WO2009031762A2 - Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same - Google Patents

Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same Download PDF

Info

Publication number
WO2009031762A2
WO2009031762A2 PCT/KR2008/004611 KR2008004611W WO2009031762A2 WO 2009031762 A2 WO2009031762 A2 WO 2009031762A2 KR 2008004611 W KR2008004611 W KR 2008004611W WO 2009031762 A2 WO2009031762 A2 WO 2009031762A2
Authority
WO
WIPO (PCT)
Prior art keywords
acid
hydroxy
seq
gene
base sequence
Prior art date
Application number
PCT/KR2008/004611
Other languages
French (fr)
Other versions
WO2009031762A3 (en
Inventor
Si Jae Park
Tae Wan Kim
Hye Ok Kang
Taek Ho Yang
Sang Yup Lee
Original Assignee
Lg Chem, Ltd.
Korea Advanced Institute Of Science And Technology
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
Priority claimed from KR1020080068607A external-priority patent/KR101037354B1/en
Application filed by Lg Chem, Ltd., Korea Advanced Institute Of Science And Technology filed Critical Lg Chem, Ltd.
Priority to CN200880105983.8A priority Critical patent/CN101835895B/en
Priority to US12/676,832 priority patent/US8420357B2/en
Publication of WO2009031762A2 publication Critical patent/WO2009031762A2/en
Publication of WO2009031762A3 publication Critical patent/WO2009031762A3/en

