# Patent

## Title
Recombinant cell and method for producing isoprene

## Abstract

An object of the present invention is to provide a series of techniques for producing isoprene from methanol or the like. Provided is a recombinant cell prepared by introducing a gene encoding isoprene synthase, into a host cell which is a methylotroph, wherein the gene is expressed in the host cell, and the recombinant cell is capable of producing isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. Preferably, it has at least one C1 carbon assimilating pathway selected from the group consisting of a serine pathway, a ribulose monophosphate pathway, and a xylulose monophosphate pathway as a fixing pathway of formaldehyde. Also provided is a method for producing isoprene using the recombinant cell.

## Background
TECHNICAL FIELD
The present invention relates to a recombinant cell capable of producing isoprene from methanol or the like, and a method for producing isoprene using the recombinant cell.
BACKGROUND ART
Isoprene is a monomer raw material for synthetic polyisoprene, and is an important material, in particular, in the tire industry. In recent years, the technique for conversion from a production process of basic chemicals relying on petroleum to a production process from renewable resources such as plant resources has been developed and practical realization thereof is steadily progressing. Also regarding isoprene, for example, a production technique from saccharides as a raw material by recombinant Escherichia coli is known (Patent Documents 1 and 2, and Non-patent Document 1).
On the other hand, among C1 compounds, methanol is produced at a low cost from natural gas, synthetic gas which is a mixed gas of carbon monooxide, carbon dioxide and hydrogen obtained from biomass and wastes such as municipal wastes, and so on. Natural gas is focused as a next-generation energy source because it abundantly exists in fossil resources, and generates a relatively small amount of CO2, and transition from conventional petroleum to natural gas is progressing. Methanol is easy to handle and stock because of its water solubility and so on, and is also suited as a carbon source in microbial culture.
A methylotroph is a general name for a C1 compound assimilating microorganism that uses a carbon compound not having a C—C bond in the molecule, e.g., methane, methanol, methylamine, dimethylamine, trimethylamine or the like as a sole carbon source or energy source. Any microorganisms called methanotroph, methane-oxidizing bacteria, methanol assimilating bacteria, methanol assimilating yeast, methanol assimilating microorganism belong to methylotrophs.
Central metabolism of methylotroph is a reaction of converting formaldehyde into an organic matter having a C—C bond after converting methanol to formaldehyde. As shown in the FIGURE, as a carbon assimilation metabolism pathway via formaldehyde, a serine pathway, a ribulose monophosphate pathway (RuMP pathway), and a xylulose monophosphate pathway (XuMP pathway) can be recited. Methylotrophs classified into bacteria (methylotrophic bacteria) have a serine pathway or a RUMP pathway. On the other hand, methylotrophs classified into yeast (methylotrophic yeast) has a XuMP pathway.
Methylotrophic bacteria are classified into obligate methylotrophs and facultative methylotrophs capable of using other carbon compound according to the difference in methanol requirement.
PRIOR ART DOCUMENTS
Patent Document


