JP2017055702A - Recombinant cell, method for producing recombinant cell, and method for producing shyobunol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technology for producing shyobunol from syngas and methanol using recombinant cells.SOLUTION: The present invention provides a recombinant cell having the ability of synthesizing farnesyl diphosphate, the cell comprising a gene encoding a shyobunol synthase or a functional fragment thereof, expressing the gene, and capable of producing shyobunol from at least one C1 compound selected from carbon monoxide, carbon dioxide, formic acid, methane, and methanol. The present invention also provides a recombinant cell having (i) the ability of synthesizing isopentenyl diphosphate by a non-mevalonate pathway or (ii) function of synthesizing acetyl CoA from methyl tetrahydrofolic acid, carbon monoxide, and CoA and capable of producing shyobunol from at least one C1 compound selected from carbon monoxide, carbon dioxide, formic acid, methane, and methanol. The present invention further provides a method for producing shyobunol, where the recombinant cell is a syngas-utilizing bacterium or a methylotroph and the recombinant cell is allowed to produce shyobunol from the C1 compound.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a recombinant cell, a method for producing the recombinant cell, and a method for producing shobunol.

  Sesquiterpene is a generic name for compounds that follow the isoprene rule with 15 carbons, using farnesyl diphosphate (FPP) in which three molecules of isopentenyl diphosphate (IPP) are condensed as biosynthetic precursors. Currently, over 3000 types of sesquiterpenes are known. Sesquiterpenes have a fragrance like roses and citrus fruits, and are often used in perfumes. It also acts as a physiologically active substance and is used in pharmaceuticals.

  Shyobunol, a kind of sesquiterpene, is useful as a fragrance, a cosmetic, a pharmaceutical product and an intermediate thereof, and a resin raw material.

  The following pathways can be considered as the biosynthesis pathway of shobunol. First, geranyl diphosphate (GPP) is biosynthesized from isopentenyl diphosphate (IPP) by the action of geranyl diphosphate (GPP) synthase. Is done. Subsequently, farnesyl diphosphate (FPP) is biosynthesized from geranyl diphosphate (GPP) by the action of farnesyl diphosphate (FPP) synthase. Then, shobunol is biosynthesized from farnesyl diphosphate (FPP) by the action of shovenol synthase. However, at present, the shovenol synthase has not yet been identified.

  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. Patent Document 2 describes a technique for producing isoprene using a recombinant cell produced from a synthesis gas assimilating microorganism. However, neither technology produces shobunol.

  Among the C1 compounds, methanol is produced at low cost from natural gas and synthesis gas that is a mixed gas of carbon monoxide, carbon dioxide, and hydrogen obtained from waste such as biomass and municipal waste. Methanol is easy to handle and store, such as being soluble in water, and is also suitable as a carbon source for microbial culture.

  Methylotroph is a carbon compound that does not have a C—C bond in the molecule, such as methane, methanol, methylamine, dimethylamine, trimethylamine, etc., as the sole carbon source and energy source. It is a generic name for microorganisms. Microorganisms called methanotroph, methane oxidizing bacteria, methanol-assimilating bacteria, methanol-assimilating yeast, methanol-assimilating microorganisms, etc. all belong to methylotrophs.

  A methylotroph uses, as a central metabolism, a reaction in which methanol is converted into formaldehyde and then formaldehyde is converted into an organic substance having a C—C bond. Serine pathway, ribulose monophosphate pathway (RuMP pathway), and xylulose monophosphate pathway (XuMP pathway) are known as carbon assimilation metabolic pathways via formaldehyde. Methylotrophs classified as bacteria (methylotrophic bacteria) possess a serine cycle or RuMP pathway. On the other hand, methylotrophs (methylotrophic yeast) classified as yeast possess the XuMP pathway.

  Moreover, methylotrophic bacteria are classified into obligate methylotroph and facultative methylotroph that can use other carbon compounds because of the difference in methanol requirement.

  Patent Document 3 describes a technique for producing isoprene using recombinant cells prepared from methylotrophs. However, this technique does not produce Shobunol.

Special table 2011-509691 gazette International Publication No. 2014/066511 International Publication No. 2014/104202

  In view of the above situation, an object of the present invention is to provide a technique capable of producing shovenol from synthesis gas or methanol using recombinant cells.

  The inventors of the present invention have constructed a technology that can produce shoubnol from synthesis gas or methanol using recombinant cells using a gene encoding a newly isolated shobnol synthase.

  One aspect of the present invention is a recombinant cell having the ability to synthesize farnesyl diphosphate, having a gene encoding a shovenol synthase or a functional fragment thereof, wherein the gene is expressed, carbon monoxide, A recombinant cell capable of producing shoubnol from at least one C1 compound selected from the group consisting of carbon dioxide, formic acid, methane, and methanol.

  Shobunol synthase has the effect of converting farnesyl diphosphate (FPP) into shovenol. The recombinant cell of this aspect has the ability to synthesize farnesyl diphosphate, has a gene encoding a shovenol synthase or a functional fragment thereof, and the gene is expressed. Then, shobunol can be produced from at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol. The recombinant cell of this aspect can convert farnesyl diphosphate synthesized in the cell into shobunol by the action of shobunol synthase. That is, according to the recombinant cell of this aspect, shobunol can be produced from the aforementioned C1 compound.

