Method for producing polyetherdiol

Abstract

To provide a novel method for producing polyether diol using an enzyme or a microbial catalyst.SOLUTION: A method for producing polyether diol 1-phosphate includes a process of phosphorylating aliphatic diol in the presence of a first kinase to produce aliphatic diol 1-phosphate, phosphorylating the aliphatic diol 1-phosphate in the presence of a second kinase to produce aliphatic diol 2-phosphate, and allowing the aliphatic diol 1-phosphate to react with the aliphatic diol 2-phosphate in the presence of 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase to obtain polyether diol 1-phosphate.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] Polyether diols represented 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). However, since 1,4-butanediol as a raw material needs to be converted into THF once, a dehydration cyclization reaction under high temperature and high pressure is required; since a high reaction temperature is also required for the polymerization reaction, it is a process with a high environmental load; and 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.

[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 known 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., and the bioproduction of polyethers by microbial or enzyme-catalyzed polymerization is known. However, a method for producing a polyether diol by microbial or enzyme-catalyzed polymerization is not known.

Prior Art Documents

Patent Documents

[0004] <0OO0023>

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

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

[0006] After diligent research, the inventors discovered that 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase has the ability to ether-bond aliphatic diol 1-phosphate and aliphatic diol 2-phosphate. They also discovered an enzyme that catalyzes the phosphorylation of aliphatic diols and used this enzyme to complete an enzymatic polymerization method for polyetherdiols using aliphatic diols as raw materials.

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

[12] . [1] A method for producing an aliphatic diol 1-phosphate, comprising the step of phosphorylating an aliphatic diol in the presence of a phosphorylation enzyme. [2] Phosphorylation enzymes, a) Contains the amino acid sequence shown in any of Sequence IDs 1-2, or b) The method according to [1], comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 1 to 2, and having enzymatic activity that catalyzes the monophosphorylation of aliphatic diols. [3] A method for producing aliphatic diol 2-phosphate, comprising the step of phosphorylating aliphatic diol 1-phosphate in the presence of a phosphorylation enzyme. [4] Phosphorylation enzymes, c) Contains an amino acid sequence shown in any of Sequence IDs 3-7, or d) The method according to [3], comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 3 to 7, and having enzymatic activity that catalyzes the diphosphorylation of aliphatic diol 1-phosphate. [5] A method for producing polyetherdiol 1-phosphate, comprising the step of reacting aliphatic diol 1-phosphate with aliphatic diol 2-phosphate in the presence of 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase to obtain polyetherdiol 1-phosphate. A method for producing a polyetherdiol, comprising the steps of producing polyetherdiol 1-phosphate according to the method described in [6] [5], and dephosphorylating the polyetherdiol 1-phosphate to obtain a polyetherdiol. A method for producing polyetherdiol 2-phosphate, comprising the steps of producing polyetherdiol 1-phosphate according to the method described in [7] [5], and phosphorylating the polyetherdiol 1-phosphate in the presence of a phosphorylation enzyme to obtain polyetherdiol 2-phosphate. A method for producing polyetherdiol 1-phosphate, comprising the steps of producing polyetherdiol 2-phosphate according to the method described in [8] [7], and reacting the polyetherdiol 2-phosphate with aliphatic diol 1-phosphate in the presence of 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase to obtain polyetherdiol 1-phosphate. A method for producing a polyetherdiol, comprising the steps of producing polyetherdiol 1-phosphate according to the method described in [9] [8], and dephosphorylating the polyetherdiol 1-phosphate to obtain a polyetherdiol.

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

[11] A microorganism comprising genes encoding a first phosphorylation enzyme, a second phosphorylation enzyme, and 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, respectively: The first phosphorylation enzyme described above, a) Contains the amino acid sequence shown in any of Sequence IDs 1-2, or b) At least one enzyme having an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 1-2, and having enzymatic activity that catalyzes the monophosphorylation of aliphatic diols. The second phosphorylation enzyme described above, c) Contains an amino acid sequence shown in any of Sequence IDs 3-7, or d) At least one enzyme having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 3 to 7, and that has enzymatic activity to catalyze the diphosphorylation of aliphatic diol 1-phosphate, The aforementioned 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, e) Contains an amino acid sequence shown in any of Sequence IDs 8-12, or f) The microorganism comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 8 to 12, and at least one enzyme having 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase activity.

[12] A method for producing polyetherdiol 1-phosphate, comprising the step of culturing the microorganism described in

[11] in the presence of an aliphatic diol to obtain polyetherdiol 1-phosphate. [Effects of the Invention]

[0008] Polymerization reactions catalyzed by microorganisms or enzymes allow for polymerization under milder conditions compared to polymerization reactions using organometallic catalysts, thus reducing environmental impact. Furthermore, the strict substrate specificity of enzymes 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 2-phosphate (1,4-butanediol-2-phosphate) by LC / MS. [Figure 3] Confirmation of ether conjugate (PTMG dimer-1-phosphate) by LC / MS.

Mode for Carrying Out the Invention

[0010] 1. Method for Producing Aliphatic Diol 1-Phosphate The present invention provides a method for producing aliphatic diol 1-phosphate by phosphorylating an aliphatic diol with a phosphorylating enzyme.

[0011] In the present invention, the "aliphatic diol" is a saturated or unsaturated fatty acid diol, which may be linear or branched.

[0012] The fatty acid diol is preferably a fatty acid diol having 2 to 14 carbon atoms, more preferably 2 to 12 carbon atoms, still more preferably 2 to 8 carbon atoms, and even more preferably 2 to 6 carbon atoms.

[0013] Preferably, the fatty acid diol is a saturated linear fatty acid diol, such as linear alkylene glycols, specifically, ethylene glycol, 1,3-propylene glycol, 1,4-butanediol, 1,6-hexanediol, 1,10-decanediol, 1,12-dodecanediol, 1,14-tetradecanediol, etc. Among them, 1,4-butanediol is more preferable.

[0014] The "phosphorylating enzyme" used in the present invention is not particularly limited as long as it has an enzyme activity that catalyzes the phosphorylation of aliphatic diol. For example, mevalonate kinase, glycerol kinase, ethanolamine kinase, 3-phosphomevalonate kinase, etc. can be mentioned.

[0015] The origin of the "phosphorylating enzyme" is not particularly limited, and examples include those derived from eukaryotes and those derived from bacteria such as eubacteria.