Links

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
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • 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.)
    • C12N9/1025—Acyltransferases (2.3)
    • 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 recombinant microorganism capable of producing a polylactate (PLA) or PLA copolymer from sucrose and a method of producing a PLA or PLA copolymer from sucrose using the microorganism.
  • PLA polylactate
  • PLA copolymer from sucrose
  • Polylactate is a typical biodegradable polymer derived from lactate that is highly applicable commercially and biomedically. Although preparation of PLA presently involves polymerization of lactate produced by fermenting microorganisms, only PLA with a low molecular weight of about 1000 to 5000 daltons is obtained by direct polymerization of lactate. In order to synthesize PLA with a molecular weight of 100,000 daltons or higher, PLA with a low molecular weight obtained by direct polymerization of lactate may be polymerized using a chain coupling agent. In this method, however, the entire process becomes complicated due to addition of an organic solvent or the chain coupling agent, which are not easy to remove.
  • a presently commercially available process of preparing high-molecular weight PLA may include converting lactate into lactide and synthesizing PLA using ring-opening polycondensation of lactide rings.
  • PLA is synthesized by chemical synthesis of lactate, a PLA homopolymer is easily obtained, but a PLA copolymer composed of various types of monomers is difficult to synthesize and commercially unavailable.
  • polyhydroxyalkanoate is polyester stored by microorganisms as energy or a carbon source when there are excessive carbon sources and a lack of other nutrients such as phosphorus (P), nitrogen (N), magnesium (Mg), and oxygen (O), etc. Since PHA has similar physical properties to conventional synthetic polymers from petroleum and exhibits complete biodegradability, it is being recognized as a substitute for conventional synthetic plastics. In order to produce PHAs using microorganisms, enzymes converting microbial metabolic products into a PHA monomer and a PHA synthase for synthesizing a PHA polymer using the PHA monomer are needed.
  • the present invention is directed to a microorganism capable of producing a polylactate (PLA) or PLA copolymer using sucrose as a substrate and a method of producing a PLA or PLA copolymer using the same.
  • One aspect of the present invention provides a method of producing a polylactate (PLA) or hydroxyalkanoate-lactate copolymer from sucrose.
  • the method includes culturing or growing a cell or plant in an environment containing lactate and sucrose or an environment containing lactate, sucrose, and hydroxyalkanoate.
  • the cell or plant contains a gene of an enzyme converting lactate into lactyl-CoA and a gene of a polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate and is able to produce a PLA or hydroxyalkanoate-lactate copolymer using sucrose as a substrate. Thereafter, the PLA or hydroxyalkanoate-lactate copolymer is retrieved from the cell or plant.
  • the present inventors successfully synthesized PLA and PLA copolymer using a propionyl-CoA transferase from Clostridium propionicum for providing lactyl-CoA, and a mutant of a PHA synthase from Pseudomonas sp. 6-19 using lactyl-CoA as a substrate, as disclosed in Korean Patent Application No. 10-2006-0116234. Furthermore, the present inventors attempted to produce PLA and PLA copolymer using a low-cost substrate, sucrose, in order to economically produce biodegradable polymers.
  • the present inventors transformed E.coli using sucrose as a low-cost substrate with a plasmid expressing a propionyl-CoA transferase from Clostridium propionicum and a PHA synthase from Pseudomonas sp. 6-19, and found that PLA and PLA copolymer could be efficiently produced from sucrose using the transformed recombinant E.coli. This led them to complete the present invention.
  • the cell or plant capable of producing the PLA or PLA copolymer may be obtained by transforming a cell or plant that does not include at least one of: (a) the gene of the enzyme converting lactate into lactyl- CoA and (b) the gene of the PHA synthase using lactyl-CoA as the substrate with at least one of (a) and (b).
  • the cell or plant capable of producing the PLA or hydroxyalkanoate-lactate copolymer may be obtained by transforming a cell or plant not having (a) and (b) with at least one of (a) and (b), or by transforming a cell or plant not having (a) but having (b) with (a), but the present invention is not limited thereto.
  • the cell capable of producing the PLA or hydroxyalkanoate-lactate copolymer may be obtained by amplifying the included one of (a) and (b) and transforming the cell with the absent one of (a) and (b).
  • hydroxyalkanoate of the hydroxyalkanoate-lactate copolymer may be at least one selected from the group consisting of 3 -hydroxy butyrate, 3- hydroxyvalerate, 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-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, 4- hydroxyvaleric acid, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4- hydroxyoctanoic acid, 4-hydroxydecanoic acid, 5 -hydroxy valeric acid, 5-hydroxyhexanoic acid, 6-hydroxydodecano
  • the gene of the enzyme converting lactate into lactyl-CoA may be a propionyl-CoA transferase (pet) gene. More specifically, the gene of the enzyme converting lactate into lactyl-CoA may be a pet gene from Clostridium propionicum.
  • the gene of the enzyme converting lactate into lactyl-CoA may have one selected from the group consisting of: a base sequence (CpPCT) of SEQ ID NO: 1; a base sequence (CpPCT522) obtained by mutating T78C, T669C, Al 125G and Tl 158C in the base sequence of SEQ ID NO: 1 ; a base sequence (CpPCT512) obtained by mutating A 1200G in the base sequence of SEQ ID NO:1; a base sequence (CpPCT531) obtained by mutating A 1200G in the base sequence of SEQ ID NO:1 and mutating Gly335Asp in an amino-acid sequence of SEQ ID NO: 2; a base sequence (CpPCT533) obtained by mutating T669C, Al 125G and Tl 158Ca in the base sequence of SEQ ID NO:1 and mutating Asp65Gly in the amino-acid sequence of SEQ
  • the cell or plant according to the present invention may include the gene of the PHA synthase using lactyl-CoA as the substrate, which is a gene of a PHA synthase from
  • the gene of the PHA synthase using lactyl-CoA as the substrate may be a gene having an amino-acid sequence of SEQ ID NO: 4 or a gene having a base sequence corresponding to a mutant amino acid sequence in which at least one selected from the group consisting of El 3OD, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, and Q481R is mutated in the amino-acid sequence of SEQ ID NO: 4.
  • the phaClp s6-19 400 gene may be the gene of a PHA synthase from Pseudomonas sp. 6-19 that is preferable for producing the PLA or PLA copolymer using sucrose.
  • the cell or plant according to the present invention may further include a gene of an enzyme for producing hydroxyacyl-CoA from sucrose. Since a recombinant cell or plant further including the gene of the enzyme for producing hydroxyacyl-CoA from sucrose can produce hydroxyacyl-CoA on its own, even if hydroxyalkanoate is not included in a medium, hydroxyalkanoate-lactate copolymer may be produced with high yield.
  • the enzymes for producing hydroxyacyl-CoA from sucrose may be a ketothiolase and an acetoacetyl-CoA reductase, but the present invention is not limited thereto.
  • the ketothiolase and acetoacetyl-CoA reductase may be derived from Ralstonia eutropha.
  • PLA or hydroxyalkanoate-lactate copolymer may be a bacterium, particularly, E. coli.
  • the present invention provides a cell or plant that is transformed with a recombinant vector for producing a PLA or PLA copolymer containing a gene of an enzyme converting lactate into lactyl-CoA and a gene of a polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate and uses sucrose as a substrate.
  • the cell or plant may further include a gene of an enzyme for producing 3-hydroxybutyl-CoA from sucrose.
  • a vector refers to a DNA construct containing a DNA sequence operably linked to a suitable control sequence capable of expressing DNA in an appropriate host.
  • the vector may be a plasmid vector, a bacteriophage vector, a cosmid vector, or a yeast artificial chromosome (YAC) vector.
  • a plasmid vector may be used.
  • a typical plasmid vector that serves the purpose of the invention includes (a) a replication origin that allows efficient replication such that each host cell includes several hundred plasmid vectors, (b) an antibiotic-resistance gene that allows selection of a host cell transformed into a plasmid vector, and (c) a restriction enzyme cleavage site in which a foreign DNA fragment may be inserted. Even if there is no appropriate restriction enzyme cleavage site, a vector may be easily ligated with foreign DNA by an ordinary method using a synthetic oligonucleotide adaptor or a linker.
  • the vector After the ligation process, the vector has to be transformed into an appropriate host cell.
  • the host cell may be a prokaryotic cell or a eukaryotic cell.
  • the host cell is a prokaryotic cell.
  • the prokaryotic cell may be a microorganism having one of the above-described three genes or a microorganism having none of the above-described three genes, for example, E.coli.
  • the E.coli may include E.coli strain DH5a, E.coli strain JMlOl, E.coli K12, E.coli W3110, E.coli X1776, E.coli XLl -Blue (Stratagene), E.coli B, etc.
  • E.coli strains such as FMBlOl, NM522,
  • NM538, and NM539 and other prokaryotic species and genera may be also used.
  • Agrobacterium sp. strains such as Agrobacterium A4, bacilli such as Bacillus subtilis, and other enterobacteria such as Salmonella typhimurium or Serratia marcescens, may be used as host cells.
  • Known eukaryotic host cells such as yeast and mold, insect cells such as spodoptera frugiperda (SF9), animal cells such as Chinese hamster ovary (CHO) cells and mouse cells, and tissue-cultured human and plant cells may be used as host cells.
  • the vector When the vector is transformed into an appropriate host, the vector is capable of replicating or functioning irrespective of a host genome or, in some cases, being integrated with the genome itself.
  • the transformed gene in order to raise an expression level of a transformed gene in a host cell, the transformed gene must be operably linked to an expression control sequence that performs transcription and translation functions in a selected expression host.
  • the expression control sequence and the transformed gene are included in a single expression vector including both a bacterial selectable marker and a replication origin.
  • the expression vector When an expression host is a eukaryotic cell, the expression vector must further include an expression marker that is useful in the eukaryotic expression host.
  • control sequence means a DNA sequence essential for expression of an operably linked coding sequence in a specific host.
  • the control sequence includes a promoter required for transcription, an arbitrary operator sequence for controlling the transcription, a sequence for encoding an appropriate mRNA ribosome binding site (RBS), and a sequence for controlling termination of transcription and translation.
  • a control sequence suitable for a prokaryote includes a promoter, a random operator sequence, and an RBS.
  • a control sequence suitable for a eukaryotic cell includes a promoter, a polyadenylation signal, and an enhancer. The most important factor that affects the expression amount of a gene in a plasmid is a promoter.
  • a SRa promoter or a cytomegalovirus promoter may be used as a high-expression promoter.
  • any one of various expression control sequences may be used for a vector.
  • the expression control sequence may be, for example, early and late promoters of SV40 or adenovirus, the lac system, the trp system, the tac system, the trc system, T3 and T7 promoters, the major operator and promoter regions of phage ⁇ , the control region of fd code protein, the promoters of 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of phosphatase, for example, the promoters, e.g., Pho5, the promoter of a yeast ⁇ -mating system, other sequences with configurations or derivations known to control expression of a prokaryotic or eukaryotic cell or their viruses, and various combinations thereof.
  • nucleic acid When a nucleic acid is disposed in a functional relationship with another nucleic acid sequence, it is operably linked to the nucleic acid sequence.
  • An appropriate molecule e.g., a transcription-activating protein
  • DNA of a pre-sequence or a secretory leader is operably linked to DNA of polypeptide when it is expressed as a pre-protein that participates in secretion of polypeptide; a promoter or enhancer is operably linked to a coding sequence when it affects transcription of the coding sequence; an RBS is operably linked to a coding sequence when it affects transcription of the coding sequence; or the RBS is operably linked to the coding sequence when it is disposed to facilitate translation.
  • "operably linked sequences" means that DNA sequences being linked are contiguous, and in case of the secretory leader contiguous and in a reading frame. However, an enhancer does not have to contact a coding sequence.
  • Linkage between sequences may be performed by ligation in a convenient restriction enzyme site. However, when there is no restriction enzyme site, a synthetic oligonucleotide adaptor or a linker may be used according to an ordinary method.
  • a synthetic oligonucleotide adaptor or a linker may be used according to an ordinary method.
  • all vectors and expression control sequences do not equally function to express DNA sequences according to the present invention.
  • all hosts do not equally function with respect to the same expression system.