    Patent Document 1: WO 2009/076676
    Patent Document 2: WO 2009/132220


Non-Patent Document


    Non-patent Document 1: Yang J., et al., PLoSOne 2012, 7(4), e33509


DISCLOSURE OF INVENTION
Technical Problem
Regarding the production process from renewable resources, most of the conventional techniques including the aforementioned isoprene production technique are production methods by microorganisms relying on organic substances, in particular, saccharides, glycerol or oil components. However, for covering the global production quantity of a large number of basic chemicals derived from petroleum, the amounts of currently available saccharides, glycerin and oil components derived from plant resources and the like will be necessarily insufficient for carbon sources of microorganisms. In other words, the production amounts of basic chemicals by microorganisms relying on saccharides or oil components is limited also in the future. These processes also have a fear of competition with foods.
Application examples of methylotrophs include production techniques of SCP (single cell protein), biodegradable plastic, amino acid and so on from methanol. However, there has been no case that a methylotroph is applied to production of basic chemicals derived from petroleum such as isoprene. As described above, all of the monomer compounds utilized in general polymer material products rely on petroleum at present, however, the possibility that petroleum having the quality equivalent to that of currently used petroleum is supplied is very low, and development of a new, efficient, alternative process is urgently demanded. Although a technique of saccharifying hard biomass including cellulose, hemicellulose, lignin and the like is examined for the purpose of ensuring carbon sources for production of chemicals by microorganisms, an enzyme treatment for saccharification is required, and a significant problem in terms of the cost arises.
In light of the above, it is an object of the present invention to provide a series of techniques capable of producing isoprene from methanol or the like.
Solution to Problem
One aspect of the present invention for solving the aforementioned problem is a recombinant cell prepared by introducing a gene encoding isoprene synthase, into a host cell which is a methylotroph, wherein the gene is expressed in the host cell, and the recombinant cell is capable of producing isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
Isoprene synthase has the function of converting dimethylallyl diphosphate (DMAPP) which is an isomer of isopentenyl diphosphate (IPP) into isoprene. Structure conversion between isopentenyl diphosphate and dimethylallyl diphosphate is catalyzed by isopentenyl diphosphate isomerase. Isopentenyl diphosphate isomerase exists in any organism.
The recombinant cell of the present aspect is prepared by introducing a gene encoding isoprene synthase, into a host cell which is a methylotroph, and the gene is expressed in the host cell. And it is able to produce isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. The recombinant cell of the present aspect is able to convert dimethylallyl diphosphate and isopentenyl diphosphate synthesized in the cell into isoprene by the action of isoprene synthase. That is, according to the recombinant cell of the present aspect, it is possible to produce isoprene from the aforementioned C1 compound.
Preferably, the recombinant cell has at least one C1 carbon assimilating pathway selected from the group consisting of a serine pathway, a ribulose monophosphate pathway, and a xylulose monophosphate pathway as a fixing pathway of formaldehyde.
Preferably, a gene encoding 3-hexulose-6-phosphate synthase and a gene encoding 6-phospho-3-hexuloisomerase are further introduced, and the genes are expressed in the host cell.
With such a configuration, formaldehyde fixing ability by the ribulose monophosphate pathway is imparted or enhanced.
Preferably, the host cell is bacterium or yeast.
Preferably, the yeast belongs to genus Pichia, genus Hansenula, or genus Candida. 
Other aspect of the present invention for solving a similar problem is a recombinant cell prepared by introducing a gene imparting the function of converting methanol and/or formic acid into formaldehyde, a gene imparting formaldehyde fixing ability, and a gene encoding isoprene synthase, into a host cell, wherein the genes are expressed in the host cell, and the recombinant cell is capable of producing isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
The recombinant cell of the present aspect is prepared by introducing “gene imparting the function of converting methanol and/or formic acid into formaldehyde”, “gene imparting formaldehyde fixing ability”, and “gene encoding isoprene synthase” into a host cell, and these genes are expressed in the host cell. And it is able to produce isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
That is, since the recombinant cell of the present aspect is prepared by introducing “gene imparting the function of converting methanol and/or formic acid into formaldehyde”, and “gene imparting formaldehyde fixing ability”, it has characteristics similar to those of a methylotroph. And, since “gene encoding isoprene synthase” is introduced, isoprene synthase can be expressed in the cell. As a result, it is possible to convert isopentenyl diphosphate synthesized in the cell into isoprene. That is, also by the recombinant cell of the present aspect, it is possible to produce isoprene from the aforementioned C1 compound.
Preferably, the recombinant cell has at least one C1 carbon assimilating pathway selected from the group consisting of a serine pathway, a ribulose monophosphate pathway, and a xylulose monophosphate pathway as a fixing pathway of formaldehyde.
Another aspect of the present invention for solving a similar problem is a recombinant cell prepared by introducing a gene imparting the function of converting methanol and/or formic acid into formaldehyde and a gene encoding isoprene synthase, into a host cell having a ribulose monophosphate pathway, wherein the genes are expressed in the host cell, and the recombinant cell is capable of producing isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
The present aspect corresponds to the form wherein, for example, a non-methylotroph having a ribulose monophosphate pathway is a host cell.
Preferably, the gene imparting the function of converting methanol into formaldehyde is a gene encoding methanol dehydrogenase or alcohol oxydase, and the gene imparting the function of converting formic acid into formaldehyde is a gene encoding formaldehyde dehydrogenase.
Both of methanol dehydrogenase and alcohol dehydrogenase have the function of converting methanol into formaldehyde. Also, formaldehyde dehydrogenase has the function of converting formic acid into formaldehyde. Any of these enzymes is one of the methane metabolism enzymes in methylotrophs belonging to bacteria. On the other hand, methylotrophs belonging to yeast does not have methane oxidizing activity, but has the function of converting methanol into formaldehyde by the action of alcohol oxydase. Also yeast has the enzymatic activity of converting formic acid into formaldehyde.
Preferably, a gene imparting the function of converting methane into methanol is further introduced, and the gene is expressed in the host cell.
Preferably, the gene imparting the function of converting methane into methanol is a gene encoding methane monooxygenase.
Methane monooxygenase has the function of converting methane into methanol. Also methane monooxygenase is one of methane metabolism enzymes in methylotrophs.
Preferably, a gene encoding 3-hexulose-6-phosphate synthase and a gene encoding 6-phospho-3-hexuloisomerase are further introduced, and the genes are expressed in the host cell.
Preferably, the host cell is bacterium or yeast.
Preferably, the host cell has isopentenyl diphosphate synthesis ability by a mevalonate pathway, and a gene encoding at least one enzyme acting in a mevalonate pathway and/or a gene encoding a group of enzymes acting in a non-mevalonate pathway are/is further introduced, and the gene is expressed in the host cell.
With such a configuration, synthesis ability of IPP which is a supply source of DMAPP is enhanced, and IPP and DMAPP are efficiently supplied. As a result, the recombinant cell of the present aspect has still higher isoprene productivity.
Preferably, the gene encoding at least one enzyme acting in a mevalonate pathway is derived from actinomycete.
Preferably, the host cell has isopentenyl diphosphate synthesis ability by a non-mevalonate pathway, and a gene encoding a group of enzymes acting in a mevalonate pathway and/or a gene encoding at least one enzyme acting in a non-mevalonate pathway are/is further introduced, and the gene is expressed in the host cell.
With such a configuration, synthesis ability of IPP which is a supply source of DMAPP is enhanced, and IPP and DMAPP are efficiently supplied. As a result, the recombinant cell of the present aspect has still higher isoprene productivity.
Preferably, the gene encoding at least one enzyme acting in a non-mevalonate pathway is derived from those other than the host cell.
Preferably, the isoprene synthase is derived from plants.
Preferably, the gene encoding isoprene synthase encodes a protein of the following (a), (b) or (c).
(a) a protein having an amino acid sequence represented by SEQ ID NO: 2;
(b) a protein having 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 isoprene synthase activity;
(c) a protein having an amino acid sequence having a homology of 60% or more with the amino acid sequence represented by SEQ ID NO: 2, and having isoprene synthase activity.
Preferably, a gene encoding isopentenyl diphosphate isomerase is further introduced, and the gene is expressed in the host cell.
Since the direct substrate for isoprene synthase is dimethylallyl diphosphate (DMAPP), conversion from IPP to DMAPP is enhanced also by enhancing isopentenyl diphosphate isomerase activity, and the production efficiency of isoprene is improved.
Preferably, a treatment of suppressing an expression amount of geranyl pyrohosphate synthase, neryl pyrophosphate synthase, or farnesyl pyrophosphate synthase is conducted.
IPP can be converted into geranyl pyrohosphate (GPP), neryl pyrophosphate (NPP), or farnesyl pyrophosphate (FPP). And the recombinant cell of the present aspect is subjected to a treatment of suppressing an expression amount of the geranyl pyrohosphate synthase (GPP synthase), neryl pyrophosphate synthase (NPP synthase), or farnesyl synthase (FPP synthase). With such a configuration, waste of IPP which is a supply source of DMAPP is suppressed, and the isoprene productivity is further increased.
Other aspect of the present invention for solving a similar problem is a recombinant cell prepared by introducing a gene imparting the function of converting methanol and/or formic acid to formaldehyde and a gene imparting formaldehyde fixing ability into a host cell having isoprene synthase, wherein the genes are expressed in the host cell, and the recombinant cell is capable of producing isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
The recombinant cell of the present aspect is prepared by introducing “gene imparting the function of converting methanol and/or formic acid into formaldehyde” and “gene imparting formaldehyde fixing ability” into a host cell having isoprene synthase, and the genes are expressed in the host cell. And it is able to produce isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
That is, since the recombinant cell of the present aspect is prepared by introducing “gene imparting the function of converting methanol and/or formic acid into formaldehyde”, and “gene imparting formaldehyde fixing ability”, it has characteristics similar to those of a methylotroph. And since the host cell itself has isoprene synthase, dimethylallyl diphosphate and isopentenyl diphosphate can be converted into isoprene by the action of isoprene synthase. That is, also by the recombinant cell of the present aspect, it is possible to produce isoprene from the aforementioned C1 compound.
Preferably, the gene imparting the function of converting methanol into formaldehyde is a gene encoding methanol dehydrogenase or alcohol oxydase, and the gene imparting the function of converting formic acid into formaldehyde is a gene encoding formaldehyde dehydrogenase.
Preferably, a gene imparting the function of converting methane into methanol is further introduced, and the gene is expressed in the host cell.
Preferably, the gene imparting the function of converting methane into methanol is a gene encoding methane monooxygenase.
Preferably, the recombinant cell has at least one C1 carbon assimilating pathway selected from the group consisting of a serine pathway, a ribulose monophosphate pathway, and a xylulose monophosphate pathway as a fixing pathway of formaldehyde.
Preferably, a gene encoding 3-hexulose-6-phosphate synthase and a gene encoding 6-phospho-3-hexuloisomerase are further introduced, and the genes are expressed in the host cell.
Preferably, the host cell is bacterium or yeast.
Preferably, the host cell has isopentenyl diphosphate synthesis ability by a mevalonate pathway, and a gene encoding at least one enzyme acting in a mevalonate pathway, and/or a gene encoding a group of enzymes acting in a non-mevalonate pathway are/is further introduced, and the gene is expressed in the host cell.
Preferably, the gene encoding at least one enzyme acting in a mevalonate pathway is derived from actinomycete.
Preferably, the host cell has isopentenyl diphosphate synthesis ability by a non-mevalonate pathway, and a gene encoding a group of enzymes acting in a mevalonate pathway, and/or a gene encoding at least one enzyme acting in a non-mevalonate pathway are/is further introduced, and the gene is expressed in the host cell.
Preferably, the gene encoding at least one enzyme acting in a non-mevalonate pathway is derived from those other than the host cell.
Preferably, a gene encoding isoprene synthase is further introduced, and the gene is expressed in the host cell.
With such a configuration, an expression amount of isoprene synthase in the host cell is enhanced, and the isoprene productivity is further increased.
Preferably, the isoprene synthase is derived from plants.
Preferably, the gene encoding isoprene synthase encodes a protein of the following (a), (b) or (c).
(a) a protein having an amino acid sequence represented by SEQ ID NO: 2;
(b) a protein having 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 isoprene synthase activity;
(c) a protein having an amino acid sequence having a homology of 60% or more with the amino acid sequence represented by SEQ ID NO: 2, and having isoprene synthase activity.
Preferably, a gene encoding isopentenyl diphosphate isomerase is further introduced, and the gene is expressed in the host cell.
Preferably, a treatment of suppressing the expression amount of geranyl pyrohosphate synthase, neryl pyrophosphate synthase, or farnesyl pyrophosphate synthase is conducted.
Another aspect of the present invention is a method for producing isoprene including culturing the aforementioned recombinant cell by using at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide as a carbon source, to cause the recombinant cell to produce isoprene.
The present aspect relates to a method for producing isoprene. In the present aspect, the recombinant cell is caused to produce isoprene by culturing the aforementioned recombinant cell by using at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide as a carbon source. According to the present aspect, it is possible to produce isoprene from methanol or the like.
Another aspect of the present invention is a method for producing isoprene including bringing at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide into contact with the aforementioned recombinant cell, to cause the recombinant cell to produce isoprene from the C1 compound.
In the present aspect, isoprene is produced from a C1 compound by bringing at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide into contact with the aforementioned recombinant cell. The present aspect also makes it possible to produce isoprene from methanol or the like.
Advantageous Effect of Invention
According to the recombinant cell of the present invention, it is possible to produce isoprene from methane, methanol, methylamine, formic acid, formaldehyde, or formamide.
According to the method for producing isoprene of the present invention, it is similarly possible to produce isoprene from methane, methanol, methylamine, formic acid, formaldehyde, or formamide.



