Patent Grant
9677096
The 30,069 byte text file titled “SequenceListing2.txt” (Creation Date: Aug. 20, 2015) is hereby incorporated by reference.
The present invention relates to a manufacturing method for 1,4-butanediol, a microbe, and a gene.
In recent years, attention is paid to a compound manufacturing process with a renewable source as a raw material from the viewpoint of depletion of fossil resource, a countermeasure for global warming, or the like. In particular, a so-called bio-refinery has widely been studied wherein a variety of compounds as raw materials for a polymer or compounds as raw materials for a chemical product are manufactured in biochemical processes with biomass as raw materials.
For a compound that is expected for raw material conversion of biomass, 1,4-butanediol is provided. 1,4-butanediol is widely used as a synthetic raw material for a precision organic chemical product, monomer units of a polyester and an engineering plastic, or the like, and a market size thereof is large. For that reason, demand for a method of manufacturing 1,4-butanediol efficiently in a biochemical process with a renewable source such as biomass as a raw material is increased.
For manufacturing methods for 1,4-butanediol that use a biochemical process, there are provided, for example, methods described in patent documents 1 and 2 and non-patent document 1.
[Patent Document 1] Japanese Patent No. 4380704 specification
[Patent Document 2] International Publication No. 2008/115840 official gazette
[Non-patent document 1] Harry Yim et al., Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol, Nature Chemical Biology, 7, 445-452 (2011).
However, methods described in patent documents 1 and 2 and non-patent document 1 are complicated processes.
Against a problem as described above, there is provided a new manufacturing method for 1,4-butanediol that is capable of obtaining 1,4-butanediol economically.
The present invention includes the following.
[1] A method of manufacturing 1,4-butanediol by using a microbe and/or a culture thereof, through the following processes:
(1) a process of converting acetyl-CoA into acetoacetyl-CoA;
(2) a process of converting acetoacetyl-CoA into 3-hydroxybutyryl-CoA;
(3) a process of converting 3-hydroxybutyryl-CoA into crotonyl-CoA;
(4) a process of converting crotonyl-CoA into 4-hydroxybutyryl-CoA; and
(5) a process of converting 4-hydroxybutyryl-CoA into 1,4-butanediol,
wherein the microbe in the manufacturing method for 1,4-butanediol
includes any one of genes among:
(a) a gene that has a base sequence of sequence number 1;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 1, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 1; and
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 1 on a stringent condition;
as a gene that codes an enzyme that catalyzes a process of (4) described above; and
includes any one or more genes among:
(d) a gene that has a base sequence of any one of sequence numbers 2 to 9;
(e) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of any one of sequence numbers 2 to 9, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to an original base sequence thereof; and
(f) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence of any one of sequence numbers 2 to 9 on a stringent condition;
as a gene that codes an enzyme that catalyzes any one of processes of (1)-(3) and (5) described above.
[2] A microbe that has a capability of manufacturing 1,4-butanediol through the following processes:
(1) a process of converting acetyl-CoA into acetoacetyl-CoA;
(2) a process of converting acetoacetyl-CoA into 3-hydroxybutyryl-CoA;
(3) a process of converting 3-hydroxybutyryl-CoA into crotonyl-CoA;
(4) a process of converting crotonyl-CoA into 4-hydroxybutyryl-CoA; and
(5) a process of converting 4-hydroxybutyryl-CoA into 1,4-butanediol,
wherein the microbe
includes any one of genes among:
(a) a gene that has a base sequence of sequence number 1;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 1, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 1; and
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 1 on a stringent condition;
as a gene that codes an enzyme that catalyzes a process of (4) described above; and
includes any one or more genes among:
(d) a gene that has a base sequence of any one of sequence numbers 2 to 9;
(e) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of any one of sequence numbers 2 to 9, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to an original base sequence thereof; and
(f) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence of any one of sequence numbers 2 to 9 on a stringent condition;
as a gene that codes an enzyme that catalyzes any one of processes of (1)-(3) and (5) described above.