[0016] Specifically, "phosphorylation enzymes" that can be used include those derived from the genera Staphylococcus, Escherichia, Klebsiella, Halobacterium, Pseudomonas, Firmicutes, Arabidopsis, Saccharomyces, Bos, and Mus.

[0017] Specific examples of the "phosphorylation enzyme" used in the present invention include, for example, an enzyme (protein) containing the amino acid sequence shown in any of SEQ ID NOs: 1 to 2.

[0018] The phosphorylation enzyme shown in Sequence ID No. 1 is registered in public databases as Mevalonate kinase (Uniprot ID: Q2G0I8) from Staphylococcus aureus, and the phosphorylation enzyme shown in Sequence ID No. 2 is registered in public databases as Glycerol kinase (Uniprot ID: B7UNP6) from Escherichia coli O127:H6.

[0019] The "phosphorylation enzyme" used in the present invention is not limited to those having the above sequence, but also includes enzymes (proteins) that have an amino acid sequence having approximately 60% or more homology or identity with the amino acid sequence described in any of Sequence IDs 1 to 2, preferably approximately 70% or more, more preferably approximately 80% or more, even more preferably approximately 90% or more, particularly preferably approximately 95% or more, and most preferably approximately 98% or more, and that have enzymatic activity to catalyze the phosphorylation of aliphatic diols.

[0020] Furthermore, the "phosphorylation enzymes" used in the present invention also include enzymes (proteins) that have enzymatic activity to catalyze the phosphorylation of aliphatic diols, and which include an amino acid sequence in which one or several amino acids, specifically 1 to 20, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 2, are deleted, substituted, or added in the amino acid sequence described in any of Sequence ID No. 1 to 2.

[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) An amino acid sequence of a protein having approximately 60% or more, preferably approximately 70% or more, more preferably approximately 80% or more, even more preferably approximately 90% or more, particularly preferably approximately 95% or more, and most preferably approximately 98% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 1 to 2, and having enzymatic activity that catalyzes the phosphorylation of aliphatic diols.

[0022] The genes for "phosphorylation enzymes" used in the present invention also include genes that, for example, hybridize under stringent conditions with a polynucleotide having a base sequence encoding the amino acid sequence shown in any of Sequence IDs 1 to 2, and that encode a protein having enzymatic activity to catalyze the phosphorylation of aliphatic diols. Preferably, the above genes are derived from the genus Staphylococcu or Escherichia.

[0023] Stringent conditions include, for example, a condition in which a nylon membrane immobilized with DNA is incubated with a probe at 65°C for 20 hours in a solution containing 6×SSC (1×SSC is made by dissolving 8.76g of sodium chloride and 4.41g of sodium citrate in 1 liter of water), 1% SDS, 100 μg / ml salmon sperm DNA, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, and 0.1% Ficol, and hybridization is performed, but is not limited to this. Those skilled in the art can set hybridization conditions by taking into account not only the salt concentration and temperature of such buffers, but also other conditions such as probe concentration, probe length, and reaction time. Examples of washing conditions after hybridization include "2×SSC, 0.1%SDS, 42°C" and "1×SSC, 0.1%SDS, 37°C," while more stringent conditions include "1×SSC, 0.1%SDS, 65°C" and "0.5×SSC, 0.1%SDS, 50°C."

[0024] 2. A method for producing aliphatic diol 2-phosphate. The present invention provides a method for producing aliphatic diol 2-phosphate by phosphorylating aliphatic diol 1-phosphate with a phosphorylation enzyme.

[0025] The aliphatic diol constituting "aliphatic diol 1-phosphate" is as described in item 1 above. As aliphatic diol 1-phosphate, the aliphatic diol 1-phosphate obtained by the method described in item 1 above can be used.

[0026] The "phosphorylation enzyme" used in this invention is not particularly limited as long as it has enzymatic activity that catalyzes the phosphorylation of aliphatic diol 1-phosphate. Examples include 5-phosphomevalonate kinase and isopentenyl phosphate kinase.

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

[0028] Specifically, the "phosphorylation enzymes" that can be used are those derived from the genera Arabidopsis, Oryza, Nitrosopumilus, Panicum, Branchiostoma, Methanofervidicoccus, Methanocaldococcus, Haloferax, Thermoplasma, Streptomyces, Thermanaerothrix, Longilinea, Flexilinea, Scytonema, and Archaea.

[0029] Specific examples of the "phosphorylation enzyme" used in the present invention include, for example, enzymes (proteins) containing the amino acid sequence shown in any of SEQ ID NOs: 3 to 7.

[0030] The phosphorylation enzyme shown in Sequence ID No. 3 is registered in public databases as Isopentenyl phosphate kinase from Nitrosopumilus adriaticus (Uniprot ID: A0A0D5C472), the phosphorylation enzyme shown in Sequence ID No. 4 is registered as Isopentenyl phosphate kinase from Archaea Phylum (Uniprot ID: A0A7C1LM50), the phosphorylation enzyme shown in Sequence ID No. 5 is registered as Isopentenyl phosphate kinase from Panicum miliaceum (Uniprot ID: A0A3L6SU37), the phosphorylation enzyme shown in Sequence ID No. 6 is registered as Isopentenyl phosphate kinase from Branchiostoma belcheri (Uniprot ID: A0A6P4ZPW9), and the phosphorylation enzyme shown in Sequence ID No. 7 is registered as Isopentenyl phosphate kinase from Methanofervidicoccus abyssi (Uniprot ID: A0A401HP04).

[0031] The "phosphorylation enzyme" used in the present invention is not limited to those having the above sequence, but also includes enzymes (proteins) that have an amino acid sequence having approximately 60% or more, preferably approximately 70% or more, more preferably approximately 80% or more, even more preferably approximately 90% or more, particularly preferably approximately 95% or more, and most preferably approximately 98% or more homology or identity with the amino acid sequence described in any of SEQ ID NOs: 3 to 7, and that have enzymatic activity to catalyze the 2-phosphorylation of aliphatic diol 1-phosphate.

[0032] Furthermore, the "phosphorylation enzymes" used in the present invention also include enzymes (proteins) that have enzymatic activity to catalyze the diphosphorylation of aliphatic diol 1-phosphate, and that include an amino acid sequence in which one or several amino acids, specifically 1 to 20, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 2, are deleted, substituted, or added in the amino acid sequence described in any of Sequence IDs 3 to 7.

