WO2018159965A1 - Procédé de production de polyhydroxyalcanoate à l'aide de 2-hydroxyisocaproate-coa transférase - Google Patents

Procédé de production de polyhydroxyalcanoate à l'aide de 2-hydroxyisocaproate-coa transférase Download PDF

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WO2018159965A1
WO2018159965A1 PCT/KR2018/002305 KR2018002305W WO2018159965A1 WO 2018159965 A1 WO2018159965 A1 WO 2018159965A1 KR 2018002305 W KR2018002305 W KR 2018002305W WO 2018159965 A1 WO2018159965 A1 WO 2018159965A1
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gene encoding
monomer
amino acid
polyhydroxyalkanoate
acid sequence
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PCT/KR2018/002305
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Korean (ko)
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이상엽
양정은
박시재
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한국과학기술원
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Priority to BR112019017457A priority Critical patent/BR112019017457A2/pt
Priority to EP18760488.9A priority patent/EP3594350A4/fr
Priority to JP2019545788A priority patent/JP6854909B2/ja
Priority to CN201880014308.8A priority patent/CN110382699B/zh
Priority to US16/483,426 priority patent/US10961521B2/en
Publication of WO2018159965A1 publication Critical patent/WO2018159965A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
    • C08G63/688—Polyesters containing atoms other than carbon, hydrogen and oxygen containing sulfur
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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

Definitions

  • the present invention relates to a method for producing a polyhydroxyalkanoate containing an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) as a monomer, more particularly 2-hydroxyiso.
  • a gene encoding a caproate-CoA transferase and a gene encoding a polyhydroxyalkanoate synthetase have been introduced, and have a polyhydroxyalkanoate generating ability containing an aromatic monomer or a long chain 2-HA as a monomer.
  • PHAs Polyhydroxyalkanoates
  • These polymers are thermoplastics with biodegradable and biocompatible properties, and can have similar industrial properties as petroleum-based polymers, enabling a variety of industrial biomedical applications and resulting from renewable resources (Lee, SY Biotechnol. Bioeng . 49: 1 1996).
  • PHAs are classified into short-chain-length PHAs (SCL-PHAs) having a short carbon number and medium-chain-length PHAs (MCL-PHAs) having a long carbon number according to side chain lengths.
  • SCL-PHAs short-chain-length PHAs
  • MCL-PHAs medium-chain-length PHAs
  • Suitable examples of copolymers of certain two monomers include poly PHB-co-3-hydroxyhexanoate (Brandl et al., Int. J. Biol. Macromol . 11:49, 1989; A mos & McInerey, Arch Microbiol. , 155: 103, 1991; US 5,292,860).
  • Biosynthesis of PHA undergoes a process in which hydroxy acids are converted to hydroxyacyl-CoA by CoA-transferase or CoA-ligase, and the converted hydroxyacyl-CoA is polymerized by PHA synthase.
  • the activity for 2-hydroxyacyl-CoA is much lower than for 3-hydroxyacyl-CoA.
  • the inventors of Pseudomonas sp. 6-19 PHA synthase was genetically engineered to use lactyl-CoA, a type of 2-hydroxyacyl-CoA, as a substrate (WO08 / 062996; Yang et al., Biotechnol.
  • PhaC1ps6-19 has a wide variety of substrate specificities and converts various types of 2-hydroxy acids to 2-hydroxyacyl-CoA because it can use lactyl-CoA, a type of 2-hydroxyacyl-CoA, as a substrate. Developing a feasible system would allow the synthesis of new PHAs containing different types of 2-hydroxy acids.
  • the present inventors have made intensive efforts to develop a method for biosynthesis of PHA containing a new 2-hydroxy acid.
  • the enzyme converts 2-hydroxy acid using acetyl-CoA into 2-hydroxyacyl-CoA.
  • screening and using the enzyme it was confirmed that it is possible to produce a variety of 2-hydroxyacyl-CoA under in vitro conditions, it was confirmed that it is possible to produce a variety of PHA by using this, to complete the present invention .
  • An object of the present invention is to provide a recombinant microorganism having a polyhydroxyalkanoate producing ability containing an aromatic monomer or a long chain 2-HA as a monomer.
  • Another object of the present invention to provide a method for producing a polyhydroxyalkanoate containing an aromatic monomer or a long chain 2-HA as a monomer using the recombinant microorganism.
  • the present invention provides a gene encoding 2-hydroxyisocaproate-CoA transferase and a gene encoding polyhydroxyalkanoate synthase in a microorganism having acetyl-CoA generating ability from a carbon source.
  • a recombinant microorganism having a polyhydroxyalkanoate-producing ability which is introduced and contains an aromatic monomer or a long chain 2-HA as a monomer.
  • the present invention also comprises the steps of (a) culturing the recombinant microorganism to produce a polyhydroxyalkanoate containing an aromatic monomer or a long chain 2-HA as a monomer; And (b) obtaining a polyhydroxyalkanoate containing the produced aromatic monomer or long chain 2-HA as a monomer.
  • the present invention also relates to a gene encoding 2-hydroxyisocaproate-CoA transferase, a gene encoding polyhydroxyalkanoate synthase, DAHP (3-de) in a microorganism having acetyl-CoA generating ability from a carbon source.
  • Oxy-D-arabino-heptulsonate-7-phosphate) synthetase gene encoding corismate mutase / prephenate dehydrogenase and D-lactate dehydrogenase
  • a recombinant microorganism having a polyhydroxyalkanoate-producing ability containing a phenyl lactate as a monomer and amplified gene is provided.
