Method for producing polyetherdiol

Abstract

To provide a novel method for producing polyetherdiols using enzymes or microbial catalysts. [Solution] In the presence of the first phosphorylation enzyme, the aliphatic diol is phosphorylated, and the aliphatic diol 1-ly A phosphoryl acid is produced, and in the presence of a second phosphorylation enzyme, the aliphatic diol 1-phosphate is phosphorylated. , an aliphatic diol 2-phosphate is produced, and the aliphatic diol 1-phosphate and the aliphatic diol 3-(o-geranylgeranyl)-glycerol 2-phosphate is converted to 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase in the presence of 1-phosphate synthase. The process includes a step of reacting with to obtain polyetherdiol 1-phosphate, A method for producing 1-phosphate.

Description

Technical Field

[0001] The present invention relates to a method for producing polyether diol. More specifically, it relates to a method for producing polyether diol using an aliphatic diol as a raw material by an enzymatic polymerization method.

Background Art

[0002] Polyether diols typified by polytetramethylene ether glycol (PTMG) are used as raw materials for polyurethane resins and polyester resins. The industrial production method of PTMG is by cationic ring-opening polymerization of tetrahydrofuran (THF), but it requires a dehydration cyclization reaction under high temperature and high pressure to convert the raw material 1,4-butanediol into THF once; since a high reaction temperature is also required for the polymerization reaction, it is a process with a high environmental load; since it is difficult to control the degree of polymerization, it becomes a mixture of a large number of molecules with different degrees of polymerization and has a wide molecular weight distribution, and there are these problems. The polymerization reaction also requires a high reaction temperature, so it is a process with a high environmental load; since it is difficult to control the degree of polymerization, it becomes a mixture of a large number of molecules with different degrees of polymerization and has a wide molecular weight distribution, and there are these problems. The polymerization reaction also requires a high reaction temperature, so it is a process with a high environmental load; since it is difficult to control the degree of polymerization, it becomes a mixture of a large number of molecules with different degrees of polymerization and has a wide molecular weight distribution, and there are these problems.

[0003] As a method for producing a polymer with a small environmental load, there is a method using an enzyme or a microorganism. There is a method for obtaining a polyhydroxyalkanoate copolymer by polymerizing 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH) using a microorganism (Patent Document 1), etc. The bioproduction of polyethers by microbial and enzyme-catalyzed polymerization is known. However, a production method of polyether diol by microbial or enzyme-catalyzed polymerization is not known. <了 How to polymerize 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH) using microorganisms to obtain a polyhydroxyalkanoate copolymer (Patent Document 1), etc., the bioproduction of polyethers by microbial and enzyme-catalyzed polymerization is known. However, a production method of polyether diol by microbial or enzyme-catalyzed polymerization is not known. How to polymerize 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH) using microorganisms to obtain a polyhydroxyalkanoate copolymer (Patent Document 1), etc., the bioproduction of polyethers by microbial and enzyme-catalyzed polymerization is known. However, a production method of polyether diol by microbial or enzyme-catalyzed polymerization is not known. How to polymerize 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH) using microorganisms to obtain a polyhydroxyalkanoate copolymer (Patent Document 1), etc., the bioproduction of polyethers by microbial and enzyme-catalyzed polymerization is known. However, a production method of polyether diol by microbial or enzyme-catalyzed polymerization is not known. How to polymerize 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH) using microorganisms to obtain a polyhydroxyalkanoate copolymer (Patent Document 1), etc., the bioproduction of polyethers by microbial and enzyme-catalyzed polymerization is known. However, a production method of polyether diol by microbial or enzyme-catalyzed polymerization is not known. How to polymerize 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH) using microorganisms to obtain a polyhydroxyalkanoate copolymer (Patent Document 1), etc., the bioproduction of polyethers by microbial and enzyme-catalyzed polymerization is known. However, a production method of polyether diol by microbial or enzyme-catalyzed polymerization is not known. How to polymerize 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH) using microorganisms to obtain a polyhydroxyalkanoate copolymer (Patent Document 1), etc., the bioproduction of polyethers by microbial and enzyme-catalyzed polymerization is known. However, a production method of polyether diol by microbial or enzyme-catalyzed polymerization is not known. How to polymerize 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH) using microorganisms to obtain a polyhydroxyalkanoate copolymer (Patent Document 1), etc., the bioproduction of polyethers by microbial and enzyme-catalyzed polymerization is known. However, a production method of polyether diol by microbial or enzyme-catalyzed polymerization is not known. How to polymerize 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH) using microorganisms to obtain a polyhydroxyalkanoate copolymer (Patent Document 1), etc., the bioproduction of polyethers by microbial and enzyme-catalyzed polymerization is known. However, a production method of polyether diol by microbial or enzyme-catalyzed polymerization is not known. How to polymerize 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HH) using microorganisms to obtain a polyhydroxyalkanoate copolymer (Patent Document 1), etc., the bioproduction of polyethers by microbial and enzyme-catalyzed polymerization is known. However, a production method of polyether diol by microbial or enzyme-catalyzed polymerization is not known.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] The object of the present invention is to provide a method for producing polyetherdiols using enzymes or microbial catalysts. It is about doing it. [Means for solving the problem]

[0006] After diligent research, the inventors concluded that 3-(o-geranylgeranyl)-glycerol 1-phosphate The ability of synthase to ether-link aliphatic diol 1-phosphate and aliphatic diol 2-phosphate. They discovered that it possesses power. They also discovered an enzyme that catalyzes the phosphorylation of aliphatic diols, and We have perfected an enzymatic polymerization method for polyether diols using aliphatic diols as raw materials, using the enzyme. Set.

[0007] The present invention is based on the above findings and relates to the following [1] to

[12] . [1] A step comprising phosphorylating an aliphatic diol in the presence of a phosphorylase, A method for producing 1-phosphate. [2] Phosphorylation enzymes, a) Contains the amino acid sequence shown in any of Sequence IDs 1-2, or b) Having 80% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 1-2 It contains an amino acid sequence and has enzymatic activity that catalyzes the monophosphorylation of aliphatic diols. The method described in [1]. [3] A step comprising phosphorylating an aliphatic diol 1-phosphate in the presence of a phosphorylation enzyme, A method for producing aliphatic diol 2-phosphate. [4] Phosphorylation enzymes, c) Contains an amino acid sequence shown in any of Sequence IDs 3-7, or d) Having 80% or more sequence identity with the amino acid sequence shown in any of sequence numbers 3-7. It contains an amino acid sequence and is an enzyme that catalyzes the diphosphorylation of aliphatic diol 1-phosphate. The method described in [3], which has the property of [3]. [5] 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, aliphatic Reacting diol 1-phosphate with aliphatic diol 2-phosphate yields polyetherdiol 1 A method for producing polyetherdiol 1-phosphate, comprising the step of obtaining phosphoric acid. Polyetherdiol 1-phosphate is produced according to the method described in [6] [5], and The process of dephosphorylating polyetherdiol 1-phosphate to obtain polyetherdiol is described below. A method for producing polyetherdiols, including Polyetherdiol 1-phosphate is produced according to the method described in [7] [5], and The polyetherdiol 1-phosphate is phosphorylated in the presence of a phosphorylation enzyme to form a polyether A method for producing polyetherdiol 2-phosphate, comprising the step of obtaining diol 2-phosphate. Polyetherdiol 2-phosphate is produced according to the method described in [8] [7], and Polyetherdiol 2-phosphate and aliphatic diol 1-phosphate are combined into 3-(o-geranyl phosphate) The reaction in the presence of lanyl-glycerol 1-phosphate synthase leads to polyetherdiol 1- A method for producing polyetherdiol 1-phosphate, comprising the step of obtaining phosphoric acid. Polyetherdiol 1-phosphate is produced according to the method described in [9] [8], and The process of dephosphorylating polyetherdiol 1-phosphate to obtain polyetherdiol is described below. A method for producing polyetherdiols, including

