WO2011074842A2 - Micro-organisme recombinant dans la production d'acide polylactique ou de copolymère d'acide polylactique à partir de glycérol et méthode de production d'acide polylactique ou de copolymère d'acide polylactique à partir de glycérol à l'aide dudit micro-organisme - Google Patents

Micro-organisme recombinant dans la production d'acide polylactique ou de copolymère d'acide polylactique à partir de glycérol et méthode de production d'acide polylactique ou de copolymère d'acide polylactique à partir de glycérol à l'aide dudit micro-organisme Download PDF

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WO2011074842A2
WO2011074842A2 PCT/KR2010/008901 KR2010008901W WO2011074842A2 WO 2011074842 A2 WO2011074842 A2 WO 2011074842A2 KR 2010008901 W KR2010008901 W KR 2010008901W WO 2011074842 A2 WO2011074842 A2 WO 2011074842A2
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acid
hydroxy
seq
coa
lactate
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WO2011074842A3 (fr
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김태완
강혜옥
양택호
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(주)Lg화학
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
    • 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

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  • the present invention relates to recombinant microorganisms capable of producing polylactic acid or polylactic acid copolymers from glycerol and to methods of producing polylactic acid or lactic acid copolymers from glycerol using such microorganisms.
  • Polylactate (PLA) is a representative biodegradable polymer derived from lactate and is a polymer having high applicability as a general purpose polymer or a medical polymer.
  • PLA is produced by polymerizing lactate produced by microbial fermentation, but only low molecular weight (1000-5000 Daltons) PLA is produced by the direct polymerization of lactate.
  • the addition of ring agents complicates the process and also has the disadvantage that it is not easy to remove them.
  • commercially available high molecular weight PLA production process is used to convert the lactate to lactide (lactide), and then synthesize the PLA through the ring-opening condensation reaction of the lactide ring.
  • PLA homopolymers can be easily obtained when PLA is synthesized using lactate through chemical synthesis, but the synthesis of PLA copolymers having various monomer compositions is difficult and commercially useful.
  • polyhydroxyalkanoaste is a polyester (microorganisms) that accumulate inside as energy or carbon storage material when there is an excessive carbon source and lacks other nutrients such as phosphorus, nitrogen, magnesium and oxygen. polyester). PHA is regarded as a material to replace conventional synthetic plastics because it has properties similar to synthetic polymers derived from petroleum and shows complete biodegradability.
  • the problem to be solved by the present invention is to provide a microorganism capable of producing a lactate polymer or copolymer using glycerol as a substrate and a method for producing a lactate polymer or copolymer using these microorganisms.
  • the present invention includes the gene of the enzyme for converting lactate to lactyl-CoA and the gene of polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate,
  • Cells or plants having the ability to produce lactate polymers or hydroxyalkanoate-lactate copolymers capable of using glycerol as substrates include lactate and glycerol; Or cultivated or grown in an environment containing lactate, glycerol and hydroxyalkanoate;
  • the inventors of the present invention have found that polyhydroxyalcohol of Pseudomonas sp. 6-19 using propionyl-CoA transferase derived from Clostridium propionicum to provide lactyl-CoA and lactyl-CoA produced thereby as a substrate. Variants of canoate synthase have been used to successfully synthesize lactate polymers and copolymers (Korean Patent Application No. 10-2006-0116234).
  • the present inventors was to prepare a lactate polymer and a copolymer using a matrix of glycerol, a low cost for the economical production of the biodegradable polymer, and thus, the present inventors have found that the E. coli using the substrate, glycerol-cost Clostridium propionicum It was transformed with a plasmid expressing propionyl-CoA transferase derived from polyhydroxyalkanoate synthase of Pseudomonas sp. 6-19 and transformed with E. coli. It was confirmed from the present invention that lactate polymers and copolymers can be efficiently produced, thereby completing the present invention.
