WO2024101063A1 - ポリヒドロキシアルカン酸の製造方法 - Google Patents

ポリヒドロキシアルカン酸の製造方法 Download PDF

Info

Publication number
WO2024101063A1
WO2024101063A1 PCT/JP2023/037027 JP2023037027W WO2024101063A1 WO 2024101063 A1 WO2024101063 A1 WO 2024101063A1 JP 2023037027 W JP2023037027 W JP 2023037027W WO 2024101063 A1 WO2024101063 A1 WO 2024101063A1
Authority
WO
WIPO (PCT)
Prior art keywords
acid
ctg
pha
gaa
cag
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2023/037027
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
丈治 柘植
シヴァシャンカリ エム ラマムザイ
佑宜 宮原
智義 山本
優 小澤
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Teijin Ltd
Tokyo Institute of Technology NUC
Original Assignee
Teijin Ltd
Tokyo Institute of Technology NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Teijin Ltd, Tokyo Institute of Technology NUC filed Critical Teijin Ltd
Priority to JP2024557257A priority Critical patent/JPWO2024101063A1/ja
Publication of WO2024101063A1 publication Critical patent/WO2024101063A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • the present invention relates to a method for producing polyhydroxyalkanoic acid by a microorganism using a specific class I polyhydroxyalkanoic acid polymerase gene, and to a mutant polyhydroxyalkanoic acid polymerase derived from Plesiomonas shigelloides.
  • PHAs Polyhydroxyalkanoates
  • PHA a homopolymer with (R)-3-hydroxybutanoic acid (3HB) as a constituent unit
  • P(3HB) a homopolymer with (R)-3-hydroxybutanoic acid
  • P(3HB) a homopolymer with (R)-3-hydroxybutanoic acid
  • P(3HB) a homopolymer with (R)-3-hydroxybutanoic acid
  • P(3HB) is crystalline, which allows for shorter processing times, but it is hard and brittle, making it less practical.
  • the polymer deteriorates during molding processing, for example by becoming low molecular weight when melted, making it unsuitable for industrial production.
  • various copolymers of 3HB and other monomers have been developed (Non-Patent Documents 1-4).
  • PHA synthases are important enzymes involved in the polymerization of 3-hydroxyalkanoic acids (Non-Patent Document 5) and are classified into four classes based on their substrate specificity and subunit composition (Non-Patent Document 6).
  • Class I PHA synthases and class II PHA synthases are homodimers of PHA synthase subunits.
  • class I PHA synthases derived from Ralstonia eutropha mainly polymerize short chain-length monomers (3-5 carbon atoms)
  • class II PHA synthases derived from Pseudomonas aeruginosa and Pseudomonas putida polymerize medium chain-length monomers (6-14 carbon atoms).
  • class III PHA synthases from Allochromatium vinosum and Synechocystis sp. PCC 6803, and class IV PHA synthases from Bacillus megaterium and Bacillus cereus are composed of two heterosubunits and polymerize short-chain length monomers.
  • Aeromonas caviae-derived PHA synthase (hereinafter, appropriately referred to as "PhaC Ac ”) can synthesize a copolymer of 3HB and 3-hydroxyhexanoic acid (hereinafter, appropriately referred to as "P(3HB-co-3HHx)”) from vegetable oils and fatty acids (Non-Patent Documents 7 to 11). Aeromonas caviae-derived PHA synthase has a broad substrate specificity and is capable of synthesizing high molecular weight PHA, making it one of the excellent polymerases and widely used in the synthesis of PHA. In addition, a mutant in which a mutation has been introduced into Aeromonas caviae-derived PHA synthase has also been developed (Non-Patent Document 10), making it possible to increase the production of PHA.
  • PhaC Ac Aeromonas caviae-derived PHA synthase
  • P(3HB-co-3HHx) 3-hydroxyhexanoic acid
  • Sudesh K Abe H, Doi Y., Prog Polym Sci 2000; 25: 1503-55.
  • a specific class I PHA synthase can synthesize a high molecular weight PHA as compared with the PHA synthase derived from Aeromonas caviae, and have completed the present invention.
  • the PHA synthase derived from Plesiomonas shigelloides hereinafter, appropriately referred to as "PhaC Ps ”
  • mutant PhaC Ps having specific mutations introduced therein are particularly excellent as PHA synthases in that they have a relatively broad substrate specificity and a high polymerization ability, and have completed the present invention.
  • the present invention provides the following: (1) A method for producing polyhydroxyalkanoic acid, comprising the steps of introducing a gene for a class I polyhydroxyalkanoic acid polymerase (excluding polyhydroxyalkanoic acid polymerase derived from Aeromonas caviae) into a microorganism and culturing the microorganism into which the gene has been introduced in the presence of a carbon source. (2) The method according to (1), wherein the class I polyhydroxyalkanoate synthase is selected from the group consisting of the genus Ferrimonas, the genus Shewanella, the genus Plesiomonas, and the genus Vibrio.
  • the class I polyhydroxyalkanoate synthase is selected from the group consisting of Ferrimonas marina, Shewanella pealeana, Plesiomonas shigelloides, and Vibrio metschnikovii.
  • the class I polyhydroxyalkanoate synthase gene has a sequence identity of less than 60% with a polyhydroxyalkanoate synthase gene derived from Aeromonas caviae.
  • the class I polyhydroxyalkanoate synthase is a polyhydroxyalkanoate synthase derived from Plesiomonas shigelloides.
  • the class I polyhydroxyalkanoate synthase is a polyhydroxyalkanoate synthase derived from Plesiomonas shigelloides and has an N175G mutation.
  • the microorganism is a microorganism selected from the group consisting of the genera Escherichia, Pseudomonas, Aeromonas, Alcaligenes, Bacillus, Corynebacterium, Brevibacterium, Serratia, and Vibrio, or a hydrogen-oxidizing bacterium.
  • the microorganism is Escherichia coli.
  • Figure 1 shows a phylogenetic tree of class I PHA synthases.
  • the PHA synthase sequences were aligned using Clustal W, and a neighbor-joining tree was generated using Genetyx software.
