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
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 Download PDFInfo
- Publication number
- KR101085960B1 KR101085960B1 KR1020090030133A KR20090030133A KR101085960B1 KR 101085960 B1 KR101085960 B1 KR 101085960B1 KR 1020090030133 A KR1020090030133 A KR 1020090030133A KR 20090030133 A KR20090030133 A KR 20090030133A KR 101085960 B1 KR101085960 B1 KR 101085960B1
- Authority
- KR
- South Korea
- Prior art keywords
- acid
- hydroxy
- glycolate
- lactate
- spp
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
- C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
- C08G63/08—Lactones or lactides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
- C12P7/20—Glycerol
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
본 발명은 락테이트(lactate)와 글라이콜레이트(glycolate)를 각각 lactyl-CoA와 glycolyl-CoA로 전환시키는 프로피오네이트 CoA-트랜스퍼라아제(propionate CoA transferase, Pct) 유전자와 lactyl-CoA와 glycolyl-CoA를 기질로 사용하는 폴리하이드록시알카노에이트(polyhydroxyalkanoate, PHA) 합성효소 유전자를 동시에 가지는 세포 또는 식물을 배양 또는 재배하는 것을 특징으로 하는 락테이트-글라이콜레이트 공중합체 및 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체의 제조방법에 관한 것이다.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.
본 발명에 따르면, 세포 또는 식물을 이용하여 락테이트-글리콜레이트 공중합체를 제조하는 것이 가능할 뿐만 아니라, 글라이콜레이트, 락테이트 및 다양한 하이드록시알카노에이트를 모노머로 함유하고 있는 다양한 종류의 폴리에스터를 제조하는 것이 가능하다.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.
락테이트-글라이콜레이트 공중합체, 프로피오네이트 CoA-트랜스퍼라아제, 폴리하이드록시알카노에이트(PHA) 합성효소 Lactate-glycolate copolymers, propionate CoA-transferases, polyhydroxyalkanoate (PHA) synthase
Description
본 발명은 락테이트(lactate)와 글라이콜레이트(glycolate)를 각각 lactyl-CoA와 glycolyl-CoA로 전환시키는 프로피오네이트 CoA-트랜스퍼라아제(propionate CoA transferase, Pct) 유전자와 lactyl-CoA와 glycolyl-CoA를 기질로 사용하는 폴리하이드록시알카노에이트(polyhydroxyalkanoate, PHA) 합성효소 유전자를 동시에 가지는 세포 또는 식물을 배양 또는 재배하는 것을 특징으로 하는 락테이트-글라이콜레이트 공중합체 및 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체의 제조방법에 관한 것이다.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.
락테이트-글라이콜레이트 공중합체 [Poly(lactate-co-glycolate), PLGA]는 락테이트(lactate)와 글라이콜레이트(glycolate)로부터 유래된 대표적인 생분해성 고분자로서 범용고분자 혹은 의료용 고분자로서의 응용성이 높은 고분자이다. 현재 PLGA는 락테이트와 글라이콜레이트의 직접중합반응에 의해 제조될 수 있으나, 상기 반응을 통해서는 주로 낮은 분자량(1000-5000 달톤)의 PLGA만이 생성된다. 100,000 달톤 이상의 높은 분자량의 PLGA는 락타이드(lactide)와 글라이콜라이드(glycolide)의 링 개환축합반응을 통해 합성되어질 수 있다. 락타이드와 글라이콜라이드는 각각 락테이트와 글라이콜레이트의 고리 디에스테르(cyclic diester)로서, 각각 락테이트 올리고머와 글라이콜레이트 올리고머의 열분해에 의해 만들어진다. 링 개환축합반응에는 2-에틸헥사논산 주석(tin(II) 2-ethylhexanoate), 주석 알콕사이드(tin(II) alkoxide), 또는 알루미늄 이소프로폭사이드(aluminum isopropoxide) 등의 촉매의 사용이 요구되어진다. 직접중합으로 얻어진 낮은 분자량의 고분자로부터 chain coupling agent를 이용하여 보다 분자량이 높은 고분자로 중합하는 방법이 있으나 chain coupling agent를 이용하기 때문에 고분자량의 PLGA를 제조하는 방법은 유기용제(solvent)나 chain coupling agent의 첨가로 인해 공정이 복잡해지고, 또한 이들을 제거가 쉽지 않다는 단점이 있다. 현재 상용화되고 있는 고분자량의 PLGA 생산공정은 락테이트와 글라이콜레이트를 각각 락타이드와 글라이콜라이드로 전환한 다음, 락타이드와 글라이콜라이드의 링 개환축합반응을 통해 PLGA를 합성하는 방법이 사용되고 있다. 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. .
한편, PHA는 과도한 탄소원이 존재하면서 인, 질소, 마그네슘, 산소 등의 다른 영양분이 부족할 때, 미생물이 에너지나 탄소원 저장물질로 그 내부에 축적하는 폴리에스터(polyester)이다. PHA는 기존의 석유로부터 유래된 합성고분자와 비슷한 물성을 가지면서 완전한 생분해성을 보이기 때문에 기존의 합성 플라스틱을 대체할 물질로 인식되고 있다. 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.
기존에 알려진 PHA는 대표적으로 짧은 탄소수를 가진 SCL-PHA (short-chain-length PHA)와 긴 탄소수를 가진 MCL-PHA (medium-chain-length PHA)로 나눌 수 있다. PHA를 합성하는 유전자는 Ralstonia eutropha, Pseudomonas 등으로부터 클로닝되었으며 재조합 미생물을 통해 다양한 종류의 모노머로 구성된 PHA가 합성되었다 (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).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).
미생물에서 PHA를 생산하기 위해서는 미생물의 대사산물을 PHA 모노머로 전환해 주는 효소와 PHA 모노머를 이용하여 PHA 고분자를 합성하는 PHA 합성효소(synthase)가 필수적이다. PHA 합성효소는 hydroxyacyl-CoA를 기질로 사용하여 PHA를 합성하며, PHA의 기질인 hydroxyacyl-CoA를 제공할 수 있는 효소로는 Ralstonia eutropha 등으로부터 클로닝된 β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), Pseudomonas로부터 클로닝된 3-hydroxydecanoyl-ACP:CoA transferase (PhaG), Aeromonas caviae와 Pseudomonas aeruginosa로부터 유래된 (R)-specific enoyl-CoA hydratase (PhaJ) (Fukui et al., J. Bacteriol., 180:667, 1998; Tsuge et al., FEMS Microbiol. Lett., 184:193, 2000), 대장균과 Pseudomonas aeruginosa 등으로부터 유래된 3-ketoacyl-ACP reductase (FabG) 등이 알려져 있다 (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). 이러한 효소들을 이용하여 다양한 탄소위치(주로 3, 4, 5, 6번 위치)가 hydroxylation된 하이드록시알카노에이트를 이용하여 다양한 종류의 PHA를 합성해 왔다.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).
하지만 2번 위치가 hydroxylation된 하이드록시알카노에이트에 대한 PHA 합성효소의 반응성은 거의 없는 것으로 보고되어 있다 (Zhang et al., Appl. Microbiol. Biotechnol., 56:131, 2001; Valentin and Steinbuchel, Appl. Microbiol. Biotechnol., 40:699, 1994). In vitro에서 PHA synthase의 glycolyl-CoA에 대해 반응성을 분석한 보고는 있었지만, glycolyl-CoA에 대한 PHA synthase의 반응성은 매우 미약하였다 (Zhang et al., Appl. Microbiol. Biotechnol., 56:131, 2001; Valentin and Steinbuchel, Appl. Microbiol. Biotechnol., 40:699, 1994). 즉, 2번-탄소위치가 hydroxylation된 글라이콜레이트와 같은 하이드록시알카노에이트는 PHA synthase의 기질특이성에 적합하지 않아, 자연적으로 혹은 재조합 세포나 식물을 이용하여 PHA 및 그 공중합체를 제조한 예는 없다.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.
2-hydroxy acid 모노머로서 글라이콜레이트를 포함하는 폴리하이드록시알카노에이트(PHA) 공중합체에 대한 특허가 보고되어진 적은 있으나(US 2003/0211131 A1), 이는 다양한 PHA 모노머 중의 하나로 글라이콜레이트를 포함시킨 것에 그 의 미가 있을 뿐, 폴리락테이트(PLA) 또는 폴리글라이콜레이트(PGA)의 공중합체인 락테이트-글라이콜레이트 공중합체(PLGA) 및 이의 다양한 하이드록시알카노에이트와의 공중합체[poly(lactate-co-glycolate-co-hydroxyalkanoate)]의 생합성과는 차별화된다. 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.
본 발명자들은 락테이트와 글라이콜레이트를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소인 Clostridium propionicum 유래 프로피오네이트 CoA-트랜스퍼라아제(Pct) 유전자와 lactyl-CoA와 glycolyl-CoA를 기질로 사용할 수 있는 폴리하이드록시알카노에이트(PHA) 합성효소 유전자로 형질전환된 재조합 대장균을 포도당(glucose)과 글라이콜레이트 또는 포도당과 글라이콜레이트, 하이드록시알카노에이트가 포함된 생산배지에서 배양하는 경우, poly(lactate-co-glycolate) 공중합체 및 poly(lactate-co-glycolate-co-hydroxyalkanoate) 공중합체가 생성되는 것을 확인하고 본 발명을 완성하게 되었다. 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.
