Patent Application
20100222545
The present invention relates to copolymer comprising 4-hydroxybutyrate monomer unit and lactate monomer unit or copolymer comprising 4-hydroxybutyrate monomer unit, lactate monomer unit and 3-hydroxyalkanoate monomer unit, and a method for manufacturing such polymer.
Polylactate (PLA) is a typical biodegradable polymer originated from lactate, which has a variety of applications as a common or a medical polymer. At present, PLA is being prepared by polymerizing lactate which is produced by fermenting microorganisms, but only low molecular weight PLA (1000-5000 dalton) is produced by direct polymerization of lactate. To synthesize high molecular weight (>100,000 dalton) of PLA, a method polymerizing low molecular weight PLA obtained by direct polymerization of lactate with a chain coupling agent can be used. However, it has disadvantages like that the process for preparing PLA of high molecular weight is complicated due to the addition of a solvent or a chain coupling agent, and also it isn't easy to remove them. At present, in the process for preparing commercially available PLA of high molecular weight, a method, in which lactate is converted into lactide to synthesize PLA by cyclodehydration of the lactide ring, is being used.
Meanwhile, polyhydroxyalkanoate (PHA) is a polyester which microorganisms accumulate therein as a carbon and energy storage compound when other nutritive elements, for example, phosphorus, nitrogen, magnesium, oxygen, are deficient while the carbon source is in excess. PHA is recognized as an alternative material for synthesized plastics since it has similar properties to synthetic polymers originating from petroleum, and, at the same time, shows an excellent biodegradation property.
The existing PHA is divided into SCL-PHA (short-chain-length PHA) having short carbon chains and MCL-PHA (medium-chain-length PHA) having long carbon chains. A gene synthesizing PHA was cloned from Ralstonia eutropha, Pseudomonas sp. Microorganism, and PHA consisting of various monomers was synthesized by recombinant microorganisms (Qi et al., FEMS Microbiol. Lett., 157:155, 1997; Qi et al., FEMS Microbiol. Lett., 167:89, 1998; Langenbach et al., FEMS Microbiol. Lett., 150:303, 1997; WO 01/55436; U.S. Pat. No. 6,143,952; WO 98/54329; and WO 99/61624).
To produce PHA in microorganisms, an enzyme which converts microorganisms' metabolites into a PHA monomer and PHA synthase which synthesizes a PHA polymer using the PHA monomers are required. PHA synthase synthesizes PHA using hydroxyacyl-CoA as a substrate and alpha-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), cloned from Ralstonia eutropha etc., 3-hydroxydecanoyl-ACP:CoA transferase (PhaG) cloned from Pseudomonas sp., (R)-specific enoyl-CoA hydratase (PhaJ) derived from Aeromonas caviae and Pseudomonas aeruginosa (Fukui et al., J. Bacteriol., 180:667, 1998; Tsage et al., FEMS Microbiol. Lett., 184:193, 2000), 3-ketoacyl-ACP reductase (FabG) derived from E. coli, Pseudomonas aeruginosa, etc. (Taguchi et al., FEMS Microbiol. Lett., 176:183, 1999; Ren et al., J. Bacteriol., 182:2978, 2000; Park et al., FEMS Microbiol. Lett., 214:217, 2002), phosphotransbutylase (Ptb) and butyrate kinase (Buk) derived from Clostridium acetobutyricum (Liu and Steinbuchel, Appl Environ Microbiol, 66:739, 2000), Cat2 derived from Clostridium kluyveri (Hein et al. FEMS Microbiol. Lett., 15:411, 1997), etc. are known as enzymes capable of generating hydroxyacyl-CoA which is a substrate of PHA. Various kinds of PHAs have been synthesized with these enzymes using hydroxyalkanoates hydroxylated at various positions in the carbon chain (mainly the 3, 4, 5, and 6 positions).
However, it has been reported that it has little PHA synthase activity on hydroxyalkanoate which is hydroxylated at the 2-position (Zhang et al., Appl. Microbiol. Biotechnol., 56:131, 2001; Valentin and Steinbuchel, Appl. Microbiol. Biotechnol., 40:699, 1994). Thus far, there have been reports of PHA synthase activity on lactyl-CoA measured in vitro, but PHA synthase activity on lactyl-CoA is very weak (Zhang et al., Appl. Microbiol. Biotechnol., 56:131, 2001; Valentin and Steinbuchel, Appl. Microbiol. Biotechnol., 40:699, 1994). That is, there are no examples of natural production or production by recombinant cells of PHA and its copolymers because a hydroalkanoate, such as lactate hydroxylated at the 2-carbon position, is not a suitable substrate for PHA synthase.
