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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12Y402/01—Hydro-lyases (4.2.1)
  • C12Y402/01017—Enoyl-CoA hydratase (4.2.1.17), i.e. crotonase
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  • C12Y402/00—Carbon-oxygen lyases (4.2)
  • C12Y402/01—Hydro-lyases (4.2.1)
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

• the present invention relates to a transformed microorganism capable of producing a copolymerized polyhydroxyalkanoic acid containing 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms, and a method for producing a copolymerized polyhydroxyalkanoic acid using the transformed microorganism.
• PHA Polyhydroxyalkanoates
• Known monomer units that make up PHA include, for example, 3-hydroxybutyric acid (abbreviation: 3HB), 3-hydroxyvaleric acid (abbreviation: 3HV), 3-hydroxyhexanoic acid (abbreviation: 3HH), 3-hydroxyoctanoic acid (abbreviation: 3HO), 3-hydroxydecanoic acid (abbreviation: 3HD), and 3-hydroxydodecanoic acid (abbreviation: 3HDD).
• 3HB 3-hydroxybutyric acid
• 3HV 3-hydroxyvaleric acid
• 3HH 3-hydroxyhexanoic acid
• 3HO 3-hydroxyoctanoic acid
• 3HD 3-hydroxydecanoic acid
• 3HDD 3-hydroxydodecanoic acid
• 3HDD 3-hydroxydodecanoic acid
• PHAs containing 3-hydroxyalkanoic acid monomer units with 8 or more carbon atoms are known as PHAs with low Tg.
• Tg of a PHA tends to decrease as the content of monomers with a large number of carbon atoms increases.
• PHA synthase is classified into four classes, Class 1, Class 2, Class 3, and Class 4, based on the substrate specificity and subunit structure of the enzyme. According to this document, PhaC classified into Class 1, Class 3, and Class 4 have polymerization activity for 3-hydroxyalkanoic acids with 3 to 5 carbon atoms, while Class 2 PhaC has polymerization activity for 3-hydroxyalkanoic acids with 6 to 14 carbon atoms.
• known methods for producing copolymerized PHAs containing 3HA monomer units with 8 or more carbon atoms include culturing Pseudomonas bacteria or culturing transformed microorganisms into which a gene encoding Class 2 PhaC derived from Pseudomonas bacteria or a mutant thereof has been introduced.
• Non-Patent Documents 2 and 3 describe the production of copolymerized PHA containing medium-chain length 3 HA monomer units by culturing transformants into which genes encoding Class 2 PhaC derived from the genus Pseudomonas were introduced, such as PhaC (PhaC1H9) derived from Pseudomonas sp. H9, PhaC (PhaC1po) derived from Pseudomonas putida Gpo1, and PhaC (PhaC1pm) derived from Pseudomonas mendocina.
• PhaC PhaC (PhaC1H9) derived from Pseudomonas sp. H9
• PhaC (PhaC1po) derived from Pseudomonas putida Gpo1
• PhaC (PhaC1pm) derived from Pseudomonas mendocina.
• Patent Documents 1 and 2 also describe the production of copolymerized PHA containing three medium-chain-length HA monomer units by culturing a transformant into which a gene encoding a mutant of Class 2 PhaC (PhaC1ps) derived from Pseudomonas sp. 61-3 strain was introduced.
• PhaC1ps Class 2 PhaC
• Patent Document 3 describes the production of a copolymer PHA containing 3-hydroxyhexanoate units with 6 carbon atoms by culturing a transformant into which the genes for CO9 synthase or D12 synthase, which is a PhaC of unknown class derived from the actinomycete Rhodococcus aetherivorans I24, have been introduced.
• the inventors have found that in transformed microorganisms into which the above-mentioned Class 2 PhaC derived from Pseudomonas bacteria or mutants thereof, or genes encoding CO9 synthase or D12 synthase derived from the actinomycete Rhodococcus aetherivorans have been introduced, the content of 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms contained in the copolymerized polyhydroxyalkanoic acid produced is not always high, and there is room for improvement in this regard.
• the present invention aims to provide a transformed microorganism capable of producing a copolymerized polyhydroxyalkanoic acid containing a high content of 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms, and a method for producing a copolymerized PHA using the transformed microorganism.
• a copolymerized PHA containing a high content of medium-chain-length 3HA monomer units can be produced by culturing a transformed microorganism into which a foreign gene encoding Class 2 PhaC derived from the genus Mycobacterium, Mycolicibacterium, or Nocardioides has been introduced, and thus completed the present invention.
• the present invention relates to a transformed microorganism capable of producing a copolymerized polyhydroxyalkanoic acid containing a 3-hydroxyalkanoic acid monomer unit having 8 or more carbon atoms
• the present invention relates to a transformed microorganism having a foreign gene encoding a polyhydroxyalkanoate synthase consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 4, or a foreign gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 4 and having polyhydroxyalkanoate synthase activity.
• the present invention also relates to a method for producing a culture medium comprising the steps of: culturing the transformed microorganism in the presence of a carbon source; and recovering from the transformed microorganism a copolymerized polyhydroxyalkanoic acid containing a 3-hydroxyalkanoic acid monomer unit having 8 or more carbon atoms.
• the present invention also relates to a method for producing a copolymerized polyhydroxyalkanoic acid.
• the present invention it is possible to provide a transformed microorganism capable of producing a copolymerized polyhydroxyalkanoic acid containing a high content of 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms.
• a copolymerized PHA containing a high content of 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms by fermentation.
• the resulting copolymer PHA has the advantage that it can have better mechanical properties in a low-temperature environment, as compared with a PHA composed only of 3-hydroxyalkanoic acid monomer units having 7 or less carbon atoms.
• the transformed microorganism disclosed herein is a transformed microorganism capable of producing a copolymerized polyhydroxyalkanoic acid containing 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms.