  • the polylactate or lactate copolymer (hydroxyalkanoate-lactate copolymer: a cell or plant having a poly (hydroxyalkanoate-co-lactate) production ability (a) converts lactate to lactyl-CoA A cell or plant that does not contain any one or more of the gene of the enzyme and (b) the gene of PHA synthase using r-lactyl-CoA as a substrate, the gene of any one or more of (a) and (b) Can be obtained by transforming a cell or plant without (a) and (b) genes into (a) and (b) genes, or (a) without a gene (b) A cell or plant having a gene may be obtained by transformation with the gene (a), but is not limited thereto, for example, in a cell having any one of the above (a) and (b) genes. Amplifying genes and children It is also within the scope of the present invention to transform into other genes.
  • the hydroxyalkanoate in the hydroxyalkanoate-lactate copolymer is 3-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxy 4-hydroxybutyrate, medium chain length (D) -3-hydroxycarboxylic acid with 6 to 14 carbon atoms, 2-hydroxypropionic acid , 3-hydroxypropionic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid ), 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid (3 -hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid (3-hydroxyhexadecanoic acid), 4-hydroxyvaleric acid, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4-hydroxy Octanoic acid (4-hydroxyoctanoic acid), 4-hydroxydecanoic acid, 5-hydroxyvaleric acid, 5-hydroxy
  • propionyl-CoA transferase gene ( pct ) may be used, and more specifically, such lactate may be lactyl-CoA.
  • the gene of the enzyme converting to may be a propionyl-CoA transferase gene derived from Clostridium propionicum .
  • the gene of the enzyme for converting lactate to lactyl-CoA is the nucleotide sequence of SEQ ID NO: 1 (CpPCT); Nucleotide sequences of which T78C, T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1 (CpPCT522); A nucleotide sequence of A1200G mutated from the nucleotide sequence of SEQ ID NO: 1 (CpPCT512); A1200G is mutated in the nucleotide sequence of SEQ ID NO: 1 and Gly335Asp is mutated in the amino acid sequence of SEQ ID NO: 2 (CpPCT531); A base sequence (CpPCT533) in which T669C, A1125G and T1158C are mutated in the nucleotide sequence of SEQ ID NO: 1, and Asp65Gly is mutated in the amino acid sequence of SEQ
  • the CpPct540 gene is a gene of the Clostridium propionicum- derived propionyl-CoA transferase variant which is more preferred for producing lactate polymers or copolymers using glycerol.
  • the cell or plant according to the invention also comprises a gene of PHA synthase (Polyhydroxyalkanoate synthase) using the lactyl-CoA as a substrate.
  • PHA synthase genes include the amino acid sequence of SEQ ID NO: 4 which is a PHA synthase derived from Pseudomonas genus 6-19; Or a gene having a base sequence corresponding to an amino acid sequence comprising one or more mutations selected from the group consisting of E130D, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, and Q481R in the amino acid sequence of SEQ ID NO: 4 And the like can be used.
  • the phaC1 Ps6-19 337 gene is a gene of the PHA synthase variant derived from Pseudomonas 6-19 which is more preferable for producing lactate polymers or copolymers using glycerol.
  • the cell or plant according to the present invention may further include a gene of an enzyme that generates hydroxyacyl-CoA from glycerol.
  • Recombinant cells or plants that additionally contain the gene of the enzyme that produces hydroxyacyl-CoA from glycerol can produce hydroxyacyl-CoA by itself, so do not include hydroxyalkanoate in the medium. If not, it is possible to produce hydroxyalkanoate-lactate copolymers in high yields.
  • the enzyme for producing hydroxyacyl-CoA from the glycerol may be ketothiolase and acetoacetyl-CoA reductase, but is not limited thereto.
  • the ketothiolase and acetoacetyl-CoA reductase are preferably derived from Ralstonia eutropha .
  • the cells having the ability to produce lactate polymers or hydroxyalkanoate-lactate copolymers can be bacteria, in particular E. coli.
  • the invention also relates to lactate polymers or copolymers comprising genes of enzymes that convert lactate to lactyl-CoA and genes of polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate.
  • lactate polymers or copolymers comprising genes of enzymes that convert lactate to lactyl-CoA and genes of polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate.
