprene units. In the present specification, prenyl transferase refers to a new isoprenyl that has increased by 1 unit of isoprene units by catalyzing a condensation reaction between IPP and isoprenyl diphosphate (n isoprene units) (allylic substrate). A generic term for enzymes that synthesize diphosphate (n + 1 isoprene units).

Prenyltransferase is a basic precursor of each terpenoid by linking isoprene units, geranyl diphosphate (GPP: C10), farnesyl diphosphate (FPP: C15), geranylgeranyl diphosphate (GGPP: C20). , An enzyme group that synthesizes various isoprenyl diphosphates such as geranyl farnesyl diphosphate (GFPP: C25), hexaprenyl diphosphate (HPP: C30), and is located in the mainstream of terpene penoid biosynthesis . Isoprenyl diphosphates produced by various prenyl transferases are shown in FIG. In the present specification, examples of prenyl transferase include geranyl diphosphate synthase, farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, geranyl farnesyl diphosphate synthase, hexaprenyl diphosphate synthase, and the like. Examples thereof include trans-isoprenyl diphosphate synthase and cis-prenyl transferase (cis-isoprenyl diphosphate synthase in FIG. 2).

As a result of various investigations in order to develop a stable natural rubber source other than the adult tree of Para rubber tree, the present inventors have found that even among isoprenoid-producing plants, they are linear and have a relatively high molecular weight. We paid attention to Sequoia spp. Because it produces rubber (polyisoprenoid) and has a strong fertility. Farnesyl diphosphate and geranylgeranyl diphosphate, which are presumed to be substrates when various isoprenoids and polyisoprenoids are biosynthesized, among the enzymes involved in the isoprenoid biosynthetic pathway in Sedum moth Since the enzyme responsible for biosynthesis of dimethylallyl diphosphate is expected to play a particularly important role, each protein responsible for biosynthesis of these substances was analyzed.

Specifically, farnesyl diphosphate synthase (FPPS) involved in biosynthesis of farnesyl diphosphate (FPP), geranylgeranyl diphosphate synthase (GGPPS) involved in biosynthesis of geranylgeranyl diphosphate (GGPP), iso Isopentenyl diphosphate isomerase (IDI) involved in isomerization of pentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), and cis-prenyl transferase (cis-type prenyl transferase) involved in biosynthesis Focusing on the four types of enzyme proteins, the base sequences and amino acid sequences of these four types were clarified by analyzing the genes encoding these four types of enzyme proteins derived from Sequoia spp.

Furthermore, when a confirmation test was performed using Escherichia coli of each of the transformants into which each of the four types of genes that had been clarified was introduced, it was also clear that these four types of enzyme proteins each had a predetermined enzyme activity. It became. Therefore, by introducing each of these four genes into an isoprenoid-producing plant, each of them exhibits a predetermined enzyme activity.

Since the above four types of enzyme proteins are enzyme proteins derived from Aedes albopictus producing relatively high molecular weight polyisoprenoids, they have a very high probability of having superior properties compared to the same types of enzyme proteins derived from other plants. In addition, by introducing each of these four genes into an isoprenoid-producing plant, each of them shows a predetermined enzyme activity, and the protein (enzyme) catalyzes and increases the amount of biosynthetic compound biosynthesized, The productivity of isoprenoids and polyisoprenoids can also be improved. In the present specification, to improve the productivity of isoprenoids and polyisoprenoids means to improve the production of isoprenoids and polyisoprenoids.

In the present specification, farnesyl diphosphate synthase (FPPS) is farnesyl diphosphate (GPP) using isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP) as substrates. FPP) is an enzyme that catalyzes a biosynthetic reaction.
Moreover, in this specification, geranylgeranyl diphosphate synthase (GGPPS) is isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Is an enzyme that catalyzes the biosynthesis reaction of geranylgeranyl diphosphate (GGPP).
Also herein, isopentenyl diphosphate isomerase (IDI) catalyzes the isomerization reaction between isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP). It is an enzyme.
In the present specification, cis-prenyltransferase (cis-type prenyltransferase) is an enzyme that catalyzes a reaction for extending the chain length of an isoprenoid compound to a cis-type. Here, in this specification, an isoprenoid compound means a compound having an isoprene unit (C ₅ H ₈ ). A cis-type isoprenoid is a compound having an isoprenoid compound in which isoprene units are bonded in a cis-type, and examples thereof include cis-isoprenyl diphosphate.

(protein)
The protein of the present invention is a protein shown in any of the following [A1], [B1], [C1], and [D1].
[A1] A protein consisting of the amino acid sequence of amino acid numbers 1-333 represented by SEQ ID NO: 1 [B1] A protein consisting of the amino acid sequence of amino acid numbers 1-360 represented by SEQ ID NO: 3 [C1] represented by SEQ ID NO: 5 A protein comprising the amino acid sequence of amino acid number 1-302 [D1] a protein comprising the amino acid sequence of amino acid number 1-263 represented by SEQ ID NO: 7

The proteins represented by [A1], [B1], [C1], and [D1] are farnesyl diphosphate synthase (FPPS), geranylgeranyl diphosphate synthase (GGPPS), isopentenyl diphosphate isomerase (IDI), respectively. It is a cis-prenyl transferase (cis-type prenyl transferase).

Further, it is known that a protein may have an inherent function even when it includes substitution, deletion, insertion, or addition of one or more amino acids in the original amino acid sequence. Therefore, the protein of the present invention is also a protein shown in any of the following [A2], [B2], [C2], and [D2].
[A2] The amino acid sequence of amino acid Nos. 1-333 represented by SEQ ID No. 1 consists of a sequence containing substitution, deletion, insertion, and / or addition of one or more amino acids and has an inherent function The protein [B2] is an amino acid sequence of amino acid Nos. 1-360 represented by SEQ ID No. 3 and consists of a sequence containing substitution, deletion, insertion, and / or addition of one or a plurality of amino acids, and has an inherent function Protein [C2] having the sequence having amino acid number 1-302 amino acid sequence represented by SEQ ID NO: 5 and comprising one or more amino acid substitutions, deletions, insertions, and / or additions, and has the inherent function Protein [D2] having the amino acid sequence of amino acid number 1-263 represented by SEQ ID NO: 7, substitution or deletion of one or more amino acids, A protein comprising a sequence containing an insertion and / or addition and having an inherent function. In this specification, the intrinsic function of the protein represented by SEQ ID NO: 1 is farnesyl diphosphate synthase (FPPS). Is an enzyme activity that catalyzes a reaction inherently, ie, a reaction using isopentenyl diphosphate and dimethylallyl diphosphate as substrates, or a reaction using isopentenyl diphosphate and geranyl diphosphate as substrates.
Here, in the present specification, the function inherent to the protein represented by SEQ ID NO: 3 is the function inherent to geranylgeranyl diphosphate synthase (GGPPS), that is, isopentenyl diphosphate and dimethylallyl diphosphate. Is an enzyme activity that catalyzes a reaction in which isopentenyl diphosphate and geranyl diphosphate are used as substrates, or a reaction in which isopentenyl diphosphate and farnesyl diphosphate are used as substrates.
Here, in the present specification, the function inherent to the protein represented by SEQ ID NO: 5 is the function inherent to isopentenyl diphosphate isomerase (IDI), that is, isopentenyl diphosphate or dimethylallyl diphosphate. It is an enzyme activity that catalyzes a reaction to isomerize the aldehyde.
Here, in the present specification, the function inherent to the protein represented by SEQ ID NO: 7 is the function inherent to cis-prenyltransferase (cis-type prenyltransferase), that is, the chain length of the isoprenoid compound is extended to cis-type. It is an enzyme activity that catalyzes the reaction to be carried out.