• copolymerized polyhydroxyalkanoic acid The copolymerized polyhydroxyalkanoic acid (hereinafter also referred to as copolymerized PHA) produced by the transformed microorganism according to the present disclosure is a copolymer of a 3-hydroxyalkanoic acid having 8 or more carbon atoms (hereinafter also referred to as medium-chain-length 3HA) and another hydroxyalkanoic acid copolymerizable with the medium-chain-length 3HA.
• medium-chain-length 3HA the mechanical properties of the copolymerized PHA in a low-temperature environment can be improved.
• the upper limit of the number of carbon atoms in the medium chain length 3HA is not particularly limited, but may be, for example, 14 or less, or 12 or less.
• medium-chain-length 3HA examples include 3-hydroxyoctanoic acid (carbon number 8, abbreviation: 3HO), 3-hydroxynonanoic acid (carbon number 9), 3-hydroxydecanoic acid (carbon number 10, abbreviation: 3HD), 3-hydroxyundecanoic acid (carbon number 11), 3-hydroxydodecanoic acid (carbon number 12, abbreviation: 3HDD), 3-hydroxytetradecanoic acid (carbon number 14), etc.
• the copolymer PHA may contain only one type of these medium-chain-length 3HAs, or may contain two or more types.
• the copolymer PHA preferably contains at least one type of medium chain length 3HA selected from the group consisting of 3HO, 3HD, and 3HDD.
• the medium chain length 3HA may contain only 3HO and/or 3HD, or may contain only 3HO. In particular, it is preferable that the medium chain length 3HA contains at least 3HO.
• the other hydroxyalkanoic acid copolymerized with the medium chain length 3HA is not particularly limited, but may be a 3-hydroxyalkanoic acid having 7 or less carbon atoms, such as 3-hydroxybutyric acid (4 carbon atoms, abbreviated as 3HB), 3-hydroxyvaleric acid (5 carbon atoms), 3-hydroxyhexanoic acid (6 carbon atoms, abbreviated as 3HH), 3-hydroxyheptanoic acid (7 carbon atoms), etc.
• the other hydroxyalkanoic acid may be a hydroxyalkanoic acid other than 3-hydroxyalkanoic acid, such as 2-hydroxyalkanoic acid, 4-hydroxyalkanoic acid, 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid, etc.
• the copolymerized PHA may contain only one type of these other hydroxyalkanoic acids, or may contain two or more types.
• the copolymerized PHA may contain 3HB and/or 3HH as other hydroxyalkanoic acids, or may contain 3HB and 3HH.
• the content ratio (3HB:3HH) is not particularly limited, but may be, for example, approximately 3:1 to 1:30, or 2:1 to 1:10 on a molar basis.
• the copolymer PHA may contain at least one type of medium chain length 3HA selected from the group consisting of 3HO, 3HD, and 3HDD, and 3HB and/or 3HH, or may contain at least one type of medium chain length 3HA selected from the group consisting of 3HO, 3HD, and 3HDD, and 3HB and 3HH.
• the total content of medium-chain length 3HA monomer units in the copolymerized PHA is preferably 1 mol% or more, more preferably 5 mol% or more, and even more preferably 10 mol% or more, from the viewpoint of improving the mechanical properties of the copolymerized PHA in a low-temperature environment. It may also be 20 mol% or more, 30 mol% or more, or 40 mol% or more.
• the upper limit of the total content of the medium chain length 3HA monomer units in the copolymerized PHA is not particularly limited, but may be, for example, 90 mol% or less, 70 mol% or less, 50 mol% or less, 30 mol% or less, or 20 mol% or less.
• the total content of the medium-chain length 3HA monomer units in the copolymerized PHA can be adjusted by changing the composition of the transformed microorganism according to the present disclosure, the carbon source, the culture conditions, etc.
• Hosts for transformed microorganisms are not particularly limited, and examples thereof include the genus Cupriavidus, such as Cupriavidus necator, the genus Alcaligenes, such as Alcaligenes latas, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas resinovorans, Pseudomonas oleovorans, and the like.
• Cupriavidus such as Cupriavidus necator
• Alcaligenes such as Alcaligenes latas
• Pseudomonas putida Pseudomonas fluorescens
• Pseudomonas aeruginosa Pseudomonas resinovorans
• Pseudomonas oleovorans Pseudomonas oleovorans
• the genus examples include the genus Pseudomonas, such as Bacillus megaterium, the genus Bacillus megaterium, the genus Azotobacter, the genus Nocardia, the genus Aeromonas, such as Aeromonas caviae and Aeromonas hydrophila, the genus Ralstonia, the genus Wautersia, and the genus Comamonas.
• Pseudomonas such as Bacillus megaterium, the genus Bacillus megaterium, the genus Azotobacter, the genus Nocardia
• Aeromonas such as Aeromonas caviae and Aeromonas hydrophila
• the genus Ralstonia such as Aeromonas caviae and Aeromonas hydrophila
• the genus Ralstonia such as Aeromonas caviae and Aeromonas hydrophila
• the genus Ralstonia such as Aeromonas cavia
• the bacteria may be gram-negative bacteria such as Escherichia genus, gram-positive bacteria such as Bacillus genus, or yeasts such as Saccharomyces genus, Yarrowia genus, or Candida genus.
• the host is preferably a bacterium capable of accumulating a large amount of PHA, more preferably a bacterium belonging to the genus Cupriavidus, and particularly preferably Cupriavidus necator.
• the transformed microorganism according to the present disclosure has a foreign gene encoding a polyhydroxyalkanoic acid synthase (hereinafter also referred to as PHA synthase) consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 4, or a variant thereof.
• PHA synthase polyhydroxyalkanoic acid synthase
• the transformed microorganism into which this gene has been introduced makes it possible to produce a copolymerized polyhydroxyalkanoic acid containing a high content of 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms.
