JP2000245482A - Production of monoterpene and microorganism therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new transformed microorganism having both of a gene encoding a polypeptide having activities of synthetic enzyme of geranyl diphosphate, and a synthetic enzyme gene of monoterpene, and usable for production or the like of the monoterpene useful for an insecticide, a painkiller, a flavoring agent or the like. SOLUTION: This microorganism is the new transformed microorganism having both of a gene encoding a polypeptide having activities of synthetic enzyme of geranyl diphosphate, and a synthetic enzyme gene of monoterpene, and capable of producing the monoterpene synthesized by the synthetic enzyme of the monoterpene. The microorganism is useful for production of the monoterpene having microbicidal, insecticidal and paregoric activities or the like, further having effect as an agent for aromatherapy, and capable of being applied for the development of chemicals for terminating pathogenic microbes and pests, and the painkiller, the development of a flavoring agent, or the like. The transformed microorganism is obtained by integrating the gene encoding the polypeptide having the synthetic enzyme activities of the geranyl diphosphate, and the synthetic enzyme gene of the monoterpene to different vectors respectively, and inserting the obtained two kinds of recombinant vectors into microorganisms such as Escherichia coli.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a monoterpene by a genetic engineering technique and a transformed microorganism used for the method.

[0002]

2. Description of the Related Art Monoterpene is a general term for compounds having 10 carbon atoms and conforming to the isoprene rule, wherein dimethylallyl diphosphate and isopentenyl diphosphate are condensed. Fragrances obtained from leaves, flowers and fruits of many plants, components of essential oils, and part of insect pheromones are known. The pharmacological properties of monoterpenes have fungicidal, insect repellent and analgesic effects, and are effective as allomer therapy. Therefore, the production of monoterpenes can be applied to the development of medicines and analgesics for controlling pathogenic microorganisms and pests, and the development of fragrances.

[0003] In order to produce such industrially useful monoterpene, it is of course advantageous if it can be mass-produced using a microorganism such as Escherichia coli by genetic engineering techniques. Conventionally, some monoterpene synthase genes such as limonene synthase gene are known (Colby, SM, Alonso, WR, Katahira, E.
J., McGavey, DJ, and Croteau, R. (1993) 4S-Limo
nene Synthase from the Oil Glands of Spearmint (Men
tha spicata). J. Biol. Chem. 268, pp. 23016-2302
4), Some are cloned. However, even if the thus-cloned monoterpene synthase gene is inserted into an expression vector to transform a host microorganism such as Escherichia coli, the resulting transformed microorganism does not produce the desired monoterpene. For this reason, a transformed microorganism producing monoterpene has not yet been obtained.

[0004]

Accordingly, an object of the present invention is to provide a method for producing a sufficient amount of monoterpene by a microorganism by a genetic engineering technique, and a transformed microorganism for the same.

[0005]

SUMMARY OF THE INVENTION The present inventors have determined that a microorganism transformed with a recombinant vector containing a monoterpene synthase gene does not produce the monoterpene because geranyl nitrate, a precursor of the monoterpene, is not produced. It was conceived that the concentration of phosphate was low in the host microbial cells. Geranyl diphosphate synthase, which produces geranyl diphosphate, has been reported to be partially purified in plants (Hiede, L. et al.
and Berger, U. (1989) Parcial purification and Pro
perties of Granyl Pyrophosphate Synthase from Lith
ospermumerythrorhizon Cell Cultures. Archiv. Bioch
em. Biophys. 273, 331-338), but the geranyl diphosphate synthase gene has not yet been identified. The present inventors have found that farnesyl diphosphate synthase that produces C15 farnesyl diphosphate also has geranyl diphosphate synthase activity that produces C10 geranyl diphosphate. Then, by introducing such a gene having geranyl diphosphate synthase activity and a monoterpene synthase gene into a host microorganism, it has been conceived that a transformed microorganism producing the desired monoterpene can be obtained. The present inventors have succeeded in producing a monoterpene-producing transformed microorganism and completed the present invention.

That is, the present invention comprises a gene encoding a polypeptide having geranyl diphosphate synthase activity and a monoterpene synthase gene, and produces a monoterpene synthesized by the monoterpene synthase. A transformed microorganism is provided. The present invention also provides a method for producing a monoterpene, which comprises culturing the microorganism of the present invention and collecting the produced monoterpene.

[0007]

As described above, the transformed microorganism of the present invention has a gene encoding a polypeptide having geranyl diphosphate synthase activity and a monoterpene synthase gene.

Here, the polypeptide having geranyl diphosphate synthase activity may be any polypeptide having geranyl diphosphate synthase activity, not only an enzyme called geranyl diphosphate synthase, but also other polypeptides. Enzymes and the like referred to by enzyme names are also included. For example, in the following example, a gene encoding farnesyl diphosphate synthase that synthesizes C15 farnesyl diphosphate is used, but farnesyl diphosphate synthase also has geranyl diphosphate synthase activity. Therefore, such an enzyme can also be used.

A preferred example of the amino acid sequence of a polypeptide having geranyl diphosphate synthase activity and a base sequence encoding the same are shown in SEQ ID NO: 1 in the sequence listing. The nucleotide sequence shown in SEQ ID NO: 1 is Bacillus stearothermophilu
1 shows the nucleotide sequence of a gene encoding farnesyl diphosphate synthase derived from s.

[0010] The gene encoding the polypeptide having geranyl diphosphate synthase activity includes one or more amino acids substituted or deleted in the amino acid sequence shown in SEQ ID NO: 1; Alternatively, a gene encoding an amino acid sequence having a plurality of amino acids inserted or added and having geranyl diphosphate synthase activity can also be used. Here, the nucleotide sequence of these modified genes is 70% or more, more preferably 9% or more, of the nucleotide sequence described in SEQ ID NO: 1.
It is preferable that the homology is 0% or more, more preferably 95% or more. Also, these modified genes are
A nucleic acid having the nucleotide sequence represented by SEQ ID NO: 1 is used under stringent conditions (that is, 5 × Denhardt's reagen).
t, 6x SSC, 0.5% SDS or 0.1% SDS using a common hybridization solution at 50 to 65 ° C).

As a specific example of such a modified gene, a gene encoding a polypeptide in which serine at position 82 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with phenylalanine (codon tct is substituted with ttc) is used. Examples using such genes are described below.

As apparent from the above, the term "gene" (similarly in the case of the following monoterpene synthase gene) in the present specification means not only a natural gene but also a gene into which a mutation has been artificially introduced. Is also included. Also, "gene"
May include not only nucleic acids such as DNA and RNA, but also those containing artificially modified nucleic acids and those containing other substances capable of encoding amino acid sequences in the same manner as nucleic acids.

The gene having the nucleotide sequence represented by SEQ ID NO: 1 is contained in the genome of Bacillus stearothermophilus, and since the nucleotide sequence is represented by SEQ ID NO: 1, the genome of Bacillus stearothermophilus is used as a template. It can be easily prepared by PCR or the like.