  • the present invention also comprises the steps of (a) culturing the recombinant microorganism, to produce a polyhydroxyalkanoate containing phenyl lactate as a monomer; And (b) obtaining a polyhydroxyalkanoate containing the produced phenyllactate as a monomer. It provides a method for producing polyhydroxyalkanoate containing a phenyllactate as a monomer.
  • the present invention also relates to a gene encoding 2-hydroxyisocaproate-CoA transferase, a gene encoding polyhydroxyalkanoate synthase, DAHP (3-de) in a microorganism having acetyl-CoA generating ability from a carbon source.
  • Oxy-D-arabino-heptulsonate-7-phosphate) synthetase gene encoding corismate mutase / prephenate dehydrogenase and D-lactate dehydrogenase
  • a recombinant microorganism having hydroxyalkanoate producing ability.
  • the present invention also comprises the steps of (a) culturing the recombinant microorganism to produce a polyhydroxyalkanoate containing mandelate as a monomer; And (b) obtaining a polyhydroxyalkanoate containing the produced mandelate as a monomer, thereby providing a method for preparing polyhydroxyalkanoate containing a mandelate as a monomer.
  • Figure 1 shows the biosynthetic metabolic pathway of the aromatic polyester according to the present invention
  • A is the metabolic pathway when using FldA (cinnamoyl-CoA: phenyllactate CoA-transferase)
  • B is HadA (2- Metabolic pathways using hydroxyisocaproate-CoA transferase).
  • Figure 2 shows the result of comparing the amino acid sequence homology of HadA and FldA.
  • Figure 4 shows that HadA uses acetyl-CoA as CoA donor, mandelate, 4-hydroxymandelate, phenyllactate, 4-hydroxyphenyllactate, 2-hydroxy-4-phenylbutyrate, 3-hydroxy
  • acetyl-CoA as CoA donor
  • mandelate 4-hydroxymandelate
  • phenyllactate 4-hydroxyphenyllactate
  • 2-hydroxy-4-phenylbutyrate 3-hydroxy
  • the conversion of oxy-3-phenylpropionate and 4-hydroxy benzoic acid to the corresponding CoA derivatives was confirmed by LC-MS analysis after in vitro enzyme assay.
  • Figure 6 shows the results of increasing the production of D-phenyl lactate by performing metabolic analysis according to in silico genome scale metabolic flux analysis.
  • Figure 7 shows the results of the analysis of poly (3HB-co-D-phenyl lactate) produced by E. coli XB201TBAL.
  • 10A and 10B show the production of poly (3HB-co-D-phenyllactate) by fed-batch fermentation of E. coli XB201TBAL strain expressing AroGfbr, PheAfbr, FldH, HadA and PhaC1437 in MR medium containing 3HB.
  • C and d are fed by a fed-batch fermentation of the E. coli XB201TBAL strain expressing PhaAB under the AroGfbr, PheAfbr, FldH, HadA, PhaC1437 and BBa_J23114 promoters without addition of 3HB to obtain poly (3HB-co-D-phenyllactate).
  • Aromatic polyesters are essential plastics produced mainly from petroleum.
  • an aromatic polyester or a long chain 2-hydroxyalkanoate is prepared from glucose by metabolic engineering E. coli expressing polyhydroxyalkanoate (PHA) synthase and coenzyme A (CoA) transferase.
  • PHA polyhydroxyalkanoate
  • CoA coenzyme A
  • cinnamoyl-CoA phenyllactate CoA-transferase (FldA) and 4-Cuma have been identified by in vitro assays to produce PHAs containing phenyllactate as aromatic polyesters.
  • an E. coli engineered to have an optimal metabolic pathway for D-phenyllactate production in order to prepare an E. coli engineered to have an optimal metabolic pathway for D-phenyllactate production, over-expression of feedback resistance aroG, pheA and fldH genes in tyrR deficient E. coli, and competitive metabolism
  • the pathways (pflB, poxB, adhE and frdB) were deleted and the tyrB and aspC genes were further deleted following in silico genome scale metabolic flux analysis.
  • the metabolically engineered Escherichia coli produced 1.62 g / L of D-phenyllactate.
  • the present invention in a microorganism having the ability to produce acetyl-CoA from a carbon source, a gene encoding 2-hydroxyisocaproate-CoA transferase and a gene encoding a polyhydroxyalkanoate synthase is introduced
  • the present invention relates to a recombinant microorganism having a polyhydroxyalkanoate generating ability containing an aromatic monomer or a long chain 2-HA as a monomer.
  • the long chain 2-HA means 2-hydroxyalkanoate having 6 to 8 carbon atoms.
  • the aromatic monomer or long-chain 2-HA monomer is 2-hydroxyisocaproate, 2-hydroxyhexanoate, 2-hydroxyoctanoate, phenyl lactate, 2-hydroxy-4 It may be characterized in that it is selected from the group consisting of -phenylbutyrate, 3-hydroxy-3-phenylpropionate, 4-hydroxybenzoic acid and mandelate.
  • the polyhydroxyalkanoate synthase is Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp. It may be characterized as a PHA synthase derived from a strain selected from the group consisting of 6-19 or a variant enzyme of PHA synthase having an amino acid sequence selected from:
  • amino acid sequence comprising one or more variants selected from the group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: 2;
  • Amino acid sequence (C1335) wherein E130D, S325T, L412M, S477G and Q481M are mutated in amino acid sequence of SEQ ID NO: 2;
  • the 2-hydroxyisocaproate-CoA transferase may be characterized as hadA derived from Clostridium difficile 630.
  • the 2-hydroxyisocaproate-CoA transferase may be characterized by using acetyl-CoA as CoA donor.
  • the microorganism of the present invention is a gene encoding ⁇ -ketothiolase involved in 3-hydroxybutyryl-CoA biosynthesis and a gene encoding acetoacetyl-CoA reductase in order to produce a polymer without externally supplying 3HB. It may be characterized by being introduced further.
