JP2014155455A - RECOMBINANT CELLS AND METHOD FOR PRODUCING CROTONYL-CoA OR CROTYL ALCOHOL - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a series of technologies to produce crotonyl-CoA or crotyl alcohol from methanol etc.SOLUTION: A recombinant cell is prepared by introducing to a methylotroph host cell a gene encoding at least one enzyme selected from the group consisting of acetoacetyl-CoA thiolase, 3-hydroxybutyl-CoA dehydrogenase, and 3-hydroxybutyrate-CoA dehydratase to be expressed in the host cell. The recombinant cell can produce crotonyl-CoA from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde and formamide.

Description

  The present invention relates to a recombinant cell capable of producing crotonyl CoA or crotyl alcohol from methanol or the like, and a method for producing crotonyl CoA or crotyl alcohol using the recombinant cell.

  Crotyl alcohol and Crotonyl-CoA are organic compounds that can be an important raw material for butadiene as a monomer for synthetic rubber, and are particularly important materials in the tire industry. In recent years, the development and practical application of technology for converting from the production process of basic chemicals that depend on petroleum to the production process of microorganisms from renewable resources such as plant resources has been steadily progressing. A biosynthetic hypothesis for achieving its production by microorganisms has also been shown for butadiene (Patent Document 1).

  An example of the hypothesis pathway of crotyl alcohol described in Patent Document 1 is shown in FIG. As shown in FIG. 1, crotyl alcohol can be biosynthesized using, for example, acetyl CoA as a starting material. In this route, first, acetyl CoA is converted to crotonyl CoA via acetoacetyl CoA and 3-hydroxybutyric acid CoA. Enzymes that catalyze each reaction are (a) acetoacetyl-CoA thiolase, (b) 3-hydroxybutyryl-CoA dehydrogenase, (c) 3- Hydroxybutyrate CoA dehydratase (3-Hydroxybutyryl-CoA dehydratase) (FIG. 1).

  Furthermore, crotonyl CoA is converted to crotyl alcohol via crotonaldehyde. Enzymes that catalyze each reaction are (d) crotonyl-CoA reductase (aldehyde-forming) and (e) crotonaldehyde reductase (alcohol forming), respectively (Fig. 1).

  There is also a route where crotonyl CoA can be converted directly to crotyl alcohol. This reaction is catalyzed by (f) Crotonyl-CoA reductase (alcohol forming) (FIG. 1).

  Furthermore, there is a pathway in which crotonyl CoA is converted to crotyl alcohol via crotonic acid and crotonaldehyde. Enzymes that catalyze each reaction are (g) crotonyl-CoA hydrolase, (h) crotonate reductase, and (e) crotonaldehyde reductase, respectively (FIG. 1). .

By the way, among C1 compounds, methanol is inexpensively produced from natural gas and synthesis gas, which is a mixed gas of carbon monoxide, carbon dioxide, and hydrogen obtained by incineration of waste such as biomass and municipal waste. Is done. Natural gas is abundant in fossil resources, and the amount of CO ₂ generated is relatively small. Therefore, natural gas is attracting attention as a next-generation energy source, and the transition from conventional oil to natural gas is progressing. 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. Many bacterial methylotrophs can assimilate methane, and these are often called mesanotrophs.

  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. As shown in FIG. 2, 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.

International Publication No. 2011/140171

  Regarding the production process from renewable resources, most of the prior art is a production method using microorganisms, which depends on organic substances, particularly sugar, glycerol or oil components. However, the amount of sugar, glycerin and oil components that can be used at present from plant resources is insufficient as a carbon source for microorganisms in order to cover the global production of many basic chemicals derived from petroleum. Is essential. That is, the amount of basic chemicals produced by microorganisms that depend on carbohydrates and oil components is limited in the future. In addition, such a process is also concerned about competition with food.

  In view of the above situation, an object of the present invention is to provide a series of techniques for producing crotonyl CoA or crotyl alcohol, which can be a raw material of butadiene, from methanol or the like.

  One aspect of the present invention for solving the above-described problems is that a host cell that is a methylotroph is divided into a group consisting of acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, and 3-hydroxybutyrate CoA dehydratase. A gene encoding at least one enzyme selected from the group is introduced, the gene is expressed in the host cell, and at least selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide It is a recombinant cell capable of producing crotonyl CoA from one C1 compound.

The present invention relates to a recombinant cell capable of producing crotonyl CoA.
As shown in FIG. 1, crotonyl CoA can be biosynthesized from acetyl CoA. The recombinant cell of the present invention comprises an enzyme group acting in a biosynthetic pathway from acetyl CoA to crotonyl CoA, that is, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, and 3-hydroxybutyrate CoA dehydra. A gene encoding at least one enzyme selected from the group consisting of a tase is introduced into a host cell that is a methylotroph, and the gene is expressed in the host cell. Then, crotonyl CoA can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
According to the recombinant cell of the present invention, the above-mentioned C1 is obtained via acetyl CoA based on the “function of converting methanol and / or formic acid into formaldehyde” and “formaldehyde immobilization ability” inherent to methylotrophs. Crotonyl CoA can be produced (expressed) from a compound.

  Another aspect of the present invention for solving the same problem is that a host cell that is a methylotroph is treated with acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA reduction. A gene encoding at least one enzyme selected from the group consisting of an enzyme (aldehyde producing type) and a crotonaldehyde reductase (alcohol producing type) is introduced, and the gene is expressed in the host cell, , A recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of methanol, methylamine, formic acid, formaldehyde, and formamide.

The present invention relates to a recombinant cell capable of producing crotyl alcohol.
As shown in FIG. 1, crotyl alcohol can be biosynthesized from acetyl CoA via crotonyl CoA and crotonaldehyde. The recombinant cell of the present invention is a group of enzymes acting in the biosynthetic pathway from acetyl CoA to crotyl alcohol, that is, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydrogenase. A gene encoding at least one enzyme selected from the group consisting of hydratase, crotonyl-CoA reductase (aldehyde-generating type), and crotonaldehyde reductase (alcohol-generating type) is introduced into a host cell that is a methylotroph And the gene is expressed in the host cell. Then, crotyl alcohol can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
According to the recombinant cell of the present invention, methylotrophic inherently has the "function of converting methanol and / or formic acid into formaldehyde" and "formaldehyde immobilization ability", via acetyl CoA and crotonyl CoA, Crotyl alcohol can be produced (expressed) from the aforementioned C1 compound.