[10] 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, e) comprising an amino acid sequence represented by any one of SEQ ID NOs: 8 to 12, or f) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 8 to 12, and having catalytic activity for ether condensation of aliphatic diol 1-phosphate and aliphatic diol 2-phosphate and being a method according to any one of [5] to [9]. Method

[11] A microorganism comprising genes encoding a first phosphorylase, a second phosphorylase, and 3-(o -geranylgeranyl)-glycerol 1-phosphate synthase, respectively, wherein: The first phosphorylase is a) comprising an amino acid sequence represented by any one of SEQ ID NOs: 1 to 2, or b) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 2, and having enzyme activity for catalyzing 1-phosphorylation of an aliphatic diol and being at least one enzyme, The second phosphorylase is c) comprising an amino acid sequence represented by any one of SEQ ID NOs: 3 to 7, or d) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 3 to 7, and having enzyme activity for catalyzing 2-phosphorylation of aliphatic diol 1-phosphate and being at least one enzyme, The 3-(o -geranylgeranyl)-glycerol 1-phosphate synthase is e) comprising an amino acid sequence represented by any one of SEQ ID NOs: 8 to 12, or f) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 8 to 12, and having 3-(o -geranylgeranyl)-glycerol 1-phosphate synthase activity and being at least one enzyme, and being the microorganism as described above.

[12] The microorganisms described in

[11] were cultured in the presence of aliphatic diols to obtain polyether diols. A method for producing polyetherdiol 1-phosphate, comprising a step of obtaining all 1-phosphate. [Effects of the Invention]

[0008] Polymerization reactions catalyzed by microorganisms or enzymes are milder compared to polymerization reactions using organometallic catalysts. Polymerization is possible under certain conditions, and environmental impact can be reduced. Furthermore, the enzyme's strict substrate specificity... This property enables the synthesis of polymers with a narrow molecular weight distribution. [Brief explanation of the drawing]

[0009] [Figure 1] Confirmation of monophosphoric oxide (1,4-butanediol-1phosphoric oxide) by LC / MS. [Figure 2] Confirmation of diphosphorus oxide (1,4-butanediol-2phosphorus oxide) by LC / MS. [Figure 3] Confirmation of the ether-bound compound (PTMG dimer-1 phosphorus oxide) by LC / MS. [Modes for carrying out the invention]

[0010] 1. Method for producing aliphatic diol 1-phosphate This invention involves phosphorylating an aliphatic diol with a phosphorylation enzyme to obtain an aliphatic diol 1- This invention provides a method for producing phosphoric acid.

[0011] In the present invention, "aliphatic diol" is a saturated or unsaturated fatty acid diol, It can be chain-like or branched.

[0012] Fatty acid diols preferably have 2 to 14 carbon atoms, more preferably 2 to 12 carbon atoms, Preferably, it is a fatty acid diol having 2 to 8 carbon atoms, and more preferably, 2 to 6 carbon atoms.

[0013] Preferably, the fatty acid diol is a saturated linear fatty acid diol, for example, linear Alkylene glycols, specifically ethylene glycol, 1,3-propylene glycol Coal, 1,4-butanediol, 1,6-hexanediol, 1,10-decanediol Examples include 1,12-dodecanediol and 1,14-tetradecanediol. However, 1,4-butanediol is more preferred.

[0014] The "phosphorylation enzyme" used in this invention is an enzyme that catalyzes the phosphorylation of aliphatic diols. It is not particularly limited as long as it has sexual properties. For example, mevalonate kinase, glycerol Examples include ethanolamine kinase, 3-phosphomevalonate kinase, etc. It is possible.

[0015] The origin of "phosphorylation enzymes" is not particularly limited; for example, they can originate from eukaryotes, bacteria, etc. Examples include those derived from bacteria.

[0016] Specifically, "phosphorylation enzymes" include those from the genera Staphylococcus, Escherichia, and Klebs. iella spp., Halobacterium spp., Pseudomonas spp., Firmicutes spp., Arabidopsis spp., Sacchar You can use materials from the genera Omyces, Bos, and Mus.

[0017] Specific examples of the "phosphorylation enzyme" used in this invention include, for example, SEQ ID NOs: 1-2 Enzymes (proteins) containing the amino acid sequence shown by either of the following can be listed.

[0018] The phosphorylation enzyme shown in Sequence ID No. 1 is Mevalonate kin from Staphylococcus aureus. The phosphorylation enzyme indicated by sequence number 2, as ase (Uniprot ID: Q2G0I8), is Escherichia Glycerol kinase (Uniprot ID: B7UNP6) derived from coli O127:H6, respectively, is publicly available. It is registered in the database.

[0019] The "phosphorylation enzyme" used in this invention is not limited to those having the above sequence. Not, but more than 60% of the amino acid sequence described in any of SEQ ID NOs: 1-2, preferably about 7% 0% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably amino acids having approximately 95% or more homology or identity, most preferably approximately 98% or more. An enzyme (protein) that contains a sequence and has enzymatic activity that catalyzes the phosphorylation of aliphatic diols. It also includes (the quality).

[0020] Furthermore, the "phosphorylation enzyme" used in this invention is one of the enzymes described in any of Sequence IDs 1 to 2. In the amino acid sequence, one or several, specifically 1 to 20, preferably 1 to 10 1, more preferably 1 to 5, even more preferably 1 to 2 amino acids are deleted, substituted or It contains an added amino acid sequence and has enzymatic activity that catalyzes the phosphorylation of aliphatic diols. It also contains enzymes (proteins).

[0021] The gene for the "phosphorylation enzyme" used in this invention encodes the following amino acid sequence. a) The amino acid sequence shown in any of Sequence IDs 1-2, or b) The amino acid sequence shown in any of SEQ ID NOs: 1-2 and approximately 60% or more, preferably approximately 7% 0% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably It has sequence identity of approximately 95% or more, most preferably approximately 98% or more, and aliphatic dio The amino acid sequence of a protein that has enzymatic activity to catalyze the phosphorylation of a molecule.

[0022] The "phosphorylation enzyme" gene used in this invention may include, for example, one of sequence numbers 1 to 2. Polynucleotides and strings having a base sequence that encodes the amino acid sequence shown by Enzyme activity that hybridizes under optimal conditions and catalyzes the phosphorylation of aliphatic diols Genes containing base sequences that encode proteins having sex are also genes for the phosphorylation enzyme of the present invention. It is included in the offspring. Preferably, the above gene is of the genus Staphylococcu or Escherichia. ru.

[0023] Stringent conditions include, for example, a nylon membrane on which DNA is immobilized, and 6×SSC(1× SSC is a solution of 8.76g of sodium chloride and 4.41g of sodium citrate dissolved in 1 liter of water. 1% SDS, 100 μg / ml salmon sperm DNA, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolid The probe was kept in a solution containing 0.1% Ficol at 65°C for 20 hours to hybridize. While we can list the conditions for performing dilation, we are not limited to these. If you are a professional, in addition to conditions such as the salt concentration and temperature of the buffer, other factors will also be considered. Taking into account various conditions such as ion concentration, probe length, and reaction time, the hybridization conditions It is possible to set the following conditions for cleaning after hybridization: for example, "2×S SC, 0.1% SDS, 42°C, "1×SSC, 0.1% SDS, 37°C", and more stringent conditions For example, conditions such as "1×SSC, 0.1%SDS, 65℃" and "0.5×SSC, 0.1%SDS, 50℃" It can be listed.

[0024] 2. A method for producing aliphatic diol 2-phosphate. This invention involves phosphorylating aliphatic diol 1-phosphate with a phosphorylation enzyme, thereby This invention provides a method for producing all-2-phosphate.