• the PHA synthase shown in SEQ ID NO: 1 or 2 is Class 2 PhaC derived from the genus Mycobacterium.
• the PHA synthase shown in SEQ ID NO:3 is Class 2 PhaC derived from the genus Mycolicibacterium.
• the PHA synthase shown in SEQ ID NO:4 is a Class 2 PhaC derived from the genus Nocardioides. No transformed microorganisms into which genes encoding these PHA synthases have been introduced have been reported so far.
• a PHA synthase mutant consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 4 refers to a protein consisting of an amino acid sequence having 90% or more sequence identity to an amino acid sequence shown in any one of SEQ ID NOs: 1 to 4 and having PHA synthase activity.
• the sequence identity is preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.
• the number of PHA synthase genes to be introduced may be one or more. Furthermore, when multiple PHA synthase genes are introduced, they may be the same gene or different genes.
• the method for introducing the PHA synthase gene into the host is not particularly limited, but examples include a method in which the target gene is directly inserted or replaced on the host chromosome, a method in which the target gene is directly inserted or replaced on a megaplasmid carried by the host, or a method in which the target gene is placed on a vector such as a plasmid, phage, or phagemid and then introduced into the host. Two or more of these methods may be used in combination.
• a method of directly inserting or replacing the target gene on the host chromosome or on a megaplasmid possessed by the host is preferred, and a method of directly inserting or replacing the target gene on the host chromosome is more preferred.
• the "gene expression regulatory sequence” may be a DNA sequence containing a base sequence that controls the transcription amount of the gene (e.g., a promoter sequence) and/or a base sequence that controls the translation amount of messenger RNA transcribed from the gene (e.g., a Shine-Dalgarno sequence).
• a base sequence that controls the transcription amount of the gene e.g., a promoter sequence
• a base sequence that controls the translation amount of messenger RNA transcribed from the gene e.g., a Shine-Dalgarno sequence.
• any base sequence existing in nature may be used, or an artificially constructed or modified base sequence may be used.
• Foreign genes can be introduced by methods well known to those skilled in the art. Representative methods include a method that utilizes the mechanism of transposon and homologous recombination (Ohman et al., J. Bacteriol., 162:1068-1074 (1985)), a method based on site-specific integration caused by the mechanism of homologous recombination and loss by second-stage homologous recombination (Noti et al., Methods Enzymol., 154:197-217 (1987)), and a method based on the mechanism of Bacillus subtilis. A method of introducing the sacB gene derived from S.
• Plasmid vectors can be prepared by linking a DNA fragment having the base sequence of the foreign gene to a plasmid vector such as pCUP2.
• the promoter for expressing the introduced gene is not particularly limited.
• the promoter of the phaC1 gene of Capribia necator, the promoter of the phaP1 gene, the trp promoter, the lac promoter, the lacUV5 promoter, the trc promoter, the tic promoter, the tac promoter derived from Escherichia coli, or the lacN17 promoter having an artificially produced modified base sequence derived from Escherichia coli, the lacN19 promoter having an artificially produced modified base sequence derived from Escherichia coli, etc. can be used.
• the transformed microorganism may be one into which a gene encoding a protein having R-specific enoyl-CoA hydratase activity that recognizes enoyl-CoA having 8 or more carbon atoms as a substrate has been introduced, or one that has been transformed so that expression of the gene is enhanced. This increases the amount of 3HA monomers having 8 or more carbon atoms produced, and the content of 3HA monomer units having 8 or more carbon atoms in the copolymerized PHA produced can be further increased.
• protein with R-specific enoyl-CoA hydratase activity refers to a protein that has the enzyme activity to generate (R)-3-hydroxyacyl-CoA, which is a PHA monomer, using enoyl-CoA, an intermediate in the ⁇ -oxidation system of fatty acids, as a substrate.
• enoyl-CoA with 8 or more carbon atoms as a substrate, the protein increases the amount of (R)-3-hydroxyacyl-CoA with 8 or more carbon atoms converted, which is thought to result in a higher content of 3HA monomer units with 8 or more carbon atoms in the copolymerized PHA.
• Proteins with R-specific enoyl-CoA hydratase activity that recognizes enoyl-CoA with 8 or more carbon atoms as a substrate include, but are not limited to, bacterial R-specific enoyl-CoA hydratase (PhaJ) and eukaryotic multifunctional enzyme type 2 (MFE2).
• PhaJ bacterial R-specific enoyl-CoA hydratase
• MFE2 eukaryotic multifunctional enzyme type 2
• PhaJ is not particularly limited, and examples thereof include bacteria of the genus Pseudomonas and actinomycetes, etc.
• PhaJ derived from the genus Capriavidus has low activity for enoyl-CoA having 8 or more carbon atoms.
• the origin of MFE2 is not particularly limited, but examples thereof include Drosophila melanogaster and Yarrowia lipolytica.
• a protein having R-specific enoyl-CoA hydratase activity that recognizes enoyl-CoA having 8 or more carbon atoms as a substrate is preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 5, 6, or 7, or a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 5, 6, or 7.
• the sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more.
• a gene encoding a protein having R-specific enoyl-CoA hydratase activity that recognizes enoyl-CoA having 8 or more carbon atoms as a substrate may be introduced into a transformed microorganism, or the transformed microorganism may be transformed so that expression of the gene is enhanced.
• the gene can be introduced by the method described above.
• the expression regulatory sequence promoter sequence and/or SD sequence
• the expression regulatory sequence can be modified to enhance expression of the gene.
• enhanced gene expression refers to a state in which the transcription amount of the target gene or the expression amount of the polypeptide encoded by the target gene is increased compared to a strain in which expression of the target gene is not enhanced.
• the amount of increase is not particularly limited, but it is sufficient if it is more than 1-fold compared to a strain in which expression of the target gene is not enhanced, and is preferably an increase of 1.1-fold or more, more preferably 1.2-fold or more, even more preferably 1.5-fold or more, and even more preferably 2-fold or more.
