JP6325862B2 - Recombinant cells and method for producing cyclic monoterpenes - Google Patents

Description

  The present invention relates to a recombinant cell capable of producing a cyclic monoterpene from a specific C1 compound such as carbon monoxide, and a method for producing a cyclic monoterpene using the recombinant cell.

  Monoterpene is a 10-carbon isoprene rule with geranyl diphosphate (GPP), which is a condensate of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), as a biosynthetic precursor. Is a generic term for compounds according to Currently, over 900 types of monoterpenes are known.

  Cyclic monoterpenes have fragrances such as roses and citrus fruits and are often used in perfumes. For example, limonene is an aroma component contained in citrus fruits such as lemon, and is also used as a solvent and an adhesive raw material. Menthol has a refreshing fragrance and is used as a refreshing agent in confectionery and pharmaceuticals. On the other hand, in the resin industry, β-pinene (α-Pinene), α-pinene (α-Pinene), limonene, α-ferrondrene (α-Phellandrene) and the like have been studied as raw materials for monomers such as adhesives and transparent resins. (Non-Patent Document 1).

  Synthesis gas (Syngas) is a mixed gas composed mainly of carbon monoxide, carbon dioxide, and hydrogen, which is efficiently obtained from waste, natural gas, and coal by the action of a metal catalyst at high temperature and high pressure. It is. In the field of C1 chemistry using a metal catalyst starting from synthesis gas, a process has been developed for producing liquid chemicals such as methanol, formic acid and formaldehyde at low cost and in large quantities.

  Carbon monoxide, carbon dioxide, and hydrogen are contained in synthesis gas derived from waste, factory exhaust gas, natural gas, or coal-derived synthesis gas, and can be used almost permanently. However, there are very few examples of chemical production by microorganisms using C1 carbon sources such as synthesis gas. At present, development is progressing only in the production of ethanol, 2,3-butanediol and the like from synthesis gas. In particular, there are few reports on the use of synthetic gas assimilation materials by recombinants. Patent Document 1 discloses a technique for producing isopropanol using a recombinant Escherichia coli. In this technique, a plurality of CO metabolizing enzyme genes are introduced into Escherichia coli to impart syngas assimilation ability, and isopropanol is produced from syngas. However, this technology does not produce cyclic monoterpenes. In addition, it is difficult to efficiently and efficiently express the CO metabolizing enzyme gene group originally possessed by a syngas-utilizing microorganism in E. coli, and the recombinant E. coli having CO metabolism is selected because of its low CO tolerance. The system used cannot be expected to have high productivity of the target substance.

  Β-Phellandrene, which is a kind of cyclic monoterpene, is expected to be used as a new polymer material. β-Ferlandolene may give a higher molecular weight polymer than α-Ferlandolene. However, when β-ferrandolene is obtained by a synthetic chemical method, formation of α-ferrandolene, which is an isomer, is unavoidable, and separation of these isomers is extremely difficult (Non-patent Document 2). ). For this reason, it is difficult to obtain high-purity β-ferrandolene, which makes it difficult to examine the polymer physical properties of β-ferrandolene.

  On the other hand, the biosynthetic pathway of β-ferrandrene includes geranyl diphosphate (IPP) to geranyl diphosphate (IPP) by the action of geranyl diphosphate (GPP) synthase or neryl diphosphate (NPP) synthase. GPP) or neryl diphosphate (NPP) is biosynthesized. Subsequently, β-ferrandolene is biosynthesized from GPP or NPP by the action of β-ferrandolene synthase. And tomato and lavender, β-ferrandrene synthase has been found (Non-patent Documents 3 and 4).

  Patent Document 2 describes a method for producing a monoterpene using a transformant of a C1 metabolic host cell into which a nucleic acid encoding a cyclic terpene synthase (cyclic terpene synthase) has been introduced. As an example, an experimental example in which limonene was produced using a transformant of the genus Methylomonas was described. There is also a reference to β-ferrandolene synthase, but specific examples and methods for obtaining β-ferrandolene synthase and its gene, as well as specific configurations and construction methods for transformants capable of producing β-ferrandolene are shown. Not. In addition, this technique does not use carbon monoxide, carbon dioxide, and hydrogen or waste-derived synthesis gas, factory exhaust gas, natural gas, or coal-derived synthesis gas as a carbon source.

  Patent Document 3 describes a method for producing a terpene using a transformant of C1 metabolic host cells, and there is an example of producing a kind of farnesene of sesquiterpene. However, no practical production method of cyclic monoterpenes using C1 metabolic host cells has been shown.

Special table 2011-509691 gazette Special table 2005-500805 gazette International Publication No. 2013/180584

Satou K., et al., Green Chemistry 2006, 8, 878-882 Mori K., Tetrahedron: Asymmetry 2006, 17, 2133-2142 Demissie, Z.A., et al., Planta. 2011, 233, 685-96. Schilmiller, A. L., et al., Proc Natl Acad Sci U S A., 2009, 106, 10865-70.

  In view of the above situation, an object of the present invention is to provide a series of techniques for obtaining a large amount of cyclic monoterpene from a C1 carbon source such as synthesis gas.

The above-mentioned one aspect for solving the problem, a cyclic monoterpene possible production recombinant cells, host cells having isopentenyl diphosphate synthesis ability of non-mevalonate pathway, or methyl tetrahydrofolate, At least one selected from the group consisting of a nucleic acid encoding geranyl diphosphate synthase and a nucleic acid encoding neryl diphosphate synthase in a host cell having a function of synthesizing acetyl CoA from carbon monoxide and CoA A nucleic acid and a nucleic acid encoding a cyclic monoterpene synthase are introduced. These nucleic acids are expressed in the host cell, and are at least one selected from the group consisting of carbon monoxide and carbon dioxide. Recombinant cells capable of producing cyclic monoterpenes from two C1 compounds.

This aspect concerns recombinant cells capable of producing cyclic monoterpenes. The recombinant cell of this aspect is a host cell having “the ability to synthesize isopentenyl diphosphate (IPP) by the non-mevalonate pathway” or “the ability to synthesize acetyl CoA from methyltetrahydrofolate, carbon monoxide, and CoA”. Let it be a cell. Then, the host cell contains “at least one nucleic acid selected from the group consisting of a nucleic acid encoding a geranyl diphosphate (GPP) synthase and a nucleic acid encoding a neryl diphosphate (NPP) synthase”, "Nucleic acid encoding monoterpene synthase" is introduced, and these nucleic acids are expressed in host cells.
That is, in the recombinant cell of this aspect , the expression ability of GPP synthase and / or NPP synthase and the expression ability of cyclic monoterpene synthase are newly added or enhanced to the host cell.
According to the recombinant cell of this aspect , isopentenyl diphosphate (IPP) is synthesized from the aforementioned C1 compound. Then, GPP is synthesized from IPP by the action of GPP synthase expressed in the cell, and / or NPP is synthesized from IPP by the action of NPP synthase expressed in the cell. Furthermore, cyclic monoterpenes are synthesized from GPP and / or NPP by the action of cyclic monoterpene synthase expressed in cells. As a result, a large amount of cyclic monoterpene can be produced by culturing the recombinant cells of the present invention.