BRIEF DESCRIPTION OF DRAWINGS
The FIGURE shows an explanatory diagram showing a carbon assimilation metabolism pathway via formaldehyde.



DESCRIPTION OF EMBODIMENT
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”.
One aspect of the present invention is a recombinant cell prepared by introducing a gene encoding isoprene synthase into a host cell which is a methylotroph, wherein the gene is expressed in the host cell, and the recombinant cell is capable of producing isoprene 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 is a recombinant cell prepared by introducing a gene imparting the function of converting methanol and/or formic acid into formaldehyde, a gene imparting a formaldehyde fixing ability, and a gene encoding isoprene synthase into a host cell, wherein the genes are expressed in the host cell, and the recombinant cell is capable of producing isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
As described above, a methylotroph is a C1 compound assimilating microorganism that uses a carbon compound not having a C—C bond in the molecule, e.g., methane, methanol, methylamine, dimethylamine, trimethylamine or the like as a sole carbon source or energy source. In general, a methylotroph originally has a carbon assimilation metabolism pathway via formaldehyde, concretely the function (pathway) of converting methanol and/or formic acid to formaldehyde, and a formaldehyde fixing ability (fixing pathway of formaldehyde).
As a fixing pathway of formaldehyde, a serine pathway, a ribulose monophosphate pathway (RUMP pathway), and a xylulose monophosphate pathway (XuMP pathway) shown in the FIGURE can be recited. In general, a methylotroph has a serine pathway, a RUMP pathway, or a XuMP pathway as a carbon assimilation metabolism pathway via formaldehyde.
Here, description will be made for each formaldehyde fixing pathwayin the FIGURE.
The important reaction for formaldehyde fixation by the serine pathway is serine generation reaction of glycine and 5,10-methylene-tetrahydrofolic acid by serine hydroxymethyltransferase. Generation of 5,10-methylene-tetrahydrofolic acid is made by binding formaldehyde to tetrahydrofolic acid. In the serine pathway, one molecule of acetyl CoA is directly generated from one molecule of formaldehyde.
The important reactions for formaldehyde fixation by the RUMP pathway are generation reaction of D-arabino-3-hexulose-6-phosphate from ribulose 5-phosphate (Ru5P) and formaldehyde by 3-hexulose-6-phosphate synthase (hereinafter, also abbreviated as “HPS”), and generation reaction of fructose-6-phosphate (F6P) from D-arabino-3-hexulose-6-phosphate by 6-phosphate-3-hexuloisomerase (hereinafter, also abbreviated as “PHI”).
F6P and the like generated in this pathway are also supplied to a glycolytic pathway to subsequently generate acetyl CoA, glyceraldehyde 3-phosaphate (G3P) and pyruvic acid. In the case of F6P, one molecule of F6P is converted to two molecules of G3P, and then two molecules of acetyl CoA are generated via two molecules of pyruvic acid.
The important reaction for formaldehyde fixation by the XuNP pathway is generation reaction of dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) from xylulose-5-phosphate (Xu5P) and formaldehyde by dihydroxyacetone synthase. G3P generated in this pathway is also supplied to the glycolytic pathway, and converted to pyruvic acid and acetyl CoA. Dihydroxyacetone is also supplied to the glycolytic pathway by phosphorylation, and can be converted to G3P, pyruvic acid, and acetyl CoA.
The recombinant cell of the present invention is capable of producing isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. For example, a recombinant cell having methanol dehydrogenase or alcohol oxydase is able to convert methanol to formaldehyde.
A recombinant cell having methane monooxydase in addition to methanol dehydrogenase or alcohol oxydase is able to convert methane to methanol, and then convert methanol to formaldehyde.
Further, a recombinant cell having formaldehyde dehydrogenase is able to convert formic acid to formaldehyde.
In general, methylotrophs classified into bacteria (methylotrophic bacteria) are able to synthesize formaldehyde from methane or methanol because they have methane monooxygenase and methanol dehydrogenase. Further, methylotrophs classified into yeast (methylotrophic yeast) are able to synthesize formaldehyde from methanol because they have alcohol oxydase. Further, methylotrophs have formaldehyde dehydrogenase, and are able to convert formic acid to formaldehyde.
The aforementioned 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, and N,N′-dimethyl-4-nitrosoaniline (DMNA)-dependent methanol oxide reductase (Park H. et al., Microbiology 2010, 156, 463-471) found in methylotrophs of gram positive bacteria. Conversion from methanol to formaldehyde in yeast is usually catalyzed by oxygen-dependent alcohol oxydase.
Also a recombinant cell having amine oxidase or a methylamine dehydrogenase is able to convert methylamine to formaldehyde. These enzymes are known to be inherent in some methylotrophs and Arthrobacter bacteria (Anthony C., The Biochemistry of Methylotroph, 1982, Academic Press Inc.)
Also, enzymes that convert formamide to formaldehyde are found in some microorganisms (Anthony C., The Biochemistry of Methylotroph, 1982, Academic Press Inc.)
Additionally, isoprene can be produced via formaldehyde.
While the kind of methylotrophs to be used as a host cell is not particularly limited, for example, those classified into bacteria or yeast can be employed.
Examples of methylotrophic bacteria include bacteria belonging to genus Methylacidphilum, genus Methylosinus, genus Methylocystis, genus Methylobacterium, genus Methylocella, genus Methylococcus, genus Methylomonas, genus Methylobacter, genus Methylobacillus, genus Methylophilus, genus Methylotenera, genus Methylovorus, genus Methylomicrobium, genus Methylophaga, genus Methylophilaceae, genus Methyloversatilis, genus Mycobacterium, genus Arthrobacter, genus Bacillus, genus Beggiatoa, genus Burkholderia, genus Granulibacter, genus Hyphomicrobium, genus Pseudomonas, genus Achromobactor, genus Paracoccus, genus Crenothrix, genus Clonothrix, genus Rhodobacter, genus Rhodocyclaceae, genus Silicibacter, genus Thiomicrospira, and genus Verrucomicrobia. 
Examples of methylotrophic yeasts include yeast belonging to genus Pichia, genus Candida, genus Saccharomyces, genus Hansenula, genus Torulopsis, and genus Kloeckera. Examples of Pichia yeasts include P. haplophila, P. pastoris, P. trehalophila, and P. lindnerii. Examples of Candida yeasts include C. parapsilosis, C. methanolica, C. boidinii, and C. alcomigas. Example of Saccharomyces yeast includes Saccharomyces metha-nonfoams. Examples of Hansenula yeasts include H. wickerhamii, H. capsulata, H. glucozyma, H. henricii, H. minuta, H. nonfermentans, H. philodendra, and H. polymorpha. Examples of Torulopsis yeasts include T. methanolovescens, T. glabrata, T. nemodendra, T. pinus, T. methanofloat, T. enokii, T. menthanophiles, T. methanosorbosa, and T. methanodomercqii. 
When the host cell is a non-methylotroph, it is necessary to impart at least “the function of converting methanol and/or formic acid to formaldehyde” because the host cell does not always have a pathway of converting methanol or the like to formaldehyde. Further, it is preferred to impart “the function of converting methane to methanol”. Impartation of such functions can be achieved by introducing a gene encoding the aforementioned enzyme into the host cell.
For example, as a gene imparting the 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 oxydase (for example, EC1.13.13) can be used. As a gene imparting the function of converting formic acid to formaldehyde, a gene encoding formaldehyde dehydrogenase (e.g., EC1.2.1.46) can be used. Further, as a gene imparting the function of converting methane to methanol, a gene encoding methane monooxygenase can be used.
Also a plasmid imparting methanol assimilability is known. For example, methanol assimilability of Bacillus methanolicus relies on a plasmid encoding a group of enzymes involved in methanol metabolism (Brautaset T. et al., J. Bacteriology 2004, 186(5), 1229-1238). By introducing such a plasmid to a related non-methylotroph, it is possible to impart methanol assimilability. Further, by modifying such a plasmid, it is possible to impart methanol assimilability to various non-methylotrophs.
In the manner as described above, by imparting “the function of converting methanol and/or formic acid to formaldehyde”, and further imparting “the formaldehyde fixing ability” to a non-methylotroph, it becomes possible to handle a non-methylotroph similarly to a methylotroph. Impartation of the formaldehyde fixing ability can be realized, for example, by introducing a gene encoding an enzyme acting in the serine pathway, RuMP pathway, or XuMP pathway into a non-methylotroph.