[0025] The aliphatic diols that make up "aliphatic diol 1-phosphate" are as described in item 1 above. Therefore, the aliphatic diol 1-phosphate is obtained by the method described in 1 above. Acid can be used.

[0026] The "phosphorylation enzyme" used in this invention catalyzes the phosphorylation of aliphatic diol 1-phosphate. It is not particularly limited as long as it has the enzyme activity to do so. For example, 5-phosphomevalone Examples include enzymes and isopentenyl phosphate kinases.

[0027] The origin of "phosphorylation enzymes" is not particularly limited; for example, they can originate from eukaryotes or archaea, or Examples include those of nuclear origin.

[0028] Specifically, "phosphorylation enzymes" include those from the genera Arabidopsis, Oryza, and Nitr. osopumilus, Panicum, Branchiostoma, Methanofervidicoccus, Methanocaldoco ccus, Haloferax, Thermoplasma, Streptomyces, Thermanaerothrix, Longili You can use materials from the genera Nea, Flexilinea, Scytonema, and Archaea.

[0029] Specific examples of the "phosphorylation enzymes" used in this invention include, for example, SEQ ID NOs: 3-7 Enzymes (proteins) containing the amino acid sequence shown by either of the following can be listed.

[0030] The phosphorylation enzyme shown in Sequence ID No. 3 above is Isopent from Nitrosopumilus adriaticus. The phosphorus shown in SEQ ID NO: 4 is represented as enyl phosphate kinase (Uniprot ID: A0A0D5C472). The oxidase is Isopentenyl phosphate kinase (Uniprot ID: A0A7C1) derived from Archaea Phylum. The phosphorylation enzyme indicated by Sequence ID No. 5 (LM50) is Isopent from Panicum miliaceum. The phosphorus shown in SEQ ID NO: 6 is represented as enyl phosphate kinase (Uniprot ID: A0A3L6SU37). The oxidase is Isopentenyl phosphate kinase (Uniprot ID) derived from Branchiostoma belcheri. The phosphorylation enzyme indicated by sequence number 7 (A0A6P4ZPW9) is Methanofervidicoccus a As isopentenyl phosphate kinase (Uniprot ID: A0A401HP04) derived from Byssi, each It is registered in a public database.

[0031] The "phosphorylation enzyme" used in this invention is not limited to those having the above sequence. Not, but more than 60% of the amino acid sequence described in any of SEQ ID NOs: 3-7, preferably about 7% 0% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferred amino acids having approximately 95% or more homology or identity, most preferably approximately 98% or more. It contains an acid sequence and has enzymatic activity that catalyzes the diphosphorylation of aliphatic diol 1-phosphate. It also includes enzymes (proteins).

[0032] Furthermore, the "phosphorylation enzyme" used in this invention is one of the enzymes described in any of Sequence IDs 3 to 7. In the amino acid sequence, one or several, specifically 1 to 20, preferably 1 to 10 1, more preferably 1 to 5, even more preferably 1 to 2 amino acids are deleted, substituted or It contains an added amino acid sequence and catalyzes the diphosphorylation of aliphatic diol 1-phosphate. This also includes enzymes (proteins) that possess enzymatic activity.

[0033] The gene for the "phosphorylation enzyme" used in this invention encodes the following amino acid sequence. c) The amino acid sequence shown in any of Sequence IDs 3-7, or d) The amino acid sequence shown in any of SEQ ID NOs: 3-7 and approximately 60% or more, preferably approximately 7 0% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferred It has a sequence identity of approximately 95% or more, most preferably approximately 98% or more, and is an aliphatic geo The amino acid sequence of a protein having enzymatic activity that catalyzes the diphosphorylation of 1-phosphate.

[0034] The gene for the "phosphorylation enzyme" used in this invention is indicated by any of SEQ ID NOs: 3 to 7. Polynucleotides having a base sequence that encodes an amino acid sequence and stringent An enzyme that hybridizes under certain conditions and catalyzes the diphosphorylation of aliphatic diol 1-phosphate. Genes containing base sequences encoding active proteins are also genes of the phosphorylation enzyme of the present invention. It is included in the gene. The stringent conditions are as described above.

[0035] The enzyme used in this invention is immobilized on a resin or a suitable carrier in accordance with methods well known in the art. Even if they are immobilized (fixed enzymes) or encapsulated (embedded) in polymers or microcapsules, Furthermore, the enzyme may be used in a state of isolation from the raw material, such as by a semipermeable membrane. The same applies even if they are present.

[0036] 3. Method for producing polyetherdiol 1-phosphate In the presence of 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, lipid A polyether diol is formed by reacting aliphatic diol 1-phosphate with aliphatic diol 2-phosphate. A method for producing 1-phosphate is also provided.

[0037] As an aliphatic diol 1-phosphate, the polyetherdiol 1-phosphate obtained in step 1 above is used. These can be used. In addition, as aliphatic diols, 2-phosphate can be obtained in step 2 above. A modified polyetherdiol 2-phosphate can be used.

[0038] The "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" used in this invention is It has catalytic activity for the ether condensation of aliphatic diol 1-phosphate and aliphatic diol 2-phosphate. This allows the aliphatic diol 1-phosphate and aliphatic diol 2-phosphate to produce a polyether. Ludiol 1-phosphate can be produced.

[0039] The "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" used in this invention is The catalytic activity for ether condensation of aliphatic diol 1-phosphate and aliphatic diol 2-phosphate is It is not particularly limited as long as it possesses the properties of heptaprenylglyceryl phosphate. Examples include tosintase and geranylgeranylglycerylphosphate synthase.

[0040] The origin of "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" is not particularly limited. For example, these include those derived from archaea, bacteria such as eubacteria, and eukaryotes. It is possible.

[0041] Specifically, "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" is , Aeropyrum, Thermococcus, Archaeoglobus, Bacteroidetes, Chitinophaga , Zunongwangia sp., Spirosoma sp., Methanothermobacter sp., Flavobacterium sp., Bacillus We use species from the genera Octadecabacter, Pustulibacterium, and the family Crocinitomicaceae. It is possible.

[0042] The "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" used in this invention For example, yeast containing the amino acid sequence shown in any of sequence numbers 8-12. One can list the basic components (proteins).

[0043] 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, as shown in Sequence ID No. 8 above. This is Heptaprenylglyceryl phosphate synthase (Uniprot ID: O) derived from Bacillus subtilis. The phosphorylation enzyme indicated by sequence number 9 (34790) is derived from Flavobacterium johnsoniae. Geranylgeranylglyceryl phosphate synthase (Uniprot ID: A5FJK8), sequence number The phosphorylation enzyme shown in No. 10 is geranylgeranylglyceryl derived from Hugenholtzia roseola. The sequence is / heptaprenylglyceryl phosphate synthase (Uniprot ID: UPI0004282B8E). The phosphorylation enzyme indicated by number 11 is Geranylgerany from Crocinitomicaceae bacterium. As shown in Sequence ID No. 12, glyceryl phosphate synthase (Uniprot ID: A0A2E4SBV2) The phosphorylation enzyme is Geranylgeranylglyceryl phosphatase derived from Pustulibacterium marinum. Each of these has been registered in a public database as te synthase (Uniprot ID: A0A1I7EWQ1). It is being done.

[0044] The "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" used in this invention is However, this is not limited to those having the above sequence, but is described in any of sequence numbers 8 to 12. The amino acid sequence is approximately 60% or more, preferably approximately 70% or more, more preferably approximately 80% or more. More preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 9 It contains an amino acid sequence with 8% or more homology or identity, and is an aliphatic diol. An enzyme (tan) that has catalytic activity for ether condensation of 1-phosphate and aliphatic diol 2-phosphate. It also includes protein.