결국, 본 발명의 목적은 세포 또는 식물을 이용하여 락테이트-글라이콜레이트 공중합체 또는 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체를 제조하기 위한, 프로피오네이트 CoA-트랜스퍼라아제 유전자와 폴리하이드록시알카노에이트 합성효소 유전자를 함유하는 세포 또는 식물을 배양 또는 재배하는 것을 특징으로 하는 락테이트-글라이콜레이트 공중합체 또는 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체의 제조방법을 제공하는데 있다.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.
상기 목적을 달성하기 위하여, 본 발명은 락테이트(lactate)와 글라이콜레이트(glycolate)를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소의 유전자 및 lactyl-CoA와 glycolyl-CoA를 기질로 사용할 수 있는 폴리하이드록시알카노에이트 합성효소 유전자를 동시에 가지는 세포 또는 식물을 배양 또는 재배하여, 배양배지에 첨가된 락테이트 및 글라이콜레이트를 이용하거나, 상기 세포 또는 식물 내에서 자체 생산된 락테이트 및 글라이콜레이트를 이용하여, 락테이트-글라이콜레이트 공중합체를 합성한 다음, 상기 배양된 세포 또는 재배된 식물로부터 락테이트-글라이콜레이트 공중합체를 회수하는 것을 특징으로 하는 락테이트-글라이콜레이트 공중합체 [poly(lactate-co-glycolate)]의 제조방법을 제공한다.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)].
또한, 본 발명은 락테이트와 글라이콜레이트롤 모노머로 함유하는 락테이트- 글라이콜레이트 공중합체 [poly(lactate-co-glycolate)]를 제공한다.The present invention also provides a lactate-glycolate copolymer [poly (lactate-co-glycolate)] containing lactate and glycolate monomers.
또한, 본 발명은 락테이트(lactate)와 글라이콜레이트(glycolate)를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소의 유전자 및 lactyl-CoA와 glycolyl-CoA를 기질로 사용할 수 있는 폴리하이드록시알카노에이트 합성효소 유전자를 동시에 가지는 세포 또는 식물을 배양 또는 재배하여, 배양배지에 첨가된 락테이트, 글라이콜레이트 및 하이드록시알카노에이트를 이용하거나, 상기 세포 또는 식물 내에서 자체 생산된 락테이트, 글라이콜레이트 및 하이드록시알카노에이트를 이용하여, 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체를 합성한 다음, 상기 배양된 세포 또는 재배된 식물로부터 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체를 회수하는 것을 특징으로 하는 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체 [poly(lactate-co-glycolate-co-hydroxyalkanoate)]의 제조방법을 제공한다.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)].
또한, 본 발명은 2-하이드록시부탄산(hydroxybutyrate), 3-하이드록시프로피온산(hydroxypropionate), 3-하이드록시부탄산(hydroxybutyrate), 3-하이드록시발레르산(hydroxyvalerate), 4-하이드록시부탄산(hydroxybutyrate), 탄소수가 6∼14개인 중간사슬 길이의 (D)-3-하이드록시카르복실산(hydroxycarboxylic acids), 3-하이드록시프로피온산(hydroxypropionic acid), 3-하이드록시헥산산(hydroxyhexanoic acid), 3-하이드록시헵탄산(hydroxyheptanoic acid), 3-하이드록시옥탄산(hydroxyoctanoic acid), 3-하이드록시노난산(hydroxynonanoic acid), 3-하이드록시데칸산(hydroxydecanoic acid), 3-하이드록시운데칸 산(hydroxyundecanoic acid), 3-하이드록시 도데칸산(hydroxydodecanoic acid), 3-하이드록시테트라데칸산(hydroxytetradecanoic acid), 3-하이드록시헥사데칸산(hydroxyhexadecanoic acid), 4-하이드록시발레르산(hydroxyvaleric acid), 4-하이드록시헥산산(hydroxyhexanoic acid), 4-하이드록시헵탄산(hydroxyheptanoic acid), 4-하이드록시옥탄산(hydroxyoctanoic acid), 4-하이드록시데칸산(hydroxydecanoic acid), 5-하이드록시발레르산(hydroxyvaleric acid), 5-하이드록시헥산산(hydroxyhexanoic acid), 6-하이드록시도데칸산(hydroxydodecanoic acid), 3-하이드록시(hydroxy)-4-펜텐산(pentenoic acid), 3-하이드록시(hydroxy)-4-trans-헥센산(hexenoic acid), 3-하이드록시(hydroxy)-4-cis-헥센산(hexenoic acid), 3-하이드록시(hydroxy)-5-헥센산(hexenoic acid), 3-하이드록시(hydroxy)-6-trans-옥텐산(octenoic acid), 3-하이드록시(hydroxy)-6-cis-옥텐산(octenoic acid), 3-하이드록시(hydroxy)-7-옥텐산(octenoic acid), 3-하이드록시(hydroxy)-8-노넨산(nonenoic acid), 3-하이드록시(hydroxy)-9-데센산(decenoic acid), 3-하이드록시(hydroxy)-5-cis-도데센산(dodecenoic acid), 3-하이드록시(hydroxy)-6-cis-도데센산(dodecenoic acid), 3-하이드록시(hydroxy)-5-cis-테트라데센산(tetradecenoic acid), 3-하이드록시(hydroxy)-7-cis-테트라데센산(tetradecenoic acid), 3-하이드록시(hydroxy)-5,8-cis-cis-테트라데센산(tetradecenoic acid), 3-하이드록시(hydroxy)-4-메틸발레르산(methylvaleric acid), 3-하이드록시(hydroxy)-4-메틸헥산산(methylhexanoic acid), 3-하이드록시(hydroxy)-5-메틸헥산산(methylhexanoic acid), 3-하이드록시(hydroxy)-6-메틸헵 탄산(methylheptanoic acid), 3-하이드록시(hydroxy)-4-메틸옥탄산(methyloctanoic acid), 3-하이드록시(hydroxy)-5-메틸옥탄산(methyloctanoic acid), 3-하이드록시(hydroxy)-6-메틸옥탄산(methyloctanoic acid), 3-하이드록시(hydroxy)-7-메틸옥탄산(methyloctanoic acid), 3-하이드록시(hydroxy)-6-메틸노난산(methylnonanoic acid), 3-하이드록시(hydroxy)-7-메틸노난산(methylnonanoic acid), 3-하이드록시(hydroxy)-8-메틸노난산(methylnonanoic acid), 3-하이드록시(hydroxy)-7-메틸데칸산(methyldecanoic acid), 3-하이드록시(hydroxy)-9-메틸데칸산(methyldecanoic acid), 3-하이드록시(hydroxy)-7-메틸-6-옥텐산(octenoic acid), 말산(malic acid), 3-하이드록시숙신산(hydroxysuccinic acid)-메틸에스테르, 3-하이드록시아디핀산(hydroxyadipinic acid)-메틸에스테르, 3-하이드록시스베린산(hydroxysuberic acid)-메틸에스테르, 3-하이드록시아젤라인산(hydroxyazelaic acid)-메틸에스테르, 3-하이드록시세바신산(hydroxysebacic acid,)-메틸에스테르, 3-하이드록시스베린산(hydroxysuberic acid)-에틸에스테르, 3-하이드록시세바신산(hydroxysebacic acid)-에틸에스테르, 3-하이드록시피메린산(hydroxypimelic acid)-프로필에스테르, 3-하이드록시세바신산(hydroxysebacic acid)-벤질에스테르, 3-하이드록시(hydroxy)-8-아세톡시옥탄산(acetoxyoctanoic acid), 3-하이드록시(hydroxy)-9-아세톡시노난산(acetoxynonanoic acid), 페녹시(phenoxy)-3-하이드록시부탄산(hydroxybutyric acid), 페녹시(phenoxy)-3-하이드록시발레르산(hydroxyvaleric acid), 페녹시(phenoxy)-3-하이드록시헵탄산(hydroxyheptanoic acid), 페녹시(phenoxy)-3-하이드록시옥탄산(hydroxyoctanoic acid), para-시아노 페녹시(cyanophenoxy)-3-하이드록시부탄산(hydroxybutyric acid), para-시아노페녹시(cyanophenoxy)-3-하이드록시발레르산(hydroxyvaleric acid), para-시아노페녹시(cyanophenoxy)-3-하이드록시헥산산(hydroxyhexanoic acid), para-니트로페녹시(nitrophenoxy)-3-하이드록시헥산산(hydroxyhexanoic acid), 3-하이드록시(hydroxy)-5-페닐발레르산(phenylvaleric acid), 3-하이드록시(hydroxy)-5-시클로헥실부탄산(cyclohexylbutyric acid), 3,12-디하이드록시도데칸산(dihydroxydodecanoic acid), 3,8-디하이드록시(dihydroxy)-5-cis-테트라데센산(tetradecenoic acid), 3-하이드록시(hydroxy)-4,5-에폭시데칸산(epoxydecanoic acid), 3-하이드록시(hydroxy)-6,7-에폭시도데칸산(epoxydodecanoic acid), 3-하이드록시(hydroxy)-8,9-에폭시(epoxy)-5,6-cis-테트라데칸산(tetradecanoic acid), 7-시아노(cyano)-3-하이드록시헵탄산(hydroxyheptanoic acid), 9-시아노(cyano)-3-하이드록시노난산(hydroxynonanoic acid), 3-하이드록시(hydroxy)-7-플루오로헵탄산(fluoroheptanoic acid), 3-하이드록시(hydroxy)-9-플루오로노난산(fluorononanoic acid), 3-하이드록시(hydroxy)-6-클로로헥산산(chlorohexanoic acid), 3-하이드록시(hydroxy)-8-클로로옥탄산(chlorooctanoic acid), 3-하이드록시(hydroxy)-6-브로모헥산산(bromohexanoic acid), 3-하이드록시(hydroxy)-8-브로모옥탄산(bromooctanoic acid), 3-하이드록시(hydroxy)-11-브로모운데칸산(bromoundecanoic acid), 3-하이드록시(hydroxy)-2-부텐산(butenoic acid), 6-하이드록시(hydroxy)-3-도데센산(dodecenoic acid), 3-하이드록시(hydroxy)-2-메틸부탄산(methylbutyric acid), 3-하이드록시(hydroxy)-2-메틸발레르산(methylvaleric acid), 및 3-하이드록시(hydroxy)-2,6-디메틸-5-헵텐산(heptenoic acid)로 구성된 군에서 선택된 하나 이상의 하이드록시알카노에이트와 락테이트 및 글라이콜레이트를 모노머로 함유하는 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체 [poly(lactate-co-glycolate-co-hydroxyalkanoate)]를 제공한다.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.