The monoterpene synthase gene used in the present invention may be any enzyme gene that synthesizes a desired monoterpene. Here, the monoterpene may be any monoterpene, and examples thereof include limonene, linalool, geraniol, 1,8-cineole, borneol, α-pinene, β-pinene, and myrcene.

Preferred examples of the monoterpene synthase gene include a gene encoding the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of limonene synthase derived from spearmint, and the nucleotide sequence described in SEQ ID NO: 2 is the nucleotide sequence of a gene encoding limonene synthase derived from spearmint. The base sequence of the spearmint limonene synthase gene shown in SEQ ID NO: 2 and the amino acid sequence encoded thereby have been reported previously (Colby et al., Supra). It differs from the amino acid sequence encoded thereby and is novel.

The gene encoding the monoterpene synthase includes one of the amino acid sequences shown in SEQ ID NO: 2
Alternatively, a gene encoding an amino acid sequence in which one or more amino acids are substituted or deleted, or one or more amino acids are inserted or added to the amino acid sequence, and which has monoterpene synthase activity is also used. Can be. Here, the nucleotide sequence of these modified genes differs from the nucleotide sequence described in SEQ ID NO: 2 by 70% or more.
More preferably 90% or more, more preferably 95%
It is preferable to have the above homology. In addition, these modified genes are combined with a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 under stringent conditions (that is, 5 ×
Denhardt's reagent, 6 x SSC, 0.5% SDS or 0.1% SDS
It is preferable that the hybridization is carried out at 50 to 65 ° C. using a general hybridization solution.

The gene having the nucleotide sequence represented by SEQ ID NO: 2 is obtained from spearmint leaves by the conventional RT-
It can be easily prepared by PCR (one specific example is described in the Examples below).

The transformed microorganism of the present invention can be obtained by incorporating the above two genes into an expression vector and transforming a host microorganism with the obtained recombinant vector. The two types of genes may be integrated into the same expression vector, or may be integrated into separate expression vectors and co-transformed with both recombinant vectors. In addition,
If the host microorganism has one of these two genes and its expression level is sufficient for monoterpene production to be satisfactory, the shortage of the two genes is deficient. Transformation may be performed using only an expression vector containing the gene in which the transformation is performed.

Any expression vector can be used as long as it can express a foreign gene inserted into a cloning site downstream of the promoter in a host cell. Generally, such expression vectors include a replication origin for replication in a host cell, a promoter that enables expression of the foreign gene in the host cell, and a foreign gene inserted downstream of the promoter. At least one restriction enzyme site for, and further preferably, a selection marker such as a drug resistance gene, which allows selection of a vector, and a terminator for terminating transcription downstream of the foreign gene insertion site,
Further, a prokaryotic vector preferably has an SD sequence between the insertion site of the foreign gene and the promoter.
Since various expression vectors are commercially available for various host microorganisms, those commercially available expression vectors can be used in the present invention.

The host microorganism may be any microorganism capable of producing a monoterpene by the above transformation. Examples of preferred host microorganisms include Escherichia coli, genus Pseudomonas, genus Bacillus, yeast, mold and the like, with Escherichia coli being particularly preferred.

The transformation operation itself is well known in this field, and a method suitable for a host cell and an expression vector to be used can be appropriately selected and used. For example, as described in the Examples below, when the host is Escherichia coli, the calcium chloride method or the like can be used.

After the transformation operation, the transformant of the present invention is selected by selecting a transformant based on the selection marker of the expression vector used, and further selecting a clone producing the desired monoterpene. Obtainable.

The transformed microorganism obtained as described above is cultured to recover the target monoterpene. The method for culturing microorganisms can be carried out using a known culture method for each microorganism. Further, the produced monoterpene depends on the properties of the host microorganism and the expression vector used,
It can be recovered from cells or culture supernatant. The recovered monoterpene can be purified or partially purified by a conventional method such as chromatography, if necessary.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below more specifically based on embodiments. However, the present invention is not limited to the following examples.

2-1 Cloning of limonene-synthesizing gene from spearmint General genetic manipulation procedures in the following experiments were performed by the method of J. Sambrook et al. (Sambrook, J., Fritsch,
EF, and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor lab
oratory, Cold Spring harbor, NY).

2-1-1 Preparation of Total RNA Total RNA was prepared by the following method. 1) Fresh spearmint grown indoors (Mentha spicat
a) Place 50 g of leaves in a mortar and crush with a pestle while adding liquid nitrogen. 2) Separate the crushed material into two 10 ml centrifuge tubes,
Add and suspend the extraction buffer (4.23 M guanidine thiocyanate, 26.4 mM trisodium citrate, 0.53% sarcosine, 100 mM 2-mercaptoethanol). 3) Centrifuge at 3,000 rpm for 10 minutes at 4 ℃. 4) Dispense the supernatant into 10 1.5 ml centrifuge tubes,
Centrifuge at 15,000 rpm for 10 minutes. 5) Transfer the supernatant to six 3.5 ml ultracentrifuge tubes, and add 1 ml of RNA cusion (5.7 M cesium chloride, 10 mM EDTA).
(pH 8.0), 0.1% diethyl pyrocarbonate (DEPC)). 6) Centrifuge at 20 ℃, 80,000 rpm for 2 hours. 7) Remove the upper layer, then wash the vicinity of the interface three times with the extraction buffer to remove impurities floating at the interface. Then remove the lower layer. 8) Wash the precipitate with 400 μl of 0.1% sodium dodecyl sulfate (SD
S)) and dissolved in the same amount of phenol: chloroform (1: 1)
Add the mixture and mix well. Centrifuge at 15,000 rpm for 3 minutes at room temperature, and transfer the supernatant to another tube. Add 400 μl of enol: chloroform (1: 1) mixture and repeat. 9) Add 40 μl of sodium acetate (pH5.2) and 880 μl of ethanol and mix well. 10) Centrifuge at 4 ℃, 15,000 rpm for 15 minutes. 11) Discard the supernatant, add 400 μl of cold 70% ethanol at -20 ℃, and centrifuge at 4 ℃, 15,000 rpm for 3 minutes. 12) Thoroughly remove the ethanol and remove the precipitate with 40 μl of 0.1% DEPC.
Dissolve in H ₂ O containing 13) Check the RNA by electrophoresis of 1 μl. Also, OD260 n
Measure the concentration in m.

