# Patent

## Title
Recombinant cell, and method for producing β-phellandrene

## Abstract

To provide a series of techniques for obtaining β-phellandrene with high purity and in a large quantity.
Provided is a recombinant cell capable of producing β-phellandrene, prepared by introducing at least one nucleic acid selected from the group consisting of a nucleic acid encoding geranyl pyrophosphate (GPP) synthase and a nucleic acid encoding neryl pyrophosphate (NPP) synthase, and a nucleic acid encoding β-phellandrene synthase into a host cell in such a manner that these nucleic acids are expressed in the host cell. Also provided is a method for producing β-phellandrene by culturing the recombinant cell to produce β-phellandrene in the recombinant cell.

## Background
TECHNICAL FIELD
The present invention relates to a recombinant cell capable of producing β-phellandrene, and a method for producing β-phellandrene using the recombinant cell.
BACKGROUND ART
Monoterpene is a generic name for compounds having 10 carbons according to the isoprene rule, biologically generated from geranyl pyrophosphate (GPP) in which dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) are condensed, as a precursor. Over 900 kinds of monoterpene are known at present.
Monoterpene has fragrances of roses or citrus fruits, and are commonly used in perfume and so on. For example, limonene is an aroma constituent contained in citrus fruits such as lemon, and is also used as a solvent or a material for an adhesive. Menthol has refreshing fragrance, and is used as a refrigerant in confectionary and pharmaceuticals. On the other hand, in the resin industry, β-pinene, α-pinene, limonene, α-phellandrene and the like are considered as monomer materials for adhesives and transparent resins (Non-Patent Document 1).
For β-phellandrene which is one kind of monoterpene, application as a new polymer material is expected. β-phellandrene can possibly provide a polymer of higher molecular weight than α-phellandrene. However, when β-phellandrene is obtained by a synthetic chemical technique, generation of α-phellandrene which is an isomer thereof is inevitable, and separation of these isomers is very difficult (Non-Patent Document 2). Therefore, it is difficult to obtain β-phellandrene with high purity, and this makes it difficult to examine the polymer properties and the like of β-phellandrene.
On the other hand, as the biosynthesis pathway of β-phellandrene, geranyl pyrophosphate (GPP) or neryl pyrophosphate (NPP) is biosynthesized from isopentenyl diphosphate (IPP) under the action of geranyl pyrophosphate (GPP) synthase or neryl pyrophosphate (NPP) synthase. Sequentially, under the action of β-phellandrene synthase, β-phellandrene is biosynthesized from GPP or NPP. In tomato and lavender, β-phellandrene synthase is found (Non-Patent Documents 3 and 4).
Patent Document 1 discloses a method for producing monoterpene using a transformant of C1 metabolism host cell into which a nucleic acid encoding cyclic terpene synthase is introduced. As an example thereof, an experiment example of producing limonene using a transformant of Methylomonas bacterium is described. Although β-phellandrene is referred, an example thereof is not given. Although β-phellandrene synthase is referred, a concrete example and an acquiring method of β-phellandrene synthase and its gene, and a concrete configuration and a construction method of a transformant capable of producing β-phellandrene are not shown.
PRIOR ART DOCUMENTS
Patent Document


    Patent Document 1: JP 2005-500805 A


Non-Patent Documents


    Non-Patent Document 1: Satou K., et al., Green Chemistry 2006, 8, 878-882
    Non-Patent Document 2: Mori K., Tetrahedron: Asymmetry 2006, 17, 2133-2142
    Non-Patent Document 3: Demissie, Z. A., et al., Planta. 2011, 233, 685-96.
    Non-Patent Document 4: Schilmiller, A. L., et al., Proc Natl Acad Sci USA., 2009, 106, 10865-70.


