KR101085960B1 - Method for Preparing Polylactate-co-glycolate or Polylactate-co-glycolate-co-hydroxyalkanoate Copolymers Using Cells or Plants Having Producing Ability of Polylactate-co-glycolate or Polylactate-co-glycolate-co-hydroxyalkanoate Copolymers - Google Patents

Abstract

The present invention provides a propionate CoA transferase (Pct) gene and lactyl-CoA and glycolyl- that convert lactate and glycolate to lactyl-CoA and glycolyl-CoA, respectively. Lactate-glycolate copolymers and lactate-glycolates characterized by culturing or cultivating cells or plants simultaneously having a polyhydroxyalkanoate (PHA) synthase gene using CoA as a substrate A method for producing a hydroxyalkanoate copolymer.

According to the present invention, not only it is possible to prepare lactate-glycolate copolymers using cells or plants, but also various kinds of polyesters containing glycolate, lactate and various hydroxyalkanoates as monomers. It is possible to prepare.

Lactate-glycolate copolymers, propionate CoA-transferases, polyhydroxyalkanoate (PHA) synthase

Description

Preparation of lactate glycolate copolymers or lactate glycolate hydroxyalkanoate copolymers using cells or plants having a lactate glycolate copolymer or lactate glycolate hydroxyalkanoate copolymer generating ability Method for Preparing Poly (lactate-co-glycolate) or Poly (lactate-co-glycolate-co-hydroxyalkanoate) Copolymers Using Cells or Plants Having Producing Ability of Poly (lactate-co-glycolate) or Poly (lactate-co- glycolate-co-hydroxyalkanoate) Copolymers}

The present invention provides a propionate CoA transferase (Pct) gene and lactyl-CoA and glycolyl- that convert lactate and glycolate to lactyl-CoA and glycolyl-CoA, respectively. Lactate-glycolate copolymers and lactate-glycolates characterized by culturing or cultivating cells or plants simultaneously having a polyhydroxyalkanoate (PHA) synthase gene using CoA as a substrate A method for producing a hydroxyalkanoate copolymer.

Lactate-glycolate copolymer [Poly (lactate-co-glycolate), PLGA] is a representative biodegradable polymer derived from lactate and glycolate (glycolate) and its application as a general purpose polymer or medical polymer It is a high polymer. Currently PLGA can be prepared by the direct polymerization of lactate and glycolate, but mainly produces only low molecular weight (1000-5000 Daltons) PLGA. PLGA of high molecular weight of 100,000 Daltons or more can be synthesized through ring-opening condensation reaction of lactide and glycolide. Lactide and glycolide are cyclic diesters of lactate and glycolate, respectively, which are produced by thermal decomposition of lactate oligomers and glycolate oligomers, respectively. The ring-opening condensation reaction requires the use of a catalyst such as 2-ethylhexanoate (tin (II) 2-ethylhexanoate), tin alkoxide (tin (II) alkoxide), or aluminum isopropoxide. . There is a method of polymerizing from a low molecular weight polymer obtained by direct polymerization to a polymer having a higher molecular weight using a chain coupling agent.However, a method of producing a high molecular weight PLGA using a chain coupling agent is an organic solvent or chain coupling. The addition of agents complicates the process and also has the disadvantage that it is not easy to remove them. Currently commercially available high molecular weight PLGA production process converts lactate and glycolate to lactide and glycolide, respectively, and then synthesizes PLGA through ring-opening condensation reaction of lactide and glycolide. .

On the other hand, PHAs are polyesters that microorganisms accumulate inside as energy or carbon source storage materials when there are excessive carbon sources and other nutrients such as phosphorus, nitrogen, magnesium, and oxygen are insufficient. PHA is regarded as a material to replace conventional synthetic plastics because it has properties similar to synthetic polymers derived from petroleum and shows complete biodegradability.

Conventionally known PHAs can be classified into short-chain-length PHAs (SCL-PHAs) and medium-chain-length PHAs (MCL-PHAs) with long carbon atoms. Genes that synthesize PHA have been cloned from Ralstonia eutropha , Pseudomonas, etc., and PHAs composed of various monomers have been synthesized through 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; US 6,143,952; WO 98/54329; WO 99/61624).

In order to produce PHA in microorganisms, enzymes for converting metabolites of microorganisms to PHA monomers and PHA synthase synthesizing PHA polymers using PHA monomers are essential. PHA synthase synthesizes PHA using hydroxyacyl-CoA as a substrate, and enzymes capable of providing hydroxyacyl-CoA, a substrate of PHA, include β-ketothiolase (PhaA) and acetoacetyl-CoA reductase (cloned from Ralstonia eutropha ). PhaB), 3-hydroxydecanoyl-ACP: CoA transferase (PhaG) cloned from Pseudomonas , ( R ) -specific enoyl-CoA hydratase (PhaJ) derived from Aeromonas caviae and Pseudomonas aeruginosa (Fukui et al., J. Bacteriol. , 180: 667, 1998; Tsuge et al., FEMS Microbiol. Lett ., 184: 193, 2000), 3-ketoacyl-ACP reductase (FabG) derived from E. coli and 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). Using these enzymes, various types of PHA have been synthesized using hydroxyalkanoate hydroxylated with various carbon positions (mostly 3, 4, 5, 6).

However, there has been little reported reactivity of PHA synthase to hydroxylated hydroxyalkanoates at position 2 (Zhang et al., Appl. Microbiol. Biotechnol ., 56: 131, 2001; Valentin and Steinbuchel, A ppl Microbiol. Biotechnol ., 40: 699, 1994). Although there have been reports of in vivo reactivity of PHA synthase to glycolyl-CoA, the reactivity of PHA synthase to glycolyl-CoA is very low (Zhang et al., Appl. Microbiol. Biotechnol ., 56: 131, 2001 Valentin and Steinbuchel, Appl. Microbiol. Biotechnol ., 40: 699, 1994). That is, hydroxyalkanoate such as glycolate with 2-carbonyl position is not suitable for the substrate specificity of PHA synthase, so that PHA and its copolymers are produced naturally or using recombinant cells or plants. There is no.

Although no patents have been reported for polyhydroxyalkanoate (PHA) copolymers containing glycolate as a 2-hydroxy acid monomer (US 2003/0211131 A1), this is one of the various PHA monomers, including glycolate. The lactate-glycolate copolymer (PLGA), which is a copolymer of polylactate (PLA) or polyglycolate (PGA), and its copolymer with various hydroxyalkanoates [poly] (lactate-co-glycolate-co-hydroxyalkanoate)] is different from the biosynthesis.

The inventors of the present invention have found that Clostridium propionicum- derived propionate CoA-transferase (Pct), an enzyme that converts lactate and glycolate to lactyl-CoA and glycolyl-CoA, respectively. Recombinant Escherichia coli transformed with the gene and the polyhydroxyalkanoate (PHA) synthase gene, which can use lactyl-CoA and glycolyl-CoA as substrates, were prepared using glucose, glycolate or glucose, glycolate, and hydride. When cultured in a production medium containing oxyalkanoate, it was confirmed that poly (lactate-co-glycolate) copolymer and poly (lactate-co-glycolate-co-hydroxyalkanoate) copolymer were produced and completed the present invention. It became.

Finally, an object of the present invention is to propionate CoA-transferase gene, to prepare lactate-glycolate copolymer or lactate-glycolate-hydroxyalkanoate copolymer using cells or plants. And lactate-glycolate copolymers or lactate-glycolate-hydroxyalkanoate copolymers characterized by culturing or culturing cells or plants containing polyhydroxyalkanoate synthase genes. To provide a method.

In order to achieve the above object, the present invention can use lactyl-CoA and glycolyl-CoA as a substrate and the gene of the enzyme for converting lactate and glycolate to lactyl-CoA and glycolyl-CoA, respectively By culturing or cultivating cells or plants having the polyhydroxyalkanoate synthase gene present at the same time, using lactate and glycolate added to the culture medium, or lactate and writing produced in-house within the cell or plant Lactate-glycolate copolymers are synthesized using lycolate, and then the lactate-glycolate copolymer is recovered from the cultured cells or cultivated plants. Provided is a method for preparing the copolymer [poly (lactate-co-glycolate)].

The present invention also provides a lactate-glycolate copolymer [poly (lactate-co-glycolate)] containing lactate and glycolate monomers.