In order to maintain the enzyme activity, one or more amino acids, preferably 1 to 67 amino acids, more preferably 1 to 67 amino acids in the amino acid sequence of amino acid numbers 1 to 333 represented by SEQ ID NO: 1. Substitution or deletion of 50 amino acids, more preferably 1 to 33 amino acids, particularly preferably 1 to 17 amino acids, most preferably 1 to 7 amino acids, and most preferably 1 to 3 amino acids It is preferably an amino acid sequence containing an insertion, and / or addition.

Moreover, in order to maintain enzyme activity, in the amino acid sequence of amino acid number 1-360 represented by sequence number 3, it is 1 or more amino acids, Preferably it is 1-72 amino acids, More preferably, it is 1-54. 1 amino acid, more preferably 1 to 36 amino acids, particularly preferably 1 to 18 amino acids, most preferably 1 to 7 amino acids, most preferably 1 to 4 amino acid substitutions, deletions, An amino acid sequence containing an insertion and / or addition is preferred.

Moreover, in order to maintain enzyme activity, in the amino acid sequence of amino acid number 1-302 represented by sequence number 5, it is 1 or more amino acids, Preferably it is 1-60 amino acids, More preferably, it is 1-45. 1 amino acid, more preferably 1-30 amino acids, particularly preferably 1-15 amino acids, most preferably 1-6 amino acids, more preferably 3 amino acid substitutions, deletions, insertions, And / or an amino acid sequence containing an addition.

In order to maintain the enzyme activity, one or more amino acids, preferably 1 to 53 amino acids, more preferably 1 to 39 in the amino acid sequence of amino acid number 1-263 represented by SEQ ID NO: 7. 1 to 26 amino acids, more preferably 1 to 26 amino acids, particularly preferably 1 to 13 amino acids, most preferably 1 to 5 amino acids, and most preferably 1 to 3 amino acid substitutions, deletions, An amino acid sequence containing an insertion and / or addition is preferred.

As an example of amino acid substitution, conservative substitution is preferable, and specifically, substitution within a group in parentheses below can be mentioned. For example, (glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

In addition, it is known that a protein having an amino acid sequence having high sequence identity with the original amino acid sequence may have the same function. Therefore, the protein of the present invention is also a protein shown in any of the following [A3], [B3], [C3], and [D3].
[A3] A protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of amino acid numbers 1-333 represented by SEQ ID NO: 1 and having the same function [B3] represented by SEQ ID NO: 3 A protein [C3] consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of amino acid Nos. 1-360 [C3] and the amino acid sequence of amino acid Nos. 1-302 represented by SEQ ID No. 5 and 80 A protein [D3] consisting of an amino acid sequence having a sequence identity of at least% and having the same function [D3] Amino acid sequence having an amino acid sequence of amino acid number 1-263 represented by SEQ ID NO: 7 and at least 80% sequence identity A protein having the same function as used herein, wherein the same function as the protein represented by SEQ ID NO: 1 is referred to as farnesyl Catalyze the function of phosphate synthase (FPPS), ie, reaction using isopentenyl diphosphate and dimethylallyl diphosphate as substrates, or reaction using isopentenyl diphosphate and geranyl diphosphate as substrates Enzyme activity.
Here, in this specification, the function similar to that of the protein represented by SEQ ID NO: 3 is a function inherent to geranylgeranyl diphosphate synthase (GGPPS), that is, isopentenyl diphosphate and dimethylallyl diphosphate. Is an enzyme activity that catalyzes a reaction in which isopentenyl diphosphate and geranyl diphosphate are used as substrates, or a reaction in which isopentenyl diphosphate and farnesyl diphosphate are used as substrates.
Here, in this specification, the function similar to that of the protein represented by SEQ ID NO: 5 is a function inherent to isopentenyl diphosphate isomerase (IDI), that is, isopentenyl diphosphate or dimethylallyl diphosphate. It is an enzyme activity that catalyzes a reaction to isomerize the aldehyde.
Here, in the present specification, the function similar to that of the protein represented by SEQ ID NO: 7 is the function inherent to cis-prenyltransferase (cis-type prenyltransferase), that is, the chain length of the isoprenoid compound is extended to cis-type. It is an enzyme activity that catalyzes the reaction to be carried out.

In order to maintain the same function, that is, the enzyme activity, the sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 1, 3, 5, and 7 is 80% or more, preferably It is 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

The sequence identity of amino acid sequences and base sequences is determined by the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol. , 183, 63 (1990)].

As a method for confirming that the protein has enzyme activity, a conventionally known method can be used. For example, using E. coli or the like, a transformant into which a gene encoding the target protein has been introduced, And measuring the activity of the target protein by the respective activity measurement methods.

(gene)
The gene of the present invention is a gene encoding a protein represented by any of the above [A1] to [A3], [B1] to [B3], [C1] to [C3], [D1] to [D3] It is.