  The cyclic monoterpene refers to a monoterpene containing a cyclic structure such as a six-membered ring, and includes limonene, α-pinene, β-pinene, α-ferrandolene, β-ferrandolene, menthol, thymol, carvacrol, caren, Including sabinene, camphene, tsugen, camphor, borneol, cineole.

The biosynthetic pathway of isoprenoids is broadly divided into a mevalonate pathway (also referred to as MVA pathway) and a non-mevalonate pathway (also referred to as MEP pathway). The non-mevalonic acid pathway is a pathway for finally producing isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP) from glyceraldehyde 3-phosphate and pyruvic acid. The host cell according to one embodiment of this aspect has an ability to synthesize isopentenyl diphosphate through a non-mevalonate pathway.
In addition, as a cell having a “function of synthesizing acetyl CoA from methyltetrahydrofolate, carbon monoxide, and CoA” which is a host cell according to another embodiment of this aspect, the acetyl CoA pathway (Wood-Ljungdahl pathway shown in FIG. And anaerobic microorganisms having a methanol pathway.

  Preferably, it has carbon monoxide dehydrogenase.

  Carbon monoxide dehydrogenase (eg, EC 1.2.99.2/1.2.7.4) (CO dehydrogenase, CODH) is a process that converts carbon monoxide and protons from carbon monoxide and water. It has a function of generating carbon monoxide and water from carbon dioxide and protons, and the reverse reaction. Carbon monoxide dehydrogenase is one of the enzymes that acts in the acetyl-CoA pathway (FIG. 1).

Preferably, said host cell, Ru Clostridium bacteria or Moorella bacteria der.

  Preferably, the host cell is Clostridium ljungdahlii or Clostridium autoethanogenum.

  Preferably, a gas mainly containing carbon monoxide, a gas mainly containing carbon monoxide and hydrogen, a gas mainly containing carbon dioxide and hydrogen, or carbon monoxide, carbon dioxide and hydrogen are mainly used. Cyclic monoterpenes can be produced from the constituent gases.

Preferably, the nucleic acid encoding geranyl diphosphate synthase encodes the following protein (a), (b) or (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a protein comprising 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 the activity of geranyl diphosphate synthase,
(C) a protein having an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having the activity of geranyl diphosphate synthase.

Preferably, the nucleic acid encoding neryl diphosphate synthase encodes the following protein (d), (e) or (f).
(D) a protein comprising the amino acid sequence represented by SEQ ID NO: 4,
(E) a protein comprising 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 the activity of neryl diphosphate synthase,
(F) A protein having an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having the activity of neryl diphosphate synthase.

  Preferably, a nucleic acid encoding at least one enzyme acting in the isopentenyl diphosphate synthesis pathway is further introduced, and the nucleic acid is expressed in the host cell.

  With this configuration, IPP serving as a substrate for GPP synthase or NPP synthase is efficiently supplied.

  Preferably, the cyclic monoterpene synthase is β-ferrandolene synthase, and β-ferrandolene, 4-carene, or limonene can be produced as the cyclic monoterpene.

  According to this preferred aspect, β-ferrandolene, 4-carene, or limonene is synthesized from GPP and / or NPP by the action of β-ferrandolene synthase expressed in cells. As a result, by culturing the recombinant cell of the present invention, β-ferrandrene, 4-carene, or limonene can be produced in large quantities.

  Preferably, at least a nucleic acid encoding neryl diphosphate synthase has been introduced.

Preferably, the nucleic acid encoding β-ferrandrene synthase encodes the following protein (g), (h) or (i).
(G) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8,
(H) a protein comprising 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 the activity of β-ferrandrene synthase,
(I) A protein having an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 6 or 8, and having β-ferrandrene synthase activity.

  Preferably, a nucleic acid encoding an enzyme group that acts in the mevalonate pathway is further introduced, and further has an ability to synthesize isopentenyl diphosphate by the mevalonate pathway.

  With this configuration, IPP serving as a substrate for GPP synthase or NPP synthase is synthesized from both the mevalonate pathway and the non-mevalonate pathway, and IPP is efficiently supplied. As a result, the recombinant cell of the present invention has a higher ability to produce cyclic monoterpenes.

  Preferably, the mevalonate pathway is a yeast, prokaryotic, or actinomycete mevalonate pathway.

  Preferably, a nucleic acid encoding at least one enzyme acting in the non-mevalonate pathway is further introduced and the nucleic acid is expressed in the host cell.

  With this configuration, the ability to synthesize IPP through the non-mevalonate pathway is enhanced. As a result, the recombinant cell of the present invention has a higher ability to produce cyclic monoterpenes.

  Preferably, the non-mevalonate pathway is a non-mevalonate pathway other than a host cell.

  Preferably, the nucleic acid introduced into the host cell is integrated into the genome of the host cell.

  Preferably, the nucleic acid introduced into the host cell is incorporated into a plasmid.

  Preferably, 10 mg or more of cyclic monoterpene can be produced per 1 g of wet cells of recombinant cells.

  Preferably, 10 mg or more of β-ferrandrene can be produced per 1 g of wet cells of recombinant cells.

  Preferably, 6 mg or more of 4-carene can be produced per 1 g of wet cells of recombinant cells.

  Preferably, 2 mg or more of limonene can be produced per 1 g of wet cells of recombinant cells.

In another aspect, the above-described recombinant cells are cultured using at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide as a carbon source, and cyclic monoterpenes are produced in the recombinant cells. This is a production method of a cyclic monoterpene to be produced.

This aspect relates to a method for producing a cyclic monoterpene. In this aspect , by culturing the above-described recombinant cell using at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide as a carbon source, the recombinant cell can produce a cyclic monoterpene. . According to this aspect , for example, a cyclic monoterpene can be produced from a synthesis gas containing carbon monoxide and carbon dioxide.

In another aspect, the above-described recombinant cell is contacted with at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide, and the recombinant cell is caused to produce a cyclic monoterpene from the C1 compound. This is a production method for cyclic monoterpenes.

In this aspect , the above-described recombinant cell is contacted with at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide, and a cyclic monoterpene is produced from the C1 compound. Also according to this aspect , for example, a cyclic monoterpene can be produced from a synthesis gas containing carbon monoxide and carbon dioxide.

  Preferably, a gas mainly containing carbon monoxide, a gas mainly containing carbon monoxide and hydrogen, a gas mainly containing carbon dioxide and hydrogen, or carbon monoxide, carbon dioxide and hydrogen are mainly used. The component gas is provided to the recombinant cell.

  “Providing gas to a recombinant cell” means giving gas to the recombinant cell as a carbon source or the like, or bringing the gas into contact with the recombinant cell.

  Preferably, formic acid or methanol is further provided to the recombinant cell.

  “Providing gas and methanol to a recombinant cell” means that gas and methanol are supplied to the recombinant cell as a carbon source or the like, or the gas and methanol are brought into contact with the recombinant cell. The same applies to formic acid.

  Preferably, the recombinant cell is a host cell made of Clostridium bacteria or Moorella bacteria.

  Preferably, the cyclic monoterpene synthase is β-ferrandolene synthase, and β-ferrandolene, 4-carene, or limonene is produced as the cyclic monoterpene.

  Preferably, 10 mg or more of cyclic monoterpene is produced per 1 g of wet cells of recombinant cells.

  Preferably, 10 mg or more of β-ferrandrene is produced per 1 g of wet cells of the recombinant cells.