  • the present invention comprises the steps of (a) culturing the recombinant microorganism to produce a polyhydroxyalkanoate containing an aromatic monomer or long chain 2-HA as a monomer; And (b) obtaining a polyhydroxyalkanoate containing the produced aromatic monomer or long chain 2-HA as a monomer. It relates to a method for producing a canoate.
  • Pct Propionyl-CoA transferase
  • Pctcp Propionyl-CoA transferase
  • polyesters comprising 2-hydroxy acids such as glycolic acid, lactic acid, 2-hydroxybutyl acid, 2-hydroxyisovalorate and various hydroxy acids. Since it is used, Pct540 can be said to have a broad substrate spectrum with respect to carbon number and hydroxyl position.
  • Pct540 was found to have no catalytic activity for phenyllactate and mandelate, and therefore, in the present invention, for the production of aromatic copolymers, a novel CoA-transition capable of activating CoA derivatives corresponding to aromatic compounds We wanted to find an enzyme.
  • Cinnamoyl-CoA Phenyllactate CoA-transferase (FldA) of Clostridium sporogenes has been reported to convert phenyllactate to phenyllactyl-CoA using cinnamoyl-CoA as a CoA donor (Dickert, S. et al., Eur. J. Biochem . 267: 3874, 2000). Cinnamoyl-CoA is a non-natural metabolite of Escherichia coli. Therefore, to confirm the use of acetyl-CoA, a rich metabolite in cells, as a CoA donor, Clostridium botulinum A str. FldA from ATCC 3502 was tested. However, C. botulinum A str. It was confirmed that FldA of ATCC 3502 does not have the catalytic activity of generating phenyllactyl-CoA using acetyl-CoA as CoA donor.
  • CoA ligase (4CL) plays an important role in phenylpropanoid metabolism, which produces precursors of plant secondary metabolites such as lignin, flavonoids and phytoalexin (Kaneko, M. et al, J. Bacteriol. 185: 20, 2003). Therefore, in one aspect of the present invention, a biosynthetic pathway for synthesizing cinnamoyl-CoA from cinnamate was designed by introducing 4CL.
  • the 4CL variant was used to convert cinnamates to cinnamoyl-CoA and cinnamoyl-CoA was used as CoA donor of FldA for phenyllactyl-CoA formation.
  • phenyllactyl-CoA was successfully synthesized by sequential reaction of 4CL and FldA in vitro .
  • 4CL and FldA could be used for the production of phenyllactyl-CoA and that it could be used for producing aromatic polyesters.
  • 4-hydroxyphenyllactate another promising aromatic monomer, was also converted to 4-hydroxyphenyllactyl-CoA by in vitro sequential reaction of 4CL variant with FldA.
  • Pseudomonas sp. MBEL 6-19 PHA synthase (PhaCPs6-19) variants were expressed in Escherichia coli XL1-Blue overexpressing AroGfbr, PAL, 4CL, FldA and Pct540.
  • the prepared recombinant strain was cultured in MR medium to which 20 g / L glucose, 1 g / L D-phenyllactate and 1 g / L sodium 3-hydroxybutyrate (3HB) were added.
  • Sodium 3-hydroxybutyrate (3HB) was converted by Pct540 to 3HB-CoA, which is the preferred substrate of PhaC, and was added to enhance the production of the polymer because it allows the production of sufficient amounts of PHA.
  • E. coli XL1-Blue which expresses different PHA synthase variants, can produce varying amounts of poly (D-lactate-co-3HB-co-D-phenyllactate) with different monomer compositions.
  • PhaC1437 containing four amino acid substitutions (E130D, S325T, S477G and Q481K) in the PhaC variant was poly (18.3 mol% D-lactate-co-76.9 mol% 3HB-co-4.8 mol% D-phenyl Lactate) was produced at 7.8% by weight of the dry cell weight to identify the most suitable PhaC variant.
  • E. coli was manipulated to generate D- phenyllactate from glucose in vivo .
  • Biosynthesis of aromatic compounds begins with the synthesis of 3-deoxy-D-arabino-heptulsonate-7-phosphate (DAHP), which is combined with phosphoenolpyruvate (PEP) by DAHP synthase. It is produced by the condensation of ellithrose-4-phosphate (E4P). The resulting DAHP is converted to phenylpyruvate (PPA) followed by D-lactate dihydrogenase (FldH) to D-phenyllactate (FIG. 1). Metabolic pathways for aromatic compound biosynthesis are known to be complexly controlled by various inhibitory mechanisms.
  • AroGfbr AroG (D146N)] and PheAfbr [PheA (T326P)] were constructed to release feedback inhibition by L-phenylalanine (Zhou, HY et al., Bioresour. Technol. 101: 4151, 2010; Kikuchi, Y. et al., Appl. Environ.Microbiol. 63: 761, 1997).
  • Escherichia coli XL1-Blue expressing FldH of ATCC 3502 produced 0.372 g / L of D-phenyllactate from 15.2 g / L of glucose.
  • Overexpression of PAL, 4CL, FldA, Pct540 and PhaC1437 of the strain was poly (16.8 mol% D-lactate-co-80.8 mol% 3HB-co-1.6 mol% D-phenyllactate-co-0.8 mol% D -4-hydroxyphenyllactate) was increased to 12.8% by weight of the dry cell weight.
  • an enzyme having a broad spectrum of aromatic substrate was found using acetyl-CoA as CoA donor.
  • An array similarity analysis was performed to identify homologous enzymes for FldA and 2-isocaprenoyl-CoA: 2-hydroxyisocapronate of Clostridium difficile having at least 48% amino acid sequence identity with FldA among various FldAs of different origins.
