WO2007018062A1 - Gène de monoterpène synthétase d'eucalyptus globulus - Google Patents

Gène de monoterpène synthétase d'eucalyptus globulus Download PDF

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WO2007018062A1
WO2007018062A1 PCT/JP2006/315107 JP2006315107W WO2007018062A1 WO 2007018062 A1 WO2007018062 A1 WO 2007018062A1 JP 2006315107 W JP2006315107 W JP 2006315107W WO 2007018062 A1 WO2007018062 A1 WO 2007018062A1
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amino acid
acid sequence
protein
monoterpene
gene
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PCT/JP2006/315107
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English (en)
Japanese (ja)
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Shinichi Yano
Kazufumi Yazaki
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National Institute Of Advanced Industrial Science And Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes

Definitions

  • the present invention relates to a monoterpene synthase protein and a gene encoding the same.
  • terpenes also referred to as terpenes or terpenoids
  • monoterpenes sesquiterpenes
  • diterpenes triterpenes
  • tetraterpenes tetraterpenes
  • sesterterpenes etc. containing multiple C5H8 isoprene units in addition to isoprene with 5 carbon atoms.
  • IPP isopenterunilic acid
  • DMAPP dimethylallyl diphosphate
  • GFP gera-runiphosphate
  • FPP farnesyl diphosphate
  • GGPP gera-lugella diphosphate
  • G FPP geralfalfa Nesyl diphosphate
  • 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.
  • 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.
  • monoterpenes over 400 kinds of compounds such as limonene, binene, menthol, camphor, myrcene, gera-ol, cineole, etc. are known.
  • various plant powers have been isolated from genes encoding terpene synthases that catalyze the production of various monoterpenes from gal-runilic acid (GPP).
  • GPP gal-runilic acid
  • 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).
  • 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
  • An object of the present invention is to provide a novel monoterpene synthase and a gene encoding the same.
  • 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.
  • a monoterpene synthase protein which is one of the following (a) to (d).
  • 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.
  • a monoterpene synthase gene which is any one of the following (a) to (! 1).
  • 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.
  • 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.
  • 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.
  • monoterpenes such as 1,8-cineole and myrcene can be preferably produced.
  • 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.
  • FIG. 1 is a graph showing the homology (%) between a Eucalyptus monoterpene synthase gene and a known terpenoid synthase gene.
  • 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
  • the peak of 24.2 coincided with the retention period of gala-all standard.
  • 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.
  • the gene of the present invention 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. .
  • 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.
  • 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.
  • 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.
  • 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 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).
  • 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).
  • a stop codon eg, TGA, TAA, TAG, etc.
  • 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.
  • 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.
  • 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%. .
  • the washing conditions of the filter after hybridization are 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.
  • 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.
  • the "gene” may contain a modified base, which may be DNA or RNA.
  • DNA includes at least genomic DNA and cDNA
  • RNA includes mRNA and synthetic RNA.
  • 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!
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. .
  • a mutation introduction method such as site-directed mutagenesis.
  • a known method such as the Kunkel method or the Gapped duplex method or a method equivalent thereto can be employed.
  • a mutagenesis kit for example, Mutan-K (TAKARA BIO INC.) Or Mutan-G (TA KARA BIO INC.)
  • TA KARA BIO INC. Mutan-K
  • Mutan-G TA KARA BIO INC.
  • Mutations can be introduced using LA PCR in vitro Mutagenesis series kits manufactured by TAKARA BIO INC.
  • Base sequencing can be performed by known methods such as Maxam-Gilbert's chemical modification method, dideoxynucleotide chain termination method, etc.
  • an automated base sequencer e.g., DNA sequencer PRISM377XL manufactured by ABI is used. Use it! ⁇ .
  • the gene of the present invention isolated as described above is preferably cloned into a vector to prepare a recombinant vector for subsequent operations.
  • 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.
  • 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, ⁇ , ⁇ , etc.).
  • animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can be used.
  • 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.
  • an appropriate restriction enzyme for example, a method of inserting in-frame into a vector and linking it to a vector is adopted.
  • 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.
  • a recombinant expression vector so that the gene of the present invention is expressed as a protein having good activity in the host.
  • 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.
  • the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.
  • 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.
  • 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.
  • 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.
  • a targeting vector for direct introduction into the genome of a host organism by homologous recombination.
  • the vector that can be used for this purpose include known gene targeting vectors such as Cre-loxP.
  • these targeting vectors incorporating the gene of the present invention are also encompassed by the recombinant vector of the present invention.
  • a transformant preferably a transformed cell such as a cultured cell, callus, or tissue
  • a transformant into which the eucalyptus monoterpene synthase gene of the present invention has been introduced
  • 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.
  • 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.
  • E. coli 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.
  • transformants 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.
  • the method of culturing the transformant of the present invention is carried out according to a usual method used for culturing host organisms.
  • 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.
  • a natural medium or a synthetic medium may be used.
  • antibiotics such as ampicillin or tetracycline may be added to the medium.
  • an inducer may be added to the medium as necessary.
  • an inducer 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.
  • IPTG isopylpill-1-thio-13-D-galactoside
  • IAA indole acetic acid
  • the culture conditions are not particularly limited, but are preferably performed under conditions suitable for the host organism used for transformation.
  • the cells or cells are disrupted.
  • 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.
  • cell-free translation The system may be used to produce the eucalyptus monoterpene synthase of the present invention.
  • 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.
  • 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.
  • 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.
  • 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! /.
  • 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.
  • 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.
  • Such monoterpene synthesizing activity of the eucalyptus monoterpene synthase protein of the present invention can be tested using an ordinary monoterpene synthesis reaction system.
  • 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.
  • 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.
  • Gera-Luniphosphoric acid to a degree of 100-20,000 ⁇ 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.
  • 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.
  • 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.
  • DMAPP dimethylallyl diphosphate
  • GPP geranyl diphosphate
  • 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.
  • the eucalyptus monoterpene synthase protein of the present invention preferably produces a monoterpene using geranylphosphoric acid (GPP) in the reaction system as a substrate.
  • GPP geranylphosphoric acid
  • the eucalyptus monoterpene synthase protein of the present invention can particularly produce 1,8-cineole and myrcene.
  • 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.
  • GPP gera-runiphosphate
  • 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.
  • poly (A) + RNA was obtained using Oligotex TM -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
  • 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.
  • 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 ⁇ 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 ⁇ l), then add 25 ⁇ l of black form and DMSO (dimethylsulfoxide) equivalent to 7% final concentration. And then stored at 80 ° C.
  • SM buffer 500 ⁇ l
  • DMSO dimethylsulfoxide
  • 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.
  • 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.
  • 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.
  • Example 1 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
  • KOD-Plus- (Toyobo) was used as a DNA polymerase.
  • the reaction solution was 0.2 mM dNTPs, ImM MgSO4, each primer 0.3 ⁇ , and 200 ⁇ g of type DNA according to the KOD-Plus- manual.
  • 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 ⁇ of isopropyl- ⁇ -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.
  • 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
  • DMAPP dimethylallyl diphosphate
  • GPP geranyl-phosphate
  • 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.
  • Example 1 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.
  • 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
  • sequences of SEQ ID Nos: 5, 8, and 9 are primers.
  • sequences of SEQ ID NOs: 6 and 7 are oligonucleotides forming an adapter.