• a transformed microorganism according to a preferred embodiment may be transformed so that expression of a gene encoding ⁇ -ketothiolase is suppressed.
• the ⁇ -ketothiolase has thiolysis activity for ⁇ -ketoacyl-CoA having 8 or less carbon atoms.
• the enzyme activity of ⁇ -ketothiolase can be eliminated or reduced. This suppresses decomposition of ⁇ -ketoacyl-CoA having 8 or less carbon atoms, and allows for more efficient production of copolymerized PHA containing medium-chain-length 3HA monomers.
• the expression of the ⁇ -ketothiolase gene may be suppressed in only one gene or in two or more genes.
• a transformed microorganism according to a preferred embodiment may have both suppressed expression of the ⁇ -ketothiolase gene and the introduction or enhanced expression of a gene encoding a protein having the above-mentioned R-specific enoyl-CoA hydratase activity.
• ⁇ -ketothiolase refers to an enzyme that catalyzes the reaction in the ⁇ -oxidation of fatty acids, in which ⁇ -ketoacyl-CoA undergoes thiolysis (thiol cleavage) in the presence of coenzyme A to produce fatty acyl-CoA that is two carbons shorter and acetyl-CoA.
• the ⁇ -ketothiolase gene whose expression is suppressed may be a gene encoding a ⁇ -ketothiolase having thiolysis activity for ⁇ -ketoacyl-CoA having 8 or less carbon atoms.
• the ⁇ -ketothiolase may have thiolysis activity for ⁇ -ketoacyl-CoA having 9 or more carbon atoms, in addition to thiolysis activity for ⁇ -ketoacyl-CoA having 8 or less carbon atoms.
• it may have thiolysis activity for ⁇ -ketoacyl-CoA having 4 to 8 carbon atoms, thiolysis activity for ⁇ -ketoacyl-CoA having 4 to 18 carbon atoms, or thiolysis activity for ⁇ -ketoacyl-CoA having 6 to 20 carbon atoms, but is not limited to the above.
• the gene encoding the ⁇ -ketothiolase is not particularly limited, but examples thereof include the bktB gene and the A1528 gene. Specifically, examples thereof include a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 or 9, and a gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 8 or 9.
• the sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more.
• Examples of methods for suppressing the expression of a gene encoding ⁇ -ketothiolase include a method of completely deleting the enzyme gene in a transformed microorganism, a method of inserting an entirely different gene, such as a drug resistance gene, into the sequence of the enzyme gene, or a method of deleting, substituting, adding, or inserting a part of the sequence of the enzyme gene (preferably a region involved in enzyme activity).
• Gene disruption techniques include, for example, homologous recombination techniques using a vector containing a gene or DNA for disruption, and techniques that utilize transposons.
• other disruption methods include known techniques such as the CRISPR/Cas (e.g., Cas9) system for disrupting a target gene and genome editing techniques using TALEN (Y.
• the guide RNA has a sequence that can bind to a portion of the base sequence of the ⁇ -ketothiolase gene to be destroyed, and plays a role in transporting Cas9 to the target.
• mutations such as deletion, substitution, addition, and insertion of the base sequence around the gene can be used to eliminate or reduce the enzyme activity by reducing the transcription and translation efficiency of the gene and the stability of the mRNA.
• the transformed microorganism can be cultured in the presence of a carbon source to accumulate the copolymerized PHA in the microbial cells.
• the transformed microorganism can be cultured according to a conventional microbial culture method, and may be cultured in a medium containing an appropriate carbon source.
• the medium composition, the method of adding the carbon source, the culture scale, the aeration and agitation conditions, the culture temperature, the culture time, and the like are not particularly limited.
• the carbon source is preferably added to the medium continuously or intermittently.
• any carbon source can be used as a carbon source during culture, as long as the transformed microorganism can assimilate it.
• the carbon source include, but are not limited to, sugars such as glucose, fructose, sucrose, and xylose; oils and fats such as palm oil and palm kernel oil (including palm olein, palm double olein, and palm kernel oil olein, which are low melting point fractions obtained by fractionating these oils), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, and fractionated oils thereof, or refined by-products thereof; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid, and derivatives thereof, and glycerol.
• sugars such as glucose, fructose, sucrose, and xylose
• oils and fats such as palm oil and palm kernel oil (including palm olein, palm double olein, and palm kernel oil ole
• oils and fats may be partially or entirely degraded oils.
• Degraded oils refer to oils and fats that have been thermally denatured or that have been degraded by reacting with oxygen and/or water under heating.
• the name of the oils and fats is not limited, and includes those called waste oil, discarded oil, waste edible oil, waste edible oil, waste vegetable oil, used oil, and the like.
• the transformed microorganism can utilize gases or alcohols such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, these can also be used as the carbon source.
• the carbon source preferably contains fats and oils (particularly vegetable oils) or free fatty acids.
• the carbon number of the constituent fatty acids of the oil or the free fatty acids is not particularly limited, but from the viewpoint of productivity of copolymerized PHA containing a medium-chain-length 3HA monomer, it is preferably 8 or more, more preferably 10 or more, and even more preferably 12 or more.
• a medium containing the carbon source a nitrogen source which is a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources.
• nitrogen sources include, but are not limited to, ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, and yeast extract.
• inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.
• examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C.
• the copolymerized PHA After culturing the transformed microorganism for an appropriate period of time to accumulate the copolymerized PHA within the microbial cells, the copolymerized PHA can be recovered using a known method. There are no particular limitations on the recovery method, but industrially, recovery by separation and purification in an aqueous system with low environmental impact is preferred. For example, after the end of the culture, a cell disruption solution in which cell components other than the copolymerized PHA are dissolved in water can be obtained by applying mechanical shearing force or disrupting the cells using a surfactant, alkali, enzyme, etc. The copolymerized PHA can be recovered by separating the copolymerized PHA from the aqueous phase by filtration or centrifugation of the cell disruption solution, and then drying.