U.S. Patent application publication no. 20040076982 discloses a method that lactate is made from glucose, and lactyl-CoA is biosynthesized from lactate, and 3-hydroxyalkanoate-CoA is biosynthesized from lactyl-CoA. However, the publication does not disclose a method for preparing copolymer with lactyl-CoA and 3-hydroxyalkanoate-CoA.
Accordingly, the object of the present invention is to provide a copolymer comprising 4-hydroxybutyrate monomer unit and lactate monomer unit or a copolymer comprising 4-hydroxybutyrate monomer unit, lactate monomer unit and 3-hydroxyalkanoate monomer unit.
Another object of the present invention is to provide a method for preparing the copolymer.
To achieve the object, the present invention provides a copolymer comprising lactate monomer unit and 4-hydroxybutyrate monomer unit.
The present invention also provides a copolymer comprising lactate monomer unit, 4-hydroxybutyrate monomer and 3-hydroxyalkanoate monomer unit.
More preferably, the copolymer according to the present invention is 4-hydroxybutyrate-lactate copolymer (poly(4-hydroxybutyrate-co-lactate)), 4-hydroxybutyrate-3-hydroxypropionate-lactate terpolymer (poly(4-hydroxybutyrate-co-3-hydroxypropionate-co-lactate)), 3-hydroxybutylate-4-hydroxybutyrate-lactate terpolymer (poly(3-hydroxybutyrate-co-4-hydroxybutylate-co-lactate)), or 3-hydroxybutyrate-3-hydroxypropionate-4-hydroxybutyrate-lactate tetrapolymer (poly(3-hydroxybutyrate-co-3-hydroxypropionate-co-4-hydroxybutylate-co-lactate)).
The present invention also provides a method for preparing a copolymer comprising lactate monomer unit and 4-hydroxybutyrate monomer unit, wherein the method comprises culturing a cell or plant comprising (a) a gene of enzyme converting lactate into lactyl-CoA and converting 3-hydroxyalkanoate into 3-hydroxyalkanoyl-CoA, (b) phosphotransbutylase gene, (c) butyrate kinase gene and (d) polyhydroxyalkanoate (PHA) synthase gene together.
In the present invention, the cell or plant can be obtained by transforming a cell or plant not having at least one among the (a), (b), (c) and (d) genes with the gene(s) that the cell or plant does not have among the (a), (b), (c) and (d) genes. The cell or plant can be obtained also by transforming a cell or plant in which the expression of at least one among the (a), (b), (c) and (d) genes is weak or not present with the gene(s) whose expression is week or not present.
That is, the cell or plant being able to synthesize the copolymer comprising 4-hydroxybutyrate monomer unit and lactate monomer unit can be obtained by (i) transforming a cell or plant not having any one of the genes with gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, phosphotransbutylase gene, butyrate kinase gene and PHA synthase gene, (ii) transforming a cell or plant having the gene of PHA synthase using lactyl-CoA as a substrate with gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, phosphotransbutylase gene and butyrate kinase gene, (iii) transforming a cell or plant having gene of enzyme converting lactate into lactyl-CoA with phosphotransbutylase gene, butyrate kinase gene and PHA synthase gene, (iv) transforming a cell or plant having phosphotransbutylase gene with gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, butyrate kinase gene and PHA synthase gene, (v) transforming a cell or plant having butyrate kinase gene with gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, phosphotransbutylase gene and PHA synthase gene, (vi) transforming a cell or plant having gene of PHA synthase using lactyl-CoA as a substrate and gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, with phosphotransbutylase gene and butyrate kinase gene, (vii) transforming a cell or plant having gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, and phosphotransbutylase gene with gene of PHA synthase using lactyl-CoA as a substrate and butyrate kinase gene, (viii) transforming a cell or plant having gene of enzyme converting 3-hydroxyalkanoate into 3-hydroxyalkanoyl-CoA and butyrate kinase gene with PHA synthase gene and phosphotransbutylase gene, (ix) transforming a cell or plant having gene of PHA synthase using lactyl-CoA as a substrate and butyrate kinase gene with gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, and phosphotransbutylase gene, (x) transforming a cell or plant having PHA synthase gene and phosphotransbutylase gene with gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, and butyrate kinase gene, (xi) transforming a cell or plant having phosphotransbutylase gene and butyrate kinase gene with gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, and gene of PHA synthase using lactyl-CoA as a substrate, (xii) transforming a cell or plant having gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, phosphotransbutylase gene and butyrate kinase gene with gene of PHA synthase using lactyl-CoA as a substrate, (xiii) transforming a cell or plant having phosphotransbutylase gene, butyrate kinase gene and PHA synthase gene with gene of enzyme converting lactate into lactyl-CoA, (xiv) transforming a cell or plant having gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, butyrate kinase gene and PHA synthase gene with phosphotransbutylase gene, or (xv) transforming a cell or plant having gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, phosphotransbutylase and PHA synthase gene with butyrate kinase gene. However, the scope of the present invention is not limited to the concrete examples described above.