  Preferably, 6 mg or more of 4-carene is produced per 1 g of wet cells of recombinant cells.

  Preferably, 2 mg or more of limonene is produced per 1 g of wet cells of the recombinant cells.

  Preferably, the cyclic monoterpene released outside the recombinant cell is recovered.

  Preferably, the cyclic monoterpene is recovered from the gas phase of the recombinant cell culture system.

  Bicarbonate may be used instead of carbon dioxide.

The invention according to claim 1 is a recombinant cell of a Clostridium genus bacterium capable of producing a cyclic monoterpene, wherein a nucleic acid encoding geranyl diphosphate synthase and neryl diphosphate synthesis are introduced as the introduced nucleic acid. At least one nucleic acid selected from the group consisting of nucleic acids encoding enzymes, and nucleic acids encoding β-ferrandrene synthase, these nucleic acids being expressed in the recombinant cell, carbon monoxide and A recombinant cell capable of producing β-ferrandrene as a cyclic monoterpene from at least one C1 compound selected from the group consisting of carbon dioxide.

The invention according to claim 2 is the recombinant cell according to claim 1, wherein the Clostridium genus bacterium is Clostridium ljungdahlii.

The invention according to claim 3 is a gas mainly composed of carbon monoxide, a gas mainly composed of carbon monoxide and hydrogen, a gas mainly composed of carbon dioxide and hydrogen, or carbon monoxide and carbon dioxide. The recombinant cell according to claim 1 or 2, wherein the cyclic monoterpene can be produced from a gas mainly composed of carbon and hydrogen.

In the invention described in claim 4, the nucleic acid encoding geranyl diphosphate synthase encodes the following protein (a), (b) or (c): The described recombinant cell.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a protein comprising 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 the activity of geranyl diphosphate synthase,
(C) A protein having an amino acid sequence showing 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having the activity of geranyl diphosphate synthase.

The invention according to claim 5 is the nucleic acid encoding neryl diphosphate synthase encoding the following protein (d), (e) or (f): The described recombinant cell.
(D) a protein comprising the amino acid sequence represented by SEQ ID NO: 4,
(E) a protein comprising 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 the activity of neryl diphosphate synthase,
(F) A protein having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having the activity of neryl diphosphate synthase.

In the invention described in claim 6, the nucleic acid encoding β-ferrandrene synthase encodes the following protein (g), (h) or (i): The described recombinant cell.
(G) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8,
(H) a protein comprising 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 the activity of β-ferrandrene synthase,
(I) A protein having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 6 or 8, and having β-ferrandrene synthase activity.

The invention according to claim 7 is the recombinant cell according to any one of claims 1 to 6, wherein the nucleic acid is integrated into the genome of the recombinant cell.

The invention according to claim 8 is the recombinant cell according to any one of claims 1 to 6, wherein the nucleic acid is incorporated in a plasmid.

The invention according to claim 9 is a recombinant cell of Clostridium genus bacteria capable of producing a cyclic monoterpene, wherein the introduced nucleic acid includes a nucleic acid encoding neryl diphosphate synthase, and β-ferrandolene. A nucleic acid encoding a synthase, these nucleic acids are expressed in the recombinant cell, and β- as a cyclic monoterpene from at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide. Recombinant cells capable of producing ferrandlene, 4-carene, or limonene.

The invention according to claim 10 is the recombinant cell according to claim 9, wherein the Clostridium genus bacterium is Clostridium ljungdahlii.

The invention according to claim 11 is a gas mainly composed of carbon monoxide, a gas mainly composed of carbon monoxide and hydrogen, a gas mainly composed of carbon dioxide and hydrogen, or carbon monoxide and carbon dioxide. The recombinant cell according to claim 9 or 10, wherein the cyclic monoterpene can be produced from a gas mainly composed of carbon and hydrogen.

In the invention described in claim 12, the nucleic acid encoding neryl diphosphate synthase encodes the following protein (d), (e) or (f): The described recombinant cell.
(D) a protein comprising the amino acid sequence represented by SEQ ID NO: 4,
(E) a protein comprising 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 the activity of neryl diphosphate synthase,
(F) A protein having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having the activity of neryl diphosphate synthase .

The invention according to claim 13 is the nucleic acid encoding β-ferrandolene synthase encoding the following protein (g), (h) or (i): The described recombinant cell.
(G) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8,
(H) a protein comprising 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 the activity of β-ferrandrene synthase,
(I) A protein having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 6 or 8, and having β-ferrandrene synthase activity.

The invention according to claim 14 is the recombinant cell according to any one of claims 9 to 13, wherein the nucleic acid is integrated in the genome of the recombinant cell.

The invention according to claim 15 is the recombinant cell according to any one of claims 9 to 13, wherein the nucleic acid is incorporated in a plasmid.

The invention according to claim 16 is a culture of the recombinant cell according to any one of claims 1 to 8 using at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide as a carbon source. And a method for producing a cyclic monoterpene, wherein the recombinant cell produces β-ferrandrene as a cyclic monoterpene.

The invention according to claim 17 contacts the recombinant cell according to any one of claims 1 to 8 with at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide. It is a method for producing a cyclic monoterpene, wherein β-ferrandrene is produced as a cyclic monoterpene from the C1 compound in a cell.

The invention according to claim 18 is a gas mainly composed of carbon monoxide, a gas mainly composed of carbon monoxide and hydrogen, a gas mainly composed of carbon dioxide and hydrogen, or carbon monoxide and carbon dioxide. The method for producing a cyclic monoterpene according to claim 16 or 17, wherein a gas mainly composed of carbon and hydrogen is provided to the recombinant cell.

The invention according to claim 19 is a culture of the recombinant cell according to any one of claims 9 to 15 using at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide as a carbon source. And producing the cyclic monoterpene by causing the recombinant cell to produce β-ferrandrene, 4-carene, or limonene as the cyclic monoterpene.

The invention according to claim 20 contacts the recombinant cell according to any one of claims 9 to 15 with at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide. It is a method for producing a cyclic monoterpene by causing a cell to produce β-ferrandrene, 4-carene, or limonene as a cyclic monoterpene from the C1 compound.

The invention according to claim 21 is a gas mainly composed of carbon monoxide, a gas mainly composed of carbon monoxide and hydrogen, a gas mainly composed of carbon dioxide and hydrogen, or carbon monoxide and carbon dioxide. 21. The method for producing a cyclic monoterpene according to claim 19 or 20, wherein a gas mainly composed of carbon and hydrogen is provided to the recombinant cell.

The invention according to claim 22 is the method for producing a cyclic monoterpene according to any one of claims 16 to 21, wherein the cyclic monoterpene released to the outside of the recombinant cell is recovered.

The invention according to claim 23 is the method for producing a cyclic monoterpene according to any one of claims 16 to 22, wherein the cyclic monoterpene is recovered from the gas phase of the recombinant cell culture system.

  According to the present invention, cyclic monoterpenes can be produced in high purity and in large quantities.

It is explanatory drawing showing an acetyl CoA path | route and a methanol path | route.

  Hereinafter, embodiments of the present invention will be described. In the present invention, the term “gene” can be replaced by the term “nucleic acid” or “DNA”.