[3] The gene to be used in the manufacturing method for 1,4-butanediol described in [1], which is a gene described in any one of under-mentioned (a)-(c):
(a) a gene that has a base sequence of any one of sequence numbers 1 to 9
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of any one of sequence numbers 1 to 9, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to an original base sequence thereof
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence of any one of sequence numbers 1 to 9 on a stringent condition.
It is possible to provide a new manufacturing method for 1,4-butanediol that is capable of obtaining 1,4-butanediol economically.
The present invention will be described in detail below. Here, “CoA” in the present specification means “coenzyme A”. Furthermore, “%” indicates “% by mass” unless otherwise described. “ppm” is a mass standard.
A manufacturing method for 1,4-butanediol according to an embodiment is a manufacturing method for 1,4-butanediol that relies on an enzyme reaction that uses a microbe or a culture thereof, through acetyl-CoA, acetoacetyl-CoA, 3-hydroxybutyryl-CoA, crotonyl-CoA, and 4-hydroxybutyryl-CoA.
Specifically, each enzyme reaction includes:
(1) a process of converting acetyl-CoA into acetoacetyl-CoA;
(2) a process of converting acetoacetyl-CoA into 3-hydroxybutyryl-CoA;
(3) a process of converting 3-hydroxybutyryl-CoA into crotonyl-CoA;
(4) a process of converting crotonyl-CoA into 4-hydroxybutyryl-CoA; and
(5) a process of converting 4-hydroxybutyryl-CoA into 1,4-butanediol.
The present inventors executed a variety of studies in order to improve productivity of 1,4-butanediol, and as a result, found that it was possible to obtain 1,4-butanediol at a high productivity by using a particular gene or a homolog thereof in a gene that codes an enzyme that catalyzes each reaction in a process as described above.
For a particular gene as described previously, specifically, any one of genes among:
(a) a gene that has a base sequence of sequence number 1;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 1, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 1; and
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 1 on a stringent condition;
is preferably used as a gene that codes an enzyme that catalyzes a reaction in (4) a process of converting crotonyl-CoA into 4-hydroxybutyryl-CoA.
Furthermore, any one of genes among:
(a) a gene that has a base sequence of any one of sequence numbers 2 to 9;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of any one of sequence numbers 2 to 9, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to a base sequence of sequence number 1; and
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence of any one of sequence numbers 2 to 9 on a stringent condition;
is preferably used as a gene that codes an enzyme that catalyzes a corresponding conversion reaction in any one or more processes among other processes (1), (2), (3), and (5).
In the present embodiment, a particular gene as described above includes a gene that has a base sequence that is specifically indicated in a sequence listing, and a homolog thereof. A homolog includes an ortholog and a paralog. An ortholog refers to a set of corresponding genes among species generated from a gene of a common ancestor by means of speciation and enzymes obtained from such genes. A paralog refers to corresponding genes among species generated by means of not speciation but gene duplication in an identical species and enzymes obtained from such genes. A homolog refers to genes that have an identity in sequences thereof, regardless of an ortholog or a paralog, and enzymes obtained from such genes.
More specifically, a homolog (gene) of a gene as described above refers to a gene that has a base sequence with an identity greater than or equal to 90%, preferably an identity greater than or equal to 95%, with respect to such a gene, and more preferably, a gene that is completely identical to such a gene or wherein one or several bases thereof are deleted, substituted, or added.
