WO2007018062A1

WO2007018062A1 - Eucalyptus globulus monoterpene synthetase gene

Info

Publication number: WO2007018062A1
Application number: PCT/JP2006/315107
Authority: WO
Inventors: Shinichi Yano; Kazufumi Yazaki
Original assignee: National Institute Of Advanced Industrial Science And Technology
Priority date: 2005-08-10
Filing date: 2006-07-31
Publication date: 2007-02-15
Also published as: JP2007043959A; JP4446482B2

Abstract

A monoterpene synthetase protein comprising the amino acid sequence depicted in SEQ ID NO:2 or 4 or a mature protein part thereof or a variant of the protein; a gene encoding the protein or variant; and a monoterpene synthesis method using the protein, variant or gene.

Description

Eucalyptus' Monoterpene Synthase Gene Technology

TECHNICAL FIELD [0001] The present invention relates to a monoterpene synthase protein and a gene encoding the same.

[0002] Plants are known to produce polyketides, alkaloids, terpenes and the like as secondary metabolites. Of these, terpenes (also referred to as terpenes or terpenoids) are classified into monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, sesterterpenes, etc. containing multiple C5H8 isoprene units in addition to isoprene with 5 carbon atoms. The

[0003] In terpene synthesis in plants, isopenterunilic acid (IPP) and dimethylallyl diphosphate (DMAPP) are produced through the mevalonic acid-mediated pathway or the dalyceraldehyde / pyruvate pathway (non-mevalonic acid pathway). By adding dimethylaryl diphosphate to isopenta-runiphosphate in succession, gera-runiphosphate (GPP), farnesyl diphosphate (FPP), gera-lugella diphosphate (GGPP), geralfalfa Nesyl diphosphate (G FPP) is produced sequentially. Terpene synthases dephosphorylate them and produce various terpenes via carbocation intermediates. Monoterpenes are generated from gera-lunaric acid, sesquiterpenes are generated from farnesyl diphosphate, diterpenes are generated from gera-lugera-runilic acid, and sesterterpenes are generated from gera-ruphanesyl diphosphate. Isoprene can also generate dimethylallyl diphosphate (DMAPP) power.

[0004] Monoterpenes are the main components of natural essential oils that can be taken from fruits and wood moss, and are widely used as raw materials for perfumes and solvents. As monoterpenes, over 400 kinds of compounds such as limonene, binene, menthol, camphor, myrcene, gera-ol, cineole, etc. are known. On the other hand, various plant powers have been isolated from genes encoding terpene synthases that catalyze the production of various monoterpenes from gal-runilic acid (GPP). For example, 1,8-cineole synthase genes include Citrus unshiu (Non-patent document 1), Sage (Salvia officinalis) (Non-patent document 2 and Patent document 1), Arabidopsis thaliana (Ar abidopsis thaliana) (Non-patent Document 3).

[0005] Further, among other terpene synthase genes, isoprene synthase genes have been isolated from Populus (Populus sp.) (Non-patent Document 4) and Kudu (Pueraria montana) (Non-patent Document 5). .

Patent Document 1: U.S. Pat.No. 5,891,697

^^ Patent Literature 1: Shimaaa T. et al., Isolation and charactrerization of North-Boci-ocimen e and 1,8-cineole synthase in Citrus unshiu Marc. "Plant Science, (2004) 168: p.987 -995)

Non-Patent Document 2: Wise M.L. et al., Monoterpene synthase from common sage (Salvia o fficinalis) "J. Biol. Chem., (1998) 273: p.14891— 14899

Non-Patent Document 3: Chen F., et al., "Characterization of a root-specific Arabidopsis terp ene synthase responsible for the formation of the volatile monoterpene 1,8-cineole.Plant Physiol, (2004) 135: p.1956- 1966

Non-Patent Document 4: Miller B., et al., "First isolation of an isoprene synthase gene from po plar snd successful expression of the gene in Escherichia coli. Planta, (2001) 213 (3): p.483-487

Non-Patent Document 5: Sharkey T. D., et al., "Evolution of isoprene biosynthetic pathway in Kudzu." Plant Physiol., (2005) 137: p.700-712

Disclosure of the invention

Problems to be solved by the invention

[0006] An object of the present invention is to provide a novel monoterpene synthase and a gene encoding the same.

Means for solving the problem

[0007] As a result of intensive studies to solve the above problems, the present inventors have succeeded in isolating a gene encoding a eucalyptus monoterpene synthase and, further, a recombinant protein expressed from the gene. Has been found to have an activity of catalyzing the production of myrcene, 1,8-cineol and other monoterpene compounds using GPP as a substrate, and the present invention has been completed. That is, the present invention is as follows.

[0009] [1] A monoterpene synthase protein which is one of the following (a) to (d).

(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4

(b) a protein having amino acid alignment ability comprising at least positions 38 to 582 on the amino acid sequence shown in SEQ ID NO: 2 or 4

(c) Amino acid sequence ability in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4 or an amino acid sequence comprising at least positions 38 to 582 in the amino acid sequence And a protein having monoterpene synthesis activity

(d) consisting of an amino acid sequence shown in SEQ ID NO: 2 or 4 or an amino acid sequence having at least 85% identity with an amino acid sequence comprising positions 38 to 582 on the amino acid sequence, and monoterpene synthesis Active protein

This monoterpene synthase protein preferably has at least 1,8-cineole synthesizing activity as monoterpene synthesizing activity. More preferably, the protein further has myrcene synthesis activity.

[0010] [2] A monoterpene synthase gene which is any one of the following (a) to (! 1).