In addition, the present invention is a gene of an enzyme that converts lactate and glycolate to lactyl-CoA and glycolyl-CoA, and polyhydroxyal which can use lactyl-CoA and glycolyl-CoA as a substrate. By culturing or culturing cells or plants simultaneously having a canoate synthase gene, using lactate, glycolate and hydroxyalkanoate added to the culture medium, or lactates produced in-house within the cells or plants, Lactate-glycolate-hydroxyalkanoate copolymers were synthesized using glycolate and hydroxyalkanoate, and then lactate-glycolate-hydroxy from the cultured cells or cultivated plants. Lactate-glycolate-hydroxyalkanoate copolymer characterized by recovering an alkanoate copolymer [poly (lacta te-co-glycolate-co-hydroxyalkanoate)].

In addition, the present invention is 2-hydroxybutyrate (hydroxybutyrate), 3-hydroxypropionate (hydroxypropionate), 3-hydroxybutyrate (hydroxybutyrate), 3-hydroxyvalerate (4-hydroxyvalerate), 4-hydroxybutanoic acid (hydroxybutyrate), medium chain length (D) -3-hydroxycarboxylic acids having 6 to 14 carbon atoms, 3-hydroxypropionic acid, 3-hydroxyhexanoic acid , 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyunde Hydroxyundecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, 4-hydroxyvaleric acid (hydroxyvaleric acid, 4-hydroxyhexanoic acid hexanoic 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-octenic acid, 3-hydroxy-6-cis-octenic acid, 3-hydroxy-7-octennoic 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 acid, 3-hydroxy-4-methylvaleric acid, 3-hydroxy-4-methylhexanoic acid, 3-hydroxy-5-methyl acid Hexane acid (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 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-octenic acid, malic acid, 3-hydroxysuccinic acid (hydroxysuccinic acid) -methyl ester, 3-hydroxyadipinic acid-methyl ester, 3-hydroxysuberic acid-methyl ester, 3-hydroxyazelaic acid-methyl ester , 3-hydroxysebacic acid, methyl ester, 3-hydroxysuberic acid-ethyl ester, 3-hydroxysebacic acid-ethyl ester, 3-hydroxypi Hydroxypimelic acid-propyl ester, 3-hydroxysebacic acid-benzyl ester, 3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy -9-acetoxynonanoic acid, phenoxy-3-hydroxybutanoic acid tyric acid, phenoxy-3-hydroxyvaleric acid, phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxyoctanoic acid (hydroxyoctanoic acid), para-cyanophenoxy-3-hydroxybutyric acid, para-cyanophenoxy-3-hydroxyvaleric acid, para-sia Cyanophenoxy-3-hydroxyhexanoic acid, para-nitrophenoxy-3-hydroxyhexanoic acid, 3-hydroxy-5-phenylvaleric Phenylvaleric acid, 3-hydroxy-5-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 epoxydod ecanoic acid), 3-hydroxy-8,9-epoxy-5,6-cis-tetradecanoic acid, 7-cyano-3-hydroxyheptanoic acid ( 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 Hydroxy-6-bromohexanoic acid, 3-hydroxy-8-bromooctanoic acid, 3-hydroxy-11-bromooundecanoic acid, 3-hydroxy-2-butenoic acid, 6-hydroxy-3-dodecenoic acid, 3-hydroxy-2-methylbutanoic acid (methylbutyric acid), 3-hydroxy-2-methylvaleric acid, And lactate containing one or more hydroxyalkanoates selected from the group consisting of 3-hydroxy-2,6-dimethyl-5-heptenoic acid, lactate and glycolate as monomers. Glycolate-hydroxyalkanoate copolymer [poly (lactate-co-glycolate-co-hydroxyalkanoate)] is provided.

The present invention also provides a 2-hydroxybutyrate (2HB) -lactate-glycolate copolymer [poly (2HB-co-lactate-co-glycolate)].

The present invention also relates to 2-hydroxybutyrate (2HB) -3-hydroxybutyrate (3HB) -lactate-glycolate copolymer [poly (2HB-co-3HB-co-lactate-co-glycolate)]. to provide.

According to the present invention, not only it is possible to prepare lactate-glycolate copolymers using cells or plants, but also various kinds of polyesters containing glycolate, lactate and various hydroxyalkanoates as monomers. It is possible to prepare.

In one aspect, the present invention is a gene of an enzyme that converts lactate and glycolate to lactyl-CoA and glycolyl-CoA, and polyhydride which can use lactyl-CoA and glycolyl-CoA as a substrate. Cultivation or cultivation of cells or plants having the oxyalkanoate synthase gene at the same time, using lactate and glycolate added to the culture medium, or lactate and glycolate produced in-house within the cell or plant Using the lactate-glycolate copolymer, and then recovering the lactate-glycolate copolymer from the cultured cells or cultivated plants. (lactate-co-glycolate)] and lactate-glycol containing lactate and glycolateol monomer prepared by the above method Byte relates to a copolymer [poly (lactate-co-glycolate)].

In the embodiment of the present invention, the cells were cultured in a medium containing glucose and glycolate, and lactate produced from glucose in the cells was used as a precursor, but the medium was produced using cells containing only glucose. It will be apparent to those skilled in the art that the lactate-glycolate copolymer can be prepared even by using a glycolate and lactate, and even if only lactate is added to the medium or lactate and glycolate are added, It will also be apparent to those skilled in the art that the lactate-glycolate copolymers can be prepared.

In the present invention, the cell or plant is a cell or plant containing a polyhydroxyalkanoate synthase gene, the gene of the enzyme converting lactate and glycolate to lactyl-CoA and glycolyl-CoA, respectively, transformation It may be characterized in that it is inserted into or on the chromosome.

Various microorganisms are known as cells having the gene of the PHA synthase (KR 10-250830 B1). For example, Aeromonas spp, Achromobacter spp, Acidovorax delafieldii, Acidovax facilis ,, Acinetobacter spp, Actinomyces spp, Aeromonas spp, Alcaligenes spp, Alteromonas spp, Amoebobacter spp, Aphanocapa sp., Aphanothece sp. Aquaspirillum autotrophicum, Azorhizobium caulinodans, Azospirillum sp ., Azospirillum spp, Azotobacter spp, Bacillus spp, Beggiatoa spp, Beijerinckia spp, Beneckea spp, Bordetella pertussis, Bradyrhizobium japonicum, Caryophamon latum, Caulobacter spp, Chlorogloea spp ., Chromatium microorganism of the genus, Chromobacterium spp, Clostridium spp, Comamonas spp, Corynebacterium spp, Cyanobacteria in microorganisms, Derxia spp, Desulfonema spp, Desulfosacina variabilis, Desulfovibrio sapovorans, Ectothiorhodospira spp, Ferrobacillus ferroxidans, Flavobacterium sp, Haemophilus influenzae, Halobacterium spp, Haloferax mediterranei, Hydroclathratus clathratus, Hydrogenomonas facilis, microorganisms in Hydrogenophaga, microorganisms in Hyphomicrobium, Ilyobacter delafieldii, Labrys monachus, Lamprocystis reseopersicina, Lampropedia hyalina, Legionella sp., L eptothrix discophorus, Methylobacterium spp, Methylosinus spp, Micrococcus spp, Mycobacterium spp, Nitrobacter spp, Nocardia spp, Paracoccus dentrificans, Oscillatoria limosa, Penicillium cyclopium, Photobacterium spp, Physarum ploycephalum, Pseudomonas spp, Ralstonia spp , Rhizobium spp, Rhodobacillus spp, Rhodobacter spp, Rhodococcus spp, Rhodocyclus spp, Rhodomicrobium vannielii, Rhodopseudomonas spp, Rhodospirillum spp, Sphingomonas paucimobilis, Spirillum spp, Spirulina spp, Staphylococcus spp, Stella spp , Streptomyces spp, Syntrophomonas wolfei, Thermophilic cyanobacteria, Thermus thermophilus, Thiobacillus A2, Thiobacillus spp, Thiocapsa spp, Thiocystis violacea, Vibrio parahaemolyticus, Xanthobacter autotrophicus, Xanthomonas maltophilia, Zoogloea in Etc. can be mentioned.