[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) An amino acid sequence of a protein having approximately 60% or more, preferably approximately 70% or more, more preferably approximately 80% or more, even more preferably approximately 90% or more, particularly preferably approximately 95% or more, and most preferably approximately 98% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 3 to 7, and having enzymatic activity that catalyzes the 2-phosphorylation of aliphatic diol 1-phosphate.

[0034] The genes for "phosphorylation enzymes" used in this invention also include genes that contain a base sequence encoding a protein that hybridizes under stringent conditions with a polynucleotide having a base sequence encoding the amino acid sequence shown in any of Sequence IDs 3 to 7, and that has enzymatic activity to catalyze the diphosphorylation of aliphatic diol 1-phosphate. The stringent conditions are as described above.

[0035] The enzymes used in this invention may be immobilized on a resin or a suitable carrier (immobilized enzyme) or encapsulated in a polymer or microcapsule (embedded) according to methods well known in the art. Alternatively, the enzymes may be used in isolation from the raw materials using a semipermeable membrane or the like. The same applies to the following methods.

[0036] 3. Method for producing polyetherdiol 1-phosphate The present invention also provides a method for producing polyetherdiol 1-phosphate by reacting aliphatic diol 1-phosphate with aliphatic diol 2-phosphate in the presence of 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase.

[0037] As the aliphatic diol 1-phosphate, the polyetherdiol 1-phosphate obtained in 1 above can be used. Also, as the aliphatic diol 2-phosphate, the polyetherdiol 2-phosphates obtained in 2 above can be used.

[0038] The "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" used in the present invention has catalytic activity for ether condensation of aliphatic diol 1-phosphate and aliphatic diol 2-phosphate, thereby enabling the production of polyetherdiol 1-phosphate from aliphatic diol 1-phosphate and aliphatic diol 2-phosphate.

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

[0040] The origin of "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" is not particularly limited; for example, it can be derived from archaea, bacteria such as eubacteria, or eukaryotes.

[0041] Specifically, "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" can be derived from the genera Aeropyrum, Thermococcus, Archaeoglobus, Bacteroidetes, Chitinophaga, Zunongwangia, Spirosoma, Methanothermobacter, Flavobacterium, Bacillus, Octadecabacter, Pustulibacterium, and the family Crocinitomicaceae.

[0042] Specific examples of the "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" used in the present invention include, for example, enzymes (proteins) containing the amino acid sequence shown in any of SEQ ID NOs: 8 to 12.

[0043] The 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase shown in SEQ ID NO: 8 is Heptaprenylglyceryl phosphate synthase (Uniprot ID: O34790) derived from Bacillus subtilis, the phosphorylation enzyme shown in SEQ ID NO: 9 is Geranylgeranylglyceryl phosphate synthase (Uniprot ID: A5FJK8) derived from Flavobacterium johnsoniae, the phosphorylation enzyme shown in SEQ ID NO: 10 is geranylgeranylglyceryl / heptaprenylglyceryl phosphate synthase (Uniprot ID: UPI0004282B8E) derived from Hugenholtzia roseola, and the phosphorylation enzyme shown in SEQ ID NO: 11 is Geranylgeranylglyceryl phosphate synthase (Uniprot ID: The phosphorylation enzyme indicated by Sequence ID No. 12 (A0A2E4SBV2) is registered in public databases as Geranylgeranylglyceryl phosphate synthase (Uniprot ID: A0A1I7EWQ1) derived from Pustulibacterium marinum.

[0044] The "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase" used in the present invention is not limited to having the above sequence, but also includes an amino acid sequence having about 60% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more homology or identity with the amino acid sequence described in any of SEQ ID NOs: 8 to 12, and also includes enzymes (proteins) that have catalytic activity for ether condensation of aliphatic diol 1-phosphate and aliphatic diol 2-phosphate.

[0045] Furthermore, the "phosphorylation enzymes" used in the present invention also include enzymes (proteins) that have catalytic activity for ether condensation of aliphatic diol 1-phosphate and aliphatic diol 2-phosphate, and that include an amino acid sequence in which one or several amino acids, specifically 1 to 20, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 2 amino acids, are deleted, substituted, or added in the amino acid sequence described in any of Sequence IDs 8 to 12.

[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) An amino acid sequence of a protein having approximately 60% or more, preferably approximately 70% or more, more preferably approximately 80% or more, even more preferably approximately 90% or more, and particularly preferably approximately 95% or more sequence identity with the amino acid sequence shown in any of sequence numbers 8 to 12, and having catalytic activity for ether condensation of aliphatic diol 1-phosphate and aliphatic diol 2-phosphate.

[0047] The genes for "3-(o-geranylgeranyl)-glycerol 1-phosphate synthase activity" used in the present invention also include genes that hybridize under stringent conditions with a polynucleotide having a base sequence encoding the amino acid sequence shown in any of Sequence IDs 8 to 12, and that also include a base sequence encoding a protein having catalytic activity for ether condensation of aliphatic diol 1-phosphate and aliphatic diol 2-phosphate. The stringent conditions are as described above.

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

[0049] 5. Method for producing polyetherdiol 1-phosphate The present invention also provides a method for producing polyetherdiol 1-phosphate, which includes a step of reacting polyetherdiol 2-phosphate obtained by the method described in 4 above with aliphatic diol 1-phosphate in the presence of 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase.

[0050] 6. Method for producing polyetherdiol The present invention provides a method for producing a polyetherdiol, comprising the step of dephosphorylating the polyetherdiol 1-phosphate obtained by the method described in 3 above. The present invention also provides a method for producing a polyetherdiol, comprising the step of dephosphorylating the polyetherdiol 1-phosphate obtained by the method described in 5 above.

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

[0052] 7. Microbial catalyst The present invention also provides a microorganism (microbial catalyst) containing a gene encoding at least one of the phosphorylation enzymes (first phosphorylation enzyme) described in 1. above, at least one of the phosphorylation enzymes (second phosphorylation enzyme) described in 2. above, and at least one of the 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase described in 3. above.

[0053] The microorganisms mentioned above may be non-recombinant or recombinant. That is, the genes encoding each enzyme may be endogenous genes of the microorganism, exogenous genes, or partially endogenous and partially exogenous genes.

[0054] Recombinant microorganisms (transformers) are obtained by transforming a host microorganism using an expression vector containing genes encoding each enzyme. The expression vector may contain one gene encoding each enzyme individually, or it may contain genes encoding two or more enzymes.