DISCLOSURE OF INVENTION
Technical Problem
In light of the above, an object of the present invention is to provide a series of techniques for obtaining β-phellandrene with high purity and in a large quantity.
Solutions to Problem
One aspect of the present invention for solving the aforementioned problem is a recombinant cell capable of producing β-phellandrene prepared by introducing at least one nucleic acid selected from the group consisting of a nucleic acid encoding geranyl pyrophosphate synthase and a nucleic acid encoding neryl pyrophosphate synthase, and a nucleic acid encoding β-phellandrene synthase into a host cell in such a manner that these nucleic acids are expressed in the host cell.
The present invention relates to a recombinant cell capable of producing β-phellandrene. The recombinant cell of the present invention is prepared by introducing “at least one nucleic acid selected from the group consisting of a nucleic acid encoding geranyl pyrophosphate (GPP) synthase and a nucleic acid encoding neryl pyrophosphate (NPP) synthase”, and “a nucleic acid encoding β-phellandrene synthase” into a host cell, and these nucleic acids are expressed in the host cell. In other words, in the recombinant cell of the present invention, the ability to express GPP synthase and/or NPP synthase and the ability to express β-phellandrene synthase are newly added or enhanced with respect to the host cell.
In the recombinant cell of the present invention, GPP is synthesized from isopentenyl diphosphate (IPP) under the action of GPP synthase expressed in the cell, and/or NPP is synthesized from IPP under the action of NPP synthase expressed in the cell. Further, β-phellandrene is synthesized from GPP and/or NPP under the action of β-phellandrene synthase expressed in the cell. Therefore, by culturing the recombinant cell of the present invention, it is possible to produce β-phellandrene with high purity and in large quantity.
Preferably, the host cell does not have methane monooxygenase.
Preferably, the host cell is Escherichia coli or yeast.
With this configuration, it is possible to easily culture the recombinant cell in large scale.
Preferably, 10 mg or more of β-phellandrene can be produced per 1 g of wet cells of the recombinant cell
Preferably, the nucleic acid encoding geranyl pyrophosphate synthase encodes the following protein (a), (b) or (c):
(a) protein consisting of an amino acid sequence represented by SEQ ID NO: 2,
(b) protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having geranyl pyrophosphate synthase activity, or
(c) protein consisting of an amino acid sequence having a homology of 60% or more with the amino acid sequence represented by SEQ ID NO: 2, and having geranyl pyrophosphate synthase activity.
Preferably, the nucleic acid encoding neryl pyrophosphate synthase encodes the following protein (d), (e) or (f):
(d) protein consisting of an amino acid sequence represented by SEQ ID NO: 4,
(e) protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having neryl pyrophosphate synthase activity, or
(f) protein consisting of an amino acid sequence having a homology of 60% or more with the amino acid sequence represented by SEQ ID NO: 4, and having neryl pyrophosphate synthase activity.
Preferably, the nucleic acid encoding β-phellandrene synthase encodes the following protein (g), (h) or (i):
(g) protein consisting of an amino acid sequence represented by SEQ ID NO: 6 or 8,
(h) protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 6 or 8, and having β-phellandrene synthase activity, or
(i) protein consisting of an amino acid sequence having a homology of 60% or more with the amino acid sequence represented by SEQ ID NO: 6 or 8, and having β-phellandrene synthase activity.
Preferably, a nucleic acid encoding at least one enzyme that acts in a synthesis pathway of isopentenyl diphosphate is further introduced, and the nucleic acid is expressed in the host cell.
With such a configuration, IPP which is to be a substrate for GPP synthase or NPP synthase is supplied efficiently.
Preferably, the synthesis pathway of isopentenyl diphosphate is a mevalonate pathway.
Preferably, the mevalonate pathway is a mevalonate pathway of yeast or actinomyces. 
Another aspect of the present invention is a method for producing β-phellandrene by culturing the recombinant cell to produce β-phellandrene in the recombinant cell.
The present invention relates to a method for producing β-phellandrene. In the present invention, by culturing the recombinant cell, β-phellandrene is produced in the recombinant cell. According to the present invention, it is possible to produce β-phellandrene with high purity and in large quantity.
Preferably, 10 mg or more of β-phellandrene is produced per 1 g of wet cells of the recombinant cell.
Preferably, β-phellandrene released outside the recombinant cell is collected.
Preferably, β-phellandrene is collected from a gas phase of a culture system of the recombinant cell.
Advantageous Effect of Invention
According to the present invention, it is possible to produce β-phellandrene with high purity and in large quantity.



BRIEF DESCRIPTION OF DRAWINGS
FIGS. 1A and 1B are total ion chromatograms of GC-MS analysis conducted for the gas phase fraction of the culture conducted in Example 5, wherein FIG. 1A shows the case of a control recombinant, and FIG. 1B shows the case of a recombinant capable of producing β-phellandrene.
FIGS. 2A-2C are mass spectrums showing the results of peaks in FIG. 1B identified by GC-MS, wherein FIG. 2A shows the case of peak A (β-phellandrene), FIG. 2B shows the case of peak B (limonene), and FIG. 2C shows the case of peak C (myrcene).