In the present invention, cells or plants are genes of enzymes that convert lactate and glycolate to lactyl-CoA and glycolyl-CoA and polyhydroxyalkanoates which can use lactyl-CoA and glycolyl-CoA as substrates, respectively. Enzyme genes transformed with synthetase genes or convert lactate and glycolate to lactyl-CoA and glycolyl-CoA, and polyhydroxyalkanoate synthesis using lactyl-CoA and glycolyl-CoA as substrates The enzyme gene is characterized in that it is inserted on the chromosome, the gene of the enzyme that converts lactate and glycolate to lactyl-CoA and glycolyl-CoA, respectively, the propionate CoA-transferase gene ( pct It can be characterized by the).

The gene of the enzyme for converting lactate and glycolate to lactyl-CoA and glycolyl-CoA, respectively, may comprise (a) an A1200G mutation in the nucleotide sequence of SEQ ID NO: 1; (b) T78C, T669C, A1125G and T1158C at the nucleotide sequences of SEQ ID NO: 1; (c) a Gly335Asp variant in the amino acid sequence corresponding to SEQ ID NO: 1 and an A1200G variant in the nucleotide sequence of SEQ ID NO: 1; (d) Ala243Thr variant in amino acid sequence corresponding to SEQ ID NO: 1 and A1200G in base sequence of SEQ ID NO: 1; (e) Asp65Gly mutations in the amino acid sequence corresponding to SEQ ID NO: 1 and T669C, A1125G and T1158C mutations in the nucleotide sequence of SEQ ID NO: 1; (f) an Asp257Asn variant in the amino acid sequence corresponding to SEQ ID NO: 1 and an A1200G variant in the nucleotide sequence of SEQ ID NO: 1; (g) Asp65Asn mutation in the amino acid sequence corresponding to SEQ ID NO: 1 and T669C, A1125G and T1158C mutations in the nucleotide sequence of SEQ ID NO: 1; And (h) a Propionate CoA-transferase variant containing a Thr199Ile variant in the amino acid sequence corresponding to SEQ ID NO: 1 and a mutation selected from the group consisting of T669C, A1125G, and T1158C in the nucleotide sequence of SEQ ID NO: 1 The propionate CoA-transferase variant is an enzyme that converts lactate and glycolate to lactyl-CoA and glycolyl-CoA, respectively, and propionate CoA-transfer derived from Clostridium propionicum. The lyase gene ( cp-pct ) was prepared by mutating by an error-prone PCR method (Korean Patent Publication 2009-0017252).

In the present invention, the polyhydroxyalkanoate synthase gene may be characterized as phaC1 , wherein the polyhydroxyalkanoate synthase gene is selected from the amino acid sequence of SEQ ID NO: 2, (i) S325T and Q481M; (ii) E130D, S325T and Q481M; (iii) E130D, S325T, S477R and Q481M; And (iv) a gene encoding a polyhydroxyalkanoate synthetase variant, characterized in that it has an amino acid sequence containing a mutation selected from the group consisting of E130D, S477F and Q481K (Korea Patent Publication 2008-0047279).

In the present invention, the polyhydroxyalkanoate synthase variant may use glycolyl-CoA as a substrate, Pseudomonas sp. Is a mutant of phaC1 derived from MBEL 6-19.

In another aspect, the present invention provides a gene of an enzyme that converts lactate and glycolate to lactyl-CoA and glycolyl-CoA, and polyhydrides that can use lactyl-CoA and glycolyl-CoA as substrates. Lactate, glycolate, and hydroxyalkanoate added to the culture medium by culturing or culturing cells or plants having the oxyalkanoate synthase gene at the same time, or a lock produced in-house within the cell or plant Lactate-glycolate-hydroxyalkanoate copolymers were synthesized using tate, glycolate and hydroxyalkanoate, and then lactate-glycolate- from the cultured cells or cultivated plants. Lactate-glycolate-hydroxyalkanoate copolymerization characterized by recovering the hydroxyalkanoate copolymer Method for preparing [poly (lactate-co-glycolate-co-hydroxyalkanoate)] and lactate-glycolate-hydroxyalcohol containing hydroxyalkanoate, lactate and glycolate prepared as a monomer as monomers It relates to a canoate copolymer [poly (lactate-co-glycolate-co-hydroxyalkanoate)].

In the embodiment of the present invention, the cells were cultured in a medium containing glucose, glycolate and hydroxyalkanoate, and lactate produced from glucose in the cells was used as a precursor, but glucose and hydroxyalkanoate were used as precursors. It will be appreciated by those skilled in the art that lactate-glycolate-hydroxyalkanoate copolymers can be prepared using glycolate, lactate and hydroxyalkanoate produced intracellularly using a medium containing only It is evident to the fact that the lactate-glycolate-hydroxyalkanoate copolymer can be prepared by adding only lactate to the medium or adding lactate, glycolate, and hydroxyalkanoate. It will also be apparent to those skilled in the art.

In the present invention, the hydroxyalkanoate constituting the lactate-glycolate-hydroxyalkanoate copolymer is lactate, 2-hydroxybutyrate, 3-hydroxypropionic acid (hydroxypropionate), 3-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxybutyrate, medium chain length of 6 to 14 carbon atoms (D) -3- Hydroxycarboxylic acids, 3-hydroxypropionic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid acid), 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxy Hydroxytedecanoic acid tradecanoic 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 (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-octenic acid, 3-hydroxy (hydroxy) -6-cis-octennoic acid, 3-hydroxy-7-octennoic acid, 3-hydroxy-8-nonenoic acid, 3 Hydroxy-9-decenoic acid cid), 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 (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 ( hydroxy-5-methyloctanoic acid, 3-hydroxy-6-methyloctanoic acid, 3-hydroxy-7-methyloctanoic acid, 3 -Hydroxy-6-methylnonanoic acid, 3-hydroxy-7-meth Nonanoic acid, 3-hydroxy-8-methylnonanoic acid, 3-hydroxy-7-methyldecanoic acid, 3-hydroxy -9-methyldecanoic acid, 3-hydroxy-7-methyl-6-octenic acid, malic acid, 3-hydroxysuccinic acid-methyl ester , 3-hydroxyadipinic acid-methyl ester, 3-hydroxysuberic acid-methyl ester, 3-hydroxyazelaic acid-methyl ester, 3-hydroxy sebacic acid (hydroxysebacic acid,)-methyl ester, 3-hydroxysuberic acid-ethyl ester, 3-hydroxysebacic acid-ethyl ester, 3-hydroxypimelic acid- Propyl ester, 3-hydroxysebacic acid-benzyl ester, 3-hydroxy-8-ace Acetoxyoctanoic acid, 3-hydroxy-9-acetoxynonanoic acid, phenoxy-3-hydroxybutyric acid, phenoxy-3 -Hydroxyvaleric acid, phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxyoctanoic acid, para-cyanophenoxy (cyanophenoxy) -3-hydroxybutyric acid, para-cyanophenoxy-3-hydroxyvaleric acid, para-cyanophenoxy-3-hydroxy Hexahexaic acid, para-nitrophenoxy-3-hydroxyhexanoic acid, 3-hydroxy-5-phenylvaleric acid, 3-hydroxy ( hydroxy-5-cyclohexylbutyric acid, 3,12-dihydroxydodecanoic acid, 3,8-dihydroxy-5-cis-tet 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 (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-bromooundecanoic acid, 3-hydroxy Hydroxy-2-butenoic acid, 6-hydroxy-3- Dodecenoic acid, 3-hydroxy-2-methylbutyric acid, 3-hydroxy-2-methylvaleric acid and 3-hydroxy It may be characterized in that at least one selected from the group consisting of -2,6-dimethyl-5-heptenic acid (heptenoic acid).

In the present invention, the lactate-glycolate-hydroxyalkanoate copolymer is poly (lactate-co-glycolate), poly (2HB-co-lactate-co-glycolate), poly (2HB-co-3HB -co-lactate-co-glycolate), poly (3HB-co-lactate-co-glycolate), poly (4HB-co-lactate-co-glycolate), poly (3HP-co-lactate-co-glycolate), poly (3HB-co-4HB-co-lactate-co-glycolate) and poly (3HP-co-4HB-co-lactate-co-glycolate).