  • CoA-transferase (HadA) was screened (FIG. 2).
  • HadA is an enzyme known to convert CoA into 2-hydroxyisocaproate using isocaprenoyl-CoA as a CoA donor
  • the present invention investigated whether HadA can use acetyl-CoA as a CoA donor ( 3).
  • In vitro enzyme analysis showed that HadA was able to activate phenyllactate as phenyllactyl-CoA using acetyl-CoA as a CoA donor (FIG. 4).
  • HadA also contains mandelate, 4-hydroxymandelate, phenyllactate, 4-hydroxyphenyllactate, 2-hydroxy-4-phenylbutyrate, 3-hydroxy-3-phenylpropionate and 4 It was confirmed through LC-MS analysis after in vitro assay that hydroxy benzoic acid can be converted into the corresponding CoA derivative (FIGS. 4 and 5). Therefore, HadA has the potential to produce various aromatic polyesters more efficiently by using acetyl-CoA as a CoA donor.
  • E. coli XL1-Blue strain without TyrR deletion To eliminate a path conflict with D-phenyllactate biosynthesis, in E. coli XBT, poxB (a gene encoding pyruvate oxidase), a pyruvate oxidase), pflB (a gene encoding pyruvate formate lyase), Escherichia coli XB201T was constructed by deleting adhE (gene encoding acetaldehyde dehydrogenase / alcohol dehydrogenase) and frdB (gene encoding fumarate reductase).
  • adhE gene encoding acetaldehyde dehydrogenase / alcohol dehydrogenase
  • frdB Gene encoding fumarate reductase
  • coli XB201T strains expressing AroGfbr, PheAfbr and FldH produced 0.55 g / L of D-phenyllactate from 15.7 g / L of glucose, indicating a 10% higher yield than E. coli XBT.
  • Escherichia coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA was incubated in a medium containing 20 g / L glucose and 1 g / L sodium 3HB to obtain poly (52.1 mol% 3HB-co-47.9 mol% D-phenyllactate).
  • Poly 52.1 mol% 3HB-co-47.9 mol% D-phenyllactate
  • the present invention provides a gene encoding 2-hydroxyisocaproate-CoA transferase, a gene encoding polyhydroxyalkanoate synthase, in a microorganism having acetyl-CoA generating ability from a carbon source, Gene encoding DAHP (3-deoxy-D-arabino-heptulsonate-7-phosphate) synthase, gene encoding corismate mutase / prephenate dehydrogenase and D-lactate di
  • the present invention relates to a recombinant microorganism having a polyhydroxyalkanoate-producing ability, in which a gene encoding a hydrogenase has been introduced and containing phenyllactate as a monomer.
  • the 2-hydroxyisocaproate-CoA transferase may be characterized as hadA derived from Clostridium difficile 630, the hydroxyalkanoate synthase is Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp . It may be characterized as a PHA synthase derived from a strain selected from the group consisting of 6-19 or a variant enzyme of PHA synthase having an amino acid sequence selected from:
  • amino acid sequence comprising one or more variants selected from the group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: 2;
  • Amino acid sequence (C1335) wherein E130D, S325T, L412M, S477G and Q481M are mutated in amino acid sequence of SEQ ID NO: 2;
  • the gene encoding the DAHP (3-deoxy-D-arabino-heptulsonate-7-phosphate) synthase is a gene encoding the amino acid sequence represented by SEQ ID NO: 8
  • the gene encoding the corismate mutase / prephenate dehydrogenase may be a gene encoding an amino acid sequence represented by SEQ ID NO: 9, wherein the D-lactate dehydrogeze
  • the gene encoding the claw may be a gene encoding the amino acid sequence represented by SEQ ID NO: 10.
  • the gene encoding the introduced D-lactate dehydrogenase may be characterized as being a fldH gene that replaces the ldhA gene.
  • the microorganism of the present invention is a gene encoding ⁇ -ketothiolase involved in 3-hydroxybutyryl-CoA biosynthesis and a gene encoding acetoacetyl-CoA reductase in order to produce a polymer without externally supplying 3HB. It may be characterized by being introduced further.
  • the recombinant microorganism is a tyrR gene, a gene encoding pyruvate oxidase, a gene encoding pyruvate formate lyase, a gene encoding acetaldehyde dehydrogenase, a gene encoding fumarate reductase, tyrosine amino
  • a gene encoding the transferase and a gene encoding the aspartic acid aminotransferase may be deleted.
  • the present invention relates to a method for preparing polyhydroxyalkanoate containing a phenyllactate as a monomer.
  • mandelate was tested using monomers. This is because polymandelate, the single polymer of mandelate, is a pyrolysis resistant polymer having a relatively high Tg of 100 ° C., while the material properties are similar to polystyrene. Polymandelate is chemically synthesized by ring-opening polymerization of cyclic dimer of mandelate produced in the petroleum industry.
  • Escherichia coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA was cultured in a medium containing 1 g / L sodium 3HB and 0.5 g / L D-mandelate to obtain poly (55.2 mol% 3HB-co-43 mol% D-phenyl Lactate-co-1.8 mol% D-mandelate) was prepared at a 11.6 wt% content of dry cell weight (FIGS. 8 a, b).
  • an aromatic copolymer including D-mandelate was successfully prepared using D-mandelate as a substrate, and then, to produce D-mandelate in vivo by metabolic engineering.
  • HmaS Amycolatopsis orientalis-derived hydroxymandelate synthetase
  • S. coelicolor Hydrolysis of Amycolatopsis orientalis-derived hydroxymandelate synthetase (HmaS), S. coelicolor, in E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA to produce an aromatic copolymer comprising D-mandelate from glucose Roxymandelate oxidase (Hmo) and D-mandelate dehydrogenase (Dmd) from Rhodotorula graminis were expressed.