  • PHA synthases from Aeromonas caviae (BAA21815), as well as those from Ferrimonas marina (WP_067661665), Plesiomonas shigelloides (WP_116546999), and Vibrio metschnikofii (WP_154168902)
  • PHA synthases from Delftia acidovorans BAA33155
  • Halomonas elongate WP_013333150
  • Ralstonia eutropha WP_011615085
  • Shewanella pirina (WP_012154995) were used.
  • FIG. 2 shows the sequence alignment of PHA synthase genes derived from F. marina, S. pealeana, P. shigelloides, and V. metschnikovii, and the PHA synthase gene (PhaC Ac ) derived from Aeromonas caviae.
  • the # symbol indicates the active site of the synthase, which is cysteine (C 319 ), aspartic acid (D 475 ), and histidine (H 503 ), respectively.
  • the * symbol indicates the site where the mutation is introduced.
  • FIG. 3 shows the chemical structures of representative PHAs and their precursors.
  • FIG. 4 is a diagram showing the structure of the plasmid (pBBR1-phaCsAB Re J Ac ).
  • phaC represents the PHA synthase (phaC) gene
  • phaA Re represents the 3-ketothiolase gene derived from R. eutropha H16
  • phaB Re represents the acetoacetyl-CoA reductase gene derived from R. eutropha H16
  • T Re represents the terminator region of the pha operon derived from R. eutropha H16
  • phaJ Ac represents the (R)-specific enoyl-CoA hydratase gene derived from A. caviae
  • SD represents the SD sequence
  • P lac represents the lac promoter region derived from A.
  • FIG. 5 is a schematic diagram showing the structure of an expression cassette in which a mutation (N175G) has been introduced into the phaC Ps gene.
  • P pha_Re indicates the pha promoter region derived from Ralstonia eutropha H16
  • SD indicates the SD sequence
  • phaC Ps indicates the PHA synthase gene derived from P. shigelloides
  • phaA Re indicates the 3-ketothiolase gene derived from R. eutropha H16.
  • the present invention relates to a method for producing polyhydroxyalkanoic acid, which comprises the steps of introducing a gene for a class I PHA polymerase other than polyhydroxyalkanoic acid polymerase derived from Aeromonas caviae into a microorganism and culturing the transformant into which the gene has been introduced in the presence of a carbon source and, if necessary, a precursor of a second monomer.
  • PHA polymerases other than the PHA polymerase derived from Aeromonas caviae are not particularly limited as long as they are class I PHA polymerases, and examples thereof include PHA polymerases derived from species selected from the group consisting of the genera Ferrimonas, Shewanella, Plesiomonas, and Vibrio. from the genus Pherimonas, for example, Pherimonas marina, F. balearica; from the genus Shewanella, for example, S. pealeana, S. algae, S. amazonensis, S. aquimarina, S. baltica, S. benthica, S. colwelliana, S. decolorationis, S. denitrificans, S.
  • examples of the genus Plesiomonas include Plesiomonas shigelloides; examples of the genus Vibrio include Vibrio metschnikovii, V. cholerae, V. vulnificus, V. multiplinnatiensis, V. fluvialis, V. furnissii, V. mimicus, and V. parahaemolyticus.
  • PHA synthases are classified as class I synthases, the same as the PHA synthase derived from Aeromonas caviae, their amino acid sequences have low sequence identity or homology of less than 60% with the PHA synthase derived from Aeromonas caviae.
  • the "identity" of two amino acid sequences to be compared refers to the ratio of identical amino acid residues appearing at corresponding positions when both amino acid sequences are aligned
  • the “homology” of two amino acid sequences refers to the ratio of similar amino acid residues appearing at corresponding positions when both amino acid sequences are aligned, and is obtained by appropriately aligning the two amino acid sequences to be compared, determining the identical residues present in each sequence, determining the number of matching sites, and then dividing the number of matching sites by the total number of residues in the sequence region to be compared, and multiplying the obtained number by 100.
  • it can be obtained using the BLAST (Basic Local Alignment Search Tool) program (Altschul et al., J. Mol. Biol., (1990), 215(3):403-10) or the like.
  • mutants of PHA synthase can also be preferably used in terms of increasing the amount of PHA produced.
  • Such mutants have an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the wild-type PHA synthase, and have PHA polymerization activity.
  • Such mutants have a sequence identity or homology of 85% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more to the amino acid sequence of the wild-type PHA synthase.
  • the wild-type PHA synthase derived from Plesiomonas shigelloides contains a serine at position 153, which corresponds to position 149 of the mutant (N149S) of the PHA synthase derived from Aeromonas caviae.
  • an example of a mutant of PHA synthase derived from Plesiomonas shigelloides is a mutant in which asparagine (N) at position 175, which corresponds to position 171 of a mutant (D171G) of PHA synthase derived from Aeromonas caviae, is replaced with an aliphatic amino acid, such as glycine (G), alanine (A), valine (V), leucine (L), or isoleucine (I).
  • G glycine
  • A alanine
  • V valine
  • L leucine
  • I isoleucine
  • an example of a mutant is one in which asparagine at position 175 is replaced with glycine.
  • SEQ ID NO: 4 shows the base sequence encoding a mutant in which asparagine at position 175 is replaced with glycine.
  • the PHA synthesized by the manufacturing method of the present invention is a homopolymer of PHA, i.e., P(3HB), in the absence of a precursor.
  • the PHA synthesized by the manufacturing method of the present invention is a copolymer of 3HB and a second monomer in the presence of a precursor.