Further description will be made while taking the case of imparting the RuMP pathway as an example. Impartation of the RuMP pathway can be realized, for example, by introducing the aforementioned 3-hexulose-6-phosphate synthase (HPS; e.g., EC4.1.2.43) gene and a 6-phospho-3-hexuloisomerase (PHI; e.g., EC5.3.1.27) gene. That is, ribulose 5-phosphate (Ru5P) and fluctose 6-phosphate (F6P) which are a substrate or a product of the formaldehyde fixing reaction by HPS/PHI generally exist in any organism as metabolic intermediates of the pentose phosphate pathway, and the calvin cycle. Therefore, by introducing HPS/PHI, it is possible to impart the formaldehyde fixing ability to every organism including Escherichia coli, Bacillus subtilis, and yeast.
A HPS gene and a PHI gene may be introduced to a host cell originally having the RuMP pathway. As a result, it is possible to enhance the formaldehyde fixing ability by the RUMP pathway. For example, by introducing genes encoding enzymes such as alcohol dehydrogenase such as methanol dehydrogenase (e.g., EC1.1.1.244, EC1.1.2.7), 3-hexulose 6-phosphate synthase (HPS; e.g., EC4.1.2.43), 6-phospho-3-hexuloisomerase (PHI; e.g., EC5.3.1.27) to a microorganism originally having the RuMP pathway or a pathway equivalent to the same, such as Bacillus subtilis, it is possible to impart the function of converting methanol to formaldehyde (i.e. methanol assimilability) and to enhance the formaldehyde fixing ability.
HPS gene and PHI gene may be introduced into a host cell which is a methylotroph. That is, by introducing HPS/PHI to a methylotroph having a serine pathway, a RuMP pathway, or a XuMP pathway, it is possible to enhance the formaldehyde fixing ability by the RuMP pathway. As a result, it is possible to improve the formaldehyde resistance of the recombinant cell, and to improve the resistance and assimilability to methanol and formic acid. As a result, it becomes possible to increase the culture efficiency of the recombinant cell and the production efficiency of isoprene.
On the other hand, for imparting the formaldehyde fixing ability by the serine pathway, the aforementioned serine hydroxymethyltransferase (e.g., EC2.1.2.1) gene can be employed. For example, by introducing alcohol dehydrogenase (e.g. methanol dehydrogenase) gene, 5,10-methylenetetrahydrofolate (CH2=H4F) synthase gene, and serine hydroxymethyltransferase (e.g., EC2.1.2.1) gene into a non-methylotroph, it is possible to impart the formaldehyde fixing ability by the methanol assimilability and the serine pathway.
Subsequently, isoprene synthase will be described. Isoprene synthase is not particularly limited as far as its enzyme activity can be exerted in the recombinant cell. Similarly, the gene encoding isoprene synthase is not particularly limited as far as it is normally transcribed and translated in the recombinant cell.
Isoprene synthase is found in many plants. Concrete examples of isoprene synthase include those derived from poplar (Populus nigra) (GenBank Accession No.: AM410988.1). Also those derived from Bacillus subtilis (SivyTL. et al., Biochem. Biophys. Res. Commu. 2002, 294(1), 71-5) can be recited.
SEQ ID NO: 1 represents a nucleotide sequence of a gene (DNA) encoding isoprene synthase derived from poplar, and a corresponding amino acid sequence, and SEQ ID NO: 2 represents only the amino acid sequence. DNA having the nucleotide sequence represented by SEQ ID NO: 1 is one example of gene encoding isoprene synthase.
Further, the gene encoding isoprene synthase includes at least a gene encoding a protein of the following (a), (b) or (c).
(a) a protein having an amino acid sequence represented by SEQ ID NO: 2;
(b) a protein having 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 isoprene synthase activity;
(c) a protein having an amino acid sequence having a homology of 60% or more with the amino acid sequence represented by SEQ ID NO: 2, and having isoprene synthase activity.
The homology of the amino acid sequence in (c) is more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more.
In the recombinant cell of the present invention, other gene may be further introduced in addition to the gene encoding isoprene synthase and so on. As a gene that is introduced, for example, a gene of an enzyme acting in the biosynthesis pathway of isoprenoid described below can be recited.
Every organism including the recombinant cell of the present invention has the isoprenoid biosynthesis pathway involving a mevalonate pathway (also referred to as MVA pathway) or a non-mevalonate pathway (also referred to as MEP pathway), and can synthesize isopentenyl diphosphate (IPP).
The mevalonate pathway is inherent in eukaryotes, and starts with acetyl CoA as a starting substance. Enzymes acting in the mevalonate pathway include, in the order from the upstream, acetyl CoA acetyl transferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase.
On the other hand, the non-mevalonate pathway is inherent in prokaryotes and chloroplasts and plastids, and starts with glyceraldehyde 3-phosphate and pyruvic acid as starting substances. Enzymes acting in the non-mevalonate pathway include, in the order from the upstream, DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase and HMB-PP reductase.
In one embodiment, when the host cell has IPP synthesis ability by the mevalonate pathway, (i) a gene encoding at least one enzyme acting in the mevalonate pathway, and/or (ii) a gene encoding a group of enzymes acting in the non-mevalonate pathway are/is further introduced. Introduction of the gene of (i) enhances the IPP synthesis ability by the mevalonate pathway. Also, introduction of the gene of (ii) results in synthesis of IPP from both the mevalonate pathway and the non-mevalonate pathway, and enhances the IPP synthesis ability. Enhancement of the IPP synthesis ability results in more efficient production of isoprene.
In another embodiment, when the host cell has IPP synthesis ability by the non-mevalonate pathway, (iii) a gene encoding a group of enzymes acting in the mevalonate pathway, and/or (iv) a gene encoding at least one enzyme acting in the non-mevalonate pathway are/is further introduced. Introduction of the gene of (iii) results in synthesis of IPP from both the mevalonate pathway and the non-mevalonate pathway, and therefore, enhances the IPP synthesis ability. Also, introduction of the gene of (iv) enhances the IPP synthesis ability by the non-mevalonate pathway. Enhancement of the IPP synthesis ability results in more efficient production of isoprene.
As described above, as the group of enzymes acting in the mevalonate pathway, acetyl CoA acetyl transferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase can be recited.
In the case of introducing the gene of (i), the gene to be introduced can be selected so that one or more enzymes selected from the group consisting of, for example, acetyl CoA acetyl transferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase are expressed in the host cell. For example, one or more enzymes can be selected from the group of enzymes, and a gene encoding the enzyme can be introduced into the host cell.
In the case of introducing the gene of (iii), the gene to be introduced can be selected so that the group of enzymes consisting of, for example, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase is expressed in the host cell.
As described above, the group of enzymes acting in the non-mevalonate pathway include DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase and HMB-PP reductase.
In the case of introducing the gene of (ii), the gene to be introduced can be selected so that the group of enzymes consisting of, for example, DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase and HMB-PP reductase is expressed in the host cell.
In the case of introducing the gene of (iv), the gene to be introduced can be selected so that one or more enzymes selected from the group consisting of, for example, DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase and HMB-PP reductase are expressed in the host cell. For example, one or more enzymes can be selected from the group of enzymes, and a gene encoding the enzyme can be introduced into the host cell.
The mevalonate pathway is inherent in all eukaryotes, but is also found in those other than eukaryotes. Examples of those having a mevalonate pathway other than eukaryotes include Streptomyces sp. Strain CL190 (Takagi M. et al., J. Bacteriol. 2000, 182 (15), 4153-7), and Streptomyces griseolosporeus MF730-N6 (Hamano Y. et al., Biosci. Biotechnol. Biochem. 2001, 65(7), 1627-35) which are actinomycetes. In bacteria, Lactobacillus helvecticus (Smeds A et al., DNA seq. 2001, 12(3), 187-190), Corynebacterium amycolatum, Mycobacterium marinum, Bacillus coagulans, Enterococcus faecalis, Streptococuss agalactiae, Myxococcus xanthus and so on (Lombard J. et al., Mol. Biol. Evol. 2010, 28(1), 87-99) can be recited. In archaea, genus Aeropyrum, genus Sulfolobus, genus Desulfurococcus, genus Thermoproteus, genus Halobacterium, genus Methanococcus, genus Thermococcus, genus Pyrococcus, genus Methanopyrus, genus Thermoplasma and so on (Lombard J. et al., Mol. Biol. Evol. 2010, 28 (1), 87-99) can be recited. For example, as the gene of (i) or (iii), the one derived from the foregoing organisms can be used.