  • Hmo glucose Roxymandelate oxidase
  • Dmd D-mandelate dehydrogenase
  • the present invention provides a gene encoding 2-hydroxyisocaproate-CoA transferase, a gene encoding polyhydroxyalkanoate synthase, in a microorganism having acetyl-CoA generating ability from a carbon source, Gene encoding DAHP (3-deoxy-D-arabino-heptulsonate-7-phosphate) synthase, gene encoding corismate mutase / prephenate dehydrogenase and D-lactate di Genes encoding hydrogenase, genes encoding hydroxymandelate synthase, genes encoding hydroxymandelate oxidase and genes encoding D-mandelate dehydrogenase have been introduced.
  • the present invention relates to a recombinant microorganism having a polyhydroxyalkanoate producing ability contained as a monomer.
  • the 2-hydroxyisocaproate-CoA transferase may be characterized as hadA derived from Clostridium difficile 630, the hydroxyalkanoate synthase is Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp . It may be characterized as a PHA synthase derived from a strain selected from the group consisting of 6-19 or a variant enzyme of PHA synthase having an amino acid sequence selected from:
  • amino acid sequence comprising one or more variants selected from the group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: 2;
  • Amino acid sequence (C1335) wherein E130D, S325T, L412M, S477G and Q481M are mutated in amino acid sequence of SEQ ID NO: 2;
  • the gene encoding the DAHP (3-deoxy-D-arabino-heptulsonate-7-phosphate) synthase is a gene encoding the amino acid sequence represented by SEQ ID NO: 8
  • the gene encoding the corismate mutase / prephenate dehydrogenase may be a gene encoding an amino acid sequence represented by SEQ ID NO: 9, wherein the D-lactate dehydrogeze
  • the gene encoding the claw may be a gene encoding the amino acid sequence represented by SEQ ID NO: 10.
  • the gene encoding the hydroxymandelate synthetase may be a gene encoding the amino acid sequence represented by SEQ ID NO: 11
  • the gene encoding the hydroxymandelate oxidase May be a gene encoding an amino acid sequence represented by SEQ ID NO: 12
  • the gene encoding the D-mandelate dehydrogenase may be a gene encoding an amino acid sequence represented by SEQ ID NO: 13 Can be.
  • the microorganism of the present invention is a gene encoding ⁇ -ketothiolase involved in 3-hydroxybutyryl-CoA biosynthesis and a gene encoding acetoacetyl-CoA reductase in order to produce a polymer without externally supplying 3HB. It may be characterized by being introduced further.
  • the present invention provides a method for producing a polyhydroxyalkanoate containing mandelate as a monomer by culturing the recombinant microorganism; And (b) obtaining a polyhydroxyalkanoate containing the produced mandelate as a monomer.
  • 2-hydroxyisocapro which is a long-chain 2-HA monomer 8 (2HIC), 2-hydroxyhexanoate (2HH) and 2-hydroxyoctanoate (2HO) were used as monomers to confirm polymer production capacity.
  • 2-hydroxyisocaproate It was confirmed that a copolymer containing 2-hydroxyhexanoate or 2-hydroxyoctanoate was produced, and as the concentration of 2-HA contained in the medium increased, the mole fraction of the monomer contained in the copolymer increased. was confirmed (Table 4, Table 5 and Table 6).
  • the present invention provides a recombinant microorganism having the ability to produce polyhydroxyalkanoate containing the aromatic monomer or the long-chain 2-HA as a monomer, in a further aspect, 2-hydroxyisocaproate, 2-hydroxy.
  • a bacterial platform system has been developed for the production of various aromatic polyesters.
  • the aromatic polymer production system of the present invention identifies CoA-transferases having a new broad substrate range for activating aromatic compounds into their CoA derivatives, and in vivo, PHA synthase variants capable of polymerizing their aromatic CoA derivatives.
  • the pathway to overproduce aromatic monomers was established through the design and optimization of metabolism.
  • HadA or related enzymes
  • PHA synthetase can be engineered to accommodate the desired aromatic monomer.
  • the bacterial platform system developed in the present invention can contribute to establishing a bioprocess for the production of aromatic polyesters from renewable non-food biomass.
  • vector refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host.
  • the vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, “plasmid” and “vector” are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors.
  • Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.
  • the vector should be transformed into the appropriate host cell.
  • preferred host cells are prokaryotic cells.
  • Suitable prokaryotic host cells include E. coli DH5 ⁇ , E. col JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli B, E. coli B21, and the like. It includes. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the aforementioned E.
  • strains of the genus Agrobacterium such as Agrobacterium A4, bacilli, such as Bacillus subtilis, Salmonella typhimurium or Serratia marghesen Still other enterobacteria such as marcescens and various Pseudomonas strains can be used as host cells.
  • Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation (Neumann et al., EMBO J., 1: 841, 1982) can also be used for transformation of these cells.
  • an expression vector known in the art may be used, and it is preferable to use a pET family vector (Novagen).
  • a pET family vector Novagen
  • histidine groups are bound to the ends of the expressed protein, and thus the protein can be effectively purified.
  • a general method known in the art may be used, and specifically, it may be separated by a chromatographic method using Ni-NTA His-binding resin (Novagen).
  • the recombinant vector may be characterized in that the pET-SLTI66, the host cell may be characterized in that E. coli or Agrobacterium.
  • expression “expression control sequence” in the present invention means a DNA sequence essential for the expression of a coding sequence operably linked in a particular host organism.
  • regulatory sequences include promoters for performing transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation.
  • suitable control sequences for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites.