(a) a gene having the nucleotide sequence shown in SEQ ID NO: 1 or 3

(b) a gene comprising a base sequence comprising at least positions 112 to 1746 on the base sequence shown in SEQ ID NO: 1 or 3

(c) DNA or nucleotides under stringent conditions complementary to the base sequence shown in SEQ ID NO: 1 or 3 or a base sequence comprising at least positions 112 to 1746 on the base sequence under stringent conditions; And a gene encoding a protein having monoterpene synthesis activity

(d) a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 or 3 or a nucleotide sequence comprising at least positions 112 to 1746 on the nucleotide sequence, and having monoterpene synthesis activity Gene encoding protein

(e) a gene encoding the amino acid sequence shown in SEQ ID NO: 2 or 4

(a sequence containing at least positions 38 to 582 on the amino acid sequence shown in β SEQ ID NO: 2 or 4) Gene encoding the amino acid sequence

(g) Amino acid sequence ability in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4 or an amino acid sequence comprising at least positions 38 to 582 in the amino acid sequence And a gene encoding a protein having monoterpene synthesis activity

(h) consisting of an amino acid sequence represented by SEQ ID NO: 2 or 4 or an amino acid sequence having at least 85% identity with the amino acid sequence comprising positions 38 to 582 on the amino acid sequence, and monoterpene synthesis A gene encoding a protein having activity.

The monoterpene synthase gene has at least 1,8-cineole synthesizing activity as monoterpene synthesizing activity, and more preferably encodes a protein having further myrcene synthesizing activity.

[0011] [3] A recombinant vector comprising the gene according to [2] above.

[0012] [4] A transformed cell comprising the recombinant vector according to [3] above.

[5] A method for producing a monoterpene synthase, comprising culturing the transformed cell according to the above [4] and collecting the produced protein from the obtained culture.

[6] A method for producing a monoterpene, comprising reacting the protein according to the above [1] with gera-runilic acid to separate the monoterpene from the reaction product.

By this method, monoterpenes such as 1,8-cineole and myrcene can be preferably produced.

The invention's effect

The eucalyptus monoterpene synthase protein provided in the present invention can efficiently synthesize monoterpenes such as 1,8-cineole and myrcene using geranyl diphosphate as a substrate.

Brief Description of Drawings

FIG. 1 is a graph showing the homology (%) between a Eucalyptus monoterpene synthase gene and a known terpenoid synthase gene.

[Figure 2] Figure 2 shows the results of gas chromatographic analysis of the reaction products obtained by reacting two eucalyptus monoterpene synthase genes and recombinantly expressed proteins with GPP. It is a figure which shows a fruit. The peak of 17.2 coincided with the retention time of Myrcene standard, the peak of 18.7 coincided with the retention time of 1,8-cineole standard, and the peak of 24.2 coincided with the retention period of gala-all standard.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

[0018] In the present invention, preparation of mRNA, cDNA preparation (RT-PCR), PCR, library preparation, ligation into a vector, cell transformation, DNA sequencing, primer synthesis Molecular biological 'biochemical experimental procedures such as mutagenesis, protein extraction, etc. can basically be performed according to the description in a normal experiment document. Representative examples of such experimental documents include Sambrook et al., Molecular Cloning, A laboratory manual, 2001, Eds., Sambrook, J. & Russell, DW.

[0019] 1. Eucalyptus 'monoterpene synthase protein'

The gene of the present invention (eucalyptus monoterpene synthase gene) is a gene encoding a monoterpene synthase protein that is typically isolated from eucalyptus. Examples of the eucalyptus monoterpene synthase gene of the present invention include, for example, a gene having a nucleotide sequence ability shown in SEQ ID NO: 1 or 3 derived from Eucalyptus globulus shown in Examples below. . Alternatively, the eucalyptus monoterpene synthase gene of the present invention may be a gene encoding a monoterpene synthase protein having an amino acid sequence shown in SEQ ID NO: 2 or 4. The protein encoded by the eucalyptus monoterbene synthase gene of the present invention is preferably a protein having monoterpene synthesis activity, particularly 1,8-cineole synthesis activity. More preferably, the protein also has the activity of synthesizing another monoterpene compound, which preferably also has myrcene synthesis activity.

[0020] The eucalyptus monoterpene synthase gene of the present invention may also be a gene comprising a base sequence containing a sequence encoding an amino acid sequence containing at least the mature protein portion of the eucalyptus monoterpene synthase. In general, in terpenoid synthase, the Arg-Arg sequence immediately after the signal peptide sequence is widely conserved. For example, at positions 38 and 39 on the amino acid sequence shown in SEQ ID NO: 2 or 4, From Arg-Arg sequence It is presumed that this is a signal peptide of the eucalyptus monoterpene synthase of the present invention. That is, the eucalyptus monoterpene synthase of the present invention is produced as a mature protein by cleaving the signal peptide at the predicted cleavage site located before position 38 on the amino acid sequence shown in SEQ ID NO: 2 or 4, for example. The It is demonstrated in Example 2 described later that the protein including the amino acid sequence of positions 38 to 582 on the amino acid sequence shown in SEQ ID NO: 2 or 4 and from which the signal peptide is removed has monoterpene synthesis activity. Therefore, the eucalyptus monoterpene synthase gene of the present invention is composed of a partial sequence containing a mature protein portion of the amino acid sequence shown in SEQ ID NO: 2 or 4, and is essentially composed of the 38th position on the amino acid sequence It may be an amino acid sequence having a force of 545 or more amino acid residues including at least position 582. The gene is, for example, an amino acid sequence having positions 38 to 582 on the amino acid sequence shown in SEQ ID NO: 2 or 4, or an amino acid sequence having positions 38 to 582 on the amino acid sequence shown in SEQ ID NO: 2 or 4. 'A gene encoding a sequence with methionine added to the end. The eucalyptus monoterpene synthase gene of the present invention that encodes such a mature protein portion is located at positions 112 to 1746 on the nucleotide sequence shown in SEQ ID NO: 1 or 3 (position 112 to when including a stop codon). It can also be identified as a gene having a base sequence ability comprising at least the sequence at position 1749). In addition, as shown in Example 2 described later, a eucalyptus monoterpene synthase gene in which a sequence encoding a signal peptide is removed and a start codon (for example, ATG) is added to the 5 ′ end is expressed recombinantly. Useful above. Such a recombinant protein produced by Eucalyptus monoterpene synthase gene has a methionine derived from the start codon at the N-terminus, but still retains monoterpene synthesis activity (see Example 2). See). On the other hand, in order to recombinantly express a eucalyptus 'monoterpene synthase gene that does not contain a stop codon, it is preferable to add a stop codon (eg, TGA, TAA, TAG, etc.) to the 3' end of the gene. .