As an example for enhancing IPP synthesis of a bacterium, it is preferred to introduce a group of mevalonate pathway enzymes that is not inherent in the bacterium so as to avoid the gene expression control of the host.
The enzymes acting in the non-mevalonate pathway encoded by the gene of (ii) or (iv) are preferably derived from those other than the host cell. With such a constitution, it is possible to avoid reaction suppression by a reaction product.
In the case of a host cell having a serine pathway, it is particularly preferred to introduce a gene of a group of mevalonate pathway enzymes as a foreign gene so as to directly generate acetyl CoA from the serine pathway which is a formaldehyde fixing pathway.
The enzymes acting in the mevalonate pathway or the non-mevalonate pathway encoded by the gene of (i) to (iv) may be naturally occurring enzymes or enzymes modified therefrom. For example, amino acid substitution variants of each enzyme, and polypeptides that are partial fragments of each enzyme and have equivalent enzyme activity are also applicable.
Since the direct substrate for isoprene synthase is dimethylallyl diphosphate (DMAPP), conversion from IPP to DMAPP is enhanced also by enhancing isopentenyl diphosphate isomerase activity, and the production efficiency of isoprene is improved. Accordingly, an isopentenyl diphosphate isomerase gene may further be introduced as a foreign gene. The isopentenyl diphosphate isomerase gene in this case is preferably derived from the same as or related species with the host.
By conducting a treatment of suppressing the expression amount of geranyl pyrohosphate synthase (GPP synthase), neryl pyrophosphate synthase (NPP synthase), or farnesyl pyrophosphate synthase (FPP synthase) on the recombinant, it is possible to further improve the isoprene productivity. That is, by this treatment, conversion from IPP to GPP, NPP, or FPP is suppressed, and the waste of IPP which is a supply source of DMAPP is suppressed.
As the treatment, various treatments on the gene encoding GPP synthase, NPP synthase, or FPP synthase, for example, introduction of mutation to the gene (modification of codon, e.g. knockout and so on), modification of a promotor, modification of a SD sequence and so on are recited.
A still another aspect of the present invention is a recombinant cell prepared by introducing a gene imparting the function of converting methanol and/or formic acid to formaldehyde and a gene imparting formaldehyde fixing ability into a host cell having isoprene synthase, wherein the genes are expressed in the host cell, and the recombinant cell is capable of producing isoprene from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
As a host cell having isoprene synthase that can be employed in this aspect, bacteria belonging to genus Bacillus, genus Acinetobacter, genus Agrobacterium, genus Erwinia, genus Pseudomonas and so on as described in U.S. Pat. No. 5,849,970 can be recited.
Also in this aspect, any embodiment described above can be directly applied. For example, in the present aspect, the aforementioned example can be directly applied regarding “gene imparting “the function of converting methanol and/or formic acid to formaldehyde” and “gene imparting formaldehyde fixing ability”. That is, genes respectively encoding methanol dehydrogenase, alcohol oxydase, formaldehyde dehydrogenase, methane monooxygenase and so on can be introduced into the host cell.
Also in the present aspect, a gene encoding 3-hexulose 6-phosphate synthase (HPS) and a gene encoding 6-phospho-3-hexuloisomerase (PHI) may further be introduced.
Further, also in the present aspect, a gene of enzyme that acts in the isoprenoid biosynthesis pathway may be introduced into the host cell. For example, embodiments using the genes of (i) to (iv) can also be applied to the present aspect.
In the present aspect, a gene encoding isoprene synthase may further be introduced into the host cell. According to the present embodiment, since foreign isoprene synthase is synthesized in addition to isoprene synthase that is inherent in the host cell, the isoprene productivity is further improved. The isoprene synthase gene to be introduced is not particularly limited, and for example, isoprene synthase gene derived from poplar can be used. Also, isoprene synthase gene derived from the host cell may further be introduced.
Also in this aspect, a treatment for suppressing the expression amount of geranyl pyrohosphate synthase (GPP synthase), neryl pyrophosphate synthase (NPP synthase), or farnesyl pyrophosphate synthase (FPP synthase) may be conducted on the recombinant.
The method of introducing a gene into the host cell is not particularly limited, and may be selected appropriately depending on the kind of the host cell and the like. For example, a vector that can be introduced into the host cell and can allow expression of the gene incorporated therein may be used.
For example, when the host cell is a prokaryote such as a bacterium, a vector that can self duplicate or can be incorporated in chromosome in the host cell, and contains a promoter at the position allowing transcription of the inserted gene can be used. For example, it is preferred to construct in the host cell a series of structures including a promoter, a ribosome binding sequence, the above gene and a transcription termination sequence by using the vector.
For example, as a method of incorporating into chromosome of a methylotroph bacterium, exemplified is a method of destroying a target gene in Methylobacillus flagellatus having a ribulose monophosphate pathway, and in Methylobacterium extorquencs having a serine pathway (Chistoserdova L. et al., Microbiology 2000, 146, 233-238; Chistoserdov A Y., et al., J. Bacteriol 1994, 176, 4052-4065). While these are the methods for introducing a gene into a genome using cyclic DNA, a method for introducing a gene into genome using a linear DNA is also developed in Methylophilus bacteria and the like (see JP 2004-229662 A). In general, genomic recombination is more efficient by linear DNA than by cyclic DNA when the DNA is less susceptible to degradation by the host cell. Generally, in a homologous recombination method, it is preferred to target a gene existing in multi copies on the genome likewise an inverted-repeat sequence. As a technique for introducing multi copies into a genome, a method of carrying on a transposon is also known besides the homologous recombination. As a method of introducing a gene into a methylotrophic bacterium by a plasmid, for example, pAYC32 (Chistoserdov A Y., 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), and pCM80 (Marx C J. et al., Microbiology 2001, 147, 2065-2075) which are broad host range vectors are known.
A method of introducing a gene in methylotrophic yeast is established mainly in Pichia pastoris, and vectors such as pPIC3.5K, pPIC6, pGAPZ, and pFLD (available from Invitrogen) are commercially available.
As a plasmid that can be used for gene introduction into Bacillus bacteria, pMTLBS72 (Nguyen H D. Et al., Plasmid 2005, 54(3), 241-248), pHT01 (available from Funakoshi Co., Ltd.), pHT43 (available from Funakoshi Co., Ltd.) and so on are available for Bacillus subtilis, p3STOP1623 hp (available from Funakoshi Co., Ltd.), pSPYocHhp (available from Funakoshi Co., Ltd.) and so on are available for Bacillus megaterium, and pNI DNA (available from TAKARA BIO INC.) and so on are available for Bacillus brevis. 
In introducing plural kinds of genes by using a vector, the genes may be incorporated into one vector, or incorporated into different vectors. Further, in incorporating a plurality of genes into one vector, the genes may be expressed under a common promotor, or may be expressed under different promotors. As an example of introducing plural kinds of genes, an embodiment of introducing the gene of (i) to (iv), or HPS/PHI gene in addition to “gene encoding isoprene synthase” when the host cell is a methylotroph is recited.
As describe above, while the known vectors that can be used in methylotroph and so on have been shown, the region involved in transcription control and replication regions such as promotor and terminator can be modified depending on the purpose. The modification includes change to other natural gene sequence in each host cell or its related species, and change to an artificial gene sequence.
It is possible to further improve the productivity of isoprene by combining a variation technique such as mutation or genome shuffling in addition to the modification by the gene introduction as described above.
In one aspect of the method for producing isoprene, the recombinant cell is cultured by using at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide as a carbon source, and the recombinant cell is caused to produce isoprene. Regarding these C1 compounds used as a carbon source, one compound or a combination of two or more compounds may be used. These C1 compounds are preferably used as a main carbon source, and more preferably as a sole carbon source.