  • Eukaryotic cells include promoters, polyadenylation signals, and enhancers. The factor that most influences the amount of gene expression in the plasmid is the promoter.
  • an SR ⁇ promoter As the promoter for high expression, an SR ⁇ promoter, a promoter derived from cytomegalovirus, and the like are preferably used.
  • any of a wide variety of expression control sequences can be used in the vector.
  • Useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter regions of phage lambda, fd code protein Regulatory region of, promoter for 3-phosphoglycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, promoter of yeast alpha-crossing system and gene expression of prokaryotic or eukaryotic cells or viruses Other sequences known to modulate and various combinations thereof.
  • the T7 promoter can be usefully used to express the proteins of the invention in E. coli.
  • Nucleic acids are "operably linked” when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to allow gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s).
  • the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence;
  • the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence;
  • the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation.
  • "operably linked” means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame.
  • enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.
  • heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once the expression vector is in the host cell, it can replicate independently of the host chromosomal DNA and several copies of the vector and the inserted (heterologous) DNA can be produced.
  • the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.
  • the expression control sequence and the gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the host cell is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.
  • Host cells transformed or transfected with the expression vectors described above constitute another aspect of the present invention.
  • transformation means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration.
  • transfection means that the expression vector is accepted by the host cell whether or not any coding sequence is actually expressed.
  • the relative strength of the sequence, the controllability, and the compatibility with the DNA sequences of the present invention should be considered, particularly with regard to possible secondary structures.
  • Single cell hosts may be selected from a host for the selected vector, the toxicity of the product encoded by the DNA sequence of the invention, the secretory properties, the ability to accurately fold the protein, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention from the host. It should be selected in consideration of factors such as the ease of purification.
  • one skilled in the art can select a variety of vector / expression control sequence / host combinations capable of expressing the DNA sequences of the invention in fermentation or large scale animal culture.
  • a binding method binding method
  • a panning method a panning method
  • a film emulsion method and the like can be applied.
  • E. coli was used as a recombinant microorganism, but any microorganism that produces acetyl-CoA from a carbon source can be used without limitation, and includes genus Alcaligenes, Pseudomonas, Escherichia, Ralstonia genus, Bacillus genus, Corynebacterium and the like can be used.
  • Recombinant strains, plasmids and primers used or produced in the present invention are shown in Tables 1-3.
  • Acetyl-CoA was used as CoA donor to find enzymes with broad spectrum of aromatic substrates.
  • An array similarity assay was performed to identify homologous enzymes for FldA and 2-isocaprenoyl-CoA: 2-hydroxyisocaproate of Clostridium difficile having at least 48% amino acid sequence identity with FldA among various FldAs of different origins.
  • CoA-transferase (HadA, SEQ ID NO: 1) was screened (FIG. 2).
  • a recombinant vector containing a gene encoding HadA the chromosomal DNA of Clostridium diffsile 630 strain was used as a template, and PCR was performed using HadA-hisF and HadA-hisR primers to obtain a C terminus.
  • the prepared his_HadA fragment was treated with restriction enzymes (NdeI and NotI) to the pET22b plasmid undergoing strong gene expression of the T7 promoter, followed by T4 DNA ligase, and his_HadA fragment digested with restriction enzyme and pET22b plasmid was conjugated to prepare a recombinant plasmid pET22b_hisHadA (FIG. 2).
  • restriction enzymes NdeI and NotI
  • the pET22b_hisHadA was introduced into Escherichia coli XL1-Blue (Stratagene Cloning Systems, USA), cultured, and IPTG was added to induce HadA expression, followed by Ni-NTA spin kit (Quiagen, Germany) using His-tag. HadA was purified from the culture medium at (Fig. 3a).
  • Figure 5 shows the molecular formula of the CoA conversion reaction of the various substrates that HadA can convert.
  • E. coli was engineered to produce D-phenyllactate from glucose in vivo.
  • Biosynthesis of aromatic compounds begins with the synthesis of 3-deoxy-D-arabino-heptulsonate-7-phosphate (DAHP), which is combined with phosphoenolpyruvate (PEP) by DAHP synthase. It is produced by the condensation of erythrose-4-phosphate (E4P). The resulting DAHP is converted to phenylpyruvate (PPA) followed by D-lactate dihydrogenase (FldH) to D-phenyllactate (FIG. 1). Metabolic pathways for aromatic compound biosynthesis are known to be complexly controlled by various inhibitory mechanisms.
  • the feedback inhibition resistance mutants AroGfbr [AroG (D146N)] and PheAfbr [PheA (T326P)] were constructed to release feedback inhibition by L-phenylalanine (Zhou, HY et al., Bioresour. Technol . 101: 4151, 2010; Kikuchi, Y. et al., Appl. Environ.Microbiol . 63: 761, 1997).
  • E. coli XL1-Blue expressing the FldH of ATCC 3502 was constructed.
  • 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase gene (aroG), a feedback inhibition resistance mutation, was constructed using primers AroG-F and AroG-R to construct pKM212-AroGfbr.
  • PCR product was prepared using the plasmid pTyr-a (Na, D. et al., Nature Biotechnol. 31: 170, 2013) as a template, and the PCR product was purified using pKM212 using restriction enzymes (EcoRI / HidIII).
  • PKM212-AroGfbr was constructed in conjunction with -MCS (Park, SJ et al., Metab. Eng . 20:20, 2013).
  • the pKM212-AroGfbrPheAfbr plasmid was constructed as follows. First, 991 bp DNA fragments from genomic DNA of Escherichia coli were amplified by PCR using primers PheA-F and PheAmut-R of a single mutated base (T976G). Second, a 200 bp DNA fragment was amplified from genomic DNA of Escherichia coli using a single mutated base (A976C) and PheAmut-F primer of PheA-R.