[0045] Furthermore, the "phosphorylation enzyme" used in this invention is one of those described in Sequence ID No. 8 to 12. In the amino acid sequence, one or several, specifically 1 to 20, preferably 1 to 1 0, more preferably 1 to 5, even more preferably 1 to 2 amino acids are deleted or substituted. It contains an added amino acid sequence, and aliphatic diol 1-phosphate and aliphatic diol This also includes enzymes (proteins) that have catalytic activity for ether condensation of 2-phosphate.

[0046] The gene for the "phosphorylation enzyme" used in this invention encodes the following amino acid sequence. e) The amino acid sequence shown in any of Sequence IDs 8-12, or f) About 60% or more of the amino acid sequence shown in any of sequence numbers 8-12, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferred Furthermore, it has sequence identity of approximately 95% or more, and is composed of aliphatic diol 1-phosphate and aliphatic diol. The amino acid sequence of a protein that has catalytic activity for ether condensation of 2-phosphate.

[0047] The "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase activity" used in the present invention The gene contains a base that encodes the amino acid sequence shown in one of sequence numbers 8-12. It hybridizes with polynucleotides having a sequence under stringent conditions, and is also lipid-based. It possesses catalytic activity for ether condensation of aliphatic diol 1-phosphate and aliphatic diol 2-phosphate. The gene containing the base sequence that codes for the protein is also the "3-(o-geranylgeranyl)-" of the present invention. It is included in the gene for "glycerol 1-phosphate synthase activity". Stringent conditions are As mentioned above.

[0048] 4. Method for producing polyetherdiol 2-phosphate The present invention relates to the polyetherdiol 1-phosphate obtained by the method described in 3 above, in the presence of a phosphorylation enzyme. A method for producing polyetherdiol 2-phosphate is also provided, which includes a step of phosphorylation below.

[0049] 5. Method for producing polyetherdiol 1-phosphate The present invention relates to a polyetherdiol 2-phosphate obtained by the method described in 4 above, and an aliphatic diol 1-phosphate is converted in the presence of 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase. A method for producing polyetherdiol 1-phosphate, which includes a reaction step, is also provided.

[0050] 6. Method for producing polyetherdiol The present invention relates to the process of dephosphorylating polyetherdiol 1-phosphate obtained by the method described in 3 above. The present invention provides a method for producing polyetherdiols, including the method described in 5 above. The process includes a step of dephosphorylating polyetherdiol 1-phosphate. We also provide a manufacturing method.

[0051] The "dephosphorylation" of polyetherdiol 1-phosphate is carried out using methods well known in the field. It can be done.

[0052] 7. Microbial catalyst The present invention relates to at least one of the phosphorylation enzymes (first phosphorylation enzymes) described in 1. above. , at least one of the phosphorylation enzymes (second phosphorylation enzymes) described in 2. above, and At least the 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase described in section 3. We also provide microorganisms (microbial catalysts) that contain genes encoding each of the two types.

[0053] The microorganisms of the world may be non-recombinant or recombinant. That is, Even if the genes encoding each enzyme are endogenous genes of the microorganism in question, they may be exogenous genes. It is acceptable for such genes to exist, or for them to be partially endogenous and partially exogenous.

[0054] Recombinant microorganisms (transformers) use expression vectors containing genes encoding each enzyme. It is obtained by transforming the host microorganism. The expression vector encodes each enzyme. It may contain each gene individually, or it may contain two or more genes encoding enzymes. It may also include.

[0055] Expression vectors can be prepared by known methods. Generally, a specific enzyme is expressed using a vector. A transcription promoter is inserted upstream of the encoding gene, and in some cases, a terminator is inserted downstream. You can then construct an expression cassette and insert this cassette into the expression vector. If the expression vector already contains a transcription promoter and / or terminator This involves constructing an expression cassette without modifying the transcription promoter of the vector and / or This can be done by using a terminator and inserting a gene encoding a specific enzyme in between. As mentioned above, in a single expression vector, one can include genes encoding two or more enzymes. In addition, all of those genes may be inserted under the same promoter, or under different promoters. It may be inserted under the motor. The type of promoter allows for appropriate expression in the host. While there are no particular limitations as long as it is something that enables this, for example, it can be used in an E. coli host. Possible options include the T7 promoter, trp promoter, lac promoter, and lambdaph. Examples include the PL promoter and PR promoter derived from pharmacokinetics, the tac promoter, and the trc promoter. It can be done.

[0056] The genes encoding each specified enzyme are, for example, (i) according to the base sequence information, primer (ii) enzymes It can also be obtained by synthesizing DNA organically according to its amino acid sequence information. The genes may be optimized depending on the host cell of the transformed organism.

[0057] To insert a gene encoding a specific enzyme into an expression vector, a restriction enzyme method is used. Methods using topoisomerase, etc., can be used. If necessary during insertion, A suitable linker may be added. Also, the base sequence is important for translation into amino acids. Ribosome-binding sequences such as the SD sequence and the Kozak sequence are known, and these sequences are inherited. It may be inserted upstream of the offspring. Along with the insertion, a portion of the amino acid sequence encoded by the gene may be inserted. Substitutions may be made. Additionally, the vector may contain factors (selection factors) for selecting the desired transformant. It is preferable to include markers. Select markers include drug resistance genes and nutritional requirements. Examples include sex complement genes and assimilation-conferring genes, which can be selected depending on the purpose and host. Examples of drug resistance genes used as selection markers in Enterococcus include ampicillin resistance. The genes, kanamycin gene, dihydrofolate reductase gene, and neomycin resistance gene It can be listed.

[0058] Expression vectors vary depending on the host, including plasmid DNA, bacteriophage DNA, and retrotransistors. You can use an appropriate one selected from sposon DNA, artificial chromosome DNA, etc. For example, When using Enterococcus as a host, pTrc99A (GE Healthcare Biosciences), pACYC184 ( Nippon Gene, pMW118 (Nippon Gene), pET series vector (Novagen), etc. It can be listed as follows. Also, as a vector with two or more insertion points, pETDuet-1(Novage Examples include n). Modified versions of these vectors can be used as needed. It is also possible.

[0059] Expression vectors are used to connect genes encoding specific enzymes with appropriate promoters and terminators. By inserting an expression cassette containing linked marker genes, etc., into the host genome, The expression of a specific enzyme can be enhanced. Transmutation occurs when an expression cassette is inserted into the genome. Known methods can be used to obtain the substitute. For example, homologous recombination To insert an expression cassette into the genome, use the expression cassette of the specified enzyme and any gene By performing transformation using a plasmid that has a sequence in the 'mu' region and cannot replicate within the host, A transformant can be obtained in which the entire plasmid or the expression cassette is inserted. In this process, negative selection markers such as the SacB gene (which codes for levanscuase) are incorporated. By using plasmids, or plasmids with a temperature-sensitive (ts) replication mechanism, two homologous pairs can be created. By replacing the expression cassette, it is also possible to efficiently obtain transformants in which only the expression cassette is present on the genome. It is possible. Furthermore, by performing transformation using a DNA fragment consisting only of an expression cassette, the genome can be transformed. It is also possible to obtain a transformant in which an expression cassette is inserted at a random position above.

[0060] When utilizing enzymes that the host naturally possesses in its genome (endogenous enzymes), the relevant enzyme in the genome... By replacing the promoter of a primary gene with a stronger one, its expression can also be enhanced. The promoter used in the expression vector described above is similar to the promoter used in the expression vector. It can be used.

[0061] The host microorganism is subjected to a predetermined protein expression system using an expression vector, etc. The cells that can express the enzyme are not particularly limited. For example, the large intestine. Bacteria (Escherichia coli), Bacillus subtilis, Actinomycetes (e.g., Rhodococcus) Bacteria such as those of the genera Rhodococcus and Corynebacterium; yeast (e.g.) For example, the genera Saccharomyces, Candida, and Pi Examples include filamentous fungi; plant cells; and animal cells such as insect cells and mammalian cells. Among them, Escherichia coli, Corynebacterium and Rhodococcus, and S Yeasts of the genera Calomyces, Candida, and Pichia are preferred, and Escherichia coli is also preferred. It is preferable.