Furthermore, a homolog gene includes a gene that hybridizes with a gene that has a base sequence complementary with a target gene on a stringent condition. Specifically, it is possible to acquire a gene or an enzyme that is obtained by transformation caused by such a gene, by applying a homology retrieval program (for example, BLAST or FASTA) to a publicly-known data base, or based on an ordinary method such as hybridization or polymerase chain reaction (PCR) on a stringent condition that uses a probe that is composed of at least a portion of an identified gene (DNA that is composed of a base sequence complementary with DNA that is composed of a base sequence of such a gene). Furthermore, it is possible for a person(s) skilled in the art to execute self-design by substituting a base sequence or the like. Here, for a stringent condition referred herein, there is provided, for example, a condition for executing hybridization described in a non-patent document of Molecular Cloning—A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press). More specifically, a hybridizing condition is a condition that retention with a probe in a solution that contains 6×SSC (a composition of 1×SSC: 0.15 M of sodium chloride, 0.015 M of sodium citrate, pH: 7.0), 0.5% of SDS, 5× Denhardt's solution, and 100 mg/mL of herring sperm DNA, at a constant temperature of 65° C. for 8-16 hours is executed to cause hybridization.
In the present invention, for example, a reaction is caused to proceed, by expressing (that may be co-expressing) an enzyme or a series or group of enzymes that is/are coded by each gene, that is selected as described above, in a body of a microbe provided in such a manner that a host microbe as described below is transformed by means of gene recombination.
A feature of a microbe that is used in the present embodiment, a fabrication method for the microbe, a method of use of the microbe (that is, a manufacturing method for 1,4-butanediol), an acquisition method of manufactured 1,4-butanediol, and the like, will be described below.
(A Host Microbe)
A host microbe that is used in the present embodiment is a host microbe that is capable of introducing a variety of genes as described below, and for example, it is possible to apply a gene recombination technique to a host microbe.
An example of a host microbe that is capable of introducing a gene as described above in the present embodiment is not particularly limited as long as it is possible to apply a gene recombination technique to such a microbe. From the viewpoint of industrial availability, there is provided Escherichia coli, a yeast, a coryneform bacteria, or a clostridial bacteria, as a specific example. For a yeast, there is provided Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, or the like. For a coryneform bacteria, there is provided, Corynebacterium glutamicum, Corynebacterium efficiens, Brevibacterium divaricatum, Brevibacterium saccharolyticum, Brevibacterium immariophilum, Brevibacterium lactofermentum, Brevibacterium roseum, Brevibacterium flavum, Brevibacterium thiogenitalis, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium callunae, Corynebacterium lilium, Corynebacterium mellassecola, Microbacterium ammoniaphilum, or the like. For a clostridial bacteria, there is provided Clostridium kluyveri, Clostridium acetobutylicum, Clostridium aminobutyricum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, or the like. Among these, it is preferable to use Escherichia coli, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Corynebacterium glutamicum, because transformation thereof is easy, and it is more preferable to use Escherichia coli.
Furthermore, a transformed microbe in the present embodiment may be used as a microbe culture bacterial body itself or a variety of forms of a culture thereof. Specifically, a culture of a microbe in the present embodiment includes a suspension of a microbe culture bacterial body in a medium such as a culture medium or a buffer solution, a cell-free extracted fluid from a microbe culture bacterial body, and further, a product processed by concentrating, purifying, and extracting a component that catalyzes such a reaction from such a cell-free extracted fluid or the like. A culture of a microbe in the present embodiment further includes the above-mentioned processed product of a microbe that is fixed on a poorly soluble carrier. For such a fixation carrier, there is provided a compound that forms a poorly water-soluble solid content that encloses a microbial or bacterial body as described previously or a processed product thereof, such as polyacrylamide, polyvinyl alcohol, poly-N-vinylformamide, polyallylamine, polyethyleneimine, methylcellulose, glucomannan, alginate, or carrageenan, or further a copolymer or crosslinked product thereof, or the like. One kind of these may be used singly or two or more kinds thereof may be mixed and used. Furthermore, it is also possible to use, as a culture of a microbe, a microbe or an extracted fluid or extracted component thereof that is held on an object that is preliminarily formed as a solid, such as activated carbon, a porous ceramic, glass fiber, a porous polymer molded object, or a nitrocellulose film.