  The functional fragment of schovenol synthase refers to a polypeptide having a partial sequence of schovenol synthase, which has the activity of schovenol synthase (the activity of converting farnesyl diphosphate to shobunol). .

  As a specific example of the shobunol synthase, there is a shovenol synthase derived from vetiver zizanioides described later.

  Preferably, it has (i) the ability to synthesize isopentenyl diphosphate via a non-mevalonate pathway, or (ii) the function of synthesizing acetyl CoA from methyltetrahydrofolate, carbon monoxide, and CoA, and Shobunol can be produced from at least one C1 compound selected from the group consisting of carbon, formic acid, and methanol.

  The function (i) or (ii) is generally possessed by the synthesis gas assimilating microorganism.

  Preferably, it has carbon monoxide dehydrogenase.

  Preferably, it has the activity to generate carbon dioxide and hydrogen from carbon monoxide and water.

  Preferably, it is Clostridium bacteria or Moorella bacteria.

  Preferably, it is a methylotroph that can biosynthesize shoubnol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.

  This aspect corresponds to an embodiment in which the recombinant cell is a methylotroph.

  Preferably, the formaldehyde immobilization pathway has at least one C1 carbon assimilation pathway selected from the group consisting of serine pathway, ribulose monophosphate pathway, and xylulose monophosphate pathway.

  Preferably, it further has a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase, and the gene is expressed.

  With this configuration, formaldehyde immobilization ability by the ribulose monophosphate pathway is imparted or enhanced.

  Preferably, it belongs to the genus Pichia, Hansenula, or Candida.

Preferably, the said shobunol synthetase consists of protein which is the following (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 an activity of converting farnesyl diphosphate into shovenol,
(C) a protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shobunol.

  The protein (a) is a schivenol synthase derived from vetiver.

  Preferably, it further has a gene encoding at least one enzyme that acts in the farnesyl diphosphate synthesis pathway, and the gene is expressed.

  Preferably, the farnesyl diphosphate synthesis pathway comprises the mevalonate pathway.

  Preferably, the mevalonate pathway is an actinomycete mevalonate pathway.

  Another aspect of the present invention is a method for producing a recombinant cell, the first step of providing a host cell having the ability to synthesize farnesyl diphosphate, and a Shobunol synthase or a functional fragment thereof to the host cell. At least one C1 selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol, wherein the gene is expressed in the host cell. A method for producing a recombinant cell, wherein a recombinant cell producing shoubnol from a compound is produced.

  This aspect relates to a method for producing a recombinant cell. The method for producing a recombinant cell of this aspect comprises a first step of providing a host cell having a farnesyl diphosphate synthesizing ability, and a step of introducing a gene encoding a shovenol synthase or a functional fragment thereof into the host cell. 2 steps. Then, the gene introduced in the second step is expressed in the host cell, and recombination produces shobnol from at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol. Cells. According to this aspect, a recombinant cell that produces shobunol from the above-described C1 compound can be produced.

  Preferably, the host cell has (i) the ability to synthesize isopentenyl diphosphate by a non-mevalonate pathway, or (ii) the function of synthesizing acetyl CoA from methyltetrahydrofolate, carbon monoxide, and CoA. A recombinant cell producing shobnol is produced from at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, and methanol.

  Preferably, the recombinant cell has carbon monoxide dehydrogenase.

  Preferably, the recombinant cell has an activity to generate carbon dioxide and hydrogen from carbon monoxide and water.

  Preferably, the recombinant cell is a Clostridium bacterium or a Moorella bacterium.

  Preferably, the recombinant cell is a methylotroph that can biosynthesize shovenol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.

  Preferably, the recombinant cell has at least one C1 carbon assimilation pathway selected from the group consisting of a serine pathway, a ribulose monophosphate pathway, and a xylulose monophosphate pathway as a formaldehyde immobilization pathway.

  Preferably, the method further includes a step of introducing a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase into the host cell. Expressed in

  Preferably, the recombinant cell belongs to the genus Pichia, Hansenula, or Candida.

Preferably, the said shobunol synthetase consists of protein which is the following (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 an activity of converting farnesyl diphosphate into shovenol,
(C) a protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shobunol.

  Preferably, the method further includes the step of introducing into the host cell a gene encoding at least one enzyme that acts in the farnesyl diphosphate synthesis pathway, and the gene is expressed in the host cell.

  Preferably, the farnesyl diphosphate synthesis pathway comprises the mevalonate pathway.

  Preferably, the mevalonate pathway is an actinomycete mevalonate pathway.

  Another aspect of the present invention provides the above recombinant cell or the recombinant cell produced by the above method with at least one C1 selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol. This is a method for producing shobunol, wherein a compound is brought into contact with the recombinant cell to produce shobunol from the C1 compound.

  This aspect relates to a method for producing Shobunol. In this aspect, the above-described recombinant cell is contacted with at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol, and shobunol is produced from the C1 compound. According to this aspect, shobunol can be produced from synthesis gas, methane, and methanol.

  Preferably, the recombinant cell is cultured using at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol as a carbon source, and Shobunol is obtained from the culture. get.

  According to the present invention, shobenol can be produced from carbon monoxide, carbon dioxide, formic acid, methane, or methanol. For example, it becomes possible to produce shobunol from synthetic gas containing carbon monoxide and carbon dioxide, or methanol using recombinant cells.