  • a DNA fragment of 1161bp was amplified by the primers PheA-F and PheA-R by overlap PCR.
  • the PCR product was linked to pKM212-AroGfbr prepared above using restriction enzyme (HindIII).
  • fldH D-lactate dehydrogenase
  • Codon usage frequency of the fldH gene was carried out using E. coli-optimized E. coli codon-optimized fldH gene using primers FldH-F and FldH-R and pUC57-FldHopt (GenScript, Piscataway, NJ, USA) as a template.
  • the PCR product was linked with pTrc99A (Pharmacia, Biotech, Sweden) using restriction enzymes (BamHI / HindIII) to prepare pTrc-FldH.
  • the fldH gene coupled to the trc promoter and rrnB terminator was amplified by PCR using primers Trc-F and Ter-R using pTrc-FldH as a template.
  • the amplified PCR product was linked with pACYC184KS (Korean Patent Publication No. 2015-0142304) using restriction enzymes (XhoI / SacI) to obtain pACYC-FldH.
  • PKM212-AroGfbrPheAfbr and pACYC-FldH prepared above were introduced into E. coli XL1-Blue to prepare recombinant E. coli expressing AroGfbr, PheAfbr and FldH.
  • the Escherichia coli produced 0.372 g / L of D-phenyllactate when incubated in MR medium containing 15.2 g / L of glucose.
  • the MR medium comprises 6.67 g KH 2 PO 4, 4 g (NH 4) 2 HPO 4, 0.8 g MgSO 4 .7H 2 O, 0.8 g citrate and 5 ml of trace metal solution, the trace metal solution being 0.5 M HCl: 10 g FeSO 4 ⁇ 7H 2 O, 2g CaCl 2, 2.2g ZnSO 4 .7H 2 O, 0.5g MnSO 4 .4H 2 O, 1g CuSO 4 .5H 2 O, 0.1g (NH 4) 6 Mo 7 O 24 .4H 2 O and 0.02 g Na 2 B 4 O 7 .10H 2 O.
  • E. coli XL1-Blue strains expressing the AroGfbr, PheAfbr and FldH producing small amounts of D-phenyllactate (0.372 g / L) from glucose were subjected to metabolic engineering. Yield was increased through.
  • E. coli XBT strains expressing AroGfbr, PheAfbr and FldH, which deleted TyrR, a dual transcription regulator that regulates aromatic amino acid biosynthesis were prepared.
  • the E. coli XBT strain was cultured in MR medium containing 16.4 g / L glucose and produced 0.5 g / L D-phenyllactate, which was 30% higher than the E. coli XL1-Blue strain without tyrR. Productivity was shown.
  • poxB a gene encoding pyruvate oxidase
  • pflB a gene encoding pyruvate formate lyase
  • adhE acetaldehyde dehydrogenase / alcohol dehydrogenase
  • E. coli XB201T strain produced 0.55 g / L of D-phenyllactate from 15.7 g / L of glucose, indicating a 10% higher yield than E. coli XBT.
  • metabolic analysis was performed according to in silico genome scale metabolic flux analysis to further increase D-phenyllactate production.
  • the tyrB gene encoding tyrosine aminotransferase and the aspC gene encoding aspartic acid aminotransferase were removed from the E. coli XB201T strain, and L-phenylalanine biosynthesis was reduced to D-phenyllactate. The carbon flow to the furnace was enhanced.
  • the E. coli XB201TBA strain produced as a result of the in silico flux response analysis produced 1.62 g / L of D-phenyllactate from 18.5 g / L of glucose, which greatly increased the yield of E. coli expressing AroGfbr, PheAfbr and FldH. It is 4.35 times higher than D-phenyllactate production of XL1-Blue strain.
  • chromosomal DNA of Clostridium difficile 630 strain was used as a template, and PCR was performed using HadA-sbF and HadA-ndR primers to prepare a recombinant vector.
  • a hadA gene segment encoding isocaproate-CoA transferase was constructed.
  • the amplified PCR product was linked with p619C1437-pct540 (Yang, TH et al. Biotechnol Bioeng 105: 150, 2010) using restriction enzymes (SbfI / NdeI) to obtain p619C1437-HadA.
  • P619C1437-HadA prepared above was introduced into E. coli XB201TBAL to prepare recombinant E. coli expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA.
  • the E. coli was cultured in MR medium containing 20 g / L glucose and 1 g / L sodium 3HB to convert poly (52.1 mol% 3HB-co-47.9 mol% D-phenyllactate) to a content of 15.8% by weight of the dry cell weight.
  • Produced (FIG. 7).
  • Fed-batch also produced poly (52.3 mol% 3HB-co-47.7 mol% D-phenyllactate) at a 24.3 wt% content of dry cell weight (FIGS. 10a, b).
  • Escherichia coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA was incubated in MR medium containing 1 g / L sodium 3HB and 0.5 g / L D-mandelate, resulting in poly (55.2 mol% 3HB-co-43.0 mol % D-phenyllactate-co-1.8 mol% D-mandelate) was prepared in an amount of 11.6% by weight of the dry cell weight (FIG. 8a, b), containing D-mandelate using D-mandelate as a substrate. Aromatic polymers have been successfully prepared.
  • D-mandelate was produced in vivo by metabolic engineering.
  • Amycolatopsis orientalis-derived hydroxymandelate synthetase (HmaS), S. coelicolor, hydroxymandelate oxidase in E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA to produce D-mandelate from glucose (Hmo) and D-mandelate dehydrogenase (Dmd) from Rhodotorula graminis.
  • hmaS hydroxymandelic acid synthase gene from A. orientalis was used and the codon was cloned into a synthetic vector in Escherichia coli to construct plasmid pUC57-HmaSopt (GenScript, Piscataway, NJ, USA). ).