In the case of obligate methylotrophs, basically a synthetic culture medium containing a C1 compound as a sole carbon source is used, and addition of small amounts of natural culture medium such as yeast extract, corn steep liquor, and meat extract and vitamins to this culturemedium promotes proliferation of bacteria. In the case of facultative methylotrophs, carbohydrates, lipids and the like substances other than C1 compounds may be used as a carbon source in the bacterial proliferation stage, and in this case, the carbon source can be changed to the above C1 compound in the isoprene production stage. Microorganisms can be cultured in any of aerobic, microaerobic, or anaerobic condition depending on the purpose. Any of the batch culture, feeding culture, and continuous culture can be employed.
For example, when methanol is used as a carbon source, it is typically used at a concentration of 1.0% (v/v) in the case of bacteria, or a concentration of 3.0% (v/v) or less in the case of yeasts, however, when the resistance to these is artificially modified, the culture can be also conducted with methanol of higher concentrations.
In another aspect of the method for producing isoprene of the present invention, at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide are brought into contact with the recombinant cell, and the recombinant cell allows to produce isoprene from the C1 compound. That is, regardless of whether cell division (cell proliferation) is involved or not, it is possible to produce isoprene by bringing the C1 compound into contact with the recombinant cell. For example, it is possible to continuously produce isoprene by continuously supplying an immobilized recombinant cell with the C1 compound.
Also in the present aspect, regarding these C1 compounds, only one C1 compound may be used, or a combination of two or more C1 compounds may be used.
The produced isoprene is accumulated in the cell or released outside the cell. For example, by collecting, isolating and purifying the isoprene released outside the cell, it is possible to acquire purified isoprene.
In the following, the present invention will be described more specifically by way of examples. However, the present invention is not limited to these examples.
Example 1
Introduction of isoprene synthase gene into a methylotroph having the XuMP pathway, and production of isoprene from methanol using a recombinant
In the present example, methanol assimilating yeast Pichia pastolis GS115 strain (available from Invitrogen) was used as a methylotroph having a XuMP pathway.
Using total RNA derived from leaf of poplar (Populus nigra) as a template, a gene encoding isoprene synthase (IspS) from poplar (IspS gene from poplar, SEQ ID NO: 1, GenBank Accession No.: AM410988.1) was amplified by RT-PCR using primers represented by SEQ ID NO: 3 and SEQ ID NO: 4. The obtained amplified DNA fragment was cloned into pT7-Blue T vector (available from TAKARA BIO INC.) to construct pT7IS. The pT7IS was cut with BamHI to obtain IspS gene. The obtained IspS gene was introduced into BamHI site of pPIC3.5K (available from Invitrogen) to construct a vector pPCIPS in which isoprene synthase gene is introduced.
Introduction of isoprene synthase gene by means of pPCIPS into Pichia pastoris GS115 strain was conducted according to the Invitrogen Manual “Version D 032002/25-0156”. For obtaining a multi-copy transformant, Geneticin (available from Invitrogen) tolerant strain at a concentration of 1.5 mg/mL was acquired. In this manner, methanol assimilating yeast GS115IPS strain having a plurality of copies of foreign isoprene synthase gene was constructed. As a control strain, GS11535K strain in which only pPIC3.5K was introduced having tolerance to Geneticin at a concentration of 1.5 mg/mL was obtained.
Each of GS115IPS strain and GS11535K strain was cultured aerobically at 30° C. for 64 hours in 20 mL of synthetic A culture containing methanol as a sole carbon source (containing 18 g of H3PO4, 14.28 g of K2SO4, 3.9 g of KOH, 0.9 g of CaSO4.2H2O, 11.7 g of MgSO4.7H2O, 8.4 mg of CuSO4.5H2O, 1.1 mg of KI, 4.2 mg of MnSO4H2O, 0.3 mg of NaMoO4.2H2O, 0.03 mg of H3BO3, 0.7 mg of CoCl2.6H2O, 28 mg of ZnSO4.7H2O, 91 mg of FeSO4.7H2O, 0.28 mg of biotin, 20 mL of methanol per 1 L). After collecting the cells, the cells were further cultured in 45 mL of synthetic A culture medium in a 125 mL vial sealed with a butyl rubber plug at 30° C. for another 16 hours under shaking. After end of the culture, the gas phase component was analyzed by GC/MS. Isoprene was not detected in GS11535K strain, but was detected in GS115IPS strain. These revealed that the present example allowed production of isoprene by eukaryotic microorganism (yeast) via a XuMP pathway which is one of methanol assimilating pathways.
Example 2
Introduction of methanol dehydrogenase, HPS gene, PSI gene, and isoprene synthase gene into a non-methylotroph, and production of isoprene from methanol by a recombinant
In the present example, Bacillus subtilis was used as a non-methylotroph.
(Preparation of Various Genes)
NADP-dependent methanol dehydrogenase (MDH) gene of SEQ ID NO: 5 was amplified from genomic DNA of Bacillus methanolicus (NCIMB 13114) by PCR using primers of SEQ ID NO: 7 and SEQ ID NO: 8. The amplified DNA fragment was cloned into pT7 Blue-T vector to construct pT7BMmdh.
3-hexulose 6-phosphate synthase (HPS) gene of SEQ ID NO: 9 was amplified from genomic DNA of Methylomonas aminofaciens by PCR using primers of SEQ ID NO: 11 and SEQ ID NO: 12. The amplified DNA fragment was cloned into pT7 Blue-T vector to construct pT7MAhps.
6-phospho-3-hexuloisomerase (PHI) gene of SEQ ID NO: 13 was amplified from genomic DNA of Methylomonoas aminofaciens by PCR using primers of SEQ ID NO: 15 and SEQ ID NO: 16. The amplified DNA fragment was cloned into pT7 Blue-T vector to construct pT7MAphi.
PCR was conducted by using pT7IS having isoprene synthase (IspS) gene prepared in Example 1 as a template, and primers of SEQ ID NO: 17 and SEQ ID NO: 18. The amplified DNA fragment was cloned into pT7 Blue-T vector to construct pT7IS2.
(Construction of Various Expression Vectors)
The cloning vector pT7MAhps was cut with BglII and BamHI to cut out hps gene. The hps gene was introduced into BamHI site of an expression vector for Bacillus subtilis pHT01 (MoBiTec) to prepare pHTh.
By cutting the cloning vector pT7MAphi with BglII and BamHI, PHI gene was cut out. The PHI gene was introduced into BamHI site of pHTh to prepare pHThp.
By cutting the cloning vector pT7BMmdh with BglII and BamHI, MDH gene was cut out. The MDH gene was introduced into BamHI site of pHThp to prepare pHThpm.
Further, by cutting pT7IS2 with BamHI and SmaI, IspS gene was cut out. The IspS gene was introduced into BamHI/SmaI site of pHThpm to construct pHThpmIS. The expression vector pHThpmIS has an operon in which HPS gene, PHI gene, MDH gene, and IspS gene are arranged in this order downstream of promotor of pHT01.
As a control, an expression vector pHThpIS having only HPS gene, PHI gene, and IspS gene was prepared in the same manner as described above. Similarly, an expression vector pHThpm having only HPS gene, PHI gene, and MDH gene was prepared.
According to the manual of MoBiTec “Bacillus subtilis Expression Vectors”, each expression vector was introduced into Bacillus subtilis and a recombinant (recombinant cell) was prepared. In this manner, BShpmIS strain having the expression vector pHThpmIS, BShpIS strain having the expression vector pHThpIS, and BShpm strain having the expression vector pHThpm were prepared, respectively.
Each recombinant was aerobically cultured at 37° C. in 20 mL of a methanol assimilating inductive culture medium (containing 10 mL of methanol, 3 g of ammonium phosphate, 1 g of potassium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.5 g of yeast extract, 0.01 mM IPTG, and 5 mg of chloramphenicol in 1 L of tap water). The cells were collected at the point of time when OD600 of the culture liquid reached 1.0 to 1.2. All the collected cells were added to 45 mL of a methanol assimilating inductive culture medium similar to that described above (provided that IPTG concentration was 0.1 mM), and were further cultured in a 125 mL vial sealed with a butyl rubber plug at 37° C. for 24 hours under shaking. After end of the culture, the gas phase component was analyzed by GC/MS.
Consequently, while BShpmIS strain and BShpm strain grew sufficiently, BShpIS strain not having MDH little grew.
Regarding isoprene production, isoprene was detected in BShpmIS strain. Conversion efficiency from the assimilated methanol to isoprene in BShpmIS strain was 14%. Although isoprene was slightly detected in BShpm strain, conversion efficiency from the assimilated methanol to isoprene was as small as about 0.3%.
These revealed that by introducing MDH gene, HPS gene, and PHI gene to Bacillus subtilis which is a non-methylotroph, efficient growth in a culture medium containing methanol as a main carbon source was enabled, and by introducing IspS gene, isoprene was generated efficiently.
Example 3