[0055] Expression vectors can be prepared by known methods. Generally, an expression cassette is constructed by inserting a transcription promoter upstream of the gene encoding a given enzyme, and possibly a terminator downstream, and then inserting this cassette into the expression vector. Alternatively, if the expression vector already contains a transcription promoter and / or terminator, the gene encoding the given enzyme can be inserted between them using the vector's transcription promoter and / or terminator without constructing an expression cassette. As mentioned above, when an expression vector contains two or more genes encoding enzymes, these genes may all be inserted under the same promoter or under different promoters. The type of promoter is not particularly limited as long as it enables appropriate expression in the host, but examples of promoters that can be used in the E. coli host include the T7 promoter, trp promoter, lac promoter, lambda phage-derived PL promoter and PR promoter, tac promoter, and trc promoter.

[0056] The genes encoding each specified enzyme can be obtained, for example, (i) by constructing primers according to the base sequence information and amplifying them using the genome or the like as a template, or (ii) by synthesizing DNA organically according to the amino acid sequence information of the enzyme. The genes may be optimized depending on the host cell of the transformant.

[0057] To insert a gene encoding a specific enzyme into an expression vector, methods such as restriction enzymes or topoisomerases can be used. If necessary during insertion, an appropriate linker may be added. In addition, ribosome-binding sequences such as SD sequences and Kozak sequences are known to be important nucleotide sequences for amino acid translation, and these sequences may be inserted upstream of the gene. Along with the insertion, a portion of the amino acid sequence encoded by the gene may be replaced. Furthermore, it is preferable to include factors (selection markers) in the vector for selecting the target transformant. Examples of selection markers include drug resistance genes, nutrient requirement complementation 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 E. coli include the ampicillin resistance gene, kanamycin gene, dihydrofolate reductase gene, and neomycin resistance gene.

[0058] The expression vector should be selected appropriately from plasmid DNA, bacteriophage DNA, retrotransposon DNA, or artificial chromosome DNA, depending on the host. For example, when using E. coli as the host, pTrc99A (GE Healthcare Biosciences), pACYC184 (Nippon Gene), pMW118 (Nippon Gene), and pET series vectors (Novagen) can be used. Vectors with two or more insertion sites include pETDuet-1 (Novagen). Modified versions of these vectors can also be used as needed.

[0059] An expression vector enhances the expression of a specific enzyme by inserting an expression cassette, which consists of a gene encoding that enzyme and is linked to an appropriate promoter, terminator, marker gene, etc., into the host genome. Known methods can be used to obtain a transformant with the expression cassette inserted into the genome. For example, when inserting an expression cassette into the genome by homologous recombination, the entire plasmid or a transformant with the expression cassette inserted can be obtained by performing transformation using a plasmid that has the expression cassette for the specified enzyme and the sequence of an arbitrary genomic region, and is not replicable in the host. In this case, by using a plasmid carrying a negative selection marker such as the SacB gene (encoding levansculas) or a plasmid with a temperature-sensitive (ts) replication mechanism, a transformant with only the expression cassette on the genome can be efficiently obtained through two homologous recombinations. Furthermore, by performing transformation using a DNA fragment consisting only of the expression cassette, a transformant with the expression cassette inserted at a random position on the genome can be obtained.

[0060] When utilizing enzymes that are naturally present in the host genome (endogenous enzymes), their expression can be enhanced by replacing the promoter of the enzyme gene in the genome with a stronger one. The same promoters used in the expression vectors described above can be used as promoters.

[0061] The host microorganism is not particularly limited as long as it is a cell capable of expressing a specific enzyme using a protein expression system utilizing an expression vector or the like. Examples include bacteria such as Escherichia coli, Bacillus subtilis, and actinomycetes (e.g., Rhodococcus and Corynebacterium); yeasts (e.g., Saccharomyces, Candida, and Pichia); filamentous fungi; plant cells; and animal cells such as insect cells and mammalian cells. Among these, Escherichia coli, Corynebacterium and Rhodococcus bacteria, as well as Saccharomyces, Candida, and Pichia yeasts are preferred, with Escherichia coli being more preferred.

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

[0063] The method for introducing the expression vector into the host is not particularly limited, as long as it is suitable for the host. Available methods include, for example, electroporation, calcium ion methods, spheroplast methods, lithium acetate methods, calcium phosphate methods, and lipofection methods.

[0064] The transformed cells into which the expression vector has been introduced can be cultured using a method suitable for the host cells (bacteria) to express each enzyme.

[0065] 8. Method for producing polyetherdiol 1-phosphate using microorganisms The present invention also provides a method for producing polyetherdiol 1-phosphate, which includes the step of culturing the microorganism described in 7. in the presence of an aliphatic diol to produce polyetherdiol 1-phosphate.

[0066] Microorganisms may be used as resting cells, cells with improved membrane permeability, inactivated cells, lithified cells, cell-free extracts prepared from lithified cells, and stabilized products obtained by stabilizing these.

[0067] Polyetherdiol 1-phosphate can be produced by culturing the above-mentioned transformant in a culture medium containing an aliphatic diol, and then collecting polyetherdiol 1-phosphate from the resulting culture.

[0068] In the present invention, "culture" includes any of the following: culture supernatant, cultured cells, cultured bacterial cells, or lysates of cells or bacterial cells.

[0069] The culture of microorganisms shall be carried out according to the methods normally used for culturing the host organism. The culture medium used may be either a natural or synthetic medium, as long as it contains carbon sources, nitrogen sources, inorganic salts, etc. that the host organism can utilize and can efficiently cultivate the transformants. Examples of carbon sources include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose, and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol. Examples of nitrogen sources include inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or ammonium salts of organic acids, or other nitrogen-containing compounds.

[0070] Other substances that may be used include peptone, yeast extract, meat extract, corn steep liquor, and various amino acids. Inorganic substances that may be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, copper sulfate, and calcium carbonate. Furthermore, an antifoaming agent may be added as needed to prevent foaming during culture. Compounds that induce enzyme expression may also be added to the culture medium.

[0071] The culture conditions for the microorganisms are not particularly limited as long as they do not hinder the productivity of polyetherdiol 1-phosphate and the growth of the host organism, but are usually carried out at 10°C to 40°C, preferably 20°C to 37°C, for 5 to 100 hours. pH adjustment is performed using inorganic or organic acids, alkaline solutions, etc., and for example, for E. coli, the pH is adjusted to 4 to 9.