  Another aspect of the present invention for solving the same problem is that a host cell that is a methylotroph is divided into acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, and crotonyl CoA. A gene encoding at least one enzyme selected from the group consisting of a reductase (alcohol-generating type) is introduced, the gene is expressed in the host cell, methane, methanol, methylamine, formic acid, formaldehyde, And a recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of formamide.

As mentioned above, there is also a pathway that can convert crotonyl CoA directly to crotyl alcohol (FIG. 1). In the recombinant cell of the present invention, crotonyl CoA is biosynthesized from acetyl CoA, and further, an enzyme group acting in a biosynthetic pathway in which crotonyl CoA is directly converted to crotyl alcohol, that is, acetoacetyl CoA thiolase, 3-hydroxy A gene encoding at least one enzyme selected from the group consisting of butyryl-CoA dehydrogenase, 3-hydroxybutyrate-CoA dehydratase, and crotonyl-CoA reductase (alcohol-producing type) is introduced into a host cell that is a methylotroph. And the gene is expressed in the host cell. Then, crotyl alcohol can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
Even with the recombinant cell of the present invention, the above-mentioned “function of converting methanol and / or formic acid to formaldehyde” and “formaldehyde immobilization ability” inherently possessed by methylotrophs, via acetyl CoA and crotonyl CoA, Crotyl alcohol can be produced (expressed) from the obtained C1 compound.

  Another aspect of the present invention for solving the same problem is that a host cell that is a methylotroph is made to contain acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA hydrolase. A gene encoding at least one enzyme selected from the group consisting of lysase, crotonic acid reductase, and crotonaldehyde reductase (alcohol-generating type) is introduced, and the gene is expressed in the host cell, , A recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of methanol, methylamine, formic acid, formaldehyde, and formamide.

As described above, there is also a pathway in which crotonyl CoA is converted to crotyl alcohol via crotonic acid and crotonaldehyde (FIG. 1). In the recombinant cell of the present invention, crotonyl CoA is biosynthesized from acetyl CoA, and crotonyl CoA is further converted into crotyl alcohol via crotonic acid and crotonaldehyde. Selected from the group consisting of acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA hydrolase, crotonic acid reductase, and crotonaldehyde reductase (alcohol-generating) Further, a gene encoding at least one enzyme is introduced into a host cell that is a methylotroph, and the gene is expressed in the host cell. Then, crotyl alcohol can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
Even with the recombinant cell of the present invention, the above-mentioned “function of converting methanol and / or formic acid to formaldehyde” and “formaldehyde immobilization ability” inherently possessed by methylotrophs, via acetyl CoA and crotonyl CoA, Crotyl alcohol can be produced (expressed) from the obtained C1 compound.

  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 xylose monophosphate pathway.

  Preferably, a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase are further introduced, and the gene is expressed in the host cell.

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

  Preferably, the host cell is methanol-assimilating yeast, and a gene encoding an enzyme that converts methanol into formaldehyde by dehydrogenation is further introduced, and the gene is expressed in the host cell.

  In general, yeast is highly resistant to alcohol. Therefore, in this aspect, methanol-assimilating yeast is adopted as a host, and resistance to methanol and crotyl alcohol in the recombinant cells is increased. Further, in yeast, alcohol oxidase is generally responsible for the conversion reaction from methanol to formaldehyde. For this reason, oxygen is required for the conversion reaction, and specifically, it is necessary to aerate vigorously during the culture. Therefore, in this aspect, a gene encoding “enzyme that converts methanol into formaldehyde by dehydrogenation” is introduced so that the conversion reaction from methanol to formaldehyde can be performed without relying on oxygen.

  Another aspect of the present invention for solving the same problem is that a host cell is provided with a gene that functions to convert methanol and / or formic acid into formaldehyde, a gene that imparts formaldehyde immobilization ability, and acetoacetyl-CoA. A gene encoding at least one enzyme selected from the group consisting of thiolase, 3-hydroxybutyryl CoA dehydrogenase, and 3-hydroxybutyrate CoA dehydratase, and the gene is introduced into the host cell. And is capable of producing crotonyl CoA from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.

In the recombinant cell of the present invention, “a gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and “a gene that imparts formaldehyde immobilization ability” are introduced into a host cell, and further, acetyl CoA to crotyl are introduced. Enzymes acting in the biosynthetic pathway leading to alcohol, that is, at least one enzyme selected from the group consisting of acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, and 3-hydroxybutyrate CoA dehydratase Has been introduced. Then, crotonyl CoA can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
That is, the recombinant cell of the present invention has the same characteristics as a methylotroph because a “gene that imparts a function of converting methanol and / or formic acid to formaldehyde” and a “gene that imparts formaldehyde immobilization ability” have been introduced. Have. Then, based on the “function of converting methanol and / or formic acid to formaldehyde” and “formaldehyde immobilization ability” imparted by these foreign genes, crotonyl CoA is produced from the aforementioned C1 compound via acetyl CoA. (Expression).

  Another aspect of the present invention for solving the same problem is that a host cell is provided with a gene that functions to convert methanol and / or formic acid into formaldehyde, a gene that imparts formaldehyde immobilization ability, and acetoacetyl-CoA. At least one selected from the group consisting of thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA reductase (aldehyde-generating type), and crotonaldehyde reductase (alcohol-generating type) From at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. A group capable of producing crotyl alcohol For example a cell.