또한, 본 발명은 2-하이드록시부티레이트(2HB)-락테이트-글라이콜레이트 공중합체 [poly(2HB-co-lactate-co-glycolate)]를 제공한다.The present invention also provides a 2-hydroxybutyrate (2HB) -lactate-glycolate copolymer [poly (2HB-co-lactate-co-glycolate)].
또한, 본 발명은 2-하이드록시부티레이트(2HB)-3-하이드록시부티레이트(3HB)-락테이트-글라이콜레이트 공중합체 [poly(2HB-co-3HB-co-lactate-co-glycolate)]를 제공한다.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.
본 발명에 따르면, 세포 또는 식물을 이용하여 락테이트-글리콜레이트 공중합체를 제조하는 것이 가능할 뿐만 아니라, 글라이콜레이트, 락테이트 및 다양한 하이드록시알카노에이트를 모노머로 함유하고 있는 다양한 종류의 폴리에스터를 제조하는 것이 가능하다.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.
일 관점에서, 본 발명은 락테이트(lactate)와 글라이콜레이트(glycolate)를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소의 유전자 및 lactyl-CoA와 glycolyl-CoA를 기질로 사용할 수 있는 폴리하이드록시알카노에이트 합성효소 유전 자를 동시에 가지는 세포 또는 식물을 배양 또는 재배하여, 배양배지에 첨가된 락테이트 및 글라이콜레이트를 이용하거나, 상기 세포 또는 식물 내에서 자체 생산된 락테이트 및 글라이콜레이트를 이용하여, 락테이트-글라이콜레이트 공중합체를 합성한 다음, 상기 배양된 세포 또는 재배된 식물로부터 락테이트-글라이콜레이트 공중합체를 회수하는 것을 특징으로 하는 락테이트-글라이콜레이트 공중합체 [poly(lactate-co-glycolate)]의 제조방법 및 상기 방법으로 제조된 락테이트와 글라이콜레이트롤 모노머로 함유하는 락테이트-글라이콜레이트 공중합체 [poly(lactate-co-glycolate)]에 관한 것이다.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)].
본 발명의 실시예에서는 포도당 및 글라이콜레이트를 함유하는 배지에서 상기 세포를 배양하여, 세포 내에서 포도당으로부터 생산된 락테이트를 전구체로 이용하였으나, 포도당만을 함유하는 배지를 사용하여, 세포 내에서 생산되는 글라이콜레이트와 락테이트를 이용하여도 락테이트-글라이콜레이트 공중합체를 제조할 수 있다는 것은 당업자에게 자명한 사항이며, 배지에 락테이트만을 첨가하거나, 락테이트와 글라이콜레이트를 첨가하여도 락테이트-글라이콜레이트 공중합체를 제조할 수 있다는 것 또한, 당업자에게 자명할 것이다.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.
본 발명에 있어서, 상기 세포 또는 식물은 폴리하이드록시알카노에이트 합성효소 유전자를 함유하는 세포 또는 식물에 락테이트와 글라이콜레이트를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소의 유전자가 형질전환되어 있거나 염색체 상에 삽입되어 있는 것을 특징으로 할 수 있다.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.
상기 PHA 합성효소의 유전자를 가지는 세포로는 다양한 미생물이 알려져 있 다 (KR 10-250830 B1). 예를 들면, Aeromonas 속 미생물, Achromobacter 속 미생물, Acidovorax delafieldii, Acidovax facilis,, Acinetobacter 속 미생물, Actinomyces 속 미생물, Aeromonas 속 미생물, Alcaligenes 속 미생물, Alteromonas 속 미생물, Amoebobacter 속 미생물, Aphanocapa sp., Aphanothece sp. Aquaspirillum autotrophicum, Azorhizobium caulinodans, Azospirillum sp., Azospirillum 속 미생물, Azotobacter 속 미생물, Bacillus 속 미생물, Beggiatoa 속 미생물, Beijerinckia 속 미생물, Beneckea 속 미생물, Bordetella pertussis, Bradyrhizobium japonicum, Caryophamon latum, Caulobacter 속 미생물, Chlorogloea 속 미생물, Chromatium 속 미생물, Chromobacterium 속 미생물, Clostridium 속 미생물, Comamonas 속 미생물, Corynebacterium 속 미생물, Cyanobacteria 속 미생물, Derxia 속 미생물, Desulfonema 속 미생물, Desulfosacina variabilis, Desulfovibrio sapovorans, Ectothiorhodospira 속 미생물, Ferrobacillus ferroxidans, Flavobacterium sp., Haemophilus influenzae, Halobacterium 속 미생물, Haloferax mediterranei, Hydroclathratus clathratus, Hydrogenomonas facilis, Hydrogenophaga 속 미생물, Hyphomicrobium 속 미생물, Ilyobacter delafieldii, Labrys monachus, Lamprocystis reseopersicina, Lampropedia hyalina, Legionella sp., Leptothrix discophorus, Methylobacterium 속 미생물, Methylosinus 속 미생물, Micrococcus 속 미생물, Mycobacterium 속 미생물, Nitrobacter 속 미생물, Nocardia 속 미생물, Paracoccus dentrificans, Oscillatoria limosa, Penicillium cyclopium, Photobacterium 속 미생물, Physarum ploycephalum, Pseudomonas 속 미생물, Ralstonia 속 미생물, Rhizobium 속 미생물, Rhodobacillus 속 미생물, Rhodobacter 속 미생물, Rhodococcus 속 미생물, Rhodocyclus 속 미생물, Rhodomicrobium vannielii, Rhodopseudomonas 속 미생물, Rhodospirillum 속 미생물, Sphingomonas paucimobilis, Spirillum 속 미생물, Spirulina 속 미생물, Staphylococcus 속 미생물, Stella 속 미생물, Streptomyces 속 미생물, Syntrophomonas wolfei, Thermophilic cyanobacteria, Thermus thermophilus, Thiobacillus A2, Thiobacillus 속 미생물, Thiocapsa 속 미생물, Thiocystis violacea, Vibrio parahaemolyticus, Xanthobacter autotrophicus, Xanthomonas maltophilia, Zoogloea 속 등을 들 수 있다.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.
본 발명에 있어서, 세포 또는 식물은 락테이트와 글라이콜레이트를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소의 유전자 및 lactyl-CoA와 glycolyl-CoA를 기질로 사용할 수 있는 폴리하이드록시알카노에이트 합성효소 유전자로 형질전환되거나, 락테이트와 글라이콜레이트를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소의 유전자 및 lactyl-CoA와 glycolyl-CoA를 기질로 사용할 수 있는 폴리하이드록시알카노에이트 합성효소 유전자가 염색체 상에 삽입되어 있는 것을 특징으로 할 수 있으며, 상기 락테이트와 글라이콜레이트를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소의 유전자는 프로피오네이트 CoA-트랜스퍼라아제 유전자(pct)인 것을 특징으로 할 수 있다.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).
상기 락테이트와 글라이콜레이트를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소의 유전자는 (a) 서열번호 1의 염기서열에서 A1200G 변이; (b) 서열번호 1의 염기서열에서 T78C, T669C, A1125G 및 T1158C; (c) 서열번호 1과 대응하는 아미노산 서열에서 Gly335Asp 변이 및 서열번호 1의 염기서열에서 A1200G 변이; (d) 서열번호 1과 대응하는 아미노산 서열에서 Ala243Thr 변이 및 서열번호 1의 염기서열에서 A1200G; (e) 서열번호 1과 대응하는 아미노산 서열에서 Asp65Gly 변이 및 서열번호 1의 염기서열에서 T669C, A1125G 및 T1158C 변이; (f) 서열번호 1과 대응하는 아미노산 서열에서 Asp257Asn 변이 및 서열번호 1의 염기서열에서 A1200G 변이; (g) 서열번호 1과 대응하는 아미노산 서열에서 Asp65Asn 변이 및 서열번호 1의 염기서열에서 T669C, A1125G 및 T1158C 변이; 및 (h) 서열번호 1과 대응하는 아미노산 서열에서 Thr199Ile 변이 및 서열번호 1의 염기서열에서 T669C, A1125G 및 T1158C로 구성된 군에서 선택되는 변이를 함유하는 프로피오네이트 CoA-트랜스퍼라아제 변이체를 코딩하는 유전자인 것을 특징으로 할 수 있으며, 상기 프로피오네이트 CoA-트랜스퍼라아제 변이체는 락테이트와 글라이콜레이트를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소로, Clostridium propionicum 유래 프로피오네이트 CoA-트랜스퍼라아제 유전자(cp-pct)를 error-prone PCR 방법으로 변이시켜 제조하였다 (한국특허공개 2009-0017252).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).