[0072] Culture methods include solid culture, static culture, shaking culture, and aerated stirring culture. However, when culturing Rhodococcus transformants in particular, it is preferable to culture them under aerobic conditions using shaking culture or aerated stirring culture (jar fermenter).

[0073] The obtained polyetherdiol 1-phosphate can be dephosphorylated by the method described above to produce a polyetherdiol. [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.

[0075] [Example 1] Creation of E. coli expressing 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, aliphatic diol 1-phosphorylation enzyme, and aliphatic diol 1-phosphate 2-phosphorylation enzyme. (1) Creation of a plasmid for expressing aliphatic diol monophosphorylation enzyme in E. coli Gene sequence information for mevalonate kinase (UniprotID: Q2G0I8) derived from Staphylococcus aureus and glycerol kinase (UniprotID: B7UNP6) derived from Escherichia coli O127:H6 (hereinafter referred to as J288VGA140-6 and J288VGA140-4, respectively) was obtained from the National Center for Biotechnology Information (NCBI), and the sequences were optimized for the codon frequency of the cell-free translation system PUREfrex2.0 (Gene Frontier) through gene synthesis. Plasmids were constructed by inserting the codon-optimized gene fragments of J288-VGA140-4 and J288VGA140-6 into the NdeI-XhoI region of the E. coli expression vector pET-22b(+) (Novagen). The sequences corresponding to J288-VGA140-6 and J288VGA140-4 inserted into the vector are shown in Sequence IDs 13 and 14, respectively. The introduction method involved 4 μl of digested product, 1 μl of cleaved pETDuet-1, and a DNA Ligation Kit.<Mighty Mix> After adding 5 μl of (Takara Bio) and mixing, a ligation reaction was carried out at 16°C for 1 hour. 10 μl of the ligation reaction solution was mixed with 100 μl of E. coli JM109 competent cells (Takara Bio) and left to stand on ice for 30 minutes. After incubation at 42°C for 45 seconds, it was left to stand again on ice for 5 minutes. 500 μl of SOC medium was added, and the mixture was cultured at 37°C for 1 hour with shaking at 200 rpm, after which it was spread onto LB agar medium containing 100 μg / ml ampicillin.

[0076] After culturing overnight at 37°C, the grown colonies were inoculated into LB liquid medium (containing 100 μg / ml ampicillin) and cultured at 37°C for 24 hours with shaking at 200 rpm. The culture medium was centrifuged to collect the cells, and plasmid extraction was performed using the QIAprep Spin Miniprep Kit (Qiagen) according to the attached protocol. PCR was performed again using the obtained plasmids as templates, and the position of the bands was confirmed. The obtained plasmid DNAs were named PTMG11 / pPETDuet-1 (J288-VGA140-4) and PTMG21 / pPETDuet-1 (J288-VGA140-6), respectively.

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

[0078] A0A3L6SU37 Amplification Primer: NdeI-6His.F aaaaaaCATATGCATCATCACCATCACC(Sequence ID 19) U37-XhoI.R aaaaaaCTCGAGTTACTTACTACTACGGATAATCGTACCCA(Sequence ID 20) PCR reaction solution composition: 2 μl of template DNA, 25 μl of 2×PrimeSTAR Max Premix, 2 μl each of 10 μM primers, and 19 μl of sterile water (these were mixed to a total volume of 50 μl). Reaction temperature conditions: Using a TaKaRa PCR Thermal Cylcer Dice Touch (model TP350), a cycle of 10 seconds at 98°C, 5 seconds at 58°C, and 10 seconds at 72°C was repeated 30 times. However, the incubation period at 98°C in the first cycle was 30 seconds.

[0079] The PCR amplification product was identified by separating it using 1% agarose (Agarose S: ​​Nippon Gene Co., Ltd.) gel electrophoresis, followed by visualization using GelRed (NEW ENGLAND BioLabs, also known as NEB) staining. A fragment of approximately 1.1 kb derived from A0A3L6SU37 was detected. Each obtained PCR amplification fragment was purified according to the protocol of the QIAquick PCR purification Kit (Qiagen). The purified PCR product was purified using restriction enzymes from NEB. 10 μl of PCR product was mixed with 4 μl of Cutsmart buffer (included in the kit), 1 μl of NdeI, 1 μl of XhoI, and 4 μl of sterile water. After processing at 37°C for 30 minutes, the product 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 vector are shown in SEQ ID NO: 15. The introduction method involved 4 μl of PCR digest product, 1 μl of cleaved pETDuet-1, and a DNA Ligation Kit.<Mighty Mix> After adding 5 μl of (Takara Bio) and mixing, a ligation reaction was carried out at 16°C for 1 hour. 10 μl of the ligation reaction solution was mixed with 100 μl of E. coli JM109 competent cells (Takara Bio) and left to stand on ice for 30 minutes. After incubation at 42°C for 45 seconds, it was left to stand again on ice for 5 minutes. 500 μl of SOC medium was added, and the mixture was cultured at 37°C for 1 hour with shaking at 200 rpm, after which it was spread onto LB agar medium containing 100 μg / ml ampicillin.

[0081] After culturing overnight at 37°C, the grown colonies were inoculated into LB liquid medium (containing 100 μg / ml ampicillin) and cultured at 37°C for 24 hours with shaking at 200 rpm. The culture medium was centrifuged to collect the cells, and plasmid extraction was performed using the QIAprep Spin Miniprep Kit (Qiagen) according to the attached protocol. PCR was performed again using the obtained plasmid as a template, and the position of the bands was confirmed. The obtained plasmid DNA was named PTMG31 / pPETDuet-1 (A0A3L6SU37).

[0082] (3) Preparation of a plasmid for expressing 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase in E. coli The gene fragments of geranylgeranylglyceryl / heptaprenylglyceryl phosphate synthase from Hugenholtzia roseola (UniprotID:UPI0004282B8E), geranylgeranylglyceryl phosphate synthase from Crocinitomicaceae bacterium (UniprotID:A0A2E4SBV2), and geranylgeranylglyceryl phosphate synthase from Pustulibacterium marinum (UniprotID:A0A1I7EWQ1) (hereinafter referred to as UPI0004282B8E, A0A2E4SBV2, and A0A1I7EWQ1, respectively) were amplified by PCR using the primers shown below, to which restriction enzyme NdeI sites were added to the 5' end and restriction enzyme XhoI sites were added to the 3' end. PCR was performed according to the protocol of PrimeSTAR MAX Premix (Takara Bio Inc.).