DESCRIPTION OF EMBODIMENT
The recombinant cell of the present invention is prepared by introducing at least one nucleic acid selected from the group consisting of a nucleic acid encoding geranyl pyrophosphate (GPP) synthase and a nucleic acid encoding neryl pyrophosphate (NPP) synthase, and a nucleic acid encoding β-phellandrene synthase into a host cell in such a manner that these nucleic acids are expressed in the host cell, and the recombinant cell is capable of synthesizing β-phellandrene.
GPP synthase is not particularly limited as far as it can exert its enzyme activity in the recombinant cell. The same applies also to the nucleic acid (gene) encoding GPP synthase, and the nucleic acid is not particularly limited as far as it is normally transcribed and translated in the recombinant cell.
The same applies to NPP synthase, β-phellandrene synthase, and nucleic acids encoding these enzymes.
Concrete examples of GPP synthase include those derived from Arabidopsis thaliana (GenBank Accession No.: Y17376/At2g34630; Bouvier, F., et al., Plant J., 2000, 24, 241-52.), and those derived from Mycobacterium tuberculosis (GenBank Accession No.: NP_215504; Mann, F. M., et al., FEBS Lett., 2011, 585, 549-54.).
SEQ ID NO: 1 represents a nucleotide sequence of nucleic acid (DNA) encoding GPP synthase derived from Arabidopsis thaliana, and an amino acid sequence corresponding to this, and SEQ ID NO: 2 represents only the amino acid sequence. DNA having the nucleotide sequence represented by SEQ ID NO: 1 is one example of the nucleic acid encoding GPP synthase.
Further, the nucleic acid encoding GPP synthase includes at least a nucleic acid encoding the following protein (a), (b) or (c):
(a) protein consisting of an amino acid sequence represented by SEQ ID NO: 2,
(b) protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having geranyl pyrophosphate synthase activity, or
(c) protein consisting of an amino acid sequence having a homology of 60% or more with the amino acid sequence represented by SEQ ID NO: 2, and having geranyl pyrophosphate synthase activity.
The homology of amino acid sequence in (c) is more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more.
Concrete examples of NPP synthase include those derived from tomato (Solanum lycopersicum) (GenBank Accession No.: FJ797956) and so on.
SEQ ID NO: 3 represents a nucleotide sequence of nucleic acid (DNA) encoding the NPP synthase derived from tomato and an amino acid sequence corresponding to this, and SEQ ID NO: 4 represents only the amino acid sequence. DNA having the nucleotide sequence represented by SEQ ID NO: 3 is one example of the nucleic acid encoding NPP synthase.
Further, the nucleic acid encoding NPP synthase includes at least a nucleic acid encoding the following protein (d), (e) or (f):
(d) protein consisting of an amino acid sequence represented by SEQ ID NO: 4,
(e) protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having neryl pyrophosphate synthase activity, or
(f) protein consisting of an amino acid sequence having a homology of 60% or more with the amino acid sequence represented by SEQ ID NO: 4, and having neryl pyrophosphate synthase activity.
The homology of amino acid sequence in (f) is more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more.
In the recombinant cell of the present invention, either one nucleic acid or both nucleic acids of “nucleic acid encoding geranyl pyrophosphate (GPP) synthase” and “nucleic acid encoding neryl pyrophosphate (NPP) synthase” may be introduced.
Concrete examples of β-phellandrene synthase and a nucleic acid encoding the same include those derived from tomato (Solanum lycopersicum) (GenBank Accession No.: FJ797957; Schilmiller, A. L., et al., Proc Natl Acad Sci USA., 2009, 106, 10865-70.), and those derived from lavender (Lavandula angustifolia) (GenBank Accession No.: HQ404305; Demissie, Z. A., et al., Planta, 2011, 233, 685-96).
SEQ ID NO: 5 represents a nucleotide sequence of a nucleic acid (DNA) encoding the β-phellandrene synthase derived from tomato, and a corresponding amino acid sequence, and SEQ ID NO: 6 represents only the amino acid sequence.
SEQ ID NO: 7 represents a nucleotide sequence of a nucleic acid (DNA) encoding the β-phellandrene synthase derived from lavender, and a corresponding amino acid sequence, and SEQ ID NO: 8 represents only the amino acid sequence.
DNA having the nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7 is one example of the nucleic acid encoding β-phellandrene synthase.
Further, the nucleic acid encoding β-phellandrene synthase includes at least a nucleic acid encoding the following protein (g), (h) or (i):
(g) protein consisting of an amino acid sequence represented by SEQ ID NO: 6 or 8,
(h) protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 6 or 8, and having β-phellandrene synthase activity, or
(i) protein consisting of an amino acid sequence having a homology of 60% or more with the amino acid sequence represented by SEQ ID NO: 6 or 8, and having β-phellandrene synthase activity.
The homology of amino acid sequence in (i) is more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more.
The host cell in the recombinant cell of the present invention is not particularly limited, and may be a procaryotic cell or a eucaryotic cell. Examples of a procaryotic cell include bacteria and actinomyces. Examples of bacteria include Escherichia bacteria such as Escherichia coli, Bacillus bacteria such as Bacillus subtilis, Pseudomonas bacteria, cyanobacteria, Clostridium bacteria, Corynebacterium bacteria, and Ralstonia bacteria. Among these, Escherichia coli which is easy to be cultured in large scale is particularly preferred.
Examples of a eucaryotic cell include yeast, filamentous fungus, eucaryotic microalgae, a vegetable cell, and an animal cell.
Examples of yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces uvarum, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Candida tropicalis, Candida maltosa, Candida parapsilosis, Pichia pastoris, Pichia farinosa, Pichia pinus, Pichiavarijii, Pichiafermentans, Pichia guilliermondii, Pichia stipitis, Saccharomyces elluris, Candida utilis, Candida guilliermondii, Hansenula henricii, Hansenula capsulata, Hansenula polymorpha, Hansenula saturnus, Lypomyces kononenkoae, Kluyveromyces marxianus, Candida lipolytica, Saccaromycopsis fibuligera, Saccharomycodes ludwigii, Saccharomyces kluyveri, Tremellamesenterica, Zygosaccharomyces acidofaciens, Zygosaccharomyces fermentati, Yarrowia lipolytica, and Zygosaccharomyces soja. Among these, yeast which is easy to be cultured in large scale is particularly preferably selected.
Preferably, the host cell does not have methane monooxygenase (EC 1.14.13.25).
The method for introducing the nucleic acids respectively encoding GPP synthase, NPP synthase, and β-phellandrene synthase into the host cell is not particularly limited, and can be appropriately selected depending on the kind of the host cell or the like. For example, a vector that can be introduced into the host cell and can allow expression of the nucleic acid incorporated therein may be used.