본 발명에 있어서, 폴리하이드록시알카노에이트 합성효소 유전자는 phaC1인 것을 특징으로 할 수 있으며, 상기 폴리하이드록시알카노에이트 합성효소 유전자는 서열번호 2의 아미노산 서열에서, (i) S325T 및 Q481M; (ii) E130D, S325T 및 Q481M; (iii) E130D, S325T, S477R 및 Q481M; 및 (iv) E130D, S477F 및 Q481K로 구성된 군에서 선택되는 변이를 함유하는 아미노산 서열을 가지는 것을 특징으로 하 는 폴리하이드록시알카노에이트 합성효소 변이체를 코딩하는 유전자인 것을 특징으로 할 수 있다 (한국특허공개 2008-0047279).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).
본 발명에 있어서, 상기 폴리하이드록시알카노에이트 합성효소 변이체는 glycolyl-CoA를 기질로 사용할 수 있으며, Pseudomonas sp. MBEL 6-19 유래 phaC1 의 돌연변이체이다.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.
다른 관점에서, 본 발명은 락테이트(lactate)와 글라이콜레이트(glycolate)를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소의 유전자 및 lactyl-CoA와 glycolyl-CoA를 기질로 사용할 수 있는 폴리하이드록시알카노에이트 합성효소 유전자를 동시에 가지는 세포 또는 식물을 배양 또는 재배하여, 배양배지에 첨가된 락테이트, 글라이콜레이트 및 하이드록시알카노에이트를 이용하거나, 상기 세포 또는 식물 내에서 자체 생산된 락테이트, 글라이콜레이트 및 하이드록시알카노에이트를 이용하여, 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체를 합성한 다음, 상기 배양된 세포 또는 재배된 식물로부터 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체를 회수하는 것을 특징으로 하는 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체 [poly(lactate-co-glycolate-co-hydroxyalkanoate)]의 제조방법 및 상기 방법으로 제조된 하이드록시알카노에이트, 락테이트 및 글라이콜레이트를 모노머로 함유하는 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체 [poly(lactate-co-glycolate-co-hydroxyalkanoate)]에 관한 것이다.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)].
본 발명의 실시예에서는 포도당, 글라이콜레이트 및 하이드록시알카노에이트 를 함유하는 배지에서 상기 세포를 배양하여, 세포 내에서 포도당으로부터 생산된 락테이트를 전구체로 이용하였으나, 포도당과 하이드록시알카노에이트만을 함유하는 배지를 사용하여, 세포 내에서 생산되는 글라이콜레이트, 락테이트 및 하이드록시알카노에이트를 이용하여도 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체를 제조할 수 있다는 것은 당업자에게 자명한 사항이며, 배지에 락테이트만을 첨가하거나, 락테이트와 글라이콜레이트, 하이드록시알카노에이트를 첨가하여도 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체를 제조할 수 있다는 것 또한, 당업자에게 자명할 것이다.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.
본 발명에 있어서, 상기 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체를 구성하는 하이드록시알카노에이트는 락테이트(lactate), 2-하이드록시부탄산(hydroxybutyrate), 3-하이드록시프로피온산(hydroxypropionate), 3-하이드록시부탄산(hydroxybutyrate), 3-하이드록시발레르산(hydroxyvalerate), 4-하이드록시부탄산(hydroxybutyrate), 탄소수가 6~14개인 중간사슬 길이의 (D)-3-하이드록시카르복실산(hydroxycarboxylic acids), 3-하이드록시프로피온산(hydroxypropionic acid), 3-하이드록시헥산산(hydroxyhexanoic acid), 3-하이드록시헵탄산(hydroxyheptanoic acid), 3-하이드록시옥탄산(hydroxyoctanoic acid), 3-하이드록시노난산(hydroxynonanoic acid), 3-하이드록시데칸산(hydroxydecanoic acid), 3-하이드록시운데칸산(hydroxyundecanoic acid), 3-하이드록시 도데칸산(hydroxydodecanoic acid), 3-하이드록시테트라데칸산(hydroxytetradecanoic acid), 3-하이드록시헥사데칸산(hydroxyhexadecanoic acid), 4-하이드록시발레르 산(hydroxyvaleric acid), 4-하이드록시헥산산(hydroxyhexanoic acid), 4-하이드록시헵탄산(hydroxyheptanoic acid), 4-하이드록시옥탄산(hydroxyoctanoic acid), 4-하이드록시데칸산(hydroxydecanoic acid), 5-하이드록시발레르산(hydroxyvaleric acid), 5-하이드록시헥산산(hydroxyhexanoic acid), 6-하이드록시도데칸산(hydroxydodecanoic acid), 3-하이드록시(hydroxy)-4-펜텐산(pentenoic acid), 3-하이드록시(hydroxy)-4-trans-헥센산(hexenoic acid), 3-하이드록시(hydroxy)-4-cis-헥센산(hexenoic acid), 3-하이드록시(hydroxy)-5-헥센산(hexenoic acid), 3-하이드록시(hydroxy)-6-trans-옥텐산(octenoic acid), 3-하이드록시(hydroxy)-6-cis-옥텐산(octenoic acid), 3-하이드록시(hydroxy)-7-옥텐산(octenoic acid), 3-하이드록시(hydroxy)-8-노넨산(nonenoic acid), 3-하이드록시(hydroxy)-9-데센산(decenoic acid), 3-하이드록시(hydroxy)-5-cis-도데센산(dodecenoic acid), 3-하이드록시(hydroxy)-6-cis-도데센산(dodecenoic acid), 3-하이드록시(hydroxy)-5-cis-테트라데센산(tetradecenoic acid), 3-하이드록시(hydroxy)-7-cis-테트라데센산(tetradecenoic acid), 3-하이드록시(hydroxy)-5,8-cis-cis-테트라데센산(tetradecenoic acid), 3-하이드록시(hydroxy)-4-메틸발레르산(methylvaleric acid), 3-하이드록시(hydroxy)-4-메틸헥산산(methylhexanoic acid), 3-하이드록시(hydroxy)-5-메틸헥산산(methylhexanoic acid), 3-하이드록시(hydroxy)-6-메틸헵탄산(methylheptanoic acid), 3-하이드록시(hydroxy)-4-메틸옥탄산(methyloctanoic acid), 3-하이드록시(hydroxy)-5-메틸옥탄산(methyloctanoic acid), 3-하이드록시(hydroxy)-6-메틸옥탄산(methyloctanoic acid), 3-하이드록시(hydroxy)-7-메틸옥 탄산(methyloctanoic acid), 3-하이드록시(hydroxy)-6-메틸노난산(methylnonanoic acid), 3-하이드록시(hydroxy)-7-메틸노난산(methylnonanoic acid), 3-하이드록시(hydroxy)-8-메틸노난산(methylnonanoic acid), 3-하이드록시(hydroxy)-7-메틸데칸산(methyldecanoic acid), 3-하이드록시(hydroxy)-9-메틸데칸산(methyldecanoic acid), 3-하이드록시(hydroxy)-7-메틸-6-옥텐산(octenoic acid), 말산(malic acid), 3-하이드록시숙신산(hydroxysuccinic acid)-메틸에스테르, 3-하이드록시아디핀산(hydroxyadipinic acid)-메틸에스테르, 3-하이드록시스베린산(hydroxysuberic acid)-메틸에스테르, 3-하이드록시아젤라인산(hydroxyazelaic acid)-메틸에스테르, 3-하이드록시세바신산(hydroxysebacic acid,)-메틸에스테르, 3-하이드록시스베린산(hydroxysuberic acid)-에틸에스테르, 3-하이드록시세바신산(hydroxysebacic acid)-에틸에스테르, 3-하이드록시피메린산(hydroxypimelic acid)-프로필에스테르, 3-하이드록시세바신산(hydroxysebacic acid)-벤질에스테르, 3-하이드록시(hydroxy)-8-아세톡시옥탄산(acetoxyoctanoic acid), 3-하이드록시(hydroxy)-9-아세톡시노난산(acetoxynonanoic acid), 페녹시(phenoxy)-3-하이드록시부탄산(hydroxybutyric acid), 페녹시(phenoxy)-3-하이드록시발레르산(hydroxyvaleric acid), 페녹시(phenoxy)-3-하이드록시헵탄산(hydroxyheptanoic acid), 페녹시(phenoxy)-3-하이드록시옥탄산(hydroxyoctanoic acid), para-시아노페녹시(cyanophenoxy)-3-하이드록시부탄산(hydroxybutyric acid), para-시아노페녹시(cyanophenoxy)-3-하이드록시발레르산(hydroxyvaleric acid), para-시아노페녹시(cyanophenoxy)-3-하이드록시헥산산(hydroxyhexanoic acid), para-니트로페녹 시(nitrophenoxy)-3-하이드록시헥산산(hydroxyhexanoic acid), 3-하이드록시(hydroxy)-5-페닐발레르산(phenylvaleric acid), 3-하이드록시(hydroxy)-5-시클로헥실부탄산(cyclohexylbutyric acid), 3,12-디하이드록시도데칸산(dihydroxydodecanoic acid), 3,8-디하이드록시(dihydroxy)-5-cis-테트라데센산(tetradecenoic acid), 3-하이드록시(hydroxy)-4,5-에폭시데칸산(epoxydecanoic acid), 3-하이드록시(hydroxy)-6,7-에폭시도데칸산(epoxydodecanoic acid), 3-하이드록시(hydroxy)-8,9-에폭시(epoxy)-5,6-cis-테트라데칸산(tetradecanoic acid), 7-시아노(cyano)-3-하이드록시헵탄산(hydroxyheptanoic acid), 9-시아노(cyano)-3-하이드록시노난산(hydroxynonanoic acid), 3-하이드록시(hydroxy)-7-플루오로헵탄산(fluoroheptanoic acid), 3-하이드록시(hydroxy)-9-플루오로노난산(fluorononanoic acid), 3-하이드록시(hydroxy)-6-클로로헥산산(chlorohexanoic acid), 3-하이드록시(hydroxy)-8-클로로옥탄산(chlorooctanoic acid), 3-하이드록시(hydroxy)-6-브로모헥산산(bromohexanoic acid), 3-하이드록시(hydroxy)-8-브로모옥탄산(bromooctanoic acid), 3-하이드록시(hydroxy)-11-브로모운데칸산(bromoundecanoic acid), 3-하이드록시(hydroxy)-2-부텐산(butenoic acid), 6-하이드록시(hydroxy)-3-도데센산(dodecenoic acid), 3-하이드록시(hydroxy)-2-메틸부탄산(methylbutyric acid), 3-하이드록시(hydroxy)-2-메틸발레르산(methylvaleric acid) 및 3-하이드록시(hydroxy)-2,6-디메틸-5-헵텐산(heptenoic acid)로 구성된 군에서 선택된 하나 이상인 것을 특징으로 할 수 있다.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).