  The recombinant cell of the present invention can produce a cyclic monoterpene. The recombinant cell of the present invention can be used as a host cell having the ability to synthesize isopentenyl diphosphate by a non-mevalonate pathway or a host cell having a function of synthesizing acetyl CoA from methyltetrahydrofolate, carbon monoxide, and CoA. At least one nucleic acid selected from the group consisting of a nucleic acid encoding a diphosphate synthase and a nucleic acid encoding a neryl diphosphate synthase, and a nucleic acid encoding a cyclic monoterpene synthase, These nucleic acids are expressed in the host cell and can produce a cyclic monoterpene from at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide.

  First, the host cell will be described. The host cell in the recombinant cell of the present invention is “a host cell having the ability to synthesize IPP by the non-mevalonate pathway” or “a host cell having a function of synthesizing acetyl CoA from methyltetrahydrofolate, carbon monoxide, and CoA”. is there.

  As described above, generally, the synthesis route of IPP is roughly divided into two types, a mevalonate pathway (MVA pathway) and a non-mevalonate pathway (MEP pathway). The mevalonate pathway is provided by eukaryotes and starts from acetyl CoA. Enzymes acting in the mevalonate pathway include, in order from the upstream, acetyl CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl diphosphate isomerase Is mentioned.

On the other hand, the non-mevalonic acid pathway is provided in prokaryotes, chloroplasts, and plastids, and starts from glyceraldehyde 3-phosphate and pyruvic acid. The enzymes that act in the non-mevalonate pathway include DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphoditidyl-2-C-methyl-D- in order from the upstream. Examples include erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase, and HMB-PP reductase.
In one embodiment of the present invention, “a host cell having the ability to synthesize IPP through a non-mevalonate pathway” is used.

On the other hand, for host cells having “a function of synthesizing acetyl CoA from methyltetrahydrofolate, carbon monoxide, and CoA”, methyltetrahydrofolate ([CH ₃ ] -THF), carbon monoxide (CO), and CoA are used. The pathway for synthesizing acetyl CoA is, for example, included in the acetyl CoA pathway (Wood-Ljungdahl pathway) and the methanol pathway (Methanol pathway) shown in FIG.

As shown in FIG. 1, in the acetyl CoA pathway, carbon dioxide (CO ₂ ) is reduced separately to carbon monoxide (CO) and a methyl cation source in two pathways. Then, using these two carbon sources as substrates, the thiol group of CoA (indicated as HSCoA in FIG. 1) is acetylated to synthesize one molecule of acetyl CoA. In the acetyl-CoA pathway, enzymes such as acetyl-CoA synthase (ACS), methyltransferase, carbon monoxide dehydrogenase (CODH), formate dehydrogenase (Formate dehydrogenase, FDH) are acting. . Note that the path from formyl tetrahydrofolate ([CHO] -THF) to the [CH _3] -THF is called methyl branch (Methyl branch).
On the other hand, the methanol pathway includes a pathway for converting methanol into formaldehyde (HCHO) and further formic acid (HCOOH) and a pathway for deriving [CH ₃ ] -THF from methanol.
That is, the route for synthesizing acetyl CoA from methyltetrahydrofolate ([CH ₃ ] -THF), carbon monoxide (CO), and CoA is common to the acetyl CoA route and the methanol route.

  In a preferred embodiment, the recombinant cell has carbon monoxide dehydrogenase (CODH). Specifically, a cell that grows mainly by carbon monoxide metabolism, that is, by the function of carbon monoxide dehydrogenase to generate carbon dioxide and protons from carbon monoxide and water is preferable. Anaerobic microorganisms having an acetyl-CoA pathway and a methanol pathway have carbon monoxide dehydrogenase (CODH).

The anaerobic microorganisms include Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium ragsdalei (Kopke M. et al., Appl. Environ. Microbiol. 2011, 77 (15), 5467-5475), Moorella thermoacetica (same as Clostridium thermoaceticum (Pierce EG. Et al., Environ. Microbiol. 2008, 10, 2550-2573), Acetobacterium woodii (Dilling S. et al., Appl. Environ. Microbiol. 2007, 73 (11), 3630-3636), etc. Typical examples include Clostridium bacteria, Moorella bacteria, or Acetobacterium bacteria. In particular, Clostridium bacteria have established host-vector systems and culture methods and are suitable as host cells in the present invention.
The above five Clostridium bacteria, Moorella bacteria, or Acetobacterium bacteria are known as representative examples of syngas-utilizing microorganisms.

  Other than Clostridium, Moorella, and Acetobacterium, Carboxydocella sporoducens sp. Nov. (Slepova TV. Et al., Inter. J. Sys. Evol. Microbiol. 2006, 56, 797-800), Rhodopseudomonas gelatinosa (Uffen RL, J. Bacteriol. 1983, 155 (3), 956-965), Eubacterium limosum (Roh H. et al., J. Bacteriol. 2011, 193 (1), 307-308), Butyribacterium methylotrophicum (Lynd , LH. Et al., J. Bacteriol. 1983, 153 (3), 1415-1423) can be used as host cells.

The above-mentioned bacterial growth and CODH activity are all oxygen-sensitive, but oxygen-insensitive CODH is also known. For example, Oligotropha carboxidovorans (Schubel, U. et al., J. Bacteriol., 1995, 2197-2203), Bradyrhizobium japonicum (Lorite MJ. Et al., Appl. Environ. Microbiol., 2000, 66 (5), 1871 In other bacterial species, oxygen-insensitive CODH exists (King GM et al., Appl. Environ. Microbiol. 2003, 69 (12), 7257-7265). There is also oxygen-insensitive CODH in the genus Ralsotonia, which is an aerobic hydrogen-oxidizing bacterium (NCBI Gene ID: 4249199, 8019399).
As described above, bacteria having CODH exist widely, and a host cell used in the present invention can be appropriately selected from them. For example, CO, CO / H ₂ (a gas containing CO and H ₂ as main components), or CO / CO ₂ / H ₂ (a gas containing CO, CO ₂ and H ₂ as main components) is the only carbon source and Using a selective medium as an energy source, bacteria having CODH that can be used as host cells can be isolated under anaerobic, microaerobic, or aerobic conditions.

  Next, GPP synthase, NPP synthase, cyclic monoterpene synthase, and nucleic acids encoding these enzymes will be described.

The GPP synthase is not particularly limited as long as it can exhibit the enzyme activity in a recombinant cell. The same applies to the nucleic acid (gene) encoding GPP synthase as long as it is normally transcribed and translated in a recombinant cell.
The same applies to NPP synthase, cyclic monoterpene synthase, and nucleic acids encoding them.

Specific 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.), Those derived from Mycobacterium tuberculosis; (GenBank Accession No .: NP — 215504; Mann, FM, et al., FEBS Lett., 2011, 585, 549-54.), And the like.
SEQ ID NO: 1 shows the amino acid sequence corresponding to the nucleotide sequence of the nucleic acid (DNA) encoding the Arabidopsis thaliana-derived GPP synthase, and SEQ ID NO: 2 shows only the amino acid sequence. DNA having the base sequence represented by SEQ ID NO: 1 is an example of a nucleic acid encoding GPP synthase.

Furthermore, the nucleic acid encoding the GPP synthase includes at least a nucleic acid encoding the following protein (a), (b), or (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a protein comprising 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 the activity of geranyl diphosphate synthase,
(C) a protein having an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having the activity of geranyl diphosphate synthase.
The homology of the amino acid sequence in (c) is more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more.