The eucalyptus monoterpene synthase gene of the present invention has one or more (preferably 1 to 40) amino acid sequences represented by SEQ ID NO: 2 or 4 or an amino acid sequence containing at least positions 38 to 582 on the amino acid sequence. Individual amino acids, more preferably 1 to 10, more preferably several (1 to 5) amino acids have been deleted, substituted or added, It may be a gene encoding a protein having monoterpene synthesis activity.

[0022] Further, the eucalyptus monoterpene synthase gene of the present invention is complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 3, or a nucleotide sequence comprising at least the region of positions 112 to 1746 on the nucleotide sequence. It may be a gene that codes for a protein that is hybridized under stringent conditions with DNA that also has a base sequence ability and has monoterpene synthesis activity. Here, stringent conditions refer to conditions under which so-called specific nucleic acid hybrids are formed, and specific examples thereof include a sodium salt concentration of preferably 50 to 750 mM, more preferably 300 to 750 mM. The reaction temperature is preferably 50 ° C to 70 ° C, more preferably 55 to 65 ° C, and the formamide concentration is preferably 20 to 50%, more preferably 35 to 45%. . Further, the washing conditions of the filter after hybridization. The sodium salt concentration is preferably 50 to 600 mM, more preferably 300 to 600 mM, the temperature is 50 to 70 ° C, preferably 55 to 70 ° C, more preferably. The conditions at 60 to 65 ° C. can also be included in the “stringent conditions” in the present invention.

[0023] Alternatively, the eucalyptus monoterpene synthase gene of the present invention preferably has a base sequence represented by SEQ ID NO: 1 or 3, or a partial base sequence comprising at least positions 112 to 1746 on the base sequence, preferably 90% or more, More preferably, it may be a gene encoding a protein having a nucleotide sequence ability of 98% or more identity and having monoterpene synthesis activity. The eucalyptus monoterpene synthase gene of the present invention also preferably has an amino acid sequence shown in SEQ ID NO: 2 or 4, or a partial amino acid sequence containing at least positions 38 to 582 on the amino acid sequence, preferably 85% or more, more preferably It may be a gene consisting of an amino acid sequence showing 98% or more identity and encoding a protein having monoterpene synthesis activity.

[0024] In the present invention, the "gene" may contain a modified base, which may be DNA or RNA. Here, DNA includes at least genomic DNA and cDNA, and RNA includes mRNA and synthetic RNA. In the present invention, the “gene” may be a nucleic acid fragment having a base sequence not including a start codon and a stop codon. The “gene” of the present invention may include an untranslated region (UTR) sequence and the like!

[0025] The gene of the present invention (eucalyptus' monoterpene synthase gene) It can be isolated from eucalyptus globulus, but it may also be isolated from eucalyptus other than eucalyptus globulus. Examples of such eucalyptus include Eucalyptus camaldulensis, Eucalyptus grandis, Eucalyptus grandis, Eucalyptus tereticornis, Eucalyptus tereticornis, Eucalyptus rudis, Eucalyptus. Sanolegenti (Eucalyptus s argent ii), Eucalyptus 'Radius (Eucalyptus radiata), Eucalyptus' Citri odora, Eucalyptus smithi (Eucalyptus smithi, Eucalyptus vininal is), Eucalyptus vininal is (Eucalyptus polybractea), Eucalyptus' Ives (Eucalypt us dieves), etc. Furthermore, the gene of the present invention may be isolated from plants, fungi, Bacillus subtilis, etc. other than eucalyptus. The terpene synthase gene is a natural product derived from eucalyptus or plants other than eucalyptus. It may be a gene derived from natural or may be artificially modified.

The eucalyptus monoterpene synthase gene of the present invention can be isolated by a conventional method based on the nucleotide sequence of SEQ ID NO: 1 or 3 or the amino acid sequence of SEQ ID NO: 2 or 4. For example, an mRNA, cDNA, cDNA library or genomic DNA library preferably prepared from eucalyptus (for example, Eucalyptus globul us) or a non-eucalyptus plant, fungus, Bacillus subtilis or the like, prepared in a conventional manner. The gene of the present invention is obtained as a DNA amplified fragment by PCR using a eucalyptus monoterpene synthesis enzyme gene-specific primer set designed based on the sequences of SEQ ID NOS: 1-4. can do. The obtained DNA amplified fragment is preferably extracted and purified by a conventional method. Alternatively, a eucalyptus 'monoterpene synthase gene-specific probe is designed and prepared from the nucleotide sequence of SEQ ID NO: 1 or 3, and is prepared from eucalyptus (for example, eucalyptus' globulus) or plants other than eucalyptus, fungi, Bacillus subtilis, etc. The gene of the present invention can also be obtained as a clone by hybridizing with a cDNA library or a genomic DNA library that has been prepared. The gene of the present invention may be synthesized using a chemical synthesis method.