본 발명에 있어서, 상기 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체는 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) 및 poly(3HP-co-4HB-co-lactate-co-glycolate)로 구성된 군에서 선택되는 것을 특징으로 할 수 있다.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).
이 경우, 탄소원으로 글라이콜레이트 또는 하이드록시알카노에이트와 글라이콜레이트를 사용하면, 락테이트-글라이콜레이트 공중합체 또는 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체 [poly(hydroxyalkanoate-co-glycolate)]의 제조가 가능하다. 이 경우, 탄소원으로 지방산(fatty acid)과 글라이콜레이트를 사용하면, 탄소수 3~12의 3HA와 글라이콜레이트의 공중합체 [poly(3HA-co-glycolate)]를 제조하는 것이 가능하다.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.
또 다른 관점에서, 본 발명은 2-하이드록시부티레이트(2HB)-락테이트-글라이콜레이트 공중합체 [poly(2HB-co-lactate-co-glycolate)]에 관한 것이다.In another aspect, the present invention relates to a 2-hydroxybutyrate (2HB) -lactate-glycolate copolymer [poly (2HB-co-lactate-co-glycolate)].
또 다른 관점에서, 본 발명은 2-하이드록시부티레이트-3-하이드록시부티레이트-락테이트-글라이콜레이트 공중합체 [poly(2HB-co-3HB-co-lactate-co-glycolate)]에 관한 것이다.In another aspect, the present invention relates to a 2-hydroxybutyrate-3-hydroxybutyrate-lactate-glycolate copolymer [poly (2HB-co-3HB-co-lactate-co-glycolate)].
본 발명에 있어서, 용어 "벡터 (vector)"는 적합한 숙주 내에서 DNA를 발현시킬 수 있는 적합한 조절 서열에 작동가능하게 연결된 DNA 서열을 함유하는 DNA 제조물을 의미한다. 본 발명에서, 상기 벡터로는 플라스미드 벡터, 박테리오파지 벡터, 코스미드 벡터, YAC(Yeast Artificial Chromosome) 벡터 등이 사용될 수 있 다. 본 발명의 목적상, 플라스미드 벡터를 이용하는게 바람직하다. 그러한 목적에 사용될 수 있는 전형적인 플라스미드 벡터는 (a) 숙주세포당 수백 개의 플라스미드 벡터를 포함하도록 복제가 효율적으로 이루어지도록 하는 복제 개시점, (b) 플라스미드 벡터로 형질전환된 숙주세포가 선발될 수 있도록 하는 항생제 내성 유전자 및 (c) 외래 DNA 절편이 삽입될 수 있는 제한효소 절단부위를 포함하는 구조를 지니고 있다. 적절한 제한효소 절단부위가 존재하지 않을지라도, 통상의 방법에 따른 합성 올리고뉴클레오타이드 어댑터(adaptor) 또는 링커(linker)를 사용하면 벡터와 외래 DNA를 용이하게 라이게이션(ligation)할 수 있다. 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.
라이게이션 후에, 벡터는 적절한 숙주세포로 형질전환되어야 한다. 본 발명에 바람직한 숙주세포는 원핵 또는 진핵생물 세포일 수 있다. 선호되는 숙주세포는 원핵세포이다. 적합한 원핵세포는 전술한 PHA 합성효소의 유전자를 가지는 미생물뿐만 아니라, 대장균과 같이 PHA 합성효소의 유전자를 가지지 않는 미생물이 모두 사용가능하다. 또한, 상기 PHA 합성효소의 유전자를 가지는 미생물로써 PHA 합성효소의 유전자로 형질전환된 대장균을 사용할 수도 있다. 바람직한 대장균은 E.coli DH5a, E.coli JM101, E.coli K12, E.coli W3110, E.coli X1776, E.coli XL1-Blue(Stratagene) 및 E.coli B 등을 포함한다. 그러나 FMB101, NM522, NM538 및 NM539와 같은 E. coli 균주 및 다른 원핵생물의 종(speices) 및 속(genera) 등도 또한 사용될 수 있다. 전술한 E. coli 및 PHA 합성효소의 유전자를 가지는 미생물에 덧붙여, 아그로박테리움 A4와 같은 아그로박테리움 속 균주, 바실루스 섭틸리스(Bacillus subtilis)와 같은 바실리(bacilli), 살모넬라 타이피뮤리 움(Salmonella typhimurium) 또는 세라티아 마르게센스(Serratia marcescens)와 같은 또 다른 장내세균 등이 숙주세포로서 이용될 수 있다. 효모와 곰팡이 같은 주지의 진핵숙주세포, 스포도프테라 프루기페르다(SF9)와 같은 곤충세포, CHO 및 생쥐 세포와 같은 동물세포, 조직배양된 인간세포 및 식물세포도 사용될 수 있다. 적당한 숙주로 형질전환되면, 벡터는 숙주 게놈과 무관하게 복제하고 기능할 수 있거나, 또는 일부 경우에 게놈 그 자체에 통합될 수 있다. 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.
당업계에 주지된 바와 같이, 숙주세포에서 형질전환 유전자의 발현 수준을 높이기 위해서는, 해당 유전자가 선택된 발현 숙주 내에서 기능을 발휘하는 전사 및 해독 발현 조절 서열에 작동가능하도록 연결되어야만 한다. 바람직하게는 발현 조절서열 및 해당 유전자는 세균 선택 마커 및 복제 개시점 (replication origin)을 같이 포함하고 있는 하나의 발현 벡터 내에 포함되게 된다. 발현 숙주가 진핵세포인 경우에는, 발현 벡터는 진핵 발현 숙주 내에서 유용한 발현 마커를 더 포함하여야만 한다.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.
상술한 발현 벡터에 의해 형질전환 세포는 본 발명의 또 다른 측면을 구성한다. 본원 명세서에 사용된 용어 "형질전환"은 DNA를 숙주로 도입하여 DNA가 염색체외 인자로서 또는 염색체 통합완성에 의해 복제가능하게 되는 것을 의미한다. 물론 모든 벡터와 발현 조절 서열이 본 발명의 DNA 서열을 발현하는데 모두 동등하게 기능을 발휘하지는 않는다는 것을 이해하여야만 한다. 마찬가지로 모든 숙주가 동일한 발현 시스템에 대해 동일하게 기능을 발휘하지는 않는다. 그러나, 당업자라면 과도한 실험적 부담없이 본 발명의 범위를 벗어나지 않는 채로 여러 벡터, 발현 조절 서열 및 숙주 중에서 적절한 선택을 할 수 있다. 예를 들어, 벡터를 선택함에 있어서는 숙주를 고려하여야 하는데, 이는 벡터가 그 안에서 복제되어야만 하기 때문이다. 벡터의 복제 수, 복제 수를 조절할 수 있는 능력 및 당해 벡터에 의해 코딩되는 다른 단백질, 예를 들어 항생제 마커의 발현도 또한 고려되어야만 한다. 이들 변수의 범위내에서, 당업자는 본 발명에 적합한 각종 벡터/발현 조절 서열/숙주 조합을 선정할 수 있다.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.
본 발명에서 상기 유전자를 숙주세포의 염색체상에 삽입하는 방법으로는 통상적으로 알려진 유전자조작방법을 사용할 수 있으며, 일례로는 레트로바이러스 벡터, 아데노바이러스 벡터, 아데노-연관 바이러스 벡터, 헤르페스 심플렉스 바이러스 벡터, 폭스바이러스 벡터, 렌티바이러스 벡터 또는 비바이러스성 벡터를 이용하는 방법을 들 수 있다.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.