It is explanatory drawing showing the biosynthetic pathway from the chemical structure of a shobunol and a farnesyl diphosphate. It is the photograph showing the root and leaf of the vetiver (Vetiveria zizanioides) used in the Example. It is a chromatogram showing the result of gas chromatography. It is a mass spectrum showing the result of mass spectrometry.

  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”.

In the present invention, a gene encoding a shovenol synthase or a functional fragment thereof is used. As an example of a shobunol synthetase, the shobunol synthetase which consists of the protein of following (a), (b) or (c) is mentioned.
(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 an activity of converting farnesyl diphosphate into shovenol,
(C) a protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shobunol.

  SEQ ID NO: 1 is a base sequence of a gene (cDNA) encoding a shovenol synthase derived from vetiver (Vetiveria zizanioides) and a corresponding amino acid sequence. SEQ ID NO: 2 is an excerpt of only the amino acid sequence of SEQ ID NO: 1. That is, SEQ ID NO: 2 represents the amino acid sequence of schivenol synthase derived from vetiver.

  The shovenol synthase used in the present invention comprises 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 farnesyl diphosphate is converted to shobunol. Includes proteins with activity to convert. The number of amino acids deleted, substituted or added is 1 to 20, preferably 1 to 15, more preferably 1 to 10, and still more preferably 1 to 5.

  The shovenol synthase used in the present invention is a protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shovenol. Include. The homology with the amino acid sequence represented by SEQ ID NO: 2 is 80% or more, preferably 85% or more, more preferably 90% or more, and further preferably 95% or more.

  In the present invention, a gene encoding a functional fragment of shovenol synthase can be used. As described above, a functional fragment of schovenol synthase is a polypeptide having a partial sequence of schovenol synthase, and the activity of schovenol synthase (activity to convert farnesyl diphosphate to shobnol). It refers to what you have. For example, a polypeptide of the above-mentioned schovenol synthase and having an activity of converting farnesyl diphosphate into schovenol is a functional fragment of schovenol synthase.

  FIG. 1 shows the chemical structure of shobunol and the biosynthetic pathway from farnesyl diphosphate. The shovenol synthase used in the present invention catalyzes the reaction shown in FIG.

The present invention is a recombinant cell having the ability to synthesize farnesyl diphosphate, has a gene encoding a shovenol synthase or a functional fragment thereof, and the gene is expressed, and carbon monoxide, carbon dioxide, formic acid A recombinant cell capable of producing shovenol from at least one C1 compound selected from the group consisting of methane, and methanol.
The present invention also provides a method for producing a recombinant cell, the first step of providing a host cell having a farnesyl diphosphate synthesizing ability, and encoding the schovenol synthase or a functional fragment thereof in the host cell. A second step of introducing a gene, wherein the gene is expressed in the host cell and is expressed from at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol. The present invention includes a method for producing a recombinant cell for producing a recombinant cell that produces anol.

  The gene introduced into the host cell may be a gene that the host cell does not originally have, that is, an exogenous gene, or a gene that the host cell originally has, that is, an endogenous gene. Also good. The latter corresponds to so-called self-cloning.

  In one embodiment, the recombinant cell has (i) the ability to synthesize isopentenyl diphosphate via a non-mevalonate pathway, or (ii) the ability to synthesize acetyl CoA from methyltetrahydrofolate, carbon monoxide, and CoA. And can produce shobunol from at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, and methanol. For example, a host cell into which a Shobunol synthase gene is introduced has the function (i) or (ii). The functions (i) and (ii) are generally provided by the synthesis gas assimilating microorganism. Hereinafter, this embodiment (first embodiment) will be described in detail.

  In general, the synthesis pathway of isopentenyl diphosphate (IPP) is roughly divided into two routes, the mevalonate pathway (MVA pathway) and the 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 (GAP) 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.

  It is preferable that the recombinant cell used in this embodiment further 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. Examples of such cells include anaerobic microorganisms having an acetyl-CoA pathway (Wood-Ljungdahl pathway) and a methanol pathway.

The anaerobic microorganisms include Clostridium ljungdahlii, Clostridium autoethanogenumn, Clostridium carboxidivorans, Clostridium ragsdalei (Kopke M. et al., Appl. Environ. Microbiol. 2011, 77 (15), 5467-5475), Moorella thermoacetica (same as Clostridium thermoacetic ) (Pierce EG. Et al., Environ. Microbiol. 2008, 10, 2550-2573) and the like, typical examples include Clostridium bacteria or Moorella 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 kinds of Clostridium bacteria or Moorella bacteria are known as representative examples of syngas assimilating microorganisms.

  Other than Clostridium and Moorella bacteria, 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.

  The gene encoding the shovenol synthase may be modified to a codon that is easily transcribed in the host cell. For example, if the host cell is a Clostridium bacterium, the codon of the gene to be introduced can be modified based on the information on the codon usage of the Clostridium bacterium.

  In the recombinant cell of the present invention, other genes may be further introduced in addition to the shovenol synthase gene. In one embodiment, a gene encoding at least one enzyme that acts in the farnesyl diphosphate synthesis pathway is further introduced.