The gene of the present invention is also a gene shown in any of the following [A4], [B4], [C4], [D4], or any of [A5], [B5], [C5], [D5]. is there.
[A4] DNA consisting of the base sequence of base numbers 1-999 represented by SEQ ID NO: 2
[B4] DNA consisting of the nucleotide sequence of nucleotide numbers 1-180 represented by SEQ ID NO: 4
[C4] DNA consisting of the nucleotide sequence of nucleotide numbers 1-906 represented by SEQ ID NO: 6
[D4] DNA consisting of the base sequence of base numbers 1 to 789 represented by SEQ ID NO: 8
[A5] DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence of base numbers 1-999 represented by SEQ ID NO: 2 and encodes a protein having an inherent function
[B5] DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence of base numbers 1-1080 represented by SEQ ID NO: 4 and encodes a protein having an inherent function
[C5] DNA which hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence of base numbers 1-906 represented by SEQ ID NO: 6 and which encodes a protein having an inherent function
[D5] DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence of base numbers 1 to 789 represented by SEQ ID NO: 8 and encodes a protein having an inherent function
Here, in this specification, the function originally possessed by the protein encoded by the gene represented by SEQ ID NO: 2 is the same as the function inherent to the protein represented by SEQ ID NO: 1 described above.
Here, in this specification, the function originally possessed by the protein encoded by the gene represented by SEQ ID NO: 4 is the same as the function inherent to the protein represented by SEQ ID NO: 3 described above.
Here, in this specification, the function originally possessed by the protein encoded by the gene represented by SEQ ID NO: 6 is the same as the function inherently possessed by the protein represented by SEQ ID NO: 5 described above.
Here, in this specification, the function originally possessed by the protein encoded by the gene represented by SEQ ID NO: 8 is the same as the function originally possessed by the protein represented by SEQ ID NO: 7.

The term “hybridize” as used herein refers to a step in which DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific nucleotide sequence or a part of the DNA sequence is useful as a probe for Northern or Southern blot analysis, or can be used as an oligonucleotide primer for PCR (Polymerase Chain Reaction) analysis. It may be DNA. Examples of DNA used as a probe include DNA of at least 100 bases, preferably 200 bases or more, more preferably 500 bases or more, but may be DNA of at least 10 bases, preferably 15 bases or more. .

Methods of DNA hybridization experiments are well known, for example, Molecular Cloning 2nd Edition, 3rd Edition (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual. In addition to those described in Molecular), hybridization conditions can be determined and experiments can be performed according to a number of other standard textbooks.

The above stringent conditions include, for example, a filter in which DNA is immobilized and probe DNA, 50% formamide, 5 × SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6). ) After incubation overnight at 42 ° C. in a solution containing 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / l denatured salmon sperm DNA, for example 0.2 × SSC solution at about 65 ° C. While the conditions for washing the filter can be raised, lower stringent conditions can also be used. Stringent conditions can be changed by adjusting the concentration of formamide (the lower the formamide concentration, the lower the stringency), and changing the salt concentration and temperature conditions. As low stringent conditions, for example, 6 × SSCE (20 × SSCE is 3 mol / l sodium chloride, 0.2 mol / l sodium dihydrogen phosphate, 0.02 mol / l EDTA, pH 7.4), 0 After overnight incubation at 37 ° C. in a solution containing 5% SDS, 30% formamide, 100 μg / l denatured salmon sperm DNA, 50 ° C. 1 × SSC, 0.1% SDS solution The conditions for using and washing can be raised. Further, examples of lower stringent conditions include conditions under which hybridization is performed using a solution having a high salt concentration (for example, 5 × SSC) under the above-described low stringency conditions and then washed.

The various conditions described above can also be set by adding or changing a blocking reagent used to suppress the background of hybridization experiments. The addition of the blocking reagent described above may be accompanied by a change in hybridization conditions in order to adapt the conditions.

As DNA that can hybridize under the above-mentioned stringent conditions, for example, when calculated based on the above parameters using a program such as BLAST and FASTA, the DNA of base number 1-999 represented by SEQ ID NO: 2 is used. Examples thereof include DNA consisting of a base sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 98% or more, particularly preferably 99% or more. . Examples of the base sequences represented by SEQ ID NOs: 4, 6, and 8 also include DNAs each having a base sequence having the sequence identity in the above range.

As a method for confirming that the DNA that hybridizes with the above-described DNA under stringent conditions is a DNA encoding a protein having a predetermined enzyme activity, a conventionally known method can be used. The target protein is expressed by a transformant into which a gene encoding the target protein has been introduced, and the activity of the target protein is determined by the respective activity measurement methods.

In addition, as a method for identifying the amino acid sequence and base sequence of the above protein, a conventionally known method can be used. For example, total RNA is extracted from a growing plant, mRNA is purified, and cDNA is synthesized by reverse transcription reaction. To do. Next, based on the amino acid sequence of a known plant-derived protein corresponding to the target protein, degenerate primers are designed, RT-PCR is performed, DNA fragments are partially amplified, and the sequence is partially identified. To do. Subsequently, RACE method etc. are performed and a full length base sequence and amino acid sequence are identified. The RACE method (Rapid Amplification of cDNA Ends method) means that when the cDNA base sequence is partially known, PCR is performed based on the base sequence information of the known region, and the unknown region up to the end of the cDNA is determined. In this cloning method, full-length cDNA can be cloned by PCR without preparing a cDNA library.
The degenerate primer is preferably prepared from a plant-derived sequence having a sequence site highly common with the target protein.

(Transformant)
Expression of a predetermined protein by introducing the gene of the present invention (the gene encoding the above protein or the genes described in [A4] to [D4] and [A5] to [D5] above) into an isoprenoid-producing plant Thus, a transformed organism (transformant) is obtained. In the transformant, when a predetermined protein is expressed, a predetermined enzyme activity of the protein is newly obtained in the introduced isoprenoid-producing plant, and the protein (enzyme) catalyzes and biosynthesizes. The amount of compound biosynthesis can be improved. Furthermore, since the partial reaction in the isoprenoid biosynthetic pathway in the introduced isoprenoid-producing plant is promoted, the productivity of isoprenoids and polyisoprenoids can also be improved.