[0027] The gene of the present invention may be prepared by modifying a gene obtained from a natural source or a synthesized gene using a mutation introduction method such as site-directed mutagenesis. . In order to introduce a mutation into a gene, a known method such as the Kunkel method or the Gapped duplex method or a method equivalent thereto can be employed. For example, using a mutagenesis kit (for example, Mutan-K (TAKARA BIO INC.) Or Mutan-G (TA KARA BIO INC.)) Using site-directed mutagenesis, or Mutations can be introduced using LA PCR in vitro Mutagenesis series kits manufactured by TAKARA BIO INC.

[0028] It is preferable to confirm the sequence of the obtained eucalyptus monoterpene synthase gene DNA fragment by base sequencing. Base sequencing can be performed by known methods such as Maxam-Gilbert's chemical modification method, dideoxynucleotide chain termination method, etc.Usually, an automated base sequencer (e.g., DNA sequencer PRISM377XL manufactured by ABI) is used. Use it!ヽ.

[0029] 2. Production of assembly vector

The gene of the present invention isolated as described above is preferably cloned into a vector to prepare a recombinant vector for subsequent operations.

[0030] The recombinant vector of the present invention can be obtained by ligating (inserting) the eucalyptus monoterpene synthase gene of the present invention into an appropriate vector. The vector for inserting the eucalyptus / monoterpene synthase gene of the present invention is not particularly limited as long as it can be replicated in the host, and examples thereof include plasmid DNA and phage DNA.

[0031] Plasmid DNA includes plasmids derived from E. coli (eg, pET22b (+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast Plasmids derived from the origin (eg, YEpl3, YCp50, etc.) and the like, and phage DNAs include fly phages (Charon4A, Charon21A, EMBL3, EMBL4, gtl0, gtll, λλ, λΖΑΡΠ, etc.). In addition, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can be used.

In order to insert the eucalyptus monoterpene synthase gene of the present invention into a vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, and then the restriction enzyme site of the appropriate vector DNA or the multicloning site. For example, a method of inserting in-frame into a vector and linking it to a vector is adopted. [0033] The recombinant vector containing the eucalyptus monoterpene synthase gene of the present invention may be prepared as a recombinant expression vector so that the gene of the present invention is expressed as a protein having good activity in the host. Favored ,. In order to produce this recombinant expression vector, various commercially available expression vectors corresponding to various host organisms can be used. Expression vectors usually include transcriptional promoters, terminators, ribosome binding sites, and other various elements essential for expression in the host organism, as well as selectable markers that indicate that the vector is retained in the cell and the vector. Useful sequences such as polylinkers for inserting genes in the correct orientation, cis-elements such as enhancers, splicing signals, poly A addition signals, and ribosome binding sequences (SD sequences) are connected as necessary. Examples of the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.

[0034] The eucalyptus monoterpene synthase gene of the present invention is linked to the vector as described above in a position and orientation so that it can be appropriately expressed. For the purpose of obtaining an active recombinant protein by expressing the eucalyptus monoterpene synthase gene of the present invention in prokaryotes such as Escherichia coli, the sequence encoding the signal peptide is removed, and the initiation codon is replaced. It is preferable to prepare a DNA fragment having a base sequence added to the 5 ′ end and insert it into an expression vector.

[0035] The gene of the present invention may also be prepared in the form of a targeting vector for direct introduction into the genome of a host organism by homologous recombination. Examples of the vector that can be used for this purpose include known gene targeting vectors such as Cre-loxP. In the present specification, these targeting vectors incorporating the gene of the present invention are also encompassed by the recombinant vector of the present invention.

[0036] 3. Manufacture of shaped cocoon and production of eucalyptus' monoterpene synthase using the shaped candy

In the present invention, a transformant (preferably a transformed cell such as a cultured cell, callus, or tissue) into which the eucalyptus monoterpene synthase gene of the present invention has been introduced is prepared and cultured to produce a eucalyptus mono Terpene synthase can be produced. The present invention provides such a transformant and a eucalyptus / monoterpene compound using the transformant. It also relates to a method for producing a synthetic enzyme.

[0037] For transformation, any of bacteria such as Escherichia coli and Bacillus subtilis, yeast cells, insect cells, animal cells (eg, mammalian cells), plant cells and the like may be used. In the present invention, it is particularly preferable to use E. coli.

[0038] For transformation, generally used techniques such as calcium phosphate method, electoral position method, lipofusion method, particle gun method, PEG method and the like can be applied. Selection of transformants can be performed according to a conventional method, but is usually performed using a selectable marker or reporter protein incorporated in the used recombinant vector.

[0039] The method of culturing the transformant of the present invention is carried out according to a usual method used for culturing host organisms. For example, the culture medium for transformants obtained using microorganisms such as E. coli and yeast cells as a host contains a carbon source, nitrogen source, and inorganic salts that can be assimilated by the host microorganism. As long as it is a medium that can efficiently perform the above, either a natural medium or a synthetic medium may be used. If necessary, antibiotics such as ampicillin or tetracycline may be added to the medium.

[0040] When cultivating a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when cultivating microorganisms transformed with an expression vector using the Lac promoter, isopylpill-1-thio-13-D-galactoside (IPTG) was transformed with an expression vector using the trp promoter. When culturing microorganisms, indole acetic acid (IAA) or the like may be added to the medium.

[0041] The culture conditions are not particularly limited, but are preferably performed under conditions suitable for the host organism used for transformation.

[0042] After the cultivation, when the expressed eucalyptus monoterpene synthase protein is produced in the cells or cells, the cells or cells are disrupted. On the other hand, when the protein is secreted outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like to obtain a supernatant. The resulting liquid contains the eucalyptus monoterpene synthase of the present invention. In the present invention, instead of performing transformation, cell-free translation The system may be used to produce the eucalyptus monoterpene synthase of the present invention.