  As described above, in the biosynthesis pathway of shobunol, geranyl diphosphate (GPP) is first biosynthesized from isopentenyl diphosphate (IPP) by the action of geranyl diphosphate (GPP) synthase. Subsequently, farnesyl diphosphate (FPP) is biosynthesized from geranyl diphosphate (GPP) by the action of farnesyl diphosphate (FPP) synthase. Then, shobunol is biosynthesized from farnesyl diphosphate (FPP) by the action of shovenol synthase. Accordingly, examples of enzymes that act in the farnesyl diphosphate synthesis pathway include GPP synthase and FPP synthase.

  Other examples of enzymes that act in the farnesyl diphosphate synthesis pathway include the aforementioned enzymes that act in the mevalonate pathway (MVA pathway) or non-mevalonate pathway (MEP pathway), which are the synthesis pathways of IPP. It is done.

  In one embodiment, a gene 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 such a 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, biosynthesis of shobunol 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 gene to be introduced may be selected. These genes 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 gene encoding at least one enzyme that acts in the non-mevalonate pathway is further introduced and the gene is expressed in the cell. Also in this embodiment, the ability to synthesize IPP is enhanced, and as a result, the biosynthesis of shobunol is performed more efficiently. The gene to be introduced may be only one type or two or more types.
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 gene 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 genes 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 (functional fragment) that is a partial fragment of each enzyme and has the same enzyme activity.

A method for introducing a gene 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 gene can be used.
For example, when the host cell is a prokaryotic organism such as a bacterium, the vector can be replicated in the host cell or can be integrated into a chromosome, and can be transcribed into the inserted gene (nucleic acid, DNA). Those containing a promoter can be used. For example, it is preferable to construct a series of components comprising a promoter, a ribosome binding sequence, the above gene, 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 a Moorella bacterium), a shuttle vector pIMP1 (Mermelstein LD et al., Bio / technology 1992, 10, 190) between Clostridium bacterium and Escherichia coli is described. -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.

  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) .

  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.

  Further, 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 types 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 "genes encoding enzymes that act in the mevalonate pathway" and "non-mevalonate pathways" in addition to "genes encoding shobunol synthase or functional fragments thereof" And a gene that encodes at least one enzyme acting in the above.

  In addition to the introduction of foreign genes as described above, mutations and genome shuffling can be further performed to breed strains with significantly improved Shobunol productivity.

  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 another embodiment, the recombinant cell is a methylotroph that can biosynthesize shovenol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. Hereinafter, this embodiment (second embodiment) will be described in detail.

  As described above, methylotroph uses, as a central metabolism, a reaction of converting formaldehyde into an organic substance having a C—C bond after converting methanol into formaldehyde. Serine pathway, ribulose monophosphate pathway (RuMP pathway), and xylulose monophosphate pathway (XuMP pathway) are known as carbon assimilation metabolic pathways via formaldehyde. Methylotrophic bacteria possess a serine cycle or RuMP pathway. On the other hand, methylotrophic yeast possesses the XuMP pathway.

  Examples of the combination of a host cell and a transgene for obtaining the above recombinant cell which is a methylotroph include the following.

  One example is a combination where the host cell is a methylotroph and the transgene is a shovenol synthase gene. This is the most basic combination.

Even if the host cell is a non-methylotroph, it can be treated in the same manner as a methylotroph by introducing a necessary gene. An example of one combination is that the host cell is a non-methylotroph and the transgene is a gene that imparts a function of converting methanol and / or formic acid into formaldehyde, a gene that imparts formaldehyde immobilization ability, and schovenol synthase. A combination that is a gene. That is, even if the host cell is a non-methylotroph, a recombinant cell that is a methylotroph is introduced by introducing a gene that imparts a function of converting methanol and / or formic acid into formaldehyde and a gene that imparts formaldehyde immobilization ability. Can be obtained.
As a similar example, there is a combination in which the host cell is a non-methylotroph having a ribulose monophosphate pathway, and the transgene is a gene that imparts a function of converting methanol and / or formic acid into formaldehyde and a shovenol synthase gene. It is done.

  Use a gene encoding methanol dehydrogenase (for example, EC1.1.1.244, EC1.1.2.7) or a gene encoding alcohol oxidase (for example, EC1.13.13) as a gene that gives the function of converting methanol to formaldehyde Can do. Moreover, a gene encoding formaldehyde dehydrogenase (for example, EC1.2.1.46) can be used as a gene that imparts a function of converting formic acid into formaldehyde. Furthermore, a gene encoding methane monooxygenase can be used as a gene imparting the function of converting methane to methanol.

  As a gene that imparts formaldehyde immobilization ability, for example, a gene encoding an enzyme that acts in the serine pathway, RuMP pathway, or XuMP pathway described above can be used.

  When conferring formaldehyde immobilization ability by the serine pathway, the above-mentioned serine hydroxymethyltransferase (for example, EC2.1.2.1) gene can be used. For example, non-methylotrophs include alcohol dehydrogenase genes such as methanol dehydrogenase, 5,10-methylenetetrahydrofolate (CH2 = H4F) synthase gene, and serine hydroxymethyltransferase (eg EC2.1.2. 1) By introducing a gene or the like, it is possible to impart methanol utilization and formaldehyde immobilization ability by serine pathway.