The host used for the transformant is not particularly limited as long as it is a plant capable of producing an isoprenoid. For example, a Hevea genus such as Hevea brasiliensis; (Sonchus brachyotus) Sonchus spp such as;. goldenrod (Solidago altissima), goldenrod (. Solidago virgaurea subsp asiatica), Miyama goldenrod (. Solidago virgaurea subsp leipcarpa), tung moth Mine goldenrod (Solidago virgaurea subsp leipc .... Rpaf paludosa), giant goldenrod (Solidago virgaurea subsp gigantea), Solidago gigantea (Solidago gigantea Ait var leiophylla Fernald) Solidago genus and the like; sunflower (Helianthus annuus), Shirota et sunflower (Helianthus argophyllus), Helianthus Atororubensu (Helianthus atrorubens ), Sunflower (Helianthus debilis), kohimawari (Helianthus decapetalus), giant sunflower (Helianthus giganteus) and other genus Helianthus; dandelion (T raxacum), Ezotanpopo (Taraxacum venustum H.Koidz), Shinano dandelion (Taraxacum hondoense Nakai), Kanto dandelion (Taraxacum platycarpum Dahlst), Kansai dandelion (Taraxacum japonicum), dandelion (Taraxacum officinale Weber), Russian dandelion (Taraxacum koksaghyz) such as Taraxacum genus; Ficus carica, Indian rubber tree (Ficus elastica), Eurythius (Ficus pumila L. ), Ficus erecta Thumb., Ficus ampelas Burm.f., Ficus bengetensis Merr., Ficus irisana Elm.F. The genus Ficus such as Ficus septica Burm.f., Ficus benghalensis; the genus Parthenium argentatum, the genus Parthium hysterophorus, the thaliana tuca serriola), Bengal linden, and the like. Among these, a plant belonging to at least one genus selected from the group consisting of the genus Hevea, Sonchus, Taraxacum, and Parhenium is preferable.

As the expression vector, one that can autonomously replicate in the host cell or can be integrated into a chromosome and that contains a promoter at a position where the recombinant DNA can be transcribed can be used.

Examples of expression vectors include pBI vectors, pUC vectors, Ti plasmids, tobacco mosaic virus vectors, and the like.
Any promoter may be used as long as it functions in plant cells. For example, cauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promoter, nopaline synthase gene promoter, and tobacco mosaic virus 35S. Examples thereof include promoters and rice-derived actin gene promoters.

In addition, it is preferable to use an expression vector having a promoter that is specifically expressed in a tissue in which an isoprenoid compound is biosynthesized, such as a milk duct. By specifically expressing the polyisoprenoid in the biosynthesized tissue, it is possible to suppress harmful effects such as delayed growth of plants.

As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, a method using Agrobacterium (Japanese Patent Application Laid-Open No. 59-140885, Japanese Patent Application Laid-Open No. 59-14088). Sho 60-70080, WO 94/00977), electroporation method (Japanese Patent Laid-Open No. Sho 60-251887), method using particle gun (gene gun) (Patent No. 2606856, Patent No. 2517813), etc. be able to.

The transformant (transformed plant cell) can be obtained by the above method or the like.

The present invention also provides an isoprenoid-producing plant into which the gene of the present invention has been introduced. The isoprenoid-producing plant is not particularly limited as long as it is an isoprenoid-producing plant having transformed plant cells. For example, not only transformed plant cells obtained by the above-described method, but also their progeny or clones, and further subculture them. It is a concept that includes all of the progeny plants obtained. Once a transformed plant cell into which the above DNA or vector has been introduced into the genome is obtained, offspring or clones can be obtained from the transformed plant cell by sexual reproduction, asexual reproduction, tissue culture, cell culture, cell fusion, etc. It is possible to obtain. In addition, a propagation material (eg, seed, fruit, cut ear, tuber, tuber, strain, adventitious bud, adventitious embryo, callus, protoplast, etc.) is obtained from the transformed plant cell or its progeny or clone. The isoprenoid-producing plant can be mass-produced.

A method for regenerating plant bodies from transformed plant cells is, for example, the method of Toi et al. (Japanese Patent Application No. 11-127005) in Eucalyptus, and the method of Fujimura et al. (Fujimura et al. (1995), Plant Tissue Culture Lett. , Vol. 2: p74-), for corn, the method of Shirrito et al. (Shillito et al. (1989), Bio / Technology, vol. 7: p581-), for potato, the method of Visser et al. (Visser et al. (1989), Theor. Appl. Genet., Vol. 78: p589-), and in Arabidopsis, the method of Akama et al. (Akama et al. (1992), Plant Cell Rep., Vol. 12: p7-) is known. Regenerating plants from the transformed plant cells with reference to these.

In the regenerated plant body, the expression of the target protein gene can be confirmed by using a well-known technique. For example, the expression of the target protein may be analyzed by Western blot.

As a method for obtaining seeds from the above transformed plant body, for example, the transformed plant body is rooted in an appropriate medium, and the rooted body is transplanted into a pot containing water-containing soil. The seeds are obtained by growing them under appropriate cultivation conditions and finally forming seeds. In addition, as a method for obtaining a plant from seeds, for example, a plant is obtained by sowing seeds derived from a transformed plant obtained as described above in water-containing soil and growing them under appropriate cultivation conditions. You can get a body.

In the present invention, by producing an isoprenoid or a polyisoprenoid using an isoprenoid-producing plant into which the gene of the present invention has been introduced, the productivity of the isoprenoid or polyisoprenoid can be improved. Specifically, transformed plant cells obtained by the above method, callus obtained from transformed plant cells, cells re-differentiated from the callus, etc. are cultured in an appropriate medium or regenerated from transformed plant cells. Isoprenoids and polyisoprenoids can be produced by growing the transformed plants obtained, the plants obtained from the seeds obtained from the transformed plants, and the like under appropriate cultivation conditions. In the transformant of the present invention, the reaction of a part of the biosynthetic pathway of isoprenoids and polyisoprenoids is improved by the introduced protein. Therefore, the biosynthetic amount of the compound synthesized by the protein (enzyme) is increased. It is possible to improve the productivity of isoprenoids, and it is also possible to improve the productivity of polyisoprenoids.

In this specification, polyisoprenoids is a general term for structured polymer with isoprene units (C 5 _{H 8).} Examples of polyisoprenoids include monoterpenes (C ₁₀ ), sesquiterpenes (C ₁₅ ), diterpenes (C ₂₀ ), sesterterpenes (C ₂₅ ), triterpenes (C ₃₀ ), tetraterpenes (C ₄₀ ), and natural rubber. A polymer is mentioned. In the present specification, isoprenoids, means a compound having an isoprene unit (C 5 _{H 8),} is a concept including polyisoprenoid.

As described above, in the present invention, the amino acid sequence and base sequence of farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, isopentenyl diphosphate isomerase, and cis-prenyltransferase in Solidago radish were found. In addition, the amount of biosynthesis of a specific compound in the biosynthesis pathway of isoprenoids can be improved by transformants into which a gene encoding a protein having a specific enzyme activity is introduced, which in turn improves the productivity of isoprenoids. The productivity of isoprenoids can also be improved.

Specifically, farnesyl is obtained by introducing a gene encoding the protein described in any of [A1] to [A3] or the gene described in any of [A4] and [A5] into an isoprenoid-producing plant. The biosynthetic amount of diphosphate can be improved, and the productivity of isoprenoids can be improved, and the productivity of polyisoprenoids can also be improved.