한편, 식물체의 형질전환은 아그로박테리움이나 바이러스 벡터 등을 이용한 통상의 방법에 의해 달성할 수 있다. 예컨대, 본 발명에 따른 유전자를 함유하는 재조합벡터로 아그로박테리움 속 미생물을 형질전환시킨 다음, 상기 형질전환된 아그로박테리움 속 미생물을 대상 식물의 조직 등에 감염시켜 형질전환 식물을 수득할 수 있다. 예를 들면, 형질전환된 식물을 이용하여 PHA를 제조하는 것에 관한 선행특허(WO 94/11519; US 6,103,956)와 동일 내지 유사한 방법으로 본 발명에 적합한 형질전환 식물을 수득할 수 있다.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.
본 발명에서 이용가능한 형질전환 대상 식물로는 담배, 토마토, 고추, 콩, 벼, 옥수수 등을 들 수 있으나, 이에 한정되는 것은 아니다. 또한, 형질전환에 사 용되는 식물이 유성번식 식물이라 할지라도, 조직배양 등에 의해 무성적으로 반복생식 시킬 수 있다는 것은 당업자에게 자명하다 할 것이다.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.
이하 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의하여 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. 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. .
특히 하기 실시예에서는 PHA 합성효소의 기질인 lactyl-CoA와 glycolyl-CoA를 제공하기 위하여, 락테이트와 글라이콜레이트를 각각 lactyl-CoA와 glycolyl-CoA로 전환하는 효소의 유전자로 Clostridium propionicum 유래 propionate CoA transferase (pct)를 사용하였지만, 다른 미생물 유래의 transferase 유전자를 사용하여도 동일한 결과를 얻을 수 있다는 것은 본 발명이 속하는 분야에서 통상적인 지식을 가진 자에게 자명하다 할 것이다.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.
특히, 하기 실시예에서는 lactyl-CoA와 glycolyl-CoA를 기질로 사용할 수 있는 PHA 합성효소의 유전자로 Pseudomonas sp. MBEL 6-19 유래 phaC1 만을 예시하였지만, Wautersia eutropha, Alcaligenes latus, Sinorhizobium meliloti, Bacillus megaterium, Chromatium vinosum 등 다양한 종류의 미생물로부터 유래된 기질특이성이 비슷한 PHA 합성효소 등을 사용하여도 동일한 결과를 얻을 수 있다는 것은 본 발명이 속하는 분야에서 통상적인 지식을 가진 자에게 자명하다 할 것이다.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.
또한 하기 실시예에서는 PHA 합성효소 유전자를 가지지 않는 미생물 및 PHA 합성효소 유전자를 가지는 미생물로 각각 대장균만을 예시하였지만, 다른 종류의 세포(박테리아, 효모, 곰팡이, 동식물 세포) 및 식물을 사용하여도 동일한 결과를 얻을 수 있다는 것 역시 본 발명이 속하는 분야에서 통상적인 지식을 가진 자에게 자명하다 할 것이다.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.
실시예1: Poly(lactate-co-glycolate) 및 Poly(lactate-co-glycolate-co-hydroxyalkanoate) 제조용 재조합벡터의 제작Example 1 Preparation of Recombinant Vector for Preparation of Poly (lactate-co-glycolate) and Poly (lactate-co-glycolate-co-hydroxyalkanoate)
1-1. pPs619C1300-CPPCT 재조합 벡터의 제작1-1. Construction of pPs619C1300-CPPCT Recombinant Vector
lactyl-CoA와 glycolyl-CoA를 기질로 사용할 수 있는 효소로서 Pseudomonas sp. MBEL 6-19(KCTC 11027BP) 유래의 PHA 합성효소의 변이체를 사용하였다(한국공개특허 2008-0047279). 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. MBEL 6-19(KCTC 11027BP) 유래의 PHA 합성효소(phaC1Ps6-19)유전자를 분리하기 위하여, Pseudomonas sp. MBEL 6-19의 전체 DNA를 추출하고, phaC1Ps6-19유전자 서열(송애진, Master's Thesis, Department of Chemical and Biomolecular Engineering, KAIST, 2004)에 기반하여, 서열번호 3 및 4의 염기서열을 가지는 프라이머를 제작하고, 상기 추출한 전체 DNA를 주형으로 하여, PCR을 수행하였다. 얻어진 PCR 산물을 전기영동하여, phaC1Ps6-19유전자에 해당하는 1.7 kb 크기의 유전자 절편을 확인하고, phaC1Ps6-19유전자를 수득하였다. 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.
서열번호 3: 5- GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG -3SEQ ID NO: 5- GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG -3
서열번호 4 : 5- CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC -3 SEQ ID NO: 5-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3
phaC1Ps6-19합성효소를 발현시키기 위하여 단량체공급효소와 합성효소가 같이 발현되는 오페론 형태의 항시적 발현 시스템을 하기와 같은 방법으로 제작하였다. 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.
pSYL105 벡터(Lee et al., Biotech. Bioeng., 1994, 44:1337-1347)에서 Ralstonia eutropha H16 유래의 PHB 생산 오페론이 함유된 DNA 절편을 BamHI/EcoRI으로 절단하여, pBluescript II (Stratagene)의 BamHI/EcoRI 인식부위에 삽입함으로써 pReCAB 재조합 벡터를 제조하였다.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.
pReCAB 벡터는 PHA 합성효소(phaCRE)와 단량체공급효소(phaARE&phaBRE)가 PHB 오페론 프로모터에 의해 항시적으로 발현되며, 대장균에서도 잘 작동된다고 알려져 있다(Lee et al., Biotech.Bioeng., 44:1337,1994). pReCAB벡터를 BstBI/SbfI으로 절단하여 R.eutropha H16 PHA 합성효소 (phaCRE)를 제거한 다음, 상기에서 수득한 phaC1Ps6-19유전자를 BstBI/SbfI 인식부위에 삽입함으로써 pPs619C1-ReAB 재조합 벡터를 제조하였다. 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.
BstBI/SbfI 인식부위가 각각 양끝에 하나씩만 포함된 phaC1Ps6-19합성효소 유전자 절편을 만들기 위해 우선 내재하고 있는 BstBI 위치를 SDM(site directed mutagenesis) 방법으로 아미노산의 변환없이 제거하였고, BstBI/SbfI 인식부위를 첨가하기 위해 서열번호 5 및 6, 서열번호 7 및 8, 서열번호 9 및 10의 염기서열을 가지는 프라이머를 이용하여 오버랩핑 PCR을 수행하였다. 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.
서열번호 5: 5- atg ccc gga gcc ggt tcg aa - 3 SEQ ID NO: 5- atg ccc gga gcc ggt tcg aa-3
서열번호 6: 5- CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC - 3 SEQ ID NO: 5- CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC-3
서열번호 7: 5- GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG - 3 SEQ ID NO: 5- GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG-3
서열번호 8: 5- CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC - 3 SEQ ID NO: 5- CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3
서열번호 9: 5- GTA CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG - 3 SEQ ID NO: 5- GTA CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG-3
서열번호 10: 5- aac ggg agg gaa cct gca gg - 3 SEQ ID NO: 10: 5- aac ggg agg gaa cct gca gg-3
상기 phaC1Ps6-19합성효소의 PHB 합성 여부를 확인하기 위하여, pPs619C1-ReAB 재조합 벡터를 E.coli XL-1Blue(Stratagene, USA)에 형질전환 시키고, 이를 PHB 검출배지(LB agar, glucose 20g/L, Nile red 0.5μg/ml)에서 생육시킨 결과 PHB 생성이 관찰되지 않았다. 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).
SCL(short chain length) 활성에 영향을 미치는 아미노산 위치 3 곳을 아미노산 서열 배열분석을 통해 찾았고, 서열번호 11~16의 프라이머를 사용한 SDM 방법을 이용하여 하기 표 1과 같은 phaC1Ps6-19합성효소 변이체들을 만들었다. 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.
서열번호 11: 5- CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC- 3 SEQ ID NO: 5- CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC- 3
서열번호 12: 5- GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG- 3 SEQ ID NO: 12- 5-GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG- 3
서열번호 13: 5- CGA GCA GCG GGC ATA TC A TGA GCA TCC TGA ACC CGC- 3 SEQ ID NO: 5- CGA GCA GCG GGC ATA TC A TGA GCA TCC TGA ACC CGC- 3
서열번호 14: 5- GCG GGT TCA GGA TGC TCA TGA TAT GCC CGC TGC TCG- 3 SEQ ID NO: 14 5- GCG GGT TCA GGA TGC TCA TGA TAT GCC CGC TGC TCG-3
서열번호 15: 5- atc aac ctc atg acc gat gcg atg gcg ccg acc- 3 SEQ ID NO: 15 5- atc aac ctc atg acc gat gcg atg gcg ccg acc-3
서열번호 16: 5- ggt cgg cgc cat cgc atc ggt cat gag gtt gat- 3 SEQ ID NO 16: 5- ggt cgg cgc cat cgc atc ggt cat gag gtt gat- 3
이들 재조합 벡터를 E.coli XL-1Blue에 형질전환 시키고, 이를 PHB 검출배지 (LB agar, glucose 20g/L, Nile red 0.5μg/ml)에서 생육시켰다. 그 결과, pPs619C1200-ReAB로 형질전환된 E.coli XL-1Blue와 pPs619C1300-ReAB로 형질전환된 E.coli XL-1Blue에서 모두 PHB 생성을 확인할 수 있었다.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.