Preferably, in the present invention, the gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, is propionyl-CoA transferase gene (pct).
Preferably, in the present invention, the phosphotransbutylase (Ptb) gene is derived from Clostridium acetobutyricum.
Preferably, in the present invention, the butyrate kinase (Buk) gene is derived from Clostridium acetobutyricum.
In the present invention, Cat2 gene derived from Clostridium kluyveri may be used instead of the Ptb gene and buk gene. The Cat2 gene is a enzyme converting 4-hydroxybutyrate into 4-hydroxybutyryl-CoA like the Ptb gene and buk gene. Preferably, the nucleotide sequence of Cat2 gene is SEQ ID No: 30.
Furthermore, in case that a gene encoding PHA synthase for which lactyl-CoA is a substrate is phaC, the cells or plants are transformed with a recombinant vector comprising pct, ptb and buk gene. At the same time, the cells or plants are transformed with a vector comprising phaC, or phaC is inserted into a chromosome. In addition, in case that a gene encoding PHA synthase for which lactyl-CoA is a substrate is phaC, the cells or plants are transformed with a recombinant vector comprising pct gene. At the same time, the cells or plants are transformed with a vector comprising phaC, or phaC is inserted into a chromosome.
As is known in the art, various microorganisms have a gene encoding PHA synthase (Korea Patent issued No. 10-250830). The following are examples of such microorganisms: microorganisms of the genus Achromobacter that include Achromobacter sp., Achromobacter xylosoxidans, etc., microorganisms of the genus Acinetobacter that include Acidovorax delafieldii, Acidovax facilis, Acinetobacter sp., Acinetobacter calcoaceticus, Acinetobacter lwoffii, etc., microorganisms of the genus Aeromonas that include Actinomyces sp., Aeromonas caviae, Aeromonas hydrophila, Aeromonas salmonicida, etc., microorganisms of the genus Alcaligenes that include Alcaligenes aestus, Alcaligenes denitrificans, Alcaligenes eutrophus (after renamed as Ralstonia eutropha, it is renamed as Wautersia eutropha), Alcaligenes faecalis, Alcaligenes latus, Alcaligenes pacificus, Alcaligenes paradoxus, Alcaligenes venestus, etc., microorganisms of the genus Amoebobacter that include Alteromonas macleodii, Amoebobacter roseu, Amoebobacter pendens, etc., microorganisms of the genus Azospirillum that include Aphanocapa sp., Aphanothece sp., Aquaspirillum autotrophicum, Azorhizobium caulinodans, Azospirillum sp., Azospirillum brasilense, Azospirillum lipoferum, etc., microorganisms of the genus Azotobacter that include Azotobacter sp., Azotobacter agilis, Azotobacter chroococcum, Azotobacter macrocytogenes, Azotobacter vinelandii, etc., microorganisms of the genus Bacillus that include Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus subtillus, Bacillus thuringiensis, etc., microorganisms of the genus Beggiatoa that include Beggiatoa sp., Beggiatoa alba, etc., microorganisms of the genus Beijerinckia that include Beijerinckia indicus, Beijerinckia mobilis, etc., microorganisms of the genus Beneckea that include Beneckea natrigens, Beneckea pelagia, etc., microorganisms of the genus Caulobacter that include Bordetella pertussis, Bradyrhizobium japonicum, Caryophamon latum, Caulobacter bacteroides, Caulobacter crescentus, etc., microorganisms of the genus Chlorogloea that include Chloroflexus aurantiacus, Chlorogloea fritschii, etc., microorganisms of the genus Chromatium that include Chromatium minutissimum, Chromatium okenii, Chromatium tepidum, etc., microorganisms of the genus Chromobacterium that include Chromobacterium violaceum, etc., microorganisms of the genus Clostridium that include Clostridium botulinum, Clostridium sphenoides, etc., microorganisms of the genus Comamonas that include Comamonas acidovorans, Comamonas