(A Transformed Microbe)
A host microbe that is used in the present embodiment is a host microbe that is capable of introducing a variety of genes as described below, wherein it is possible to apply, for example, a gene recombination technique to such a host microbe. Specifically, such a host microbe further has each enzyme system of an enzyme reaction system that is capable of producing 1,4-butanediol through acetyl-CoA, acetoacetyl-CoA, 3-hydroxybutyryl-CoA, crotonyl-CoA, and 4-hydroxybutyryl-CoA, in addition to an enzyme system that is intrinsically possessed thereby. An enzyme system in a manufacturing method for 1,4-butanediol according to the present embodiment and a gene that codes each enzyme system will be described below.
[A Gene that Catalyzes a Reaction that Converts Acetyl-CoA into Acetoacetyl-CoA]
For a gene that codes an enzyme that catalyzes a reaction that converts acetyl-CoA into acetoacetyl-CoA in the present embodiment,
(a) a gene that has a base sequence of sequence number 2;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 2, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 2; or
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 2 on a stringent condition;
is preferably used that is provided by the inventors.
Furthermore, for a gene that codes an enzyme that catalyzes a reaction that converts acetyl-CoA into acetoacetyl-CoA in the present embodiment,
(a) a gene that has a base sequence of sequence number 3;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 3, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 3; or
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 3 on a stringent condition;
is preferably used that is provided by the inventors.
[A Gene that Catalyzes a Reaction that Converts Acetoacetyl-CoA into 3-hydroxybutyryl-CoA]
For a gene that codes an enzyme that catalyzes a reaction that converts acetoacetyl-CoA into 3-hydroxybutyryl-CoA in the present embodiment,
(a) a gene that has a base sequence of sequence number 4;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 4, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 4; or
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 4 on a stringent condition;
is preferably used that is provided by the inventors.
Furthermore, for a gene that codes an enzyme that catalyzes a reaction that converts acetoacetyl-CoA into 3-hydroxybutyryl-CoA in the present embodiment,
(a) a gene that has a base sequence of sequence number 5;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 5, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 5; or
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 5 on a stringent condition;
is preferably used that is provided by the inventors.
[A Gene that Codes an Enzyme that Catalyzes a Reaction that Converts 3-hydroxybutyryl-CoA into Crotonyl-CoA]
For a gene that codes an enzyme that catalyzes a reaction that converts 3-hydroxybutyryl-CoA into crotonyl-CoA in the present embodiment,
(a) a gene that has a base sequence of sequence number 6;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 6, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 6; or
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 6 on a stringent condition;
is preferably used that is provided by the inventors.
Furthermore, for a gene that codes an enzyme that catalyzes a reaction that converts 3-hydroxybutyryl-CoA into crotonyl-CoA in the present embodiment,
(a) a gene that has a base sequence of sequence number 7;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 7, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 7; or
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 7 on a stringent condition;
is preferably used that is provided by the inventors.
[A Gene that Codes an Enzyme that Catalyzes a Reaction that Converts Crotonyl-CoA into 4-hydroxybutyryl-CoA]
For a gene that codes an enzyme that catalyzes a reaction that converts crotonyl-CoA into 4-hydroxybutyryl-CoA in the present embodiment,
(a) a gene that has a base sequence of sequence number 1;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 1, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 1; or
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 1 on a stringent condition;
is preferably used that is provided by the inventors.
[A Gene that Codes an Enzyme that Catalyzes a Reaction that Converts 4-hydroxybutyryl-CoA into 1,4-butanediol]
For a gene that codes an enzyme that catalyzes a reaction that converts 4-hydroxybutyryl-CoA into 1,4-butanediol in the present embodiment,
(a) a gene that has a base sequence of sequence number 8;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 8, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 8; or
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 8 on a stringent condition;
is preferably used that is provided by the inventors.
Furthermore, for a gene that codes an enzyme that catalyzes a reaction that converts 4-hydroxybutyryl-CoA into 1,4-butanediol in the present embodiment,
(a) a gene that has a base sequence of sequence number 9;
(b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 9, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 9; or
(c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 9 on a stringent condition;
is preferably used that is provided by the inventors.