Preparation of a methylotroph having a serine pathway into which MVA pathway gene and isoprene synthase gene are introduced, and isoprene production from methanol by a recombinant
In the present example, an isoprene producing strain was prepared by using Methylobacterium extorquens (ATCC 55366) as a methylotroph having a serine pathway, and introducing IspS gene and a mevalonate pathway gene cluster derived from actinomycete into this strain.
A DNA fragment containing IspS gene derived from poplar was prepared by PCR using pT7IS prepared in Example 1 as a template, and primers represented by SEQ ID NO: 19 and SEQ ID NO: 20. This DNA fragment was cloned into pT7-Blue T vector to prepare pT7IS3.
A DNA fragment containing a gene encoding a group of a mevalonate pathway enzymes of S. griseolosporeus (SEQ ID NO: 23) was amplified by PCR using genomic DNA of actinomycete, Streptomyces griseolosporeus (Kitasatospora griseola) as a template, and primers represented by SEQ ID NO: 21 and SEQ ID NO: 22. This DNA fragment includes a gene cluster encoding mevalonate kinase, mevalonate diphosphate decarboxylase, Phosphomevalonate kinase, IPP isomerase, HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (HMGR), and HMG-CoA synthase. The obtained amplified DNA fragment was cloned into pT7-Blue T vector to construct pT7SMV.
The pT7IS3 was cut with BamHI and KpnI to obtain IspS gene. The IspS gene was introduced into BamHI/KpnI site of pCM80 (Marx C J. et al., Microbiology 2001, 147, 2065-2075) which is a broad host range vector to prepare pC80IS. The pT7SMV was cut with KpnI to obtain a fragment containing an actinobacterial MVA pathway gene and a terminator sequence. The fragment was introduced into KpnI site of the pC80IS to construct pC80ISMV. The expression vector pC80ISMV has IspS gene and a group of genes of actinobacterial MVA pathway enzymes downstream of the promotor.
The expression vector pC80IS was introduced into M. extorquens by electroporation to obtain ME-IS strain. The expression vector pC80ISMV was introduced into M. extorquens by electroporation to obtain ME-ISMV strain. As a control, the expression vector pCM80 was introduced into M. extorquens by electroporation to obtain ME-CM80 strain.
ME-IS strain, ME-ISMV strain, or ME-CM80 strain was aerobically cultured at 30° C. in 20 mL of synthetic B culture medium containing methanol as a sole carbon source (containing 18 g of H3PO4, 14.28 g of K2SO4, 3.9 g of KOH, 0.9 g of CaSO4, 2H2O, 11.7 g of MgSO4.7H2O, 8.4 mg of CuSO4.5H2O, 1.1 mg of KI, 4.2 mg of MnSO4H2O, 0.3 mg of NaMoO4.2H2O, 0.03 mg of H3BO3, 0.7 mg of CoCl2.6H2O, 28 mg of ZnSO4.7H2O, 91 mg of FeSO4.7H2O, 0.28 mg of biotin, 5 mL of methanol, and 10 mg of tetracycline per 1 L). The cells were collected at the point of time when OD600 of the culture liquid reached 1.0 to 1.2. All the collected cells were added to 45 mL of synthetic B culture medium as described above, and were cultured in a 125 mL vial sealed with a butyl rubber plug at 30° C. for 16 hours under shaking. After end of the culture, the gas phase component was analyzed by GC/MS.
Consequently, isoprene was detected in ME-IS strain and ME-ISMV strain. Conversion efficiency from the assimilated methanol to isoprene was 8% in ME-IS strain and 27% in ME-ISMV strain. In ME-CM80 strain, isoprene was not detected.
These revealed that by introducing MVA pathway gene and isoprene synthase gene into a methylotroph having a serine pathway, it was possible to efficiently produce isoprene from methanol.
Example 4
Introduction of isoprene synthase gene into methylotroph having a RUMP pathway and isoprene production from methanol using a recombinant
In the present example, an isoprene producing strain was prepared by using Methylophilus methylotrophus (ATCC 53528) as a methylotroph having a RuMP pathway, and introducing IspS gene into this strain.
A DNA fragment containing IspS gene was amplified by PCR using pT7IS prepared in Example 1 as a template and primers of SEQ ID NO: 19 and SEQ ID NO: 24. This DNA fragment was cloned into pT7Blue-T vector to prepare pT7IspS4. The pT7IspS4 was cut with BamHI and KpnI to obtain a DNA fragment containing IspS gene and a terminator sequence. This DNA fragment was introduced into BamHI/KpnI site of pCM80 (Example 3) to construct an IspS expression vector pM80IS. The pM80IS was introduced to M. methylotrophus by electroporation to obtain MM-80IS strain.
Also a gene cluster including Escherichia coli IDI (isopentenyl diphosphate isomerase) gene and popular IspS gene (SEQ ID NO: 25) was introduced into BamHI/KpnI site of the pCM80 to construct pC80IDIS. The pC80IDIS was introduced into M. methylotrophus by electroporation to obtain MM-80IDIS strain.
As a control, the pCM80 was introduced into M. methylotrophus by electroporation to obtain MM-80 strain.
MM-80IS strain, MM-80IDIS strain, or MM-80 strain was aerobically cultured at 37° C. in 20 mL of synthetic B culture medium containing methanol as a sole carbon source used in Example 3 (provided that the methanol concentration was set at 1% (v/v)). The cells were collected at the point of time when OD600 of the culture liquid reached 1.0 to 1.2. All the collected cells were added to 45 mL of synthetic B culture medium as described above, and were cultured in a 125 mL vial sealed with a butyl rubber plug at 37° C. for 16 hours under shaking. After end of the culture, the gas phase component was analyzed by GC/MS.
Consequently, isoprene was detected in MM-80IS strain and MM-80IDIS strain. Conversion efficiency from the assimilated methanol to isoprene was 12% in MM-80IS strain, and 19% in MM-80IDIS strain. On the other hand, isoprene was not detected in MM-80 strain.
These revealed that by introducing an isoprene synthase gene into a methylotroph having a RUMP pathway, it was possible to efficiently produce isoprene from methanol. It was also revealed that isoprene synthesis was promoted by introduction of IDI gene.
Example 5
Introduction of methanol dehydrogenase(MDH) gene, HPS gene, PHI gene, and IspS gene into Escherichia coli, and production of isoprene from methanol by a recombinant
A DNA fragment (hpmIS) containing HPS gene, PHI gene, MDH gene and IspS gene was amplified by PCR using the expression vector pHThpmIS prepared in Example 2 as a template, and primers of SEQ ID NOs: 26 and 27. This amplified DNA fragment was cloned into pT7Blue-T vector to prepare pT7hpmIS. The pT7hpmIS was cut with NcoI and BamHI to obtain a DNA fragment containing hpmIS gene. This DNA fragment was introduced into NcoI/BamHI site of pET23d (available from Novagen) to construct expression vector pThpmIS in Escherichia coli. The expression vector pThpmIS was introduced into Escherichia coli Rosetta (DE3) (available from Novagen) to obtain Escherichia coli RHPMI strain.
A DNA fragment (hpIS) containing HPS gene, PHI gene, and IspS gene was amplified by PCR using the expression vector pHThpIS prepared in Example 2 as a template and primers of SEQ ID NOs: 26 and 27. This amplified DNA fragment was cloned into pT7Blue-T vector to prepare pT7hpIS. The pT7hpIS was cut with NcoI and BamHI to obtain a DNA fragment containing hpIS gene. This DNA fragment was introduced into NcoI/BamHI site of pET23d (available from Novagen) to construct expression vector pThpIS in Escherichia coli. The expression vector pThpIS was introduced into Escherichia coli Rosetta (DE3) to obtain Escherichia coli RHPI strain.
A DNA fragment (hpm) containing HPS gene, PHI gene, and MDH gene was amplified by PCR using the expression vector pHThpm prepared in Example 2 as a template and primers of SEQ ID NOs: 26 and 28. This amplified DNA fragment was cloned into pT7Blue-T vector to prepare pT7hpm. The pT7hpm was cut with NcoI and BamHI to obtain a DNA fragment containing hpm gene. This DNA fragment was introduced into NcoI/BamHI site of pET23d (available from Novagen) to construct expression vector pThpm in Escherichia coli. The expression vector pThpm was introduced into Escherichia coli Rosetta (DE3) to obtain Escherichia coli RHPM strain.
Each recombinant Escherichia coli was cultured aerobically at 37° C. in 20 mL of methanol assimilative synthetic C culture medium containing 0.05 mM IPTG (containing 18 g of H3PO4, 14.28 g of K2SO4, 3.9 g of KOH, 0.9 g of CaSO4.2H2O, 11.7 g of MgSO4.7H2O, 8.4 mg of CuSO4.5H2O, 1.1 mg of KI, 4.2 mg of MnSO4H2O, 0.3 mg of NaMoO4.2H2O, 0.03 mg of H3BO3, 0.7 mg of CoCl2.6H2O, 28 mg of ZnSO4.7H2O, 91 mg of FeSO4.7H2O, 0.28 mg of biotin, 5 mL of methanol, 34 mg of chloramphenicol, and 100 mg of ampicillin per 1 L). The cells were collected at the point of time when OD600 of the culture liquid reached 1.0 to 1.2. All the collected cells were added to 45 mL of synthetic C culture medium of the same composition described above, and were cultured in a 125 mL vial sealed with a butyl rubber plug at 37° C. for 24 hours under shaking. After end of the culture, the gas phase component was analyzed by GC/MS.
Isoprene was detected only in RHPMI strain. Conversion efficiency from the assimilated methanol to isoprene in RHPMI strain was 14%. The RHPI strain was completely unable to grow. The RHPM strain was able to grow, but generation of isoprene was not detected.