In this case, when glycolate or hydroxyalkanoate and glycolate are used as the carbon source, the lactate-glycolate copolymer or the lactate-glycolate-hydroxyalkanoate copolymer [poly (hydroxyalkanoate- co-glycolate)]. In this case, when fatty acid and glycolate are used as the carbon source, it is possible to prepare a copolymer [poly (3HA-co-glycolate)] having 3 to 12 carbon atoms and glycolate.

In another aspect, the present invention relates to a 2-hydroxybutyrate (2HB) -lactate-glycolate copolymer [poly (2HB-co-lactate-co-glycolate)].

In another aspect, the present invention relates to a 2-hydroxybutyrate-3-hydroxybutyrate-lactate-glycolate copolymer [poly (2HB-co-3HB-co-lactate-co-glycolate)].

In the present invention, the term "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. In the present invention, a plasmid vector, a bacteriophage vector, a cosmid vector, a YAC (Yeast Artificial Chromosome) vector, and the like may be used. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for such purposes include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. Preferred host cells for the present invention may be prokaryotic or eukaryotic cells. Preferred host cells are prokaryotic cells. Suitable prokaryotic cells can be used not only in the microorganism having the gene of the PHA synthase described above, but also in the microorganism having no PHA synthase gene, such as E. coli. In addition, E. coli transformed with a PHA synthase gene may be used as a microorganism having the PHA synthase gene. Preferred E. coli include E. coli DH5a, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL1-Blue (Stratagene), E. coli B and the like. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the microorganisms having the genes of E. coli and PHA synthases described above, Agrobacterium strains such as Agrobacterium A4, bacilli such as Bacillus subtilis , Salmonella typhimurium ( Another enterobacteria such as Salmonella typhimurium ) or Serratia marcescens may be used as the host cell. Known eukaryotic host cells such as yeast and fungi, insect cells such as Spodoptera fruitgiper (SF9), animal cells such as CHO and mouse cells, tissue cultured human cells and plant cells can also be used. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself.

As is well known in the art, to raise the expression level of a transgene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the chosen expression host. Preferably, the expression control sequence and the gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the expression host is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.

The transformed cells by the above-described expression vector constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. Within the scope of these variables, one skilled in the art can select various vector / expression control sequence / host combinations suitable for the present invention.

As the method for inserting the gene on the chromosome of the host cell in the present invention can be used a commonly known genetic engineering method, for example retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector , Poxvirus vectors, lentiviral vectors or non-viral vectors.

On the other hand, transformation of plants can be achieved by conventional methods using Agrobacterium, viral vectors and the like. For example, after transforming the microorganism of the genus Agrobacterium with a recombinant vector containing the gene according to the present invention, the transformed plant can be obtained by infecting the transformed Agrobacterium microorganisms in the tissues of the target plant. For example, a transgenic plant suitable for the present invention can be obtained by the same or similar method as in the prior patent (WO 94/11519; US 6,103,956) for producing PHA using the transformed plant.

Plants to be transformed usable in the present invention include tobacco, tomato, pepper, soybean, rice, corn, and the like, but is not limited thereto. In addition, even if the plant used for transformation is a sexual propagation plant, it will be apparent to those skilled in the art that it can be repeatedly reproduced by tissue culture.

Through the following examples will be described the present invention in more detail. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

In particular, the following examples in order to provide the substrate for the PHA synthase lactyl-CoA and glycolyl-CoA, lactate and glycolate each lactyl-CoA and a gene of an enzyme that converted into glycolyl-CoA Clostridium propionicum-derived propionate CoA Although transferase ( pct ) was used, it would be apparent to those skilled in the art that the same result can be obtained using transferase genes derived from other microorganisms.

In particular, in the following examples, Pseudomonas sp. As a gene of PHA synthase which can use lactyl-CoA and glycolyl-CoA as a substrate. Although only phaC1 derived from MBEL 6-19 is exemplified, the same results can be obtained by using PHA synthetase with similar substrate specificity derived from various microorganisms such as Wautersia eutropha, Alcaligenes latus , Sinorhizobium meliloti , Bacillus megaterium , and Chromatium vinosum . It will be apparent to those skilled in the art to which the present invention pertains.

In addition, in the following examples, only E. coli was exemplified as a microorganism having no PHA synthase gene and a microorganism having a PHA synthase gene, but the same result was obtained using other kinds of cells (bacteria, yeast, fungi, flora and fauna cells) and plants. It will also be apparent to those skilled in the art that the present invention can be obtained.

Example 1 Preparation of Recombinant Vector for Preparation of Poly (lactate-co-glycolate) and Poly (lactate-co-glycolate-co-hydroxyalkanoate)

1-1. Construction of pPs619C1300-CPPCT Recombinant Vector

As enzymes that can use lactyl-CoA and glycolyl-CoA as substrates, Pseudomonas sp. A variant of PHA synthase derived from MBEL 6-19 (KCTC 11027BP) was used (Korean Patent Publication No. 2008-0047279).

Pseudomonas sp. To isolate the PHA synthase (phaC1Ps6-19) gene from MBEL 6-19 (KCTC 11027BP), Pseudomonas sp. Extracting the entire DNA of MBEL 6-19, and based on the phaC1Ps6-19 gene sequence (Song Ae-jin, Master's Thesis, Department of Chemical and Biomolecular Engineering, KAIST, 2004), primers having the nucleotide sequences of SEQ ID NOs: 3 and 4 PCR was performed using the extracted whole DNA as a template. The obtained PCR product was electrophoresed to identify a 1.7 kb gene fragment corresponding to the phaC1Ps6-19 gene, thereby obtaining a phaC1Ps6-19 gene.

SEQ ID NO: 5- GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG -3

SEQ ID NO: 5-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3

In order to express phaC1Ps6-19 synthase, a operon-type constitutive expression system in which a monomer feed enzyme and a synthetase were expressed together was constructed as follows.

DNA fragments containing PHB-producing operons from Ralstonia eutropha H16 from pSYL105 vector (Lee et al. , Biotech. Bioeng ., 1994, 44: 1337-1347) were digested with BamHI / EcoRI and BamHI of pBluescript II (Stratagene) PReCAB recombinant vector was prepared by inserting in / EcoRI recognition site.

The pReCAB vector is known to express PHA synthase (phaCRE) and monomer feedase (phaARE & phaBRE) at all times by the PHB operon promoter and work well in Escherichia coli (Lee et al ., Biotech . Bioeng ., 44: 1337, 1994). The pReCAB vector was digested with BstBI / SbfI to remove R.eutropha H16 PHA synthase (phaCRE), and then the pPs619C1-ReAB recombinant vector was prepared by inserting the phaC1Ps6-19 gene obtained above into the BstBI / SbfI recognition site.

In order to make a phaC1Ps6-19 synthase gene fragment containing only one BstBI / SbfI recognition site at each end, the BstBI position, which is inherent in the BstBI / SbfI recognition site, was removed without using amino acid conversion by the SDM (site directed mutagenesis) method. In order to add the overlapping PCR was performed using a primer having a nucleotide sequence of SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10.

SEQ ID NO: 5- atg ccc gga gcc ggt tcg aa-3

SEQ ID NO: 5- CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC-3

SEQ ID NO: 5- GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG-3

SEQ ID NO: 5- CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3

SEQ ID NO: 5- GTA CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG-3

       SEQ ID NO: 10: 5- aac ggg agg gaa cct gca gg-3

In order to confirm whether or not the phaC1Ps6-19 synthetase is synthesized with PHB, pPs619C1-ReAB recombinant vector is transformed into E. coli XL-1Blue (Stratagene, USA), and the PHB detection medium (LB agar, glucose 20g / L, No growth of PHB was observed when grown in Nile red 0.5μg / ml).

Three amino acid positions affecting SCL (short chain length) activity were found through amino acid sequence sequencing, and the phaC1Ps6-19 synthetase variants as shown in Table 1 using the SDM method using the primers of SEQ ID NOs: 11-16 made.