  • PHA polyhydroxyalkanoate
  • vector refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host.
  • a plasmid vector a bacteriophage vector, a cosmid vector, a YAC (Yeast Artificial Chromosome) vector, and the like may be used. Preference is given to using plasmid vectors for the purposes of the present invention.
  • Typical plasmid vectors that can be used for such purposes 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.
  • Preferred host cells for the present invention may be prokaryotic or eukaryotic cells.
  • Preferred host cells are prokaryotic cells. Suitable prokaryotic cells can be used as well as microorganisms having any of the three genes described above, as well as microorganisms which do not have all of these genes such as E. coli.
  • Preferred E. coli include E. coli DH5a, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL1-Blue (Stratagene), E. coli B and the like. However, E.
  • coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used.
  • Agrobacterium A4 and Agrobacterium sp Bacillus subtilis (Bacillus subtilis) and Bashile (bacilli)
  • S. typhimurium Salmonella such as Another enterobacteria such as typhimurium
  • Serratia marcescens may be used as the host cell.
  • eukaryotic host cells such as yeast and fungi, insect cells such as Spodoptera fruitgiper (SF9), animal cells such as CHO and mouse cells, tissue cultured human cells and plant cells can also be used.
  • yeast and fungi insect cells such as Spodoptera fruitgiper (SF9)
  • animal cells such as CHO and mouse cells
  • tissue cultured human cells and plant cells can also be used.
  • the vector Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases integrate into the genome itself.
  • the gene in order to raise the expression level of a transgene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function within the selected host.
  • the expression control sequence and the gene of interest are included in one expression vector containing the bacterial selection marker and the replication origin together. If the expression host is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.
  • expression control sequence refers to 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 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 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 region of phage lambda, fd Regulatory regions of the code protein, promoters for 3-phosphoglycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, promoters of the yeast alpha-crossing system and prokaryotic or eukaryotic cells or viruses thereof And other sequences of constitution and induction known to modulate the expression of the genes, and various combinations thereof.
  • 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 enable 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 as described above.
  • prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al ., Supra.
  • electroporation (Neumann et al., EMBO J., 1: 841 (1982)) can also be used to transform these cells.
  • transformation of plants can be achieved by conventional methods using Agrobacterium, viral vectors and the like.
  • the transformed plant after transforming the microorganism of the genus Agrobacterium with a recombinant vector containing the gene according to the present invention, the transformed plant can be obtained by infecting the transformed Agrobacterium microorganisms in the tissues of the target plant.
  • a transgenic plant suitable for the present invention can be obtained by the same or similar method as in the prior patent (WO 94/11519; US 6,103,956) for producing PHA using the transformed plant.
  • the term "explant” refers to a slice of tissue cut out of a plant, and includes cotyledon or hypocotyl.
  • the explants of the plant used in the method of the present invention may be cotyledons or hypocotyls, it is more preferable to use the cotyledons obtained by germinating in MS medium after disinfecting and washing the seeds of the plants.
  • Plants to be transformed usable in the present invention include tobacco, tomato, pepper, soybean, rice, corn, and the like, but is not limited thereto.
  • plant used for transformation is a sexually propagating plant, it will be apparent to those skilled in the art that it can be repeatedly reproduced by tissue culture or the like.
  • the present invention provides cells or plants capable of efficiently producing lactate polymers or copolymers from glycerol, which is a low-cost substrate, and the production of lactate polymers or copolymers comprising cultivating or culturing such cells or plants. Provide a method.
  • Figure 1 shows the pPs619C1337-CPPCT540 vector.
  • pPs619C1337CPPCT540 plasmid and pMCS104ReAB were prepared, respectively.
  • the pPs619C1337CPPCT540 plasmid and pMCS104ReAB were designed to express two major enzymes. These enzymes are essential enzymes for the biosynthesis of the biodegradable polymers polylactate and poly (3-hydroxybutyrate-co-lactate) in Escherichia coli, Pseudomonas sp.