In the recombinant cell of the present invention, a “gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and a “gene that imparts formaldehyde immobilization ability” are introduced into a host cell, and further, acetyl CoA to crotonyl CoA. And enzymes acting in the biosynthetic pathway from crotonaldehyde to crotyl alcohol, that is, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA reductase ( A gene encoding at least one enzyme selected from the group consisting of an aldehyde-forming type) and a crotonaldehyde reductase (alcohol-generating type). Then, crotyl alcohol can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
That is, the recombinant cell of the present invention also has the same characteristics as a methylotroph because a “gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and a “gene that imparts formaldehyde immobilization ability” have been introduced. Have. Then, based on the “function of converting methanol and / or formic acid into formaldehyde” and “formaldehyde immobilization ability” imparted by these foreign genes, acetyl CoA and crotonyl CoA are used to clot from the above-mentioned C1 compound. Chill alcohol can be produced (expressed).

  Another aspect of the present invention for solving the same problem is that a host cell is provided with a gene that functions to convert methanol and / or formic acid into formaldehyde, a gene that imparts formaldehyde immobilization ability, and acetoacetyl-CoA. A gene encoding at least one enzyme selected from the group consisting of thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, and crotonyl CoA reductase (alcohol-generating type) A recombinant cell in which the gene is expressed in a host cell and can produce crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide It is.

In the recombinant cell of the present invention, a “gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and a “gene that imparts formaldehyde immobilization ability” are introduced into a host cell, and further, acetyl CoA to crotonyl CoA. A group of enzymes that act in the biosynthetic pathway leading to crotyl alcohol, ie, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, and crotonyl CoA reductase (alcohol production) A gene encoding at least one enzyme selected from the group consisting of: Then, crotyl alcohol can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
That is, the recombinant cell of the present invention also has the same characteristics as a methylotroph because a “gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and a “gene that imparts formaldehyde immobilization ability” have been introduced. Have. Then, based on the “function of converting methanol and / or formic acid into formaldehyde” and “formaldehyde immobilization ability” imparted by these foreign genes, acetyl CoA and crotonyl CoA are used to clot from the above-mentioned C1 compound. Chill alcohol can be produced (expressed).

  Another aspect of the present invention for solving the same problem is that a host cell is provided with a gene that functions to convert methanol and / or formic acid into formaldehyde, a gene that imparts formaldehyde immobilization ability, and acetoacetyl-CoA. At least one selected from the group consisting of thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA hydrolase, crotonic acid reductase, and crotonaldehyde reductase (alcohol producing type) A gene encoding one enzyme, and the gene is expressed in a host cell and is cloned from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. Can produce chilled alcohol It is a recombinant cell.

In the recombinant cell of the present invention, a “gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and a “gene that imparts formaldehyde immobilization ability” are introduced into a host cell, and further, acetyl CoA to crotonyl CoA. , Crotonic acid, and enzymes acting in the biosynthetic pathway from crotonaldehyde to crotyl alcohol, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA hydro A gene encoding at least one enzyme selected from the group consisting of lyase, crotonic acid reductase, and crotonaldehyde reductase (alcohol-producing type) has been introduced. Then, crotyl alcohol can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
That is, the recombinant cell of the present invention also has the same characteristics as a methylotroph because a “gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and a “gene that imparts formaldehyde immobilization ability” have been introduced. Have. Then, based on the “function of converting methanol and / or formic acid into formaldehyde” and “formaldehyde immobilization ability” imparted by these foreign genes, acetyl CoA and crotonyl CoA are used to clot from the above-mentioned C1 compound. Chill alcohol can be produced (expressed).

  Preferably, the gene imparting formaldehyde immobilization ability is a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase.

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

  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 xylose monophosphate pathway.

  Another aspect of the present invention for solving the same problem is that a host cell having a ribulose monophosphate pathway has a gene for imparting a function of converting methanol and / or formic acid to formaldehyde, an acetoacetyl CoA thiolase, 3- A gene encoding at least one enzyme selected from the group consisting of hydroxybutyryl CoA dehydrogenase and 3-hydroxybutyrate CoA dehydratase, and the gene is expressed in the host cell; A recombinant cell capable of producing crotonyl CoA from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.

  Another aspect of the present invention for solving the same problem is that a host cell having a ribulose monophosphate pathway has a gene for imparting a function of converting methanol and / or formic acid to formaldehyde, an acetoacetyl CoA thiolase, 3- Codes at least one enzyme selected from the group consisting of hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA reductase (aldehyde-generating type), and crotonaldehyde reductase (alcohol-generating type) And the gene is expressed in the host cell to produce crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. It is a recombinant cell that is possible.

  Another aspect of the present invention for solving the same problem is that a host cell having a ribulose monophosphate pathway has a gene for imparting a function of converting methanol and / or formic acid to formaldehyde, an acetoacetyl CoA thiolase, 3- A gene encoding at least one enzyme selected from the group consisting of hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, and crotonyl CoA reductase (alcohol-generating type), A recombinant cell in which a gene is expressed in a host cell and can produce crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.

  Another aspect of the present invention for solving the same problem is that a host cell having a ribulose monophosphate pathway has a gene for imparting a function of converting methanol and / or formic acid to formaldehyde, an acetoacetyl CoA thiolase, 3- Codes at least one enzyme selected from the group consisting of hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA hydrolase, crotonic acid reductase, and crotonaldehyde reductase (alcohol producing type) Crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide is expressed in the host cell. Recombinant cells that can be produced It is.

  These aspects correspond to, for example, an embodiment in which a non-methylotroph having a ribulose monophosphate pathway is a host cell.

  Preferably, a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase are further introduced, and the gene is expressed in the host cell.

  Preferably, the gene imparting the function of converting methanol to formaldehyde is a gene encoding methanol dehydrogenase or alcohol oxidase, and the gene imparting the function of converting formic acid to formaldehyde is a gene encoding formaldehyde dehydrogenase.

  Both methanol dehydrogenase and alcohol dehydrogenase have an action of converting methanol into formaldehyde. Formaldehyde dehydrogenase has a function of converting formic acid into formaldehyde. These enzymes are all methane metabolizing enzymes in methylotrophs belonging to bacteria. On the other hand, methylotrophs belonging to yeast do not have methane oxidation activity, but have an action of converting methanol into formaldehyde by the action of alcohol oxidase. Yeast also has an enzymatic activity to convert formic acid into formaldehyde.