SEQ ID NO: 5- CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC- 3

SEQ ID NO: 12- 5-GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG- 3

SEQ ID NO: 5- CGA GCA GCG GGC ATA TC A TGA GCA TCC TGA ACC CGC- 3

SEQ ID NO: 14 5- GCG GGT TCA GGA TGC TCA TGA TAT GCC CGC TGC TCG-3

SEQ ID NO: 15 5- atc aac ctc atg acc gat gcg atg gcg ccg acc-3

SEQ ID NO 16: 5- ggt cgg cgc cat cgc atc ggt cat gag gtt gat- 3

phaC1 _Ps6-19 synthase variant Recombinant vector Nucleic Acid Substitution Amino acid substitutions primer pPs619C1200-ReAB AGC → ACC S325T SEQ ID NO: 11/12 CAG → ATG Q481M SEQ ID NO: 13/14 pPs619C1300-ReAB GAA → GAT E130D SEQ ID NO: 15/16 AGC → ACC S325T SEQ ID NO: 11/12 CAG → ATG Q481M SEQ ID NO: 13/14

These recombinant vectors were transformed into E. coli XL-1Blue and grown in PHB detection medium (LB agar, glucose 20g / L, Nile red 0.5μg / ml). As a result, PHB production was confirmed in both E. coli XL-1Blue transformed with pPs619C1200-ReAB and E. coli XL-1Blue transformed with pPs619C1300-ReAB.

That is, 3HB-CoA is generated from glucose by monomer supply enzymes phaARE and phaBRE, and phaC1Ps6-19 synthase SCL variants (phaC1Ps6-19200 & phaC1Ps6-19300) synthesize PHB using the substrate as a substrate.

In order to construct a system for the constant expression of an operon in which a propionate CoA transferase for providing lactyl-CoA and glycolyl-CoA, which are monomers necessary for synthesizing a lactate-glycolate copolymer, is expressed together Propionate CoA transferase (CP-PCT) from Clostridium propionicum was used.

CP-PCT used a fragment obtained by PCR chromosomal DNA of Clostridium propionicum using primers of SEQ ID NO: 17 and SEQ ID NO: 18. At this time, the Nde I site originally present in the wild type CP-PCT was removed using the SDM method for ease of cloning.

SEQ ID NO: 17: 5-GGAATTCATGAGAAAGGTTCCCATTATTACCGCAGATGA-3

SEQ ID NO: 18 5-gc tctaga tta gga ctt cat ttc ctt cag acc cat taa gcc ttc tg-3

In addition, overlapping PCR was performed using primers having the nucleotide sequences of SEQ ID NOs: 19 and 20 to add Sbf I / Nde I recognition sites.

SEQ ID NO: 19 5-agg cct gca ggc gga taa caa ttt cac aca gg- 3

SEQ ID NO: 20 5-gcc cat atg tct aga tta gga ctt cat ttc c- 3

The pPs619C1300-ReAB vector containing phaC1Ps6-19 synthase SCL variant phaC1 _Ps6-19 300 was cleaved with Sbf I / Nde I to remove the monomer feed enzyme (phaARE & phaBRE) derived from Ralstonia eutrophus H16, and then the PCR cloned CP- The pPs619C1300-CPPCT recombinant vector was prepared by inserting the PCT gene into the Sbf I / Nde I recognition site (FIG. 1).

1-2. Construction of pPs619C1300-CPPCT540 Recombinant Vector

CP-PCT is known to be highly toxic due to severe metabolic disorders when expressed in E. coli. In general, the expression of CP-PCT by IPTG using the tac promoter or T7 promoter, which is widely used for expression of recombinant proteins, is recombined simultaneously with the addition of inducers. E. coli all died.

Thus, in order to prepare Clostridium propionicum propionate CoA transferase variants, lactate-glycolate copolymers and lactate-glycolate-hydroxyal using a constantly expressed system that is weakly expressed but continuously expressed as the microorganism grows The synthesis of the canoate copolymer was successful.

To introduce random mutagenesis into the CP-PCT gene, pPs619C1300-CPPCT prepared in 1-1 above was used as a template, and Mn2 + was added using primers of SEQ ID NOs: 21 and 22, and the concentration difference of dNTPs was decreased. Error-prone PCR was performed under the present conditions.

SEQ ID NO: 21 5-CGCCGGCAGGCCTGCAGG-3

SEQ ID NO: 22 5-GGCAGGTCAGCCCATATGTC-3

Thereafter, PCR was performed under normal conditions using the primers of SEQ ID NOs: 21 and 22 to amplify the PCR fragment containing the random mutation.

The pPs619C1300-CPPCT vector containing phaC1Ps6-19 synthase SCL variant phaC1Ps6-19300 was digested with SbfI / Nde I to remove wild type cp-pct, and then the amplified mutant PCR fragment was inserted into the Sbf I / Nde I recognition site. Ligation mixture was prepared and introduced into E. coli JM109 to produce a CP-PCT library of ~ 10 ⁵ scale.

The prepared CP-PCT library was grown in a polymer detection medium (LB agar, glucose 20g / L, 3HB 1g / L, Nile red 0.5μg / ml) for 3 days and then screened to determine whether the polymer was produced. More than 80 candidates were selected first. These candidates were subjected to liquid culture (LB agar, glucose 20g / L, 3HB 1g / L, ampicillin 100mg / L, 37 ° C) for 4 days under the conditions where the polymer was produced, and the final two individuals were analyzed by FACS (Florescence Activated Cell Sorting) analysis. Was selected.

Gene sequences were analyzed to find mutation positions of the prepared CP-PCT variants, and the results are shown in Table 2 below.

CP-PCT Primary Variants CP-PCT variant version Nucleic Acid Substitution CP-PCT Variant 512 A1200G CP-PCT Variant 522 T78C, T669C, A1125G, T1158C

Based on the final selected mutants (CP-PCT Variant 512, CP-PCT Variant 522), the random mutation was performed again by the method of Error-prone PCR to obtain various CP-PCT variants. Among them, there were CP-PCT variants 532 and 540 as shown in Table 3 below.

CP-PCT Secondary Variants CP-PCT variant version Mutations Silent mutations CP-PCT Variant 532 Ala243Thr A1200G CP-PCT Variant 540 Val193Ala T78C, T669C, A1125G, T1158C

Thereafter, PCR was performed under general conditions using the primers of SEQ ID NOs: 19 and 20 to amplify the PCR fragment containing the CpPct540 mutation. The pPs619C1300-CPPCT vector to remove the CPPCT section, taken on SbfI / Nde I, the amplified CpPct532 PCR fragment Sbf I / Nde I made that ligation mixture into the recognition site to prepare a pPs619C1300-CPPCT540 vector (Korea Pat. Published 2009-0017252).

       1-3. Construction of pPs619C1310-CPPCT540 Recombinant Vector

Lactate-glycolate copolymers using lactyl-CoA and glycolyl-CoA as substrates using variants of polyhydroxyalkanoate synthase of Pseudomonas sp.MBEL 6-19 And a system capable of preparing lactate-glycolate-hydroxyalkanoate copolymers.

Based on the phaC1Ps6-19 synthase variant (phaC1Ps6-19300) prepared in 1-1 above, the genus Pseudomonas having the amino acid sequence of E130D, S477F and Q481K was mutated using the SDM method using the primers of SEQ ID NOs: 23 and 24. PHA synthase variant (phaC1Ps6-19310) derived from MBEL 6-19 was prepared (Korean Patent Publication No. 2008-0047279).

SEQ ID NO: 23: 5'-gaa ttc gtg ctg tcg agc cgc ggg cat atc- 3 '

SEQ ID NO: 24 5'-gat atg ccc gcg gct cga cag cac gaa ttc-3

The recombinant vector (Fig. 2, pPs619C1310-CPPCT540) obtained therefrom was transformed into E. coli JM109, and the polymer detection medium containing 3HB (LB agar, glucose 20g / L, 3HB 2g / L, Nile red 0.5μg / growth in ml) confirmed the formation of polymers.

Example 2: Preparation of Recombinant Escherichia Coli and Preparation of Lactate-Glycolate Copolymer

PPs619C1310-CpPCT540 prepared in Example 1 was introduced into E. coli XL1-Blue to produce recombinant E. coli XL1-Blue (pPs619C1310-CpPCT540), and then flask culture was performed. First, recombinant E. coli XL1-Blue (pPs619C1310-CpPCT540) was incubated in MR medium containing 20 g / L glucose and 2 g / L glycolate for 3 days. The composition of the MR medium is as follows: 6.67 g of KH ₂ PO ₄ , ₄ g of (NH ₄ ) ₂ HPO ₄ , 0.8 g of MgSO ₄ · 7H ₂ O, 0.8 g of citric acid, and 5 mL of trace metal solution. Trace metal solution (per liter): 5 M HCl 5 mL, FeSO ₄ 7H ₂ O 10 g, CaCl _{2 2} g, ZnSO ₄ 7H ₂ O 2.2 g, MnSO ₄ 4H ₂ O 0.5 g, CuSO ₄ 5H ₂ O 1 g, (NH ₄ ) 6 Mo ₇ O ₂ .4H ₂ O 0.1 g, and Na ₂ B ₄ O ₂ .10H ₂ O 0.02 g.