[0027] 2-1-2 Preparation of poly (A) ⁺ RNA of the prepared poly (A) ⁺ RNA by using the oligotex-dT 30 (super) (Japan Synthetic Rubber + manufactured by Nippon Roche), as follows: went. 1) In a 1.5 ml centrifuge tube, add 12 μg / 95 μl total R
Add NA and 600 μl oligotex-dT 30 (super), mix well, and heat at 65 ° C for 5 minutes. 2) Cool in ice for 3 minutes. 3) Add 70μl of 5M NaCl, mix well, and incubate at 37 ℃ for 10 minutes. 4) Centrifuge at room temperature at 15,000 rpm for 3 minutes. 5) Remove the supernatant and remove the precipitate with 500 μl of NS buffer (0.5
M NaCl, 0.1% SDS, 0.1% DEPC). Then 37
Incubate at ° C for 5 minutes. 6) Centrifuge at room temperature at 15,000 rpm for 3 minutes. 7) Remove the supernatant and suspend the precipitate with 200 μl of H ₂ O containing 0.1% DEPC. 8) Incubate at 65 ° C for 5 minutes. Then 3 in ice
Let cool for a minute. 9) Centrifuge at 15,000 rpm for 3 minutes at room temperature. 10) Collect the supernatant and repeat 8) and 9). 200 μl of precipitate
Suspend the cells in H ₂ O containing 0.1% DEPC, and repeat steps 8) and 9) in the same manner. 11) Extract the supernatant twice with phenol: chloroform. 12) Add 20 μl μl sodium acetate to the supernatant after extraction.
(pH5.2) and 440 μl of ethanol, and mix well. 13) Centrifuge at 15,000 rpm for 15 minutes at 4 ℃. 14) Discard the supernatant, add 400 μl of cold 70% ethanol at -20 ℃, and centrifuge at 4 ℃, 15,000 rpm for 3 minutes. 15) Thoroughly remove ethanol, and precipitate 30 μl of 0.1% DEPC.
Dissolve in H ₂ O containing This was used as an mRNA solution for cDNA synthesis.

2-1-3 Synthesis of cDNA cDNA was synthesized using a ZAP-cDNA Synthesis Kit (manufactured by STRATAGENE) as follows. First strand DNA synthesis 1) 28 μl of 3.8 μg mRNA solution in a 1.5 ml centrifuge tube,
5 μl of 10 × 1st strand buffer, 3 μl of first strand methyl nucleotide mixture, 2 μl of 1.4 μg / μl of linker primer (5′-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTT
TTTTTTTTTTTT-3 '), 7.5 μl DEPC treated H2O, 1 μl 4
0 units / μl RNase block ribonuclease inhibitor), and mix gently. 2) Leave at room temperature for 10 minutes to anneal the primer and template (mRNA obtained above). 3) Add 3.5 μl of reverse transcriptase MMLV-RTase (200 U / μl). Mix gently. 4) Incubate at 37 ° C for 1 hour. 5) Add 20 μl of 10x2nd strand buffer, 2nd strand nucleotide mixture, 108.2 μl of H ₂ O, 3.5 μl of Rnase H to the reaction solution.
(0.9 U / μl), 10 μl DNA polymerase (10 U / μl)
And incubate at 16 ° C for 90 minutes. Then immediately place on ice. 6) Add 23 μl of Blunting dNTP mix, 2 μl of cloned Pfu DNA polymerase (2.5 units / μl) to the reaction solution,
Mix well and react at 72 ° C for 30 minutes. 7) Return to room temperature, add 200 µl phenol: chloroform (1: 1) and mix well. 8) Centrifuge at room temperature at 15,000 rpm for 2 minutes. Transfer the supernatant to another tube. 9) Add an equal volume of chloroform and mix well. 10) Centrifuge at room temperature at 15,000 rpm for 2 minutes. Transfer the supernatant to another tube. 11) Add 20 μl of 3M sodium acetate (pH 5.2) and 400 μl of ethanol and mix well. Then leave at -20 ° C overnight. 12) Centrifuge at 4 ℃, 15,000 rpm for 60 minutes. 13) Discard the supernatant, add 500 μl of 80% ethanol,
Centrifuge at 15,000 rpm for 2 minutes at room temperature. 14) Thoroughly remove ethanol and remove 9 μl of EcoRI
Dissolve in the Adapter solution. 1 μl of this was used as a template for RT-PCR.

2-1-4 Cloning of limonene synthase gene by PCR PCR amplification and purification of the PCR product were carried out by the following procedures. 1) Using the cDNA prepared from the spearmint as a template, the primer for cloning the limonene synthase gene was previously cloned by Croteau et al. S1 primer: 5'-ATGGCTCTC
AAAGTGTTAAGTG-3 'and S3 primer for 3' terminal sequence: 5'-TCATGCAAAGGGCTCGAATAAGGTTC-3 '. 2) PCR was performed under the following conditions. PCR reaction at 94 ° C for 15 seconds, 5
32 cycles of 2 seconds at 5 ° C and 30 seconds at 74 ° C were performed. Each (adenosine, thymine, guanosine, cytosine) deoxynucleotide triphosphate was added to a final concentration of 200 μM. Primers are 20 pmo each for S1 and S2
l Used. MgCl ₂ was added to a final concentration of 1 mM. DNA polymerase is KOD DNA polymerase (TOYOBO C
O., LTD) was used in 2.5 units. 1 μl of cDNA was used. The total volume of the PCR reaction solution was adjusted to 50 μl with H ₂ O. 3) After the PCR reaction was completed, 5 μl was subjected to agarol gel electrophoresis to confirm the PCR product. 4) Add 355 μl of H ₂ O to the remaining reaction mixture and add another 400 μl.
Add 1 phenol: chloroform (1: 1) mixture and mix well. Centrifuge at room temperature at 15,000 rpm for 3 minutes, and transfer the supernatant to another tube. 5) Add 40 μl of 3M sodium acetate (pH 5.2) and 880 μl of ethanol and mix well. 6) Centrifuge at 15,000 rpm for 15 minutes at 4 ℃. 7) Discard the supernatant, add 400 µl of -20 ° C cold 80% ethanol, and centrifuge at 4 ° C, 15,000 rpm for 3 minutes. 8) Thoroughly remove the ethanol and remove the precipitate with 15 μl of H ₂ O
Dissolve in 9) Perform electrophoresis on the entire volume. 10) Cut out the band of the desired size with a razor and
The DNA was transferred to a centrifuge tube having a volume of ml, and DNA was recovered from the gel using Microcon (Amicon).

2-1-5 Phosphorylation of 5′-end of PCR product The 5′-end phosphorylation of the PCR product was carried out by the following method. 1) To 100 μl of the DNA solution recovered from the gel, add 58 μl of H2O, 40
Add 1 μl of 5x kinase buffer, 2 μl of T4 polynucleotide kinase (10 units / μl), mix, and mix.
Incubate at 30 ° C for 30 minutes. 2) Immediately, the enzyme was inactivated by incubation at 70 ° C for 10 minutes. 3) Add 200 μl of H ₂ O, then add 400 μl of a phenol: chloroform (1: 1) mixture and mix well.
Centrifuge at room temperature at 15,000 rpm for 3 minutes, and transfer the supernatant to another tube. 4) Add 40 μl of 3M sodium acetate (pH 5.2) and 880 μl of ethanol, and mix well. 6) Centrifuge at 15,000 rpm for 15 minutes at 4 ℃. 7) Discard the supernatant, add 400 µl of -20 ° C cold 80% ethanol, and centrifuge at 4 ° C, 15,000 rpm for 3 minutes. 8) Thoroughly remove the ethanol and remove the precipitate with 10 μl of H2O.
Dissolve in 5 μl of this was used as a ligation insert.