  • one skilled in the art may appropriately select from among various vectors, expression control sequences, and hosts without either departing from the scope of the present invention or bearing excessive experimental burden. For example, a vector must be selected considering a host because the vector must be replicated in the host.
  • the copy number and copy number control of the vector and expression of other protein (e.g., an antibiotic marker) encoded by the corresponding vector must be also considered.
  • other protein e.g., an antibiotic marker
  • One skilled in the art may select an appropriate combination out of various vectors, expression control sequences, and hosts within appropriate ranges of the above- described variables.
  • a eukaryotic cell may be easily accomplished using a calcium chloride method described in section 1.82, supra by Sambrook et al.
  • electroporation may be employed for transformation of these cells(Neumann et al., EMBO J., 1:841(1982)).
  • transformation of a plant may be achieved by an ordinary method using Agrobacterium, a virus vector, etc.
  • a microorganism is transformed with a recombinant vector containing a gene according to the present invention, and the transformed Agrobacterium sp. microorganism may infect tissue of a target plant, thereby obtaining a transformed plant.
  • a transformed plant according to the present invention may be obtained in the same manner as or in a similar manner to a method of producing PHA using a transformed plant disclosed in WO 94/11519 or US 6,103,956.
  • production of a transfected plant may involve (a) pre-culturing an explant of a target plant and co-culturing the explant with transformed agrobacterium for transfection of the plant; (b) culturing the transfected explant in a callus-inducing medium to obtain a callus; and (c) cleaving the callus and culturing the cleaved callus in a shoot- inducing medium to obtain a shoot.
  • the term "explant” refers to a tissue fragment cut off from a plant and includes a cotyledon or a hypocotyl.
  • the cotyledon or hypocotyl may be used as an explant of a plant used for the method of the present invention.
  • the cotyledon which is obtained by disinfecting and cleaning a seed of a plant and germinating the seed in a Murashige and Skoog (MS) medium, is preferably used.
  • target plants to be transformed may be tobacco, tomatoes, red peppers, beans, rice plants, corn, etc, but the present invention is not limited thereto. Also, it is known to one skilled in the art that even if a plant used for transformation is sexually reproducible, it can be asexually reproduced by tissue culture, etc. [Advantageous Effects]
  • FIG. 1 is a schematic diagram of a process of preparing a recombinant expression vector containing a mutant gene of a polyhydroxyalkanoate (PHA) synthase from
  • Pseudomonas sp. 6-19 a mutant gene of a propionyl-CoA transferase from Clostridium propionicum, and a gene of a phaAB from Ralstonia eutropha according to an example of the present invention.
  • a recombinant E.coli W transformed with a pPs619C1400CPPCT532ReAB plasmid was prepared.
  • the E.coli strain W (ATCC9637) according to the present Example may metabolize sucrose as a carbon source.
  • the pPs619C1400CPPCT532ReAB plasmid prepared for the present invention was constructed to express four kinds of major enzymes.
  • the major enzymes are essential for biosynthesis of poly(3-hydroxybutyrate-co-lactate), which is a biodegradable polymer, in the E. coli.
  • the major enzymes may be phaCl Ps6- i 9 400, which is a polymerase from Pseudomonas sp.
  • KCTC 11027BP a propionyl-CoA transferase from Clostridium propionicum (CP-PCT), which is an enzyme transferring CoA from acetyl-CoA to lactate to be converted into lactyl-CoA
  • CP-PCT Clostridium propionicum
  • ketothiolase(phaA RE ) and an acetoacetyl-CoA reductase (phaB RE ) from Ralstonia eutropha which are enzymes for synthesizing 3- hydroxybutyl-CoA from sucrose.
  • the pPs619C1400CPPCT532ReAB plasmid includes phaClp s6- j9400, CPPCT532, phaARE and phaB RE genes respectively encoding the above major enzymes (refer to FIG. 1).
  • the phaClPs ⁇ - 19400 and CPPCT532 genes are respectively obtained by mutating a gene of phaC Ip 56-19 , which is a PHA synthase from Pseudomonas sp. of SEQ ID NO: 3, and a gene of a CP-PCT of SEQ ID NO: 1, to be advantageous for PLA and PLA copolymer.
  • the pPs619C1400CPPCT532ReAB plasmid is prepared to constitutively express all four genes in the recombinant E. coli.
  • Example 1-1 Preparation of a substrate-specific mutant of a PHA synthase from Pseudomonas sp. 6-19
  • a Type II PHA synthase is known as a medium-chain-length PHA (MCL-PHA) synthase for polymerizing a substrate having relatively many carbon atoms, and the MCL-PHA synthase is expected to be very useful in production of a PLA copolymer.
  • MCL-PHA medium-chain-length PHA
  • SEQ ID NO:6 5'-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3'
  • pSYL105 vector From a pSYL105 vector (Lee et al, Biotech. Bioeng., 1994, 44:1337-1347), a DNA fragment containing a PHB producing operon from Ralstonia eutropha Hl 6 was cleaved with BamHI/EcoRI, and then inserted into a BamHI/EcoRI restriction site of pBluescript II (Stratagene), thereby preparing a pReCAB recombinant vector. It is known that the pReCAB vector constitutively expresses a PHA synthase
  • phase R E and monomer-supplying enzymes (phaA RE and phaBRg) by a PHB operon promoter, and also effectively operated in E.coli (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347).
  • the pReCAB vector was cleaved with BstBI/Sbfl to remove a R. eutropha Hl 6 PHA synthase (phaC R ⁇ ), and the phaClp s6- i 9 gene was inserted into a BstBI/Sbfl restriction site, thereby preparing a pPs619C 1 -ReAB recombinant vector.
  • SEQ ID NO: 8 5'- CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC -3'
  • SEQ ID NO: 9 5 1 - GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG -3'
  • SEQ ID NO: 10 5'- CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC -3 1
  • SEQ ID NO: 11 5'- GTA CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG -3'
  • sequence of the phaClp s6-19 gene of the prepared pPs619Cl-ReAB recombinant vector was confirmed by sequencing and represented as SEQ ID NO: 3, and a sequence of an amino acid encoded by the base sequence of SEQ ID NO: 3 was represented as SEQ ID NO: 4.
  • the pPs619Cl- ReAB recombinant vector was transformed into E.coli XL-lBlue(Stratagene), and then the transformed E.coli was grown in a PHB detection medium (Luria Bertani (LB) agar, glucose 20g/L, Nile red 0.5 ⁇ g/ml). As a result, PHB production was not detected.
  • a PHB detection medium Lia Bertani (LB) agar, glucose 20g/L, Nile red 0.5 ⁇ g/ml.
  • SEQ ID NO: 14 5'- GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG- 3'
  • SEQ ID NO: 16 5'- GCG GGT TCA GGA TGC TCA TGA TAT GCC CGC TGC TCG- 3'
  • SEQ ID NO: 17 5'- ate aac etc atg ace gat gcg atg gcg ccg ace- 3'
  • SEQ ID NO: 18 5'- ggt egg cgc cat cgc ate ggt cat gag gtt gat- 3'
  • the recombinant vectors were transformed into E.coli XL-I Blue and grown in a PHB detection medium (LB agar, glucose 20g/L, Nile red 0.5 ⁇ g/ml).
  • a cp-pct gene was used for the cp-pct gene.
  • a DNA fragment obtained by polymerizing chromosomal DNA of Clostridium propionicum was cloned by PCR using primers of SEQ ID NOs: 19 and 20.
  • SEQ ID NO: 19 5 '-ggaattcATGAGAA AGGTTCCC ATTATTACCGC AGATGA
  • SEQ ID NO: 20 5'-gc tctaga tta gga ctt cat ttc ctt cag ace cat taa gcc ttc tg
  • overlapping PCR was performed using primers of SEQ ID NOs: 21 and 22 in order to add a Sbfl/Ndel restriction site.
  • SEQ ID NO: 21 5'-agg cct gca ggc gga taa caa ttt cac aca gg- 3'
  • SEQ ID NO: 22 5 '-gcc cat atg tct aga tta gga ctt cat ttc c- 3 '
  • SEQ ID NO: 17 5 '-ate aac etc atg ace gat gcg atg gcg ccg ace- 3'
  • SEQ ID NO: 18 5' -ggt egg cgc cat cgc ate ggt cat gag gtt gat- 3'
  • SEQ ID NO: 13 5'-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC Aces'
  • SEQ ID NO: 14 5'- GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG- 3'
  • SEQ ID NO: 27 5'- gaa ttc gtg ctg teg age ttt ggg cat ate- 3'
  • SEQ ID NO: 28 5'- gat atg ccc aaa get cga cag cac gaa ttc- 3'
  • SEQ ID NO: 29 5'-gaa ttc gtg ctg teg age tat ggg cat ate- 3'
  • SEQ ID NO: 30 5'-gat atg ccc ata get cga cag cac gaa ttc- 3'
  • SEQ ID NO: 33 5'-ggg cat ate aaa age ate ctg aac ccg c- 3'
  • SEQ ID NO: 34 5'-gcg ggt tea gga tgc ttt tga tat gee c- 3' Q481M
  • SEQ ID NO: 35 5'-ggg cat ate atg age ate ctg aac ccg c- 3'
  • SEQ ID NO: 36 5'-gcg ggt tea gga tgc tea tga tat gcc c- 3' Q481R
  • Example 1-2 Preparation and screening of a library of a mutant of propionyl- CoA transferase from Clostridium propionicum
  • SEQ ID NO: 39 5'- cgc egg cag gcc tgc agg -3'
  • SEQ ID NO: 40 5'- ggc agg tea gcc cat atg tc -3'
  • PCR was performed under common conditions using the primer of SEQ ID NOs: 39 and 40.
  • coli JMl 09 thereby preparing a CP-PCT library with a scale of about -lO" 5 .
  • the prepared CP-PCT library was incubated for 3 days in a polymer detection medium (LB agar, glucose 20g/L, 3HB lg/L, Nile red 0.5 ⁇ g/ml) and screened to confirm whether a polymer was produced, thereby primarily selecting up to 80 candidates.
  • the candidates were grown for 4 days in a liquid medium (LB agar, glucose 20g/L, 3HB lg/L, ampicillin 100mg/L, 37 "C) under polymer-generating conditions and analyzed by florescence activated cell sorting (FACS), thereby selecting two final samples.
  • FACS florescence activated cell sorting
  • a PCR method was performed under common conditions using the primers of SEQ ID NOs: 39 and 40.
  • the PPS619C1300-CPPCT vector was cleaved with Sbfl/Ndel to remove a wild-type CP-PCT gene, and the amplified CpPct532 PCR fragment was inserted into a Sbfl/Ndel restriction site to produce a ligation mixture, thereby producing the pPs619C1300-CPPCT532 vector used in the following Example 1-3.
  • Example 1-3 Preparation of the pPs619C1300-CPPCT532 vector
  • a PHA synthase mutant (phaCl Ps6-19 400) from Pseudomonas sp. 6-19, which has an amino-acid sequence in which E130D, S325T, S477R, and Q481M were mutated, was prepared using the phaClp S6 -i 9 synthase mutant (phaClp s6- i 9 300) by SDM using primers of SEQ ID NOs: 41 and 42.
  • E.coli JMl 09 and grown in a 3HB-containing polymer detection medium (LB agar, glucose 20g/L, 3HB 2g/L, Nile red 0.5 ⁇ g/ml). As a result, polymer production was detected.
  • a 3HB-containing polymer detection medium LB agar, glucose 20g/L, 3HB 2g/L, Nile red 0.5 ⁇ g/ml.
  • Example 1-4 Preparation of a pPs619C1400CPPCT532ReAB plasmid and transformation of E.coli
  • PCR cloning was performed with respect to a PHB - producting promoter pReC and phaA RE and phaB RE genes from Ralstonia eutropha Hl 6 on a pSYL105 vector (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347) using primers of SEQ ID NOs: 43 and 44 and primers of SEQ ID NOs: 45 and 46, respectively.
  • SEQ ID NO: 43 5'- aga cat atg caa gta cct tgc cga cat eta tg- 3'
  • SEQ ID NO: 44 5'- gat gac aac gtc agt cat gat ttg att gtc tct ctg - 3'
  • SEQ ID NO: 45 5'- cag aga gac aat caa ate atg act gac gtt gtc ate - 3'
  • SEQ ID NO: 46 5'- gca ggt cag cc ata tgc ag - 3'
  • a PCR reactant was purified on an agarose gel, and the overlapping PCR was performed using the PCR reactant as a template and the primers of SEQ ID NOs: 43 and
  • the PCR reactant and the pPs619C1400CPPCT532 vector were cleaved with Ndel, and the PCR reactant (pReC-phaABRE) was inserted into a Ndel restriction site of the pPs619C1400CPPCT532 vector, thereby completing a pPs619C1400CPPCT532ReAB vector as shown in FIG. 1.
  • the PHB- producting promoter pReC was additionally inserted into the front portions of the phaA RE and phaBRE genes in order to further promote transcription of the target phaARE and phaBRE genes.
  • the recombinant vector (pPs619C1400CPPCT532ReAB) was transformed into E.coli JM 109, and grown in a 3HB-free polymer detection medium (LB agar, glucose 20g/L, Nile red 0.5 ⁇ g/ml). As a result, polymer production could be observed. Thus, it could be confirmed that the phaA RE and phaB RE genes additionally inserted into the pPs619C1400CPPCT532 vector were expressed and operated normally.
  • a 3HB-free polymer detection medium LB agar, glucose 20g/L, Nile red 0.5 ⁇ g/ml
  • Example 2 Preparation of poly(3-hydroxybutyrate-co-lactate) (P(3HB-co- LA)) from a recombinant E.coli using sucrose as a carbon source
  • a colony on an LB agar plate containing 100 mg/L of ampicillin as an antibiotic was inoculated into 3mL of a LB medium containing 100 mg/L of ampicillin and cultured for 12 hours at 30 ° C , while being stirred at 200rpm.
  • ImL of the culture solution was inoculated into each of 10OmL methyl red (MR) medium and 10OmL LB medium, which contains 100 mg/L of ampicillin, 20 g/L of sucrose, and in some cases, 2 g/L of 3- hydroxybutyrate. Thereafter, each culture solution was cultured at about 30 ° C for 4 days, while being stirred at 200 rpm.
  • the initial pH of the MR medium was adjusted to pH 7 using 10 N NaOH.
  • the medium samples used in the present culturing process are shown in the following Table 8.
  • biomasses were retrieved from the culture solutions.
  • the retrieved cells were cleaned 3 times with distilled water and dried for 24 hours in a dryer maintained at about 100 ° C.
  • the dried cells were partially collected and underwent gas chromatographic (GC) analysis, thereby measuring the content of P(3HB-co-LA) synthesized in cells.
  • Standard materials used for the GC analysis were a P(3HB-co-3HV) copolymer (including about 12% by weight 3HV) and a PLA homopolymer.
  • the GC analysis results are shown in the following Table 9. As can be seen from Table 9, biosynthesis of a P(3HB-co-LA) copolymer was enabled in both the MR and LB media irrespective of the addition of 3HB.
  • the biosynthesis of the P(3HB-co-LA) copolymer was more efficient in the MR medium than in the LB medium. Therefore, the P(3HB-co-LA) copolymer could be produced from sucrose using the recombinant E. coli according to the present invention. [Table 8]