Specific examples of NPP synthase include those derived from tomato (Solanum lycopersicum) (GenBank Accession No .: FJ797956).
SEQ ID NO: 3 shows the amino acid sequence corresponding to the base sequence of the nucleic acid (DNA) encoding the tomato-derived NPP synthetase, and SEQ ID NO: 4 shows only the amino acid sequence. The DNA having the base sequence represented by SEQ ID NO: 3 is an example of a nucleic acid encoding NPP synthase.

Furthermore, the nucleic acid encoding NPP synthase includes at least a nucleic acid encoding the following protein (d), (e), or (f).
(D) a protein comprising the amino acid sequence represented by SEQ ID NO: 4,
(E) a protein comprising 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 the activity of neryl diphosphate synthase,
(F) A protein having an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having the activity of neryl diphosphate synthase.
The amino acid sequence homology in (f) is more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more.

  In the recombinant cell of the present invention, only one nucleic acid is introduced for “nucleic acid encoding geranyl diphosphate (GPP) synthase” and “nucleic acid encoding neryl diphosphate (NPP) synthase”. May have been introduced, or both nucleic acids may be introduced.

  An example of a cyclic monoterpene synthase is β-ferrandrene synthase. By the action of β-ferrandolene synthase, β-ferrandolene is synthesized from GPP and / or NPP. Also, 4-carene and limonene can be synthesized as by-products.

Specific examples of β-ferrandrene synthase and nucleic acid encoding the same include those derived from tomato (Solanum lycopersicum) (GenBank Accession No .: FJ797957; Schilmiller, AL, et al., Proc Natl Acad Sci US A., 2009, 106, 10865-70.), Lavender (Lavandula angustifolia) (GenBank Accession No .: HQ404305; Demissie, ZA, et al., Planta, 2011 ,. 233, 685-96), etc. .
SEQ ID NO: 5 shows the amino acid sequence corresponding to the base sequence of the nucleic acid (DNA) encoding the tomato-derived β-ferrandrene synthase, and SEQ ID NO: 6 shows only the amino acid sequence.
SEQ ID NO: 7 shows the amino acid sequence corresponding to the base sequence of the nucleic acid (DNA) encoding the lavender-derived β-ferrandrene synthase, and SEQ ID NO: 8 shows only the amino acid sequence.
The DNA having the base sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7 is an example of a nucleic acid encoding β-ferrandrene synthase.

Furthermore, the nucleic acid encoding β-ferrandrene synthase includes at least a nucleic acid encoding the following protein (g), (h) or (i).
(G) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8,
(H) a protein comprising 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 the activity of β-ferrandrene synthase,
(I) A protein having an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 6 or 8, and having β-ferrandrene synthase activity.
The homology of the amino acid sequence in (i) is more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more.

  In the recombinant cell of the present invention, other nucleic acids may be further introduced in addition to the nucleic acids encoding GPP synthase, NPP synthase, and cyclic monoterpene synthase. In one embodiment, a nucleic acid encoding an enzyme group that acts in the mevalonate pathway is further introduced, and further has an ability to synthesize IPP through the mevalonate pathway. According to this configuration, since IPP is synthesized from both the mevalonate pathway and the non-mevalonate pathway, the ability to synthesize IPP is enhanced, and as a result, production of cyclic monoterpenes is performed more efficiently.

  As described above, enzymes acting in the mevalonate pathway include acetyl CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl dilin Acid isomerase is mentioned. Among these, for example, an enzyme group consisting of HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase is expressed in the host cell. The nucleic acid to be introduced may be selected. These nucleic acids can also be modified into codons that are easily transcribed in the host cell.

The mevalonate pathway is possessed by all eukaryotes, but has also been found in prokaryotes. Prokaryotes that have a 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).
Examples of bacteria include 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, etc. J. et al., Mol. Biol. Evol. 2010, 28 (1), 87-99).
In the archaea, Aeropyrum genus, Sulfolobus genus, Desulfurococcus genus, Thermoproteus genus, Halobacterium genus, Methanococcus genus, Thermococcus genus, Pyrococcus genus, Methanopyrus genus, Thermoplasma genus, etc. (Lombard J. et al., Mol. Biol. Evol. 2010, 28 (1), 87-99).

  The origin of the enzyme group acting in the mevalonate pathway is not particularly limited, but the enzyme group acting in the mevalonate pathway of yeast is preferable. In addition, enzymes that act in the mevalonate pathway of actinomycetes are also preferably employed.

In another embodiment, a nucleic acid encoding at least one enzyme that acts in the non-mevalonate pathway is further introduced and the nucleic acid is expressed in the host cell. Also in the present embodiment, the ability to synthesize IPP is enhanced, and as a result, production of cyclic monoterpenes is performed more efficiently. The nucleic acid to be introduced may be only one kind or two or more kinds.
As mentioned above, enzymes that act in the non-mevalonate pathway include DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphoditidyl-2-C-methyl-D. -Erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase, HMB-PP reductase. For example, one or more enzymes may be selected from these enzyme groups and a nucleic acid encoding the enzyme may be introduced into the host cell.
In addition, the enzyme that acts in the non-mevalonate pathway is preferably derived from sources other than the host cell. With this configuration, reaction suppression due to the reaction product can be avoided.
These nucleic acids can also be modified into codons that are easily transcribed in the host cell.

  These enzymes acting in the mevalonate pathway or the non-mevalonate pathway may be naturally occurring or a modified form of each enzyme. For example, it may be an amino acid substitution mutant of each enzyme or a polypeptide that is a partial fragment of each enzyme and has the same enzyme activity.

The method for introducing a nucleic acid into a host cell is not particularly limited, and may be appropriately selected depending on the type of the host cell. For example, a vector that can be introduced into a host cell and can express an integrated nucleic acid can be used.
For example, when the host cell is a prokaryotic organism such as a bacterium, the vector is a promoter that can replicate autonomously in the host cell or can be integrated into a chromosome and can transcribe the inserted nucleic acid (DNA). What is contained can be used. For example, it is preferable to construct a series of components comprising a promoter, a ribosome binding sequence, the nucleic acid (DNA), and a transcription termination sequence in the host cell using the vector.

  When the host cell is a Clostridium bacterium (including closely related species such as the Moorella bacterium), the shuttle vector pIMP1 (Mermelstein LD et al., Bio / technology 1992, 10, 190-) 195) can be used. This shuttle vector is a fusion vector of pUC9 (ATCC 37252) and pIM13 (Projan SJ et al., J. Bacteriol. 1987, 169 (11), 5131-5139) isolated from Bacillus subtilis. It is held stably even within. Another example of a shuttle vector between Clostridium bacteria and E. coli is pSOS95 (GenBank: AY187686.1).