[0062] Examples of E. coli include strains K12 and B, as well as their wild-type derivatives. The strains are W3110, JM109, XL1-Blue (e.g., XL1-BlueMRF'), K802, C600, and BL. Examples include strains 21, BL21(DE3), and BN8.

[0063] The method for introducing the expression vector into the host is not particularly limited, as long as it is a method suitable for the host. There is no such method. Available methods include, for example, electroporation and calcium ion Methods using phosphate, spheroplast method, lithium acetate method, calcium phosphate method, lipofer One example is the action method.

[0064] The transformants into which the expression vector has been introduced are then processed using a method suitable for the host cells (bacteria). You can then culture the cells and express each enzyme.

[0065] 8. Method for producing polyetherdiol 1-phosphate using microorganisms The present invention involves culturing the microorganism described in 7. in the presence of an aliphatic diol, thereby producing a polyether diol. A method for producing polyetherdiol 1-phosphate, which includes a step for producing all 1-phosphate, is also proposed. To provide.

[0066] The microorganisms include resting cells, cells with improved membrane permeability, inactivated cells, and lysated bacteria prepared from the microorganisms. Cell-free extracts prepared from the cells, lysated cells, and stabilization treatments performed on these cells. It may be used as a processed material.

[0067] Polyetherdiol 1-phosphate is added to the above transformant in a culture medium containing an aliphatic diol. The culture is cultured, and polyetherdiol 1-phosphate is collected from the resulting culture to produce it. It can be made.

[0068] In the present invention, "culture" includes culture supernatant, cultured cells, cultured bacterial cells, or cells or It includes all of the fragments of the bacterial cells.

[0069] The culture of microorganisms is carried out according to the methods normally used for culturing host organisms. The culture medium used is the host culture medium. It contains carbon sources, nitrogen sources, inorganic salts, etc. that the main bacterium can utilize, enabling efficient cultivation of transformants. Any culture medium that can be used may be either a natural medium or a synthetic medium. For example, glucose, galactose, fructose, sucrose, raffinose and starch Carbohydrates such as ethanol, organic acids such as acetic acid and propionic acid, and organic acids such as ethanol and propanol. Examples include ammonium chlorides. Nitrogen sources include ammonia, ammonium chloride, and ammonium sulfate. Inorganic acids such as ammonium acetate and ammonium phosphate, or organic acids such as ammonium Examples include nium salts or other nitrogen-containing compounds.

[0070] Other ingredients include peptone, yeast extract, meat extract, corn steep liquor, and various amino acids. You may also use monopotassium phosphate, dipotassium phosphate, and phosphate. Magnesium, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate Examples include copper sulfate and calcium carbonate. In addition, if necessary, to prevent foaming during cultivation. An antifoaming agent may be added. Furthermore, a compound that induces enzyme expression may be added to the culture medium. That's good too.

[0071] The microbial culture conditions hinder the productivity of polyetherdiol 1-phosphate and the growth of the host organism. While not particularly limited under conditions where it is not possible, typically 10°C to 40°C, preferably 20°C. The process is carried out at 37°C for 5 to 100 hours. pH adjustment is performed using inorganic or organic acids, alkaline solutions, etc. For example, if using E. coli, adjust the pH to 4-9.

[0072] Culture methods include solid culture, static culture, shaking culture, and aerated stirring culture. In particular, when culturing Rhodococcus transformants, shake culture or aerated stirring culture ( It is preferable to culture the cells under aerobic conditions using a fermenter.

[0073] The resulting polyetherdiol 1-phosphate can be dephosphorylated using the method already described. This allows for the production of polyetherdiols. [Examples]

[0074] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. It is not something that can be determined.

[0075] [Example 1] 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase and aliphatic dio Creation of E. coli expressing 1-phosphorylation enzyme and 2-phosphorylation enzyme of aliphatic diols. (1) Creation of a plasmid for expressing aliphatic diol monophosphorylation enzyme in E. coli Mevalonate kinase (UniprotID:Q2G0I8) derived from Staphylococcus aureus, and Escheri Glycerol kinase derived from Chia coli O127:H6 (UniprotID: B7UNP6) (hereinafter, each J288VGA140- 6. Gene sequence information of J288VGA140-4) is available at the National Center for Biotechnology Inf. Obtained from ormation (NCBI), the cell-free translation system PUREfrex2.0 (GeneFronte) was created through gene synthesis. Optimized for the codon frequency of (i). Genetics of codon-optimized J288-VGA140-4 and J288VGA140-6. The sub-fragment was inserted into the NdeI-XhoI site of the E. coli expression vector pET-22b(+) (Novagen). Mido was created. The sequences corresponding to J288-VGA140-6 and J288VGA140-4 inserted into the vector were... These are shown in Sequence IDs 13 and 14, respectively. The introduction method is 4 μl of digested product and 1 μl of cleaved pETDuet-1. l, DNA Ligation Kit<Mighty Mix> (Takara Bio) Add 5 μl and mix, then bake at 16°C. A ligation reaction was performed for 1 hour. 10 μl of the ligation reaction solution was then mixed with E. coli JM109 compound. Mix with 100 μl of tent cells (Takara Bio) and let stand on ice for 30 minutes. Incubate at 42°C for 45 seconds. After incubation, the mixture was left to stand on ice for 5 minutes. 500 μl of SOC medium was added, and the mixture was incubated at 37°C for 1 hour. After shaking culture at 0 rpm, the samples were spread onto LB agar medium containing 100 μg / ml ampicillin.

[0076] After culturing overnight at 37°C, the grown colonies were transferred to LB liquid medium (ampicillin 100 μg / ml). The bacteria were inoculated into (containing) and cultured at 37°C for 24 hours with shaking at 200 rpm. The culture solution was centrifuged to extract the bacterial cells. Collect the samples and use the QIAprep Spin Miniprep Kit (Qiagen) according to the attached protocol. Smid extraction was performed. Using the obtained plasmid as a template, PCR was performed again to determine the band position. Confirmed. The obtained plasmid DNA was identified as PTMG11 / pPETDuet-1(J288-VGA140-4). It was named PTMG21 / pPETDuet-1(J288-VGA140-6).

[0077] (2) Creation of a plasmid for expressing the 2-phosphorylation enzyme of aliphatic diol 1-phosphate in E. coli Isopentenyl phosphate kinase (UniprotID:A0A3L6SU37) derived from Panicum miliaceum The gene fragment (referred to as A0A3L6SU37 below) can be amplified by PCR using the primer shown below. A restriction enzyme NdeI site was added to the 5' end and a restriction enzyme XhoI site to the 3' end. PCR was performed. The procedure followed the protocol for PrimeSTAR MAX Premix (Takara Bio).

[0078] A0A3L6SU37 Amplification Primer: NdeI-6His.F aaaaaaCATATGCATCATCACCATCACC(Sequence ID 19) U37-XhoI.R aaaaaaCTCGAGTTACTTACTACTACGGATAATCGTACCCA(Sequence ID 20) PCR reaction solution composition: Template DNA 2 μl, 2×PrimeSTAR Max Premix 25 μl, 10 μM primers 2 μl each, sterile water 1 9 μl (These were mixed to make a total volume of 50 μl.) Reaction temperature conditions: Using a TaKaRa PCR Thermal Cylcer Dice Touch (model TP350), the following steps were performed: 10 seconds at 98°C, and 5 seconds at 58°C. The cycle of 10 seconds at 72°C and then emptying was repeated 30 times. However, the 98°C temperature during the first cycle was not maintained. The time was set to 30 seconds.