  • the pUC57-HmaSopt was linked to pKM212-MCS using restriction enzymes (EcoRI / KpnI).
  • EcoRI / KpnI restriction enzymes
  • To construct pKM212-HmaSHmo S. coelicolor's hydroxymandelate oxidase gene (hmo) was synthesized by codon-optimized hmo gene (GenScript, Piscataway, NJ, USA), and primers Hmo-F and Hmo-R were used. Amplified by PCR. The PCR product was linked to pKM212-HmaS using restriction enzymes (KpnI / BamHI) to prepare pKM212-HmaSHmo.
  • pUC57-Dmd containing the E. coli codon optimized dmd gene was synthesized to generate pKM212-HmaSHmoDmd (GenScript, Piscataway, NJ, USA) and E. coli codon optimized by PCR with primers Dmd-F and Dmd-R R. graminis D-mandelate dehydrogenase gene (dmd) was amplified.
  • the PCR product was linked to pKM212-HmaSHmo using restriction enzymes (BamHI / SbfI) to prepare pKM212-HmaSHmoDmd.
  • PKM212-HmaSHmoDmd prepared above was introduced into E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA, to prepare a recombinant strain having mandelate production capacity.
  • Aromatic polymer production was confirmed using 3-hydroxy-3-phenylpropionate (3HPh) as another possible aromatic monomer.
  • Escherichia coli XB201TBAL strains were cultured in a medium containing 20 g / L glucose, 0.5 g / L 3-hydroxy-3-phenylpropionic acid and 1 g / L sodium 3HB to obtain a poly (33.3 mol% 3HB-co-18.0 mol% D-phenyl Lactate-co-48.7 mol% 3HPh) was confirmed to produce 14.7 wt% of the dry cell weight (Figure 8c, d).
  • Copolymers containing hydroxyisocaproate, 2-hydroxyhexanoate or 2-hydroxyoctanoate were produced.
  • concentration of 2-HA contained in the medium increases, the mole fraction of the monomer contained in the copolymer increases (Table 4, Table 5 and Table 6).
  • PhaAB Five different plasmids expressing PhaAB were constructed under five promoters of different intensities (SEQ ID NOs: 89-93) and introduced into XB201TBAL strains expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA.
  • pH-stat culture of E. coli XB201TBAL strain expressing PhaAB under the AroGfbr, PheAfbr, FldH, HadA, PhaC1437 and BBa_J23114 promoters was performed without 3HB feeding.
  • poly (67.6 mol% 3HB-co-32.4 mol% D-phenyllactate) having a polymer content of 43.8 wt% of dry cell weight was produced at 2.5 g / L (FIG. 10C, d).
  • the ldhA gene of the E. coli XB201TBA chromosome was replaced with the fldH gene to further optimize the production of aromatic polyhydroxyalkanoate to optimize the gene expression system.
  • the native promoter of the ldhA gene was replaced with a strong trc promoter to increase the expression of the fldH gene.
  • a fed-batch fermentation was performed using a pulse feeding method to supply glucose.
  • coli XB201TBAF strains expressing PhaAB under the AroGfbr, PheAfbr, FldH, HadA, PhaC1437, and BBa_J23114 promoters are poly (69.1 mol% 3HB-co-38.1 mol% D-phenyllactate) having a polymer content of 55.0% by weight of dry cell weight.
  • PHA Polyhydroxyalkanoate
  • monomer composition were determined by GC or GC-MS.
  • the collected cells were washed three times with distilled water, and then lyophilized for 24 hours, and the PHAs of the lyophilized cells were converted to the corresponding hydroxymethyl esters by acid catalyzed metanolysis.
  • the resulting methyl ester was obtained using GC (Agilent 6890N, Agilent, USA) equipped with an Agilent 7683 autoinjector, frame ionization detector and fused silica capillary column (ATTM-Wax, 30 m, ID 0.53 mm, thickness 1.20 ⁇ m, Alltech, USA). Analyzed.
  • the polymer was extracted by chloroform extraction and purified in cells using solvent extraction.
  • the structure, molecular weight and thermal properties of the polymers were measured using nuclear magnetic resonance (NMR), gel permeation chromatography (GPC) and differential scanning calorimetry (DSC), respectively.
  • Figure 7 shows the results of the analysis of the poly (3HB-co-D-phenyl lactate) produced by E. coli XB201TBAL
  • Figure 8 shows the poly (3HB-co-D- produced by E. coli XB201TBAL Phenyllactate-co-D-mandelate) is shown.
  • a biodegradable polymer containing an aromatic monomer or a long chain 2-HA as a monomer can be produced.

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Abstract

La présente invention concerne un microorganisme recombiné auquel un gène codant la 2-hydroxyisocaproate-CoA transférase et un gène codant la polyhydroxyalcanoate synthase sont introduits et qui a un potentiel de production de polyhydroxyalcanoate portant un monomère aromatique ou un monomère 2-HA à longue chaîne et un procédé de production de polyhydroxyalcanoate portant un monomère aromatique ou un monomère 2-HA à chaîne longue, à l'aide du microorganisme recombiné. Selon la présente invention, un polymère biodégradable portant un monomère aromatique ou un monomère 2-HA à longue chaîne peut être produit.
PCT/KR2018/002305 2017-02-28 2018-02-26 Procédé de production de polyhydroxyalcanoate à l'aide de 2-hydroxyisocaproate-coa transférase WO2018159965A1 (fr)

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BR112019017457A BR112019017457A2 (pt) 2017-02-28 2018-02-26 micro-organismo recombinante e método para a produção de poli-hidroxialcanoato transferase
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