[0043] "Cell-free translation system" refers to a suspension obtained by mechanically disrupting the cell structure of a host organism such as Escherichia coli, and a reagent such as an amino acid necessary for translation added to the suspension. It constitutes an in vitro transcription translation system or an in vitro translation system. As cell-free translation systems, kits that can be used advantageously are commercially available.

[0044] The produced eucalyptus monoterpene synthase is a general biochemical method used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, Isolate and purify the medium strength of the above-mentioned culture (cell disruption fluid, culture fluid, or supernatant thereof) or cell-free translation system by using fifty chromatography alone or in appropriate combination. be able to. In some cases, however, the culture supernatant or lysate supernatant collected or concentrated using a centrifugal separator, ultrafiltration filter, or the like, or the supernatant thereof is further dialyzed after ammonium sulfate fractionation. The obtained solution may be used as a crude enzyme solution as it is, for example, in a confirmation test for monoterpene synthesis activity.

[0045] 4. Activity of Eucalyptus 'Monoterpene Synthase Protein' Confirmation Test

The eucalyptus monoterpene synthase protein of the present invention obtained as described above has monoterpene synthesis activity. The eucalyptus monoterpene synthase protein of the present invention is a protein having an amino acid sequence ability represented by SEQ ID NO: 2 or 4, for example. The protein of the present invention may further be a protein having an amino acid sequence ability from which the signal peptide is substantially removed, including at least positions 38 to 582 on the amino acid sequence shown in SEQ ID NO: 2 or 4. In particular, the protein of the present invention may be a protein having an amino acid sequence of positions 38 to 582 on the amino acid sequence shown in SEQ ID NO: 2 or 4! /. Furthermore, the protein of the present invention is an amino acid sequence comprising at least positions 38 to 582 on the amino acid sequence shown in SEQ ID NO: 2 or 4 or amino acid sequence thereof (preferably an amino acid having a sequence power of positions 38 to 582) Sequence), one or more (preferably 1 to 40, more preferably 1 to 10, more preferably several (1 to 5)) amino acids have been deleted, substituted or appended. It may be a protein having an amino acid sequence ability and having monoterpene synthesis activity. Furthermore, the protein of the present invention is an amino acid sequence represented by SEQ ID NO: 2 or 4. Or an amino acid sequence having at least 85%, more preferably 98% or more identity with an amino acid sequence comprising at least positions 38 to 582 in the amino acid sequence, and a protein having monoterpene synthesis activity. Anyway.

[0046] Such eucalyptus monoterpene synthase protein of the present invention has at least 1,8-cineole synthesizing activity as monoterpene synthesizing activity. That is, the Uri monoterpene synthase protein of the present invention may be 1,8-cineole synthase. The protein of the present invention preferably further has myrcene synthesis activity. The eucalyptus monoterpene synthase protein of the present invention also has monoterpene synthesis activity other than 1,8-cineole synthesis activity and myrcene synthesis activity, for example, limonene synthesis activity, hy-pinene synthesis activity, —pinene synthesis activity, -It may have terpinene synthesis activity, terpinolene synthesis activity, ferrandylene synthesis activity and the like. The protein of the present invention can synthesize monoterpenes such as 1,8-cineole and myrcene by using gera-runilic acid as a substrate.

[0047] Such monoterpene synthesizing activity of the eucalyptus monoterpene synthase protein of the present invention can be tested using an ordinary monoterpene synthesis reaction system.

[0048] As a specific test example, first, an expression vector incorporating a eucalyptus / monoterpene synthase gene was introduced into Escherichia coli, and the resulting transformed cells were cultured (with an inducer such as IPTG). Induced transgene expression), disrupted cells by sonication, etc. to prepare a crude protein solution, and separated the soluble fraction from the crude protein solution by centrifugation at 3000 g for 20 minutes. To do. Since this soluble fraction contains Eucalyptus monoterpene synthase protein, this soluble fraction is then used to measure enzyme activity. Of course, the monoterpene synthase protein may be further isolated and purified from this soluble fraction and used for measuring the enzyme activity.

[0049] For enzyme activity measurement, MgCl was added to 0.1 to 5 ml of its soluble fraction solution containing eucalyptus monoterpene synthase to a final concentration of 1 to 50 mM, and the final concentration as a substrate was further increased.

2

Gera-Luniphosphoric acid (GPP) to a degree of 100-20,000 μΜ is added to the reaction system. After incubating at about 30 ° C. to 40 ° C., the reaction product is extracted and concentrated with hexane and analyzed by gas chromatography. Known 1,8-cineole synthase is 1,8 from geranyl diphosphate. Since it is known to have an activity of catalyzing the conversion to cineol, 1,8-cineole is detected as a reaction product in this gas chromatographic analysis. The eucalyptus monoterpene synthase protein of the invention is shown to have the activity of catalyzing the conversion of geraruniphosphate to 1,8-cineole. In addition, recombinantly expressed eucalyptus by being detected as a myrcene force reaction product that can be generated from a carbocation intermediate generally generated by the dephosphorylation of gera-lunalic acid catalyzed by monoterpene synthase. Monoterpene synthase protein power is shown to further have S-myrcene synthesis activity. In addition, since monoterpenes are all known to be generated from gera-lunaric acid in vivo, if additional monoterpenes are detected as reaction products, recombinantly expressed eucalyptus monoterpenes It is suggested that the synthetic enzyme protein also has an activity to synthesize the detected monoterpene.