2-1-6 Ligation and transfer
Formation 1) Plasmid vector is pBluescript II SK⁺ (STRATAG
ENE) (digested with EcoRV)
The one dephosphorylated after the treatment with the enzyme was used. This is pS
Abbreviated as K / EcoRV. 2) Place 1 μl of pSK / EcoRV in a 1.5 ml centrifuge tube on ice.
(20 ng), 5 μl phosphorylated PCR product, 3 μl lig
ation high (TOYOBO) and react at 16 ° C for 2 hours
I let it. 3) Add 74 μl H_TwoO, 10 μl of 10 x KCM (1M KCl,
 0.3M CaCl_Two, 0.5M MgCl _Two), 7 μl of 30% polyethylene
Add glycol and mix. 4) 100 μl of competent cells E. coli DH5α (Bethesda
(Commercially available from Research), mix and leave on ice for 20 minutes
You. 5) Leave at room temperature for 10 minutes. 6) Add 1 ml of 1xLB medium and incubate at 37 ° C for 1 hour.
To 7) 1xLB agar containing ampicillin at a final concentration of 50 μg / ml
Spread an appropriate amount onto a plate and incubate at 37 ° C overnight
I do.

2-1-7 Preparation of Plasmid and Cleavage and Subcloning with Restriction Enzyme Preparation of a plasmid containing the limonene synthase gene and cleavage and subcloning with the restriction enzyme were performed as follows. 1) A single colony obtained by transformation was picked up with a platinum loop, inoculated in 10 ml of 1 × LB medium containing ampicillin, and cultured with shaking at 37 ° C. overnight. 2) Plasmid preparation is performed by alkaline method or wizard plasmi
d Preparation kit (PROMEGA) was used. 3) The prepared plasmid was digested with various restriction enzymes to prepare restriction enzyme cleavage maps. In addition, fragments cut by various enzymes were separated by agarose gel electrophoresis, and the respective fragments were recovered from the gel, recyclized or inserted into another plasmid vector, and subcloned by Escherichia coli.

2-1-8 Determination of Nucleotide Sequence The nucleotide sequence of the cloned limonene synthase gene was determined as follows. 1) A plasmid was prepared from E. coli obtained by subcloning. 2) Prepared plasmid is dye terminator cycle sequence
A sequencing reaction was performed using a sing kit (Perkin Elmer ABI). 3) The sample subjected to the sequencing reaction was Genetic a
Using nalyzer 310 (manufactured by PerkinElmer ABI),
The nucleotide sequence was determined. 4) The determined nucleotide sequence is used for genetic information analysis software Gene
Analysis was performed using tix MAC ver.9 (Software Development Co., Ltd.). The determined nucleotide sequence and the deduced amino acid sequence encoded thereby are shown in SEQ ID NO: 2 in the sequence listing. The nucleotide sequence of the obtained limonene synthase gene was
Compared to the base sequence of limonene synthase gene of spearmint cloned by (supra) et al., The nucleotide sequence differs by 42 bases, and the amino acid sequence also differs by 15 amino acid residues. Therefore, the limonene synthase gene cloned in this example and the limonene synthase encoded by the limonene synthase gene are both novel.

2-2 Measurement of limonene synthase activity 2-2-1 Construction of limonene-producing plasmid It has been experimentally confirmed that geranyl diphosphate, which is a substrate of a limonene synthesis gene, is synthesized for limonene production. The construction of a plasmid that co-expresses the gene and the limonene synthesis gene cloned in this study was performed as follows.

A farnesyl diphosphate synthase gene (BSFPPs) was prepared from Bacillus stearothermophilus (commercially available from ATCC, accession number ATCC 10149) and the plasmid pTV118N
(Commercially available from Takara Shuzo Co., Ltd.) to prepare a recombinant vector. This is Koyama, T., Obata, S., Osab
e, M., Takeshita, A., Yokoyama, K., Uchida, M., Ni
shino, T and Ogura K. (1993) Thermostable Farnesyl
Diphosphate Synthaseof Bacillus stearothermophilu
s: Molecular Cloning, Sequence Determination, Over
production, and Purification. J. Biochem. 113, 355
Performed by the method described in -363. That is, Bacill
Using the genomic DNA of us stearothermophilus ATCC 10149 as a template, the following sequence was used as a primer: 5′-GAG
GAGGAGTAAGCCATGGCGCAGCTTTCA-3 'and 5'-CGACCATTAAAAG
Using two types of oligonucleotides having CTTAACGCCCGCCCTTG-3 ′, BSFPPs were amplified by PCR in a conventional manner, and the amplified BSFPPs were ligated with NcoI, Hi of plasmid pTV118N.
Insert the recombinant vector (pTV118N + Wild-B
SFPPs). The nucleotide sequence of the obtained BSFPPs and the deduced amino acid sequence encoded thereby are shown in SEQ ID NO: 1 in the sequence listing.
Shown in

The construction of pTV118N + Mutant-BSFPPs was made by introducing a site-specific mutation into pTV118N + Wild-BSFPPs by the Kunkel method. The reagent used for the Kunkel method was Mutan K (Takara Shuzo) which is commercially available as a kit. 1. Plasmid pTV118N + Wild-BSFPPs was used for E. coli CJ236 [F ',
dut ^-, ung ^-] was introduced into (Takara Shuzo commercially available from, Ltd.). 2. Single-stranded DNA is transformed into bacteriophage M
Prepared by infecting 13KO7. (The resulting prepared D
Deoxyuracil (dU) is introduced into NA. 3. The prepared single-stranded DNA is a 5'-terminal phosphorylated oligonucleotide DNA primer (S82F) 5'-CATACGTACTTCTTG
Annealed with ATTCATGATGATTTG-3 '(This primer has a serine TCT at position 82 of the wild-type and a phenylalanine T
(The BspHI restriction enzyme site TCATGA has been introduced as a silent mutation for the purpose of discriminating mutations from TC substitutions.) 4. Double-stranded using E. coli DNA ligase and T4 DNA polymerase. 5. E. coli BMH71-18 mutS using double-stranded DNA
Was transformed. 6. A plasmid was prepared from the obtained transformant (pTV1
18N + Mutant-BSFPPs). Clones into which the mutation was introduced by cleavage with the restriction enzyme BspHI were identified. 7. The entire nucleotide sequence of the BSFPPs-encoding region of the plasmid was determined, and the presence or absence of the mutation was confirmed. As a result, the serine TCT at position 82 of the wild-type BSFPPs shown in SEQ ID NO: 2 was replaced with phenylalanine TTC, and the other parts were as shown in SEQ ID NO: 2.