The culture solution was centrifuged to recover the cells, lyophilized, and the polymer material accumulated in the cells was recovered using chloroform. As a result of analyzing the monomer composition of the polymer by gas chromatography, it was confirmed that P (6 mol% LA-co-94 mol% GA). At this time, the lactate monomer (LA) is provided from the lactate produced inside of E. coli from glucose as a carbon source. Cells of the recombinant E. coli strains synthesized lactate-glycolate copolymers were observed under a microscope (Fig. 6).

Example 3 Preparation of Recombinant Escherichia Coli and Preparation of Lactate-Glycolate-Hydroxyalkanoate Copolymer

PPs619C1310-CpPCT540 prepared in Example 1 was introduced into E. coli XL1-Blue to produce recombinant E. coli XL1-Blue (pPs619C1310-CpPCT540), and then flask culture was performed.

First, recombinant E. coli XL1-Blue (pPs619C1310-CpPCT540) was added to MR medium containing 20 g / L glucose, 2 g / L glycolate, and 2 g / L 2-hydroxybutyrate (2HB), or 20 g / L glucose. (Glucose) and 2g / L glycolate, 2g / L 2-hydroxybutyrate (2HB) and 2g / L 3-hydroxybutyrate (3HB) containing the culture medium for 3 days. The culture solution was centrifuged to recover the cells, lyophilized, and the polymer materials accumulated in the cells were recovered using chloroform. As a result of analyzing the monomer composition of the polymer by gas chromatography, P (53mol% 2HB-co-17mol% LA-co-30mol% GA) and P (30mol% 2HB-co-43mol% 3HB-co-9mol% LA-, respectively co-22mol% GA). At this time, the lactate monomer (LA) is provided from the lactate produced inside of E. coli from glucose as a carbon source. Cells of the prepared E. coli strains synthesized from the 2HB-lactate-glycolate copolymer or the 2HB-3HB-lactate-glycolate copolymer were observed under a microscope (FIGS. 7 and 8).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a clot ridium propionicum- derived pct gene and Pseudomonas sp. The construction process and gene map of the recombinant plasmid pPs619C1300-CpPCT containing MBEL 6-19 derived phaC1 mutant are shown.

Figure 2 is a Clost ridium propionicum- derived pct mutant and Pseudomonas sp. The construction process and gene map of the recombinant plasmid pPs619C1310-CpPCT540 containing MBEL 6-19 derived phaC1 mutant are shown.

      3 is a schematic diagram showing a route for synthesizing a glycolate polymer (PGA) using glucose and glycolate.

      4 is a diagram illustrating a route for synthesizing a lactate-glycolate copolymer (PLGA) using glucose and glycolate.

      5 is a diagram illustrating a route for synthesizing a glycolate-lactate-3HB-2HB copolymer using glucose, glycolate, 3HB, and 2HB.

      Figure 6 is a micrograph of E. coli XL1-Blue (pPs619C1310-CpPCT540) synthesized P (LA-co-GA).

Figure 7 is a micrograph of E. coli XL1-Blue (pPs619C1310-CpPCT540) synthesized P (LA-co-GA-co-2HB).

Figure 8 is a micrograph of E. coli XL1-Blue (pPs619C1310-CpPCT540) synthesized P (LA-co-GA-co-2HB-co-3HB).

<110> Korea Advance Institute Science and Technology          LG CHEM, LTD <120> Method for Preparing Poly (lactate-co-glycolate) or          Poly (lactate-co-glycolate-co-hydroxyalkanoate) Copolymers Using          Cells or Plants Having Producing Ability of          Poly (lactate-co-glycolate) or          Poly (lactate-co-glycolate-co-hydroxyalkanoate) Copolymers <130> P09-B032 <160> 24 <170> KopatentIn 1.71 <210> 1 <211> 1572 <212> DNA <213> Clostridium propionicum <400> 1 atgagaaagg ttcccattat taccgcagat gaggctgcaa agcttattaa agacggtgat 60 acagttacaa caagtggttt cgttggaaat gcaatccctg aggctcttga tagagctgta 120 gaaaaaagat tcttagaaac aggcgaaccc aaaaacatta cctatgttta ttgtggttct 180 caaggtaaca gagacggaag aggtgctgag cactttgctc atgaaggcct tttaaaacgt 240 tacatcgctg gtcactgggc tacagttcct gctttgggta aaatggctat ggaaaataaa 300 atggaagcat ataatgtatc tcagggtgca ttgtgtcatt tgttccgtga tatagcttct 360 cataagccag gcgtatttac aaaggtaggt atcggtactt tcattgaccc cagaaatggc 420 ggcggtaaag taaatgatat taccaaagaa gatattgttg aattggtaga gattaagggt 480 caggaatatt tattctaccc tgcttttcct attcatgtag ctcttattcg tggtacttac 540 gctgatgaaa gcggaaatat cacatttgag aaagaagttg ctcctctgga aggaacttca 600 gtatgccagg ctgttaaaaa cagtggcggt atcgttgtag ttcaggttga aagagtagta 660 aaagctggta ctcttgaccc tcgtcatgta aaagttccag gaatttatgt tgactatgtt 720 gttgttgctg acccagaaga tcatcagcaa tctttagatt gtgaatatga tcctgcatta 780 tcaggcgagc atagaagacc tgaagttgtt ggagaaccac ttcctttgag tgcaaagaaa 840 gttattggtc gtcgtggtgc cattgaatta gaaaaagatg ttgctgtaaa tttaggtgtt 900 ggtgcgcctg aatatgtagc aagtgttgct gatgaagaag gtatcgttga ttttatgact 960 ttaactgctg aaagtggtgc tattggtggt gttcctgctg gtggcgttcg ctttggtgct 1020 tcttataatg cggatgcatt gatcgatcaa ggttatcaat tcgattacta tgatggcggc 1080 ggcttagacc tttgctattt aggcttagct gaatgcgatg aaaaaggcaa tatcaacgtt 1140 tcaagatttg gccctcgtat cgctggttgt ggtggtttca tcaacattac acagaataca 1200 cctaaggtat tcttctgtgg tactttcaca gcaggtggct taaaggttaa aattgaagat 1260 ggcaaggtta ttattgttca agaaggcaag cagaaaaaat tcttgaaagc tgttgagcag 1320 attacattca atggtgacgt tgcacttgct aataagcaac aagtaactta tattacagaa 1380 agatgcgtat tccttttgaa ggaagatggt ttgcacttat ctgaaattgc acctggtatt 1440 gatttgcaga cacagattct tgacgttatg gattttgcac ctattattga cagagatgca 1500 aacggccaaa tcaaattgat ggacgctgct ttgtttgcag aaggcttaat gggtctgaag 1560 gaaatgaagt cc 1572 <210> 2 <211> 559 <212> PRT Pseudomonas sp. 6-19 <400> 2 Met Ser Asn Lys Ser Asn Asp Glu Leu Lys Tyr Gln Ala Ser Glu Asn   1 5 10 15 Thr Leu Gly Leu Asn Pro Val Val Gly Leu Arg Gly Lys Asp Leu Leu              20 25 30 Ala Ser Ala Arg Met Val Leu Arg Gln Ala Ile Lys Gln Pro Val His          35 40 45 Ser Val Lys His Val Ala His Phe Gly Leu Glu Leu Lys Asn Val Leu      50 55 60 Leu Gly Lys Ser Gly Leu Gln Pro Thr Ser Asp Asp Arg Arg Phe Ala  65 70 75 80 Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Leu Gln Thr                  85 90 95 Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Trp Ile Asp Glu Ser Asn             100 105 110 Leu Ala Pro Lys Asp Val Ala Arg Gly His Phe Val Ile Asn Leu Met         115 120 125 Thr Glu Ala Met Ala Pro Thr Asn Thr Ala Ala Asn Pro Ala Ala Val     130 135 140 Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Ser 145 150 155 160 His Leu Ala Lys Asp Leu Val His Asn Gly Gly Met Pro Ser Gln Val                 165 170 175 Asn Met Gly Ala Phe Glu Val Gly Lys Ser Leu Gly Val Thr Glu Gly             180 185 190 Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Lys Pro         195 200 205 Thr Thr Glu Gln Val Tyr Glu Arg Pro Leu Leu Val Val Pro Pro Gln     210 215 220 Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser Leu Ala 225 230 235 240 Arg Phe Cys Leu Arg Asn Asn Val Gln Thr Phe Ile Val Ser Trp Arg                 245 250 255 Asn Pro Thr Lys Glu Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Glu             260 265 270 Ala Leu Lys Glu Ala Val Asp Val Val Thr Ala Ile Thr Gly Ser Lys         275 280 285 Asp Val Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala     290 295 300 Leu Leu Gly His Tyr Ala Ala Ile Gly Glu Asn Lys Val Asn Ala Leu 305 310 315 320 Thr Leu Leu Val Ser Val Leu Asp Thr Thr Leu Asp Ser Asp Val Ala                 325 330 335 Leu Phe Val Asn Glu Gln Thr Leu Glu Ala Ala Lys Arg His Ser Tyr             340 345 350 Gln Ala Gly Val Leu Glu Gly Arg Asp Met Ala Lys Val Phe Ala Trp         355 360 365 Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu     370 375 380 Leu Gly Asn Glu Pro Pro Val Phe Asp Ile Leu Phe Trp Asn Asn Asp 385 390 395 400 Thr Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Ile Glu Leu Phe                 405 410 415 Lys Asn Asn Pro Leu Ile Arg Pro Asn Ala Leu Glu Val Cys Gly Thr             420 425 430 Pro Ile Asp Leu Lys Gln Val Thr Ala Asp Ile Phe Ser Leu Ala Gly         435 440 445 Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cys Tyr Lys Ser Ala Gln     450 455 460 Leu Phe Gly Gly Asn Val Glu Phe Val Leu Ser Ser Ser Gly His Ile 465 470 475 480 Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ser Arg Tyr Met Thr                 485 490 495 Ser Thr Glu Val Ala Glu Asn Ala Asp Glu Trp Gln Ala Asn Ala Thr             500 505 510 Lys His Thr Asp Ser Trp Trp Leu His Trp Gln Ala Trp Gln Ala Gln         515 520 525 Arg Ser Gly Glu Leu Lys Lys Ser Pro Thr Lys Leu Gly Ser Lys Ala     530 535 540 Tyr Pro Ala Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg 545 550 555 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gagagacaat caaatcatga gtaacaagag taacg 35 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cactcatgca agcgtcaccg ttcgtgcacg tac 33 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atgcccggag ccggttcgaa 20 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cgttactctt gttactcatg atttgattgt ctctc 35 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gagagacaat caaatcatga gtaacaagag taacg 35 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cactcatgca agcgtcaccg ttcgtgcacg tac 33 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gtacgtgcac gaacggtgac gcttgcatga gtg 33 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 aacgggaggg aacctgcagg 20 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctgaccttgc tggtgaccgt gcttgatacc acc 33 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ggtggtatca agcacggtca ccagcaaggt cag 33 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 cgagcagcgg gcatatcatg agcatcctga acccgc 36 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gcgggttcag gatgctcatg atatgcccgc tgctcg 36 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 atcaacctca tgaccgatgc gatggcgccg acc 33 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ggtcggcgcc atcgcatcgg tcatgaggtt gat 33 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ggaattcatg agaaaggttc ccattattac cgcagatga 39 <210> 18 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gctctagatt aggacttcat ttccttcaga cccattaagc cttctg 46 <210> 19 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 aggcctgcag gcggataaca atttcacaca gg 32 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gcccatatgt ctagattagg acttcatttc c 31 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 cgccggcagg cctgcagg 18 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 ggcaggtcag cccatatgtc 20 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gaattcgtgc tgtcgagccg cgggcatatc 30 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 gatatgcccg cggctcgaca gcacgaattc 30  