  • the "second monomer” refers to a monomer other than 3HB among the components constituting the copolymer with 3HB produced by the manufacturing method of the present invention.
  • the second monomer one or more monomers selected mainly from monomers having 3 to 6 carbon atoms can be used.
  • Examples of the second monomer are 3-hydroxypropionic acid (3HP), 3-hydroxy-2-methylbutanoic acid (3H2MB), 3-hydroxy-4-methylvaleric acid (3H4MV), 3-hydroxypivalic acid (3HPi), 3-hydroxyhexanoic acid (3HHx), 3-hydroxy-2-methylpropionic acid (3H2MP), etc.
  • Some monomers with 3 to 6 carbon atoms have isomers due to the asymmetric center at the C2 and/or C3 positions, such as 3-hydroxy-4-methylvaleric acid, 3-hydroxy-2-methylbutanoic acid, and 3-hydroxyhexanoic acid, and such isomers are also included in the second monomer.
  • precursor refers to a precursor of said second monomer.
  • the precursor of the second monomer 3-hydroxypropionic acid (3HP) is, for example, propionic acid
  • the precursor of the second monomer 3-hydroxy-4-methylvaleric acid (3H4MV) is, for example, 4-methylvaleric acid
  • the precursor of the second monomer 3-hydroxypivalic acid (3HPi) is, for example, 3-hydroxypivalic acid
  • the precursor of the second monomer 3-hydroxyhexanoic acid (3HHx) is, for example, hexanoic acid
  • the precursor of the second monomer 3-hydroxy-2-methylpropionic acid (3H2MP) is, for example, 3-hydroxy-2-methylpropionic acid.
  • the precursor of 3H2MP may also be methacrylic acid.
  • Methacrylic acid is metabolized to methacrylic-CoA
  • methacrylic-CoA is metabolized to 3H2MP-CoA.
  • Precursors to the second monomer, 3-hydroxy-2-methylbutanoic acid (3H2MB) include, for example, tiglic acid ((E)-2-methyl-2-butenoic acid), angelic acid, 2-methylbutanoic acid, 3-hydroxy-2-methylbutanoic acid, isoleucine, or mixtures thereof.
  • examples of copolymers of 3HB and a second monomer include copolymers of 3-hydroxybutanoic acid and 3-hydroxyhexanoic acid (P(3HB-co-3HHx)), copolymers of 3-hydroxybutanoic acid and 3-hydroxy-4-methylvaleric acid (P(3HB-co-3H4MV)), copolymers of 3-hydroxybutanoic acid and 3-hydroxy-2-methylbutanoic acid (P(3HB-co-3H2MB)), copolymers of 3-hydroxybutanoic acid and 3-hydroxypivalic acid (P(3HB-co-3HPi)), and copolymers of 3-hydroxybutanoic acid and 3-hydroxy-2-methylpropionic acid (P(3HB-co-3H2MP)) ( Figure 3).
  • the precursors may be added to the culture medium together with the carbon source or after several hours of culture in the presence of the carbon source.
  • Precursors are generally highly toxic to cells, thus inhibiting cell growth and reducing PHA accumulation in microorganisms.
  • it is preferred to introduce the precursors into the culture medium several hours after substantial cell growth has been achieved (Furutate S, Kamoi J, Nomura CT et al., NPG Asia Mater 2021; 13: 1-11. DOI: 10.1038/s41427-021-00296-x).
  • the amount of the precursor added is preferably in the range of about 0.1 to about 5.0 g/L, more preferably in the range of about 0.5 to about 2.0 g/L. In addition, it is preferable to add these amounts intermittently in several portions to enable better uptake of the precursor for the accumulation of the second monomer and to eliminate or reduce unexpected effects on the cells.
  • a transformant into which the PHA polymerase gene of the present invention has been introduced can be produced by any method.
  • a plasmid vector or artificial chromosome into which the PHA polymerase gene of the present invention has been introduced may be introduced into a host microorganism, or the PHA polymerase gene of the present invention may be introduced into DNA such as a chromosome, plasmid, or megaplasmid carried by the host microorganism using known gene recombination techniques.
  • the former method involves inserting the PHA polymerase gene of the present invention and a known monomer supply gene into a broad host range vector for expressing a gene of interest in the host to obtain a plasmid, which is then introduced into the host microorganism.
  • Phasin is known to co-localize with PHA granules in bacterial cells, and is thought to be involved in the formation and stabilization of PHA granules.
  • mutants having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid region from the N-terminus to the 20th amino acid are also preferably used.
  • Feicin mutants similarly improve PHA productivity.
  • the feicin gene in the present invention is not particularly limited as long as it is a feicin gene derived from a biological species different from the host of the transformed microorganism in the present invention.
  • the feicin gene may be derived from organisms of the genera Ralstonia, Cupriavidus, Woutersia, Alcaligenes, Aeromonas, or Pseudomonas, or may be a mutant thereof.
  • the feicin gene is derived from a microorganism of the genus Aeromonas, or a mutant thereof, and more preferably, the feicin gene is derived from Aeromonas caviae, or a mutant thereof.
  • the mutant may be a base sequence encoding a feicin in which one or more amino acid residues have been deleted, added, inserted, or substituted.
  • Such a mutant has a sequence identity or identity of 85% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more to the amino acid sequence of wild-type feicin.
  • a broad-host-range vector for expressing a gene of interest in a host a known vector having a promoter, a ribosome-binding site, a gene cloning site, a terminator, etc. can be used. Examples include vectors pBBR1MCS-2, pJRD215, and pLA2917, which have a mob region with mobilizing activity.
  • Known monomer supply genes include, for example, phbA, phbB, phaA, and phaB from Ralstonia eutropha (Peoples, O. P. and Sinskey, A. J., J. Biol. Chem. 264: 15293-15297 (1989)) and phaJ from Aeromonas caviae (Fukui, T. and Doi, Y., J. Bacteriol. 179: 4821-4830 (1997)).