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Provided are a microorganism capable of producing a polylactate (PLA) or PLA copolymer from sucrose and a method of producing the PLA or PLA copolymer from sucrose using the same. A gene of an enzyme converting lactate into lactyl-CoA and a gene of a polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate are introduced into a microorganism capable of using sucrose as a substrate, and the microorganism is cultured using sucrose as the substrate, thereby allowing efficient production of a PLA or PLA copolymer.

Description

RECOMBINANT MICROORGANISM CAPABLE OF PRODUCING POLYLACTATE
OR POLYLACTATE COPOLYMER FROM SUCROSE AND METHOD FOR PRODUCING POLYLACTATE OR POLYLACTATE COPOLYMER FROM SUCROSE
USING THE SAME
[Technical Field]
The present invention relates to a recombinant microorganism capable of producing a polylactate (PLA) or PLA copolymer from sucrose and a method of producing a PLA or PLA copolymer from sucrose using the microorganism. [Background Art]
Polylactate (PLA) is a typical biodegradable polymer derived from lactate that is highly applicable commercially and biomedically. Although preparation of PLA presently involves polymerization of lactate produced by fermenting microorganisms, only PLA with a low molecular weight of about 1000 to 5000 daltons is obtained by direct polymerization of lactate. In order to synthesize PLA with a molecular weight of 100,000 daltons or higher, PLA with a low molecular weight obtained by direct polymerization of lactate may be polymerized using a chain coupling agent. In this method, however, the entire process becomes complicated due to addition of an organic solvent or the chain coupling agent, which are not easy to remove. A presently commercially available process of preparing high-molecular weight PLA may include converting lactate into lactide and synthesizing PLA using ring-opening polycondensation of lactide rings. When PLA is synthesized by chemical synthesis of lactate, a PLA homopolymer is easily obtained, but a PLA copolymer composed of various types of monomers is difficult to synthesize and commercially unavailable.
Meanwhile, polyhydroxyalkanoate (PHA) is polyester stored by microorganisms as energy or a carbon source when there are excessive carbon sources and a lack of other nutrients such as phosphorus (P), nitrogen (N), magnesium (Mg), and oxygen (O), etc. Since PHA has similar physical properties to conventional synthetic polymers from petroleum and exhibits complete biodegradability, it is being recognized as a substitute for conventional synthetic plastics. In order to produce PHAs using microorganisms, enzymes converting microbial metabolic products into a PHA monomer and a PHA synthase for synthesizing a PHA polymer using the PHA monomer are needed. When synthesizing PLA and PLA copolymer using microorganisms, the same system is required, and enzymes for providing lactyl-CoA are needed in addition to an enzyme for providing hydroxyacyl-CoA, which is an original substrate of a PHA synthase.
Furthermore, it is very important to adopt a low-cost substrate in order to economically produce biodegradable polymers. In particular, it is necessary to develop a technique of producing a PLA or PLA copolymer using sucrose as a low-cost substrate. [Disclosure] [Technical Problem]
The present invention is directed to a microorganism capable of producing a polylactate (PLA) or PLA copolymer using sucrose as a substrate and a method of producing a PLA or PLA copolymer using the same. [Technical Solution]
One aspect of the present invention provides a method of producing a polylactate (PLA) or hydroxyalkanoate-lactate copolymer from sucrose. The method includes culturing or growing a cell or plant in an environment containing lactate and sucrose or an environment containing lactate, sucrose, and hydroxyalkanoate. The cell or plant contains a gene of an enzyme converting lactate into lactyl-CoA and a gene of a polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate and is able to produce a PLA or hydroxyalkanoate-lactate copolymer using sucrose as a substrate. Thereafter, the PLA or hydroxyalkanoate-lactate copolymer is retrieved from the cell or plant.
The present inventors successfully synthesized PLA and PLA copolymer using a propionyl-CoA transferase from Clostridium propionicum for providing lactyl-CoA, and a mutant of a PHA synthase from Pseudomonas sp. 6-19 using lactyl-CoA as a substrate, as disclosed in Korean Patent Application No. 10-2006-0116234. Furthermore, the present inventors attempted to produce PLA and PLA copolymer using a low-cost substrate, sucrose, in order to economically produce biodegradable polymers. As a result, the present inventors transformed E.coli using sucrose as a low-cost substrate with a plasmid expressing a propionyl-CoA transferase from Clostridium propionicum and a PHA synthase from Pseudomonas sp. 6-19, and found that PLA and PLA copolymer could be efficiently produced from sucrose using the transformed recombinant E.coli. This led them to complete the present invention.
The cell or plant capable of producing the PLA or PLA copolymer (hydroxyalkanoate-lactate copolymer) may be obtained by transforming a cell or plant that does not include at least one of: (a) the gene of the enzyme converting lactate into lactyl- CoA and (b) the gene of the PHA synthase using lactyl-CoA as the substrate with at least one of (a) and (b). That is, the cell or plant capable of producing the PLA or hydroxyalkanoate-lactate copolymer may be obtained by transforming a cell or plant not having (a) and (b) with at least one of (a) and (b), or by transforming a cell or plant not having (a) but having (b) with (a), but the present invention is not limited thereto. For example, according to the present invention, when a cell has one of (a) and (b), the cell capable of producing the PLA or hydroxyalkanoate-lactate copolymer may be obtained by amplifying the included one of (a) and (b) and transforming the cell with the absent one of (a) and (b). In the present invention, hydroxyalkanoate of the hydroxyalkanoate-lactate copolymer may be at least one selected from the group consisting of 3 -hydroxy butyrate, 3- hydroxyvalerate, 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-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, 4- hydroxyvaleric acid, 4-hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4- hydroxyoctanoic acid, 4-hydroxydecanoic acid, 5 -hydroxy valeric acid, 5-hydroxyhexanoic acid, 6-hydroxydodecanoic acid, 3- hydroxy-4-pentenoic acid, 3-hydroxy-4-trans-hexenoic acid, 3-hydroxy-4-cis-hexenoic acid, 3-hydroxy-5-hexenoic acid, 3-hydroxy-6-trans- octenoic acid, 3-hydroxy-6-cis-octenoic acid, 3-hydroxy-7-octenoic acid, 3-hydroxy-8- nonenoic acid, 3-hydroxy-9-decenoic acid, 3-hydroxy-5-cis-dodecenoic acid, 3-hydroxy-6- cis-dodecenoic acid, 3-hydroxy-5-cis-tetradecenoic acid, 3-hydroxy-7-cis-tetradecenoic acid, 3-hydroxy-5,8-cis-cis-tetradecenoic acid, 3-hydroxy-4-methylvaleric acid, 3- hydroxy-4-methylhexanoic acid, 3-hydroxy-5-methylhexanoic acid, 3-hydroxy-6- methylheptanoic acid, 3-hydroxy-4-methyloctanoic acid, S-hydroxy-S-methyloctanoic acid, 3-hydroxy-6- methyloctanoic acid, 3-hydroxy-7-methyloctanoic acid, 3-hydroxy-6- methylnonanoic acid, 3-hydroxy-7-methylnonanoic acid, 3-hydroxy-8-methylnonanoic acid, 3-hydroxy-7-methyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, 3-hydroxy-7- methyl-6-octenoic acid, malic acid, 3 -hydroxy succinic acid-methylester, 3- hydroxyadipinic acid-methylester, 3 -hydroxy suberic acid-methylester, 3-hydroxyazelaic acid-methylester, 3 -hydroxy sebacic acid-methylester, 3 -hydroxy suberic acid-ethylester, 3- hydroxysebacic acid-ethylester, 3-hydroxypimelic acid-propylester, 3-hydroxysebacic acid-benzylester, S-hydroxy-δ-acetoxyoctanoic acid, 3-hydroxy-9-acetoxynonanoic acid, phenoxy-3-hydroxybutyric acid, . phenoxy-3 -hydroxy valeric acid, phenoxy-3 - hydroxyheptanoic acid, phenoxy-3 -hydroxyoctanoic acid, para-cyanophenoxy-3- hydroxybutyric acid, para-cyanophenoxy-3-hydroxyvaleric acid, para-cyanophenoxy-3- hydroxyhexanoic acid, para-nitrophenoxy-3-hydroxyhexanoic acid, 3-hydroxy-5- phenylvaleric acid, S-hydroxy-S-cyclohexylbutyric acid, 3,12-dihydroxydodecanoic acid, 3,8-dihydroxy-5-cis-tetradecenoic acid, 3-hydroxy-4,5-epoxydecanoic acid, 3-hydroxy- 6,7-epoxydodecanoic acid, 3-hydroxy-8,9-epoxy-5,6-cis-tetradecanoic acid, 7-cyano-3- hydroxyheptanoic acid, 9-cyano-3-hydroxynonanoic acid, 3-hydroxy-7-fluoroheptanoic acid, 3-hydroxy-9-fluorononanoic acid, 3-hydroxy-6-chlorohexanoic acid, 3-hydroxy-8- chlorooctanoic acid, 3-hydroxy-6-bromohexanoic acid, 3-hydroxy-8-bromooctanoic acid, 3 -hydroxy- 11-bromoundecanoic acid, 3-hydroxy-2-butenoic acid, 6-hydroxy-3-dodecenoic acid, 3-hydroxy-2-methylbutyric acid, 3-hydroxy-2-methylvaleric acid, and 3-hydroxy-2,6- dimethyl-5-heptenoic acid, but the present invention is not limited thereto. In the present invention, the gene of the enzyme converting lactate into lactyl-CoA may be a propionyl-CoA transferase (pet) gene. More specifically, the gene of the enzyme converting lactate into lactyl-CoA may be a pet gene from Clostridium propionicum. In a specific example of the present invention, the gene of the enzyme converting lactate into lactyl-CoA may have one selected from the group consisting of: a base sequence (CpPCT) of SEQ ID NO: 1; a base sequence (CpPCT522) obtained by mutating T78C, T669C, Al 125G and Tl 158C in the base sequence of SEQ ID NO: 1 ; a base sequence (CpPCT512) obtained by mutating A 1200G in the base sequence of SEQ ID NO:1; a base sequence (CpPCT531) obtained by mutating A 1200G in the base sequence of SEQ ID NO:1 and mutating Gly335Asp in an amino-acid sequence of SEQ ID NO: 2; a base sequence (CpPCT533) obtained by mutating T669C, Al 125G and Tl 158Ca in the base sequence of SEQ ID NO:1 and mutating Asp65Gly in the amino-acid sequence of SEQ ID NO: 2; a base sequence (CpPCT535) obtained by mutating T669C, A1125G, and T1158C in the base sequence of SEQ ID NO: 1 and mutating Asp65Asn in the amino-acid sequence of SEQ ID NO: 2; a base sequence (CpPCT537) obtained by mutating T669C, Al 125G, and Tl 158C in the base sequence of SEQ ID NO: 1 and mutating Thrl99Ile in the amino-acid sequence of SEQ ID NO: 2; a base sequence (CpPCT532) obtained by mutating A1200G in the base sequence of SEQ ID NO: 1 and mutating Ala243Thr in the amino-acid sequence of SEQ ID NO: 2; a base sequence (CpPCT534) obtained by mutating A1200G in the base sequence of SEQ ID NO: 1 and mutating Asp257Asn in the amino-acid sequence of SEQ ID NO: 2; and a base sequence (CpPCT540) obtained by mutating T78C, T669C, A1125G and T1158C in the base sequence of SEQ ID NO: 1 and mutating VaIl 93AIa in the amino-acid sequence of SEQ ID NO: 2. In particular, the CpPCT532 gene may be a mutant gene of a propionyl-CoA transferase from Clostridium propionicum that is preferable for producing a PLA or PLA copolymer using sucrose.
The cell or plant according to the present invention may include the gene of the PHA synthase using lactyl-CoA as the substrate, which is a gene of a PHA synthase from
Pseudomonas sp. 6-19. The gene of the PHA synthase using lactyl-CoA as the substrate may be a gene having an amino-acid sequence of SEQ ID NO: 4 or a gene having a base sequence corresponding to a mutant amino acid sequence in which at least one selected from the group consisting of El 3OD, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, and Q481R is mutated in the amino-acid sequence of SEQ ID NO: 4.
In particular, the phaClps6-19400 gene may be the gene of a PHA synthase from Pseudomonas sp. 6-19 that is preferable for producing the PLA or PLA copolymer using sucrose.
Also, the cell or plant according to the present invention may further include a gene of an enzyme for producing hydroxyacyl-CoA from sucrose. Since a recombinant cell or plant further including the gene of the enzyme for producing hydroxyacyl-CoA from sucrose can produce hydroxyacyl-CoA on its own, even if hydroxyalkanoate is not included in a medium, hydroxyalkanoate-lactate copolymer may be produced with high yield. In a specific example of the present invention, the enzymes for producing hydroxyacyl-CoA from sucrose may be a ketothiolase and an acetoacetyl-CoA reductase, but the present invention is not limited thereto. The ketothiolase and acetoacetyl-CoA reductase may be derived from Ralstonia eutropha.
In a specific example of the present invention, the cell capable of producing the
PLA or hydroxyalkanoate-lactate copolymer may be a bacterium, particularly, E. coli. The present invention provides a cell or plant that is transformed with a recombinant vector for producing a PLA or PLA copolymer containing a gene of an enzyme converting lactate into lactyl-CoA and a gene of a polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate and uses sucrose as a substrate. The cell or plant may further include a gene of an enzyme for producing 3-hydroxybutyl-CoA from sucrose.