PTV118N + Wild-BSFPPs and pTV118N + Muta
The nt-BSFPPs were cleaved with the enzyme Pvu II, which cuts and cuts at two positions, and the resultant was subjected to agarose gel electrophoresis to separate DNA fragments, and the fragments encoding BSFPPs were recovered. This fragment was inserted into the ScaI site of the plasmid pACYC184 (commercially available from Nippon Gene Co., Ltd.). At this time, two types of plasmids having different directions were obtained. Called with -r,
Plasmids inserted counterclockwise are denoted with -l.
The two plasmids generated in the wild type and mutant type, respectively, are pA
CYC184 + Wild-BSFPPs-r, pACYC184 + Wild-BSFPPs-l, pACY
C184 + Mutant-BSFPPs-r and pACYC184 + Mutant-BSFPPs-l were used in the experiments. The above operation is schematically shown in FIG.

Next, each plasmid was transformed into Escherichia coli DH5α.
Was introduced. Plasmid pBluescript II SK ⁺⁺ into which limonene synthesis gene was inserted into these transformed Escherichia coli
Introduced limonene. Therefore, every E. coli carries two types of plasmids. As a control, pACYC1
DH5α (strain J described below) transformed with two plasmids, 84 vector and Bluescript II SK ⁺ vector, was used.

Each of these transformants was A strain (DH5α
/ pACYC184 + Wild-BSFPPs-r and pBluescript II SK + + li
monene (This display shows that plasmid pACYC184 + Wild-BSFPPs-r and plasmid pBluescript II
SK ⁺ + means containing limonene, below,
Same)), B strain (DH5α / pACYC184 + Wild-BSFPPs-r and pBl
uescript II SK ⁺ ), C strain (DH5α / pACYC184 + Wild-BSFPPs-l
and pBluescript IISK ⁺ + limonene, strain D (DH5α / pACYC1
84 + Wild-BSFPPs-l and pBluescript II SK ⁺ , E strain (DH5α /
pACYC184 + Mutant-BSFPPs-r and pBluescript II SK + + l
imonene), F strain (DH5α / pACYC184 + Mutant-BSFPPs-r and p
Bluescript II SK ⁺ ), G strain (DH5α / pACYC184 + Mutant-BSFP
Ps-l and pBluescript II SK ⁺ + limonene), H strain (DH5α
/ pACYC184 + Mutant-BSFPPs-l and pBluescript II SK ⁺ ),
I strain (DH5α / pACYC184 and pBluescript II SK ⁺ + limon
ene), J strain (DH5α / pACYC184 and pBluescript II SK ⁺ )
And used for the experiment.

2-2-2 Limonene synthase assay The enzyme activity of limonene synthase was analyzed by the method of Steele (Steele
ele, CL, Crock. J., Bohlmann J., and Croteau, R.
(1998) Sesquiterpene Synthases from GrandFir (Abi
es grandis). J. Biol. Chem. 273, 2078-2089). 1) Stored in a 30% glycerol solution at -85 ° C.
60 μg / m E. coli harboring limonene-producing plasmid
lxLB agar plate containing 1 l of ampicillin and 10 mg / ml tetracycline, streaked with platinum loops and inoculated at 37 ° C.
Incubate for about 20 hours. 2) A single colony was scraped with a platinum loop, and 10 μm of 60 μg / ml ampicillin and 10 mg / ml tetracycline were added.
l Inoculate 1xLB liquid medium with shaking at 30 ℃ overnight. 3) On the next day, add 10 μl of 1M IPTG, and culture with shaking overnight. 4) Transfer the entire volume to a 12 ml centrifuge tube and store at 4 ℃, 3000 rp
Centrifuge for 5 minutes at m for collection. 5) Discard the medium and suspend the cells in 20 mM Tris-HCl (pH 7.5). 6) Transfer cells to 1 ml of sonication buffer (20 mM MOPS
Resuspend the cells in O (pH 7.0), 1 mM EDTA, 1 mM DTT, 10% (v / v) glycerol, and disrupt the cells using an ultrasonic cell disrupter. 7) Transfer the crushed material to a 1.5 ml centrifuge tube, and
Centrifuge at 0 rpm for 20 minutes. 8) The supernatant was used as a crude extract for limonene synthase assay. 9) The limonene synthase assay was performed using the following two reaction solutions. 1. Each final concentration is 20 mM β-hydroxy-4-morpholinepropanesulfonic acid: MOPSO (pH7.0),
10% glycerol (v / v), 15 mM MgCl ₂ , 1 mM DTT, 50
μM [1- ³ H] geranyl diphosphate (GPP), 40 crude extracts of [mu] l, the reaction solution was adjusted to 1ml with H ₂ O. 2. Each final concentration
20 mM MOPSO (pH7.0), 10% glycerol (v / v), 15 mM
MgCl ₂ , 1 mM DTT, 100 μM dimethylallyl diphosphate (DM
^{APP), 5 μM [1- 14} C] isopentenyl diphosphate (IPP),
40 μl crude extract, reaction mixture adjusted to 1 ml with H ₂ O. 10) Add the reaction solution to a 10 ml glass centrifuge tube with a screw cap, mix, and overlay with 1 ml of pentane to prevent the enzyme product from evaporating. 11) Incubate at 30 ℃ for 3 hours. 12) Add 1ml of pentane, mix well, room temperature, 3,000
Centrifuge at rpm for 10 minutes. 13) Place Kiesel gel 60F thin-layer chromatography plate in a 1 ml plastic syringe with a cotton stopper inside the outlet in advance.
254 (Merck) and filled with silica gel powder and anhydrous magnesium sulfate. Add pentane to the syringe and wash by centrifugation. Repeat this operation again. Add 500 μl of the centrifuged pentane layer to the syringe and insert the syringe into a 10 ml centrifuge tube. Add 300 μl pentane to the syringe, room temperature, 3,000
Centrifuge at rpm for 10 minutes. 14) The solution recovered in the centrifuge tube is subjected to a liquid scintillation counter to measure radioactivity.