[0050] On the other hand, as an example of a control experiment, in the above reaction system, dimethylallyl diphosphate (DMAPP) was added instead of geranyl diphosphate (GPP), and the mixture was similarly incubated at about 30 ° C to 40 ° C. The volatile components in the reaction system's headspace are extracted with a solid phase microextraction fiber, etc., and analyzed by gas chromatography. Known isoprene synthases are known to have the activity of catalyzing the production of dimethylallyl diphosphate power isoprene. Therefore, since isoprene is not detected as a reaction product in this gas chromatographic analysis, the recombinantly expressed eucalyptus monoterpene synthase protein has an activity of catalyzing the production of isoprene from dimethylallyl diphosphate. It is shown that it is not an isoprene synthase.

[0051] As confirmed above, the eucalyptus monoterpene synthase protein of the present invention preferably produces a monoterpene using geranylphosphoric acid (GPP) in the reaction system as a substrate. Can do. The eucalyptus monoterpene synthase protein of the present invention can particularly produce 1,8-cineole and myrcene. Accordingly, the present invention provides a monoterpene by reacting the eucalyptus monoterpene synthase protein or the protein expressed from the eucalyptus monoterpene synthase gene of the present invention with gera-runiphosphate (GPP) as a substrate. (Especially 1,8-cineole and Z or myrcene) It also relates to a process for producing monoterpenes, characterized in that it is separated from the reactants. Separation of the produced monoterpene from the reactant may be performed using a conventional extraction / purification method applicable to the monoterpene, for example, by gas chromatography after partitioning with an organic solvent, or solid phase For example, a direct introduction method to gas chromatography using a solid-phase micro extraction fiber may be used. For details on the extraction, separation, and purification of monoterpenes, see Takao Minamikawa and Seiichi Yoshida, “Methods for Secondary Metabolism of Higher Plants” (Academic Publishing Center), Jun Nakajima, New Basic Biochemical Experiments (2) Extraction 'purification' analysis (see general textbooks such as Maruzen, Methods in Enzymology, vol.111, Steroids and Isoprenoids Part B, Edited by: JH Law and HC Rilling, Academic Press (1985).

[0052] The recombinant expression vector containing the eucalyptus monoterpene synthase gene of the present invention can also be particularly preferably used as a monoterpene production vector suitable for this method.

[0053] This specification includes the contents described in Japanese Patent Application No. 2005-232143, which is the basis of the priority claim of the present application. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Example

[0054] Hereinafter, the present invention will be described with reference to examples and drawings, but the scope of the present invention is not limited to these examples.

[Example 1] Eucalyptus' Acquisition of monoterpene synthase gene

1. Total RNA extraction

Seedlings 2 to 4 leaves of Eucalyptus globulus in the second year of seedling Fresh green l.Og leaves collected from August in Japan under high temperature and high light intensity were liquefied with liquid nitrogen in a mortar. Crushed. Total RNA extraction basically followed the method of Suzuki et al. (BioTechniques, (2 003) 34 (5): 988-990, 992-993). The ground product is suspended in 5 ml of extraction buffer (500 mM sodium isoascorbate, lOOmM Tris-HCl pH 8.0, 10 mM EDTA, 5% (v / v) 2-mercaptoethanol, 2% (v / v) SDS). , And extracted three times with an equal amount of black mouth form-isoamyl alcohol (24: 1). The supernatant obtained in this way was halved to 66.7% (w / v) gua-dimu Cyanate, equal amount of water-saturated phenol, 15% amount of 3M sodium acetate pH 5.2, and left at room temperature for 3 minutes, then half amount of phenol form-isoamyl alcohol (24 1) was added and mixed. This was then left for 15 minutes in ice and then centrifuged (15000 × g, 15 minutes, 4 ° C.). To the resulting supernatant, add 1/3 amount of 1.2M NaCl-0.8M sodium citrate, 2/3 amount of isopropanol, mix, let stand at room temperature for 10 minutes, then centrifuge (15000 Xg, 15 Min, 4 ° C). The obtained precipitate was washed twice with 75% ethanol, air-dried, and dissolved in Tris-ED TA buffer.

[0056] 2. Preparation of cDNA library

From the total RNA obtained, poly (A) + RNA was obtained using Oligotex ™ -dT30 <Super> mRNA purification kit (From Toal RNA) (Takara Bio). Next, using this as a material, a cDNA library was prepared using a cDNA synthesis kit (Stratagene). First, using the obtained polv (A) ⁺ RNA 4 g as a template, reverse transcription with the following Oligo (dT) anchor primer

18

Enzymes were used to synthesize first strand cDNA using 5-methyl-dCTP. Further, after second strand cDNA synthesis, the ends were blunted and the following EcoRI adapter was ligated to it.

[0057]-OligoCdT) Anchor primer:

18

5,-(GA) ACTAGTCTCCGAG (t) V-3, (SEQ ID NO: 5)

10 18

• EcoRI adapter

5 '-OH- AATTCGGCACGAGG-3' (SEQ ID NO: 6)

3 '--- GCCGTGCTCC p-5' (SEQ ID NO: 7)

The EcoRI adapter-ligated DNA fragment was phosphorylated at the end, cleaved with restriction enzyme Xhol, and low molecular weight DNA was removed using a spin column (Clontech CHROMA SPIN TE-1000). Subsequently, phenol / chloroform purification and ethanol precipitation were followed by ligation with λ zapII (EcoRI-Xhol digested) phage vector (Stratagene); LPapx vector was subjected to MaxPlax Lambda Packaging Extracts (EPICEN TRE). After in vitro packaging reaction, add SM buffer (500 μl), then add 25 μl of black form and DMSO (dimethylsulfoxide) equivalent to 7% final concentration. And then stored at 80 ° C.