For example, when the host cell is a prokaryote such as a bacterium, a vector that can self duplicate or can be incorporated in chromosome in the host cell, and contains a promoter at the position allowing transcription of the inserted nucleic acid (DNA) can be used. For example, it is preferred to construct in the host cell a series of structures including a promoter, a ribosome binding sequence, the above nucleic acid (DNA) and a transcription termination sequence by using the vector.
As a vector that can be used when the host cell is E. coli, a so-called multi copy type vector is preferred, and plasmid having a replication origin derived from ColE1, for example, pUC-series plasmids, pBR322-series plasmids or derivatives thereof are recited. More concrete examples include pUC19 [Gene, 33, 103 (1985)], pUC18, pBR322, pHelix1 (available from Roche Diagnostics), pKK233-2 (available from Amersham-Pharmacia Biotech), pSE280 (available from Invitrogen), pGEMEX-1 (available from Promega), pQE-8 (available from QIAGEN), pET-3 (available from Novagen), pBluescriptII SK(+), pBluescript II KS(+) (available from Stratagene), pSTV28 (available from TAKARA BIO INC), and pUC118 (available from TAKARA BIO INC). The promoter on the vector may be any promoter as far as it can operate in the host cell such as E. coli. For example, promoters derived from E. coli or phage such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter or the like, and T7 promoter or the like, and promoters that are artificially designed and modified, such as tac promoter and lacT7 promoter may be used.
Basically the same applies to the case where the host cell is yeast, and the vector is preferably structured to have a replication system for stable conservation in the host yeast, a promoter capable of transcribing the inserted nucleic acid (DNA), and a terminator sequence. For example, the vector may be a plasmid that can be replicated in the host yeast or can be incorporated into the host chromosome. The vector can encode expression of repeated copies of a desired DNA sequence each separated by a selective cutting site.
While the kind of the promoter that can operate in yeast is not particularly limited, for example, promoter of isocitrate lyase gene, AOX1 promoter, GAPDH promoter, PHO5 promoter, glyceraldehyde-3-phosphate dehydrogenase (TDH3) promoter, ADHI promoter, MFα1 promoter, and GAL10 promoter can be used.
When the nucleic acids are introduced into a host cell by using a vector, the nucleic acids may be incorporated into one vector, or may be incorporated into different vectors. When a plurality of nucleic acids are incorporated into one vector, the nucleic acids may be expressed under a common promoter, or the nucleic acids may be expressed under respective promoters.
When the host cell has α-phellandrene synthase, it is preferred that α-phellandrene synthase of the host cell is knocked out in advance. However, since vegetables are only the organisms that have α-phellandrene synthase in general, this operation is not usually required when the host cell is a bacterium or yeast.
In the recombinant cell of the present invention, other nucleic acid may be introduced in addition to the nucleic acids respectively encoding GPP synthase, NPP synthase, and β-phellandrene synthase. In one embodiment, a nucleic acid encoding at least one enzyme that acts in the synthesis pathway of isopentenyl diphosphate (IPP) is further introduced, and the nucleic acid is expressed in the host cell. The introduced nucleic acid may be one kind or two or more kinds.
In general, the synthesis pathway of IPP is classified into a mevalonate pathway (MVA pathway) and a non-mevalonate pathway (MEP pathway).
The mevalonate pathway is possessed by a eukaryote, and starts with acetyl CoA. Enzymes acting in the mevalonate pathway include acetyl CoA acetyl transferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase in this order from the upstream side.
On the other hand, the non-mevalonate pathway is possessed by a prokaryote, a chloroplast, and a plastid, and starts with glyceraldehyde 3-phosphate (GAP) and pyruvic acid. Enzymes acting in the non-mevalonate pathway include DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase, and HMB-PP reductase in this order from the upstream side.
The “enzyme acting in the IPP synthesis pathway” encoded by the nucleic acid further introduced in the present embodiment is preferably an enzyme acting in the mevalonate pathway.
The enzyme group acting in the mevalonate pathway includes acetyl CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase. The nucleic acid to be introduced may be selected so that an enzyme group consisting of, for example, HMG-CoAsynthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase among these is expressed in the host cell.
While the mevalonate pathway is possessed by every eukaryote, it is also found in other organisms than eukaryotes. Examples of other organisms than eukaryotes having the mevalonate pathway include Streptomyces sp. Strain CL190 (Takagi M. et al., J. Bacteriol. 2000, 182(15), 4153-7), Streptomyces griseolosporeus MF730-N6 (Hamano Y. et al., Biosci. Biotechnol. Biochem. 2001, 65(7), 1627-35) in actinomyces. 
In bacteria, Lactobacillus helvecticus (Smeds A et al., DNA seq. 2001, 12(3), 187-190), Corynebacterium amycolatum, Mycobacterium marinum, Bacillus coagulans, Enterococcus faecalis, Streptococuss agalactiae, Myxococcus xanthus and the like are recited (Lombard J. et al., Mol. Biol. Evol. 2010, 28(1), 87-99).
In archaea, genus Aeropyrum, genus Sulfolobus, genus Desulfurococcus, genus Thermoproteus, genus Halobacterium, genus Methanococcus, genus Thermococcus, genus Pyrococcus, genus Methanopyrus, genus Thermoplasma and so on are recited (Lombard J. et al., Mol. Biol. Evol. 2010, 28(1), 87-99).
The source of the enzyme group acting in the mevalonate pathway is not particularly limited, however, the enzyme group acting in the mevalonate pathway of yeast or actinomyces is particularly preferably employed.
These enzymes may be naturally occurring enzymes or enzymes modified therefrom. For example, amino acid substitution variants of each enzyme, and polypeptides that are partial fragments of each enzyme and have equivalent enzyme activity are also applicable.
The method for culturing the recombinant cell of the present invention is not particularly limited, and may be appropriately conducted depending on the kind of the host cell and the like.
When the recombinant cell is aerobic or obligately anaerobic, for example, culturing in a liquid medium under aeration and stirring can be conducted.
When the recombinant cell is strictly anaerobic, for example, culturing can be conducted while the gas phase is replaced with gas such as high-purity or deoxidized nitrogen, and an appropriate amount of a reducing agent such as sodium sulfide, cysteine or the like is added to the culture liquid.