2-3 Limonene by Genetically Modified Escherichia coli
The production of limonene using a genetically modified Escherichia coli into which a limonene synthesis gene was introduced was analyzed by the following procedure. 1) Recombinant A, C, E and G strains containing limonene-producing plasmids stored in a glycerol solution at -85 ° C are LB agar plates containing 100 μg / ml ampicillin and tetracycline. Was streaked with a platinum loop, inoculated, and cultured at 37 ° C. overnight. 2) Pick a single colony with a loop and inoculate it in 10 ml of LB broth containing ampicillin and tetracycline.
Cultured with shaking at ℃ overnight. 3) 500 μl of the culture solution was transferred to a fresh LB medium containing 50 ml, and cultured with shaking at 30 ° C. until the OD600 nm reached 0.2. 4) 150 μl of 100 mM IPTG was added, followed by shaking culture at 30 ° C. for 3 hours. 5) Transfer the culture into a 50 ml centrifuge tube, and 5,000 rp at 4 ° C.
The cells were collected by centrifugation at m for 5 minutes. 6) The medium was discarded, and the cells were stored frozen at -85 ° C until the next operation. 7) 2 ml of TE buffer was added to suspend the cells. 8) 2 ml of 10 mg / ml lysozyme was added and allowed to stand on ice for 10 minutes. 9) 0.4 g of NaCl was added and mixed well with a vortex mixer. 10) 2 ml of dichloromethane was added and mixed well with a vortex mixer for 2 minutes. 11) Centrifuged at room temperature at 3,000 rpm for 5 minutes. 12) Transfer the lower layer to a 2 ml microtube, add 0.1 g of anhydrous sodium sulfate, mix well with a vortex mixer, and then leave for 10 minutes. 13) Centrifuge gently, transfer to vial bin, cap, and wrap with parafilm. 14) Samples transferred to Bayerbin were stored at 4 ° C until subjected to gas chromatographic analysis.

2-4 Gas Chromatographic Analysis of Limonene The analysis machine and the analysis conditions were as follows. Machine used Shimadzu Gas Chromatograph GC-17AAFw ver.3 Column DB-WAX (0.53 mm x 30 m 1.0 μm) Detector w-FID analysis Split method Sample injection volume 1.0 μl Temperature Column temperature 50 ℃ (0 min) -10 ℃ / min−220 (0 min) Inlet temperature 200 ℃ Detector temperature 250 ℃

3 Results 1) As a result of measurement of limonene synthase activity using a crude enzyme solution from limonene-producing genetically modified E. coli, all E. coli strains (A, C , E, G, I) showed limonene synthase activity. However, the activity was not shown in the Escherichia coli J strain into which the limonene synthesis gene had not been introduced. 2) The limonene synthase activity was higher in E. coli E and G strains transfected with the limonene synthase gene and the mutant FPP synthesis gene, followed by E. coli strains A and I transfected with the limonene synthase gene and the pTV118N vector. Next, Escherichia coli A and C strains in which the mutant gene of the FPP synthetic gene was introduced were high (FIG. 2). 3) As a result of gas chromatography analysis, limonene was detected in all limonene-producing Escherichia coli strains into which the limonene synthesis gene was introduced (FIG. 3). Therefore, this method established a method for producing limonene using genetically modified Escherichia coli for the first time. 4) The production of limonene is highest in the C strain, followed by A, E and
The output was higher in the order of G (FIG. 3).

[0044]

According to the present invention, a transformed microorganism producing monoterpene is provided for the first time. By using the microorganism of the present invention, various industrially useful monoterpenes can be industrially mass-produced. In addition, the transformed microorganism of the present invention produces monoterpene synthase.
Can also produce monoterpenes.

[0045]

[Sequence List] SEQUENCE LISTING <110> Sozoteki Seibutsu Kogaku Kenkyujo Co., Ltd. <120> Method for Producing Monoterpene and Microorganism Producing the Same <130> 99587 <160> 8

<210> 1 <211> 894 <212> DNA <213> Bacillus stearothermophilus <400> 1 atg gcg cag ctt tca gtt gaa cag ttt ctc aac gag caa aaa cag gcg 48 Met Ala Gln Leu Ser Val Glu Gln Phe Leu Asn Glu Gln Lys Gln Ala 1 5 10 15 gtg gaa aca gcg ctc tcc cgt tat ata gag cgc tta gaa ggg ccg gcg 96 Val Glu Thr Ala Leu Ser Arg Tyr Ile Glu Arg Leu Glu Gly Pro Ala 20 25 30 aag ctg aaa aag gcg atg gcg tac tca ttg gag gcc ggc ggc aaa cga 144 Lys Leu Lys Lys Ala Met Ala Tyr Ser Leu Glu Ala Gly Gly Lys Arg 35 40 45 atc cgt ccg ttg ctg ctt ctg tcc acc gtt cgg ggc ctc Ile Arg Pro Leu Leu Leu Leu Ser Thr Val Arg Ala Leu Gly Lys Asp 50 55 60 ccg gcg gtc gga ttg ccc gtc gcc tgc gcg att gaa atg atc cat acg 240 Pro Ala Val Gly Leu Pro Val Ala Cys Ala Ile Glu Met Ile His Thr 65 70 75 80 tac tct ttg atc cat gat gat ttg ccg agc atg gac aac gat gat ttg 288 Tyr Ser Leu Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Leu 85 90 95 cgg cgc ggc aag ccg acg aac cat aaa gtg ttc ggc gag gcg atg gcc 336 Arg Arg Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Ala Met Ala 100 105 110 atc ttg gcg ggg gac ggg ttg ttg acg tac gcg ttt caa ttg atc acc 384 Ile Leu Ala Gly Asp Gly Leu Leu Thr Tyr Ala Pln Leu Ile Thr 115 120 125 gaa atc gac gat gag cgc atc cct cct tcc gtc cgg ctt cgg ctc atc 432 Glu Ile Asp Asp Glu Arg Ile Pro Pro Ser Val Arg Leu Arg Leu Ile 130 135 140 gaa cgg ctg gcg aaa gcg gccgt ccg gaa ggg atg gtc gcc ggt cag 480 Glu Arg Leu Ala Lys Ala Ala Gly Pro Glu Gly Met Val Ala Gly Gln 145 150 155 160 gca gcc gat atg gaa gga gag ggg aaa acg ctg acg ctt tcg gag ctc 528 Ala Glu Gly Glu Gly Lys Thr Leu Thr Leu Ser Glu Leu 165 170 175 gaa tac att cat cgg cat aaa acc ggg aaa atg ctg caa tac agc gtg 576 Glu Tyr Ile His Arg His Lys Thr Gly Lys Met Leu Gln Tyr Ser Val 180 185 190 cac gcc ggc gcc ttg atc ggc ggc gct gat gcc cgg caa acg cgg gag 624 His Ala Gly Ala Leu Ile Gly Gly Ala Asp Ala Arg Gln Thr Arg Glu 195 200 205 ctt gac gaa ttc gcc gcc cat cta ttc ctt gcc att cgc gat 672 Leu Asp Glu Phe Ala Ala His Leu Gly Leu Ala Phe Gln Ile Arg Asp 210 215 220 gat att ctc gat att gaa ggg gca gaa gaa aaa atc ggc aag ccg gtc 720 Asp Ile Leu Asp Ile Glu Glu Lys Ile Gly Lys Pro Val 225 230 235 240 ggc agc gac caa agc aac aac aaa gcg acg tat cca gcg ttg ctg tcg 768 Gly Ser Asp Gln Ser Asn Asn Lys Ala Thr Tyr Pro Ala Leu Leu Ser 245 250 255 ctt gcc ggc gc aag gaa aag ttg gcg ttc cat atc gag gcg gcg cag 816 Leu Ala Gly Ala Lys Glu Lys Leu Ala Phe His Ile Glu Ala Ala Gln 260 265 270 cgc cat tta cgg aac gcc gac gtt gac ggc gcc gcg ccc ccc ccc His Leu Arg Asn Ala Asp Val Asp Gly Ala Ala Leu Ala Tyr Ile 275 280 285 tgc gaa ctg gtc gcc gcc cgc gac cat taa 894 Cys Glu Leu Val Ala Ala Arg Asp His 290 295