Moreover, by introducing the gene encoding the protein according to any one of [B1] to [B3] or the gene according to any one of [B4] and [B5] into an isoprenoid-producing plant, geranylgeranyl diphosphate The biosynthetic amount can be improved, and the productivity of isoprenoids can be improved, and the productivity of polyisoprenoids can also be improved.

Further, by introducing the gene encoding the protein according to any one of [C1] to [C3] or the gene according to any one of [C4] and [C5] into an isoprenoid-producing plant, dimethylallyl diphosphorus The biosynthetic amount of acid or isopentenyl diphosphate can be improved, and the productivity of isoprenoids can be improved, and the productivity of polyisoprenoids can also be improved.

In addition, the amount of biosynthesis by introducing the gene encoding the protein according to any one of [D1] to [D3] or the gene according to any one of [D4] and [D5] into an isoprenoid-producing plant Can improve the productivity of isoprenoids, and further improve the productivity of polyisoprenoids.

In addition, the gene encoding the protein according to any one of [D1] to [D3], or the gene according to any one of [D4] and [D5] may be used to produce a relatively high molecular weight polyisoprenoid. Since these genes are genes or genes that have high sequence identity with the genes, by introducing these genes into isoprenoid-producing plants, even plants that could only biosynthesize low-molecular-weight polyisoprenoids in the past are relatively high. A molecular weight polyisoprenoid can be produced.

In the present invention, the productivity of isoprenoids and polyisoprenoids can be improved by introducing at least one gene of the above four proteins into an isoprenoid-producing plant. Among the genes encoding the above four proteins, By introducing two or more genes into an isoprenoid-producing plant, the productivity of isoprenoids and polyisoprenoids is further improved, and by introducing all four genes into an isoprenoid-producing plant, the effects of the present invention are most clearly exhibited. Is done.

The present invention will be specifically described based on examples, but the present invention is not limited to these examples.

(Identification of the amino acid sequence and base sequence of the target protein of the enzyme group involved in the isoprenoid biosynthetic pathway in Solidago radish)
Solid walrus was grown in Tendo City, Yamagata Prefecture, and total RNA was extracted from the leaves by the AGPC (acid guanidinium thiocynate-phenol-chloroform extraction) method. Then, cDNA was synthesized by reverse transcription reaction based on the extracted total RNA.
Next, based on the known amino acid sequence of a protein derived from Para rubber tree, sunflower, Arabidopsis thaliana, periwinkle or Canadian yew corresponding to the target protein, a highly common sequence site is selected, The DNA fragments of the target protein were partially amplified by carrying out RT-PCR, and the sequence of the fragment portion was identified using an ABI3100 sequencer manufactured by Applied Biosystems. .
Primers 17 to 37 shown below were newly designed from the sequence of the fragment portion, and the full-length base sequence and amino acid sequence of the target protein were identified by the RACE method. The amino acid sequences of the respective target proteins are shown in SEQ ID NOs: 1, 3, 5, and 7, and the nucleotide sequences are shown in SEQ ID NOs: 2, 4, 6, and 8, respectively.

Degenerate primers used when the target protein is farnesyl diphosphate synthase are shown below. The degenerate primer was designed based on the following known conserved region sequences.
(Primer 1: SEQ ID NO: 9)
5′-gggigiaartknaaymcngg-3 ′
(Primer 2: SEQ ID NO: 10)
5'-gtinigaygayathatga-3 '
(Primer 3: SEQ ID NO: 11)
5'-attrcitnccdatatyticc-3 '
(Primer 4: SEQ ID NO: 12)
5'-tciarwart crtcytg-3 '
(Known conserved region 1 (corresponding to primer 1): SEQ ID NO: 13)
GlyGlyLysLeuAsnArgGly
(Known conserved region 2 (corresponding to primer 2): SEQ ID NO: 14)
LeuAspAspIleMetAsp
(Known conserved region 3 (corresponding to primer 3): SEQ ID NO: 15)
GlyLysIleGlyThrAsp
(Known conserved region 4 (corresponding to primer 4): SEQ ID NO: 16)
ThrTyrPheGlnValGln

Degenerate primers used when the target protein is geranylgeranyl diphosphate synthase are shown below. The degenerate primer was designed based on the following known conserved region sequences.
(Primer 5: SEQ ID NO: 17)
5'-garatgathcayavnatg-3 '
(Primer 6: SEQ ID NO: 18)
5'-atgwsiyatiycaygayga-3 '
(Primer 7: SEQ ID NO: 19)
5'-acrtciardatrtcrtc-3 '
(Primer 8: SEQ ID NO: 20)
5'-attrctciaciacytgraa-3 '
(Known conserved region 5 (corresponding to primer 5): SEQ ID NO: 21)
GluMetIleHisThrMet
(Known conserved region 6 (corresponding to primer 6): SEQ ID NO: 22)
MetSerLeuIleHisAspAsp
(Known storage region 7 (corresponding to primer 7): SEQ ID NO: 23)
AspAspIleLeuAspVal
(Known conserved region 8 (corresponding to primer 8): SEQ ID NO: 24)
PheGlnValValAspAspIle

Degenerate primers used when the target protein is isopentenyl diphosphate isomerase are shown below. The degenerate primer was designed based on the following known conserved region sequences.
(Primer 9: SEQ ID NO: 25)
5'-aartayaaytgycaytnatgg-3 '
(Primer 10: SEQ ID NO: 26)
5'-atgttygangaygartygat-3 '
(Primer 11: SEQ ID NO: 27)
5′-gcnacytctrtnggrttngg-3 ′
(Primer 12: SEQ ID NO: 28)
5'-tcrtcytcnccccayttncc-3 '
(Known conserved region 9 (corresponding to primer 9): SEQ ID NO: 29)
LysTyrAsnCysHis
(Known storage region 10 (corresponding to primer 10): SEQ ID NO: 30)
CysHisLeuAsnGluLys
(Known conserved region 11 (corresponding to primer 11): SEQ ID NO: 31)
ProAsnProAspGluValAla
(Known storage region 12 (corresponding to primer 12): SEQ ID NO: 32)
GlyLysTrpGlyGluHis