  In general, electroporation is used for gene introduction into Clostridium bacteria, but the introduced foreign plasmid immediately after gene introduction is susceptible to degradation by the restriction enzyme Cac824I or the like and is extremely unstable. Therefore, E. coli carrying pAN1 (Mermelstein LD et al., Apply. Environ. Microbiol. 1993, 59 (4), 1077-1081) carrying the Bacillus subtilis phage Φ3T1-derived methyltransferase gene, such as ER2275 strain, It is preferable that the vector derived from pIMP1 is once amplified and methylated and then recovered from E. coli and used for transformation by electroporation. Recently, Clostridium acetobuthylicum lacking the Cac824I gene has been developed, and non-methylated vectors are also possible stably (Dong H. et al., PLoS ONE 2010, 5 (2), e9038) . In addition, a technique is known in which NEB express, an improved strain of E. coli BL21 strain, is used to amplify a vector and efficiently introduce an unmethylated vector (Leang C. et al., Appl Environ Microbiol. 2013 79 (4), 1102-9).

  Examples of promoters for heterologous gene expression in Clostridium bacteria include thl (thiolase) promoter (Perret S et al., J. Bacteriol. 2004, 186 (1), 253-257), Dha (glycerol dehydratase) promoter (Raynaud C et al., PNAS 2003, 100 (9), 5010-5015), ptb (phosphotransbutyrylase) promoter (Desai RP et al., Appl. Environ. Microbiol. 1999, 65 (3), 936-945), adc ( acetoacetate decarboxylase) promoter (Lee J et al., Appl. Environ. Microbiol. 2012, 78 (5), 1416-1423). However, the present invention is not limited thereto, and promoter region sequences used in various metabolic operons found in host cells and the like can be used.

  In addition, when a plurality of types of genes are introduced into a host cell using a vector, each gene may be incorporated into one vector or may be incorporated into separate vectors. Further, when a plurality of genes are incorporated into one vector, each gene may be expressed under a common promoter or may be expressed under different promoters. Examples of introducing multiple types of genes include, in addition to nucleic acids encoding GPP synthase, NPP synthase, and cyclic monoterpene synthase, genes encoding enzymes that act in the above acetyl-CoA pathway and methanol pathway The mode which introduce | transduces is mentioned.

  As described above, known vectors that can be used in the present invention have been shown. However, regions related to transcription control such as promoters and terminators, replication regions, and the like can be modified according to the purpose. As a modification method, it may be changed to another natural gene sequence in each host cell or its related species, or may be changed to an artificial gene sequence.

  Moreover, in addition to the above-described modification by gene transfer, by combining mutation methods such as mutation and genome shuffling, the expression level of the transgene in the host cell, the excretion function of the cyclic monoterpene in the host cell, the specific ring The productivity of cyclic monoterpenes can be further improved by improving the resistance to the monoterpenes.

  That is, in the present invention, the foreign gene may be incorporated into the genome of the host cell or may be incorporated into a plasmid.

  In one preferred embodiment, the recombinant cells are resistant to at least 400 mM cyclic monoterpenes. Such a configuration enables high production of cyclic monoterpenes. A recombinant cell having such characteristics can be obtained, for example, by subjecting a host cell to appropriate mutation treatment, selecting a host cell having the desired characteristics, and using the host cell.

In one aspect of the cyclic monoterpene production method of the present invention, the above-described recombinant cell is cultured using at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide as a carbon source, The recombinant cell is made to produce a cyclic monoterpene. About these C1 compounds used as a carbon source, only one may be used and it may be used in combination of 2 or more. Moreover, it is preferable to use these C1 compounds as a main carbon source, and it is more preferable that it is the only carbon source.
In addition, it is preferable to simultaneously provide hydrogen (H ₂ ) as an energy source.

  The method for culturing the recombinant cell of the present invention is not particularly limited, and can be appropriately performed according to the type of the host cell. When the recombinant cell is a Clostridium genus bacterium (obligate anaerobic or absolute anaerobic), for example, it is cultured under nutrient conditions consisting of inorganic salts necessary for growth and synthetic gas. The culture is preferably performed under a pressurized state of about 0.2 to 0.3 MPa (absolute pressure). Furthermore, a small amount of organic substances such as vitamins, yeast extract, corn steep liquor, and bacto tryptone may be added to improve the initial growth and the reached cell density.

  When the recombinant cell is aerobic or facultative anaerobic, for example, aeration and agitation culture using a liquid medium can be performed.

In another aspect of the production method of the cyclic monoterpene of the present invention, the above-described recombinant cell is contacted with at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide, and the recombinant cell is contacted. A cyclic monoterpene is produced from the C1 compound. That is, regardless of whether cell division (cell proliferation) is involved or not, a cyclic monoterpene can be produced by contacting the aforementioned C1 compound with a recombinant cell. For example, the above-described C1 compound can be continuously supplied to immobilized recombinant cells to continuously produce cyclic monoterpenes.
Also in this aspect, about these C1 compounds, only one may be used and it may be used in combination of 2 or more. Further, it is preferable to simultaneously contact hydrogen (H ₂ ) as an energy source.

  In a preferred embodiment, a gas mainly composed of carbon monoxide, a gas mainly composed of carbon monoxide and hydrogen, a gas mainly composed of carbon dioxide and hydrogen, or carbon monoxide, carbon dioxide and hydrogen. Is provided to the recombinant cells. That is, recombinant cells are cultured using these gases as a carbon source, or cyclic monoterpenes are produced from carbon monoxide or carbon dioxide in the gases by contacting these gases with the recombinant cells. . Again, hydrogen is used as an energy source.

  In addition to these C1 compounds, formic acid or methanol may also be provided to the recombinant cells. That is, recombinant cells are cultured using formic acid or methanol and these gases as a carbon source, or formic acid or methanol and these gases are contacted with the recombinant cells. By adding formic acid or methanol, the culture efficiency and the efficiency of cyclic monoterpene production can be improved. As an aspect of provision, for example, formic acid or methanol and these gases are simultaneously provided to recombinant cells.

  In a preferred embodiment, the cyclic monoterpene synthase is β-ferrandolene synthase, and β-ferrandolene, 4-carene, or limonene is produced as the cyclic monoterpene. That is, as described above, β-ferrandolene is synthesized from GPP and / or NPP by the action of β-ferrandrene synthase, and 4-carene and limonene can be synthesized as by-products.

  By culturing the recombinant cells of the present invention, cyclic monoterpenes can be produced in large quantities. As the production capacity of the cyclic monoterpene, a production amount of 10 mg or more of the cyclic monoterpene per 1 g of wet cells can be realized. For example, 10 mg or more of β-ferrandrene, 6 mg or more of 4-carene, and 2 mg or more of limonene can be produced per 1 g of wet cells of recombinant cells. In the case of producing β-ferrandolene by culturing the recombinant cell of the present invention, unlike the case of producing by synthetic chemical methods, the contamination of α-ferrandrene does not substantially occur.

  The produced cyclic monoterpene is accumulated intracellularly or released extracellularly. For example, using a recombinant cell having the above-mentioned Clostridium genus or Moorella genus as a host cell, the cyclic monoterpene released outside the cell is recovered, and purified and purified by distillation or the like. Formula monoterpenes can be obtained.

  Examples of methods for isolating and purifying cyclic monoterpenes from recombinant cell cultures include, for example, extracting a culture solution (culture supernatant) with an appropriate solvent such as pentane, and further using reverse-phase chromatography and gas chromatography. Etc., and can be purified to a high purity by chromatography. Since many of the cyclic monoterpenes released to the outside of the cell also evaporate in the gas phase, they can be liquefied and recovered with a cold trap or the like.