즉, 단량체 공급효소인 phaARE와 phaBRE에 의해 글루코스로부터 3HB-CoA가 생성되고, 이를 기질로 하여 phaC1Ps6-19합성효소 SCL 변이체들(phaC1Ps6-19200&phaC1Ps6-19300)이 PHB를 합성한 것이다.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.
여기에 락테이트-글라이콜레이트 공중합체 합성시 필요한 단량체인 lactyl-CoA와 glycolyl-CoA를 제공하기 위한 프로피오네이트 CoA 트랜스퍼라아제가 같이 발현되는 오페론 형태의 항시적 발현되는 시스템을 구축하기 위하여 클로스트리듐 프로피오니쿰(Clostridium propionicum) 유래의 프로피오네이트 CoA 트랜스퍼라아제(CP-PCT)를 사용하였다. 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-PCT는 Clostridium propionicum 의 염색체 DNA를 서열번호 17 및 서열번호 18의 프라이머를 이용하여 PCR하여 얻어진 단편을 사용하였다. 이 때, 원래 야생형 CP-PCT에 존재하는 NdeI site를 cloning의 용이성을 위해 SDM방법을 이용하여 제거하였다. 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.
서열번호 17: 5-GGAATTCATGAGAAAGGTTCCCATTATTACCGCAGATGA-3SEQ ID NO: 17: 5-GGAATTCATGAGAAAGGTTCCCATTATTACCGCAGATGA-3
서열번호 18: 5-gc tctaga tta gga ctt cat ttc ctt cag acc cat taa gcc ttc tg-3SEQ ID NO: 18 5-gc tctaga tta gga ctt cat ttc ctt cag acc cat taa gcc ttc tg-3
또한, SbfI/NdeI인식부위를 첨가하기 위해 서열번호 19과 20의 염기서열을 가지는 프라이머를 이용하여 오버랩핑 PCR을 수행하였다.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.
서열번호 19: 5-agg cct gca ggc gga taa caa ttt cac aca gg- 3 SEQ ID NO: 19 5-agg cct gca ggc gga taa caa ttt cac aca gg- 3
서열번호 20: 5-gcc cat atg tct aga tta gga ctt cat ttc c- 3 SEQ ID NO: 20 5-gcc cat atg tct aga tta gga ctt cat ttc c- 3
phaC1Ps6-19합성효소 SCL 변이체인 phaC1Ps6-19300를 함유한 pPs619C1300-ReAB 벡터를 SbfI/NdeI으로 절단하여 Ralstonia eutrophus H16 유래의 단량체 공급효소 (phaARE&phaBRE)를 제거한 다음, 상기 PCR 클로닝한 CP-PCT 유전자를 SbfI/NdeI인식부위에 삽입함으로써 pPs619C1300-CPPCT 재조합 벡터를 제조하였다(도 1). 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. pPs619C1300-CPPCT540 재조합 벡터의 제작1-2. Construction of pPs619C1300-CPPCT540 Recombinant Vector
CP-PCT의 경우 대장균에서 고발현될 경우 심각한 대사 장애를 일으켜 독성을 나타낸다고 알려져 있는데, 일반적으로 재조합 단백질 발현에 널리 사용되는 tac 프로모터나 T7 프로모터를 사용한 IPTG에 의한 발현유도 시스템에서는 유도제 첨가와 동시에 재조합 대장균이 모두 사멸하였다.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.
따라서, Clostridium propionicum propionate CoA transferase 변이체를 제조하기 위하여, 약하게 발현되지만 미생물 성장에 따라 지속적으로 발현되는 항시적 발현 시스템을 사용하여 락테이트-글라이콜레이트 공중합체 및 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체 합성에 성공하였다. 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.
CP-PCT 유전자에 무작위적 돌연변이(random mutagenesis)를 도입하기 위해 상기 1-1 에서 제작된 pPs619C1300-CPPCT을 주형으로 하고, 서열번호 21 및 22의 프라이머를 이용하여 Mn2+이 첨가되고 dNTPs의 농도 차이가 존재하는 조건에서 Error-prone PCR을 실시하였다.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.
서열번호 21: 5-CGCCGGCAGGCCTGCAGG-3SEQ ID NO: 21 5-CGCCGGCAGGCCTGCAGG-3
서열번호 22: 5-GGCAGGTCAGCCCATATGTC-3SEQ ID NO: 22 5-GGCAGGTCAGCCCATATGTC-3
그 후, 무작위적 돌연변이가 포함된 PCR 단편을 증폭하기 위해 상기 서열번호 21 및 22의 프라이머를 이용하여 일반 조건에서 PCR하였다. 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.
phaC1Ps6-19합성효소 SCL 변이체인 phaC1Ps6-19300를 함유한 pPs619C1300-CPPCT 벡터를 SbfI/NdeI으로 절단하여 야생형 cp-pct를제거한후, 상기 증폭된 돌연변이 PCR 단편을 SbfI/NdeI인식부위에 삽입시킨 ligation mixture를 만들어 E. coli JM109에 도입하여 ~105정도 규모의 CP-PCT 라이브러리를 제작하였다. 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.
상기 제작된 CP-PCT 라이브러리는 고분자 검출배지(LB agar, glucose 20g/L, 3HB 1g/L, Nile red 0.5μg/ml)에서 3일간 생육시킨 후 고분자 생성 여부를 확인하는 스크리닝 작업을 수행하여 ~80여 개체의 후보를 1차 선정하였다. 이들 후보를 고분자가 생성되는 조건에서 4일간 액체 배양(LB agar, glucose 20g/L, 3HB 1g/L, ampicillin 100mg/L, 37℃)하였고, FACS(Florescence Activated Cell Sorting) 분석을 통하여 최종 2개체를 선정하였다. 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.
상기 제작된 CP-PCT 변이체의 돌연변이 위치를 찾기 위해 유전자 염기서열을 분석하였고 그 결과는 다음 표 2와 같다.Gene sequences were analyzed to find mutation positions of the prepared CP-PCT variants, and the results are shown in Table 2 below.
상기 최종 선별된 돌연변이체들(CP-PCT Variant 512, CP-PCT Variant 522)을 기본으로 다시 상기 Error-prone PCR의 방법으로 무작위적 돌연변이를 수행하여 다양한 CP-PCT 변이체들을 얻을 수 있었다. 그 중, 하기 표 3에서 볼 수 있는 바와 같은 CP-PCT 변이체 532와 540이 있었다. 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.
그 후, CpPct540 돌연변이가 포함된 PCR 단편을 증폭하기 위해 상기 서열번호 19 및 20의 프라이머를 이용하여 일반조건에서 PCR하였다. 상기 pPs619C1300-CPPCT 벡터를 SbfI/NdeI으로 절단하여 CPPCT 부분을 제거한 후, 상기 증폭된 CpPct532 PCR 단편을 SbfI/NdeI인식부위에 삽입시킨 ligation mixture를 만들어 pPs619C1300-CPPCT540 벡터를 제조하였다(한국특허공개 2009-0017252).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. pPs619C1310-CPPCT540 재조합 벡터의 제작 1-3. Construction of pPs619C1310-CPPCT540 Recombinant Vector
슈도모나스 속 MBEL 6-19 (Pseudomonas sp. MBEL 6-19)의 폴리하이드록시알카노에이트 합성효소의 변이체를 사용하여, lactyl-CoA와 glycolyl-CoA를 기질로 사용하여 락테이트-글라이콜레이트 공중합체 및 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체를 제조할 수 있는 시스템을 발명하였다.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.
상기 1-1에서 제작된 phaC1Ps6-19합성효소 변이체(phaC1Ps6-19300)를 기초로 하여 서열번호 23 및 24의 프라이머를 사용한 SDM 방법을 이용하여 E130D, S477F 및 Q481K 이 변이된 아미노산 서열을 가진 Pseudomonas 속 MBEL 6-19 유래 PHA 합성효소 변이체(phaC1Ps6-19310)를 제작하였다(한국공개특허 2008-0047279). 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).
서열번호 23:5'-gaa ttc gtg ctg tcg agc cgc ggg cat atc- 3'SEQ ID NO: 23: 5'-gaa ttc gtg ctg tcg agc cgc ggg cat atc- 3 '
서열번호 24: 5'-gat atg ccc gcg gct cga cag cac gaa ttc- 3SEQ ID NO: 24 5'-gat atg ccc gcg gct cga cag cac gaa ttc-3
이로부터 얻은 재조합 벡터(도2, pPs619C1310-CPPCT540)를 E. coli JM109에 형질전환시키고, 이를 3HB가 포함된 중합체 검출배지(LB agar, glucose 20g/L, 3HB 2g/L, Nile red 0.5μg/ml)에서 생육시킨 결과, 중합체 생성을 확인할 수 있었다.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.
실시예 2:Example 2: 재조합 대장균의 제작 및 이를 이용한 락테이트-글라이콜레이트 공중합체의 제조 Preparation of Recombinant Escherichia Coli and Preparation of Lactate-Glycolate Copolymer
실시예 1에서 제작된 pPs619C1310-CpPCT540을 대장균 XL1-Blue에 도입하여 재조합 대장균 XL1-Blue(pPs619C1310-CpPCT540)를 제작한 다음, 플라스크 배양을 수행하였다. 먼저 재조합 대장균 XL1-Blue(pPs619C1310-CpPCT540)를 20g/L의 포도당(glucose)과 2g/L의 글라이콜레이트가 함유된 MR 배지에서 3일 동안 배양하였다. MR 배지의 조성은 다음과 같다: KH2PO4 6.67g, (NH4)2HPO4 4g, MgSO4·7H2O 0.8g, citric acid 0.8g, 및 trace metal solution 5mL. Trace metal solution(per liter): 5M HCl 5mL, FeSO4·7H2O 10g, CaCl2 2g, ZnSO4·7H2O 2.2g, MnSO4·4H2O 0.5g, CuSO4·5H2O 1g, (NH4)6Mo7O2·4H2O 0.1g, 및 Na2B4O2·10H2O 0.02g. 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.