Degenerate primers used when the target protein is cis-isoprenyltransferase are shown below. The degenerate primer was designed based on the following known conserved region sequences.
(Primer 13: SEQ ID NO: 33)
5′-cayrtigsiwthrtiatgggayg-3 ′
(Primer 14: SEQ ID NO: 34)
5′-atggayggiaiavgimitgggg-3 ′
(Primer 15: SEQ ID NO: 35)
5'-yciagitcnggccayar-3 '
(Primer 16: SEQ ID NO: 36)
5'-tgccayarnarraart-3 '
(Known storage region 13 (corresponding to primer 13): SEQ ID NO: 37)
HisIleAlaPheIleLeuAspGly
(Known conserved region 14 (corresponding to primer 14): SEQ ID NO: 38)
LeuTrpProGluIleGly
(Known conserved region 15 (corresponding to primer 15): SEQ ID NO: 39)
LeuAspGlyAsnArgArg
(Known conserved region 16 (corresponding to primer 16): SEQ ID NO: 40)
AsnPhePheTrpGln

The primers used in the RACE method when the target protein is farnesyl diphosphate synthase are shown below.
(Primer 17: SEQ ID NO: 41)
5'-atacacgcagagtcaaccc-3 '
(Primer 18: SEQ ID NO: 42)
5'-aaaactttccgaggagaagcc-3 '
(Primer 19: SEQ ID NO: 43)
5'-tattattgtcattattaccg-3 '
(Primer 20: SEQ ID NO: 44)
5'-tattattgtcattattaccg-3 '
(Primer 21: SEQ ID NO: 45)
5'-actccttattagtttgtgtgagg-3 '
(Primer 22: SEQ ID NO: 46)
5'-aggtcaaccctgtgtggtca-3 '
(Primer 23: SEQ ID NO: 47)
5′-cggatgattattaggtagagtctc-3 ′

The primers used in the RACE method when the target protein is geranylgeranyl diphosphate synthase are shown below.
(Primer 24: SEQ ID NO: 48)
5'-cgactggagcacgaggacac-3 '
(Primer 25: SEQ ID NO: 49)
5'-ctaacccaacaacttgtccttc-3 '

The primers used in the RACE method when the target protein is isopentenyl diphosphate isomerase are shown below.
(Primer 26: SEQ ID NO: 50)
5'-gctatttgaagagaagtactactggg-3 '
(Primer 27: SEQ ID NO: 51)
5'-gaggaagcttttagatagaactggg-3 '
(Primer 28: SEQ ID NO: 52)
5′-acttccggtttgatgagttttatcccc-3 ′
(Primer 29: SEQ ID NO: 53)
5'-gaggaagcttttagatagaactggg-3 '
(Primer 30: SEQ ID NO: 54)
5′-acttccggtttgatgagttttatcccc-3 ′
(Primer 31: SEQ ID NO: 55)
5'-aagactctggaacaaaaggtgactt-3 '
(Primer 32: SEQ ID NO: 56)
5'-tagggctttcagtgtttctttg-3 '

When the target protein is cis-isoprenyltransferase, the primers used in the RACE method are shown below.
(Primer 33: SEQ ID NO: 57)
5'-ggaggagaaaagattgaaaagtttgacaaaa-3 '
(Primer 34: SEQ ID NO: 58)
5'-agctgagaagggccatggatgc-3 '
(Primer 35: SEQ ID NO: 59)
5'-gcatgctgttcaagaatctttgtgaag-3 '
(Primer 36: SEQ ID NO: 60)
5'-caaaagattttattctttcaggattgcg-3 '
(Primer 37: SEQ ID NO: 61)
5'-ccccagctcatagcagtaccagag-3 '

(Construction of target protein expression vector)
First, a target protein expression vector was constructed in which a gene (base sequence) encoding the target protein was incorporated into the BamHI / PstI site on the multiple cloning site of pColdIII (pColdIII / SA-FPS). For ligation when incorporating the DNA fragment, Ligation high ver. 2 was used.
In the case of isopentenyl diphosphate isomerase, which is one of the target proteins, the vector is pCold ProS2, the restriction enzyme site is BamHI / SalI, and in the case of cis-prenyltransferase, the vector is pColdProS2 (pColdProS2 / SA- CPT) and restriction enzyme sites were performed at BamHI / HindIII.

(Transformant of E. coli)
Using the E. coli BL21 strain (E. coli BL21 strain) and the target protein expression vector, transformation was performed by the heat shock method. Then, the transformant was applied on an LB agar medium containing 50 μg / ml ampicillin and cultured at 20 ° C. overnight to select and obtain a transformant.
Note that E. coli BL21 strain was also used for isopentenyl diphosphate isomerase and cis-prenyltransferase, which are one of the target proteins.

(Expression of target protein)
The obtained transformant was added to a test tube containing 3 ml of LB liquid medium containing 50 μg / ml ampicillin and cultured with shaking at 37 ° C. for 5 hours. Of the obtained culture solution, 1 ml was added to a 500 ml Erlenmeyer flask containing 100 ml of LB liquid medium containing 50 μg / ml ampicillin, and cultured with shaking at 20 ° C. for 3 hours. Subsequently, isopropyl-β-thiogalactopyranoside (IPTG) was added so as to be 0.1 mmol / L, and the mixture was again cultured with shaking at 20 ° C. for 18 hours. The obtained culture solution was centrifuged to obtain wet cells. The obtained wet cells were disrupted by sonication, centrifuged again, the supernatant was collected, and the target protein was purified using HisTrap (GE Healthcare Japan). The target protein was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE).

(Measurement of target protein activity)
The presence or absence of enzyme activity of each target protein was confirmed by the measurement method shown below. The results are shown in Tables 1-4. In addition, each reference | standard (Control) was performed by what does not mix | blend the obtained target protein.

(Measurement of biosynthesis of farnesyl diphosphate)
10 μg of the target protein (protein represented by SEQ ID NO: 1) obtained by the above purification, 50 mM Tris-HCl Buffer (pH 7.5), 40 mM magnesium chloride, 25 mM 2-mercaptoethanol, 1 mM dimethylallyl diphosphate, 1 mM iso A reaction solution containing pentenyl diphosphate was prepared and reacted in a 37 ° C. water bath for 1 hour. After completion of the reaction, 100 ml of saturated brine and 500 ml of pentane were added, and the mixture was stirred and allowed to stand. Thereafter, the supernatant (pentane layer) was extracted, and the biosynthesis amount of farnesyl diphosphate was measured with a liquid scintillation counter.

(Measurement of biosynthesis of geranylgeranyl diphosphate)
Except for using the protein represented by SEQ ID NO: 3, the biosynthesis amount of geranylgeranyl diphosphate was measured under the same conditions as in the above-described measurement of the biosynthesis amount of farnesyl diphosphate.