Claims (22)

Enzyme gene that converts lactate and glycolate to lactyl-CoA and glycolyl-CoA, and polyhydroxyalkanoate synthase gene that can use lactyl-CoA and glycolyl-CoA as substrate Comprising culturing or culturing cells or plants having the same, A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. The method of claim 1, Cells or plants are transfected with a gene of an enzyme that converts lactate and glycolate to lactyl-CoA and glycolyl-CoA, respectively, in a cell or plant containing a polyhydroxyalkanoate synthase gene. Being A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. 3. The method of claim 2, Cells containing polyhydroxyalkanoate synthase genes include: Aeromonas microorganism, Achromobacter microorganism, Acidovorax delafieldii , Acidovax facilis ,, Acinetobacter microorganism, Actinomyces microorganism, Aeromonas microorganism, Alcaligenes microorganism, Alteromonas microorganism, Amoebobacter Genus microorganism, Aphanocapa sp., Aphanothece sp. Aquaspirillum autotrophicum, Azorhizobium caulinodans, Azospirillum sp ., Azospirillum spp, Azotobacter spp, Bacillus spp, Beggiatoa spp, Beijerinckia spp, Beneckea spp, Bordetella pertussis, Bradyrhizobium japonicum, Caryophamon latum, Caulobacter spp, Chlorogloea spp ., Chromatium microorganism of the genus, Chromobacterium spp, Clostridium spp, Comamonas spp, Corynebacterium spp, Cyanobacteria in microorganisms, Derxia spp, Desulfonema spp, Desulfosacina variabilis, Desulfovibrio sapovorans, Ectothiorhodospira spp, Ferrobacillus ferroxidans, Flavobacterium sp, Haemophilus influenzae, Halobacterium spp, Haloferax mediterranei, Hydroclathratus clathratus, Hydrogenomonas facilis, microorganisms in Hydrogenophaga, microorganisms in Hyphomicrobium, Ilyobacter delafieldii, Labrys monachus, Lamprocystis reseopersicina, Lampropedia hyalina, Legionella sp., L eptothrix discophorus, Methylobacterium spp, Methylosinus spp, Micrococcus spp, Mycobacterium spp, Nitrobacter spp, Nocardia spp, Paracoccus dentrificans, Oscillatoria limosa, Penicillium cyclopium, Photobacterium spp, Physarum ploycephalum, Pseudomonas spp, Ralstonia spp , Rhizobium spp, Rhodobacillus spp, Rhodobacter spp, Rhodococcus spp, Rhodocyclus spp, Rhodomicrobium vannielii, Rhodopseudomonas spp, Rhodospirillum spp, Sphingomonas paucimobilis, Spirillum spp, Spirulina spp, Staphylococcus spp, Stella spp , Streptomyces genus microorganism, Syntrophomonas wolfei, Thermophilic cyanobacteria, Thermus thermophilus, Thiobacillus A2, Thiobacillus genus microorganism, Thiocapsa genus microorganism, Thiocystis violacea , Vibrio parahaemolyticus , Xanthobacter autotrophicus, Xanthomonas maltophilia, and Zoogloea Selected from the group consisting of the genus microorganisms A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. The method of claim 1, Cells or plants are genes of enzymes that convert lactate and glycolate to lactyl-CoA and glycolyl-CoA, and polyhydroxyalkano that can use lactyl-CoA and glycolyl-CoA as substrates. Transformed with the Eight synthase gene A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. The method according to any one of claims 1 to 4, The enzyme gene for converting lactate and glycolate into lactyl-CoA and glycolyl-CoA, respectively, is the propionate CoA-transferase gene ( pct ). A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. The method according to any one of claims 1 to 4, The gene of the enzyme that converts lactate and glycolate into lactyl-CoA and glycolyl-CoA, respectively, (a) A1200G variant in the nucleotide sequence of SEQ ID NO: 1; (b) T78C, T669C, A1125G and T1158C at the nucleotide sequences of SEQ ID NO: 1; (c) a Gly335Asp variant in the amino acid sequence corresponding to SEQ ID NO: 1 and an A1200G variant in the nucleotide sequence of SEQ ID NO: 1; (d) Ala243Thr variant in amino acid sequence corresponding to SEQ ID NO: 1 and A1200G in base sequence of SEQ ID NO: 1; (e) Asp65Gly mutations in the amino acid sequence corresponding to SEQ ID NO: 1 and T669C, A1125G and T1158C mutations in the nucleotide sequence of SEQ ID NO: 1; (f) an Asp257Asn variant in the amino acid sequence corresponding to SEQ ID NO: 1 and an A1200G variant in the nucleotide sequence of SEQ ID NO: 1; (g) Asp65Asn mutation in the amino acid sequence corresponding to SEQ ID NO: 1 and T669C, A1125G and T1158C mutations in the nucleotide sequence of SEQ ID NO: 1; And (h) a gene encoding a propionate CoA-transferase variant containing a Thr199Ile mutation in the amino acid sequence corresponding to SEQ ID NO: 1 and a mutation selected from the group consisting of T669C, A1125G and T1158C in the nucleotide sequence of SEQ ID NO: 1 sign A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. The method according to any one of claims 1 to 4, The polyhydroxyalkanoate synthase gene is phaC1 A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. The method according to any one of claims 1 to 4, Polyhydroxyalkanoate synthase gene is the amino acid sequence of SEQ ID NO: 2, (i) S325T and Q481M; (ii) E130D, S325T and Q481M; (iii) E130D, S325T, S477R and Q481M; And (iv) is a gene encoding a polyhydroxyalkanoate synthase variant, characterized in that it has an amino acid sequence containing a mutation selected from the group consisting of E130D, S477F and Q481K A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. The method of claim 1, Copolymer comprising the lactate monomer and glycolate monomer further comprises a hydroxyalkanoate monomer A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. 