[0059] The library prepared as described above was subjected to a primary library size assay using Escherichia coli XLl-Blue MRF 'as a host cell, and the result was about 3.1 Xio5001. A part of this solution was used to amplify the library using XLl-Blue MRF 'as a host cell. The resulting library has a virus titer of 2.3 X 10 ^u plU / ml.

[0060] 3. Screening from cDNA library

Using the cDNA library amplified in 2 above (equivalent to 3.3 X 10 ⁶ pfo), the isoprene synthase gene of poplar (Populus alba) donated by Prof. Yazaki of the Kyoto University Survival Zone Research Institute (Kanako ¾asaki, Kazuaki Onara and Kazufumi Yazaki, Gene expression and char acterization of isoprene synthase from Populus alba. "FEBS Letters 579 (2005) p.25 14-2518) was used as a probe. A 623 bp DNA corresponding to a portion having high amino acid sequence homology between various terpenoid synthases was prepared by PCR using a DNA clone containing as a template, and this was used as a probe. The Gene Images AlkPhos Direct Labeling and Detection System (Amersham) was initially clear when hybridization and washing were performed at 55 ° C, the standard condition. No signal was obtained. Many positive clones were obtained by reducing stringency by lowering the temperature to 45 ° C. Lifting positive clones, XL1-Blue MRF 'and ExAssist helper phage (Stratagene PBluescript SK- was excised using E. coli and cultured using Escherichia coli SOLR as a host to prepare a plasmid clone, and the obtained plasmid 18 clone was subjected to DNA sequencing by a conventional method. The amino acid sequence to be encoded was estimated, and the amino acid sequence was searched for homology using the sequence database.

[0061] As a result, 15 of the 18 clones obtained were shown to contain either gene 1 or gene 2 with slightly different nucleotide sequences (gene 1: 7 clones, gene 2). 2: 8 clones). Between gene 1 and gene 2, the total length was 10 bases and the amino acid sequence level was 4 residues different from each other. From the results of the homology search, these two types of remains All genes were shown to be highly homologous to terpenoid synthesis genes in the deduced amino acid sequence. In particular, homology with the monoterpene synthase gene of tea tree (Melaleuca alternifolia) was high at 81.7% for gene 1 and 81.9% for gene 2. In addition, these two genes were then 53.1% both together with the populus (Populus alba) isoprene synthase gene and then 48.7% together with the isoprene synthase gene from Kudu (Pueraria lobata). Showed sex. In addition, when the predicted amino acid sequence in the two obtained genes and the amino acid sequence encoded by a known terpenoid synthesis gene are aligned, the conserved region of the terpenoid synthesis gene is at the amino acid sequence level. It was confirmed that it was well held.

[0062] From the above results, it was suggested that isolated genes 1 and 2 are terpenoid synthesis genes. Therefore, the isolated genes 1 and 2 were presumed to be monoterpene synthase genes, and gene 1 was named eucalyptus' monoterpene synthase gene 1 and gene 2 was named eucalyptus monoterpene synthase gene 2. The homology with known genes shown by the above homology search is shown in FIG.

[0063] However, the specific functions of the tea tree monoterpene synthase genes, which showed only high homology with these Eucalyptus monoterpene synthase genes 1 and 2, have not been elucidated. Other than that, no terpenoid synthesis system genes that showed particularly high homology were found, so the specific proteins encoded by the two isolated genes The ability to synthesize was unpredictable o

[0064] Of the 18 clones obtained above, the remaining 3 clones had a strong homology with the heat shock protein in their predicted amino acid sequences. The clone was considered to be a false positive clone because of its low homology with the encoded amino acid sequence and lack of power.

[Example 2] Preparation of expression vector and qualitative analysis based on the expression

The two genes isolated in Example 1 were incorporated into the expression vector pET22b (+) (No vagen) by a conventional method. Specifically, first, using the above clones containing gene 1 and gene 2 as templates, tagged primers (forward primer: 5'-C AACCATATGCGACGATCGGCCAATTATCAGCC-3 'self column number 8; From the 5' end, CAAC: extra tag sequence, CATATG: NdeI site, CGACGATCGGCCAATTATCAGC C: corresponding to positions 38 to 44 of SEQ ID NOs: 2 and 4], reverse primer: 5 -CGGGAAGCTT TTTATGCCGCAGGAGAAATAGG-3 'self-string number 9; From 5' end side, CGGG: extra tag sequence, AAGCTT: Hindlll site, T: 1 base inserted to remove frame, TTA: stop codon, TGCCGCAGGAGAAATAGG : Corresponding to positions 577 to 582 of SEQ ID NOs: 2 and 4]), the mature protein part (amino acid sequence from positions 38 to 582 of SEQ ID NOs: 2 and 4), excluding the signal sequence of the translated protein A DNA region containing the coding region corresponding to (SEQ ID NOS: 1 and 3 on positions 112 to 1746) and having a start codon ATG (in the Ndel site) at the 5 'end and a stop codon at the 3' end A fragment was obtained. For PCR, KOD-Plus- (Toyobo) was used as a DNA polymerase. The reaction solution was 0.2 mM dNTPs, ImM MgSO4, each primer 0.3 μΜ, and 200 μg of type DNA according to the KOD-Plus- manual.

Four

The total volume was 50 1 using the attached buffer. PCR amplification was initially performed at 94 ° C for 2 minutes, followed by 30 cycles of 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 68 ° C for 2 minutes. The obtained amplified fragment was cleaved with Ndel and Hindlll and ligated in frame to the pET22b (+) vector similarly cleaved with Ndel and Hindlll to prepare an expression vector. Each of the obtained expression vectors was introduced into E. coli origami B (DE3) (Novagen) for expression, and 10 μΜ of isopropyl-β-D-thiogalatatoside (IPTG) was added to the medium, and the temperature was adjusted to 37 ° C. Incubation for 5 hours induced the expression of the recombinant protein. An empty pET22b (+) vector was used as a control sample.