testosteroni, etc., microorganisms of the genus Corynebacterium that include Corynebacterium autotrophicum, Corynebacterium hydrocarboxydans, etc., microorganisms of the genus Derxia that include Cyanobacteria, Derxia gummosa, etc., microorganisms of the genus Desulfonema that include Desulfococcus multivorans, Desulfonema limicola, Desulfonema magnum, etc., microorganisms of the genus Ectothiorhodospira that include Desulfosacina variabilis, Desulfovibrio sapovorans, Ectothiorhodospira halochloris, Ectothiorhodospira mobilis, Ectothiorhodospira vacuolata, etc., microorganisms of the genus Halobacterium that include Ferrobacillus ferroxidans, Flavobacterium sp., Haemophilus influenzae, Halobacterium gibbonsii, Halobacterium volcanii, etc., microorganisms of the genus Hydrogenophaga that include Haloferax mediterranei, Hydroclathratus clathratus, Hydrogenomonas facilis, Hydrogenophaga flava, Hydrogenophaga pseudoflava, Hydrogenophaga taeniospiralis, etc., microorganisms of the genus Hyphomicrobium that include Hyphomicrobium vulgare, etc., microorganisms of the genus Methylbacterium that include Ilyobater delafieldii, Labrys monachus, Lamprocystis reseopersicina, Lampropedia hyaline, Legionella sp., Leptothrix discophorus, Methylbacterium AM1, Methylbacterium extorquens, etc., microorganisms of the genus Methylosinus that include Methylococcus thermophilus, Methlocystis parvus, Methylomonas methanica, Methylosinus sporium, Methylosinus trichosporium, etc., microorganisms of the genus Micrococcus that include Methylovibrio soehngenii, Micrococcus denitrificans, Micrococcus halodenitrificans, etc., microorganisms of the genus Mycobacterium that include Mycobacterium album, Mycobacterium vacae, etc., microorganisms of the genus Nitrobacter that include Nitrobacter agilis, Nitrobacter winogradskyi, etc., microorganisms of the genus Nocardia that include Nocardia alba, Nocardia asteroides, Nocardia lucida, Nocardia rubra, etc., microorganisms of the genus Photobacterium that include Paracoccus dentrificans, Oscillatoria limosa, Penicillium cyclopium, Photobacterium mandapamensis, Photobacterium phosphoreum, etc., microorganisms of the genus Pseudomonas that include Physarum ploycephalum and Pseudomonas glathei, Pseudomonas indigofera, Pseudomonas lemonieri, Pseudomonas mallei, Pseudomonas marina, Pseudomonas mixta, Pseudomonas oleovorans, Pseudomonas oxalaticus, Pseudomonas pseudoalcaligenes, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas asplenii, Pseudomonas butanovora, Pseudomonas cepacia, Pseudomonas coronafaciens, Pseudomonas dacunhae, Pseudomonas denitrificans, Pseudomonas diminuta, Pseudomonas echinoides, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas rubrilineas, Pseudomonas saccharophila, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas thermophilus, Pseudomonas viridiflava, etc., microorganisms of the genus Ralstonia, microorganisms of the genus Rhizobium that include Rhizobium hedysarum, Rhizobium lupini, Rhizobium meliloti, Rhizobium phaseoli, Rhizobium trifoli, etc., microorganisms of the genus Rhodobacillus, microorganisms of the genus Rhodobacter that include Rhodobacter capsulatus, Rhodobacter sphaeroides, etc., microorganisms of the genus Rhodococcus that include Rhodococcus rhodochrous, etc., microorganisms of the genus Rhodocyclus that include Rhodocyclus gelatinosus, Rhodocyclus tenuis, etc., microorganisms of the genus Rhodopseudomonas that include Rhodomicrobium vannielii and Rhodopseudomonas acidophila, Rhodopseudomonas capsulata, etc., microorganisms of the genus Rhodospirillum that include Rhodospirillum molischianum, Rhodospirillum rubrum, etc., microorganisms of the genus Spirillum that include Sphingomonas paucimobilis, Spirillum itersomii, Spirillum serpens, etc., microorganisms of the genus Spirulina that include