  In a recombinant cell that is a methylotroph, a gene encoding 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase (6-phosphate-3-hexuloisomerase) And a gene encoding). With this configuration, formaldehyde immobilization ability by the ribulose monophosphate pathway (RuMP pathway) is imparted or enhanced. As a result, the formaldehyde resistance of the recombinant cells can be increased, and as a result, the resistance to methanol and formic acid and the ability to assimilate can be increased. Thereby, the culture efficiency of recombinant cells and the production efficiency of shobunol can be increased.

  The type of methylotroph used as the host cell is not particularly limited, and for example, those classified into bacteria and yeasts can be employed.

  Examples of methylotrophic bacteria include, for example, Methylacidphilum, Methylosinus, Methylocystis, Methylobacterium, Methylocella, Methylococcus, Methylomonas, Methylobacter, Methylobacillus, Methylophilus, Methylotenera, Methylovorus, Genus, Methyloversatilis, Mycobacterium, Arthrobacter, Bacillus, Beggiatoa, Burkholderia, Granulibacter, Hyphomicrobium, Pseudomonas, Achromobactor, Paracoccus, Crenothrix, Clonothrix, Rhodohobacter, Rhodobacter Examples include bacteria belonging to the genus Thiomicrospira, Verrucomicrobia, and the like.

  Examples of methylotrophic yeasts include yeasts belonging to the genus Pichia, Candida, Saccharomyces, Hansenula, Torulopsis, Kloeckera, and the like. Examples of Pichia genus yeast include P. haplophila, P. pastoris, P. trehalophila, P. lindnerii, and the like. Examples of Candida yeast include C. parapsilosis, C. methanolica, C. boidinii, C. alcomigas, and the like. Examples of Saccharomyces yeast include Saccharomyces metha-nonfoams. Examples of Hansenula yeast include H. wickerhamii, H. capsulata, H. glucozyma, H. henricii, H. minuta, H. nonfermentans, H. philodendra, H. polymorpha, and the like. Examples of Torulopsis yeast include T. methanolovescens, T. glabrata, T. nemodendra, T. pinus, T. methanofloat, T. enokii, T. menthanophiles, T. methanosorbosa, T. methanodomercqii, and the like.

  In the present invention, a recombinant cell that is a methylotroph is preferably one belonging to the genus Pichia, Hansenula, or Candida.

  Also in the second embodiment, other genes may be introduced in addition to the above-mentioned genes including the shobunol synthase gene. For example, in one embodiment, a gene encoding at least one enzyme that acts in the farnesyl diphosphate synthesis pathway is further introduced. Examples of the enzyme include GPP synthase, FPP synthase, enzymes that act in the mevalonate pathway, and enzymes that act in the non-mevalonate pathway, as in the first embodiment.

  Also in the second embodiment, the method for introducing a gene 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 gene can be used.

  For example, examples of methods for integrating methylotrophic bacteria into the chromosome include Methylobacillus flagellatus having a ribulose monophosphate pathway and Methylobacterium extorquencs having a serine pathway by disrupting the target gene (Chistoserdova L. et al. , Microbiology 2000, 146, 233-238; Chistoserdov AY., Et al., J. Bacteriol 1994, 176, 4052-4065). These are gene introduction methods into the genome using circular DNA, but in the genus Methylophilus and the like, gene introduction methods into the genome using linear DNA have also been developed (Japanese Patent Application Laid-Open No. 2004-229626). . In general, when recombination by host cells is difficult, genomic recombination with linear DNA is more efficient than with circular DNA. In general, the homologous recombination method preferably targets a gene present in multiple copies on the genome, such as an inverted-repeat sequence. In addition to homologous recombination, methods for introducing multiple copies into the genome include a method for loading in a transposon. As a method for introducing a gene into a methylotrophic bacterium, for example, a broad host range vector pAYC32 (Chistoserdov AY., Et al., Plasmid 1986, 16, 161-167), pRP301 (Lane M., et al., Arch. Microbiol. 1986, 144 (1), 29-34), pBBR1, pBHR1 (Antoine R. et al., Molecular Microbiology 1992, 6, 1785-1799), pCM80 (Marx CJ. Et al., Microbiology 2001, 147, 2065-2075), etc.

  The gene introduction method in methylotrophic yeast is established mainly by Pichia pastoris, and vectors such as pPIC3.5K, pPIC6, pGAPZ, and pFLD (Invitrogen) are commercially available.

As plasmids that can be used for gene transfer into Bacillus bacteria, Bacillus subtilis includes pMTLBS72 (Nquyen HD. Et al., Plasmid 2005, 54 (3), 241-248), pHT01 (Funakoshi), pHT43 (Funakoshi). Bacillus megaterium includes p3STOP1623hp (Funakoshi), pSP _YocH hp (Funakoshi), and Bacillus brevis includes pNI DNA (Takara Bio).

  In the present invention, the above recombinant cell is contacted with at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol. It includes a method for producing shoubnol, which produces nol.

Preferably, the recombinant cell is cultured using at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol as a carbon source, and Shobunol is obtained from the culture. get. 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. For example, it can be cultured under aerobic, microaerobic or anaerobic conditions depending on the purpose. Any of batch culture, fed-batch culture, and continuous culture may be used.

  When the recombinant cell is a Clostridium genus bacterium (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.