Here, an enzyme that is coded by a gene as described above catalyzes a reaction that converts 4-hydroxybutyryl-CoA into 4-hydroxybutanal, and 1,4-butanediol is substantially and immediately derived from obtained 4-hydroxybutanal by an alcohol reductase that is normally possessed by a host microbe as described previously.
[Supply of Acetyl-CoA]
A supply method for acetyl-CoA that is a substrate in a manufacturing method for 1,4-butanediol according to the present embodiment is not particularly limited and a variety of known methods are used. For example, it is possible to be obtained from a saccharide such as glucose due to a glycolytic system of a host microbe. Furthermore, it is also possible to obtain acetyl-CoA in a pathway of β-oxidation of a lipid. Moreover, acetyl-CoA may be supplied by using a CoA transferase in such a manner that CoA is transferred to acetic acid by means of coupling with an appropriate CoA material.
(A Fabrication Method for a Microbe)
It is possible to execute introduction of a gene into a host microbe by appropriately combining and using a variety of known methods, for example, a method based on a restriction enzyme/ligation, an In-Fusion cloning method, and the like, so that a gene as described above or a part thereof is linked with an appropriate vector and an obtained recombinant vector is introduced into a host in such a manner that it is possible for a target gene to be expressed. Alternatively, it is possible to insert a target gene or a part thereof at an arbitrary position on genome by means of homologous recombination. A “part” refers to a part of each gene that is capable of expressing a protein that is coded by each gene in a case of being introduced into a host. A gene in the present invention encompasses DNA and RNA, and preferably, is DNA.
A vector that is linked with a gene as described previously is not particularly limited as long as it is possible to execute replication thereof in a host, and there are provided, for example, a plasmid, a phage, a cosmid, and the like, that are utilized for introduction of an exotic gene into Escherichia coli. For a plasmid, there is provided, for example, pHSG398, pUC18, pBR322, pSC101, pUC19, pUC118, pUC119, pACYC117, pBluescript II SK(+), pET17b, pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, pCOLADuet-1, or the like, and for a phage, there is provided, for example, λgt10, Charon 4A, EMBL-, M13mp18, M13mp19, or the like. Some of them are commercially available and it is possible to use a commercially available product (kit) directly in accordance with, or by appropriately modifying, a procedure manual thereof.
In a vector as described above, an appropriate expression promoter may be connected to such a gene upstream thereof so that an inserted gene is expressed reliably. An expression promoter to be used is not particularly limited, and it is possible for a person skilled in the art to make an appropriate selection thereof depending on a host. For example, it is also possible to utilize T7 promoter, lac promoter, trp promoter, trc promoter, or λ-PL promoter that is utilized for expression of an exotic gene in Escherichia coli, or an Nar promoter region of a nitrate reduction gene nar GHJI operon that is derived from Escherichia coli and involved with nitrate respiration, or a promoter region of Frd gene that is a gene of a nitrate reductase of Escherichia coli.
Furthermore, depending on a case, it is also preferable to destruct an intrinsic gene of a host microbe so that such a gene is not expressed. For a method of gene destruction, it is possible to use a publicly-known method that is utilized for gene destruction in Escherichia coli. Specifically, it is possible to use a method that is used for fabricating a knockout cell or the like in such a technical field, such as a method (gene targeting method) that destructs such a gene by using a vector (targeting vector) that causes homologous recombination at an arbitrary position of a target gene, a method (gene trap method) that inserts a trap vector (reporter gene that does not have a promoter) at an arbitrary position of a target gene to destruct such a gene and be caused to lose a function thereof, or a method of a combination thereof.
A position that causes homologous substitution or a position for inserting a trap vector is not particularly limited as long as such a position is to cause mutation that eliminates expression of a target gene to be destructed, and is preferably a transcription control region.
Moreover, a method for introduction of a vector as described previously into a host is not particularly limited, and it is possible to provide, for example, a method that uses a calcium ion, a protoplast method, an electroporation method, or the like, that is generally utilized for introduction of a vector into Escherichia coli.