  Preferably, a gene imparting a function of converting methane to methanol is further introduced, and the gene is expressed in the host cell.

  Preferably, the gene imparting the function of converting methane to methanol is a gene encoding methane monooxygenase.

  Methane monooxygenase has an action of converting methane to methanol. Methane monooxygenase is also one of the methane metabolizing enzymes in methylotrophs.

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

  With this configuration, the introduced gene is more stably retained in the recombinant cell.

  Preferably, it has a resistance to at least 2% (v / v) methanol.

  With this configuration, crotonyl CoA or crotyl alcohol can be produced in a larger amount.

  In another aspect of the present invention, the above recombinant cell is cultured using at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide as a carbon source, This is a method for producing crotonyl CoA or crotyl alcohol by causing a recombinant cell to produce crotonyl CoA or crotyl alcohol.

  The present invention relates to a method for producing crotonyl CoA or crotyl alcohol. In the present invention, by culturing the above-described recombinant cell using at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide as a carbon source, CoA or crotyl alcohol is produced. According to the present invention, crotonyl CoA or crotyl alcohol can be produced from methanol or the like.

  In another aspect of the present invention, the recombinant cell is contacted with at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide, and the recombinant cell is contacted with the aforementioned recombinant cell. This is a method for producing crotonyl CoA or crotyl alcohol in which crotonyl CoA or crotyl alcohol is produced from a C1 compound.

  In the present invention, the above-described recombinant cell is contacted with at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide, and from the C1 compound, crotonyl CoA or crotyl alcohol is contacted. To produce. Also according to the present invention, crotonyl CoA or crotyl alcohol can be produced from methanol or the like.

  According to the present invention, crotonyl CoA or crotyl alcohol can be produced (expressed) from methane, methanol, methylamine, formic acid, formaldehyde, or formamide using recombinant cells. For example, the recombinant cell of the present invention can be applied to butadiene production.

It is explanatory drawing showing the metabolic pathway from acetyl CoA to crotonyl CoA or crotyl alcohol. It is explanatory drawing showing the carbon assimilation metabolic pathway through formaldehyde.

  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 basically has a biosynthetic pathway from acetyl CoA to crotonyl CoA to a host cell having “a function of converting methanol and / or formic acid into formaldehyde” and “formaldehyde immobilization ability”. A gene encoding an enzyme group that acts, or a gene encoding an enzyme group that acts in a biosynthetic pathway from acetyl CoA through crotonyl CoA to crotyl alcohol is introduced.

  The host cells employed in the present invention include both host cells that are methylotrophs and a wide range of host cells including non-methylotrophs.

  As described above, methylotroph is a C1 compound that uses a carbon compound having no C—C bond in the molecule, such as methane, methanol, methylamine, dimethylamine, and trimethylamine, as a sole carbon source and energy source. It refers to a chemical microorganism. In general, methylotrophs inherently have a carbon assimilation metabolic pathway through formaldehyde, specifically, a function (route) for converting methanol and / or formic acid to formaldehyde and a formaldehyde immobilization ability (formaldehyde immobilization pathway). I have it.

  The formaldehyde immobilization pathway includes the serine pathway, ribulose monophosphate pathway (RuMP pathway), and xylulose monophosphate pathway (XuMP pathway) shown in FIG. In general, methylotrophs possess a serine pathway, a RuMP pathway, or a XuMP pathway as a carbon assimilation metabolic pathway via formaldehyde.

Here, each formaldehyde fixation path | route (FIG. 2) is demonstrated.
An important reaction for formaldehyde fixation by the serine pathway is a serine production reaction from glycine and 5,10-methylene-tetrahydrofolic acid by serine hydroxymethyltransferase. 5,10-methylene-tetrahydrofolic acid is formed by the combination of formaldehyde and tetrahydrofolic acid. In the serine pathway, one molecule of acetyl CoA is directly generated from one molecule of formaldehyde.

The important reaction for formaldehyde fixation by the RuMP pathway is ribulose 5-phosphate (Ru5P) and formaldehyde by 3-hexulose-6-phosphate synthase (hereinafter sometimes abbreviated as “HPS”). Of D-arabino 3 hexulose 6-phosphate from lysine and D-arabino 3 hexulose by 6-phospho-3-hexuloisomerase (hereinafter sometimes abbreviated as “PHI”) It is a production reaction of fructose 6-phosphate (F6P) from 6-phosphate.
F6P and the like produced by this pathway are also used for glycolysis, and then produce acetyl CoA, glyceraldehyde triphosphate (G3P), and pyruvic acid. In the case of F6P, it is converted into two molecules of G3P per molecule, and then two molecules of acetyl CoA are generated via two molecules of pyruvic acid.

  An important reaction for formaldehyde fixation by the XuMP pathway is the formation reaction of dihydroxyacetone (Du) and glyceraldehyde triphosphate (G3P) from xylulose pentaphosphate (Xu5P) and formaldehyde by dihydroxyacetone synthase. . G3P produced by this pathway is also used for glycolysis and converted to pyruvic acid and acetyl CoA. Dihydroxyacetone can also be subjected to glycolysis by phosphorylation and converted to G3P, pyruvate, and acetyl CoA.

  The recombinant cell of the present invention is capable of producing crotonyl CoA or crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. For example, in the case of a recombinant cell having methanol dehydrogenase or alcohol oxidase, methanol can be converted to formaldehyde.

  In the case of a recombinant cell having methane monooxygenase in addition to methanol dehydrogenase or alcohol oxidase, methane can be converted to methanol, and subsequently methanol can be converted to formaldehyde.

  Furthermore, in the case of recombinant cells with formaldehyde dehydrogenase, formic acid can be converted to formaldehyde.