A vector refers to a DNA construct containing a DNA sequence operably linked to a suitable control sequence capable of expressing DNA in an appropriate host. In the present invention, the vector may be a plasmid vector, a bacteriophage vector, a cosmid vector, or a yeast artificial chromosome (YAC) vector. For the purpose of the present invention, a plasmid vector may be used. A typical plasmid vector that serves the purpose of the invention includes (a) a replication origin that allows efficient replication such that each host cell includes several hundred plasmid vectors, (b) an antibiotic-resistance gene that allows selection of a host cell transformed into a plasmid vector, and (c) a restriction enzyme cleavage site in which a foreign DNA fragment may be inserted. Even if there is no appropriate restriction enzyme cleavage site, a vector may be easily ligated with foreign DNA by an ordinary method using a synthetic oligonucleotide adaptor or a linker.
After the ligation process, the vector has to be transformed into an appropriate host cell. In the present invention, the host cell may be a prokaryotic cell or a eukaryotic cell. Preferably, the host cell is a prokaryotic cell. The prokaryotic cell may be a microorganism having one of the above-described three genes or a microorganism having none of the above-described three genes, for example, E.coli. The E.coli may include E.coli strain DH5a, E.coli strain JMlOl, E.coli K12, E.coli W3110, E.coli X1776, E.coli XLl -Blue (Stratagene), E.coli B, etc. However, E.coli strains, such as FMBlOl, NM522,
NM538, and NM539, and other prokaryotic species and genera may be also used. In addition to the above-described E.coli and microorganisms having genes of the PHA synthase, Agrobacterium sp. strains such as Agrobacterium A4, bacilli such as Bacillus subtilis, and other enterobacteria such as Salmonella typhimurium or Serratia marcescens, may be used as host cells. Known eukaryotic host cells such as yeast and mold, insect cells such as spodoptera frugiperda (SF9), animal cells such as Chinese hamster ovary (CHO) cells and mouse cells, and tissue-cultured human and plant cells may be used as host cells. When the vector is transformed into an appropriate host, the vector is capable of replicating or functioning irrespective of a host genome or, in some cases, being integrated with the genome itself. As is known to one skilled in the art, in order to raise an expression level of a transformed gene in a host cell, the transformed gene must be operably linked to an expression control sequence that performs transcription and translation functions in a selected expression host. Preferably, the expression control sequence and the transformed gene are included in a single expression vector including both a bacterial selectable marker and a replication origin. When an expression host is a eukaryotic cell, the expression vector must further include an expression marker that is useful in the eukaryotic expression host.
The term "expression control sequence" means a DNA sequence essential for expression of an operably linked coding sequence in a specific host. The control sequence includes a promoter required for transcription, an arbitrary operator sequence for controlling the transcription, a sequence for encoding an appropriate mRNA ribosome binding site (RBS), and a sequence for controlling termination of transcription and translation. For example, a control sequence suitable for a prokaryote includes a promoter, a random operator sequence, and an RBS. A control sequence suitable for a eukaryotic cell includes a promoter, a polyadenylation signal, and an enhancer. The most important factor that affects the expression amount of a gene in a plasmid is a promoter. A SRa promoter or a cytomegalovirus promoter may be used as a high-expression promoter.
In order to express a DNA sequence of the present invention, any one of various expression control sequences may be used for a vector. The expression control sequence may be, for example, early and late promoters of SV40 or adenovirus, the lac system, the trp system, the tac system, the trc system, T3 and T7 promoters, the major operator and promoter regions of phage λ, the control region of fd code protein, the promoters of 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of phosphatase, for example, the promoters, e.g., Pho5, the promoter of a yeast α-mating system, other sequences with configurations or derivations known to control expression of a prokaryotic or eukaryotic cell or their viruses, and various combinations thereof.
When a nucleic acid is disposed in a functional relationship with another nucleic acid sequence, it is operably linked to the nucleic acid sequence. An appropriate molecule (e.g., a transcription-activating protein) may be a gene and control sequence(s) that are linked in such a manner as to enable expression of the gene when it couple with the control sequence(s). For example, DNA of a pre-sequence or a secretory leader is operably linked to DNA of polypeptide when it is expressed as a pre-protein that participates in secretion of polypeptide; a promoter or enhancer is operably linked to a coding sequence when it affects transcription of the coding sequence; an RBS is operably linked to a coding sequence when it affects transcription of the coding sequence; or the RBS is operably linked to the coding sequence when it is disposed to facilitate translation. In general, "operably linked sequences" means that DNA sequences being linked are contiguous, and in case of the secretory leader contiguous and in a reading frame. However, an enhancer does not have to contact a coding sequence. Linkage between sequences may be performed by ligation in a convenient restriction enzyme site. However, when there is no restriction enzyme site, a synthetic oligonucleotide adaptor or a linker may be used according to an ordinary method. Of course, it should be understood that all vectors and expression control sequences do not equally function to express DNA sequences according to the present invention. Similarly, all hosts do not equally function with respect to the same expression system. However, one skilled in the art may appropriately select from among various vectors, expression control sequences, and hosts without either departing from the scope of the present invention or bearing excessive experimental burden. For example, a vector must be selected considering a host because the vector must be replicated in the host. Specifically, the copy number and copy number control of the vector and expression of other protein (e.g., an antibiotic marker) encoded by the corresponding vector must be also considered. One skilled in the art may select an appropriate combination out of various vectors, expression control sequences, and hosts within appropriate ranges of the above- described variables.
Also, the transformation of a eukaryotic cell may be easily accomplished using a calcium chloride method described in section 1.82, supra by Sambrook et al. Alternatively, electroporation may be employed for transformation of these cells(Neumann et al., EMBO J., 1:841(1982)).
Meanwhile, transformation of a plant may be achieved by an ordinary method using Agrobacterium, a virus vector, etc. For example, a microorganism is transformed with a recombinant vector containing a gene according to the present invention, and the transformed Agrobacterium sp. microorganism may infect tissue of a target plant, thereby obtaining a transformed plant. For instance, a transformed plant according to the present invention may be obtained in the same manner as or in a similar manner to a method of producing PHA using a transformed plant disclosed in WO 94/11519 or US 6,103,956. More specifically, production of a transfected plant may involve (a) pre-culturing an explant of a target plant and co-culturing the explant with transformed agrobacterium for transfection of the plant; (b) culturing the transfected explant in a callus-inducing medium to obtain a callus; and (c) cleaving the callus and culturing the cleaved callus in a shoot- inducing medium to obtain a shoot.
In the present invention, the term "explant" refers to a tissue fragment cut off from a plant and includes a cotyledon or a hypocotyl. The cotyledon or hypocotyl may be used as an explant of a plant used for the method of the present invention. The cotyledon, which is obtained by disinfecting and cleaning a seed of a plant and germinating the seed in a Murashige and Skoog (MS) medium, is preferably used.
In the present invention, target plants to be transformed may be tobacco, tomatoes, red peppers, beans, rice plants, corn, etc, but the present invention is not limited thereto. Also, it is known to one skilled in the art that even if a plant used for transformation is sexually reproducible, it can be asexually reproduced by tissue culture, etc. [Advantageous Effects]
As explained above, the present invention provides a cell or plant capable of efficiently producing a polylactate (PLA) or PLA copolymer from sucrose and a method of producing PLA and PLA copolymer by growing or culturing the cell or plant. [Description of Drawings] FIG. 1 is a schematic diagram of a process of preparing a recombinant expression vector containing a mutant gene of a polyhydroxyalkanoate (PHA) synthase from
Pseudomonas sp. 6-19, a mutant gene of a propionyl-CoA transferase from Clostridium propionicum, and a gene of a phaAB from Ralstonia eutropha according to an example of the present invention.
[Modes of the Invention]
Hereinafter, examples of the present invention will be described in detail. However, the present invention is not limited to the examples disclosed below, but can be implemented in various modified forms. The present examples are provided to fully enable those of ordinary skill in the art to embody and practice the invention Example 1: Preparation of a recombinant E.coli
A recombinant E.coli W transformed with a pPs619C1400CPPCT532ReAB plasmid was prepared. The E.coli strain W (ATCC9637) according to the present Example may metabolize sucrose as a carbon source. The pPs619C1400CPPCT532ReAB plasmid prepared for the present invention was constructed to express four kinds of major enzymes. The major enzymes are essential for biosynthesis of poly(3-hydroxybutyrate-co-lactate), which is a biodegradable polymer, in the E. coli. The major enzymes may be phaClPs6-i9400, which is a polymerase from Pseudomonas sp. 6-19 (KCTC 11027BP), a propionyl-CoA transferase from Clostridium propionicum (CP-PCT), which is an enzyme transferring CoA from acetyl-CoA to lactate to be converted into lactyl-CoA, and a ketothiolase(phaARE) and an acetoacetyl-CoA reductase (phaBRE) from Ralstonia eutropha, which are enzymes for synthesizing 3- hydroxybutyl-CoA from sucrose. The pPs619C1400CPPCT532ReAB plasmid includes phaClps6-j9400, CPPCT532, phaARE and phaBRE genes respectively encoding the above major enzymes (refer to FIG. 1).
Also, the phaClPsό- 19400 and CPPCT532 genes are respectively obtained by mutating a gene of phaC Ip56-19, which is a PHA synthase from Pseudomonas sp. of SEQ ID NO: 3, and a gene of a CP-PCT of SEQ ID NO: 1, to be advantageous for PLA and PLA copolymer. The pPs619C1400CPPCT532ReAB plasmid is prepared to constitutively express all four genes in the recombinant E. coli.
Example 1-1: Preparation of a substrate-specific mutant of a PHA synthase from Pseudomonas sp. 6-19 Among various kinds of PHA synthases, a Type II PHA synthase is known as a medium-chain-length PHA (MCL-PHA) synthase for polymerizing a substrate having relatively many carbon atoms, and the MCL-PHA synthase is expected to be very useful in production of a PLA copolymer. Although a phaCl synthase from Pseudomonas sp. 61-3, which has a high homology with the phaClps6-19 synthase obtained according to the present invention, is the Type II synthase, it has been reported that the phaCl synthase has a relatively wide range of substrate specificity (Matsusaki et al., J. Bacteriol., 180:6459, 1998), and results of research into a mutant suitable for production of short-chain-length PHA (SCL-PHA) were reported (Takase et al., Biomacromolecules, 5:480, 2004). Based on the above research, the present inventors prepared mutants of a phaClpS6-i9 synthase by an SDM method using amino acids affecting SCL activation, as disclosed in Korean Patent Application No. 10-2006-0116234.
More specifically, in order to separate a PHA synthase ((phaClps6-i9) gene from Pseudomonas sp. 6-19(KCTC 11027BP), the total DNA of Pseudomonas sp. 6-19 was extracted, primers having base sequences of SEQ ID NOs: 5 and 6 were prepared based on the phaClps6-i9 base sequence (Ae-jin Song, Master's Thesis, Department of Chemical and Biomolecular Engineering, KAIST, 2004), and polymerase chain reaction (PCR) was performed to obtain the phaClPs6-19 gene.
SEQ ID NO: 5: 5'-GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG-3'
SEQ ID NO:6: 5'-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3'
When Agarose gel electrophoresis was performed on a PCR reactant, a 1.7-kbp gene fragment corresponding to the phaClps6-19 gene was confirmed. In order to express the phaC Ip56-19 synthase, an operon-type constitutive expression system in which a monomer supplying enzyme and a synthase are expressed together was adopted.
From a pSYL105 vector (Lee et al, Biotech. Bioeng., 1994, 44:1337-1347), a DNA fragment containing a PHB producing operon from Ralstonia eutropha Hl 6 was cleaved with BamHI/EcoRI, and then inserted into a BamHI/EcoRI restriction site of pBluescript II (Stratagene), thereby preparing a pReCAB recombinant vector. It is known that the pReCAB vector constitutively expresses a PHA synthase
(phaCRE) and monomer-supplying enzymes (phaARE and phaBRg) by a PHB operon promoter, and also effectively operated in E.coli (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347). The pReCAB vector was cleaved with BstBI/Sbfl to remove a R. eutropha Hl 6 PHA synthase (phaCRβ), and the phaClps6-i9 gene was inserted into a BstBI/Sbfl restriction site, thereby preparing a pPs619C 1 -ReAB recombinant vector.