The culture medium is not particularly limited as far as it allows growth of the recombinant cell. As a principal carbon source in the culture medium, organic carbon sources such as saccharides and protein digests are preferably used. Examples of saccharides include monosaccharide (e.g. glucose), disaccharide (e.g. maltose), oligosaccharide, polysaccharide (e.g. starch), and sugar alcohol. Examples of protein digests include peptone, triptone and casamino acids.
By culturing the recombinant cell of the present invention, it is possible to produce a large quantity of β-phellandrene. As the producibility of β-phellandrene, a production amount of 10 mg or more of β-phellandrene per 1 g of wet cells can be achieved.
When the recombinant cell of the present invention is cultured to produce β-phellandrene, mixing of α-phellandrene does not substantially occur unlike the case of production by a synthetic chemical technique.
The produced β-phellandrene is released outside the recombinant cell, or accumulated in the cell. In the present invention, any of such β-phellandrene may be collected, and β-phellandrene can be collected concretely from crushed cells, the culture liquid (culture supernatant), the gas phase of the culture system or the like. Preferably, β-phellandrene released outside the cell is collected, and concretely, it is collected from the culture liquid (culture supernatant) or the gas phase of the culture system.
As a method for isolating and purifying β-phellandrene from the culture of the recombinant cell, for example, the culture liquid (culture supernatant) is extracted with an appropriate solvent such as pentane, and is further purified to high purity by chromatography such as reversed phase chromatography or gas chromatography. Since β-phellandrene released outside the cell evaporates also in the gas phase, this may be liquefied by a cold trap or the like to collect it.
In the following, the present invention will be described more specifically by way of examples. However, the present invention is not limited to these examples.
Example 1
In the present example, a recombinant E. coli into which NPP synthase gene from tomato and β-phellandrene synthase gene from tomato were introduced was prepared, and the recombinant E. coli was cultured to produce β-phellandrene.
(1) Isolation of NPP Synthase Gene Derived from Tomato
Using total RNA derived from tomato (Solanum lycopersicum) as a template, a nucleic acid encoding NPP synthase from tomato (NPP synthase gene from tomato, SEQ ID NO: 3, GenBank Accession No.: FJ797956) was amplified by RT-PCR using primers represented by SEQ ID NO: 9 and SEQ ID NO: 10. The obtained nucleic acid was cut with NdeI and EcoRI, and introduced into pET23a vector (available from Novagen). The nucleotide sequence of the nucleic acid introduced into the vector was examined and correctness was confirmed. In this manner, vector pT21TNPP to express NPP synthase from tomato was prepared.
(2) Isolation of β-Phellandrene Synthase Gene Derived from Tomato
Using total RNA derived from tomato as a template, a nucleic acid encoding β-phellandrene synthase from tomato (β-phellandrene synthase gene from tomato, SEQ ID NO: 5, GenBank Accession No.: FJ797957) was amplified by RT-PCR using primers represented by SEQ ID NO: 11 and SEQ ID NO: 12. The obtained nucleic acid was cut with NcoI and BamHI, and introduced into pACYCDuet-1 vector (available from Novagen). The nucleotide sequence of the nucleic acid introduced into the vector was examined and correctness was confirmed. In this manner, vector pACTPD to express β-phellandrene synthase from tomato was prepared.
(3) Preparation of Recombinant E. coli Capable of Producing β-Phellandrene
The expression vectors pT23TNPP and pACTPD obtained in the above (1) and (2) were introduced into E. coli BL21 (DE3) strain, to prepare a recombinant E. coli BL21BPD1 capable of producing β-phellandrene.
As controls, a recombinant E. coli having only pET23a vector into which a nucleic acid was not inserted, and a recombinant E. coli having only pACYCDuet-1 vector into which a nucleic acid was not inserted were separately prepared.
(4) Production of β-Phellandrene
The recombinant E. coli BL21BPD1 was cultured in a 2×YT medium (1.6% (w/v) BactoTripton, 1% (w/v) Yeast Extract, 0.5% (w/v) NaCl) containing 100 μg/mL of ampicillin and 34 μg/mL of chloramphenicol at 30° C., 110 rpm (swing) for 30 hours. At this time, the system was brought into a closed system after 16 hours from start of the culturing. After end of the culture, centrifugal separation was conducted to obtain cells and a culture supernatant.
The cells were crushed, and the supernatant of the crush liquid was extracted with pentane. The extracted fraction was analyzed by gas chromatography, and β-phellandrene was detected. The gas phase fraction was analyzed, and β-phellandrene was detected also in the gas phase.
Also the culture supernatant was extracted with pentane. This extracted fraction was analyzed by gas chromatography, and β-phellandrene was detected.
On the other hand, in the control recombinant E. coli into which only pET23a vector or pACYCDuet-1 vector was introduced, β-phellandrene was not detected in any of the cells, the culture supernatant, and the gas phase.
These reveal that β-phellandrene can be produced by culturing the recombinant E. coli into which NPP synthase gene from tomato and β-phellandrene synthase gene from tomato were introduced. The produced β-phellandrene was collectible from the cells, the culture supernatant, and the gas phase.
Example 2
In the present example, a recombinant E. coli into which GPP synthase gene from Arabidopsis thaliana and β-phellandrene synthase gene from lavender were introduced was prepared, and the recombinant E. coli was cultured to produce β-phellandrene.
(1) Isolation of GPP Synthase Gene Derived from Arabidopsis thaliana 
Using total RNA derived from Arabidopsis thaliana as a template, a nucleic acid encoding GPP synthase from Arabidopsis thaliana (GPP synthase gene from Arabidopsis thaliana, SEQ ID NO: 1, GenBank Accession No.: Y17376) was amplified by RT-PCR using primers represented by SEQ ID NO: 13 and SEQ ID NO: 14. The obtained nucleic acid was cut with NdeI and EcoRI, and introduced into pET23a vector (available from Novagen). The nucleotide sequence of the nucleic acid introduced into the vector was examined and correctness was confirmed. In this manner, vector pT23AGPP to express GPP synthase from Arabidopsis thaliana was prepared.
(2) Isolation of β-Phellandrene Synthase Gene Derived from Lavender
Using total RNA derived from lavender (Lavandula angustifolia) as a template, a nucleic acid encoding β-phellandrene synthase from lavender (β-phellandrene synthase gene from lavender, SEQ ID NO: 7, GenBank Accession No: HQ404305) was amplified by RT-PCR using primers represented by SEQ ID NO: 15 and SEQ ID NO: 16. The obtained nucleic acid was cut with NcoI and EcoRI, and introduced into pACYCDuet-1 vector (available from Novagen). The nucleotide sequence of the nucleic acid introduced into the vector was examined and correctness was confirmed. In this manner, vector pACLPD to express β-phellandrene synthase from lavender was prepared.
(3) Preparation of Recombinant E. coli Capable of Producing β-Phellandrene