• [Item 1] A transformed microorganism capable of producing a copolymerized polyhydroxyalkanoic acid containing a 3-hydroxyalkanoic acid monomer unit having 8 or more carbon atoms, A transformed microorganism having a foreign gene encoding a polyhydroxyalkanoic acid synthase having an amino acid sequence shown in any one of SEQ ID NOs: 1 to 4, or a foreign gene encoding a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 4 and having polyhydroxyalkanoic acid synthase activity.
• [Item 2] 2.
• the transformed microorganism according to item 1 into which a gene encoding a protein having R-specific enoyl-CoA hydratase activity for an enoyl-CoA substrate having 8 or more carbon atoms has been introduced, or which has been transformed so that expression of the gene is enhanced.
• the gene encoding a protein having R-specific enoyl-CoA hydratase activity is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 5, 6, or 7, or a gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 5, 6, or 7.
• the genetic manipulation in this example can be carried out by the method described in Molecular Cloning (Cold Spring Harbor Laboratory Press, 1989).
• the enzymes, cloning hosts, and the like used in the genetic manipulation can be purchased from commercial suppliers and used according to their instructions.
• the enzymes used in this example are not particularly limited as long as they can be used in genetic manipulation.
• the "KNK005dZ strain” used in the following production examples is a transformed microorganism also known as the KNK005 ⁇ phaZ1,2,6 strain, in which the phaC1 gene on the chromosome of Cupriavidus necator H16 strain has been replaced with a PHA polymerase gene mutant (NSDG) derived from Aeromonas caviae, and the phaZ1,2,6 genes, which are PHA degrading enzyme genes on the chromosome, have been deleted.
• NSDG PHA polymerase gene mutant
• a plasmid for disrupting NSDG was prepared as follows.
• a DNA fragment was prepared by linking the nucleotide sequences upstream and downstream of the NSDG gene by PCR using the genomic DNA of the KNK005dZ strain as a template (SEQ ID NO: 10).
• This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-2007-259708, which had also been digested with SmiII, using DNA ligase to prepare the plasmid vector pNS2X-sacB+NSDGUD for disrupting NSDG.
• the KNK005dZ/dNSDG strain was created using the NSDG disruption plasmid vector pNS2X-sacB+NSDGUD as follows.
• Escherichia coli strain S17-1 (ATCC47055) was transformed with pNS2X-sacB+NSDGUD, and the resulting transformed microorganism was mixed and cultured with KNK005dZ strain on Nutrient Agar (Difco) medium to perform conjugative transfer.
• the cultured cells obtained were inoculated on Simmons agar medium (sodium citrate 2g/L, sodium chloride 5g/L, magnesium sulfate heptahydrate 0.2g/L, ammonium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, agar 15g/L, pH 6.8) containing 250mg/L kanamycin, and the strains that grew on the agar medium were selected to obtain a strain in which the plasmid was integrated on the chromosome of KNK005dZ strain.
• Simmons agar medium sodium citrate 2g/L, sodium chloride 5g/L, magnesium sulfate heptahydrate 0.2g/L, ammonium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, agar 15g/L, pH 6.8
• the strains that grew on the agar medium were selected to obtain a strain in which the plasmid was integrated
• a plasmid for disrupting the phaJ4a gene was prepared as follows. A DNA fragment was prepared by linking the nucleotide sequences upstream and downstream of the phaJ4a structural gene by PCR using the genomic DNA of the KNK005dZ strain as a template (SEQ ID NO: 11).
• This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with pNS2X-sacB, which had also been digested with SmiII, using DNA ligase to prepare a plasmid vector pNS2X-sacB+phaJ4aUD for disrupting phaJ4a.
• the phaJ4a gene disruption plasmid vector pNS2X-sacB+phaJ4aUD was used to create the phaJ4a gene disruption strain KNK005dZ/dNSDG/dphaJ4a in the same manner as above, using KNK005dZ/dNSDG obtained in Production Example 1 as the parent strain.
• the resulting strain was designated host 1.
• a plasmid for disrupting the bktB gene was prepared as follows. A DNA fragment was prepared by linking the nucleotide sequences upstream and downstream of the bktB structural gene by PCR using the genomic DNA of the KNK005dZ strain as a template (SEQ ID NO: 12).
• This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with pNS2X-sacB, which had also been digested with SmiI, using DNA ligase to prepare a plasmid vector pNS2X-sacB+bktBUD for disrupting the bktB gene.
• the bktB gene-disrupted strain KNK005dZ/dNSDG/dphaJ4a obtained in Production Example 2 was used as the parent strain in the same manner as above to create the bktB gene-disrupted strain KNK005dZ/dNSDG/dphaJ4a/dbktB.
• the resulting strain was designated host 2.
• a plasmid for disrupting the A1528 gene was prepared as follows.
• a DNA fragment was prepared by linking the nucleotide sequences upstream and downstream of the A1528 structural gene by PCR using the genomic DNA of the KNK005dZ strain as a template (SEQ ID NO: 13).
• This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with pNS2X-sacB, which had also been digested with SmiI, using DNA ligase to prepare a plasmid vector pNS2X-sacB+A1528UD for disrupting the A1528 gene.
• the A1528 gene disruption strain KNK005dZ/dNSDG/dphaJ4a/dbktB obtained in Production Example 3 was used as the parent strain to create the A1528 gene disruption strain KNK005dZ/dNSDG/dphaJ4a/dbktB/dA1528 in the same manner as above.
• the resulting strain was designated host 3.
• a plasmid for introducing the phaJ4pp gene was prepared as follows.