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Abstract

L'invention concerne une protéine monoterpène synthétase qui comporte la séquence d'amino-acide dépeinte dans SEQ ID NO:2 ou 4 ou une protéine mature en faisant partie ou une variante de la protéine ; un gène codant la protéine ou cette variante ; et un procédé de synthèse de monoterpène faisant appel à la protéine, à la variante ou au gène.
PCT/JP2006/315107 2005-08-10 2006-07-31 Gène de monoterpène synthétase d'eucalyptus globulus WO2007018062A1 (fr)

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WO2010124146A3 (fr) * 2009-04-23 2011-02-24 Danisco Us Inc. Structure tridimensionnelle de l'isoprène synthase et son utilisation dans la production de variants
US8173410B2 (en) 2008-04-23 2012-05-08 Danisco Us Inc. Isoprene synthase variants for improved microbial production of isoprene
US8709785B2 (en) 2007-12-13 2014-04-29 Danisco Us Inc. Compositions and methods for producing isoprene
US9163263B2 (en) 2012-05-02 2015-10-20 The Goodyear Tire & Rubber Company Identification of isoprene synthase variants with improved properties for the production of isoprene
US9273298B2 (en) 2010-10-27 2016-03-01 Danisco Us Inc. Isoprene synthase variants for improved production of isoprene
WO2017022856A1 (fr) * 2015-08-05 2017-02-09 味の素株式会社 Procédé de production de monomère isoprène

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8709785B2 (en) 2007-12-13 2014-04-29 Danisco Us Inc. Compositions and methods for producing isoprene
US9260727B2 (en) 2007-12-13 2016-02-16 Danisco Us Inc. Compositions and methods for producing isoprene
US9909144B2 (en) 2007-12-13 2018-03-06 Danisco Us Inc. Compositions and methods for producing isoprene
US10626420B2 (en) 2007-12-13 2020-04-21 Danisco Us Inc. Compositions and methods for producing isoprene
US8173410B2 (en) 2008-04-23 2012-05-08 Danisco Us Inc. Isoprene synthase variants for improved microbial production of isoprene
US8916370B2 (en) 2008-04-23 2014-12-23 Danisco Us Inc. Isoprene synthase variants for improved microbial production of isoprene
WO2010124146A3 (fr) * 2009-04-23 2011-02-24 Danisco Us Inc. Structure tridimensionnelle de l'isoprène synthase et son utilisation dans la production de variants
US8518686B2 (en) 2009-04-23 2013-08-27 Danisco Us Inc. Three-dimensional structure of isoprene synthase and its use thereof for generating variants
US9175313B2 (en) 2009-04-23 2015-11-03 Danisco Us Inc. Three-dimensional structure of isoprene synthase and its use thereof for generating variants
US9273298B2 (en) 2010-10-27 2016-03-01 Danisco Us Inc. Isoprene synthase variants for improved production of isoprene
US9163263B2 (en) 2012-05-02 2015-10-20 The Goodyear Tire & Rubber Company Identification of isoprene synthase variants with improved properties for the production of isoprene
WO2017022856A1 (fr) * 2015-08-05 2017-02-09 味の素株式会社 Procédé de production de monomère isoprène

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