[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: 2 μl of template DNA, 25 μl of 2×PrimeSTAR Max Premix, 2 μl each of 10 μM primers, and 19 μl of sterile water (these were mixed to a total volume of 50 μl). Reaction temperature conditions: Using a TaKaRa PCR Thermal Cylcer Dice Touch (model TP350), a cycle of 10 seconds at 98°C, 5 seconds at 58°C, and 10 seconds at 72°C was repeated 30 times. However, the incubation period at 98°C in the first cycle was 30 seconds.

[0084] The PCR amplification products were identified by separating them using 1% agarose (Agarose S: ​​Nippon Gene Co., Ltd.) gel electrophoresis, followed by visualization using GelRed (NEW ENGLAND BioLabs, also known as NEB) staining. Fragments of approximately 0.9kb were detected for UPI0004282B8E, and approximately 0.8kb for A0A2E4SBV2 and A0A1I7EWQ1. Each obtained PCR amplification fragment was purified according to the protocol of the QIAquick PCR purification Kit (Qiagen). The purified PCR reaction mixture was treated with restriction enzymes from NEB. 10 μl of PCR product was mixed with 4 μl of Cutsmart buffer (included in the kit), 1 μl of NdeI, 1 μl of XhoI, and 4 μl of sterile water. After processing at 37°C for 30 minutes, the mixture was purified using the QIAquick PCR purification Kit (Qiagen).

[0085] The purified digest product was similarly introduced into pETDuet-1, which had been cleaved at the NdeI-XhoI site. The sequences corresponding to UPI0004282B8E, A0A2E4SBV2, and A0A1I7EWQ1 inserted into the vector are shown in SEQ ID NOs. 16-18, respectively. The introduction method involved 4 μl of PCR digest product, 1 μl of cleaved pETDuet-1, and a DNA Ligation Kit.<Mighty Mix> After adding 5 μl of (Takara Bio) and mixing, a ligation reaction was carried out at 16°C for 1 hour. 10 μl of the ligation reaction solution was mixed with 100 μl of E. coli JM109 competent cells (Takara Bio) and left to stand on ice for 30 minutes. After incubation at 42°C for 45 seconds, it was left to stand again on ice for 5 minutes. 500 μl of SOC medium was added, and the mixture was cultured at 37°C for 1 hour with shaking at 200 rpm, after which it was spread onto LB agar medium containing 100 μg / ml ampicillin.

[0086] After culturing overnight at 37°C, the grown colonies were inoculated into LB liquid medium (containing 100 μg / ml ampicillin) and cultured at 37°C for 24 hours with shaking at 200 rpm. The culture medium was centrifuged to collect the cells, and plasmid extraction was performed using the QIAprep Spin Miniprep Kit (Qiagen) according to the attached protocol. PCR was performed again using the obtained plasmids as templates, and the position of the bands was confirmed. The obtained plasmid DNAs were named PTMG41 / pPETDuet-1 (UPI0004282B8E), PTMG42 / pPETDuet-1 (A0A2E4SBV2), and PTMG43 / pPETDuet-1 (A0A1I7EWQ1), respectively.

[0087] (4) Creation of Escherichia coli expressing 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, aliphatic diol 1-phosphorylation enzyme, and aliphatic diol 1-phosphate 2-phosphorylation enzyme. One μl of each plasmid solution obtained above—PTMG11 / pPETDuet-1 (J288-VGA140-4), PTMG21 / pPETDuet-1 (J288-VGA140-6), PTMG31 / pPETDuet-1 (A0A3L6SU37), PTMG41 / pPETDuet-1 (UPI0004282B8E), PTMG42 / pPETDuet-1 (A0A2E4SBV2), and PTMG43 / pPETDuet-1 (A0A1I7EWQ1)—was added to 100 μl of E. coli BL21 (DE3) competent cells (Biodynamics Institute), and left to stand on ice for 10 minutes. A heat shock was performed at 42°C for 45 seconds, and then left to stand on ice again for 5 minutes. 500 μl of room temperature SOC medium was added, and the culture was incubated at 37°C for 1 hour with shaking at 200 rpm. 500 μl of the culture solution after shaking was spread onto LB agar medium (containing 100 μg / ml ampicillin). The resulting strains expressing the one-phosphorylation enzymes of two aliphatic diols were named PTMG11 (J288-VGA140-4 expressing strain) and PTMG21 (J288-VGA140-6 expressing strain), the two-phosphorylation enzyme expressing aliphatic diol 1-phosphate was named PTMG31 (A0A3L6SU37 expressing strain), and strains expressing three ether-conjugating enzymes were named PTMG41 (UPI0004282B8E expressing strain), PTMG42 (A0A2E4SBV2 expressing strain), and PTMG43 (A0A1I7EWQ1 expressing strain). Colonies grown at 37°C for 24 hours were cultured in LB liquid medium (containing ampicillin 100 μg / ml) and stored in 20% glycerol solution at -80°C until purification and activity confirmation tests were performed. For comparison, the BL21(DE3) strain, into which the pPETDuet-1 empty vector was introduced, was prepared in the same manner. [Examples]

[0088] (Preculture step) Two aliphatic diol monophosphorylation enzyme expression strains, PTMG11 (J288-VGA140-4 expression strain) and PTMG21 (J288-VGA140-6 expression strain), an aliphatic diol 1-phosphate diphosphorylation enzyme expression strain, PTMG31 (A0A3L6SU37 expression strain), and three ether-conjugating enzyme expression strains, PTMG41 (UPI0004282B8E expression strain), PTMG42 (A0A2E4SBV2 expression strain), and PTMG43 (A0A1I7EWQ1 expression strain), were each pre-cultured in LB Broth (Sigma-Aldrich, NaCl 10g / l, Trypton 10g / l, Yeast Extract 5g / l), 100mg / ml Ampicillin. [4 μl was dissolved in ultrapure water to a total volume of 4 ml, and this was sterilized.] This 4 ml solution was inoculated with the bacteria and cultured at a temperature of 37 degrees Celsius with shaking at 200 rpm for 24 hours (TAITEC, BR-23FP).