• a DNA fragment (SEQ ID NO: 15) was obtained by PCR using an artificially synthesized gene as a template, which contains a base sequence including the base sequences upstream and downstream of the phaJ4a structural gene, a trc promoter having the base sequence set forth in SEQ ID NO: 14, and a gene encoding phaJ4pp having the amino acid sequence set forth in SEQ ID NO: 5.
• This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with pNS2X-sacB similarly digested with SmiI using DNA ligase to prepare a plasmid vector for introducing the phaJ4pp gene, pNS2X-sacB+phaJ4aU-trc-phaJ4pp-phaJ4aD.
• the chromosomal DNA was modified in the same manner as in the gene disruption described above using the KNK005dZ/dNSDG/dphaJ4a/dbktB obtained in Production Example 3 as the parent strain, to create the phaJ4pp gene-inserted strain KNK005dZ/dNSDG/dbktB/dphaJ4a::trc-phaJ4pp.
• the resulting strain is designated host 4.
• a plasmid for introducing the phaJ4pa gene was prepared as follows. PCR was performed using an artificially synthesized gene as a template to obtain a DNA fragment (SEQ ID NO: 17) in which the nucleotide sequence upstream of the phaJ4a structural gene and the nucleotide sequence immediately downstream thereof, the lacUV5 promoter having the nucleotide sequence set forth in SEQ ID NO: 16, and the gene encoding phaJ4pa having the amino acid sequence set forth in SEQ ID NO: 6 were linked.
• This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with pNS2X-sacB similarly digested with SmiI using DNA ligase to prepare a plasmid vector for introducing the phaJ4pa gene, pNS2X-sacB+phaJ4aU-lacUV5-phaJ4pa-phaJ4aD.
• the chromosomal DNA was modified in the same manner as in the gene disruption described above using the KNK005dZ/dNSDG/dphaJ4a/dbktB/dA1528 obtained in Production Example 4 as the parent strain, to create the phaJ4pa gene insertion strain KNK005dZ/dNSDG/dbktB/dA1528/dphaJ4a::lacUV5-phaJ4pa.
• the resulting strain is designated host 6.
• a plasmid for introducing the mfe2dm gene was prepared as follows.
• a DNA fragment (SEQ ID NO: 19) was obtained by PCR using an artificial synthetic gene as a template, which contains the nucleotide sequence upstream of the phaJ4a structural gene and the nucleotide sequence immediately following downstream of that nucleotide sequence, the poel promoter having the nucleotide sequence set forth in SEQ ID NO: 18, and a gene encoding Mfe2dm having the amino acid sequence set forth in SEQ ID NO: 7.
• This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with pNS2X-sacB similarly digested with SmiI using DNA ligase to prepare a plasmid vector pNS2X-sacB+phaJ4aU-poel-mfe2dm-phaJ4aD for introducing the mfe2dm gene.
• the chromosomal DNA was modified in the same manner as in the gene disruption described above, using the KNK005dZ/dNSDG/dphaJ4a/dbktB/dA1528 obtained in Production Example 4 as the parent strain, to create the mfe2dm gene insertion strain KNK005dZ/dNSDG/dbktB/dA1528/dphaJ4a::poe1-mfe2dm.
• the resulting strain is designated host 7.
• an artificially synthesized DNA fragment (SEQ ID NO: 32) containing a base sequence encoding the amino acid sequence of STQK shown in SEQ ID NO: 22 was digested with the restriction enzymes MunI and SpeI. This DNA fragment was ligated using DNA ligase to the pCUP2 vector described in JP 2007-259708 A that had been digested with MunI and SpeI to create the pCUP2-STQK plasmid.
• PCR was performed on the lacUV5 promoter using pCUP2-PlacUV5-phaCRe described in JP 2020-58391 A as a template and the DNAs shown in SEQ ID NO:20 and SEQ ID NO:21 as primers, and the resulting DNA fragment was digested with MunI.
• This DNA fragment was ligated with pCUP2-STQK digested with MunI using DNA ligase to create pCUP2-lacUV5-STQK, in which the STQK gene sequence was ligated downstream of the lacUV5 promoter.
• PhaC from Rhodococcus PhaCrw represented by SEQ ID NO: 28, PhaC1ra (CO9 synthase, disclosed in Patent Document 3) as shown in SEQ ID NO:29; PhaC2ra (D12 synthase, disclosed in Patent Document 3) as shown in SEQ ID NO:30.
• PhaC from the genus Gordonia PhaCgs shown in sequence number 31.
• PhaC from Mycobacterium genus PhaCmc as shown in SEQ ID NO: 1; PhaCmp as shown in sequence number 2.
• PhaC from Mycolicibacterium genus PhaCmi shown in sequence number 3.
• PhaC from Nocardioides genus PhaCna represented by SEQ ID NO: 4
• the following plasmids for introducing the PhaC gene were prepared in the same manner as in Production Example 9, except that a base sequence shown in any one of SEQ ID NOs: 33 to 45 was used instead of the base sequence shown in SEQ ID NO: 32 as an artificially synthesized DNA fragment containing a base sequence encoding PhaC, in which the STQK gene in the plasmid pCUP2-lacUV5-STQK for introducing the STQK gene was replaced with each of the PhaC genes described above.
• pCUP2-lacUV5-STSRQR pCUP2-lacUV5-EDSCSR
• pCUP2-lacUV5-phaC1H9 pCUP2-lacUV5-phaC1po
• pCUP2-lacUV5-phaC1pm pCUP2-lacUV5-phaCrw
• pCUP2-lacUV5-phaC1ra pCUP2-lacUV5-phaC2ra
• pCUP2-lacUV5-phaCgs pCUP2-lacUV5-phaCmc
• pCUP2-lacUV5-phaCmp pCUP2-lacUV5-phaCmi
• pCUP2-lacUV5-phaCna pCUP2-lacUV5-phaCna.