In order to prepare a phaClps6-i9 synthase gene fragment having only a BstBI/Sbfl restriction site at an either end, an endogenous BstBI site was removed by site-directed mutagenesis (SDM) without changes of amino acids, and the BstBI/Sbfl restriction site was added by overlapping PCR using primers having base sequences of SEQ ID NOs: 7 and 8, 9 and 10, and 11 and 12.
SEQ ID NO: 7: 5'- atg ccc gga gcc ggt teg aa -3'
SEQ ID NO: 8: 5'- CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC -3'
SEQ ID NO: 9: 51- GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG -3'
SEQ ID NO: 10: 5'- CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC -31 SEQ ID NO: 11: 5'- GTA CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG -3'
SEQ ID NO: 12: 5'- aac ggg agg gaa cct gca gg - 3'
The sequence of the phaClps6-19 gene of the prepared pPs619Cl-ReAB recombinant vector was confirmed by sequencing and represented as SEQ ID NO: 3, and a sequence of an amino acid encoded by the base sequence of SEQ ID NO: 3 was represented as SEQ ID NO: 4.
In order to check if the phaClps6-i9 synthase produces PHB or not, the pPs619Cl- ReAB recombinant vector was transformed into E.coli XL-lBlue(Stratagene), and then the transformed E.coli was grown in a PHB detection medium (Luria Bertani (LB) agar, glucose 20g/L, Nile red 0.5 μ g/ml). As a result, PHB production was not detected.
Three amino-acid sites affecting SCL activity were found via amino-acid sequence analysis, and mutants of a phaClps6-19 synthase were prepared by SDM using primers of SEQ ID NOs: 13 to 18 as shown in Table 1.
[Table 1]
Figure imgf000019_0001
S325T
SEQ ID NO: 13: 5'- CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC Aces'
SEQ ID NO: 14: 5'- GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG- 3'
Q481M
SEQ ID NO: 15: 5'- CGA GCA GCG GGC ATA TC A TGA GCA TCC TGA ACC CGC- 3'
SEQ ID NO: 16: 5'- GCG GGT TCA GGA TGC TCA TGA TAT GCC CGC TGC TCG- 3'
E130D
SEQ ID NO: 17: 5'- ate aac etc atg ace gat gcg atg gcg ccg ace- 3' SEQ ID NO: 18: 5'- ggt egg cgc cat cgc ate ggt cat gag gtt gat- 3' The recombinant vectors were transformed into E.coli XL-I Blue and grown in a PHB detection medium (LB agar, glucose 20g/L, Nile red 0.5μ g/ml). As a result, production of PHB could be confirmed in both E.coli XL-I Blue transformed with pPs619C1200-ReAB and E.coli XL-lBlue transformed with pPs619C1300-ReAB. That is, 3HB-CoA was produced from glucose due to monomer-supplying enzymes, phaARE and phaBRE, and phaClPs6-19 synthase SCL mutants (phaClPs6-i9200 & phaClPs6-i9300) synthesized PHB using 3HB-CoA as a substrate.
Here, in order to construct an operon-type constitutive expression system expressing a propionyl-CoA transferase from Clostridium propionicum (CP-PCT) for providing lactyl-CoA functioning as a monomer required for synthesis of PLA and PLA copolymer, a cp-pct gene was used. For the cp-pct gene, a DNA fragment obtained by polymerizing chromosomal DNA of Clostridium propionicum was cloned by PCR using primers of SEQ ID NOs: 19 and 20. In this case, an Ndel site existing in a wild-type cp- pct gene was removed by SDM to facilitate cloning. SEQ ID NO: 19: 5 '-ggaattcATGAGAA AGGTTCCC ATTATTACCGC AGATGA
SEQ ID NO: 20: 5'-gc tctaga tta gga ctt cat ttc ctt cag ace cat taa gcc ttc tg
Also, overlapping PCR was performed using primers of SEQ ID NOs: 21 and 22 in order to add a Sbfl/Ndel restriction site.
SEQ ID NO: 21 : 5'-agg cct gca ggc gga taa caa ttt cac aca gg- 3' SEQ ID NO: 22: 5 '-gcc cat atg tct aga tta gga ctt cat ttc c- 3 '
A pPs619C1300-ReAB vector containing phaClps6-19300, which is a phaClps6-19 synthase SCL mutant, was cleaved with Sbfl/Ndel to remove the monomer-supplying enzymes (phaARE and phaBRE) from Ralstonia eutropha Hl 6, and the PCR-cloned cp-pct gene was inserted into the Sbfl/Ndel restriction site, thereby producing a pPs619C1300- CPPCT recombinant vector.
Similarly, various PHA synthase mutants were produced using the following primers. The produced mutants were arranged in the following Tables 2 through 5.
E130D
SEQ ID NO: 17: 5 '-ate aac etc atg ace gat gcg atg gcg ccg ace- 3' SEQ ID NO: 18: 5' -ggt egg cgc cat cgc ate ggt cat gag gtt gat- 3'
S325T
SEQ ID NO: 13: 5'-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC Aces' SEQ ID NO: 14: 5'- GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG- 3'
S477R
SEQ ID NO: 23: 5'-gaa ttc gtg ctg teg age cgc ggg cat ate- 3'
SEQ ID NO: 24: 5'-gat atg ccc gcg get cga cag cac gaa ttc- 3' S477H
SEQ ID NO: 25: 5'-gaa ttc gtg ctg teg age cat ggg cat ate- 3'
SEQ ID NO: 26: 5'-gat atg ccc atg get cga cag cac gaa ttc- 3'
S477F
SEQ ID NO: 27: 5'- gaa ttc gtg ctg teg age ttt ggg cat ate- 3' SEQ ID NO: 28: 5'- gat atg ccc aaa get cga cag cac gaa ttc- 3'
S477Y
SEQ ID NO: 29: 5'-gaa ttc gtg ctg teg age tat ggg cat ate- 3'
SEQ ID NO: 30: 5'-gat atg ccc ata get cga cag cac gaa ttc- 3'
S477G SEQ ID NO: 31 : 5'-gaa ttc gtg ctg teg age ggc ggg cat ate- 3'
SEQ ID NO: 32: 5'-gat atg ccc gee get cga cag cac gaa ttc- 3'
Q481K
SEQ ID NO: 33: 5'-ggg cat ate aaa age ate ctg aac ccg c- 3'
SEQ ID NO: 34: 5'-gcg ggt tea gga tgc ttt tga tat gee c- 3' Q481M SEQ ID NO: 35: 5'-ggg cat ate atg age ate ctg aac ccg c- 3' SEQ ID NO: 36: 5'-gcg ggt tea gga tgc tea tga tat gcc c- 3' Q481R SEQ ID NO: 37: 5'-ggg cat ate cgc age ate ctg aac ccg c- 3' SEQ ID NO: 38: 5'-gcg ggt tea gga tgc tgc gga tat gcc c- 3' [Table 2]
Figure imgf000022_0001
[Table 3]
Figure imgf000022_0002
Figure imgf000023_0001
[Table 4]
Figure imgf000023_0002
Figure imgf000024_0001
[Table 5]
Figure imgf000024_0002
Example 1-2: Preparation and screening of a library of a mutant of propionyl- CoA transferase from Clostridium propionicum
As is known, when CP-PCT is overexpressed in E.coli, it causes serious metabolic disorder and exhibits toxicity. In general, all recombinant E.coli were dead at the same time with addition of an inducer in an isopropyl-β -D-thio-galactoside (IPTG)-inducible protein expression system using a tac promoter or T7 promoter, which is widely used to express recombinant proteins. For this reason, synthesis of PLA and PLA copolymer was succeeded using a constitutive expression system weakly but continuously expressing a gene with growth of a microorganism. In order to introduce random mutation on a cp-pct gene, PPS619C1300-CPPCT disclosed in Korean Patent Application No. 10-2006-0116234 was used as a template, Mn2+ was added using primers of SEQ ID NOs: 39 and 40, and error-prone PCR was performed under conditions in which the concentrations of dNTPs varied.
SEQ ID NO: 39: 5'- cgc egg cag gcc tgc agg -3' SEQ ID NO: 40: 5'- ggc agg tea gcc cat atg tc -3'
Thereafter, in order to amplify a randomly mutated PCR fragment, PCR was performed under common conditions using the primer of SEQ ID NOs: 39 and 40. A pPs619C1300- CPPCT vector containing phaClpS6-i9300, which is a phaClps6-i9 synthase, was cleaved with Sbfl/Ndel to remove a wild-type cp-pct gene, and the amplified mutated PCR fragment was inserted into a Sbfl/Ndel restriction site to produce a ligation mixture. The ligation mixture was introduced to E. coli JMl 09, thereby preparing a CP-PCT library with a scale of about -lO"5. The prepared CP-PCT library was incubated for 3 days in a polymer detection medium (LB agar, glucose 20g/L, 3HB lg/L, Nile red 0.5μ g/ml) and screened to confirm whether a polymer was produced, thereby primarily selecting up to 80 candidates. The candidates were grown for 4 days in a liquid medium (LB agar, glucose 20g/L, 3HB lg/L, ampicillin 100mg/L, 37 "C) under polymer-generating conditions and analyzed by florescence activated cell sorting (FACS), thereby selecting two final samples. In order to find out the positions of the prepared CP-PCT mutants, gene sequence analysis of the CP-PCT mutants was performed as shown in Table 6. [Table 6]
Figure imgf000026_0001
Random mutagenesis was performed on the finally selected mutants 512 and 522 using the above-described error-prone PCR, thereby obtaining the following CP-PCT mutants 531- 537. [Table 7]
Figure imgf000026_0002
Thereafter, in order to amplify a PCR fragment including the CpPct532 mutant, a PCR method was performed under common conditions using the primers of SEQ ID NOs: 39 and 40. The PPS619C1300-CPPCT vector was cleaved with Sbfl/Ndel to remove a wild-type CP-PCT gene, and the amplified CpPct532 PCR fragment was inserted into a Sbfl/Ndel restriction site to produce a ligation mixture, thereby producing the pPs619C1300-CPPCT532 vector used in the following Example 1-3.
Example 1-3: Preparation of the pPs619C1300-CPPCT532 vector
A PHA synthase mutant (phaClPs6-19400) from Pseudomonas sp. 6-19, which has an amino-acid sequence in which E130D, S325T, S477R, and Q481M were mutated, was prepared using the phaClpS6-i9 synthase mutant (phaClps6-i9300) by SDM using primers of SEQ ID NOs: 41 and 42.
SEQ ID NO: 41: 5'- ttc gtg ctg teg age aga ggg cat ate- 3' SEQ ID NO: 42: 5'- gat atg ccc tct get cga cag cac gaa- 3' The resultant recombinant vector (pPsό 19C 1400-CPPCT532) was transformed into
E.coli JMl 09 and grown in a 3HB-containing polymer detection medium (LB agar, glucose 20g/L, 3HB 2g/L, Nile red 0.5μ g/ml). As a result, polymer production was detected.
Example 1-4: Preparation of a pPs619C1400CPPCT532ReAB plasmid and transformation of E.coli
In order to produce plasmids containing phaClps6-i9400, CPPCT532, phaARE, and phaBRE genes respectively encoding enzymes required for efficient production of PLA and PLA copolymer from sucrose, PCR cloning was performed with respect to a PHB - producting promoter pReC and phaARE and phaBRE genes from Ralstonia eutropha Hl 6 on a pSYL105 vector (Lee et al., Biotech. Bioeng., 1994, 44:1337-1347) using primers of SEQ ID NOs: 43 and 44 and primers of SEQ ID NOs: 45 and 46, respectively. SEQ ID NO: 43: 5'- aga cat atg caa gta cct tgc cga cat eta tg- 3' SEQ ID NO: 44: 5'- gat gac aac gtc agt cat gat ttg att gtc tct ctg - 3'
SEQ ID NO: 45: 5'- cag aga gac aat caa ate atg act gac gtt gtc ate - 3' SEQ ID NO: 46: 5'- gca ggt cag ccc ata tgc ag - 3'
A PCR reactant was purified on an agarose gel, and the overlapping PCR was performed using the PCR reactant as a template and the primers of SEQ ID NOs: 43 and
46. The PCR reactant and the pPs619C1400CPPCT532 vector were cleaved with Ndel, and the PCR reactant (pReC-phaABRE) was inserted into a Ndel restriction site of the pPs619C1400CPPCT532 vector, thereby completing a pPs619C1400CPPCT532ReAB vector as shown in FIG. 1. In the pPs619C1400CPPCT532ReAB vector, the PHB- producting promoter (pReC) was additionally inserted into the front portions of the phaARE and phaBRE genes in order to further promote transcription of the target phaARE and phaBRE genes.
The recombinant vector (pPs619C1400CPPCT532ReAB) was transformed into E.coli JM 109, and grown in a 3HB-free polymer detection medium (LB agar, glucose 20g/L, Nile red 0.5μ g/ml). As a result, polymer production could be observed. Thus, it could be confirmed that the phaARE and phaBRE genes additionally inserted into the pPs619C1400CPPCT532 vector were expressed and operated normally.
Example 2: Preparation of poly(3-hydroxybutyrate-co-lactate) (P(3HB-co- LA)) from a recombinant E.coli using sucrose as a carbon source
A colony on an LB agar plate containing 100 mg/L of ampicillin as an antibiotic was inoculated into 3mL of a LB medium containing 100 mg/L of ampicillin and cultured for 12 hours at 30°C , while being stirred at 200rpm. ImL of the culture solution was inoculated into each of 10OmL methyl red (MR) medium and 10OmL LB medium, which contains 100 mg/L of ampicillin, 20 g/L of sucrose, and in some cases, 2 g/L of 3- hydroxybutyrate. Thereafter, each culture solution was cultured at about 30°C for 4 days, while being stirred at 200 rpm. The initial pH of the MR medium was adjusted to pH 7 using 10 N NaOH. The medium samples used in the present culturing process are shown in the following Table 8.
After the culture was finished, biomasses were retrieved from the culture solutions. The retrieved cells were cleaned 3 times with distilled water and dried for 24 hours in a dryer maintained at about 100°C. The dried cells were partially collected and underwent gas chromatographic (GC) analysis, thereby measuring the content of P(3HB-co-LA) synthesized in cells. Standard materials used for the GC analysis were a P(3HB-co-3HV) copolymer (including about 12% by weight 3HV) and a PLA homopolymer. The GC analysis results are shown in the following Table 9. As can be seen from Table 9, biosynthesis of a P(3HB-co-LA) copolymer was enabled in both the MR and LB media irrespective of the addition of 3HB. Also, it could be confirmed that the biosynthesis of the P(3HB-co-LA) copolymer was more efficient in the MR medium than in the LB medium. Therefore, the P(3HB-co-LA) copolymer could be produced from sucrose using the recombinant E. coli according to the present invention. [Table 8]
Figure imgf000029_0001
Figure imgf000030_0001
Tracer (/L): 1Og of FeSO4-H2O ; 2.25g of ZnSO4-H2O; Ig of CuSO4-H2O; 0.5g of MnSO4-H2O; 2g of CaCl2-H2O; 0.23g Of Na2B4O7-H2O; O.lg of (NH4)6Mo7O24; 1OmL of 35 % HCl. [Table 9]
Figure imgf000030_0002
* Su: 20 g/L of sucrose
** 3HB: 2g/L of 3-hydroxybutyrate
While the invention has been shown and described with reference to certain examples thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