(Measurement of biosynthesis of dimethylallyl diphosphate)
3 μg of the target protein (protein represented by SEQ ID NO: 5) obtained by the above purification, 50 mM Tris-HCl Buffer (pH 7.0), 5 mM magnesium chloride, 50 mM 2-mercaptoethanol, 1.25 μM isopentenyl diphosphate were added. A reaction solution was prepared and reacted in a 37 ° C. water bath for 30 minutes. After completion of the reaction, 200 μl of saturated saline and 400 μl of 1 mol / L HCl were added, and after stirring, the mixture was further reacted in a 37 ° C. water bath for 20 minutes. Thereafter, 600 μl of diethyl ether was added, the supernatant (diethyl ether layer) was extracted, and the biosynthetic amount of dimethylallyl diphosphate was measured with a liquid scintillation counter.

(Measurement of biosynthesis of cis-isoprenyl diphosphate which is a cis-type isoprenoid)
10 μg of the target protein (protein represented by SEQ ID NO: 7) obtained by the above purification, 50 mM Tris-HCl Buffer (pH 7.5), 40 mM magnesium chloride, 25 mM 2-mercaptoethanol, 1 mM farnesyl diphosphate, 15 μM isopentenyl A reaction solution containing diphosphoric acid was prepared and reacted in a 37 ° C. water bath for 1 hour. After completion of the reaction, 1 ml of saturated brine was added and stirred, and then 1 ml of diethyl ether was added, stirred and then allowed to stand. Thereafter, the supernatant (diethyl ether layer) was removed, 1 ml of 1-butanol was added, and the mixture was stirred and allowed to stand. Thereafter, the supernatant (1-butanol layer) was removed, 1 ml of a toluene / hexane (1: 1) solution was added, and the mixture was allowed to stand after stirring. Thereafter, the supernatant (toluene / hexane layer) was removed, and the biosynthetic amount of cis-isoprenyl diphosphate was measured with a liquid scintillation counter.

From the results of Tables 1 to 4, it is clear that each of the target proteins has an inherent enzyme activity, each of which is a group of enzymes, such as farnesyl diphosphate synthase and geranylgeranyl diphosphate, which are involved in the isoprenoid biosynthesis pathway in Sequoia litura. It became clear that they were synthase, isopentenyl diphosphate isomerase, and cis-isoprenyl transferase.

In addition, in the transformant (transformed isoprenoid-producing plant) into which the gene encoding the target protein obtained in the present invention is introduced, the amount of biosynthesis of each compound biosynthesized by the target protein is improved. It was also supported to do. Furthermore, it was confirmed that the productivity of isoprenoids and polyisoprenoids was improved in the transformed isoprenoid-producing plant.

(Sequence table free text)
SEQ ID NO: 1: Amino acid sequence of farnesyl diphosphate synthase derived from Streptomyces sp. SEQ ID NO: 2: nucleotide sequence of a gene encoding farnesyl diphosphate synthase derived from Streptomyces sp. SEQ ID NO: 3: Amino acid sequence SEQ ID NO: 4: Nucleotide sequence of a gene encoding geranylgeranyl diphosphate synthase derived from Sequoia serrata SEQ ID NO: 5 Amino acid sequence of isopentenyl diphosphate isomerase derived from Sequoia litura The nucleotide sequence of the gene encoding phosphate isomerase SEQ ID NO: 7: The amino acid sequence of cis-isoprenyltransferase derived from Sequoia litura, SEQ ID NO: 8: Seitakawa Auda Saw derived cis- isoprenyl transferase encoding gene nucleotide sequence SEQ ID NO: 9: Primer 1
Sequence number 10: Primer 2
Sequence number 11: Primer 3
Sequence number 12: Primer 4
SEQ ID NO: 13: known storage region 1
SEQ ID NO: 14: known storage region 2
SEQ ID NO: 15: known storage region 3
SEQ ID NO: 16: known storage region 4
Sequence number 17: Primer 5
Sequence number 18: Primer 6
Sequence number 19: Primer 7
Sequence number 20: Primer 8
SEQ ID NO: 21: known storage region 5
SEQ ID NO: 22: known storage region 6
SEQ ID NO: 23: known storage region 7
SEQ ID NO: 24: known storage region 8
Sequence number 25: Primer 9
SEQ ID NO: 26: Primer 10
SEQ ID NO: 27: Primer 11
SEQ ID NO: 28: primer 12
SEQ ID NO: 29: known storage region 9
SEQ ID NO: 30: known storage region 10
SEQ ID NO: 31: known storage region 11
SEQ ID NO: 32: known storage region 12
Sequence number 33: Primer 13
SEQ ID NO: 34: Primer 14
Sequence number 35: Primer 15
Sequence number 36: Primer 16
SEQ ID NO: 37: known storage region 13
SEQ ID NO: 38: known storage region 14
SEQ ID NO: 39: known storage region 15
SEQ ID NO: 40: known storage region 16
SEQ ID NO: 41: primer 17
SEQ ID NO: 42: primer 18
SEQ ID NO: 43: Primer 19
SEQ ID NO: 44: Primer 20
Sequence number 45: Primer 21
SEQ ID NO: 46: Primer 22
SEQ ID NO: 47: Primer 23
Sequence number 48: Primer 24
SEQ ID NO: 49: Primer 25
Sequence number 50: Primer 26
Sequence number 51: Primer 27
SEQ ID NO: 52: Primer 28
SEQ ID NO: 53: Primer 29
Sequence number 54: Primer 30
Sequence number 55: Primer 31
Sequence number 56: Primer 32
Sequence number 57: Primer 33
Sequence number 58: Primer 34
Sequence number 59: Primer 35
SEQ ID NO: 60: primer 36
Sequence number 61: Primer 37

Claims (24)