• host 2 Keratinogen activator 1 (KNK005dZ/dNSDG/dphaJ4a/dbktB) was cultured overnight in 5 ml Nutrient Broth medium. The resulting culture solution was quickly cooled on ice, the cells were collected and thoroughly washed with ice-cold distilled water, and the resulting cells were suspended in 2 ml of distilled water. 1 ml of the cell solution was mixed with each plasmid solution and injected into a cuvette for electroporation. Electroporation was performed using a MicroPulser electroporator (Bio-Rad) under conditions of voltage 1.5 kV, resistance 800 ⁇ , and current 25 ⁇ F.
• Bio-Rad MicroPulser electroporator
• the cell solution was collected, 1 ml of Nutrient Broth medium was added, and the mixture was cultured at 30 ° C for 3 hours. The resulting culture solution was applied to Nutrient Agar medium containing 100 mg / l kanamycin sulfate. The mixture was cultured at 30°C for two days, and strains containing each plasmid were obtained from the resulting colonies.
• composition of the seed medium was 1% (w/v) meat extract, 1% (w/v) Bacto-Trypton, 0.2% (w/v) yeast extract, 0.9% (w/v) disodium hydrogen phosphate dodecahydrate, 0.15% (w/v) potassium dihydrogen phosphate, 50 ⁇ g/L kanamycin, pH 6.8.
• the composition of the PHA production medium used for PHA production was 1.1% (w/v) disodium hydrogen phosphate dodecahydrate, 0.19% (w/v) potassium dihydrogen phosphate, 1.29% (w/v) ammonium sulfate, 0.1% (w/v) magnesium sulfate heptahydrate, 0.5% (v/v) trace metal salt solution (1.6% (w/v) iron chloride (II) hexahydrate, 1% (w/v) calcium chloride dihydrate, 0.02% (w/v) cobalt chloride hexahydrate, 0.016% (w/v) copper sulfate pentahydrate, and 0.012% (w/v) nickel chloride hexahydrate dissolved in 0.1 N hydrochloric acid), and 50 ⁇ g/L kanamycin. Palm kernel oil was used as the carbon source at a concentration of 1.0% (w/v).
• the PHA flask production culture method was as follows. First, 50 ⁇ l of glycerol stock of the strain was inoculated into 5 ml of seed medium and cultured for 16 hours, and the resulting culture liquid was used as the seed medium. Next, 0.5 ml of the seed culture liquid was inoculated into a 500 ml Sakaguchi flask containing 50 ml of PHA production medium. The operating conditions were a culture temperature of 30°C and an agitation speed of 130 rpm, and the culture was carried out for 72 hours. After the culture was completed, the bacterial cells were collected from the culture liquid, washed with ethanol, and vacuum dried, and the dry bacterial weight was measured.
• PHA production volume and monomer composition analysis The yield and monomer composition of the resulting PHA were analyzed by gas chromatography. After the culture was completed, 1 ml of a sulfuric acid-methanol mixture (15:85) and 1 ml of chloroform were added to about 20 mg of the dried cells collected, the mixture was sealed, and heated at 100° C. for 140 minutes to methylate the PHA contained in the dried cells. After cooling, 0.5 ml of pure water was added to the mixture and allowed to stand for about 30 minutes to separate into two layers.
• the lower chloroform layer was then collected and passed through a filter, after which the PHA was analyzed by capillary gas chromatography to quantify 3HB (carbon number 4), 3HH (carbon number 6), 3HO (carbon number 8), 3HD (carbon number 10), and 3HDD (carbon number 12), and the polymerization ratio of each monomer was calculated.
• the amount of PHA contained in the dry cells and the PHA content (wt%) were calculated from the amount of polymerization of each monomer.
• the amount of PHA produced in the culture solution (g/L) was calculated from the dry cell weight (DCW) and the PHA content (wt%) of the culture solution.
• the gas chromatograph used was a Shimadzu GC-2014AF/SPL, and the capillary column used was a Frontier Labs Ultra ALLOYUA1 (MS/HT)-15M-0.25F (column length 15 m, column inner diameter 0.25 mm, liquid film thickness 0.25 ⁇ m). He was used as the carrier gas, the column inlet pressure was 43.8 kPa, and 1 ⁇ l of the sample was injected.
• the temperature conditions were as follows: the initial temperature was kept at 50° C. for 2 minutes, then the temperature was increased from 50° C. to 275° C. at a rate of 22.5° C./min, and then 275° C. was held for 10 minutes.
• Table 1 the production amount and monomer composition ratio of the obtained PHA are shown in Table 1.
• Comparative Example 2 PHA production by STSRQR
• the strains obtained by introducing the plasmid pCUP2-lacUV5-STSRQR into each of the hosts 2 to 5 were cultured in the same manner as in Comparative Example 1, and the PHA production amount and monomer composition ratio were calculated in the same manner as in Comparative Example 1.
• the obtained PHA production amount and monomer composition ratio are shown in Table 2.
• Comparative Examples 7 to 10 PHA production by PhaC1ra, PhaC2ra, PhaCrw, or PhaCgs
• Strains obtained by introducing plasmid pCUP2-lacUV5-phaC1ra, pCUP2-lacUV5-phaC2ra, pCUP2-lacUV5-phaCrw, or pCUP2-lacUV5-phaCgs into host 5 were cultured in the same manner as in Comparative Example 1, and the PHA production amount and monomer composition ratio were calculated in the same manner as in Comparative Example 1.
• the obtained PHA production amount and monomer composition ratio are shown in Table 7.
• Example 1 PHA production by PhaCmc
• the strains obtained by introducing the plasmid pCUP2-lacUV5-phaCmc into each of the hosts 1 to 7 were cultured in the same manner as in Comparative Example 1, and the PHA production amount and monomer composition ratio were calculated in the same manner as in Comparative Example 1.