A target gene together with a promoter is inserted into a sequence homologous to a sequence on a genome and such a nucleic acid fragment is introduced into a cell by means of electroporation to cause homologous recombination, so that it is possible to practice a method that inserts a target gene at an arbitrary position on a genome by means of homologous recombination. As a nucleic acid fragment wherein a drug resistance gene is linked with a target gene is used in a case of introduction into a genome, it is possible to readily choose a strain wherein homologous recombination is caused. Furthermore, it is also possible to introduce a target gene by means of homologous recombination in such a manner that a gene wherein a gene that is lethal on a particular condition is linked with a drug resistance gene is inserted into a genome by means of homologous recombination in a method as described above and subsequently the drug resistance gene and the gene that is lethal on a particular condition are replaced thereby.
Moreover, a method that selects a recombinant microbe with an introduced target gene is not particularly limited, and it is preferable to be based on a technique that is capable of readily selecting only a recombinant microbe with an introduced target gene.
(A culturing method and an acquisition method for obtained 1,4-butanediol)
For example, a reaction in the present invention is most conveniently achieved in such a manner that a transformed body is cultured in a nutritive medium such as an LB medium at a temperature of 15° C.-40° C., desirably 18° C.-37° C. for about 24 hours, subsequently implanted to a medium with a normal carbon source, for example, a carbon source that is 0.01-50%, desirably 0.1-30%, of glucose, and continuously cultured at a similar temperature for about 1 hour-200 hours, wherein 1,4-butanediol is stored in a culture solution in such a process. Furthermore, a carbon source may be added continuously or intermittently depending on consumption of a carbon source that is caused by proliferation of a bacteria or proceeding of a reaction, and in such a case, a concentration of a carbon source in a reaction fluid is not limited to one described previously.
As a medium carbon source for culturing a microbe, it is possible to use, for example, a saccharide such as glucose, sucrose, or fructose, a polyol such as glycerol, an organic substance such as ethanol, acetic acid, citric acid, succinic acid, lactic acid, benzoic acid, or a fatty acid, or an alkali metal salt thereof, an aliphatic hydrocarbon such as an n-paraffin, an aromatic hydrocarbon, or a natural organic substance such as peptone, meat extract, fish extract, soybean flour, or bran, singly or in combination thereof, at a concentration of normally 0.01%-30%, desirably about 0.1%-20%.
For a medium nitrogen source for culturing a microbe, it is possible to use, for example, an inorganic nitrogen compound such as ammonium sulfate, ammonium phosphate, sodium nitrate, or potassium nitrate, a nitrogen-containing organic substance such as urea or uric acid, or a natural organic substance such as peptone, meat extract, fish extract, or soybean flour, singly or in combination thereof, at a concentration of normally 0.01%-20%, desirably about 0.1%-10%.
Moreover, it is possible to add a phosphate such as potassium dihydrogen phosphate, or a metal salt such as magnesium sulfate, ferrous sulfate, calcium acetate, manganese chloride, copper sulfate, zinc sulfate, cobalt sulfate, or nickel sulfate, as necessary, for growth of a bacteria or improvement of an enzyme activity. A concentration for addition thereof is different depending on a culturing condition, and normally, is 0.01%-5% for a phosphate, 10 ppm-1% for a magnesium salt, or about 0.1 ppm-1,000 ppm for other compounds. Furthermore, for a supply source of a vitamin, an amino acid, a nucleic acid, or the like, it is possible to add, for example, about 1 ppm-100 ppm of yeast extract, casamino acid, or a yeast nucleic acid, depending on a selected medium, for growth of a bacteria or improvement of an enzyme activity.