10. The method of claim 9, The hydroxyalkanoate is 2-hydroxybutyrate, 3-hydroxypropionate, 3-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxy Butanoic acid, medium chain length (D) -3-hydroxycarboxylic acids having 6 to 14 carbon atoms, 3-hydroxypropionic acid, 3-hydroxyhexanoic acid), 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxy acid Hydroxyundecanoic acid, 3-hydroxy dodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, 4-hydroxy valeric acid ( hydroxyvaleric acid), 4-hydroxy 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 Hydroxy-6-trans-octennoic acid, 3-hydroxy-6-cis-octenic acid, 3-hydroxy-7-octennoic acid ), 3-hydroxy-8-nonenoic acid, 3-hydroxy-9-decenoic acid, 3-hydroxy-5-cis-dodecenoic acid (dodecenoic acid), 3-hydroxy-6-cis-dodecenoic acid, 3-hydroxy 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 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 (methyloctanoic acid), 3-hydroxy-6-methylnonanoic acid, 3-hydroxy-7-methylnonanoic acid, 3-hydroxy-8 Methylnonanoic acid, 3-hydroxy-7-methyldecanoic acid (m ethyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, 3-hydroxy-7-methyl-6-octenic acid, malic acid, 3 -Hydroxysuccinic acid -methyl ester, 3-hydroxyadipinic acid -methyl ester, 3-hydroxysuberic acid -methyl ester, 3-hydroxyazelaic acid ) -Methyl ester, 3-hydroxysebacic acid, -methyl ester, 3-hydroxysuberic acid-ethyl ester, 3-hydroxysebacic acid-ethyl ester, 3 Hydroxypimelic acid-propyl ester, 3-hydroxysebacic acid-benzyl ester, 3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy Hydroxy-9-acetoxynonanoic acid, phenoxy-3-hydroxy Hydroxybutyric acid, phenoxy-3-hydroxyvaleric acid, phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxy 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, 3-hydroxy-5-cyclohexylbutyric acid, 3,12-dihydroxydodecanoic acid, 3,8-dihydroxy (dihydroxy) -5-cis-tetradecenoic acid, 3-hydroxy-4,5-epoxydecanoic acid, 3-hydroxy-6,7-epoxy Deoxydodecanoic acid, 3-hydroxy-8,9-epoxy-5,6-cis-tetradecanoic acid, 7-cyano-3-hydroxy Heptanoic acid (hydroxyheptanoic acid), 9-cyano-3-hydroxynonanoic acid, 3-hydroxy-7-fluoroheptanoic acid, 3-hydroxy ( 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-bromodecane Acid (bromoundecanoic acid), 3-hydroxy-2-butenoic acid, 6-hydroxy-3-dodecenoic acid, 3-hydroxy-2- Methylbutyric acid, 3-hydroxy-2-methylvaleric acid (me thylvaleric acid) and at least one selected from the group consisting of 3-hydroxy-2,6-dimethyl-5-heptenoic acid A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. 10. The method of claim 9, The copolymer is poly (2HB-co-lactate-co-glycolate), poly (2HB-co-3HB-co-lactate-co-glycolate), poly (3HB-co-lactate-co-glycolate), poly (4HB -co-lactate-co-glycolate), poly (3HP-co-lactate-co-glycolate), poly (3HB-co-4HB-co-lactate-co-glycolate) and poly (3HP-co-4HB-co- lactate-co-glycolate) A method for producing a copolymer comprising a lactate monomer and a glycolate monomer. A copolymer comprising a lactate monomer and a glycolate monomer. The method of claim 12, The copolymer is 2-hydroxybutyrate, 3-hydroxypropionate, 3-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxybutanoic acid hydroxybutyrate, medium chain length (D) -3-hydroxycarboxylic acids having 6 to 14 carbon atoms, 3-hydroxypropionic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxy undecanoic acid (hydroxyundecanoic acid), 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxyhexadecanoic acid, 4-hydroxyvaleric acid , 4-hydroxyhexanoic acid hexanoic 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-octenic acid, 3-hydroxy-6-cis-octenic acid, 3-hydroxy-7-octennoic 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 acid, 3-hydroxy-4-methylvaleric acid, 3-hydroxy-4-methylhexanoic acid, 3-hydroxy-5-methyl acid Hexane acid (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 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-octenic acid, malic acid, 3-hydroxysuccinic acid (hydroxysuccinic acid) -methyl ester, 3-hydroxyadipinic acid-methyl ester, 3-hydroxysuberic acid-methyl ester, 3-hydroxyazelaic acid-methyl ester , 3-hydroxysebacic acid, methyl ester, 3-hydroxysuberic acid-ethyl ester, 3-hydroxysebacic acid-ethyl ester, 3-hydroxypi Hydroxypimelic acid-propyl ester, 3-hydroxysebacic acid-benzyl ester, 3-hydroxy-8-acetoxyoctanoic acid, 3-hydroxy -9-acetoxynonanoic acid, phenoxy-3-hydroxybutanoic acid (hydroxybut yric acid, phenoxy-3-hydroxyvaleric acid, phenoxy-3-hydroxyheptanoic acid, phenoxy-3-hydroxyoctanoic acid (hydroxyoctanoic acid), para-cyanophenoxy-3-hydroxybutyric acid, para-cyanophenoxy-3-hydroxyvaleric acid, para-sia Cyanophenoxy-3-hydroxyhexanoic acid, para-nitrophenoxy-3-hydroxyhexanoic acid, 3-hydroxy-5-phenylvaleric Phenylvaleric acid, 3-hydroxy-5-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 epoxydodec anoic acid), 3-hydroxy-8,9-epoxy-5,6-cis-tetradecanoic acid, 7-cyano-3-hydroxyheptanoic acid ( 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 Hydroxy-6-bromohexanoic acid, 3-hydroxy-8-bromooctanoic acid, 3-hydroxy-11-bromooundecanoic acid, 3-hydroxy-2-butenoic acid, 6-hydroxy-3-dodecenoic acid, 3-hydroxy-2-methylbutanoic acid (methylbutyric acid), 3-hydroxy-2-methylvaleric acid, 3-hydroxy (hydroxy) -2,6- dimethyl-5-heptenyl tensan copolymer comprises from the group consisting of (heptenoic acid) by addition of one or more hydroxy alkanoates selected. The method of claim 13, The copolymer is 2-hydroxybutyrate (2HB) -lactate-glycolate copolymer [poly (2HB-co-lactate-co-glycolate)]. The method of claim 13, The copolymer is 2-hydroxybutyrate-3-hydroxybutyrate-lactate-glycolate copolymer [poly (2HB-co-3HB-co-lactate-co-glycolate)]. delete delete delete delete delete delete delete

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