[CLAIMS] [Claim 1 ]
A method of producing a polylactate (PLA) or hydroxyalkanoate-lactate copolymer from sucrose, comprising: culturing or growing a cell or plant in an environment containing lactate and sucrose or an environment containing lactate, sucrose, and hydroxyalkanoate, wherein the cell or plant comprises a gene of an enzyme converting lactate into lactyl-CoA and a gene of a polyhydroxyalkanoate (PHA) synthase using lactyl-CoA as a substrate, and is capable of producing the PLA or hydroxyalkanoate-lactate copolymer using sucrose as a substrate; and harvesting the PLA or hydroxyalkanoate-lactate copolymer from the cell or plant. [Claim 2]
The method according to claim 1, wherein the cell or plant is obtained by transforming a cell or plant that does not include at least one of the gene of the enzyme converting lactate into lactyl-CoA and the gene of the PHA synthase using lactyl-CoA as the substrate and is capable of using sucrose as a substrate, with at least one of the gene of the enzyme converting lactate into lactyl-CoA and the gene of the PHA synthase using lactyl-CoA as the substrate. [Claim 3] The method according to claim 1, wherein hydroxyalkanoate of the hydroxyalkanoate-lactate copolymer is at least one selected from the group consisting of 3- hydroxybutyrate, 3 -hydroxy valerate, 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 -hydroxy decanoic acid, 3- hydroxyundecanoic acid, 3 -hydroxy dodecanoic acid, 3-hydroxytetradecanoic acid, 3- hydroxyhexadecanoic acid, 4-hydroxyvaleric acid, 4-hydroxyhexanoic acid, 4- hydroxyheptanoic acid, 4-hydroxyoctanoic acid, 4-hydroxydecanoic acid, 5- hydroxyvaleric acid, 5-hydroxyhexanoic acid, 6-hydroxydodecanoic acid, 3- hydroxy-4- pentenoic acid, 3-hydroxy-4-trans-hexenoic acid, 3-hydroxy-4-cis-hexenoic acid, 3- hydroxy-5-hexenoic acid, 3-hydroxy-6-trans-octenoic acid, 3-hydroxy-6-cis-octenoic acid, 3-hydroxy-7-octenoic acid, 3-hydroxy-8-nonenoic acid, 3-hydroxy-9-decenoic acid, 3- hydroxy-5-cis-dodecenoic acid, 3-hydroxy-6-cis-dodecenoic acid, 3-hydroxy-5-cis- tetradecenoic acid, 3-hydroxy-7-cis-tetradecenoic acid, 3-hydroxy-5,8-cis-cis-tetradecenoic acid, 3-hydroxy-4-methylvaleric acid, 3-hydroxy-4-methylhexanoic acid, 3-hydroxy-5- methylhexanoic acid, 3-hydroxy-6-methylheptanoic acid, 3-hydroxy-4-methyloctanoic acid, 3-hydroxy-5-methyloctanoic acid, 3-hydroxy-6- methyloctanoic acid, 3-hydroxy-7- methyloctanoic acid, 3-hydroxy-6-methylnonanoic acid, 3-hydroxy-7-methylnonanoic acid, 3-hydroxy-8-methylnonanoic acid, 3-hydroxy-7-methyldecanoic acid, 3-hydroxy-9- methyldecanoic acid, 3-hydroxy-7-methyl-6-octenoic acid, malic acid, 3 -hydroxy succinic acid-methylester, 3 -hydroxy adipinic acid-methylester, 3-hydroxysuberic acid-methylester, 3-hydroxyazelaic acid-methylester, 3 -hydroxy sebacic acid-methylester, 3-hydroxysuberic acid-ethylester, 3 -hydroxy sebacic acid-ethylester, 3-hydroxypimelic acid-propylester, 3- hydroxysebacic acid-benzylester, 3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy-9- acetoxynonanoic acid, phenoxy-3-hydroxybutyric acid, phenoxy-3 -hydroxy valeric acid, phenoxy-3 -hydroxyheptanoic acid, phenoxy-3-hydroxyoctanoic acid, para-cyanophenoxy-
3-hydroxybutyric acid, para-cyanophenoxy-3 -hydroxy valeric acid, para-cyanophenoxy-3- hydroxyhexanoic acid, para-nitrophenoxy-3-hydroxyhexanoic acid, 3-hydroxy-5- phenylvaleric acid, S-hydroxy-S-cyclohexylbutyric acid, 3,12-dihydroxydodecanoic acid,
Sjδ-dihydroxy-S-cis-tetradecenoic acid, 3-hydroxy-4,5-epoxydecanoic acid, 3-hydroxy-
6,7-epoxydodecanoic acid, 3-hydroxy-8,9-epoxy-5,6-cis-tetradecanoic acid, 7-cyano-3- hydroxyheptanoic acid, 9-cyano-3-hydroxynonanoic acid, 3-hydroxy-7-fluoroheptanoic acid, 3-hydroxy-9-fluorononanoic acid, 3-hydroxy-6-chlorohexanoic acid, 3-hydroxy-8- chlorooctanoic acid, 3-hydroxy-6-bromohexanoic acid, 3-hydroxy-8-bromooctanoic acid,
3 -hydroxy- 11-bromoundecanoic acid, 3-hydroxy-2-butenoic acid, 6-hydroxy-3-dodecenoic acid, 3-hydroxy-2-methylbutyric acid, 3 -hydroxy-2-methyl valeric acid, and 3-hydroxy-2,6- dimethyl-5-heptenoic acid.
[Claim 4]
The method according to claim 1, wherein the gene of the enzyme converting lactate into lactyl-CoA is a propionyl-CoA transferase gene (pet).
[Claim 5] The method according to claim 1, wherein the gene of the enzyme converting lactate into lactyl-CoA is a pet gene from Clostridium propionicum.
[Claim 6]
The method according to claim 1, wherein the gene of the enzyme converting lactate into lactyl-CoA has one selected from the group consisting of: a base sequence of SEQ ID NO: 1 ; a base sequence obtained by mutating T78C, T669C, Al 125G, and Tl 158C in the base sequence of SEQ ID NO: 1 ; a base sequence obtained by mutating Al 200G in the base sequence of SEQ ID NO: 1; a base sequence obtained by mutating Al 200G in the base sequence of SEQ ID NO: 1 and mutating Gly335Asp in an amino-acid sequence of SEQ ID NO: 2; a base sequence obtained by mutating T669C, Al 125G, and Tl 158Ca in the base sequence of SEQ ID NO: 1 and mutating Asp65Gly in the amino-acid sequence of SEQ ID NO: 2; a base sequence obtained by mutating T669C, Al 125 G, and T1158C in the base sequence of SEQ ID NO: 1 and mutating Asp65Asn in an amino-acid sequence with SEQ ID NO: 2; a base sequence obtained by mutating T669C, A1125G, and T1158C in the base sequence of SEQ ID NO: 1 and mutating Thrl99Ile in the amino-acid sequence of SEQ ID NO: 2; a base sequence obtained by mutating A 1200G in the base sequence of SEQ ID NO: 1 and mutating Ala243Thr in the amino-acid sequence of SEQ ID NO: 2; a base sequence obtained by mutating A 1200G in the base sequence of SEQ ID NO: 1 and mutating Asp257Asn in the amino-acid sequence of SEQ ID NO: 2; and a base sequence obtained by mutating T78C, T669C, A1125G, and T1158C in the base sequence of SEQ ID NO: 1 and mutating Vall93Ala in the amino-acid sequence of SEQ ID NO: 2.
[Claim 7]
The method according to claim 6, wherein the gene of the enzyme converting lactate into lactyl-CoA has a base sequence obtained by mutating A 1200G in the base sequence of SEQ ID NO: 1 and mutating Ala243Thr in the amino-acid sequence of SEQ ID NO: 2.
[Claim 8]
The method according to claim 1 , wherein the gene of the PHA synthase using lactyl-CoA as the substrate is a gene of a PHA synthase from pseudomonas sp. 6-19.
[Claim 9]
The method according to claim 1, wherein the gene of the PHA synthase using lactyl-CoA as the substrate has a base sequence corresponding to: an amino-acid sequence of SEQ ID NO: 4; or an amino-acid sequence in which at least one selected from the group consisting of
E130D, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K, and Q481R is mutated in SEQ ID NO: 4.
[Claim 10]
The method according to claim 1, wherein the gene of the PHA synthase using lactyl-CoA as the substrate has a base sequence corresponding to an amino-acid sequence in which E130D, S325T, S477R, and Q481M are mutated in SEQ ID NO: 4.
[Claim 11 ]
The method according to any one of claims 1 to 3, wherein the cell or plant further comprises a gene of an enzyme producing hydroxyacyl-CoA from sucrose.
[Claim 12]
The method according to claim 11, wherein the enzyme producing hydroxyacyl- CoA from sucrose is a ketothiolase and an acetoacetyl-CoA reductase.
[Claim 13] The method according to claim 12, wherein the ketothiolase and the acetoacetyl-oA reductase are derived from Ralstonia eutropha. [Claim 14]
The method according to any one of claims 1 to 9, wherein the cell is a bacterium. [Claim 15]
The method according to claim 14, wherein the bacterium is an E.coli.
PCT/KR2008/004611 2007-09-07 2008-08-08 Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same WO2009031762A2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN200880105983.8A CN101835895B (en) 2007-09-07 2008-08-08 Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same
US12/676,832 US8420357B2 (en) 2007-09-07 2008-08-08 Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20070091063 2007-09-07
KR10-2007-0091063 2007-09-07
KR10-2008-0068607 2008-07-15
KR1020080068607A KR101037354B1 (en) 2007-09-07 2008-07-15 Recombinant microorganism able to produce polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same