It is desirable to adjust a pH of a medium to 4.5-9, desirably 5-8. Furthermore, it is useful to fractionate from a culture solution by a method such as centrifugation or membrane filtration, and again suspend and react in water that contains a reaction raw material, physiological saline, a buffer that has a pH adjusted to be comparable to a pH for culturing and composed of phosphoric acid, acetic acid, boric acid, tris(hydroxymethyl)aminomethane, or the like, or a salt thereof, or the like, a microbial or bacterial body that is preliminarily cultured in a medium as described previously, in order to reduce an impurity in a reaction fluid and simplify subsequent fractionation of a product. Although a pH during a reaction is normally capable of being retained in a case where a buffer with a sufficient concentration is used, it is desirable to be execute appropriate adjustment by using sodium hydroxide, ammonia, or the like so as to provide a similar pH in a case where deviation from a pH as described above is caused by proceeding of a reaction.
In a case where 1,4-butanediol is stored in a reaction fluid and a reaction rate is lowered thereby, a method is preferable that adds water, physiological saline, a reaction buffer, or the like, into such a reaction fluid depending on a concentration of a product to execute continuous dilution thereof. Furthermore, at a point of time when a reaction rate is lowered, a bacteria is fractionated and a supernatant is recovered as a product solution and a fractionated bacteria is again returned to a solution or suspension that contains a reaction raw material, so that it is possible to recover such a reaction rate. It is possible to execute such an operation continuously or even batch-wise by using a centrifuge, a separation film, or the like.
It is possible to execute separation, recovery, and purification of 1,4-butanediol produced in a reaction fluid by using separation, recovery, and purification means for a general organic compound, after a bacterial body is eliminated from such a reaction fluid by means of centrifugation at a point of time when an amount of produced 1,4-butanediol reaches a substantial amount, or for such a reaction fluid directly. For example, extraction from a filtrate provided in such a manner that a bacterial body and others are eliminated from a culture solution is executed by using an appropriate organic solvent. 1,4-butanediol is obtained at a high purity by directly executing distillation for such an extract, as well as, further executing extraction with an appropriate solvent again, executing purification that uses chromatography on silica gel or the like, or applying multistep distillation or the like thereto.
(Practical Examples and Comparative Examples)
Next, the present invention will be described in more detail by describing practical examples.
Table 1 illustrates a summary of an envisaged reaction process, an enzyme that catalyzes each reaction process, and a gene that codes such an enzyme in Practical Examples 1 to 3 and Comparative Example 1. Here, a sequence number for a gene corresponds to a sequence number in a sequence listing.
acetobutylicum
acetobutylicum
acetobutylicum
aminobutylicum
beijerinckii strain
A blunt end fragment was prepared by an ordinary method in such a manner that sequences that corresponded to 15 base pairs at an upstream side and a downstream side that contained CAT at an upstream side and ATG at a downstream side of a NdeI site among multi-cloning sites of expression vector pET17b (produced by Novagen, Inc.), respectively, were added to a 5′-end side and a 3′-end side upstream and downstream a gene sequence indicated by sequence number 10, respectively. Ligation of this fragment and a fragment provided by NdeI-treating pET17b (produced by Novagen, Inc.) was executed by In-Fusion HD Cloning Kit (produced by TAKARA BIO INC.) to obtain plasmid pETBD10.
A gene sequence indicated by sequence number 12 was inserted into an NdeI site of pET17b as a target by a method similar to that for pETBD10 to obtain plasmid pETBD12 that contained sequence 12.
An EcoRI site positioned downstream a termination codon of sequence 10 of pETBD10 and derived from multi-cloning sites of pET17b was cleaved by a restriction enzyme treatment to prepare a ring-opened fragment of pETBD10. Then, a fragment was prepared by means of PCR such that sequences that corresponded to 15 bp at an upstream side and 15 bp at a downstream side that contained an EcoRI site of pETBD10 described previously were added upstream and downstream a region of sequence 12 of pETBD12 and a region that contained T7 promoter derived from pET17b upstream thereof. Ligation of two obtained fragments was executed by In-Fusion HD Cloning Kit to obtain plasmid pETBD10-12 that contained sequences 10 and 12.