  In general, methylotrophs (methylotrophic bacteria) classified as bacteria have methane monooxygenase and methanol dehydrogenase, so that formaldehyde can be synthesized from methane or methanol. In addition, methylotrophs (methylotrophic yeasts) classified as yeast have alcohol oxidase, so that formaldehyde can be synthesized from methanol. Moreover, methylotroph has formaldehyde dehydrogenase and can convert formic acid into formaldehyde.

  The methanol dehydrogenase includes pyrroloquinoline quinone (PQQ) -dependent methanol dehydrogenase found in methylotrophs of gram-negative bacteria, NAD (P) -dependent methanol dehydrogenase and alcohol dehydrogenase found in methylotrophs of gram-positive bacteria, gram-positive Contains DMNA (N, N'-dimethyl-4-nitrosoaniline) -dependent methanol oxidoreductase (Park H. et al., Microbiology 2010, 156, 463-471) found in bacterial methylotrophs, from methanol in yeast Conversion to formaldehyde is usually catalyzed by alcohol oxidase, which is oxygen dependent.

In the case of a recombinant cell having amine oxidase or methylamine dehydrogenase, methylamine can be converted to formaldehyde. These enzymes are known to be possessed by some methylotrophs and bacteria belonging to the genus Arthrobacter (Anthony C., The Biochemistry of Methylotroph, 1982, Academic Press Inc.).
An enzyme that converts formamide to formaldehyde has been found in some microorganisms (Anthony C., The Biochemistry of Methylotroph, 1982, Academic Press Inc.).

  Then, crotonyl CoA or crotyl alcohol can be produced via formaldehyde.

  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.

  When the host cell is a non-methylotroph, it does not necessarily have a pathway for converting methanol or the like to formaldehyde, so it is necessary to provide at least a “function for converting methanol and / or formic acid to formaldehyde”. . Furthermore, it is preferable to provide “a function of converting methane to methanol”. These functions can be imparted by introducing a gene encoding the above-described enzyme into a host cell.

  For example, as a gene imparting a function of converting methanol to formaldehyde, 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) Can be used. 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.

  Moreover, a plasmid that imparts methanol assimilation is known. For example, the assimilation ability of Bacillus methanolicus depends on a plasmid encoding an enzyme group involved in methanol metabolism (Brautaset T. et al., J. Bacteriology 2004, 186 (5), 1229-1238). By introducing such a plasmid into a related non-methylotroph, it is possible to confer methanol assimilation ability. Furthermore, by modifying such a plasmid, it is possible to impart methanol-assimilating properties to various non-methylotrophs.

  As described above, non-methylotrophs are treated in the same way as methylotrophs by providing them with the function of converting methanol and / or formic acid into formaldehyde, and further by providing the ability to immobilize formaldehyde. Is possible. The formaldehyde-immobilizing ability can be imparted, for example, by introducing a gene encoding an enzyme that acts in the serine pathway, RuMP pathway, or XuMP pathway described above into a non-methylotroph.

  In one preferred embodiment, the host cell is a methanol-assimilating yeast, a gene encoding an enzyme that converts methanol into formaldehyde by dehydrogenation is further introduced, and the gene is expressed in the host cell. For example, a target recombinant cell can be obtained by using a Pichia genus yeast having methanol assimilation property as a host cell and further introducing a methanol dehydrogenase gene. According to this embodiment, it is possible to obtain a recombinant cell capable of producing crotonyl CoA or crotyl alcohol, which has high alcohol resistance and can convert methanol into formaldehyde without depending on oxygen.

  The case where a RuMP route is assigned will be further described as an example. The provision of the RuMP pathway is, for example, the above-described 3-hexulose 6-phosphate synthase (HPS; eg EC4.1.2.43) gene and 6-phospho-3-hexoisomerase (PHI; eg EC5.3.1.27). This can be realized by introducing a gene. That is, ribulose 5-phosphate (Ru5P) and fructose 6-phosphate (F6P), which are substrates or products of formaldehyde immobilization reaction by HPS / PHI, are all biological organisms as metabolic intermediates of the pentose phosphate pathway and the calvin cycle. Exists universally. Therefore, by introducing HPS / PHI, it is possible to impart formaldehyde fixing ability to all organisms including Escherichia coli, Bacillus subtilis, yeast and the like.

  You may introduce | transduce a HPS gene and a PHI gene into the host cell which originally has RuMP pathway. Thereby, the ability to fix formaldehyde by the RuMP pathway can be enhanced. For example, an alcohol dehydrogenase such as methanol dehydrogenase (for example, EC1.1.1.244, EC1.1.2.7), 3-hexulose, or the like, which has a RuMP pathway or a pathogen of the same quality as Bacillus subtilis. By introducing a gene encoding an enzyme such as 6-phosphate synthase (HPS; eg EC4.1.2.43), 6-phospho-3-hexoisomerase (PHI; eg EC5.3.1.27), methanol In addition to imparting a function of converting aldehyde into formaldehyde (that is, methanol assimilation), the ability to fix formaldehyde can be enhanced.

  In addition, you may introduce | transduce an HPS gene and a PHI gene into the host cell which is a methylotroph. That is, by introducing HPS / PHI into a methylotroph having a serine pathway, RuMP pathway, or XuMP pathway, the ability to immobilize formaldehyde by the RuMP pathway can be 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. This makes it possible to improve the culture efficiency of recombinant cells and the production efficiency of crotonyl CoA or crotyl alcohol.

  On the other hand, 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 the recombinant cell of the present invention, a gene encoding an enzyme group that acts in a biosynthetic pathway from acetyl CoA to crotonyl CoA (hereinafter sometimes referred to as “crotonyl CoA biosynthesis-related enzyme (group)”), or A gene encoding an enzyme group acting in a biosynthetic pathway from acetyl CoA to crotyl alcohol (hereinafter sometimes referred to as “crotyl alcohol biosynthesis-related enzyme (group)”) has been introduced. The enzymes shown in (a) to (c) of FIG. 1 correspond to crotonyl CoA biosynthesis-related enzymes. The enzymes shown in FIGS. 1A to 1E correspond to crotyl alcohol biosynthesis-related enzymes.