<210> 2 <211> 1800 <212> DNA <213> Mentha spicata <400> 2 atg gct ctc aaa gtg tta agt gtt gca act caa atg gcg att cct agc 48 Met Ala Leu Lys Val Leu Ser Val Ala Thr Gln Met Ala Ile Pro Ser 1 5 10 15 aag cta acg aga tgt ctt cca ccc tca cac ttg aaa tct tct cca aaa 96 Lys Leu Thr Arg Cys Leu Pro Pro Ser His Leu Lys Ser Ser Pro Lys 20 25 30 ttg tta tct agc act aac agt agt agt cgg tct cgc ctc cgt gtg tat 144 Leu Leu Ser Ser Thr Asn Ser Ser Ser Arg Ser Arg Leu Arg Val Tyr 35 40 45 tgc tcc tcc tcg caa ctc act act gag aga cga tcc gga aac tac aac 192 Cys Ser Ser Ser Gln Leu Thr Thr Glu Arg Arg Ser Gly Asn Tyr Asn 50 55 60 cct tct cgt tgg gat gtc gaa ttc atc caa tcc ctc cac agt gat tat 240 Pro Ser Arg Trp Asp Val Glu Phe Ile Gln Ser Leu His Ser Asp Tyr 65 70 75 80 gag gag gac aaa cac gcg att agg gct tct gag ctg gtc act ttg gtg 288 Glu Glu Asp Lys His Ala Ile Arg Ala Ser Glu Leu Val Thr Leu Val 85 90 95 aag atg gaa ttg gag aaa gaa acg gat cat att cga caa ctt gag ttg 336 Lys Me t Glu Leu Glu Lys Glu Thr Asp His Ile Arg Gln Leu Glu Leu 100 105 110 atc gat gac tta cag agg atg ggg ctg tcc gat cat ttc cag aat gag 384 Ile Asp Asp Leu Gln Arg Met Gly Leu Ser Asp His Phe Gln Asn Glu 115 120 125 ttc aaa gaa atc ttg tcc tct ata tat ctc gac cat cac tat tac aag 432 Phe Lys Glu Ile Leu Ser Ser Ile Tyr Leu Asp His His Tyr Tyr Lys 130 135 140 aac cct ttt cca aaa gaa gaa agg gat ctc tac tcc aca tct ctt gca 480 Asn Pro Phe Pro Lys Glu Glu Arg Asp Leu Tyr Ser Thr Ser Leu Ala 145 150 155 160 ttt agg ctc ctc aga gaa cat ggt ttt caa gtc gca caa gag gta ttc 528 Phe Arg Leu Leu Arg Glu His Gly Phe Gln Val Ala Gln Glu Val Phe 165 170 175 gac agt ttc aag aac gag gag ggt gag ttc aaa gaa agc ctt agc gac 576 Asp Ser Phe Lys Asn Glu Glu Gly Glu Phe Lys Glu Ser Leu Ser Asp 180 185 190 gac act aaa gga ttg ttg caa ctg tat gaa gct tcc ttt ctg ttg acg 624 Asp Thr Lys Gly Leu Leu Gln Leu Tyr Glu Ala Ser Phe Leu Leu Thr 195 200 205 gaa ggc gaa acc acg ctc gag tca gcg agg cca tt t 672 Glu Gly Glu Thr Thr Leu Glu Ser Ala Arg Glu Phe Ala Thr Lys Phe 210 215 220 ttg gag gaa aga gtg aac gag ggt ggt gtt gat ggc gac ctt tta aca 720 Leu Glu Glu Arg Val Asn Glu Gly Gly Val Asp Gly Asp Leu Leu Thr 225 230 235 240 aga atc gca tat tct ttg gac atc cca ctt cat tgg agg gtt aaa agg 768 Arg Ile Ala Tyr Ser Leu Asp Ile Pro Leu His Trp Arg Val Lys Arg 245 250 255 cca aat gca cct gcg tgg atc gaa tgg tat agg aag agg ccc gac atg 816 Pro Asn Ala Pro Ala Trp Ile Glu Trp Tyr Arg Lys Arg Pro Asp Met 260 265 270 aat cca gta gtg ttg gag ctt gcc ata ctc gac tta aat att gtt caa 864 Asn Val Val Leu Glu Leu Ala Ile Leu Asp Leu Asn Ile Val Gln 275 280 285 gca caa ttt caa gaa gag ctc aaa gaa tcc ttc agg tgg tgg aga aat 912 Ala Gln Phe Gln Glu Glu Leu Lys Glu Ser Phe Arg Trp Trp Arg 295 300 act ggt ttt gtt gag aag ctg ccc ttc gca agg gat aga ttg gtg gaa 960 Thr Gly Phe Val Glu Lys Leu Pro Phe Ala Arg Asp Arg Leu Val Glu 305 310 315 320 tgc tac ttt tgg aat act ggg atc atc gagca c gt cag cat gca agt 1008 Cys Tyr Phe Trp Asn Thr Gly Ile Ile Glu Pro Arg Gln His Ala Ser 325 330 335 gca agg ata atg atg ggc aaa gtc aac gct ctg att acg gtg atc gat 1056 Ala Arg Ile Met Met Gly Lys Val Asn Ala Leu Ile Thr Val Ile Asp 340 345 350 gat att tat gat gtc tac ggc acc tta gaa gaa ctc gaa caa ttc aca 1104 Asp Ile Tyr Asp Val Tyr Gly Thr Leu Glu Glu Leu Glu Gln Phe Thr 355c 360 at gaact cgg aga tgg gat ata gac tca atc gac caa ctt ccc gat 1152 Glu Leu Ile Arg Arg Trp Asp Ile Asp Ser Ile Asp Gln Leu Pro Asp 370 375 380 tac atg caa ctg tgc ttt ctt gca ctc aac aac ttc gtc gat Tyr Met Gln Leu Cys Phe Leu Ala Leu Asn Asn Phe Val Asp Asp Thr 385 390 395 400 tcg tac gat gtt atg aag gag aaa ggc gtc aac gtt ata ccc tac ctg 1248 Ser Tyr Asp Val Met Lys Glu Lys Gly Val Asn Val Ile Pro Tyr Leu 405 410 415 cgg caa tcg tgg gtg gat ttg gcg gat aag tat atg gta gag gca cgg 1296 Arg Gln Ser Trp Val Asp Leu Ala Asp Lys Tyr Met Val Glu Ala Arg 420 425 430 tgg ttc tac ggc gga ca a cca agt ttg gaa gag tat ttg gag aac 1344 Trp Phe Tyr Gly Gly His Lys Pro Ser Leu Glu Glu Tyr Leu Glu Asn 435 440 445 tca tgg cag tcg ata agt ggg ccc tgt atg tta acg cac ata ttc ttc1392 Ser Ser Ile Ser Gly Pro Cys Met Leu Thr His Ile Phe Phe 450 455 460 cga gta aca gat tcg ttc aca aag gag acc gtc gac agt ttg tac aaa 1440 Arg Val Thr Asp Ser Phe Thr Lys Glu Thr Val Asp Ser Leu Tyr Lys 465 470 475 480 tac cac gat tta gtt cgc tgg tca tcc ttc gtt ctg cgg ctt gct gac 1488 Tyr His Asp Leu Val Arg Trp Ser Ser Phe Val Leu Arg Leu Ala Asp 485 490 495 495 gat ctg gga acc tcg gtg gaga gag gt ggc gat gtg ccg aaa 1536 Asp Leu Gly Thr Ser Val Glu Glu Val Ser Arg Gly Asp Val Pro Lys 500 505 510 tca ctt cag tgc tac atg agt gac tac gat gca tcg gag gcg gag gcg 1584 Ser Leu Gln Cys Tyr Met Ser Asp Tyr Asp Ala Ser Glu Ala Glu Ala 515 520 525 cgg aag cac gtg aaa tgg ctg ata gcg gag gtg tgg aag aag atg aat 1632 Arg Lys His Val Lys Trp Leu Ile Ala Glu Val Trp Lys Lys Met Asn 530 535 gg g agg gtg tcg aag gat tct cca ttt ggc aaa gat ttt ata gga 1680 Ala Glu Arg Val Ser Lys Asp Ser Pro Phe Gly Lys Asp Phe Ile Gly 545 550 555 560 tgt gca gtt gat tta gga agg atg gcg cag ttg atg tac gga 1728 Cys Ala Val Asp Leu Gly Arg Met Ala Gln Leu Met Tyr His Asn Gly 565 570 575 gat ggg cac ggc aca caa cat cct ata ata cat caa caa atg acc aga 1776 Asp Gly His Gly Thr Gln His Pro Ile Ile His Gln Gln Met Thr Arg 580 585 590 acc tta ttc gag ccc ttt gca tga 1800 Thr Leu Phe Glu Pro Phe Ala 595