[0079] Confirmation of PCR amplification products is done using 1% agarose (Agarose S: ​​Nippon Gene Co., Ltd.). After separation by electrophoresis, staining with GelRed (NEW ENGLAND BioLabs, formerly known as NEB) By visualizing the data, a fragment of approximately 1.1kb originating from A0A3L6SU37 was detected. Each PCR amplification fragment was processed according to the protocol of the QIAquick PCR purification Kit (Qiagen). Purified. For the purified PCR at Hannō Station, NEB restriction enzymes were used. 10 μl of PCR product was mixed with Cutsmart enzymes. Add 4 μl of ferment (included in the kit), 1 μl of NdeI, 1 μl of XhoI, and 4 μl of sterile water. Bake at 37°C for 30 minutes. After processing, the sample was purified using the QIAquick PCR Purification Kit (Qiagen).

[0080] The purified digest product was introduced into pETDuet-1, which had similarly cleaved the NdeI-XhoI site. The sequences corresponding to A0A3L6SU37 inserted into the sequence are shown in sequence number 15. The installation method is PC 4 μl of R digested product, 1 μl of cleaved pETDuet-1, DNA Ligation Kit<Mighty Mix> (Takara) After adding 5 μl of bio-based solution and mixing, the ligation reaction was carried out at 16°C for 1 hour. Mix 10 μl of the reaction solution with 100 μl of E. coli JM109 competent cells (Takara Bio). Then, it was left to stand on ice for 30 minutes. After incubating at 42°C for 45 seconds, it was left to stand on ice again for 5 minutes. Then, 500 μl of SOC medium was added, and the culture was performed at 37°C for 1 hour with shaking at 200 rpm, followed by 100 μg / ml A The sample was spread onto LB agar plate containing mphicillin.

[0081] After culturing overnight at 37°C, the grown colonies were transferred to LB liquid medium (ampicillin 100 μg / ml). The bacteria were inoculated into (containing) and cultured at 37°C for 24 hours with shaking at 200 rpm. The culture solution was centrifuged to extract the bacterial cells. Collect the samples and use the QIAprep Spin Miniprep Kit (Qiagen) according to the attached protocol. Smid extraction was performed. Using the obtained plasmid as a template, PCR was performed again to determine the band position. Confirmed. The obtained plasmid DNA was named PTMG31 / pPETDuet-1(A0A3L6SU37).

[0082] (3) 3-(o-geranylgeranil)-glycerol for E. coli, for 1-phosphate synthase expression Plus Mido creation geranylgeranylglyceryl / heptaprenylglyceryl phosphate s from Hugenholtzia roseola ynthase (UniprotID:UPI0004282B8E) and Geranylg derived from Crocinitomicaceae bacterium eranylglyceryl phosphate synthase (UniprotID:A0A2E4SBV2) and Pustulibacterium Geranylgeranylglyceryl phosphate synthase (UniprotID:A0A1I7EWQ1)( The following gene fragments (hereinafter referred to as UPI0004282B8E, A0A2E4SBV2, and A0A1I7EWQ1 respectively) are as follows: PCR amplification with the primer shown results in a restriction enzyme NdeI site at the 5' end and a site at the 3' end. Restriction enzyme XhoI sites were added. PCR was performed using PrimeSTAR MAX Premix (Takara Bio). We followed the instructions.

[0083] UPI0004282B8E amplification primer: NdeI-6His.F aaaaaaCATATGCATCATCACCATCACC(Sequence ID 19) B8E-XhoI.R aaaaaaCTCGAGTTACGGAGTCTGAGGGAAGG(Sequence ID 21) Primer for amplification of A0A2E4SBV2: NdeI-6His.F aaaaaaCATATGCATCATCACCATCACC(Sequence ID 19) BV2-XhoI.R aaaaaaCTCGAGTTAAGAGGCCAGGGTGCC(Sequence ID 22) A0A1I7EWQ1 amplification primer: NdeI-6His.F aaaaaaCATATGCATCATCACCATCACC(Sequence ID 19) WQ1-XhoI.R aaaaaaCTCGAGTTAAATATTTTTCAGTTCGTTATAAAAGTC(Sequence ID 23) PCR reaction solution composition: Template DNA 2 μl, 2×PrimeSTAR Max Premix 25 μl, 10 μM primers 2 μl each, sterile water 1 9 μl (These were mixed to make a total volume of 50 μl.) Reaction temperature conditions: Using a TaKaRa PCR Thermal Cylcer Dice Touch (model TP350), the following steps were performed: 10 seconds at 98°C, and 5 seconds at 58°C. The cycle of 10 seconds at 72°C and then emptying was repeated 30 times. However, the 98°C temperature during the first cycle was not maintained. The time was set to 30 seconds.

[0084] Confirmation of PCR amplification products is done using 1% agarose (Agarose S: ​​Nippon Gene Co., Ltd.). After separation by electrophoresis, staining with GelRed (NEW ENGLAND BioLabs, also known as NEB) This is done by visualization, and UPI0004282B8E is approximately 0.9kb, A0A2E4SBV2 and A0A1I7EWQ1 are A fragment of approximately 0.8kb was detected. Each obtained PCR-amplified fragment was subjected to QIAquick PCR purification. The solution was purified according to the protocol of Qiagen. The purified PCR reaction mixture was then treated with NEB restriction yeast. A sample was used. 10 μl of PCR product, 4 μl of Cutsmart buffer (included in the kit), 1 μl of NdeI, Xho I added 1 μl and 4 μl of sterile water. After processing at 37°C for 30 minutes, QIAquick PCR purification K It was refined by IT (Qiagen Corporation).

[0085] The purified digest product was introduced into pETDuet-1, which had similarly cleaved the NdeI-XhoI site. The sequences corresponding to UPI0004282B8E, A0A2E4SBV2, and A0A1I7EWQ1 inserted into the sequence are respectively The method is shown in numbers 16-18. The introduction method is 4 μl of PCR digest product, 1 μl of cleaved pETDuet-1, and DNA Ligat Ion Kit<Mighty Mix> (Takara Bio) Add 5 μl and mix, then leave at 16°C for 1 hour. A ligation reaction was performed. 10 μl of the ligation reaction solution was placed in E. coli JM109 competent cells. Mix with 100 μl of Takara Bio and let stand on ice for 30 minutes. Incubate at 42°C for 45 seconds. Afterward, it was left to stand on ice for 5 minutes. 500 μl of SOC medium was added, and it was shaken at 200 rpm for 1 hour at 37°C. After culturing, the samples were spread onto LB agar medium containing 100 μg / ml ampicillin.

[0086] After culturing overnight at 37°C, the grown colonies were transferred to LB liquid medium (ampicillin 100 μg / ml). The bacteria were inoculated into (containing) and cultured at 37°C for 24 hours with shaking at 200 rpm. The culture solution was centrifuged to extract the bacterial cells. Collect the samples and use the QIAprep Spin Miniprep Kit (Qiagen) according to the attached protocol. Smid extraction was performed. Using the obtained plasmid as a template, PCR was performed again to determine the band position. Confirmed. The obtained plasmid DNAs were identified as PTMG41 / pPETDuet-1(UPI0004282B8E) respectively. These were named PTMG42 / pPETDuet-1(A0A2E4SBV2) and PTMG43 / pPETDuet-1(A0A1I7EWQ1).