  In one aspect, the group of crotonyl CoA biosynthesis-related enzymes includes (a) acetoacetyl CoA thiolase, (b) 3-hydroxybutyryl CoA dehydrogenase, and (c) 3-hydroxybutyrate CoA dehydratase. A gene encoding at least one selected enzyme has been introduced into the host cell. 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 another aspect, crotyl alcohol biosynthesis-related enzymes include (a) acetoacetyl CoA thiolase, (b) 3-hydroxybutyryl CoA dehydrogenase, (c) 3-hydroxybutyrate CoA dehydratase, (d A gene encoding at least one enzyme selected from the group consisting of:) crotonyl CoA reductase (aldehyde-generating type) and (e) crotonaldehyde reductase (alcohol-generating type) has been introduced into the host cell. 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 still another aspect, crotyl alcohol biosynthesis-related enzymes include (a) acetoacetyl CoA thiolase, (b) 3-hydroxybutyryl CoA dehydrogenase, (c) 3-hydroxybutyrate CoA dehydratase, and (F) A gene encoding at least one enzyme selected from the group consisting of crotonyl CoA reductase (alcohol-producing type) is introduced into a host cell. 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 still another aspect, crotyl alcohol biosynthesis-related enzyme groups include (a) acetoacetyl CoA thiolase, (b) 3-hydroxybutyryl CoA dehydrogenase, (c) 3-hydroxybutyrate CoA dehydratase, ( g) A gene encoding at least one enzyme selected from the group consisting of crotonyl CoA hydrolase, (h) crotonic acid reductase, and (e) crotonaldehyde reductase (alcohol-producing type) is introduced into a host cell. ing. 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.

  These enzymes (crotonyl CoA biosynthesis-related enzyme and crotyl alcohol biosynthesis-related enzyme) are not particularly limited as long as the enzyme activity can be exhibited in a recombinant cell. The same applies to genes encoding these enzymes, and there is no particular limitation as long as it is normally transcribed and translated in a recombinant cell. It may be derived from the host cell or may be derived from other sources.

  Specific examples of the crotonyl CoA biosynthesis-related enzyme, crotyl alcohol biosynthesis-related enzyme, and genes thereof include those disclosed in Patent Document 1. More specifically, the following enzymes are mentioned. In addition, when the host cell originally has the enzyme shown below, an enzyme gene that is more excellent in substrate specificity, molecular activity, and stability, that is, having a higher Kcat / Km value or the like may be introduced. In this case, the enzyme gene includes a gene encoding a modified enzyme originally possessed by the host cell.

(A) Acetoacetyl-CoA thiolase (also called Acetyl-CoA acyltransferase) (for example, EC 2.3.1.9)
Examples of genes (both indicated by UniProtKB / Swiss-Prot No.): P76461 (E. coli-atoB); P45359 (Clostridium acetobutylicum-thlA); P45855 (Bacillus subtilis-mmgA); Q18AR0 (Clostridium difficile-thlA); Q9I2A8 (Pseudomonas aeruginosa-atoB) etc.

(B) 3-hydroxybutyryl-CoA dehydrogenase (for example, EC 1.1.1.157, 1.1.1.35, etc.)
Examples of genes (both indicated by UniProtKB / Swiss-Prot No.): P52041 (Clostridium acetobutylicum-hbd); P45364 (Clostridium difficile-hbd); P77339 (Escherichia coli-fadJ); O53753 (Mycobacterium tuberculosis-fadB2), etc.

(C) 3-hydroxybutyryl-CoA dehydratase (also called crotonase) (for example, EC 4.2.1.55, etc.)
Examples of genes (both indicated by UniProtKB / Swiss-Prot No.): P52046 (Clostridium acetobutylicum-crt); P45361 (Clostridium difficile-crt); B9J125 (Bacillus cereus-crt), etc.

(D) Crotonyl-CoA reductase (aldehyde forming).
This enzyme is an enzyme belonging to acyl-CoA reductase, and those showing crotonyl-CoA reductive activity correspond to, for example, EC 1.2.1.50, 1.2.1.n2, 1.2.1.76, etc. in the enzyme classification.
Examples of genes (both expressed as UniProtKB / Swiss-Prot No.): P94129 (Acinetobacter baylyi-acr1); P23113 (Photorhabdus luminescens-luxC); P08639 (Vibrio harveyi -luxC); Q97GS8 (Clostridium acetobutylicum-947) (Clostridium kluyveri-sucD) etc.

(E) Crotonaldehyde reductase (alcohol producing type)
This enzyme is an alcohol dehydrogenase (e.g., EC 1.1.1.2, 1.1.1.-, etc.) that acts on alcohol having about C2-C14.
Examples of genes (both expressed as UniProtKB / Swiss-Prot No.): Q9F1R1 (Acinetobacter sp.-Alr-A); Q46856 (Escherichia coli-yqhD); Q04944 (Clostridium acetobutylicum-bdhA); Q04945 ((Clostridium acetobutylic bdhB) etc.

(F) Crotonyl CoA reductase (alcohol-generating type)
This enzyme can directly generate crotyl alcohol from crotonyl CoA, and belongs to a group of enzymes (for example, EC 1.1.1.1, 1.2.1.10, etc.) that convert acyl CoA to the corresponding alcohol.
Examples of genes (both indicated by UniProtKB / Swiss-Prot No.): Q9ANR5 (Clostridium acetobutylicum-adhE2); D8GU53 (Clostridium ljungdahlii-adhE2); P0A9Q7 (Escherichia coli-adhE), etc.

(G) Crotonyl-CoA hydrolase
This enzyme belongs to an enzyme group (for example, EC 3.1.2.4, 3.1.2.1, etc.) that converts acyl CoA into the corresponding carboxylic acid.
Examples of genes (both indicated by UniProtKB / Swiss-Prot No.): Q5XIE6 (Rattus norvegicus-hibch); Q6NVY1 (Homo sapiens-hibch); A2VDC2 (Xenopus laevis-hibch); P32316 (Saccharomyces cerevisiae-ACH1)

(H) Crotonate reductase
This enzyme also produces crotonaldehyde from crotonic acid by a kind of activity of aldehyde dehydrase, that is, reverse reaction of crotonaldehyde dehydrogenase. This activity belongs to EC 1.2.1.22, 1.2.1.3, etc., for example in the enzyme classification.
Examples of genes (both indicated by UniProtKB / Swiss-Prot No.): Q58806 (Methanococcus jannaschii-LDH); P25553 (Escherichia coli-aldA); P12693 (Pseudomonas oleovorans-alkH); Q6RKB1 (Nocardia iowensis-car), etc.