  • glucose when glucose, a sugar, is used as a carbon source in the absence of a precursor, glucose is converted to pyruvic acid and then to acetyl-CoA, which is then dimerized (PhaA) by ⁇ -ketothiolase (PhaA) and reduced by acetoacetyl-CoA reductase (PhaB) to be converted to 3HB-CoA, resulting in the synthesis of a homopolymer of 3-hydroxybutanoic acid (P(3HB)).
  • PhaA dimerized
  • PhaA dimerized
  • PhaA ⁇ -ketothiolase
  • PhaB acetoacetyl-CoA reductase
  • tiglic acid when glucose is used as a carbon source in the presence of tiglic acid as a precursor, for example, tiglic acid is converted to tiglyl-CoA, which is then converted to 3H2MB-CoA by enoyl-CoA hydratase (PhaJ).
  • PhaJ enoyl-CoA hydratase
  • the monomers supplied via the 3HB-CoA synthesis pathway and the 3H2MB-CoA synthesis pathway are used as substrates for PHA synthase (PhaC), synthesizing P(3HB-co-3H2MB).
  • the host microorganism used in the present invention is not particularly limited as long as it has good growth properties when sugars or fats and oils are used as carbon sources, the strain has high stability, and the cells can be relatively easily separated from the culture medium.
  • microorganisms of the genera Escherichia, Pseudomonas, Aeromonas, Ralstonia, Alcaligenes, Bacillus, Corynebacterium, Brevibacterium, Serratia, and Vibrio are preferred. More preferably, the genus Escherichia.
  • An example of a microorganism of the genus Escherichia is Escherichia coli (hereinafter referred to as "E. coli" where appropriate).
  • the host microorganism used in the present invention may be a hydrogen-oxidizing bacterium (also called “hydrogen bacteria”).
  • “Hydrogen-oxidizing bacteria” refers to bacteria that oxidize free hydrogen and perform carbon dioxide assimilation by utilizing the energy generated by the reaction.
  • Examples of hydrogen-oxidizing bacteria include Ralstonia genus bacteria such as Ralstonia eutropha, Alcaligenes genus bacteria such as Alcaligenes latus, and Hydrogenovibrio genus bacteria such as Hydrogenovibrio marinus.
  • Ralstonia eutropha is preferred because it can grow under conditions in which gaseous carbon dioxide or carbonate is the sole carbon source, its entire genome information has been analyzed, and a gene recombination method has been established (Cramm, R. et al., J. Mol. Microbiol. Biotechnol., 16, 38-52(2009)).
  • Examples of Ralstonia eutropha include the Ralstonia eutropha strain H16 (ATCC17699) and the Ralstonia eutropha strain PHB - 4 (DSM541).
  • Carbon sources that can be used include, for example, sugars, carboxylic acids, oils and fats, and carbon dioxide.
  • sugars include glucose, fructose, galactose, xylose, arabinose, saccharose, maltose, starch, and starch hydrolysates.
  • carboxylic acids include acetic acid and lactic acid.
  • oils and fats are vegetable oils, such as soybean oil, corn oil, cottonseed oil, peanut oil, coconut oil, palm oil, palm kernel oil, and fractionated oils thereof, such as palm double olein oil (low boiling fraction obtained by fractionating palm oil twice without a solvent), palm kernel oil olein (low boiling fraction obtained by fractionating palm kernel oil once without a solvent), and synthetic oils obtained by chemically or biochemically treating these oils and fats or their fractions, or mixed oils thereof.
  • oils and fats are vegetable oils, such as soybean oil, corn oil, cottonseed oil, peanut oil, coconut oil, palm oil, palm kernel oil, and fractionated oils thereof, such as palm double olein oil (low boiling fraction obtained by fractionating palm oil twice without a solvent), palm kernel oil olein (low boiling fraction obtained by fractionating palm kernel oil once without a solvent), and synthetic oils obtained by chemically or biochemically treating these oils and fats or their fractions, or mixed oils thereof.
  • carbon dioxide examples include air that is bubbled and enriched with carbon dioxide.
  • a mixed gas containing hydrogen (H 2 ), oxygen (O 2 ), and carbon dioxide (CO 2 ) can be used as the carbon dioxide.
  • the mixed gas may contain other components such as ammonia, nitrogen, hydrocarbons, carbon monoxide, formaldehyde, water vapor, etc.
  • the proportion of carbon dioxide in the mixed gas is, for example, 1 to 20% (v/v), preferably 3 to 20% (v/v), more preferably 5 to 15% (v/v).
  • glucose concentrations may cause catabolic repression of PHA biosynthetic genes induced by isopropyl- ⁇ -thiogalactopyranoside (IPTG) (Furutate S, Kamoi J, Nomura CT et al., NPG Asia Mater 2021; 13: 1-11. DOI: 10.1038/s41427-021-00296-x), so it is preferable to keep the glucose concentration at a minimum to promote cell growth only.
  • the amount of glucose added is preferably in the range of about 1 to about 20 g/L, more preferably in the range of about 3 to about 10 g/L, and even more preferably in the range of about 3 to about 8 g/L. In addition, it is preferable to add these amounts intermittently in several portions to enable better uptake of precursors for second monomer accumulation and to eliminate or reduce unexpected effects on the cells.
  • the culture temperature is a temperature at which the bacteria can grow, preferably 15 to 40°C, particularly preferably 20 to 40°C, and further preferably 28 to 34°C.
  • the culture time is not particularly limited, but for example, 1 to 7 days is preferable for batch culture, and continuous culture is also possible.
  • the culture medium is not particularly limited as long as it can be utilized by the host of the present invention.
  • a medium containing a nitrogen source, inorganic salts, other organic nutrient sources, and the like in addition to a carbon source can be used.
  • the nitrogen source include ammonia, ammonium salts such as ammonium chloride, ammonium sulfate, and diammonium hydrogen phosphate, peptone, meat extract, and yeast extract.