Spirulina jenneri, Spirulina maxima, Spirulina subsaksa, etc., microorganisms of the genus Staphylococcus that include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus xylosus, etc., microorganisms of the genus Stella that include Stella humosa, Stella vacuolata, etc., microorganisms of the genus Streptomyces that include Streptomyces antibioticus, Streptomyces coelicolor, etc., microorganisms of the genus Thiobacillus that include Syntrophomonas wolfei, Thermophilic cyanobacteria, Thermus thermophilus, Thiobacillus A2, Thiobacillus acidophilus, Thiobacillus versutus, etc., microorganisms of the genus Thiocapsa that include Thiocapsa pfennigii, etc., microorganisms of the genus Zoogloea that include Thiocystis violacea, Vibrio parahaemolyticus, Xanthobacter autotrophicus, Xanthomonas maltophilia, Zoogloea ramigera, etc.
Preferably, the polyhydroxyalkanoate (PHA) synthase gene of the present invention is phaC1ps6-19 originated from Pseudomonas sp. 6-19. More preferably, the PHA synthase gene encodes the amino acid sequence of SEQ ID NO: 8 having mutations of: a) S325T and Q481M; b) E130D and Q481K; c) S325T and Q481K; d) E130D and Q481M; e) E130D and Q481R; f) E130D, S325T and Q481M; g) E130D, S325T and Q481K; h) E130D, S477R and Q481K; i) E130D, S477R and Q481M; j) E130D, S477R and Q481R; k) E130D, S477H and Q481K; 1) E130D, S477H and Q481M; m) E130D, S477H and Q481R; n) E130D, S477F and Q481K; o) E130D, S477F and Q481M; p) E130D, S477F and Q481R; q) E130D, S477Y and Q481K; r) E130D, S477Y and Q481M; s) E130D, S477Y and Q481R; t) E130D, S325T, S477R and Q481M; u) E130D, S325T, S477R and Q481K; v) E130D, S325T, S477F and Q481M; w) E130D, S325T, S477G and Q481M; or x) E130D, S325T, S477F and Q481K. These PHA synthase mutants are more preferable in aspect of using lactyl-CoA as a substrate.
In the present invention, the cell is preferably a microorganism. More preferably, the microorganism is E. Coli.
In the present invention, the cell or plant having the gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, phosphotransbutylase gene, butyrate kinase gene and polyhydroxyalkanoate (PHA) synthase gene together can be cultured in a medium comprising at least one selected from the group consisting of 4-hydroxybutyrate, 3-hydroxypropionate and 3-hydroxybutyrate to produce a copolymer comprising 4-hydroxybutyrate monomer unit, lactate monomer unit and optionally 3-hydroxyalkanoate. If the cell or plant can biosynthesize lactate, 4-hydroxybutyrate and 3-hydroxyalkanoate from other carbon sources such as glucose, citric acid, etc., there may be no need to further add 4-hydroxybutyrate, lactate and so on to the medium.
For example, poly(4-hydroxybutyrate-co-3-hydroxypropionate-co-lactate) can be preparing by culturing the cell or plant in a medium further comprising 4-hydroxybutyrate (4-HB) and 3-hydroxypropionate (3-HP).
For example, 3-hydroxybutyrate-3-hydroxypropionate-4-hydroxybutyrate-lactate tetrapolymer can be prepared by culturing the cell or plant in a medium further comprising 4-hydroxybutyrate (3-HP), 3-hydroxypropionate (3-HP) and 3-hydroxybutyrate (3-HB).
Transformation of plants for preparing plant comprising genes of transferase and synthase can be achieved by conventional methods using Agrobacterium or virus vectors. For example, transformed plants are obtained by transforming an Agrobacterium with a recombinant vector containing the inventive gene and infecting a tissue, etc. of the target plant with the transformed Agrobacterium. More specifically, the transformed plant can be prepared by pre-culturing an explant of plant of interest, and then transforming the explant by co-cultivating the explant and a transformed Agrobacterium; culturing said infected explants to induce callus; and excising obtained callus, and culturing it in shoot-inducing medium.