Shobanol can also be produced by simply contacting the aforementioned C1 compound with a recombinant cell. For example, the C1 compound can be continuously supplied to immobilized recombinant cells to produce shobunol continuously. Also in this embodiment, 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 and hydrogen or a gas mainly composed of carbon dioxide and hydrogen is provided to the recombinant cell. That is, recombinant cells are cultured using these gases as a carbon source, or these gases are brought into contact with the recombinant cells to produce shovenol from carbon monoxide or carbon dioxide in the gases. Again, hydrogen is used as an energy source.

  Formic acid and / or methanol can be provided to the recombinant cells to produce shovenol from formic acid and / or methanol. In other words, in addition to carbon monoxide or carbon dioxide, or by culturing recombinant cells using formic acid or methanol as a carbon source, or by contacting formic acid or methanol with recombinant cells, Can also produce Shobunol.

  Shobunol biosynthesized by the recombinant is released out of the recombinant cell or accumulated in the cell. In the present invention, any shobunol may be recovered. Specifically, it can be recovered from a microbial cell disruption product, a culture solution (culture supernatant), a gas phase of a culture system, or the like. Preferably, Shobunol released to the outside of the cell is recovered, and specifically, recovered from the culture medium (culture supernatant) or the gas phase of the culture system.

  When recombinant cells are obligate methylotrophs, it is fundamental to use a synthetic medium with C1 compound as the only carbon source, but this includes natural media such as yeast extract, corn steep liquor, meat extract and vitamins. The growth of bacteria is also promoted by adding a small amount. In the case of facultative methylotrophs, substances other than C1 compounds such as carbohydrates and lipids may be used as the carbon source in the cell growth stage, and the carbon source may be changed to the C1 compound in the shovenol production stage. For example, when methanol is used as the carbon source, it is usually used at a concentration of 1.0% (v / v) for bacteria and at a concentration of 3.0% (v / v) or less for yeast. When the resistance to is improved, it can be cultured at higher concentrations.

  Here, when a recombinant cell has an acetyl-CoA pathway (Wood-Ljungdahl pathway) and a methanol pathway (Methanol pathway), a combination of carbon monoxide, carbon dioxide, formic acid, and methanol that can be provided to the recombinant cell will be described. To do.

In the synthesis of acetyl CoA by the acetyl CoA pathway, methyl transferase, corrinoid iron-sulfur protein (CoFeS-P), acetyl CoA synthase (Acetyl-CoA synthase, ACS), and CODH by the action of COA, methyl tetrahydrofolate ( The synthesis process of acetyl CoA from methyltetrahydrofolate, [CH ₃ ] -THF), and CO is essential (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 as a carbon source in addition to CO and CO ₂ is necessary for CO metabolism, that is, tetrahydrofolate content in the methyl branch of the acetyl CoA pathway and CO metabolism It is known to increase the activity of CODH, formate dehydrogenase (FDH) and hydrogenase (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 recombinant cell has an acetyl CoA pathway and a methanol pathway, shobunol can be produced using the following gas or liquid.
・ 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 cell of the present invention is cultured not for the purpose of producing shobunol but exclusively for the purpose of increasing the number of cells, the above C1 compound (carbon monoxide, carbon dioxide, formic acid, methane, or methanol) is used as carbon. There is no need to use it as a 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 these Examples.

  In this example, the base sequence of the vetiver-derived schovenol synthase gene was determined, recombinant Escherichia coli into which the gene was introduced was prepared, and the recombinant Escherichia coli was cultured to perform biosynthesis of schovenol.

(1) Analysis of transcripts of vetiver and determination of the base sequence of schovenol synthase gene (VzTPS1) The root and leaf tissues (FIG. 2) of vetiver zizanioides were cut out and frozen in liquid nitrogen. Total RNA was extracted using a bead crusher (FastPrep 24, MP Biomedicals) and FastRNA Pro Green Kit (MP Biomedicals). Using the Agilent 2100 Bioanalyzer (Agilent Technology) and the Agilent RNA6000 Nano Kit (Agilent Technology), RNA quality was quantitatively evaluated by RNA Integrity Number (RIN), and RIN = Eight or more samples were subjected to subsequent experiments.

  5 μg each of total RNA obtained from roots and leaves was provided to Hokkaido System Science, and RNA sequence analysis, sequence data mixing assembly, and homology search using BlastX were performed. As a result of sequencing and mixed assembly, 177,472 contig sequences were obtained. As a result of homology search, a gene fragment showing 52% homology with Zea mays terpene synthase 7 (Genbank accession DAA38747) and 54%, Aegilops tauschii (+)-delta-cadinene synthase (Genbank accession EMT11542) ( SEQ ID NO: 1) was found. This gene fragment was named VzTPS1. VzTPS1 contained a 1521 bp open reading frame (ORF) and encoded a protein predicted to have 506 amino acid residues, a molecular weight of 58.3 kDa, and a pI = 5.8. The deduced amino acid sequence (SEQ ID NO: 2) of VzTPS1 contained a motif conserved in many terpene synthases, such as RxR and DDxxD. Further, it was predicted that the N-terminal amino acid sequence of VzTPS1 does not contain a plastid translocation sequence.