  • PhaC1 Ps6-19 337 a polymer polymerase derived from 6-19 (KCTC 11027BP), propionyl-CoA transferase derived from Clostridium propionicum , an enzyme that transfers CoA from acetyl-CoA to lactate and converts it into lactic-CoA (propionyl-CoA transferase, CPPCT), and ketothiolase (phaA RE ) and acetoacetyl-CoA reductase (phaB RE ) from Ralstonia eutropha , enzymes that synthesize 3-hydroxybutyl-CoA from glycerol.
  • propionyl-CoA transferase derived from Clostridium propionicum
  • ketothiolase phaA RE
  • the pPs619C1337CPPCT540 plasmid contains phaC1 Ps6-19 337 and CPPCT540 genes (FIG. 1), and the pMCS104ReAB plasmid contains phaA RE and phaB RE genes (FIG. 2).
  • SEQ ID NO: 3 Pseudomonas sp. PHA synthase derived from phaC1 Ps6-19 gene and propionyl-CoA transferase (CPPCT) gene derived from Clostridium propionicum of SEQ ID NO: 1 genes mutated to favor lactate polymer and copolymer, respectively . All of the genes contained on each plasmid were constructed to be constitutively expressed in recombinant E. coli.
  • Example 1-1 Pseudomonas sp. Substrate Specificity Variation of 6-19-derived PHA Synthetase
  • Type II PHA synthase is known as medium-chain-length PHA synthase that polymerizes relatively long carbon substrates, and this MCL-PHA synthase produces lactate copolymers. It is expected to be very useful.
  • Pseudomonas sp. Highly homologous to phaC1 Ps6-19 synthase obtained in the present invention.
  • 61-3-derived phaC1 synthase is a Type II synthase, but has been reported to have a relatively broad range of substrate specificities (Matsusaki et al ., J. Bacteriol ., 180: 6459, 1998), and SCL-PHA (short-chain).
  • Pseudomonas sp To isolate PHA synthase (phaC1 Ps6-19 ) gene from 6-19 (KCTC 11027BP), Pseudomonas sp. To extract the entire DNA of 6-19, to prepare a primer having the base sequence of SEQ ID NO: 5 and 6 based on the phaC1 Ps6-19 base sequence (Song Aejin, Master's Thesis, Department of Chemical and Biomolecular Engineering, KAIST, 2004) , PCR was performed to obtain phaC1 Ps6-19 gene.
  • DNA fragments containing PHB-producing operons from Ralstonia eutropha H16 from pSYL105 vector were digested with BamHI / EcoRI and BamHI from pBluescript II (Stratagene) PReCAB recombinant vector was prepared by inserting in / EcoRI recognition site.
  • the pReCAB vector is known to express PHA synthase (phaC RE ) and monomer feeder enzymes (phaA RE & phaB RE ) at all times by the PHB operon promoter and to work well in Escherichia coli (Lee et al ., Biotech. Bioeng . , 1994, 44: 1337-1347.
  • the pReCAB vector was digested with BstBI / SbfI to remove R.eutropha H16 PHA synthase (phaC RE ), and then the pPs619C1-ReAB recombinant vector was prepared by inserting the phaC1 Ps6-19 gene obtained above into the BstBI / SbfI recognition site. .
  • the nucleotide sequence of the phaC1 Ps6-19 gene of the prepared pPs619C1-ReAB recombinant vector was confirmed by sequencing, and is represented by SEQ ID NO: 3, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 4.
  • PPs619C1-ReAB recombinant vector was transformed into E. coli XL-1Blue (Stratagene) to confirm PHB synthesis of the phaC1 Ps6-19 synthase , and this was detected by PHB detection medium (LB agar, glucose 20g / L, Nile red). At 0.5 ⁇ g / ml), no PHB production was observed.
  • Propionyl-CoA derived from Clostridium propionicum to construct a system of constant expression of the operon form in which CP-PCT is expressed together to provide lactyl-CoA, which is a monomer required for the synthesis of PLA and PLA copolymers.