The term “explant,” as used herein, means a tissue fragment cut from a plant, and includes cotyledon or hypocotyl. Cotyledon or hypocotyls can be used as the explant of the present invention. It is more preferable to use cotyledon obtained by disinfecting and washing seeds of the plant, and germinating it in MS medium.
Transformed plants useful for the present invention include, but are not limited to, tobacco, tomato, red peppers, beans, nice, and corn. Also, even though a transformed plant is one that propagates sexually, it will be obvious to a person skilled in the art that such a plant can be reproduced asexually using plant tissue culture, etc.
Hereinafter, the present invention is described in considerable detail. The following examples are offered by way of illustration to help those skilled in the art understand the present invention, and are not intended to limit the scope of the invention.
Recombinant plasmids, pPs619C1300-CPPCT and pTacCpPctNCvEC, comprising pct gene and PHA synthase gene are constructed to prepare a copolymer comprising 4-hydroxybutyrate unit and lactate unit.
(1) Construction of Plasmid pPs619C1300-CPPCT
Propionyl-CoA transferase (CP-PCT) gene derived from Clostridium propionicum was used as the pct gene, and PHA synthase gene derived from Pseudomonas sp. 6-19 was used as the PHA synthase gene.
The operon of constitutive expression system expressing PHA synthase and CP-PCT together was constructed like
NdeI restriction enzyme site of wild CP-PCT was removed by SDM method for easiness of cloning. In addition, overlapping PCR was performed with the primers of SEQ ID NO: 3 and 4 to add SbfI/NdeI recognition site.
To separate the gene of PHA synthase (phaC1ps6-19) originated from Pseudomonas sp. 6-19 (KCTC 11027BP), total DNA of Pseudomonas sp. 6-19 was extracted, and the primers of SEQ ID NO: 5 and 6 were prepared based on the sequence of phaC1es6-19 gene (Ae-jin Song, Master's Thesis, Department of Chemical and Biomolecular Engineering, KAIST, 2004) and PCR was performed to get the gene of phaC1ps6-19. The nucleotide sequence of phaC1Ps6-19 gene is shown in SEQ ID NO: 7, from which the amino acid sequence evaluated is shown in SEQ ID NO: 8.
The above obtained phaC1Ps6-19 gene was inserted into BstBI/SbfI site of pBluescript II (Stratagene Co., USA) to make pPs619C1 recombinant vector. BstBI sites contained inside were removed by SDM (site directed mutagenesis) method without mutation of amino acid to make phaC1ps6-19 synthase gene fragment having two BstBI/SbfI sites only at the both ends, and overlapping PCR were performed with the primers of SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, and SEQ ID NO: 13 and 14 to add BstBI/SbfI-recognition site.
Three positions (130, 325, and 481) of amino acid affecting SCL (short-chain-length PHA) synthesis activity of phaC1Ps6-19 synthase were found out through amino acid sequence alignment analysis, and pPs619C1300 comprising the gene encoding the mutant having mutations of E130D, S325T and Q481M in the amino acid sequence phaC1ps6-19 synthase was constructed by SDM method. The phaC1Ps6-19 synthase mutant was shown in table 1 below.
The obtained pPs619C1300 vector was excised with SbfI/NdeI, and the cloned CP-PCT gene was inserted into SbfI/NdeI recognition site to construct the pPs619C1300-CPPCT recombinant vector (
(2) Construction of pTacCpPctNCvEC Plasmid
pTac99A vector (Park and Lee, J. Bacteriol. 185, 5391-5397, 2003) was cut with SspI to get a gene fragment comprising Tac promoter and transcription terminator, and the fragment was inserted into pTrc99A (Pharmacia Biotech, Sweden) exercised with restriction enzyme SspI to make pTaclac vector. phaEC gene was amplified with the chromosome DNA of Chromatium vinosum (DSMZ180) as template and the primers of SEQ ID NO: 21 and 22.