## Claims

1. A recombinant cell prepared by introducing a gene encoding isoprene synthase, into a host cell which is methylotroph, wherein:
the methylotroph host cell belongs to genus Pichia, genus Methylobacterium, or genus Methylophilus, 
the gene encoding isoprene synthase is expressed in the host cell,
the recombinant cell is prepared by further introducing into the host cell six additional genes encoding HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase,
the six additional genes are expressed in the host cell,
the recombinant cell produces isoprene from methanol, and
the conversion efficiency of methanol to isoprene is 27% or more.

2. The recombinant cell according to claim 1, wherein the gene encoding at least one enzyme acting in a mevalonate pathway is derived from actinomycete.

3. The recombinant cell according to claim 1, wherein a gene encoding isopentenyl diphosphate isomerase is further introduced, and the gene is expressed in the host cell.

4. The recombinant cell according to claim 1, wherein a treatment of suppressing an expression amount of geranyl pyrohosphate synthase, neryl pyrophosphate synthase, or farnesyl pyrophosphate synthase is conducted.

5. The recombinant cell according to claim 1,
wherein the gene encoding isoprene synthase encodes a protein of the following (a), (b) or (c):
(a) a protein having the amino acid sequence of SEQ ID NO: 2;
(b) a protein having an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2, and having isoprene synthase activity; or
(c) a protein having an amino acid sequence having a homology of 90% or more with the amino acid sequence of SEQ ID NO: 2, and having isoprene synthase activity.

6. A method for producing isoprene comprising culturing the recombinant cell according to claim 1 by using methanol as a carbon source, to cause the recombinant cell to produce isoprene.

7. A method for producing isoprene comprising bringing methanol into contact with the recombinant cell according to claim 1, to cause the recombinant cell to produce isoprene from the methanol.