The expression vectors pT23AGPP and pACLPD obtained in the above (1) and (2) were introduced into E. coli BL21 (DE3) strain, to prepare a recombinant E. coli BL21BPD2 capable of producing 3-phellandrene.
As controls, a recombinant E. coli having only pET23a vector into which a nucleic acid was not inserted, and a recombinant E. coli having only pACYCDuet-1 vector into which a nucleic acid was not inserted were separately prepared.
(4) Production of β-Phellandrene
Recombinant E. coli BL21BPD2 was cultured in a 2×YT medium containing 100 μg/mL of ampicillin and 34 μg/mL of chloramphenicol at 30° C., 110 rpm (swing) for 30 hours. At this time, the system was brought into a closed system after 16 hours from start of the culturing. After end of the culture, centrifugal separation was conducted to obtain cells and a culture supernatant.
The cells were crushed, and the supernatant of the crush liquid was extracted with pentane. The extracted fraction was analyzed by gas chromatography, and β-phellandrene was detected.
Also the culture supernatant was extracted with pentane. This extracted fraction was analyzed by gas chromatography, and β-phellandrene was detected. Also the gas phase fraction was analyzed, and β-phellandrene was detected in the gas phase.
On the other hand, in the control recombinant E. coli into which only pET23a vector or pACYCDuet-1 vector was introduced, β-phellandrene was not detected in any of the cells, the culture supernatant, and the gas phase.
These reveal that β-phellandrene can be produced by culturing the recombinant E. coli into which GPP synthase gene from Arabidopsis thaliana and β-phellandrene synthase gene from lavender were introduced. The produced β-phellandrene was collectible from the cells, the culture supernatant, and the gas phase.
Example 3
In this example, a recombinant yeast into which NPP synthase gene from tomato and β-phellandrene synthase gene from tomato were introduced was prepared, and the recombinant yeast was cultured to produce β-phellandrene.
(1) Isolation of NPP Synthase Gene Derived from Tomato
Using total RNA derived from tomato as a template, a nucleic acid encoding NPP synthase from tomato (NPP synthase gene from tomato, SEQ ID NO: 3, GenBank Accession No.: FJ797956) was amplified by RT-PCR using primers represented by SEQ ID NO: 13 and SEQ ID NO: 14. The obtained nucleic acid was cut with BamHI, and then introduced into pPIC3.5K vector (available from Invitrogen). The nucleotide sequence of the nucleic acid introduced into the vector was examined and correctness was confirmed. In this manner, vector pP3.5TNPP to express NPP synthase from tomato was prepared.
(2) Isolation of β-Phellandrene Synthase Gene Derived from Tomato
Using total RNA derived from tomato as a template, a nucleic acid encoding β-phellandrene synthase from tomato (β-phellandrene synthase gene from tomato, SEQ ID NO: 5, GenBank Accession No.: FJ797957) was amplified by RT-PCR using primers represented by SEQ ID NO: 19 and SEQ ID NO: 20. The obtained nucleic acid was cut with BamHI, and then introduced into pPIC3.5K vector (available from Invitrogen). The nucleotide sequence of the nucleic acid introduced into the vector was examined and correctness was confirmed. In this manner, vector pP3.5TPD to express β-phellandrene synthase from tomato was prepared.
(3) Preparation of Recombinant Yeast Capable of Producing β-Phellandrene
Methanol assimilating yeast Pichia pastoris GS115 strain (available from Invitrogen) was transformed with a mixture of equal parts of the expression vectors pP3.5TNPP and pP3.5TPD obtained in the above (1) and (2). The transformation was conducted according to the manual (No. 25-0156, No. 25-0043) provided from Invitrogen. To obtain multiple copies of transductant, Geneticin (available from Invitrogen) tolerant strain at a concentration of 1.5 mg/mL was obtained. In this manner, a recombinant yeast GSNP-1 having respective multiple copies of NPP synthase gene from tomato and β-phellandrene synthase gene from tomato was obtained.
As a control, Geneticin (available from Invitrogen) tolerant strain at a concentration of 1.5 mg/mL having only pPIC3.5K vector into which a nucleic acid was not inserted was obtained (recombinant yeast GS115).
(4) Production of β-Phellandrene
After pre-culturing GSNP-1 in a MGY medium (1.34% (w/v) YNB, 1% (w/v) glycerol, 4×10−5% (w/v) biotin; YMB: 13.4% (w/v) yeast nitrogen base, 10% (w/v)ammonium sulfate) (manual of Invitrogen No. 25-0043), cells were collected, and the collected cells were main-cultured in a Fermentation basal salts medium (85% Phosphoric acid 26.7 ml, Calcium sulfate 0.093% (w/v), Potassium sulfate 1.82% (w/v), Magnesiumsulfate-7H2O 1.49% (w/v), Potassium hydroxide 0.413% (w/v), Glycerol 4.0% (w/v)) (manual of Invitrogen Ver. B 053002) for 64 hours. At this time, the system was brought into a closed system after 24 hours from start of the culturing. For inducing expression of the introduced enzyme genes, 1/200-volume of 100% methanol was added to the culture liquid after lapses of 24 hours and 48 hours from starting of the culture. After end of the culture, the cells and a culture supernatant were collected by centrifugal separation.
The cells were crushed by a glass bead method (manual of Invitrogen No. 25-0043) and a supernatant was obtained. A pentane extract of the supernatant was analyzed by gas chromatography, and β-phellandrene was detected.
Also the culture supernatant was extracted with pentane. The extracted fraction was analyzed by gas chromatography, and β-phellandrene was detected. Also the gas phase fraction was analyzed, and β-phellandrene was detected in the gas phase.
On the other hand, in the control recombinant yeast GS115 into which only pPIC3.5K vector was introduced, β-phellandrene was not detected in any of the cells, the culture supernatant, and the gas phase.
These reveal that β-phellandrene can be produced by culturing the recombinant yeast into which NPP synthase gene from tomato and β-phellandrene synthase gene from tomato were introduced. The produced β-phellandrene was collectible from the cells, the culture supernatant, and the gas phase.
Example 4
In this example, a recombinant yeast into which GPP synthase gene from Arabidopsis thaliana and β-phellandrene synthase gene from lavender were introduced was prepared, and the recombinant yeast was cultured to produce β-phellandrene.
(1) Isolation of GPP Synthase Gene Derived from Arabidopsis thaliana 
Using total RNA derived from Arabidopsis thaliana as a template, a nucleic acid encoding GPP synthase from Arabidopsis thaliana (GPP synthase gene from Arabidopsis thaliana, SEQ ID NO: 1, GenBank Accession No.: Y17376) was amplified by RT-PCR using primers represented by SEQ ID NO: 21 and SEQ ID NO: 22. The obtained nucleic acid was cut with BamHI, and then introduced into pPIC3.5K vector (available from Invitrogen). The nucleotide sequence of the nucleic acid introduced into the vector was examined and correctness was confirmed. In this manner, vector pP3.5AGPP to express GPP synthase from Arabidopsis thaliana was prepared.
(2) Isolation of β-Phellandrene Synthase Gene Derived from Lavender