The amplified phaEC gene was inserted into the NdeI-excised site of the pTaclac vector to make pTaclacNCvEC vector. In addition, pct gene was obtained by cutting pPs619C1300-CPPCT with EcoRI/XbaI, and the pct gene was inserted into the EcoRI/XbaI-excised pTaclacNCvEC to make pTacCpPctNCvEC (
(3) Construction of pMCSPtbBuk Plasmid
ptb and buk gene was constructed as one operon in Clostridium acetobutyricum strain, and those nucleotide sequence were shown in SEQ ID NO: 27 and 28, respectively. ptb/buk gene was amplified with the primers of SEQ ID NO: 23 and 24 from the chromosome DNA of Clostridium acetobutyricum (ATCC824).
Further, PCR using pC1300-CPPCT as template was performed with the primers of SEQ ID NO: 25 and 26 to amplify the gene of SbfI recognition site in pC1300-CPPCT.
Overlapping PCR using the gene fragment obtained with the primers of SEQ ID NO: 23 and 24 and the gene fragment obtained with the primers of SEQ ID NO: 25 and 25 as templates was performed with the primers of SEQ ID NO: 24 and 25 to finally get ptb/buk gene fragment comprising SbfI/NdeI recognition site. The obtained ptb/buk gene fragment was cut with SbfI/NdeI, and then was inserted into pC1300-CPPCT excised with the same enzyme to get pPtbBuk plasmid. pPtbBuk plasmid was cut with XmaI/XhoI to get the gene fragment comprising the promoter of R. eutropha PHA biosynthesis gene and ptb/buk gene, and the obtained gene was inserted into pBBR1MCS(NCCB 3433) cut with XmaI/XhoI to get pMCSPtbBuk plasmid (
E. coli Top 10 (Invitrogen) was transformed with the pPs619C1300-CPPCT obtained in example 1 and pMCSPtbBuk together to get E. coli Top10/pPs619C1300-CPPCT/pMCSPtbBuk.
The transformant was cultured by two steps to get 4-hydroxybutyrate-lactate copolymer as follows: First, the transformed recombinant E. coli Top10/pPs619C1300-CPPCT/pMCSPtbBuk was cultured for 24 hours in 100 mL of LB medium (Bacto™ Triptone(BD) 10 g/L, Bacto™ yeast extract(BD) 5 g/L; NaCl (amresco) 10 g/L) containing 100 mg/L of ampicillin and 30 mg/L of chloramphenicol, and then the medium was centrifuged for 15 minutes at 4° C., 1000 g to collect cells.
Collected cells was anaerobically cultured for 3 days in MR medium (Glucose 10 g, KH2PO4 6.67 g, (NH4)2HPO4 4 g, MgSO4.7H2O 0.8 g, citric acid 0.8 g and trace metal solution 5 mL per 1 L; Trace metal solution composition: 5M HCl 5 mL, FeSO4.7H2O 10 g, CaCl2 2 g, ZnSO4.7H2O 2.2 g, MnSO4.4H2O 0.5 g, CuSO4.5H2O 1 g, (NH4)6Mo7O2.4H2O 0.1 g, and Na2B4O2.10H2O 0.02 g per 1 L) further comprising 2 g/L of 4-hydroxybutyrate (4-HB) and 100 mg/L of ampicillin and 30 mg/L of chloramphenicol.
The culture medium was centrifuged for 15 minutes at 4° C., 1000 to collect cells, and the cells was washed 4 times with lots of distilled water and dried for 12 hours at 80° C. Completely dried cells was quantified, and reacted with methanol at 100° C. in chloroform solvent under the catalyst of sulfuric acid. Half volume of distilled water was added at room temperature to the chloroform, and mixed. Then, the mixture was settled until separated into two layers. In two layers, the chloroform layer dissolving methylated monomer was collected, and the ingredients of the polymer were analyzed with gas chromatography. Benzoate was used as internal standard.
As a result of the analysis, methyl-4-hydroxybutyrate and methyl-lactate were detected in E. coli Top10/pPs619C1300-CPPCT/pMCSPtbBuk transformant, which meant that new 4-hydroxybutyrate-lactate copolymer [(poly(4-hydroxybutyrate-co-lactate)] copolymer was prepared by the recombinant E. Coli. NMR result of the obtained poly(4-hydroxybutyrate-co-lactate) copolymer was shown in
4-Hydroxybutyrate-3-hydroxypropionate-lactate terpolymer was prepared according to the method of example 2 except that the collected cells was anaerobically cultured for 3 days in MR medium further containing 2 g/L of 4-hydroxybutyrate (4-HB), 2 g/L of 3-hydroxypropionate (3-HP), 100 mg/L of ampicillin and 30 mg/L of chloramphenicol instead of MR medium further containing g/L of 4-HB, 100 mg/L of ampicillin and 30 mg/L of chloramphenicol.