  In addition, the kind (number) of genes of “crotonyl CoA biosynthesis-related enzyme” to be introduced is at least one selected from the group consisting of “methane, methanol, methylamine, formic acid, formaldehyde, and formamide”. As long as it is possible to produce crotonyl CoA from the C1 compound, at least one is sufficient. Similarly, the type (number) of genes of “crotyl alcohol biosynthesis-related enzyme” to be introduced is at least selected from the group consisting of “methane, methanol, methylamine, formic acid, formaldehyde, and formamide”. As long as it is possible to produce crotyl alcohol from one C1 compound, at least one is sufficient. However, when the activity of each enzyme is enhanced by introducing two or more genes, it is expected that the productivity of crotonyl CoA or crotyl alcohol will increase. Basically, for the crotonyl CoA biosynthesis-related enzyme and crotyl alcohol biosynthesis-related enzyme that are not possessed by the host cell, a gene encoding the enzyme is introduced from the outside. In addition, when the host cell holds the enzyme but its molecular activity is low, it is preferable to introduce a gene encoding an enzyme with higher molecular activity or the like.

  The “crotonyl CoA biosynthesis-related enzyme” and “crotyl alcohol biosynthesis-related enzyme” 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.

  In the recombinant cell of the present invention, in addition to genes encoding crotonyl CoA biosynthesis-related enzymes and crotyl alcohol biosynthesis-related enzymes, other genes may be further introduced. Examples of the gene to be introduced include an enzyme gene that converts crotyl alcohol directly or indirectly into butadiene or a precursor thereof. For example, Patent Document 1 discloses conversion of crotyl alcohol to 2-butenyl-4-phosphate, 2-butenyl-4-phosphate to 2-butenyl-4-diphosphate ( 2-butenyl-4-diphosphate), crotyl alcohol to 2-butenyl-4-diphosphate, and 2-butenyl-4-diphosphate to butadiene (Butadiene) An enzyme that catalyzes is described. For example, by further introducing these enzyme genes into the recombinant cell of the present invention, a function of synthesizing butadiene from crotyl alcohol can be imparted. As a result, the recombinant cell of the present invention can produce butadiene.

  Further examples of the gene to be introduced include the above-mentioned methanol dehydrogenase gene, alcohol dehydrogenase gene, methane monooxidase gene, HPS / PHI gene, serine hydroxymethyltransferase gene, 5,10-methylenetetrahydrofolate synthase gene, and serine And hydroxymethyltransferase gene.

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 contains a promoter at a position where it can replicate autonomously in the host cell or can be integrated into a chromosome, and the inserted gene can be transcribed. 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.

  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 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. As an example of introducing a plurality of types of genes, when the host cell is a methylotroph, in addition to “a gene encoding a crotonyl CoA biosynthesis-related enzyme” and “a gene encoding a crotyl alcohol biosynthesis-related enzyme” An embodiment in which the HPS / PHI gene is introduced is mentioned.

  As described above, known vectors that can be used in methylotrophs and the like have been shown. However, regions related to transcriptional 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.

  Further, 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 crotonyl CoA or crotyl alcohol excretion function of the host cell, crotonyl CoA Alternatively, it is possible to further improve the productivity of crotonyl CoA or crotyl alcohol by improving resistance to crotyl alcohol and the like.

  In one preferred embodiment, the recombinant cell is resistant to at least 400 mM crotyl alcohol. In another preferred embodiment, the recombinant cell has a resistance to at least 2% (v / v) methanol. Such a configuration enables high production (high expression) of crotonyl CoA and crotyl alcohol. 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 method for producing crotonyl CoA or crotyl alcohol of the present invention, the above-described recombinant cell is at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. Is used as a carbon source to cause the recombinant cells to produce crotonyl CoA or crotyl alcohol. 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 the case of obligate methylotrophs, it is fundamental to use a synthetic medium containing C1 compound as the only carbon source, but a small amount of natural medium such as yeast extract, corn steep liquor, meat extract and vitamins are added to this. This also promotes the growth of bacteria. 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 is changed to the C1 compound in the production stage of crotonyl CoA or crotyl alcohol. That's fine. The microorganism 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.
For example, when methanol is used as the carbon source, it is usually used at a concentration of 1.0% (v / v) in the case of bacteria and 3.0% (v / v) or less in the case of yeast. When the resistance to is improved, it can be cultured at higher concentrations.

In another aspect of the method for producing crotonyl CoA or crotyl alcohol of the present invention, at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide is added to the above-described recombinant cell. Then, the recombinant cell is made to produce crotonyl CoA or crotyl alcohol from the C1 compound. That is, regardless of whether cell division (cell proliferation) is involved or not, the aforementioned C1 compound can be brought into contact with a recombinant cell to produce crotonyl CoA or crotyl alcohol. For example, the above-described C1 compound can be continuously supplied to immobilized recombinant cells, and crotonyl CoA or crotyl alcohol can be continuously produced.
Also in this aspect, about these C1 compounds, only one may be used and it may be used in combination of 2 or more.

  The produced crotonyl CoA and crotyl alcohol are accumulated intracellularly or released extracellularly. For example, purified crotonyl CoA or crotyl alcohol can be obtained by recovering crotonyl CoA or crotyl alcohol released outside the cell and isolating and purifying it.