<210> 3 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> linker primer used for preparing cDNA of Mentha spicata <400> 3 gagagagaga gagagagaga actagtctcg agtttttttt tttttttttt 50

<210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer used for amplifying by PCR limonene synthase cDNA from Men tha spicata <400> 4 atggctctca aagtgttaag tg 22

<210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer used for amplifying by PCR limonene synthase cDNA from Men tha spicata <400> 5 tcatgcaaag ggctcgaata aggttc 26

<210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer used for introducing mutation to farnesyl diphosphate synt hase by site-specific mutagenesis <400> 6 catacgtact tcttgattca tgatgatttg 30

<210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer used for amplifying farnesyl diphosphate synthase gene from Bacillus stearothermophilus <400> 7 gaggaggagt aagccatggc gcagctttca 30

<210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer used for amplifying farnesyl diphosphate synthase gene from Bacillus stearothermophilus <400> 8 cgaccattaa aagcttaacg cccgcccttg 30

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing a method for constructing a recombinant vector containing a farnesyl diphosphate synthase gene having geranyl diphosphate synthase activity in Examples of the present invention.

FIG. 2 is a diagram showing limonene synthase activity of various transformed microorganisms prepared in Examples of the present invention.

FIG. 3 is a diagram showing limonene production amounts of various transformed microorganisms prepared in Examples of the present invention.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Shinichi Onuma 2- 1-6, Higashicho, Senoumachi, Fukushima City, Fukushima Prefecture (72) Inventor Tokuzo Nishino 2F-15 Minamiyoshinari 2-chome, Aoba-ku, Sendai City, Miyagi Prefecture ( Reference) 4B024 AA01 AA07 BA10 BA80 CA04 CA06 DA06 EA04 GA11 HA01 4B064 AB01 AG01 CA02 CA19 CC24 DA01 DA12 4B065 AA18Y AA26X AA88Y AB01 AC14 BA02 CA03 CA29 CA44 CA47

Claims (11)

[Claims] 1. A transformed microorganism having a gene encoding a polypeptide having geranyl diphosphate synthase activity and a monoterpene synthase gene, and producing a monoterpene synthesized by the monoterpene synthase. 2. The gene encoding the polypeptide having geranyl diphosphate synthase activity and the monoterpene synthase gene are present in different recombinant vectors, and the transformed microorganism is characterized by those two types of microorganisms. The microorganism according to claim 1, which has been co-transformed with a recombinant vector. 3. The microorganism according to claim 1, wherein the microorganism is Escherichia coli. 4. The microorganism according to claim 1, wherein the gene encoding the enzyme having geranyl diphosphate synthase activity is a farnesyl diphosphate synthase gene. 5. The farnesyl diphosphate synthase gene according to claim 1, wherein the amino acid sequence shown in SEQ ID NO: 1 or one or more amino acids in the amino acid sequence is substituted or deleted, or The microorganism according to claim 4, wherein the microorganism encodes an amino acid sequence having one or more amino acids inserted or added and having geranyl diphosphate synthase activity. 6. The microorganism according to claim 5, wherein the farnesyl diphosphate synthase gene encodes a polypeptide having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 7. The farnesyl diphosphate synthase gene has the amino acid sequence of SEQ ID NO: 1 in the sequence listing.
The microorganism according to claim 5, which encodes a polypeptide in which serine at position 2 is substituted with phenylalanine. 8. The microorganism according to claim 1, wherein the monoterpene synthase gene is a limonene synthase gene. 9. The limonene synthase gene has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing or one or more amino acids in the amino acid sequence substituted or deleted, or one or more amino acids in the amino acid sequence. 9. The microorganism according to claim 8, wherein the microorganism encodes an amino acid sequence having the amino acid inserted or added having the limonene synthase activity. 10. The microorganism according to claim 9, wherein the limonene synthase gene encodes the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 11. A method for producing a monoterpene, comprising culturing the microorganism according to claim 1 and collecting the produced monoterpene.