Next, the expression-induced Escherichia coli was suspended in an extraction buffer (50 mM Tris-HC1, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT), and a crude protein solution was prepared by ultrasonic disruption. The supernatant of the crude protein solution (soluble fraction of the crude protein) was collected and used for enzyme activity measurement. Final concentration 10 m as substrate in the presence of 20 mM MgCl

2

Add dimethylallyl diphosphate (DMAPP) that becomes M or geranyl-phosphate (GPP) to a final concentration of 400 μΜ, temperature 30 ° C (temperature suitable for monoterpene synthase reaction) or 40 ° C ( The reaction was carried out at a temperature suitable for the reaction of isoprene synthase. After the reaction, the reaction product was detected by gas chromatography (Shimadzu). When DMAPP is used as a substrate, the reaction system Volatile components in the headspace were extracted using solid-phase microextraction fiber (SPELCO) and gas chromatography (temperature program: initial temperature 40 ° C [5 min hold], then heated up at 30 ° CZ min. ) And analyzed directly. On the other hand, when GPP is used as a substrate, the reaction system is extracted and concentrated with hexane, followed by gas chromatography (temperature program: initial temperature 60 ° C [10 minutes hold], then temperature is raised at 8 ° CZ). Introduced and analyzed.

[0067] As shown in the results of gas chromatography, when GPP was used as a substrate, three peaks (only in a sample derived from an E. coli culture transformed and induced by an expression vector containing gene 1 or 2 ( Retention times: 17.2 minutes, 18.7 minutes, 19.0 minutes) were detected (Figure 2). Of these, the retention times of 17.2 minutes and 18.7 minutes are the values of Myrsen's standard (Wako Pure Chemical Industries, Ltd. # 3 21-52932) and 1,8-cineole, respectively (Wako Pure Chemical Industries, Ltd. # 051-03972)). It was completely consistent with the retention time. Since the 19.0 minute retention time did not match any retention time of the sample used, the product corresponding to this peak is unidentified. However, since the peak with a retention time of 19.0 minutes is also in the region where monoterpene is eluted, and the force is also generated using GPP as a substrate, the product with a retention time of 19.0 minutes is also one of the monoterpene compounds. Was clear. On the other hand, when DMAPP was used as a substrate, no reaction product could be detected in any sample, and no peak corresponding to isoprene was detected. The same results were obtained for both samples derived from E. coli cultures that were expressed by introducing the above genes 1 or 2 derived from eucalyptus, regardless of whether the reaction temperature was 30 ° C or 40 ° C. was gotten.

[0068] Since myrcene and 1,8-cineole were produced in the reaction solution containing GPP, the expression products of genes 1 and 2 were both myrcene and 1,8-cineoate using GPP as a substrate. It has been shown to have the activity of catalyzing the formation of water. On the other hand, since the reaction products using DMAPP as a substrate were not obtained, the expression products of the above genes 1 and 2 both have an activity to catalyze the production of reaction products from DMAPP, particularly isoprene. It was shown that.

[0069] From the above results, the two genes isolated in Example 1 are both genes encoding enzymes having the activity of synthesizing monoterpenes such as 1,8-cineole and myrcene. Became clear. [0070] On the basis of the results of the above chromatography, the peak area of each reaction product was calculated as follows by FID detection.

[0071] · Peak area of retention time 17.2 minutes (Myrcene): 2028

'Peak area with retention time 18.7 minutes (1,8-cineole): 8936

• Retention time 19.0 minutes (unidentified compound) peak area: 11756

From this data, it was possible to calculate a temporary production ratio based on FID detection as Myrcene: 1,8-cineole: unidentified compound = 8.93%: 39.33%: 51.74%.

Industrial applicability

[0072] Eucalyptus' monoterpene synthase and gene encoding the same of the present invention can be used for in vitro synthesis of monoterpenes including 1,8-cineole and myrcene Sequence Listing Free Text

[0073] The sequences of SEQ ID NOs: 5, 8, and 9 are primers.

[0074] The sequences of SEQ ID NOs: 6 and 7 are oligonucleotides forming an adapter.

Claims

The scope of the claims

[1] A monoterpene synthase protein having the following (a) to (no! /, One displacement force.

(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4

[2] The protein according to claim 1, which has 1,8-cineole synthesis activity as monoterpene synthesis activity.

[3] The protein according to claim 2, further having a myrcene synthesis activity.

[4] A monoterpene synthase gene which is one of the following (a) to (! 1).

(a) a gene having the nucleotide sequence shown in SEQ ID NO: 1 or 3

(e) a gene encoding the amino acid sequence shown in SEQ ID NO: 2 or 4

(h) consisting of an amino acid sequence represented by SEQ ID NO: 2 or 4 or an amino acid sequence having at least 85% identity with the amino acid sequence comprising positions 38 to 582 on the amino acid sequence, and monoterpene synthesis Gene encoding a protein having activity

[5] The gene according to claim 4, which encodes a protein having 1,8-cineole synthesis activity as monoterpene synthesis activity.

6. The gene according to claim 5, wherein the protein further has myrcene synthesis activity.

[7] A recombinant vector comprising the gene according to any one of claims 4 to 6.

[8] A transformed cell comprising the recombinant vector according to claim 7.

[9] A method for producing a monoterpene synthase, comprising culturing the transformed cell according to claim 8, and collecting the produced protein from the obtained culture.

[10] A method for producing a monoterpene, comprising reacting the protein according to any one of claims 1 to 3 with geranyl diphosphate and separating the monoterpene from the reaction product.

[11] The method according to claim 10, wherein the monoterpene is 1,8-cineol and Z or myrcene.