The protein according to any one of [A1] to [A3] below.
[A1] a protein comprising the amino acid sequence of amino acid numbers 1-333 represented by SEQ ID NO: 1 [A2] substitution of one or more amino acids in the amino acid sequence of amino acid numbers 1-333 represented by SEQ ID NO: 1, A reaction comprising deletion, insertion, and / or addition, and using isopentenyl diphosphate and dimethylallyl diphosphate as substrates, or isopentenyl diphosphate and geranyl diphosphate as substrates Protein [A3] having an enzymatic activity that catalyzes the reaction. The protein comprises an amino acid sequence of 80% or more of the amino acid sequence of amino acid numbers 1-333 represented by SEQ ID NO: 1, and isopentenyl diphosphate and dimethyl Enzyme activity that catalyzes reactions using allyl diphosphate as a substrate or reactions using isopentenyl diphosphate and geranyl diphosphate as substrates Protein that A gene encoding the protein according to claim 1. The gene according to [A4] or [A5] below.
[A4] DNA consisting of the base sequence of base numbers 1-999 represented by SEQ ID NO: 2
[A5] Hybridizes under stringent conditions with DNA comprising a nucleotide sequence complementary to the nucleotide sequence of nucleotide numbers 1-999 represented by SEQ ID NO: 2, and isopentenyl diphosphate and dimethylallyl diphosphate Which encodes a protein having an enzyme activity that catalyzes a reaction in which the enzyme is used as a substrate or a reaction in which isopentenyl diphosphate and geranyl diphosphate are used as substrates. A transformant in which the amount of biosynthesis of farnesyl diphosphate in the plant is improved by introducing the gene according to claim 2 or 3 into the isoprenoid-producing plant. A method for improving the biosynthesis of farnesyl diphosphate in a plant by introducing the gene according to claim 2 or 3 into an isoprenoid-producing plant. The protein according to any one of [B1] to [B3] below.
[B1] a protein consisting of the amino acid sequence of amino acid numbers 1-360 represented by SEQ ID NO: 3 [B2] substitution of one or more amino acids in the amino acid sequence of amino acid numbers 1-360 represented by SEQ ID NO: 3, Reactions consisting of sequences containing deletions, insertions, and / or additions, and reactions using isopentenyl diphosphate and dimethylallyl diphosphate as substrates, reactions using isopentenyl diphosphate and geranyl diphosphate as substrates Or a protein having an enzymatic activity that catalyzes a reaction using isopentenyl diphosphate and farnesyl diphosphate as substrates [B3] and an amino acid sequence of amino acid numbers 1-360 represented by SEQ ID NO: 3 and a sequence of 80% or more A reaction comprising isopentenyl diphosphate and dimethylallyl diphosphate, each having an amino acid sequence having the same identity, and isopentenyl diphosphate Reaction of the acid with geranyl diphosphate as a substrate, or a protein having an enzyme activity that catalyzes the reaction using a isopentenyl diphosphate and farnesyl diphosphate as a substrate A gene encoding the protein according to claim 6. The gene according to [B4] or [B5] below.
[B4] DNA consisting of the nucleotide sequence of nucleotide numbers 1-180 represented by SEQ ID NO: 4
[B5] It hybridizes under stringent conditions with DNA comprising a base sequence complementary to the base sequence 1-180 represented by SEQ ID NO: 4, and isopentenyl diphosphate and dimethylallyl diphosphate A protein having an enzyme activity that catalyzes a reaction in which isopentenyl diphosphate and geranyl diphosphate are used as substrates, or a reaction in which isopentenyl diphosphate and farnesyl diphosphate are used as substrates. DNA encoding A transformant wherein the biosynthesis amount of geranylgeranyl diphosphate in the plant is improved by introducing the gene according to claim 7 or 8 into an isoprenoid-producing plant. A method for improving the biosynthesis amount of geranylgeranyl diphosphate in an isoprenoid-producing plant by introducing the gene according to claim 7 or 8 into the plant. The protein according to any one of [C1] to [C3] below.
[C1] a protein comprising the amino acid sequence of amino acid number 1-302 represented by SEQ ID NO: 5 [C2] substitution of one or more amino acids in the amino acid sequence of amino acid number 1-302 represented by SEQ ID NO: 5, A protein [C3] consisting of a sequence containing a deletion, insertion, and / or addition, and having an enzyme activity that catalyzes a reaction to isomerize isopentenyl diphosphate or dimethylallyl diphosphate [C3] represented by SEQ ID NO: 5 A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of amino acid No. 1-302, and having an enzyme activity that catalyzes a reaction for isomerizing isopentenyl diphosphate or dimethylallyl diphosphate A gene encoding the protein according to claim 11. The gene according to [C4] or [C5] below.
[C4] DNA consisting of the nucleotide sequence of nucleotide numbers 1-906 represented by SEQ ID NO: 6
[C5] It hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of nucleotide numbers 1-906 represented by SEQ ID NO: 6, and is isopentenyl diphosphate or dimethylallyl diphosphate DNA encoding a protein having an enzymatic activity that catalyzes a reaction to isomerize A transformant in which the biosynthetic amount of dimethylallyl diphosphate or isopentenyl diphosphate in the plant is improved by introducing the gene according to claim 12 or 13 into an isoprenoid-producing plant. A method for improving the amount of biosynthesis of dimethylallyl diphosphate or isopentenyl diphosphate in an isoprenoid-producing plant by introducing the gene according to claim 12 or 13 into the isoprenoid-producing plant. The protein according to any one of [D1] to [D3] below.
[D1] a protein comprising the amino acid sequence of amino acid number 1-263 represented by SEQ ID NO: 7 [D2] substitution of one or a plurality of amino acids in the amino acid sequence of amino acid number 1-263 represented by SEQ ID NO: 7; A protein consisting of a sequence containing a deletion, insertion, and / or addition, and having an enzyme activity that catalyzes a reaction for extending the chain length of an isoprenoid compound to cis type [D3] amino acid number 1- represented by SEQ ID NO: 7 A protein having an enzyme activity that catalyzes a reaction of extending the chain length of an isoprenoid compound to a cis type, comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of 263 A gene encoding the protein according to claim 16. The gene according to [D4] or [D5] below.
[D4] DNA consisting of the base sequence of base numbers 1 to 789 represented by SEQ ID NO: 8
[D5] Hybridizes with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of nucleotide numbers 1-789 represented by SEQ ID NO: 8 under stringent conditions, and extends the chain length of the isoprenoid compound to the cis type. DNA encoding a protein having enzyme activity that catalyzes the reaction A transformant in which the amount of biosynthesis in the plant is improved by introducing the gene according to claim 17 or 18 into an isoprenoid-producing plant. A method for improving the amount of biosynthesis in a plant by introducing the gene according to claim 17 or 18 into an isoprenoid-producing plant. A transformant in which the isoprenoid-producing plant has been improved by introducing the gene according to any one of claims 2, 3, 7, 8, 12, 13, 17, and 18 into the isoprenoid-producing plant. A method for improving the productivity of isoprenoids in a plant by introducing the gene according to any one of claims 2, 3, 7, 8, 12, 13, 17, and 18 into the isoprenoid-producing plant. A transformant in which the productivity of polyisoprenoid in the plant is improved by introducing the gene according to any one of claims 2, 3, 7, 8, 12, 13, 17, and 18 into the isoprenoid-producing plant. A method for improving the productivity of polyisoprenoid in a plant by introducing the gene according to any one of claims 2, 3, 7, 8, 12, 13, 17, and 18 into an isoprenoid-producing plant.

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