In some cases, bicarbonate may be used instead of carbon dioxide. That is, Clostridium bacteria and related species are known to have carbonic anhydrase (CA) (EC 4.2.1.1: H ₂ O + CO ₂ ⇔HCO ₃ ⁻ + H ⁺ ). (Braus-Stromeyer SA et al., J. Bacteriol. 1997, 179 (22), 7197-7200), a bicarbonate such as NaHCO _{3 serving} as an HCO ₃ ⁻ source can be used as the CO ₂ source.

  Here, a combination of carbon monoxide, carbon dioxide, formic acid, and methanol that can be provided to a recombinant cell when the host cell has an acetyl CoA pathway and a methanol pathway (FIG. 1) will be described.

In the synthesis of acetyl CoA by the acetyl CoA pathway, the action of methyltransferase, corrinoid iron-sulfur protein (CoFeS-P), acetyl CoA synthase (ACS), and CODH, CoA, methyl tetrahydrofolate is essential _{(methyltetrahydrofolate, [CH 3] -THF} ), and the synthesis process of acetyl CoA from CO (Ragsdale SW et al., BBRC 2008, 1784 (12), 1873-1898).

On the other hand, in the culture of Butyribacterium methylotrophicum, addition of formic acid or methanol in addition to CO or CO ₂ as a carbon source means CO metabolism, that is, tetrahydrofolate (THF) content in the methyl branch of the acetyl CoA pathway, It is also known to increase the activities of CODH, formate dehydrogenase (FDH) and hydrogenase required for CO metabolism (Kerby R. et al., J. Bacteriol. 1987, 169). (12), 5605-5609). Eubacterium limosum and the like have also been shown to obtain high growth even when CO ₂ and methanol are used as carbon sources under anaerobic conditions (Genthner BRS. Et al., Appl. Environ. Microbiol., 1987, 53 ( 3), 471-476).

  Effects of these methanol on syngas-utilizing microorganisms and genome analysis of Moorella thermoacetica (Clostridium thermoaceticum) and Clostridium ljungdahlii (Pierce E. et al., Environ. Microbiol. 2008, 10 (10), 2550-2573; From the results of Durre P. et al., PNAS 2010, 107 (29), 13087-13092), in these microbial species, the methanol pathway as shown in FIG. 1 is the acetyl-CoA pathway (Wood-Ljungdahl). It can be explained that it participates as a methyl group donor in pathway).

The fact, positive several Clostridium The genus formate dehydrogenase ^{(FDH) (EC 1.2.1.2/1.2.1.43:Formate+NAD(P) +} ⇔ CO 2 + NAD (P) H) Directional activity (CO ₂ formation from Formate) has been confirmed (Liu CL et al., J. Bacteriol. 1984, 159 (1), 375-380; Keamy JJ et al., J. Bacteriol. 1972, 109 (1), 152-161). Therefore, in these strains, when CO ₂ or CO is in a deficient state, a reaction in the direction of CO ₂ production can partially work from methanol (CH ₃ OH) or formic acid (HCOOH) (FIG. 1). . This is due to the phenomenon of increased formate hydrogenase activity and CODH activity by adding CH ₃ OH as described above (Kerby R et al., J. Bateriol. 1987, 169 (12), 5605-5609. ) Can also be explained. That is, it can grow even if formic acid (HCOOH) or methanol (CH ₃ OH) is the only carbon source.

  Even if the host cell is originally a strain having no forward activity of formate dehydrogenase, the forward activity may be imparted by genetic modification such as mutagenesis, foreign gene introduction, or genome shuffling.

From the above, when the host cell has an acetyl CoA pathway and a methanol pathway, cyclic monoterpenes can be produced using the following gases and liquids.
・ CO
・ CO ₂
・ CO / H ₂
・ CO ₂ / H ₂
・ CO / CO ₂ / H ₂
・ CO / HCOOH
・ CO ₂ / HCOOH
・ CO / CH ₃ OH
・ CO ₂ / CH ₃ OH
・ CO / H ₂ / HCOOH
・ CO ₂ / H ₂ / HCOOH
・ CO / H ₂ / CH ₃ OH
・ CO ₂ / H ₂ / CH ₃ OH
・ CO / CO ₂ / H ₂ / HCOOH
_{· CO / CO 2 / H 2} / CH 3 OH
・ CH ₃ OH / H ₂
・ HCOOH / H ₂
・ CH ₃ OH
・ HCOOH

  When the recombinant cells of the present invention are cultured not for the purpose of producing cyclic monoterpenes but exclusively for the purpose of increasing the number of cells, it is not necessary to use carbon monoxide or carbon dioxide as a carbon source. For example, the recombinant cells may be cultured using other carbon sources such as sugars and glycerin.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited only to an Example.

  In this example, β-ferrandrene, a type of cyclic monoterpene, was produced by recombinant cells of Clostridium ljungdahlii, a type of syngas-utilizing bacterium.

(1) Construction of various vectors Artificial synthetic genes represented by SEQ ID NO: 9 (3125 bp) and SEQ ID NO: 10 (3611 bp) were respectively constructed as gene clusters containing a β-ferrandrene synthase gene. SEQ ID NOs: 9 and 10 each contain a modified sequence of a tomato-derived β-ferrandolene synthase gene (GenBank Accession No .: FJ797957) and a T7 terminator, and further include BamHI and XhoI recognition sequences at both ends. SEQ ID NO: 9 contains a modified sequence of a tomato-derived NPP synthase gene (GenBank Accession No .: FJ797956). On the other hand, SEQ ID NO: 10 contains an Arabidopsis thaliana-derived GPP synthase gene (SEQ ID NO: 5, GenBank Accession No: Y17376).

  The artificially synthesized DNA (120 bp) of SEQ ID NO: 11 was cloned into the BamHI / EheI site of Clostridium / E. Coli shuttle vector pSOS95 (GenBank: AY187686.1) to obtain pSOS-MS. Further, a gene fragment obtained by restriction treatment of the artificially synthesized gene of SEQ ID NO: 9 with BamHI / XhoI was cloned into the BamHI / XhoI site of pSOS-MS to obtain pSOS-PHS-NPP. Similarly, pSOS-PHS-GPP was obtained by cloning the artificially synthesized gene of SEQ ID NO: 10 at the BamHI / XhoI site of pSOS-MS. pSOS-PHS-NPP and pSOS-PHS-GPP function as a shuttle vector in the same manner as pSOS95. NEB express (New England Biolabs) was used for the cloning.

(2) Production of recombinants capable of producing β-ferrandrene The pSOS-PHS-NPP and pSOS-PHS-GPP produced in (1) above were each electroporated into Clostridium ljungdahlii (DSM13528 / ATCC55383). The recombinant was obtained after introduction. As a control, pSOS-MS was similarly introduced into Clostridium ljungdahlii (DSM13528 / ATCC55383) to obtain a recombinant. Electroporation was performed by the method recommended in Appl. Environ. Microbiol. 2013, 79 (4): 1102-9.