• the obtained PHA production amount and monomer composition ratio are shown in Table 8.
• Example 1 In the strain of Example 1 into which the PhaCmc gene shown in SEQ ID NO:1 was introduced, PHA production was confirmed in all hosts.
• the proportion of 3HA monomers with 8 or more carbon atoms (total proportion of 3HO, 3HD, and 3HDD) in the produced PHA was 5.5 mol% in host 1, a strain in which the ⁇ -ketothiolase gene was not disrupted and phaJ4pp was not introduced.
• the proportions were 2.6 mol% and 3.1 mol%, respectively, in hosts 2 and 3, which were strains in which the ⁇ -ketothiolase gene was disrupted.
• Example 2 PHA production by PhaCmp
• the strains obtained by introducing the plasmid pCUP2-lacUV5-phaCmp into each of the hosts 1 to 7 were cultured in the same manner as in Comparative Example 1, and the PHA production amount and monomer composition ratio were calculated in the same manner as in Comparative Example 1.
• the obtained PHA production amount and monomer composition ratio are shown in Table 9.
• Example 2 In the strain of Example 2 into which the PhaCmp gene shown in SEQ ID NO:2 was introduced, PHA production was confirmed in all hosts.
• the proportion of 3HA monomers with 8 or more carbon atoms in the produced PHA was 15.9 mol% in host 1, a strain in which the ⁇ -ketothiolase gene was not disrupted and phaJ4pp was not introduced.
• the proportions were 8.1 mol% and 7.7 mol% in hosts 2 and 3, which were strains in which the ⁇ -ketothiolase gene was disrupted, respectively.
• the proportions were 48.5 mol%, 54.9 mol%, 27.3 mol%, and 62.0 mol% in hosts 4, 5, 6, and 7, into which phaJ4pp, phaJ4pa, or mfe2dm was further introduced, respectively.
• host 1 a ⁇ -ketothiolase gene non-disrupted strain
• the PHA productivity was 0.04 g/L
• hosts 2 and 3 a ⁇ -ketothiolase gene disrupted strain, were introduced, the PHA productivity was 0.20 g/L and 0.20 g/L, respectively.
• Example 3 PHA production by PhaCmi
• the strains obtained by introducing the plasmid pCUP2-lacUV5-phaCmi into each of the hosts 1 to 7 were cultured in the same manner as in Comparative Example 1, and the PHA production amount and monomer composition ratio were calculated in the same manner as in Comparative Example 1.
• the obtained PHA production amount and monomer composition ratio are shown in Table 10.
• Example 3 In the strain of Example 3 into which the PhaCmi gene shown in SEQ ID NO:3 was introduced, PHA production was confirmed in all hosts.
• the proportion of 3HA monomers with 8 or more carbon atoms in the produced PHA was 3.9 mol% in host 1, a strain in which the ⁇ -ketothiolase gene was not disrupted and phaJ4pp was not introduced.
• the proportions were 2.1 mol% and 2.7 mol% in hosts 2 and 3, which were strains in which the ⁇ -ketothiolase gene was disrupted, respectively.
• the proportions were 15.4 mol%, 15.3 mol%, 7.4 mol%, and 11.4 mol% in hosts 4, 5, 6, and 7, into which phaJ4pp, phaJ4pa, or mfe2dm was further introduced, respectively.
• host 1 a ⁇ -ketothiolase gene non-disrupted strain
• the PHA productivity was 0.47 g/L
• hosts 2 and 3 a ⁇ -ketothiolase gene disrupted strain, were introduced, the PHA productivity was 1.37 g/L and 1.70 g/L, respectively.
• Example 4 PHA production by PhaCna
• the strains obtained by introducing the plasmid pCUP2-lacUV5-phaCna into each of the hosts 1 to 7 were cultured in the same manner as in Comparative Example 1, and the PHA production amount and monomer composition ratio were calculated in the same manner as in Comparative Example 1.
• the obtained PHA production amount and monomer composition ratio are shown in Table 11.
• Example 4 In the strain of Example 4 in which the PhaCna gene shown in SEQ ID NO:4 was introduced, PHA production was confirmed in all hosts.
• the proportion of 3HA monomers with 8 or more carbon atoms in the produced PHA was 3.3 mol% in host 1, a strain in which the ⁇ -ketothiolase gene was not disrupted and phaJ4pp was not introduced.
• the proportions were 2.5 mol% and 2.2 mol% in hosts 2 and 3, which were ⁇ -ketothiolase gene disrupted strains, respectively.
• the proportions were 9.1 mol%, 10.0 mol%, 4.3 mol%, and 9.4 mol% in hosts 4, 5, 6, and 7, in which phaJ4pp, phaJ4pa, or mfe2dm was further introduced, respectively.
• host 1 a ⁇ -ketothiolase gene non-disrupted strain
• the PHA productivity was 0.08 g/L
• hosts 2 and 3 a ⁇ -ketothiolase gene disrupted strain, were introduced, the PHA productivity was 0.71 g/L and 0.61 g/L, respectively.
• Examples 1 to 4 show that strains into which the genes of PhaCmc, PhaCmp, PhaCmi, or PhaCna represented by SEQ ID NOs: 1 to 4, which are Class 2 PhaC derived from the genus Mycobacterium, Mycolicibacterium, or Nocardioides, have been introduced, are capable of producing copolymerized PHA containing 1 mol % or more of 3HA monomers having 8 or more carbon atoms.
• Table 12 shows the sequence identity between PhaCmc, PhaCmp, PhaCmi, or PhaCna represented by SEQ ID NOs: 1 to 4 used in Examples 1 to 4 and each PhaC used in Comparative Examples 1 to 10.
• Table 12 shows that the sequence identities between each of the PhaCs in Examples 1 to 4 and each of the PhaCs in Comparative Examples 1 to 10 all show low values, with even the highest being only in the 60% range.

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