[0089] (Main culture process) 4 ml of the pre-culture medium obtained above was inoculated into 200 ml (500 ml Erlenmeyer flask) of the main culture medium [LB Broth (Sigma-Aldrich, NaCl 10 g / l, Trypton 10 g / l, Yeast Extract 5 g / l), 100 mg / ml Ampicillin 200 μl dissolved in ultrapure water to a total volume of 200 ml and sterilized]. The culture was then incubated at 37°C with shaking at 200 rpm 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 Industries, Ltd.) was added to a concentration of 0.3 mM. After addition, the culture was incubated at 17°C with shaking at 200 rpm for 24 hours to induce the expression of the target protein.

[0090] (Crushing process) The culture medium obtained above was centrifuged at 5000 rpm, 4°C, for 15 minutes (Eppendorf Centrifuge 5804R) to collect the bacterial cells. The bacterial cells were resuspended in 5 ml of 20 mM sodium phosphate buffer (pH 7.4) (hereafter referred to as PBS solution). The suspension was cooled on ice and subjected to sonication three times using a BRONSON Digital Sonifier (amplitude 15%, 5 sec-on / 5 sec-off, 5 min). The suspension obtained by sonication was centrifuged at 5000 rpm, 4°C, for 15 minutes, and 5 ml of the supernatant was collected. The collected supernatant was subjected to the purification process after its activity was confirmed by the method described later.

[0091] (purification process) The collected supernatant was filtered using a DISMIC 0.2 μm syringe filter (ADVANTEC), and the filtered cell lysate was collected. The collected filtrate was purified using a HisTrap HP volume 1 ml (Cytiva). The purification process was carried out according to the protocol recommended by Cytiva.

[0092] Specifically, after washing the column with sterile water, it was equilibrated with 10 ml of PBS solution containing 20 mM imidazole (Tokyo Chemical Industries, Ltd.). Then, 5 ml of the filtered supernatant was trapped in the equilibrated column. Next, after washing the column with PBS containing 20 mM imidazole, isocratic elution was performed with 10 ml of eluents (hereinafter referred to as eluents) prepared by dissolving 50 mM, 100 mM, 200 mM, and 500 mM imidazole in PBS solution containing 0.5 M NaCl. The His-Tag purified expression protein was subjected to SDS-PAGE after its concentration was confirmed using a Nano-Drop® Spectrophotometer (ND-1000). Purified products in which a band was confirmed were desalted and concentrated using Amicon® Ultra-15 Ultracel-10K (Merck).

[0093] (1. Measurement of phosphorylation enzyme activity) The 1-phosphorylation enzyme activity was measured by using ADP (adenosine 5'-disodium diphosphate trihydrate), which is produced in the 1-phosphorylation reaction from 1,4-butanediol, to convert phosphoenolpyruvate to pyruvate via pyruvate kinase, and then converting the resulting pyruvate to lactate via L-lactate dehydrogenase. The amount of NADH added to the reaction system was consumed during this process, and the decrease in NADH was tracked over time for approximately 5-10 minutes using a microplate reader with absorbance at a wavelength of 340 nm. The reaction mixture used was as follows.

[0094] A solution was prepared by adding 3 μl of 10 mM β-NADH (Fujifilm Yeast Co., Ltd.), 3 μl of 10 mM Dithiothreitol (Fujifilm Wako Co., Ltd.), 3 μl of 1 MgCl2 (Fujifilm Wako Co., Ltd.), 3 μl of 5 mM phosphoenolpyruvic acid (Sigma-Aldrich), 1 μl of 5000 U / ml L-lactate dehydrogenase rabbit muscle-derived Type II (Sigma-Aldrich), 6 μl of 1000 U / ml pyruvate kinase rabbit muscle-derived (Sigma-Aldrich), 30 μl of 1 M Tris-HCl (pH 7.5), 3 μl of 25 mM ATP (adenosine 5'-triphosphate disodium trihydrate) (Fujifilm Wako Co., Ltd.), and 3 μl of 250 mM 1,4-butanediol (Tokyo Kasei Kogyo Co., Ltd.), and mixing the supernatant with 300 μg to make a total volume of 300 μl.

[0095] (Analysis results) Analysis revealed a decrease in absorbance at 340 nm immediately after enzyme addition in the supernatant of both PTMG11 and PTMG21 strains. In contrast, no decrease in absorbance was observed in the supernatant or sterile water of BL21(DE3), which was introduced with an empty vector for comparison. Therefore, it was confirmed that PTMG11 and PTMG21 possess the activity to monophosphorylate 1,4-butanediol.

[0096] (Polyether synthesis reaction) A total volume of 300 μl was prepared by adding 3 μl of 10 mM β-NADH (Orient Yeast Co., Ltd.), 3 μl of 10 mM Dithiothreitol (Fujifilm Wako Co., Ltd.), 30 μl of 1 M MgCl2 (Fujifilm Wako Co., Ltd.), 30 μl of 1 M Tris-HCl (pH 7.5), 3 μl of 25 mM ATP (adenosine 5'-triphosphate disodium trihydrate) (Fujifilm Wako Co., Ltd.), 30 μl of 500 mM 1,4-butanediol (Tokyo Chemical Industries, Ltd.), and 300 μg each of purified proteins J288-VGA140-4, 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 2.7 μm 2.1 × 150 mm (Agilent). The following elution methods were used. Specifically, ultrapure water containing 0.01% formic acid (Solvent A) and CH3CN containing 0.01% formic acid (Solvent B) were used for elution. The concentration gradient was eluted using isocratic elution with Solvent A mixing ratios of 10% (10 min), 20% (10.1 min to 15 min), 30% (15.1 min to 20 min), 50% (20.1 min to 25 min), and 80% (25.1 min to 30 min) (flow rate 0.2 ml / min, column temperature 40°C). 1,4-butanediol (shown product), 1-phosphorylated derivative (reactant), 2-phosphorylated derivative (reactant), and phosphorylated PTMG (reactant) were detected by Waters SQ Detector 2 using the corresponding mass-to-charge ratio (m / z).

[0098] [Table 1]

[0099] (Reactant analysis) 1. Phosphorus oxides: After the phosphorylation reaction, the molecular ion peak of 1,4-butanediol-1-phosphoric acid ([M+H]) is observed at approximately 1.5 minutes. + The detection of (=171) confirmed the formation of 1-phosphorus oxide (Figure 1). 2 Phosphoroxides After the 2-phosphorylation reaction, the molecular ion peak of 1,4-butanediol-2-phosphoric oxide ([MH]) is detected at approximately 2.1 minutes. - 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] Based on the above, we confirmed the conversion of PTMG dimers from 1,4-butanediol to aliphatic diols via the action of a 1-phosphorylation enzyme, a 2-phosphorylation enzyme of aliphatic diol 1-phosphate, and an ether-conjugating enzyme. [Industrial applicability]

[0101] According to the present invention, a method for producing polyetherdiols that can be polymerized under mild conditions and have a reduced environmental impact is provided.