Subsequently, sequence numbers 14, 16, and 17 were sequentially added to a sequence downstream sequence 12 of pETBD10-12 as a further target in a similar manner to obtain plasmid pETBD10-12-14-16-17. Here, for addition of a sequence, ring-opening of a plasmid to be subjected to insertion was executed by cleavage with a restriction enzyme in a case where such a suitable restriction enzyme site that did not cleave a sequence subjected to insertion was present on a vector, or inverse PCR from a target site for insertion in a case where such a site was absent (similarly below). Escherichia coli JM109 (DE3) strain was transformed with pETBD10-12-14-16-17 to obtain Escherichia coli pETBD10-12-14-16-17/JM109 (DE3).
A transformed body of JM109 (DE3) was obtained by a method similar to that of Comparative Example 1 in such a manner that transformation was executed with a plasmid provided in such a manner that respective genes of sequence numbers 10, 12, 14, 16, and 17 on plasmid pETBD10-12-14-16-17 were partially substituted by genes of sequence numbers 2, 4, 6, 1, and 8 that coded enzymes corresponding to enzymes that catalyzed respective processes.
Table 2 illustrates a summary of an envisaged reaction process, an enzyme that catalyzes each reaction process, and a gene that codes such an enzyme in Practical Examples 1 to 3 and Comparative Example 1. Here, a sequence number for a gene corresponds to a sequence number in a sequence listing.
Similarly to Practical Examples 1-3 and Comparative Example 1, plasmid pETBD11-13-15-16-18 was first prepared that contained gene sequences indicated by sequence numbers 11, 13, 15, 16, and 18 and thereby, Escherichia coli JM109 (DE3) strain was transformed to obtain Escherichia coli pETBD11-13-15-16-18/JM109 (DE3).
Moreover, a transformed body of JM109 (DE3) was obtained in such a manner that transformation was executed with a plasmid provided in such a manner that respective genes of sequence numbers 11, 13, 15, 16, and 18 on plasmid pETBD11-13-15-16-18 were partially substituted by genes of sequence numbers 3, 5, 7, 1, and 9 that coded enzymes corresponding to enzymes that catalyzed respective processes.
A transformed body obtained in each of the respective practical examples and comparative examples was aerobically cultured in 5 mL of an LB medium that contained 100 mg/L of ampicillin at 37° C. for 12 hours. 0.1 mL of a culture solution was implanted to 5 mL of an LB medium that contained 1% of glucose, 100 mg/L of ampicillin, and 0.2 mM of IPTG and aerobically cultured at 30° C. for 48 hours. A supernatant of a culture solution was subjected to high performance liquid chromatography (HPLC: column; Shodex SH-1011 (produced by Showa Denko K. K.), column temperature: 60° C., eluent: 25 mM sulfuric acid aqueous solution, flow rate: 0.6 mL/min, detection: differential refraction detector). Table 3 and Table 4 illustrate a relationship between a gene that composes a used plasmid for a transformed body and an amount of 1,4-butanediol produced in a culture solution.
aminobutylicum
acetobutylicum
From Table 3 and Table 4, it is possible to obtain 1,4-butanediol at a high productivity by using a particular gene and a homolog thereof, in a manufacturing method for 1,4-butanediol that uses a microbe or a culture thereof, utilizes an enzyme reaction, and includes:
(1) a process that converts acetyl-CoA into acetoacetyl-CoA;
(2) a process that converts acetoacetyl-CoA into 3-hydroxybutyryl-CoA;
(3) a process that converts 3-hydroxybutyryl-CoA into crotonyl-CoA;
(4) a process that converts crotonyl-CoA into 4-hydroxybutyryl-CoA; and
(5) a process that converts 4-hydroxybutyryl-CoA into 1,4-butanediol.
The present application claims priority based on Japanese Patent Application No. 2012-266501 filed on Dec. 5, 2012 before the Japan Patent Office and the entire contents of Japanese Patent Application No. 2012-266501 are incorporated by reference in the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2012-266501 | Dec 2012 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2013/082068 | 11/28/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/087921 | 6/12/2014 | WO | A |
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| 20150376657 A1 | Dec 2015 | US |