[0087] (4) 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase and aliphatic diol Creation of E. coli expressing 1-phosphorylation enzyme and 2-phosphorylation enzyme of aliphatic diol 1-phosphate. The PTMG11 / pPETDuet-1 (J288-VGA140-4) and PTMG21 / pPETDuet-1 (J288-VGA1) obtained above are shown above. 40-6), PTMG31 / pPETDuet-1(A0A3L6SU37), PTMG41 / pPETDuet-1(UPI0004282B8E), PTM 1 μl of each plasmid solution containing G42 / pPETDuet-1 (A0A2E4SBV2) and PTMG43 / pPETDuet-1 (A0A1I7EWQ1) This was added to 100 μl of E. coli BL21 (DE3) competent cells (Biodynamics Institute), The sample was left undisturbed on ice for 10 minutes. A heat shock was then performed at 42°C for 45 seconds, followed by another 5 minutes of undisturbed resting on ice. 500 μl of room temperature SOC medium was added, and the culture was performed at 37°C for 1 hour with shaking at 200 rpm. 500 μl of the culture medium after harvesting was spread onto LB agar medium (containing 100 μg / ml ampicillin). PTMG11 strain (J288-VGA140-4), which expresses one phosphorylation enzyme of two types of aliphatic diols obtained from [source]. (Current strain) and PTMG21 strain (J288-VGA140-6 expressing strain), 2-phosphorylation of aliphatic diol 1-phosphate Enzyme-expressing strain PTMG31 (A0A3L6SU37 expressing strain), PTMG41 strain expressing three types of ether-binding enzymes (UPI (0004282B8E expressing strain), PTMG42 strain (A0A2E4SBV2 expressing strain), PTMG43 strain (A0A1I7EWQ1 expressing strain) It was named. Colonies grown at 37°C for 24 hours were cultured in LB liquid medium (containing ampicillin 100 μg / ml). The cells were cultured in ) and stored in a 20% glycerol solution at -80°C until purification and activity confirmation tests were performed. For comparison, the BL21(DE3) strain with the pPETDuet-1 empty vector was similarly prepared. did. [Examples]

[0088] (Preculture step) PTMG11 strain (J288-VGA140-4 expressing strain) expressing one phosphorylation enzyme of two types of aliphatic diols and PTMG21 strain (J288-VGA140-6 expressing strain), aliphatic diol 1-phosphate 2-phosphorylation enzyme expressing strain P TMG31 strain (A0A3L6SU37 expressing strain), PTMG41 strain (UPI0004282B8E expressing strains of 3 types of ether-conjugating enzymes) The following strains were used in the preceding examination: (expressing strain), PTMG42 strain (A0A2E4SBV2 expressing strain), and PTMG43 strain (A0A1I7EWQ1 expressing strain). Culture medium [LB Broth (Sigma-Aldrich, NaCl 10g / l, Trypton 10g / l, Yeast Extract (5g / l), 100mg / ml Ampicillin 4μl dissolved in ultrapure water to a total volume of 4ml, then sterilized. The cells were inoculated into 4 ml of culture medium and incubated at 37 degrees Celsius with shaking at 200 rpm for 24 hours (TAITEC, BR-23FP).

[0089] (Main culture process) Add 4 ml of the pre-culture solution obtained above to the main culture medium [LB Broth (Sigma-Aldrich, NaCl 10 g / Add 10g / l Trypton, 5g / l Yeast Extract, and 200μl 100mg / ml Ampicillin to a total volume of 200ml. Dissolve in ultrapure water and sterilize. Inoculate into 200 ml (500 ml Erlenmeyer flask) and culture at the appropriate temperature. The cells were cultured at 37°C and 200 rpm with shaking until the OD600 reached 0.4. (Takasaki Scientific Instruments Co., Ltd., TB-16R) -3). Subsequently, isopropyl-β-D(-)-thiogalactopyranoside (hereinafter referred to as IPTG) ( Fujifilm Wako Pure Chemical Corporation added 0.3 mM. After addition, the mixture was heated at 17°C and 200 rp. The target protein was cultured with shaking for 24 hours in m to induce its expression.

[0090] (Crushing process) The culture medium obtained above was subjected to centrifugation at 5000 rpm, 4°C, 15 minutes (Eppendorf, Centrifug). The bacterial cells were recovered using e 5804R). The bacterial cells were then treated with 5 ml of 20 mM sodium phosphate buffer (pH 7.4). The suspension was then resuspended in PBS (hereafter referred to as PBS). The suspension was cooled on ice using a BRONSON Digital Sonifier. Ultrasonic disruption (amplitude 15%, 5 sec-on / 5 sec-off, 5 min) was performed three times using [tool name]. The suspension obtained by crushing was centrifuged at 5000 rpm, 4°C, for 15 minutes, and 5 ml of the supernatant was collected. The recovered supernatant was tested for activity using the method described later, and then subjected to the purification process.

[0091] (purification process) The recovered supernatant was filtered using a syringe filter DISMIC 0.2μm (ADVANTEC). The solution was filtered, and the filtered bacterial cell lysate was collected. The collected filtrate was then processed using HisTrap HP volume 1m³. The sample was purified using a Cytiva product. The purification process followed the protocol recommended by Cytiva. .

[0092] Specifically, after washing the column with sterile water, a solution containing 20 mM imidazole (Tokyo Chemical Industries, Ltd.) is added. The solution was equilibrated with 10 ml of PBS. Then, 5 ml of the filtered supernatant was also equilibrated. The sample was then trapped in the column. Next, the column was washed with PBS containing 20 mM imidazole, followed by 0.5 M NaCl Elutions obtained by dissolving 50 mM, 100 mM, 200 mM, and 500 mM imidazole in the PBS containing the substance (hereafter, Isocratic elution was performed using 10 ml of the elution solution. His-Tag purified expression The protein concentration was confirmed using a Nano-Drop® Spectrophotometer (ND-1000) and then SDS-P The product was subjected to AGE (Advanced Glycation Endoscopy). The purified product in which a band was confirmed was Amicon® Ultra-15 Ultracel-10K. Desalted and concentrated at Merck.

[0093] (1. Measurement of phosphorylation enzyme activity) 1-phosphorylation enzyme activity is related to the ADP produced in the 1-phosphorylation reaction from 1,4-butanediol. Using (adenosine 5'-disodium diphosphate trihydrate), phosphorylation is performed by pyruvate kinase. It converts foenolpyruvic acid to pyruvate, and the resulting pyruvate is converted to L-lactic acid. In the process of converting naDH to lactate by rogenase, the NADH added to the reaction system is consumed. The decrease in ADH was measured over time by absorbance at a wavelength of 340 nm using a microplate reader, and was approximately 5-10 The measurement was performed by monitoring the reaction for several minutes. The reaction solution used was as follows:

[0094] 10 mM β-NADH (Fujifilm Yeast Co., Ltd.) 3 μl, 10 mM Dithiothreitol (Fujifilm Fujifilm Wako Co., Ltd. 3 μl, 1 M MgCl2 (Fujifilm Wako Co., Ltd.) 3 μl, 5 mM phosphoenol Lupiruvate (Sigma-Aldrich) 3 μl, 5000 U / ml L-lactate dehydrogenase, rabbit muscle Origin: Type II (Sigma-Aldrich) 1 μl, 1000 / ml pyruvate kinase, derived from rabbit muscle. (Sigma-Aldrich) 6μl, 1M Tris-HCl (pH 7.5) 30μl, 25mM ATP (adenosine 5'-3 Disodium phosphate trihydrate (Fujifilm Wako Co., Ltd.) 3 μl, 250 mM 1,4-butanedi 3 μl of ol (Tokyo Kasei Kogyo Co., Ltd.) and 300 μg of the supernatant were prepared to make a total volume of 300 μl.

[0095] (Analysis results) Analysis revealed that in both the lysate supernatant of PTMG11 strain and PTMG21 strain, immediately after enzyme addition... Later, a decrease in absorbance at 340 nm was confirmed. On the other hand, BL21(D) with an empty vector introduced for comparison E3) No decrease in absorbance was observed in the supernatant of the lysate and in sterile water. Therefore, PTMG11 and We confirmed that PTMG21 has the activity to monophosphorylate 1,4-butanediol.