Publications (2)

Publication Number Publication Date
WO2009031762A2 true WO2009031762A2 (en) 2009-03-12
WO2009031762A3 WO2009031762A3 (en) 2009-04-30

Family

ID=40429509

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2008/004611 WO2009031762A2 (en) 2007-09-07 2008-08-08 Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same

Country Status (1)

Country Link
WO (1) WO2009031762A2 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101942181A (en) * 2009-07-06 2011-01-12 株式会社理光 The method of copolymer resin composition, moulded product and manufacturing copolymer resin composition
WO2011074842A2 (en) * 2009-12-14 2011-06-23 (주)Lg화학 Recombinant microorganism for producing polylactic acid or a polylactic acid copolymer from glycerol and method for producing polylactic acid or a polylactic acid copolymer from glycerol using the microorganism
WO2011118671A1 (en) * 2010-03-25 2011-09-29 トヨタ自動車株式会社 Genetically modified microorganism and production method for aliphatic polyester using same
EP2377945A1 (en) * 2008-10-27 2011-10-19 Toyota Jidosha Kabushiki Kaisha Process for producing polylactic acid using genetically modified bacterium
EP2636744A1 (en) * 2007-08-14 2013-09-11 LG Chem, Ltd. Mutant of propionyl-CoA transferase from Clostridium propionicum and preparing method for PLA or PLA copolymer using the same
US8541652B2 (en) 2006-11-23 2013-09-24 Lg Chem, Ltd. Mutants of PHA synthase from Pseudomonas sp. 6-19 and method for preparing lactate homopolymer or copolymer using the same
US8563281B2 (en) 2008-04-23 2013-10-22 Toyota Jidosha Kabushiki Kaisha Method for production of polyester copolymer using recombinant microorganism
CN114196609A (en) * 2021-12-31 2022-03-18 淮阴工学院 Escherichia coli engineering bacterium for synthesizing pure polylactic acid from lactic acid and preparation method and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6492134B1 (en) * 1999-03-15 2002-12-10 UNIVERSITé LAVAL Method for producing polyhydroxyalkanoates in recombinant organisms
WO2006126796A1 (en) * 2005-05-24 2006-11-30 Lg Chem, Ltd. Cells or plants having a producing ability of polylactate or its copolymers and method for preparing polylactate or its copolymers using the same
US7169598B2 (en) * 1999-12-27 2007-01-30 Canon Kabushiki Kaisha Polyhydroxyalkanoate, method for production thereof and microorganisms for use in the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6492134B1 (en) * 1999-03-15 2002-12-10 UNIVERSITé LAVAL Method for producing polyhydroxyalkanoates in recombinant organisms
US7169598B2 (en) * 1999-12-27 2007-01-30 Canon Kabushiki Kaisha Polyhydroxyalkanoate, method for production thereof and microorganisms for use in the same
WO2006126796A1 (en) * 2005-05-24 2006-11-30 Lg Chem, Ltd. Cells or plants having a producing ability of polylactate or its copolymers and method for preparing polylactate or its copolymers using the same

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KIECHLE F.L. ET AL.: 'Lactate Production by Aerobic Bacteria Grown in Cerebrospinal Fluid' CLIN. CHEM. vol. 30, no. 11, 1984, pages 1875 - 1876 *
LONG YU ET AL.: 'Polymer blends and composites from renewable resources' PROG. POLYM. SCI. vol. 31, 2006, pages 576 - 602 *
LUENGO J.M. ET AL.: 'Bio-plastics from microorganisms' CURRENT OPINION IN MICROBIOLOGY vol. 6, 2003, pages 251 - 260 *
SANG YUP LEE: 'Bacterial Polyhydroxyalkanoates' BIOTECHNOLOGY AND BIOENGINEERING vol. 49, 1996, pages 1 - 14 *
SURIYAMONGKOL P. ET AL.: 'Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants' BIOTECHNOLOGY ADVANCES vol. 25, March 2007, pages 148 - 175 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8541652B2 (en) 2006-11-23 2013-09-24 Lg Chem, Ltd. Mutants of PHA synthase from Pseudomonas sp. 6-19 and method for preparing lactate homopolymer or copolymer using the same
EP2636744A1 (en) * 2007-08-14 2013-09-11 LG Chem, Ltd. Mutant of propionyl-CoA transferase from Clostridium propionicum and preparing method for PLA or PLA copolymer using the same
US8563281B2 (en) 2008-04-23 2013-10-22 Toyota Jidosha Kabushiki Kaisha Method for production of polyester copolymer using recombinant microorganism
EP2963120A1 (en) * 2008-10-27 2016-01-06 Toyota Jidosha Kabushiki Kaisha Method for production of polylactate using recombinant microorganism
US8748137B2 (en) 2008-10-27 2014-06-10 Toyota Jidosha Kabushiki Kaisha Method for production of polylactate using recombinant microorganism
EP2377945A1 (en) * 2008-10-27 2011-10-19 Toyota Jidosha Kabushiki Kaisha Process for producing polylactic acid using genetically modified bacterium
EP2377945A4 (en) * 2008-10-27 2012-08-08 Toyota Motor Co Ltd Method for production of polylactate using recombinant microorganism
CN101942181B (en) * 2009-07-06 2013-01-16 株式会社理光 Copolymer resin composition, molded product, and method of manufacturing a copolymer resin composition
CN101942181A (en) * 2009-07-06 2011-01-12 株式会社理光 The method of copolymer resin composition, moulded product and manufacturing copolymer resin composition
WO2011074842A3 (en) * 2009-12-14 2011-11-03 (주)Lg화학 Recombinant microorganism for producing polylactic acid or a polylactic acid copolymer from glycerol and method for producing polylactic acid or a polylactic acid copolymer from glycerol using the microorganism
WO2011074842A2 (en) * 2009-12-14 2011-06-23 (주)Lg화학 Recombinant microorganism for producing polylactic acid or a polylactic acid copolymer from glycerol and method for producing polylactic acid or a polylactic acid copolymer from glycerol using the microorganism
CN102844439A (en) * 2010-03-25 2012-12-26 丰田自动车株式会社 Recombinant microorganism and method for producing aliphatic polyester by using the same
JP2011200153A (en) * 2010-03-25 2011-10-13 Toyota Motor Corp Recombinant microorganism and method for producing aliphatic polyester by using the same
CN102844439B (en) * 2010-03-25 2014-06-04 丰田自动车株式会社 Recombinant microorganism and method for producing aliphatic polyester by using the same
WO2011118671A1 (en) * 2010-03-25 2011-09-29 トヨタ自動車株式会社 Genetically modified microorganism and production method for aliphatic polyester using same
KR101458495B1 (en) * 2010-03-25 2014-11-07 도요타 지도샤（주） Genetically modified microorganism and production method for aliphatic polyester using same
US8980615B2 (en) 2010-03-25 2015-03-17 Toyota Jidosha Kabushiki Kaisha Recombinant microorganism and method for producing aliphatic polyester using the same
CN114196609A (en) * 2021-12-31 2022-03-18 淮阴工学院 Escherichia coli engineering bacterium for synthesizing pure polylactic acid from lactic acid and preparation method and application thereof
CN114196609B (en) * 2021-12-31 2023-06-23 淮阴工学院 Escherichia coli engineering bacteria for synthesizing pure polylactic acid from lactic acid, and preparation method and application thereof

Also Published As

Publication number Publication date
WO2009031762A3 (en) 2009-04-30

Similar Documents

Publication Publication Date Title
US9957533B2 (en) Mutant of propionyl-CoA transferase from Clostridium propionicum and preparing method for PLA or PLA copolymer using the same
US8420357B2 (en) Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same
US8541652B2 (en) Mutants of PHA synthase from Pseudomonas sp. 6-19 and method for preparing lactate homopolymer or copolymer using the same
KR101211767B1 (en) Mutants of PHA Synthases and Method for Preparing PLA and PLA Copolymer Using the Same
US20100136637A1 (en) Recombinant microorganism having a producing ability of polylactate or its copolymers and method for preparing polyactate or its copolymers using the same
WO2009031762A2 (en) Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same
KR101293904B1 (en) Recombinant microorganism capable of producing polylactate or polylactate copolymer from xylose and method for producing polylactate or polylactate copolymer from xylose using the same
KR101260187B1 (en) Recombinant microorganism able to produce polylactate or polylactate copolymer from glycerol and method for producing polylactate or polylactate copolymer from glycerol using the same

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200880105983.8

Country of ref document: CN

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

Ref document number: 08793125

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 12676832

Country of ref document: US

122 Ep: pct application non-entry in european phase

Ref document number: 08793125

Country of ref document: EP

Kind code of ref document: A2