  • Transferase CP-PCT
  • cp-pct used a fragment obtained by PCR chromosomal DNA of Clostridium propionicum using primers of SEQ ID NO: 19 and SEQ ID NO: 20, at this time, for ease of cloning the Nde I site existing in the wild type CP-PCT. It was removed using the SDM method.
  • SEQ ID NO: 19 5-ggaattcATGAGAAAGGTTCCCATTATTACCGCAGATGA
  • SEQ ID NO: 20 5-gc tctaga tta gga ctt cat ttc ctt cag acc cat taa gcc ttc tg
  • overlapping PCR was performed using primers having the nucleotide sequences of SEQ ID NOs: 21 and 22 to add Sbf I / Nde I recognition sites.
  • phaC1 Ps6-19 synthase remove the SCL mutant of phaC1 Ps6-19 300 a pPs619C1300-ReAB by cutting the vector with Sbf I / Nde I Ralstonia eutrophus H16 monomer feed enzymes (phaA RE & phaB RE) of origin containing the following, the The pPs619C1300-CPPCT recombinant vector was prepared by inserting the PCR cloned CP-PCT gene into the Sbf I / Nde I recognition site.
  • SEQ ID NO: 28 5'- gat atg ccc aaa gct cga cag cac gaa ttc-3 '
  • CP-PCT is known to be highly toxic due to severe metabolic disorders when expressed in E. coli.
  • the expression of CP-PCT by IPTG using the tac promoter or T7 promoter, which is widely used for expression of recombinant proteins, is recombined simultaneously with the addition of inducers. E. coli all died. This has led to the success of the synthesis of lactate polymers and lactate copolymers using a constitutive expression system that is weakly expressed but continuously expressed as the microorganism grows.
  • pPs619C1300-CPPCT Korean Patent Application No.
  • PCR was performed under normal conditions using the primers SEQ ID NOs: 39 and 40 to amplify the PCR fragment containing the random mutation.
  • phaC1 Ps6-19 synthase SCL mutant of phaC1 by cutting a pPs619C1300-CPPCT vector containing Ps6-19 300 with Sbf I / Nde I to remove the wild-type cp-pct, the amplified mutant PCR fragment Sbf I / Nde I
  • a ligation mixture was inserted into the recognition site and introduced into E. coli JM109 to produce a CP-PCT library of ⁇ 10 ⁇ 5 scale.
  • the prepared CP-PCT library was grown in polymer detection medium (LB agar, glucose 20g / L, 3HB 1g / L, Nile red 0.5 ⁇ g / ml) for 3 days and then screened to determine whether the polymer was produced. More than 80 candidates were selected first. These candidates were subjected to liquid culture (LB agar, glucose 20g / L, 3HB 1g / L, ampicillin 100mg / L, 37 ° C) for 4 days under conditions in which the polymers were produced, and FACS (Florescence Activated Cell Sorting) analysis to analyze the final two individuals. Selected. Gene sequencing was performed to find the mutation position of the prepared CP-PCT variant. The results are shown in Table 6 below.
  • PCR was performed under normal conditions using the primers SEQ ID NOs: 39 and 40 to amplify the PCR fragment containing the CpPct540 mutation.
  • the pPs619C1300-CPPCT vector Sbf I / Nde I was cut to remove the portion CPPCT, the amplified PCR fragments CpPct540 Sbf I / Nde I made the ligation mixture was inserted into the recognition site to prepare a pPs619C1300-CPPCT540 vector.
  • the phaC1 Ps6-19 synthase variant using (phaC1 Ps6-19 300) E130D, S325T , S477G , and Pseudomonas in 6-19-derived PHA having the amino acid sequence of the mutant Q481K synthase variant was prepared using the SDM method using the primers SEQ ID NOs: 31 and 32, and SEQ ID NOs: 33 and 34, and the pPs619C1337-CPPCT540 vector was constructed using the gene (FIG. 1).
  • the recombinant vector (pPs619C1337-CPPCT540) thus obtained was transformed into E. coli JM109, and then transformed into 3HB-containing polymer detecting medium (LB agar, glucose 20g / L, 3HB 2g / L, Nile red 0.5 ⁇ g / ml). As a result of the growth, the formation of the polymer was confirmed.