[0096] (Polyether synthesis reaction) 10 mM β-NADH (Orient Yeast Co., Ltd.) 3 μl, 10 mM Dithiothreitol (Fuji Foods Co., Ltd.) (Wako Corporation) 3 μl, 1 M MgCl2 (Fujifilm Wako Corporation) 1 M Tris-HCl (pH 7. 5) 30 μl, 25 mM ATP (adenosine 5'-triphosphate disodium trihydrate) (Fujifilm Wako) (Corporate) 3 μl, 500 mM 1,4-butanediol (Tokyo Chemical Industries Co., Ltd.) 30 μl, J288-VGA1 Prepare a total volume of 300 μl by adding 300 μg each of purified proteins 40-4, A0A3L6SU37, and UPI0004282B8E. The reaction was carried out at 30°C for 4 hours.

[0097] (Analysis method) Product analysis was performed using an Agilent Technologies 6460 Triple Quad LC / MS. The column used was Agilent Proshell 120 HILIC-Z 2.7μm 2.1×150mm (Agilent). The elution method was as follows. Specifically, ultrapure water containing 0.01% formic acid (Solven) was used for elution. CH3CN (Solvent B) containing tA and 0.01% formic acid was used. The concentration gradient was calculated by adjusting the mixing ratio of Solvent A to 1. 0% (10 minutes), 20% (10.1 to 15 minutes), 30% (15.1 to 20 minutes), 50% (20.1 to 25 minutes) (minutes), performed with isocratic elution of 80% (25.1 to 30 minutes) (flow rate 0.2 ml / min, ca (Rum temperature 40°C). 1,4-Butanediol (product), 1-phosphorylated product (reactant), 2-phosphorylated product (Reactant), phosphorylated PTMG (reactant) is measured by Waters SQ Detector 2 for the corresponding mass-to-charge ratio ( Detected at m / z.

[0098] [Table 1]

[0099] (Reactant analysis) 1. Phosphorus oxides: After the phosphorylation reaction, the molecular ion peak of 1,4-butanediol-1 phosphorylate is observed at approximately 1.5 minutes ([ M+H] + The detection of (=171) confirmed the formation of 1-phosphorus oxide (Figure 1). 2 Phosphates After the phosphorylation reaction, the molecular ion peak of 1,4-butanediol-2phosphorite is observed at approximately 2.1 minutes ([ MH] - The detection of (=249) confirmed the formation of two phosphorus oxides (Figure 2). Ether compound After the ether bonding reaction, the molecular ion peak of PTMG dimer-1 phosphorylate ([M+H]) is observed at approximately 3.3 minutes. + = The detection of 243) confirmed the progress of 1,4-butanediol polymerization (Figure 3).

[0100] From the above, it is concluded that 1,4-butanediol is converted to an aliphatic diol by a phosphorylation enzyme, and aliphatic di The conversion of PTMG dimers is performed by the action of all 1-phosphate diphosphorylation enzymes and ether-conjugating enzymes. I confirmed it. [Industrial applicability]

[0101] According to the present invention, polyetherdiols can be polymerized under mild conditions, reducing environmental impact. A method for producing polyetherdiols is provided.

[0102] All publications, patents, and patent applications cited herein are used as direct reference in this specification. It shall be included in the book.

[0103] [ka] JPEG2026074106000003.jpg196166JPEG2026074106000004.jpg162166 [Sequence Listing Free Text]

[0104] Sequence ID 13: Sequence inserted into the vector (J288-VGA140-6) Sequence ID 14: Array inserted into the vector (J288VGA140-4) Sequence ID 15: Sequence inserted into the vector (A0A3L6SU37) Sequence ID 16: Sequence inserted into the vector (UPI0004282B8E) Sequence ID 17: Sequence inserted into the vector (A0A2E4SBV2) Sequence ID 18: Sequence inserted into the vector (A0A1I7EWQ1) Sequence ID 19: Primer (NdeI-6His.F) Sequence ID 20: Primer (U37-XhoI.R) Sequence ID 21: Primer (B8E-XhoI.R) Sequence ID 22: Primer (BV2-XhoI.R) Sequence ID 23: Primer (WQ1-XhoI.R)

Claims

1. A step comprising phosphorylating an aliphatic diol in the presence of a phosphorylation enzyme, A method for producing 1-phosphate.

2. Phosphorylation enzymes, a) Containing the amino acid sequence shown in any of SEQ ID NOs: 1 to 2, or b) Having 80% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 1 or 2 It contains an amino acid sequence and has enzymatic activity that catalyzes the monophosphorylation of aliphatic diols. The method according to claim 1.

3. A step comprising phosphorylating an aliphatic diol 1-phosphate in the presence of a phosphorylation enzyme, A method for producing diol 2-phosphate.

4. Phosphorylation enzymes, c) Containing an amino acid sequence shown in any of SEQ ID NOs: 3 to 7, or d) Having 80% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 3-7 It contains an amino acid sequence and is an enzyme that catalyzes the diphosphorylation of aliphatic diol 1-phosphate. The method according to claim 3, which has the property of

5. 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase is produced in the presence of aliphatic dio 1-phosphate is reacted with an aliphatic diol 2-phosphate to produce a polyether diol 1-phosphate. A method for producing polyetherdiol 1-phosphate, comprising a step of obtaining an acid.

6. A polyetherdiol 1-phosphate is produced according to the method of claim 5, and the The process includes a step of dephosphorylating 1-phosphate of etherdiol to obtain a polyetherdiol. A method for producing polyetherdiol.

7. A polyetherdiol 1-phosphate is produced according to the method of claim 5, and the Lyetherdiol 1-phosphate is phosphorylated in the presence of a phosphorylase to form polyetherdiol A method for producing polyetherdiol 2-phosphate, comprising the step of obtaining polyetherdiol 2-phosphate.

8. Polyetherdiol 2-phosphate is produced according to the method of claim 7, and the 3-(o-geranylgerani) (Lu)-glycerol 1-phosphate reacts in the presence of synthase to produce polyetherdiol 1-phosphate. A method for producing polyetherdiol 1-phosphate, comprising a step of obtaining an acid.

9. A polyetherdiol 1-phosphate is produced according to the method of claim 8, and the The process includes a step of dephosphorylating 1-phosphate of etherdiol to obtain a polyetherdiol. A method for producing polyetherdiol.

10. 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, e) Containing an amino acid sequence shown in any of SEQ ID NOs: 8 to 12, or f) Having 80% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 8-12 It contains the following amino acid sequence, and aliphatic diol 1-phosphate and aliphatic diol 2-phosphate The catalytic activity for ether condensation of the following, according to any one of claims 5 to 9. method.

11. The first phosphorylation enzyme, the second phosphorylation enzyme, and 3-(o-geranylgeranyl)-glycerol A microorganism containing genes that encode each of the following 1-phosphate synthases: The first phosphorylation enzyme described above, a) Containing the amino acid sequence shown in any of SEQ ID NOs: 1 to 2, or b) Having 80% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 1 or 2 It contains an amino acid sequence and has enzymatic activity that catalyzes the monophosphorylation of aliphatic diols. It is at least one type of enzyme, The second phosphorylation enzyme described above, c) Containing an amino acid sequence shown in any of SEQ ID NOs: 3 to 7, or d) Having 80% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 3-7 It contains an amino acid sequence and is an enzyme that catalyzes the diphosphorylation of aliphatic diol 1-phosphate. It is at least one type of enzyme that has sex, The aforementioned 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, e) Containing an amino acid sequence shown in any of SEQ ID NOs: 8 to 12, or f) Having 80% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 8-12 It contains an amino acid sequence that enables the synthesis of 3-(o-geranylgeranyl)-glycerol 1-phosphate. The microorganism is at least one enzyme having enzymatic activity.

12. The microorganism described in claim 11 is cultured in the presence of an aliphatic diol to obtain a polyether diol. A method for producing polyetherdiol 1-phosphate, comprising the step of obtaining 1-phosphate.

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