  • inorganic salts examples include monopotassium phosphate, dipotassium phosphate, monosodium phosphate, dibasic sodium phosphate, magnesium hydrogen phosphate, magnesium sulfate, sodium chloride, and hydrates thereof.
  • Metal salts containing cobalt, iron, calcium, nickel, chromium, copper, etc. may also be added as trace metal components.
  • Other organic nutrient sources include, for example, amino acids such as glycine, alanine, serine, threonine, and proline; vitamins such as vitamin B1, vitamin B12, biotin, nicotinamide, pantothenic acid, and vitamin C.
  • the PHA of the present invention can be recovered from the bacterial cells, for example, by the following method. After the cultivation is completed, the bacterial cells are separated from the culture medium using a centrifuge or the like, washed with distilled water, methanol, or the like, and then dried. The copolymer is then extracted from the dried bacterial cells using an organic solvent such as chloroform. Next, bacterial components are removed from the organic solvent solution containing the copolymer by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the copolymer. The supernatant is removed from the precipitated copolymer by filtration or centrifugation, and the copolymer can be recovered by drying. The obtained copolymer can be analyzed by, for example, gas chromatography, nuclear magnetic resonance, or the like.
  • the polydispersity (PDI) of a polymer is an important factor in determining the suitability of a polymer for a particular application, such as a biodegradable plastic material, and from a strength standpoint, a polymer with a high molecular weight and low PDI is generally desirable, whereas a molecular weight that is too high limits the options for molding methods due to the high melt viscosity of the polymer.
  • the PHA synthase of the present invention can synthesize a PHA having a weight average molecular weight (Mw) of about 20 ⁇ 10 5 to about 60 ⁇ 10 5 and a PDI in the range of about 1.3 to about 5.
  • PhaC from Plesiomonas shigelloides and its N175G mutant can synthesize P(3HB) or a copolymerized PHA of 3HB and a second monomer with a Mw of more than 30 ⁇ 10 5 and a PDI of less than 1.5. Therefore, it is expected that the PHA synthesized by the production method of the present invention can be industrially used for the above-mentioned applications.
  • the class I PHA polymerases used were those from Aeromonas caviae (accession number BAA21815), Delftia acidovorans (accession number BAA33155), Pherimonas marina (accession number WP_067661665), Halomonas elongate (accession number WP_013333150), Plesiomonas shigelloides (accession number WP_116546999), Ralstonia eutropha (accession number WP_011615085), Shewanella pirina (accession number WP_012154995), and Vibrio metschnikoffii (accession number WP_154168902).
  • PhaC box sequence is typically written as G-X-C-X-G-G (X is any amino acid), and a cysteine (Cys319 in PhaC Ac ) is the active center (Nambu Y, Ishii-Hyakutake M, Harada K et al., FEBS Lett 2020; 594: 710-6.DOI: 10.1002/1873-3468.13651).
  • PhaC Ac the active center Cys319, Asp475, and His503 are known to form a catalytic triad (Tsuge T, Watanabe S, Sato S et al., Macromolecul Biosci 2007; 7: 846-54. DOI: 10.1002/mabi.200700023), which are conserved in all PhaCs.
  • PhaC from Plesiomonas shigelloides has a protein size that is approximately 30 amino acid residues larger than other PhaCs and shows relatively low sequence identity in the C-terminal region.
  • Nucleotide sequence of PhaC from Shewanella pealeana (SEQ ID NO:2) ATG GAG AGC AAA AGC CCG TTT CAG GAC GCC ATT GAT AAT GCG ATG CAA TTC GGT CAA GCA TGG ATG GAC TCC TTT GGC CAG TCT GCG CAG TCA TCC ATC GTT GAG ACT CAG GCC GAA GAT TGG GCC CAG TGG ATG CGT TCC AGT GTT GAG CAT CCA GTG AAC TCT ATC GAA CAA CAA ATG GAT TGG TGG GGT CAG CAA GTG AAC CTG TTT AAC GAC TGC ATT ATG TCG AGC CCG GCG GAG AAA GAG ACA GAT CGG CGC TTT AAA GAT CCA GCG TGG AAC GAA CAA GCG CTG TAC AAG TAT ATT AAG GAA TCG TGT AAT AAC ATT CAG GCA AGC ATT ATG TCG AGC C
  • Nucleotide sequence of mutant PhaC derived from Plesiomonas shigelloides SEQ ID NO: 4
  • E. coli LSBJ (a fadB fadJ knockout mutant of E. coli LS5218 [fadR601, atoC(Con)] (Tappel RC, Wang Q, Nomura CT, J Biosci Bioeng 2012; 113:480-6. DOI: 10.1016/j.jbiosc.2011.12.004)), which allows greater control of the repeating units in PHA biosynthesis, was used as a host for PHA biosynthesis.
  • Plasmids The plasmid pBBR1-phaCsAB Re J Ac ( FIG. 4 ) was prepared by introducing each phaC gene, the lac promoter region derived from A. caviae, the (R)-specific enoyl-CoA hydratase gene (phaJ Ac ), the 3-ketothiolase gene derived from Ralstonia eutropha H16 (phaA Re ), and the acetoacetyl-CoA reductase gene derived from Ralstonia eutropha H16 (phaB Re ) into the broad-host-range plasmid pBBR1MCS - 2 (Kovach ME, Elzer PH, Hill DS et al., Gene 1995; 166: 175-6.
  • LB medium and modified M9 medium were prepared as follows.
  • Antibiotics kanamycin: final concentration 50 ⁇ g/mL, and carbenicillin: final concentration 50 ⁇ g/mL
  • kanamycin final concentration 50 ⁇ g/mL
  • carbenicillin final concentration 50 ⁇ g/mL
  • Modified M9 medium 17.1 g of Na2HPO4.12H2O , 3 g of KH2PO4 , 0.5 g of NaCl, and 2.5 g of bacto-yeast extract (Difco Laboratories) were dissolved in 1 L of deionized water and autoclaved for 20 minutes at 121° C. 2 mL of 1 M MgSO4, 0.1 mL of 1 M CaCl2, 50 mg of kanamycin, and 50 mg of carbenicillin were added after autoclaving the liquid medium.
  • PHA biosynthetic gene expression was induced using 1 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • P(3HB) homopolymer was synthesized by adding 20 g/L glucose at the beginning of cell culture and incubating for 72 h at 30° C.
  • the total incubation time was set to 76 h, with an initial incubation (130 rpm) of 4 h at 30° C. before adding IPTG, precursor, and glucose and culturing for an additional 72 h.