  • 3HB-containing polymer detecting medium LB agar, glucose 20g / L, 3HB 2g / L, Nile red 0.5 ⁇ g / ml
  • a plasmid pMCS104ReAB was prepared to provide ⁇ -ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB) derived from R. eutropha (Pak Si Jae, PhD thesis, Department of Chemical and Biomolecular Engineering). , KAIST, 2003).
  • p10499A Park et al ., FEMS Microbiol. Lett ., 214: 217, 2002
  • cleaving phaAB gene obtained by cleaving pSYL105 (Lee et al . Biotechnol. Bioeng. 44: 1337, 1994) with PstI.
  • p10499PhaAB was produced.
  • the p10499PhaAB plasmid was digested with SspI to obtain a gene fragment containing 104 promoter and phaAB gene, and then inserted into pBBR1MCS plasmid digested with EcoRV to prepare pMCS104ReAB plasmid (FIG. 2).
  • PLA homopolymer can be biosynthesized from glycerol.
  • PLA homopolymer can be prepared from glycerol through the recombinant E. coli according to the present invention.
  • Table 9 badge Early temperament Biosynthetic Polymer Type Polymer content (weight ratio) LA content in the polymer (molar ratio) MR G2 * PLA 6.48 100 MR G2, NaL + PLA 10.00 100 MR G5 ** PLA 1.85 100 MR G5, NaL PLA 5.75 100
  • 1 mL of the culture was inoculated into 100 mL of MR medium containing 100 mg / L ampicillin and 34 mg / L chloramphenicol and 20 g / L or 50 g / L glycerol, which was then subjected to 200 rpm at 30 ° C.
  • the main culture was started while incubating at a stirring speed.
  • the culture was carried out for 4 days.
  • the initial pH was adjusted to 7 using 10 N NaOH.
  • the medium used for this culture is shown in Table 10 below.
  • Table 11 badge Early temperament Biosynthetic Polymer Type Polymer content (weight ratio) LA content in the polymer (molar ratio) MR G2 * P (3HB-LA) 53.08 64.75 MR G2, NaL + P (3HB-LA) 51.38 69.92 MR G5 ** P (3HB-LA) 40.11 53.13 MR G5, NaL P (3HB-LA) 53.37 51.94

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Abstract

La présente invention concerne un micro-organisme recombinant dans la production d'un copolymère de lactate ou d'un copolymère à l'aide de glycérol, ainsi qu'une méthode de production d'un copolymère de lactate ou d'un copolymère à l'aide de glycérol au titre de substrat, et à l'aide du micro-organisme. Selon la présente invention, les gènes de l'enzyme convertissant le lactate en lactile-CoA, et les gènes de la polyhydroxyalcanoate (PHA) synthétase employant la lactile-CoA au titre de substrat sont introduits dans le micro-organisme, qui peut employer le glycérol au titre de substrat, et le micro-organisme est ensuite mis en culture en employant le glycérol au titre de substrat pour produire de façon efficace un copolymère de lactate et un copolymère.