Claims (25)

A gene encoding at least one enzyme selected from the group consisting of acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, and 3-hydroxybutyrate CoA dehydratase is introduced into a host cell that is a methylotroph. Become
The gene is expressed in the host cell,
A recombinant cell capable of producing crotonyl CoA from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. A host cell that is a methylotroph contains acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA reductase (aldehyde-generating type), and crotonaldehyde reductase (alcohol-generating type). A gene encoding at least one enzyme selected from the group consisting of:
The gene is expressed in the host cell,
A recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. At least one selected from the group consisting of acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, and crotonyl CoA reductase (alcohol-generating type). A gene encoding one enzyme was introduced,
The gene is expressed in the host cell,
A recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. To a host cell that is a methylotroph, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase, crotonyl CoA hydrolase, crotonic acid reductase, and crotonaldehyde reductase (alcohol-producing type) A gene encoding at least one enzyme selected from the group consisting of:
The gene is expressed in the host cell,
A recombinant cell capable of producing crotyl alcohol 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 any one of claims 1 to 4, which has at least one C1 carbon assimilation pathway selected from the group consisting of serine pathway, ribulose monophosphate pathway, and xylose monophosphate pathway as a formaldehyde immobilization pathway. .   The gene encoding 3-hexulose 6-phosphate synthase and the gene encoding 6-phospho-3-hexoisomerase are further introduced, and the gene is expressed in the host cell. Recombinant cells according to 1.   The host cell is methanol-assimilating yeast, a gene encoding an enzyme that converts methanol into formaldehyde by dehydrogenation is further introduced, and the gene is expressed in the host cell. Recombinant cells. A gene that imparts a function of converting methanol and / or formic acid to formaldehyde, a gene that imparts formaldehyde immobilization ability, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, and 3-hydroxy A gene encoding at least one enzyme selected from the group consisting of butyrate-CoA dehydratase,
The gene is expressed in the host cell,
A recombinant cell capable of producing crotonyl CoA from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. A gene that imparts a function of converting methanol and / or formic acid into formaldehyde, a gene that imparts formaldehyde immobilization ability, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate A gene encoding at least one enzyme selected from the group consisting of CoA dehydratase, crotonyl CoA reductase (aldehyde producing type), and crotonaldehyde reductase (alcohol producing type),
The gene is expressed in the host cell,
A recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. A gene that imparts a function of converting methanol and / or formic acid into formaldehyde, a gene that imparts formaldehyde immobilization ability, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate A gene encoding at least one enzyme selected from the group consisting of CoA dehydratase and crotonyl CoA reductase (alcohol generating type),
The gene is expressed in the host cell,
A recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. A gene that imparts a function of converting methanol and / or formic acid into formaldehyde, a gene that imparts formaldehyde immobilization ability, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate A gene encoding at least one enzyme selected from the group consisting of CoA dehydratase, crotonyl CoA hydrolase, crotonic acid reductase, and crotonaldehyde reductase (alcohol-generating type);
The gene is expressed in the host cell,
A recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.   The group according to any one of claims 8 to 11, wherein the gene imparting formaldehyde immobilization ability is a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexuloisomerase. Replacement cells.   The recombinant cell according to any one of claims 8 to 11, which has at least one C1 carbon assimilation pathway selected from the group consisting of a serine pathway, a ribulose monophosphate pathway, and a xylose monophosphate pathway as a formaldehyde immobilization pathway. . A gene that gives a host cell having a ribulose monophosphate pathway the function of converting methanol and / or formate into formaldehyde, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, and 3-hydroxybutyrate CoA dehydra A gene encoding at least one enzyme selected from the group consisting of tases is introduced,
The gene is expressed in the host cell,
A recombinant cell capable of producing crotonyl CoA from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. Genes that give a host cell having a ribulose monophosphate pathway the function of converting methanol and / or formic acid into formaldehyde, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase A gene encoding at least one enzyme selected from the group consisting of crotonyl CoA reductase (aldehyde-generating type) and crotonaldehyde reductase (alcohol-generating type),
The gene is expressed in the host cell,
A recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. Genes that give a host cell having a ribulose monophosphate pathway the function of converting methanol and / or formic acid into formaldehyde, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase And a gene encoding at least one enzyme selected from the group consisting of crotonyl-CoA reductase (alcohol-generating type),
The gene is expressed in the host cell,
A recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. Genes that give a host cell having a ribulose monophosphate pathway the function of converting methanol and / or formic acid into formaldehyde, acetoacetyl CoA thiolase, 3-hydroxybutyryl CoA dehydrogenase, 3-hydroxybutyrate CoA dehydratase A gene encoding at least one enzyme selected from the group consisting of crotonyl CoA hydrolase, crotonic acid reductase, and crotonaldehyde reductase (alcohol-generating type),
The gene is expressed in the host cell,
A recombinant cell capable of producing crotyl alcohol from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.   A gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase are further introduced, and the gene is expressed in a host cell. The recombinant cell according to 11, 13, 14, 15, 16, or 17.   The gene imparting the function of converting methanol to formaldehyde is a gene encoding methanol dehydrogenase or alcohol oxidase, and the gene imparting the function of converting formic acid to formaldehyde is a gene encoding formaldehyde dehydrogenase. The recombinant cell according to any one of 18.   The recombinant cell according to any one of claims 8 to 19, wherein a gene imparting a function of converting methane to methanol is further introduced, and the gene is expressed in the host cell.   The recombinant cell according to claim 20, wherein the gene imparting a function of converting methane to methanol is a gene encoding methane monooxygenase.   The recombinant cell according to any one of claims 1 to 21, wherein the introduced gene is integrated into the genome of the host cell.   23. The recombinant cell according to any one of claims 1 to 22, which has a resistance to at least 2% (v / v) methanol.   The recombinant cell according to any one of claims 1 to 23 is cultured using at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide as a carbon source, A method for producing crotonyl CoA or crotyl alcohol, wherein the recombinant cell produces crotonyl CoA or crotyl alcohol.   24. The recombinant cell according to any one of claims 1 to 23 is contacted with at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide, and the recombinant cell is contacted. A method for producing crotonyl CoA or crotyl alcohol, wherein crotonyl CoA or crotyl alcohol is produced from the C1 compound.

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