  • hexanoic acid, 4-methylvaleric acid, trans-2-methylbut-2-enoic acid (tiglic acid), 2,2-dimethyl-3-hydroxypropionic acid (3-hydroxypivalic acid), and 3-hydroxy-2-methylpropionic acid were pre-converted into their respective sodium salts and used as precursors for the second monomer components: 3HHx, 3H4MV, 3H2MB, 3HPi, and 3H2MP, respectively (Tanadchangsaeng N, Pattanasupong A., Polym 2022; 14: 428.
  • the structures of the precursors and copolymers used are shown in Figure 3. These precursors are known to inhibit cell growth, and high concentrations of glucose can repress PHA biosynthetic genes induced by IPTG.
  • glucose and precursors were added intermittently to the medium (4, 28, and 52 h). A total of 7.5 g/L glucose (2.5 g/L each time) and 0.6 g/L precursors (0.2 g/L each time) were added throughout the main incubation period. Finally, cells were harvested by centrifugation and lyophilized for further analysis.
  • the culture medium was centrifuged at 6,000 ⁇ g for 10 minutes at room temperature three times (once to discard the medium and twice to wash off the remaining salt with water), and then the dry cell weight was measured and freeze-dried for about 3 days.
  • the freeze-dried product was subjected to the following analysis.
  • the methanolylated sample was cooled to room temperature, and 1 mL of deionized water was added to separate polar components from nonpolar components.
  • the nonpolar components including 3HA-methyl ester, were filtered, and an equal volume of chloroform solution containing 0.1% (w/v) methyl-n-octane as an internal standard was added to prepare the final sample for GC analysis.
  • the sample was injected through a GC capillary column InertCap 1 (30 m x 0.25 mm, GL Science). The column temperature was first held at 90°C for 2 min, then increased to 110°C at a rate of 5°C/min, and then further increased to 280°C at a rate of 20°C/min.
  • the total PHA content and 3HA monomer composition were calculated from the obtained signal peak areas.
  • Example 1 Production of P(3HB) in E. coli expressing PhaC
  • P(3HB) was synthesized using a total of five PhaCs, including four PhaCs and a mutant (N175G) of PhaC derived from Plesiomonas shigelloides. The results are shown in Table 2.
  • Table 2 also shows the results using PhaC derived from Aeromonas caviae and its double mutant (NSDG).
  • Plesiomonas shigelloides-derived PhaC and its N175G mutant were found to have a high Mw of more than 3 ⁇ 10 6 and a relatively low PDI of less than 1.5.
  • Example 2 Preparation of copolymers of 3HB and a second monomer in E. coli expressing PhaC Copolymers of 3HB and a second monomer were synthesized in the same manner as in Example 1, except that the precursors hexanoic acid, 4-methylvaleric acid, tiglic acid, or 3-hydroxypivalic acid were further added. The precursors were added intermittently to the culture medium 4 hours after substantial cell growth was achieved (0.2 g/L x 3 times). Glucose was also added intermittently (2.5 g/L x 3 times). The results are shown in Tables 3 to 6. These tables also show the results using PhaC from Aeromonas caviae and its NSDG mutant.
  • Example 3 Preparation of a Copolymer of 3HB and a Second Monomer in E. coli Expressing PhaC
  • the precursor of the second monomer, 3-hydroxy-2-methylpropionic acid was synthesized as follows. Methyl-(R)-(-)-3-hydroxyisobutanoic acid and methyl-(S)-(+)-3-hydroxyisobutanoic acid (3 g) were dissolved in 15 mL of methanol, and 2 mol equivalents of sodium hydroxide were added on ice and stirred. The mixture was then stirred in a water bath at 40°C until white powder of 3-hydroxyisobutanoic acid sodium salt was precipitated. The resulting white powder was dried at room temperature to obtain 3-hydroxy-2-methylpropionic acid.
  • the resulting powder was dissolved in ultrapure water and the pH was adjusted to 7.0-7.4 with 1M HCl.
  • the resulting solution was filtered through a 0.45 ⁇ m cellulose acetate filter to prepare a monomer precursor to be added to the culture solution. Except for using 3-hydroxy-2-methylpropionic acid as the precursor, the copolymerized PHA was synthesized in the same manner as in Example 2. The results are shown in Table 7.
  • Tables 3-7 show that all PhaCs except for PhaC from Shewanella pirina were able to copolymerize all second monomers.
  • PHAs containing ⁇ -carbon methylation units are potentially attractive biobased materials, it is very interesting that almost all PhaCs have the ability to incorporate 3H2MB, 3HPi, or 3H2MP.
  • Plesiomonas shigelloides PhaC and its N175G mutant are overall superior to Aeromonas caviae PhaC in terms of high PHA content and broad substrate specificity.
  • Example 4 Production of PHA in Recombinant Hydrogen-Oxidizing Bacteria Expressing PhaC PHA was synthesized in the same manner as in Example 2, except that a recombinant hydrogen-oxidizing bacterium (Ralstonia eutropha PHB - 4/pBBREE"_P Ac Psh) was used as the host instead of E. coli LSBJ, and fructose was used instead of glucose.
  • Ralstonia eutropha PHB - 4/pBBREE"_P Ac Psh was constructed as follows.
  • the pBBREE"P Ac NSDG vector (7.5 kb) (Watanabe Y, Ichinomiya Y, Shimada D, Saika A, Abe H, Taguchi S, Tsuge T, J. Biosci. Bioeng., 2012, 113:286-292, DOI: 10.1016/j.jbiosc.2011.10.015) was digested with restriction enzymes NdeI and BamHI, and the chemically synthesized phaC Ps gene was inserted into the NdeI and BamHI sites. This gave pBBREE"_P Ac phaC Ps , a plasmid for PHA polymerization.
  • the obtained plasmid was transformed into Ralstonia eutropha PHB - 4 (a mutant strain lacking PHB polymerization ability) to construct Ralstonia eutropha PHB - 4/pBBREE"_P Ac phaC Ps .