Using total RNA derived from lavender as a template, a nucleic acid encoding β-phellandrene synthase from lavender (β-phellandrene synthase gene from lavender, SEQ ID NO: 7, GenBank Accession No.: HQ404305) was amplified by RT-PCR using primers represented by SEQ ID NO: 23 and SEQ ID NO: 24. The obtained nucleic acid was cut with EcoRI, and then introduced into pPIC3.5K vector (available from Invitrogen). The nucleotide sequence of the nucleic acid introduced into the vector was examined and correctness was confirmed. In this manner, vector pP3.5LPD to express β-phellandrene synthase from lavender was prepared.
(3) Preparation of Recombinant Yeast Capable of Producing β-Phellandrene
Methanol assimilating yeast Pichia pastoris GS115 strain (available from Invitrogen) was transformed with a mixture of equal parts of the expression vectors pP3.5AGPP and pP3.5LPD obtained in the above (1) and (2). The transformation was conducted according to the manual (No. 25-0156, No. 25-0043) provided from Invitrogen. To obtain multiple copies of transductant, Geneticin (available from Invitrogen) tolerant strain at a concentration of 1.5 mg/mL was obtained. In this manner, a recombinant yeast GSNP-2 having respective multiple copies of GPP synthase gene from Arabidopsis thaliana and β-phellandrene synthase gene from lavender was obtained.
(4) Production of β-Phellandrene
After pre-culturing GSNP-2 in a MGY medium (manual of Invitrogen No. 25-0043), cells were collected, and the collected cells were main-cultured in a Fermentation basal salts medium (manual of Invitrogen Ver. B 053002) for 64 hours. For inducing expression of the introduced enzyme genes, 1/200-volume of 100% methanol was added to the culture liquid after lapses of 24 hours and 48 hours from starting of the culture. At this time, the system was brought into a closed system after 24 hours from start of the culturing. After end of the culture, the cells and a culture supernatant were collected by centrifugal separation.
The cells were crushed by a glass bead method (manual of Invitrogen No. 25-0043) and a supernatant was obtained. A pentane extract of the supernatant was analyzed by gas chromatography, and β-phellandrene was detected.
The culture supernatant was also extracted with pentane. The extracted fraction was analyzed by gas chromatography, and β-phellandrene was detected. Also the gas phase fraction was analyzed, and β-phellandrene was detected in the gas phase.
On the other hand, in the control recombinant yeast GS115 into which only pPIC3.5K vector was introduced (prepared in Example 3), β-phellandrene was not detected in any of the cells, the culture supernatant, and the gas phase.
These reveal that β-phellandrene can be produced by culturing the recombinant yeast into which GPP synthase gene from Arabidopsis thaliana and β-phellandrene synthase gene from lavender were introduced. The produced β-phellandrene was collectible from the cells, the culture supernatant, and the gas phase.
Example 5
In the present example, a recombinant E. coli into which NPP synthase gene from tomato and β-phellandrene synthase gene from lavender were introduced was prepared, and the recombinant E. coli was cultured, and generation of phellandrene was confirmed and a by-product was identified.
β-phellandrene synthase gene from lavender was cut out by cutting pACLPD prepared in Example 2 with NcoI and EcoRI, and introduced into a NcoI-EcoRI site of pCOLADuet-1 (available from Novagen) to construct pCODLFS. On the other hand, using pT21TNPP prepared in Example 1 as a template, PCR was conducted by using primers of SEQ ID NO: 25 and SEQ ID NO: 26 to amplify NPP synthase gene from tomato. The amplified fragment was cut with NdeI and KpnI, and introduced into the NdeI-KpnI site of pCODLFS, to construct coexpression vector pCOLDFSNS. The pCOLDFSNS was introduced into Rosetta2 (DE3) to obtain a recombinant E. coli ROFSNS that co-expresses β-phellandrene synthase from lavender and NPP synthase from tomato. As a control E. coli, a recombinant E. coli ROCOLA having only pCOLA Duet-1 was also prepared.
The recombinant E. coli ROFSNS and ROCOLA were cultured in a 2×YT medium (1.6% (w/v) Bacto Tripton, 1% (w/v) Yeast Extract, 0.5% (w/v) NaCl) containing 34 μg/mL of chloramphenicol and 15 μg/mL of kanamycin at 18° C., 110 rpm (swing) in a closed system for 24 hours. After end of the culture, the gas phase fraction was analyzed by GC-MS. As shown in FIGS. 1A and 1B, in ROFSNS, β-phellandrene was detected (peak A), and peaks B and C that seem to be by-products were also detected. On the other hand, in ROCOLA, these peaks were not detected. Identification of each peak revealed that small amounts of limonene (peak B) and myrcene (peak C) were generated as by-products in addition to β-phellandrene which is a principal product in the culture gas phase of ROFSNS as shown in FIGS. 2A-2C.
A generation amount of β-phellandrene in the gas phase by ROFSNS was 750 μg per 1 L of the culture liquid.
Example 6
In the present example, a recombinant E. coli into which mevalonate pathway gene from actinomyces, NPP synthase gene from tomato, and β-phellandrene synthase gene from lavender were introduced was prepared, and the recombinant E. coli was cultured to produce β-phellandrene.
Using genome DNA of Streptomyces griseolosporeus (Kitasatospora griseola) as a template, PCR using primers represented by SEQ ID NO: 27 and SEQ ID NO: 28 was conducted to amplify a nucleic acid encoding mevalonate pathway enzymes of S. griseolosporeus (SEQ ID NO: 29). This nucleic acid contains a gene cluster encoding mevalonate kinase, mevalonate diphosphate decarboxylase, phosphomevalonate kinase, IPP isomerase, HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (HMGR), and HMG-CoA synthase. The obtained amplified DNA fragment was cloned into pT7-Blue T vector to construct pT7SMV.
A gene fragment collected from the vector pT7SMV by cutting with NcoI and EcoRI was introduced into a NcoI-EcoRI site of pCOLA Duet-1 (available from Novagen) to construct pCSMV.
Into the NdeI-KpnI site of pCSMV, a synthetic nucleic acid of tomato NPP synthase gene, lavender β-phellandrene synthase gene operon represented by SEQ ID NO: 30 was introduced to construct pCSMVNSFS including actinomyces mevalonate pathway gene, tomato NPP synthase gene, and lavender β-phellandrene synthase gene. The expression vector pCSMVNSFS was introduced into E. coli Rosetta2 (DE3) to obtain a recombinant E. coli ROSMVNSFS.
The recombinant E. coli ROSMVNSFS was cultured in a 2×YT medium (1.6% (w/v) Bacto Tripton, 1% (w/v) Yeast Extract, 0.5% (w/v) NaCl) containing 34 μg/mL of chloramphenicol and 15 μg/mL of kanamycin at 18° C., 110 rpm (swing) in a closed system for 24 hours. After end of the culture, the gas phase fraction was analyzed by gas chromatography and β-phellandrene was detected. A generation amount of β-phellandrene in the gas phase was 8.5 mg per 1 L of the culture liquid.
These results and the generation amount of β-phellandrene in Example 5 reveal that the production amount of β-phellandrene can be increased by further introducing actinomyces MVA pathway gene.