As a result of the analysis, methyl-4-hydroxybutyrate, methyl-3-hydroxypropionate and methyl-lactate were detected in E. coli Top10/pPs619C1300-CPPCT/pMCSPtbBuk transformant, which meant that new 4-hydroxybutyrate-3-hydroxypropionate-lactate terpolymer [poly(4-hydroxybutyrate-co-3-hydroxypropionate-co-lactate)] was prepared by the recombinant E. Coli. 1H-NMR and GC-MSD results of the obtained 4-hydroxybutyrate-3-hydroxypropionate-lactate terpolymer were shown in
3-Hydroxybutyrate-4-hydroxybutyrate-lactate terpolymer was prepared according to the method of example 2 except that the collected cells was anaerobically cultured for 3 days in MR medium further containing 2 g/L of 4-hydroxybutyrate (4-HB), 1 g/L of 3-hydroxybutyrate (3-HB), 100 mg/L of ampicillin and 30 mg/L of chloramphenicol instead of MR medium further containing g/L of 4-HB, 100 mg/L of ampicillin and 30 mg/L of chloramphenicol.
As a result of the analysis, methyl-4-hydroxybutyrate, methyl-3-hydroxybutyrate and methyl-lactate were detected in E. coli Top10/pPs619C1300-CPPCT/pMCSPtbBuk transformant, which meant that 3-hydroxybutyrate-4-hydroxyburyrate-lactate terpolymer [poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-lactate)] was prepared by the recombinant E. Coli.
3-hydroxybutyrate-3-hyroxypropionate-4-hydroxybutyrate-lactate tetrapolymer was prepared according to the method of example 2 except that the collected cells was anaerobically cultured for 3 days in MR medium further containing 2 g/L of 3-hydroxybutyrate (3-HB), 2 g/L of 3-hyroxypropionate (3-HP), 1 g/L of 4-hydroxybutyrate (4-HB) and 100 mg/L of ampicillin instead of MR medium further containing 2 g/L of 4-HB, 100 mg/L of ampicillin and 30 mg/L of chloramphenicol.
As a result of the analysis, methyl-4-hydroxybutyrate, methyl-3-hydroxybutyrate, methyl-3-hydroxypropionate and methyl-lactate were detected in E. coli Top10/pPs619C1300-CPPCT/pMCSPtbBuk transformant, which meant that 3-hydroxybutyrate-3-hydroxypropionate-4-hydroxybutyrate-lactate tetrapolymer was prepared by the recombinant E. Coli. 1H-NMR and GC-MSD results of the obtained 3-hydroxybutyrate-3-hydroxypropionate-4-hydroxybutyrate-lactate tetrapolymer were shown in
Various PHA synthase mutants were prepared like the construction of the pPs619C1300 with the primers below. Obtained mutants were shown in tables 2, 3, 4 and 5.
As described and proven above, the present invention provides a copolymer comprising 4-hydroxybutyrate monomer unit and lactate monomer unit or a copolymer 4-hydroxybutyrate monomer unit, lactate monomer unit and 3-hydroxyalkanoate. The present invention also provides a method for preparing the copolymer, wherein the method comprises culturing a cell or plant comprising the gene of enzyme converting lactate and 3-hydroxyalkanoate into lactyl-CoA and 3-hydroxyalkanoyl-CoA, respectively, phosphotransbutylase gene, butyrate kinase gene and polyhydroxyalkanoate (PHA) synthase gene together. The copolymer of the present invention is a biodegradable polymer being able to be usefully used instead of conventional synthetic plastic, and the copolymer can be used for medical use.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2006-0115158 | Nov 2006 | KR | national |
| 10-2006-0115159 | Nov 2006 | KR | national |
| 10-2006-0115160 | Nov 2006 | KR | national |
| 10-2006-0115161 | Nov 2006 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR2007/005852 | 11/21/2007 | WO | 00 | 11/20/2009 |