  • Table 8 shows that PhaC derived from Plesiomonas shigelloides can synthesize P(3HB-co-3H2MB) or P(3HB-co-3H4MV) with a second monomer fraction equal to or greater than that of PhaC derived from the Aeromonas caviae NSDG mutant, even in hosts other than E. coli.
  • Table 9 shows that PHA having an ultrahigh molecular weight of 22 ⁇ 10 5 to 58 ⁇ 10 5 can be produced by using PhaC derived from Plesiomonas shigelloides.
  • Example 6 Production of PHA in Recombinant Hydrogen-Oxidizing Bacterium Expressing PhaC (1) Preparation of Ralstonia eutropha H16 ⁇ phaC1::phaC Ps (a) Construction of Plasmid pk18-phaC Ps In order to insert the phaC Ps gene represented by SEQ ID NO:3 into Ralstonia eutropha H16 ⁇ phaC1 strain (a PHA polymerization-deficient strain), the plasmid pk18-phaC Ps was constructed. Using the chemically synthesized phaC Ps gene as a template, PCR amplification was performed with primers 5 and 6 to amplify a phaC Ps gene fragment (1.9 kb).
  • the obtained DNA fragment was inserted into the SphI and EcoRI sites of pK18mobsacB (DOI: 10.1016/0378-1119(94)90324-7) to construct the plasmid pk18-phaC Ps .
  • Forward primer 5 5'-AGAGACAATCAAATCATGGCAAGTGCGAATCAGTT-3' (SEQ ID NO: 10)
  • Reverse primer 6 5'-GCACTCATGCAAGCGTCATGCCGCGTTTTCCTCGG-3' (SEQ ID NO: 11)
  • Forward primer 7 5'-aaaGAATTCCGGGCAAGTACCTTGCCGACAT-3' (SEQ ID NO: 12)
  • Reverse primer 8 5'-CACTTGCCATGATTTGATTGTCTCTCTGCCGTCAC-3' (SEQ ID NO: 13)
  • Forward primer 9 5'-CGCGGCATGACGCTTGCATGAGTGCCGGCGTGCGT-3' (SEQ ID NO: 14)
  • Reverse primer 10 5'-aaaGCATGCACTCGGCGCGCGACAGGGCGCGCTTG-3' (SEQ ID NO: 15)
  • the transformant was inoculated into LB medium (5.0 g/L yeast extract, 10 g/L bacto-tryptone, 10 g/L NaCl) containing 50 ⁇ g/mL kanamycin, and cultured overnight at 37° C. with shaking. Meanwhile, the Ralstonia eutropha H16 strain was inoculated into NR medium (2.0 g/L yeast extract, 10 g/L bacto-tryptone, 10 g/L bonito extract) and cultured overnight at 30° C. with shaking. Next, 2 mL of the culture medium of the transformant of E.
  • LB medium 5.0 g/L yeast extract, 10 g/L bacto-tryptone, 10 g/L NaCl
  • NR medium 2.0 g/L yeast extract, 10 g/L bacto-tryptone, 10 g/L bonito extract
  • coli S17-1 strain and the culture medium of Ralstonia eutropha H16 strain were collected and centrifuged at 10,000 ⁇ g for 2 minutes to prepare a cell pellet.
  • the cells were washed three times with 1 mL of NR medium to remove the antibiotic contained in the medium.
  • the washed cell pellet was suspended in 50 ⁇ L of NR medium to prepare a cell suspension in which the two types of cells were mixed. 100 ⁇ L of the cell suspension was dropped onto the NR agar medium and incubated at 30° C. for 24 hours to perform conjugation transfer.
  • the cultured cells were suspended in physiological saline, inoculated onto Son citric acid agar medium (2 g/L trisodium citrate dihydrate, 5.0 g/L NaCl, 1.0 g/L KH 2 PO 4 , 1.0 g/L NH 4 H 2 PO 4 , 0.2 g/L MgSO 4 .7H 2 O) containing 200 ⁇ g/mL kanamycin and 10 ⁇ g/mL gentamicin, and cultured for 2 days at 30° C.
  • the formed colonies were inoculated onto NR medium (2 mL) containing 200 ⁇ g/mL kanamycin, and cultured overnight at 30° C.
  • the culture solution was suspended in saline to prepare a diluted solution, which was then inoculated into MSY medium (MS medium (see Miyahara Y, Wang Chih-Ting, Ishii-Hyakutake M, Tsuge T, Bioengineering 2022, 9(10), 586. DOI: https://doi.org/10.3390/bioengineering9100586) containing 150 g/L of sucrose) and cultured at 30 ° C. for 2 days. The formed colonies were collected and colony PCR was performed using primers 7 and 10 to confirm the construction of the Ralstonia eutropha H16 ⁇ phaC1::phaC Ps strain.
  • R. eutropha H16 refers to the Ralstonia eutropha H16 strain (ATCC17699)
  • PHB - 4/pBBR1phaC Re refers to a strain obtained by introducing the plasmid pBBR1::phaCAB Re into the Ralstonia eutropha PHB - 4 strain (a mutant strain lacking PHB polymerization ability) (Miyahara Y., et al., Bioengineering 2022, 9, 586. https://doi.org/10.3390/bioengineering9100586).
  • a mass flow controller was used to supply a mixed gas of the desired gas composition.
  • the gas composition ratio was set to a hydrogen concentration of 75 vol% or more and an oxygen concentration of 7 vol% or less to avoid explosions due to hydrogen.
  • Table 10 shows that by using PhaC derived from Plesiomonas shigelloides, PHA having an ultra-high molecular weight of 50 x 105 can be produced from carbon dioxide as the sole carbon source.
  • Example 7 Production of PHA using Ralstonia eutropha H16 ⁇ phaC1::phaC Ps
  • the Ralstonia eutropha H16 ⁇ phaC1::phaC Ps strain prepared in Example 6(1) was inoculated into NR liquid medium and cultured overnight at 30°C to prepare a preculture solution. 1% of the preculture solution was inoculated into 100 mL of MS medium containing 0.5 g/L ammonium chloride, 10 g/L fructose and 1.0 g/L 3-hydroxypivalic acid as nitrogen sources and incubated at 30°C for 72 hours. Finally, the cells were collected by centrifugation and lyophilized for further analysis. The results are shown in Table 11. For comparison, the results of PHA biosynthesis using Ralstonia eutropha H16 strain (ATCC17699) are also shown for "R. eutropha H16".
  • Table 11 shows that by inserting the PhaC gene derived from Plesiomonas shigelloides into the genome of Ralstonia eutropha H16, the strain is capable of copolymerizing 3-hydroxypivalic acid and has high PHA productivity.
  • the PHA synthase of the present invention can be applied to the industrial production of various relatively high molecular weight PHAs, and the relatively high molecular weight PHAs produced can be effectively used as plastic materials such as for films.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
PCT/JP2023/037027 2022-11-08 2023-10-12 ポリヒドロキシアルカン酸の製造方法 Ceased WO2024101063A1 (ja)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2024557257A JPWO2024101063A1 (https=) 2022-11-08 2023-10-12