(3) β-Ferrandren Production Each recombinant of pSOS-PHS-NPP and pSOS-PHS-GPP obtained in (2) above was cultured at 37 ° C. under anaerobic conditions. As the medium, ATCC medium 1754 PETC medium (containing no fructose) containing 4 μg / mL clarithromycin was used. 10 mL of medium is charged in a 27 mL sealable headspace vial container, and a mixed gas of CO / CO ₂ / H ₂ = 33/33/34% (volume ratio) is used at a gas pressure of 0.25 MPa (absolute pressure) After filling and sealing with an aluminum cap, shaking culture was performed. When OD600 reached 0.9, the culture was terminated, and the gas phase was analyzed by gas chromatography (Shimadzu GCMS-QP2010 Ultra). As a result, β-ferrandrene was detected. In addition, β-ferrandrene was detected from the pSOS-PHS-NPP recombinant about 2 to 6 times the culture medium of the pSOS-PHS-GPP recombinant. Further, in pSOS-PHS-NPP, in addition to β-ferrandrene, about 60% of β-ferrandolene and about 20% of limonene were detected as cyclic monoterpene 4-carene.
On the other hand, β-ferrandrene could not be detected in the control recombinant into which only the pSOS-MS vector was introduced.

From the above, a tomato-derived NPP synthase gene, or a recombinant Clostridium ljungdahlii into which an Arabidopsis thaliana-derived GPP synthase gene and a tomato-derived β-ferrandrene synthase gene were introduced, using CO and CO ₂ as carbon sources. It was shown that β-ferrandrene can be produced by culturing. Furthermore, it was shown that β-ferrandolene was efficiently produced by combining a tomato-derived NPP synthase gene and a tomato-derived β-ferrandrene synthase gene.

Claims (23)

A recombinant cell of Clostridium bacteria capable of producing a cyclic monoterpene,
As introduced nucleic acid, and at least one nucleic acid selected from the group consisting of nucleic acids encoding nucleic acid and neryl diphosphate synthase encoding the geranyl diphosphate synthase, nucleic acids encoding a β- phellandrene synthase And
These nucleic acids are expressed in the recombinant cells,
A recombinant cell capable of producing β -ferrandrene as a cyclic monoterpene from at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide. The Clostridium genus bacterium, a recombinant cell of claim 1 which is Clostridium ljungdahli i. Gas composed mainly of carbon monoxide, gas composed mainly of carbon monoxide and hydrogen, gas composed mainly of carbon dioxide and hydrogen, or composed mainly of carbon monoxide, carbon dioxide and hydrogen. the recombinant cell according to claim 1 or 2 from a gas can be produced said cyclic monoterpenes. The recombinant cell according to any one of claims 1 to 3 , wherein the nucleic acid encoding geranyl diphosphate synthase encodes the following protein (a), (b), or (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a protein comprising 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 the activity of geranyl diphosphate synthase,
(C) A protein having an amino acid sequence having 90 % or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having the activity of geranyl diphosphate synthase. The recombinant cell according to any one of claims 1 to 4 , wherein the nucleic acid encoding neryl diphosphate synthase encodes the following protein (d), (e) or (f).
(D) a protein comprising the amino acid sequence represented by SEQ ID NO: 4,
(E) a protein comprising 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 the activity of neryl diphosphate synthase,
(F) A protein having an amino acid sequence having 90 % or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having the activity of neryl diphosphate synthase. The recombinant cell according to any one of claims 1 to 5, wherein the nucleic acid encoding β-ferrandrene synthase encodes the following protein (g), (h) or (i).
(G) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8,
(H) a protein comprising 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 the activity of β-ferrandrene synthase,
(I) A protein having an amino acid sequence having 90 % or more identity with the amino acid sequence represented by SEQ ID NO: 6 or 8, and having a β-ferrandrene synthase activity. The recombinant cell according to any one of claims 1 to 6 , wherein the nucleic acid is integrated into the genome of the recombinant cell. The recombinant cell according to any one of claims 1 to 6 , wherein the nucleic acid is incorporated into a plasmid. A recombinant cell of Clostridium bacteria capable of producing a cyclic monoterpene,
The introduced nucleic acid has a nucleic acid encoding neryl diphosphate synthase and a nucleic acid encoding β-ferrandrene synthase,
These nucleic acids are expressed in the recombinant cells,
A recombinant cell capable of producing β-ferrandrene, 4-carene, or limonene as a cyclic monoterpene from at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide. The recombinant cell according to claim 9, wherein the Clostridium bacterium is Clostridium ljungdahlii. Gas composed mainly of carbon monoxide, gas composed mainly of carbon monoxide and hydrogen, gas composed mainly of carbon dioxide and hydrogen, or composed mainly of carbon monoxide, carbon dioxide and hydrogen. The recombinant cell according to claim 9 or 10, wherein the cyclic monoterpene can be produced from gas. The recombinant cell according to any one of claims 9 to 11, wherein the nucleic acid encoding neryl diphosphate synthase encodes the following protein (d), (e) or (f).
(D) a protein comprising the amino acid sequence represented by SEQ ID NO: 4,
(E) a protein comprising 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 the activity of neryl diphosphate synthase,
(F) A protein having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having the activity of neryl diphosphate synthase . The recombinant cell according to any one of claims 9 to 12, wherein the nucleic acid encoding β-ferrandrene synthase encodes the following protein (g), (h) or (i).
(G) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8,
(H) a protein comprising 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 the activity of β-ferrandrene synthase,
(I) A protein having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 6 or 8, and having β-ferrandrene synthase activity. The recombinant cell according to any one of claims 9 to 13, wherein the nucleic acid is integrated into the genome of the recombinant cell. The recombinant cell according to any one of claims 9 to 13, wherein the nucleic acid is incorporated into a plasmid. A recombinant cell according to any one of claims 1 to 8 is cultured using at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide as a carbon source, and the recombinant cell is cyclic. A method for producing a cyclic monoterpene , wherein β-ferrandolene is produced as a monoterpene. The recombinant cell according to any one of claims 1 to 8 is contacted with at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide, and the recombinant cell is converted into a cyclic monocycle from the C1 compound. A method for producing a cyclic monoterpene in which β-ferrandolene is produced as a terpene. Gas composed mainly of carbon monoxide, gas composed mainly of carbon monoxide and hydrogen, gas composed mainly of carbon dioxide and hydrogen, or composed mainly of carbon monoxide, carbon dioxide and hydrogen. The method for producing a cyclic monoterpene according to claim 16 or 17 , wherein gas is provided to the recombinant cell. The recombinant cell according to any one of claims 9 to 15 is cultured using at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide as a carbon source, and the recombinant cell is cyclic. A method for producing a cyclic monoterpene, wherein β-ferlandol, 4-carene, or limonene is produced as a monoterpene. 16. The recombinant cell according to any one of claims 9 to 15 is contacted with at least one C1 compound selected from the group consisting of carbon monoxide and carbon dioxide. A method for producing a cyclic monoterpene, wherein β-ferrandrene, 4-carene, or limonene is produced as a terpene. Gas composed mainly of carbon monoxide, gas composed mainly of carbon monoxide and hydrogen, gas composed mainly of carbon dioxide and hydrogen, or composed mainly of carbon monoxide, carbon dioxide and hydrogen. The method for producing a cyclic monoterpene according to claim 19 or 20, wherein gas is provided to the recombinant cells. The method for producing a cyclic monoterpene according to any one of claims 16 to 21 , wherein the cyclic monoterpene released to the outside of the recombinant cell is recovered. The method for producing a cyclic monoterpene according to any one of claims 16 to 22 , wherein the cyclic monoterpene is recovered from a gas phase of a culture system of recombinant cells.

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