## Claims

1. A recombinant Escherichia coli (E. coli) or Pichia yeast cell capable of producing β-phellandrene, prepared by introducing at least one nucleic acid selected from the group consisting of a nucleic acid encoding geranyl pyrophosphate synthase and a nucleic acid encoding neryl pyrophosphate synthase, and a nucleic acid encoding β-phellandrene synthase into a host cell in such a manner that these nucleic acids are expressed in the host cell,
wherein the nucleic acid encoding geranyl pyrophosphate synthase encodes the following protein (a), (b) or (c):
(a) protein consisting of the amino acid sequence of SEQ ID NO:2,
(b) protein consisting of the amino acid sequence of SEQ ID NO:2 in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence, and having geranyl pyrophosphate synthase activity, or
(c) protein consisting of an amino acid sequence having a homology of 90% or more with SEQ ID NO:2, and having geranyl pyrophosphate synthase activity,
wherein the nucleic acid encoding neryl pyrophosphate synthase encodes the following protein (d), (e) or (f):
(d) protein consisting of the amino acid sequence of SEQ ID NO:4,
(e) protein consisting of the amino acid sequence of SEQ ID NO:4 in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence, and having neryl pyrophosphate synthase activity, or
(f) protein consisting of an amino acid sequence having a homology of 90% or more with SEQ ID NO:4, and having neryl pyrophosphate synthase activity, and
wherein the nucleic acid encoding β-phellandrene synthase encodes the following protein (g), (h) or (i):
(g) protein consisting of the amino acid sequence of SEQ ID NO:6 or 8,
(h) protein consisting of the amino acid sequence of SEQ ID NO:6 or 8 in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence, and having β-phellandrene synthase activity, or
(i) protein consisting of an amino acid sequence having a homology of 90% or more with SEQ ID NO:6 or 8, and having β-phellandrene synthase activity, and
wherein 10 mg or more of β-phellandrene can be produced per 1 g of wet cells of the recombinant cell.

2. The recombinant E. coli or Pichia yeast cell according to claim 1, wherein the host cell does not have methane monooxygenase.

3. The recombinant E. coli or Pichia yeast cell according to claim 2, wherein the host cell is Escherichia coli or yeast.

4. The recombinant E. coli or Pichia yeast cell according to claim 1, wherein a nucleic acid encoding an enzyme group including acetyl CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase acting in a mevalonate pathway of isopentenyl diphosphate is further introduced, and the nucleic acid is expressed in the host cell.

5. The recombinant E. coli or Pichia yeast cell according to claim 4, wherein the mevalonate pathway is a mevalonate pathway of yeast or actinomyces.

6. A method for producing β-phellandrene by culturing the recombinant E. coli or Pichia yeast cell according to claim 1 to produce β-phellandrene in the recombinant E. coli or Pichia yeast cell.

7. The method according to claim 6, wherein 10 mg or more of β-phellandrene is produced per 1 g of wet cells of the recombinant E. coli or Pichia yeast cell.

8. The method according to claim 6, wherein β-phellandrene released outside the recombinant E. coli or Pichia yeast cell is collected.

9. The method according to claim 6, wherein β-phellandrene is collected from a gas phase of a culture system of the recombinant E. coli or Pichia yeast cell.

10. The recombinant E. coli or Pichia yeast cell according to claim 3, wherein a nucleic acid encoding an enzyme group including acetyl CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase acting in a mevalonate pathway of isopentenyl diphosphate is further introduced, and the nucleic acid is expressed in the host cell.

11. The recombinant E. coli or Pichia yeast cell according to claim 10, wherein the mevalonate pathway is a mevalonate pathway of yeast or actinomyces.

12. A method for producing β-phellandrene by culturing the recombinant E. coli or Pichia yeast cell according to claim 3 to produce β-phellandrene in the recombinant E. coli or Pichia yeast cell.

13. A method for producing β-phellandrene by culturing the recombinant E. coli or Pichia yeast cell according to claim 4 to produce β-phellandrene in the recombinant E. coli or Pichia yeast cell.

14. A method for producing β-phellandrene by culturing the recombinant E. coli or Pichia yeast cell according to claim 10 to produce β-phellandrene in the recombinant E. coli or Pichia yeast cell.