PCT/KR2010/008901 2009-12-14 2010-12-13 Micro-organisme recombinant dans la production d'acide polylactique ou de copolymère d'acide polylactique à partir de glycérol et méthode de production d'acide polylactique ou de copolymère d'acide polylactique à partir de glycérol à l'aide dudit micro-organisme WO2011074842A2 (fr)

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KR20090124171 2009-12-14
KR10-2009-0124171 2009-12-14
KR10-2010-0127101 2010-12-13
KR1020100127101A KR101260187B1 (ko) 2009-12-14 2010-12-13 글리세롤로부터 폴리락틱산 또는 폴리락틱산 공중합체를 제조할 수 있는 재조합 미생물 및 이러한 미생물을 이용하여 글리세롤로부터 폴리락틱산 또는 락틱산 공중합체를 제조하는 방법

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8883463B2 (en) 2011-03-11 2014-11-11 Korea Advanced Institute Of Science And Technology Recombinant microorganism having ability to produce [lactate-co-glycolate] copolymer from glucose, and method for preparing [lactate-co-glycolate] copolymer using same
US10640774B2 (en) * 2016-01-28 2020-05-05 Korea Advanced Institute Of Science And Technology Recombinant microorganism having ability to produce poly(lactate-coglycolate) or copolymer thereof from xylose and method for preparing poly(lactate-coglycolate) or copolymer thereof by using same
CN115261347A (zh) * 2022-04-06 2022-11-01 深圳蓝晶生物科技有限公司 表达乙酰乙酰辅酶a还原酶变体的工程化微生物及提高pha中3-羟基己酸比例的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006126796A1 (fr) * 2005-05-24 2006-11-30 Lg Chem, Ltd. Cellules ou plantes possedant la capacite de production du polylactate ou ses copolymeres et procede de preparation du polylactate ou ses copolymeres les utilisant
WO2009022797A1 (fr) * 2007-08-14 2009-02-19 Lg Chem, Ltd. Mutant de propionyl-coa transférase dérivé de clostridium propionicum et procédé de préparation visant à obtenir un pla ou un copolymère pla à l'aide de ce mutant
WO2009031762A2 (fr) * 2007-09-07 2009-03-12 Lg Chem, Ltd. Micro-organisme recombinant permettant de produire un polyactate ou un polyactate copolymère à partir de sucrose et méthode de fabrication correspondante
WO2009091141A2 (fr) * 2008-01-16 2009-07-23 Lg Chem, Ltd. Micro-organisme recombiné ayant une aptitude à la production de polylactate ou de ses copolymères et procédé de préparation de polylactate ou de ses copolymères utilisant celui-ci

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006126796A1 (fr) * 2005-05-24 2006-11-30 Lg Chem, Ltd. Cellules ou plantes possedant la capacite de production du polylactate ou ses copolymeres et procede de preparation du polylactate ou ses copolymeres les utilisant
WO2009022797A1 (fr) * 2007-08-14 2009-02-19 Lg Chem, Ltd. Mutant de propionyl-coa transférase dérivé de clostridium propionicum et procédé de préparation visant à obtenir un pla ou un copolymère pla à l'aide de ce mutant
WO2009031762A2 (fr) * 2007-09-07 2009-03-12 Lg Chem, Ltd. Micro-organisme recombinant permettant de produire un polyactate ou un polyactate copolymère à partir de sucrose et méthode de fabrication correspondante
WO2009091141A2 (fr) * 2008-01-16 2009-07-23 Lg Chem, Ltd. Micro-organisme recombiné ayant une aptitude à la production de polylactate ou de ses copolymères et procédé de préparation de polylactate ou de ses copolymères utilisant celui-ci

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAGUCHI, S. ET AL.: 'A microbial factory for lactate-based polyesters using a lactate- polymerizing enzyme' PROC, NATL. ACAD. SCI. USA vol. 105, no. 45, 11 November 2008, pages 17323 - 17327 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8883463B2 (en) 2011-03-11 2014-11-11 Korea Advanced Institute Of Science And Technology Recombinant microorganism having ability to produce [lactate-co-glycolate] copolymer from glucose, and method for preparing [lactate-co-glycolate] copolymer using same
US10640774B2 (en) * 2016-01-28 2020-05-05 Korea Advanced Institute Of Science And Technology Recombinant microorganism having ability to produce poly(lactate-coglycolate) or copolymer thereof from xylose and method for preparing poly(lactate-coglycolate) or copolymer thereof by using same
CN115261347A (zh) * 2022-04-06 2022-11-01 深圳蓝晶生物科技有限公司 表达乙酰乙酰辅酶a还原酶变体的工程化微生物及提高pha中3-羟基己酸比例的方法
EP4279587A4 (fr) * 2022-04-06 2024-02-28 Shenzhen Bluepha Biosciences Co Ltd Micro-organisme modifié exprimant un variant d'acétoacétyl coenzyme a réductase et procédé d'augmentation de la proportion d'acide 3-hydroxyhexanoïque dans le pha

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