(2) Preparation of Shobunol synthase gene (VzTPS1) expression plasmid (pVzTPS1)
The deduced amino acid sequence of VzTPS1 (SEQ ID NO: 2) was provided to GenScript, and a VzTPS1 gene optimized for E. coli codon usage was prepared by artificial synthesis (SEQ ID NO: 3). At this time, a restriction enzyme NdeI recognition sequence was added to the 5 ′ end so as to include the initiation methionine codon ATG, and a restriction enzyme XhoI recognition sequence was added immediately below the stop codon. The prepared plasmid containing the artificially synthesized gene was digested with NdeI and XhoI, and the obtained DNA fragment was ligated to the NdeI and XhoI sites of pRSFDuet-1 plasmid (Novagen). This plasmid was named pVzTPS1.

(3) Synthesis of Shobunol by E. coli expression system using plasmid pVzTPS1
pVzTPS1 plasmid was introduced into Escherichia coli together with pAC-Mev plasmid according to the method described in Harada H, Yu F, Okamoto S, Kuzuyama T, Utsumi R, Misawa N 2009, 81: 915-925, and a liquid medium supplemented with dodecane Cultivation, production and extraction of the product were carried out. As a result of analyzing 1 μL of the extracted dodecane layer with a gas chromatograph mass spectrometer (GC-MS), an E. coli-derived sample into which pVzTPS1 plasmid was introduced was found to be an E. coli-derived sample into which pRSFDuet-1 was introduced as a control. A plurality of new peaks that could not be obtained appeared (FIG. 3). As a result of mass spectrum analysis of the peak with the longest retention time of 37.5 minutes (FIG. 2, peak 1), it was shown that this peak coincided with the spectrum of Shobunol (FIG. 4).
From the above results, it was shown that VzTPS1 is an enzyme gene that specifically synthesizes shobunol using farnesyl diphosphate as a substrate.

  Production of methylotroph having serine pathway into which MVA pathway gene and Shobunol synthase gene were introduced, and production of Shobunol from methanol by recombinants

  In this example, Methylobacterium extorquens (ATCC 55366) is used as a methylotroph having a serine pathway, and a recombinant methylotroph that produces shobnol by introducing a VzTPS1 gene and a mevalonate pathway gene cluster derived from actinomycetes into this strain. Was made.

A DNA fragment containing the vetiver-derived VzTPS1 gene was amplified using the pVzTPS1 plasmid prepared in Example 1 as a template and using the primers represented by SEQ ID NO: 4 and SEQ ID NO: 5. This DNA fragment was cloned into the pT7-Blue T vector to construct pT7VzTPS.
DNA fragment containing genes encoding S. griseolosporeus mevalonate pathway enzymes by PCR using Streptomyces griseolosporeus (Kitasatospora griseola) genomic DNA as a template and the primers represented by SEQ ID NO: 6 and SEQ ID NO: 7 (SEQ ID NO: 8) was amplified. This DNA fragment contains gene clusters encoding Mevalonate kinase, Mevalonate diphosphate decarboxylase, Phosphomevalonate kinase, IPP isomerase, HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (HMGR), and HMG-CoA synthase It is. The obtained amplified DNA fragment was cloned into the pT7-Blue T vector to construct pT7SMVA.

  The VzTPS1 gene was excised from pT7VzTPS with BamHI and KpnI and introduced into the BamHI / KpnI site of pCM80 (Marx CJ. et al., Microbiology 2001, 147, 2065-2075), which is a broad host range vector, to prepare pC80VzTPS. By cutting pT7SMVA with KpnI, a fragment containing the actinomycete MVA pathway gene and terminator sequence was excised and introduced into the KpnI site of pC80VzTPS to construct pC80VzTPSMVA. The expression vector pC80VzTPSMVA has a VzTPS1 gene and actinomycetes MVA pathway enzyme gene group downstream of the promoter. The expression vector pC80VzTPSMVA was introduced into M. extorquens by electroporation to obtain a ME-VzTPSMVA strain. As a control, the expression vector pCM80 was introduced into M. extorquens by electroporation to obtain the ME-CM80 strain.

The ME-VzTPSMVA strain or the ME-CM80 strain is synthesized from a synthetic B medium containing methanol as a sole carbon source (18 g of H ₃ PO ₄ , 14.28 g of K ₂ SO ₄ , 3.9 g of KOH, CaSO ₄ .2H ₂ O per liter). _{0.9g, MgSO 4 · 7H 2 O} 11.7g, CuSO 4 · 5H 2 O 8.4mg, KI 1.1mg, MnSO 4 H 2 O 4.2mg, NaMoO 4 · 2H 2 O 0.3mg, H 3 _{BO 3 0.03mg, CoCl 2 · 6H} 2 O 0.7mg, ZnSO 4 · 7H 2 O 28mg, FeSO 4 · 7H 2 O 91mg, at biotin 0.28 mg, methanol 5 mL, and tetracycline 10 mg.) 20 mL The cells were aerobically cultured at 30 ° C. The cells were collected when the OD600 of the culture solution was 1.0-1.2. The collected whole cells were added to 45 mL of the synthetic B medium containing 20% dodecane, and cultured with shaking at 30 ° C. for 16 hours in a 125 mL vial sealed with a butyl rubber stopper. After completion of the culture, the dodecane phase was recovered and the components were analyzed by GC / MS. GC / MS was GCMS-QP2010Ultra (Shimadzu Corporation), and the column was DB-WAX (Agilent Technologies).