[0102] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0103] [ka] JPEG0007901281000003.jpg196166JPEG0007901281000004.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 method for producing a saturated linear aliphatic diol 1-phosphate, comprising the step of contacting a first phosphorylation enzyme with a saturated linear alkylene glycol to phosphorylate it, The first phosphorylation enzyme, a) Containing the amino acid sequence shown in any of SEQ ID NOs: 1 to 2, or b) A method comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 1 to 2, and having enzymatic activity that catalyzes the monophosphorylation of saturated linear alkylene glycol.

2. The first phosphorylation enzyme, a) Containing the amino acid sequence shown in Sequence ID No. 2, or b) The method according to claim 1, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 2, and having enzymatic activity that catalyzes the monophosphorylation of saturated linear alkylene glycol.

3. The first phosphorylation enzyme, a) Containing the amino acid sequence shown in Sequence ID No. 1, or b) The method according to claim 1, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, and having enzymatic activity that catalyzes the monophosphorylation of saturated linear alkylene glycol.

4. The method according to any one of claims 1 to 3, wherein the first phosphorylation enzyme is not glycerol kinase.

5. The method according to any one of claims 1 to 4, wherein the first phosphorylation enzyme comprises mevalonate kinase, ethanolamine kinase, or 3-phosphomevalonate kinase.

6. The method according to any one of claims 1 to 5, wherein the saturated linear alkylene glycol comprises ethylene glycol, 1,3-propylene glycol, 1,4-butanediol, 1,6-hexanediol, 1,10-decanediol, 1,12-dodecanediol, or 1,14-tetradecanediol.

7. The method according to any one of claims 1 to 6, wherein the phosphorylation step includes contacting a transformant into which a gene encoding a first phosphorylation enzyme has been introduced with a saturated linear alkylene glycol.

8. A method for producing saturated linear aliphatic diol 2-phosphate, comprising the steps of obtaining saturated linear aliphatic diol 1-phosphate by the manufacturing method described in any one of claims 1 to 7, and phosphorylating the saturated linear aliphatic diol 1-phosphate by contacting a second phosphorylation enzyme with the saturated linear aliphatic diol 1-phosphate, The second phosphorylation enzyme, c) Containing an amino acid sequence shown in any of SEQ ID NOs: 3 to 7, or d) A method comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 3 to 7, and having enzymatic activity that catalyzes the diphosphorylation of saturated linear aliphatic diol 1-phosphate.

9. The second phosphorylation enzyme, c) Containing an amino acid sequence shown in any of SEQ ID NOs: 3 to 6, or d) The method according to claim 8, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 3 to 6, and having enzymatic activity that catalyzes the diphosphorylation of saturated linear aliphatic diol 1-phosphate.

10. A method for producing polyetherdiol 1-phosphate, comprising the steps of: obtaining saturated linear aliphatic diol 1-phosphate by the manufacturing method described in any one of claims 1 to 7; and reacting saturated linear aliphatic diol 1-phosphate with saturated linear aliphatic diol 2-phosphate in the presence of 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase to obtain polyetherdiol 1-phosphate, wherein 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) A method comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 8 to 12, and having catalytic activity for ether condensation of saturated linear aliphatic diol 1-phosphate and saturated linear aliphatic diol 2-phosphate.

11. A method for producing a polyetherdiol, comprising the steps of producing polyetherdiol 1-phosphate according to the method of claim 10, and dephosphorylating the polyetherdiol 1-phosphate to obtain a polyetherdiol.

12. A method for producing polyetherdiol 2-phosphate, comprising the steps of producing polyetherdiol 1-phosphate according to the method of claim 10, and phosphorylating the polyetherdiol 1-phosphate by contacting it with a phosphorylation enzyme to obtain polyetherdiol 2-phosphate, wherein The phosphorylation enzyme, c) Containing an amino acid sequence shown in any of SEQ ID NOs: 3 to 7, or d) A method for producing an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 3 to 7, and that has enzymatic activity to catalyze the diphosphorylation of polyetherdiol 1-phosphate.

13. A method for producing polyetherdiol 1-phosphate, comprising the steps of producing polyetherdiol 2-phosphate according to the method of claim 12, and reacting the polyetherdiol 2-phosphate with saturated linear aliphatic diol 1-phosphate in the presence of 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase to obtain polyetherdiol 1-phosphate, wherein 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) A method comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 8 to 12, and having catalytic activity for ether condensation of saturated linear aliphatic diol 1-phosphate and saturated linear aliphatic diol 2-phosphate.

14. A method for producing a polyetherdiol, comprising the steps of producing polyetherdiol 1-phosphate according to the method of claim 13, and dephosphorylating the polyetherdiol 1-phosphate to obtain a polyetherdiol.

15. 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, e) Containing an amino acid sequence shown in any of SEQ ID NOs: 8 to 11, or f) The method according to any one of claims 10 to 14, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 8 to 11, and having catalytic activity for ether condensation of saturated linear aliphatic diol 1-phosphate and saturated linear aliphatic diol 2-phosphate.

16. A microorganism comprising genes encoding a first phosphorylation enzyme, a second phosphorylation enzyme, and 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase, respectively: The first phosphorylation enzyme described above, a) Containing the amino acid sequence shown in any of SEQ ID NOs: 1 to 2, or b) At least one enzyme having an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 1 to 2, and having enzymatic activity that catalyzes the monophosphorylation of saturated linear aliphatic diols, The second phosphorylation enzyme described above, c) Containing an amino acid sequence shown in any of SEQ ID NOs: 3 to 7, or d) At least one enzyme having an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 3 to 7, and having enzymatic activity that catalyzes the diphosphorylation of saturated linear aliphatic diol 1-phosphate, 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) The microorganism, wherein it contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in any of Sequence IDs 8 to 12, and is at least one enzyme having 3-(o-geranylgeranyl)-glycerol 1-phosphate synthase activity.

17. A method for producing polyetherdiol 1-phosphate, comprising the step of culturing the microorganism described in claim 16 in the presence of a saturated linear aliphatic diol to obtain polyetherdiol 1-phosphate.

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