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2022179023 2022-11-08
JP2022-179023 2022-11-08

Publications (1)

Publication Number Publication Date
WO2024101063A1 true WO2024101063A1 (ja) 2024-05-16

Family

ID=91032817

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2023/037027 Ceased WO2024101063A1 (ja) 2022-11-08 2023-10-12 ポリヒドロキシアルカン酸の製造方法

Country Status (2)

Country Link
JP (1) JPWO2024101063A1 (https=)
WO (1) WO2024101063A1 (https=)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007125004A (ja) * 2005-10-03 2007-05-24 Tokyo Institute Of Technology ポリヒドロキシアルカン酸の製造法
WO2009147918A1 (ja) * 2008-06-05 2009-12-10 国立大学法人東京工業大学 ポリヒドロキシアルカン酸共重合体及びその製造法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007125004A (ja) * 2005-10-03 2007-05-24 Tokyo Institute Of Technology ポリヒドロキシアルカン酸の製造法
WO2009147918A1 (ja) * 2008-06-05 2009-12-10 国立大学法人東京工業大学 ポリヒドロキシアルカン酸共重合体及びその製造法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 3 June 2024 (2024-06-03), ANONYMOUS: "class I poly(R)-hydroxyalkanoic acid synthase [Ferrimonas marina]", XP093170298, Database accession no. WP_067661665 *
DATABASE PROTEIN 3 June 2024 (2024-06-03), ANONYMOUS: "class I poly(R)-hydroxyalkanoic acid synthase [Plesiomonas shigelloides]", XP093170300, Database accession no. WP_116546999 *
DATABASE PROTEIN 3 June 2024 (2024-06-03), ANONYMOUS: "class I poly(R)-hydroxyalkanoic acid synthase [Vibrio metschnikovii]", XP093170297, Database accession no. WP_154168902 *
NUMATA KEIJI, MORISAKI KUMIKO; TOMIZAWA SATOSHI; OHTANI MISATO; DEMURA TAKU; MIYAZAKI MASAYUKI; NOGI YUICHI; DEGUCHI SHIGERU; DOI : "Synthesis of poly- and oligo(hydroxyalkanoate)s by deep-sea bacteria, Colwellia spp., Moritella spp., and Shewanella spp", POLYMER JOURNAL, NATURE PUBLISHING GROUP UK, LONDON, vol. 45, no. 10, 1 October 2013 (2013-10-01), London , pages 1094 - 1100, XP093170293, ISSN: 0032-3896, DOI: 10.1038/pj.2013.25 *
SIVASHANKARI RAMAMOORTHI M, MIERZATI MAIERWUFU; MIYAHARA YUKI; MIZUNO SHOJI; NOMURA CHRISTOPHER T.; TAGUCHI SEIICHI; ABE HIDEKI; T: "Exploring Class I polyhydroxyalkanoate synthases with broad substrate specificity for polymerization of structurally diverse monomer units", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, FRONTIERS RESEARCH FOUNDATION, CH, vol. 11, CH , XP093170303, ISSN: 2296-4185, DOI: 10.3389/fbioe.2023.1114946 *

Also Published As

Publication number Publication date
JPWO2024101063A1 (https=) 2024-05-16

Similar Documents

Publication Publication Date Title
Meng et al. Production and characterization of poly (3-hydroxypropionate-co-4-hydroxybutyrate) with fully controllable structures by recombinant Escherichia coli containing an engineered pathway
CN113583922B (zh) 一种以低盐培养基培养嗜盐菌生产pha的方法
US9090898B2 (en) Green process and compositions for producing poly(5HV) and 5 carbon chemicals
Tan et al. Evaluation of BP-M-CPF4 polyhydroxyalkanoate (PHA) synthase on the production of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil using Cupriavidus necator transformants
US7384766B2 (en) Gene-substituted microorganisms, and production method of polyesters using the same
WO2018021046A1 (ja) ３ｈｈ単位含有共重合ｐｈａを生産する形質転換体、及び当該ｐｈａの製造方法
Qiu et al. Production of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) from gluconate and glucose by recombinant Aeromonas hydrophila and Pseudomonas putida
WO2009145164A1 (ja) 改良されたポリヒドロキシアルカノエート生産微生物及びそれを用いたポリヒドロキシアルカノエートの製造方法
Al-Kaddo et al. Production of P (3HB-co-4HB) copolymer with high 4HB molar fraction by Burkholderia contaminans Kad1 PHA synthase
JP6313708B2 (ja) 高分子量ｐｈａ生産微生物とそれを用いた高分子量ｐｈａの製造方法
US20140378646A1 (en) UTILIZATION OF THE NOVEL, ENVIRONMENTAL ISOLATES PSEUDOMONAS sp. IPB-B26 AND N-128 FOR THE EFFICIENT HIGH YIELD PRODUCTION OF mcl/lcl-PHAs
JP6854909B2 (ja) ２−ヒドロキシイソカプロエート−ＣｏＡ転移酵素を用いたポリヒドロキシアルカノエートの製造方法
JP6853787B2 (ja) スクロース資化性を有するｐｈａ生産微生物、及び該微生物を用いたｐｈａの製造方法
CN120699861A (zh) 一种合成高比例4-羟基丁酸聚羟基脂肪酸酯的菌株及其构建方法
WO2024101063A1 (ja) ポリヒドロキシアルカン酸の製造方法
JP4960033B2 (ja) 酵素活性を低下させた微生物を用いる共重合ポリエステルの製造方法
CN102171356A (zh) 用微生物聚合羟基乙酸的方法
JP6078839B2 (ja) 変異型ポリヒドロキシアルカン酸結合タンパク質およびそれを用いたポリヒドロキシアルカン酸の製造方法
EP2620494A1 (en) Synthesis of polyhydroxyalkanoates (pha) with thioester groups in the side chain
JP7764034B2 (ja) ブロック共重合体、及びその製造方法
JP2008086238A (ja) ポリヒドロキシアルカノエートの製造方法
US20110118434A1 (en) Method for polymerising glycolic acid with microorganisms
US12281349B2 (en) Recombinant vector comprising gene for synthesizing high molecular weight copolymer, transformant and method for producing polymers using transformant, recombinant strains having gene for synthesizing high molecular weight copolymers in genome and method for producing polymers using recombinant strain
JP2025097098A (ja) ポリヒドロキシアルカン酸、組換え水素酸化細菌、及びポリヒドロキシアルカン酸の製造方法
WO2025173696A1 (ja) ポリヒドロキシアルカン酸の製造方法、及び形質転換微生物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23888416

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2024557257

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23888416

Country of ref document: EP

Kind code of ref document: A1