  As a result, Shobunol was significantly detected in the ME-VzTPSMVA strain as compared with the ME-CM80 strain. From the above, it was shown that by introducing the MVA pathway gene and the shovenol synthase gene into a methylotroph having a serine pathway, it is possible to efficiently produce shovenol from methanol.

Claims (28)

  A recombinant cell having the ability to synthesize farnesyl diphosphate, comprising a gene encoding a shovenol synthase or a functional fragment thereof, wherein the gene is expressed, carbon monoxide, carbon dioxide, formic acid, methane, and A recombinant cell capable of producing shoubnol from at least one C1 compound selected from the group consisting of methanol. (I) has the ability to synthesize isopentenyl diphosphate by a non-mevalonate pathway, or (ii) synthesizes acetyl CoA from methyltetrahydrofolate, carbon monoxide, and CoA,
The recombinant cell according to claim 1, wherein shobunol can be produced from at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, and methanol.   The recombinant cell according to claim 1 or 2, which has carbon monoxide dehydrogenase.   The recombinant cell according to any one of claims 1 to 3, which has an activity of generating carbon dioxide and hydrogen from carbon monoxide and water.   The recombinant cell according to any one of claims 1 to 4, which is a Clostridium bacterium or a Moorella genus bacterium.   2. The recombinant cell according to claim 1, which is a methylotroph capable of biosynthesizing shovenol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.   The recombinant cell according to claim 6, wherein the formaldehyde immobilization pathway has at least one C1 carbon assimilation pathway selected from the group consisting of a serine pathway, a ribulose monophosphate pathway, and a xylulose monophosphate pathway.   The recombinant cell according to claim 6 or 7, further comprising a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase, wherein the gene is expressed.   The recombinant cell according to any one of claims 6 to 8, which belongs to the genus Pichia, Hansenula, or Candida. The recombinant cell according to any one of claims 1 to 9, wherein the shovenol synthase is composed of 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 an activity of converting farnesyl diphosphate into shovenol,
(C) a protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shobunol.   The recombinant cell according to any one of claims 1 to 10, further comprising a gene encoding at least one enzyme that acts in a synthetic pathway of farnesyl diphosphate, wherein the gene is expressed.   The recombinant cell according to claim 11, wherein the synthesis route of farnesyl diphosphate includes a mevalonate pathway.   The recombinant cell according to claim 12, wherein the mevalonate pathway is an actinomycete mevalonate pathway. A method for producing a recombinant cell, comprising:
Providing a host cell having the ability to synthesize farnesyl diphosphate; and
A second step of introducing a gene encoding a shovenol synthase or a functional fragment thereof into the host cell,
Recombination for producing a recombinant cell in which the gene is expressed in the host cell and produces shobunol from at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol A method for producing cells. The host cell has (i) the ability to synthesize isopentenyl diphosphate by a non-mevalonate pathway, or (ii) the function of synthesizing acetyl CoA from methyltetrahydrofolate, carbon monoxide, and CoA,
The method for producing a recombinant cell according to claim 14, wherein a recombinant cell producing shobnol is produced from at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid and methanol.   The method for producing a recombinant cell according to claim 14 or 15, wherein the recombinant cell has carbon monoxide dehydrogenase.   The method for producing a recombinant cell according to any one of claims 14 to 16, wherein the recombinant cell has an activity of generating carbon dioxide and hydrogen from carbon monoxide and water.   The method for producing a recombinant cell according to any one of claims 14 to 17, wherein the recombinant cell is a Clostridium bacterium or a Moorella genus bacterium.   The recombinant cell according to claim 14, wherein the recombinant cell is a methylotroph that can biosynthesize shovenol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. Manufacturing method.   The combination according to claim 19, wherein the recombinant cell has at least one C1 carbon assimilation pathway selected from the group consisting of serine pathway, ribulose monophosphate pathway, and xylulose monophosphate pathway as a formaldehyde immobilization pathway. Method for producing replacement cells.   20. The method further comprises the step of introducing a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase, and the gene is expressed in the host cell. Or the method for producing a recombinant cell according to 20,   The method for producing a recombinant cell according to any one of claims 19 to 21, wherein the recombinant cell belongs to the genus Pichia, Hansenula, or Candida. The method for producing a recombinant cell according to any one of claims 14 to 22, wherein the shovenol synthase is composed of 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 an activity of converting farnesyl diphosphate into shovenol,
(C) a protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shobunol.   24. The recombination according to any one of claims 14 to 23, further comprising the step of introducing a gene encoding at least one enzyme acting in the synthetic pathway of farnesyl diphosphate, wherein the gene is expressed in the host cell. A method for producing cells.   The method for producing a recombinant cell according to claim 24, wherein the synthesis route of farnesyl diphosphate includes the mevalonate pathway.   The method for producing a recombinant cell according to claim 25, wherein the mevalonate pathway is an actinomycete mevalonate pathway.   A recombinant cell according to any one of claims 1 to 13 or a recombinant cell produced by the method according to any one of claims 14 to 26, from carbon monoxide, carbon dioxide, formic acid, methane, and methanol. A method for producing shobunol, wherein at least one C1 compound selected from the group consisting of the above-mentioned groups is brought into contact with each other, and the recombinant cell is made to produce shobunol from the C1 compound.   The recombinant cell is cultured using at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, methane, and methanol as a carbon source, and shobunol is obtained from the culture. Item 28. A method for producing Shobunol according to Item 27.