상기 배양액을 원심분리하여 균체를 회수하고, 동결건조시킨 다음, 클로로포름을 이용하여 균체에 축적된 고분자 물질을 회수하였다. 고분자의 모노머 조성을 가스크로마토그래피로 분석한 결과, P(6mol% LA-co-94mol% GA) 임을 확인하였다. 이 때, 락테이트 모노머(LA)는 탄소원인 포도당으로부터 대장균 내부에서 자체 생성된 락테이트로부터 제공된다. 락테이트-글라이콜레이트 공중합체를 합성한 재조합 대장균 균주의 세포를 현미경으로 관찰하였다 (도 6). 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).
실시예 3: 재조합 대장균의 제작 및 이를 이용한 락테이트-글라이콜레이트-하이드록시알카노에이트 공중합체의 제조Example 3 Preparation of Recombinant Escherichia Coli and Preparation of Lactate-Glycolate-Hydroxyalkanoate Copolymer
실시예 1에서 제작된 pPs619C1310-CpPCT540을 대장균 XL1-Blue에 도입하여 재조합 대장균 XL1-Blue(pPs619C1310-CpPCT540)를 제작한 다음, 플라스크 배양을 수행하였다. 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.
먼저 재조합 대장균 XL1-Blue(pPs619C1310-CpPCT540)를 20g/L의 포도당(glucose)과 2g/L의 글라이콜레이트 및 2g/L의 2-hydroxybutyrate (2HB)가 함유된 MR배지 또는 20g/L의 포도당(glucose)과 2g/L의 글라이콜레이트, 2g/L의 2-hydroxybutyrate (2HB) 및 2g/L의 3-hydroxybutyrate (3HB)가 함유된 MR배지에서 3일간 배양시켰다. 상기 배양액을 원심분리하여 균체를 회수하고, 동결건조시킨 다음, 클로로포름을 이용하여 각각 균체에 축적된 고분자 물질을 회수하였다. 고분자의 모노머 조성을 가스크로마토그래피로 분석한 결과, 각각 P(53mol% 2HB-co-17mol% LA-co-30mol% GA) 및 P(30mol% 2HB-co-43mol% 3HB-co-9mol% LA-co-22mol% GA)임을 확인하였다. 이 때, 락테이트 모노머(LA)는 탄소원인 포도당으로부터 대장균 내부에서 자체 생성된 락테이트로부터 제공된다. 2HB-락테이트-글라이콜레이트 공중합체 또는 2HB-3HB-락테이트-글라이콜레이트 공중합체를 합성한 재조한 대장균 균주의 세포를 현미경으로 관찰하였다 (도 7 및 도 8). 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).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.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.
도 1은 Clostridium propionicum 유래 pct 유전자와 Pseudomonas sp. MBEL 6-19 유래 phaC1 돌연변이체를 포함하는 재조합 플라스미드 pPs619C1300-CpPCT의 제작 과정 및 유전자 지도를 나타낸 것이다. 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.
도 2는 Clostridium propionicum 유래 pct 돌연변이체와 Pseudomonas sp. MBEL 6-19 유래 phaC1 돌연변이체를 포함하는 재조합 플라스미드 pPs619C1310-CpPCT540의 제작 과정 및 유전자 지도를 나타낸 것이다. 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.
도 3은 포도당, 글라이콜레이트를 이용하여 글라이콜레이트 중합체(PGA)를 합성하는 경로를 나타낸 모식도이다. 3 is a schematic diagram showing a route for synthesizing a glycolate polymer (PGA) using glucose and glycolate.
도 4는 포도당, 글라이콜레이트를 이용하여 락테이트-글라이콜레이트 공중합체(PLGA)를 합성하는 경로를 모식하는 그림이다. 4 is a diagram illustrating a route for synthesizing a lactate-glycolate copolymer (PLGA) using glucose and glycolate.
도 5는 포도당, 글라이콜레이트, 3HB, 2HB 를 이용하여 글라이콜레이트-락테이트-3HB-2HB 공중합체를 합성하는 경로를 모식하는 그림이다. 5 is a diagram illustrating a route for synthesizing a glycolate-lactate-3HB-2HB copolymer using glucose, glycolate, 3HB, and 2HB.
도 6은 P(LA-co-GA)를 합성한 대장균 XL1-Blue (pPs619C1310-CpPCT540)의 현미경사진이다. Figure 6 is a micrograph of E. coli XL1-Blue (pPs619C1310-CpPCT540) synthesized P (LA-co-GA).
도 7은 P(LA-co-GA-co-2HB)를 합성한 대장균 XL1-Blue (pPs619C1310-CpPCT540)의 현미경 사진이다.Figure 7 is a micrograph of E. coli XL1-Blue (pPs619C1310-CpPCT540) synthesized P (LA-co-GA-co-2HB).
도 8은 P(LA-co-GA-co-2HB-co-3HB)를 합성한 대장균 XL1-Blue (pPs619C1310-CpPCT540)의 현미경사진이다.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 <213> 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 <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)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020090030133A KR101085960B1 (en) | 2009-04-08 | 2009-04-08 | 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 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020090030133A KR101085960B1 (en) | 2009-04-08 | 2009-04-08 | 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 |
Publications (2)
Publication Number | Publication Date |
---|---|
KR20100111766A KR20100111766A (en) | 2010-10-18 |
KR101085960B1 true KR101085960B1 (en) | 2011-11-22 |
Family
ID=43131876
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
KR1020090030133A KR101085960B1 (en) | 2009-04-08 | 2009-04-08 | 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 |
Country Status (1)
Country | Link |
---|---|
KR (1) | KR101085960B1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20160092401A (en) | 2015-01-27 | 2016-08-04 | 한국수산자원관리공단 | Artifical fishing reef with replacing type loess block |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012064043A2 (en) | 2010-11-08 | 2012-05-18 | 에스케이케미칼 주식회사 | Polylactic acid resin, preparation method thereof, and packaging film comprising same |
KR101575585B1 (en) | 2011-03-11 | 2015-12-21 | 한국과학기술원 | Recombinant Microorganism Having Producing Poly(lactate-co-glycolate) from Glucose and Preparing Method of Poly(lactate-co-glycolate) Using Thereof |
KR101774431B1 (en) * | 2016-01-28 | 2017-09-05 | 한국과학기술원 | Recombinant Microorganism Having Ability Producing Poly(lactate-co-glycolate) and Its Copolymers from Xylose and Preparing Method of Poly(lactate-co-glycolate) and its copolymers Using Thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006501858A (en) | 2002-05-10 | 2006-01-19 | メタボリックス,インコーポレイテッド | Bioabsorbable polymer containing 2-hydroxy acid monomer |
-
2009
- 2009-04-08 KR KR1020090030133A patent/KR101085960B1/en active IP Right Grant
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006501858A (en) | 2002-05-10 | 2006-01-19 | メタボリックス,インコーポレイテッド | Bioabsorbable polymer containing 2-hydroxy acid monomer |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20160092401A (en) | 2015-01-27 | 2016-08-04 | 한국수산자원관리공단 | Artifical fishing reef with replacing type loess block |
Also Published As
Publication number | Publication date |
---|---|
KR20100111766A (en) | 2010-10-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR100957777B1 (en) | Mutants of PHA synthase from Pseudomonas sp. 6-19 and method for preparing lactate homopolymer or copolymer using the same | |
KR100979694B1 (en) | Cells or Plants Having an Producing Ability of Polylactate or Its Copolymers and Method for Preparing Polylactate or Its Copolymers Using the Same | |
KR101045572B1 (en) | Propionyl-CoA Transferase Variants Derived from Cructostilridium Substances and Processes for Producing Lactate Polymers or Lactate Copolymers Using the Variants | |
KR101037354B1 (en) | Recombinant microorganism able to produce polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same | |
KR100957773B1 (en) | Novel Copolymer of [Poly3-hydroxypropionate-co-lactate] and Method for Preparing the Same | |
JP5626735B2 (en) | Recombinant Ralstonia eutropha having ability to produce polylactic acid or polylactic acid copolymer and method for producing polylactic acid or polylactic acid copolymer using the same | |
BRPI0718979A2 (en) | COPOLYMER AND METHOD FOR PREPARING THE SAME | |
KR20090078925A (en) | Recombinant microorganism having a producing ability of polylactate or its copolymers and method for preparing polylactate or its copolymers using the same | |
KR101085960B1 (en) | 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 | |
EP2818561A1 (en) | Utilization of the novel, environmental isolates Pseudomonas sp. IPB-B26 and N-128 for the efficient high yield production of mcl/lcl-PHAs | |
JP2008086238A (en) | Method for producing polyhydroxyalkanoate | |
KR100926492B1 (en) | Novel Copolymer of [poly3-hydroxybutyrate-co-4-hydroxybutyrate-co-lactate] and Method for Preparing the Same | |
KR100957775B1 (en) | Novel Copolymer of [poly3-hydroxybutyrate-co-MCL 3-hydroxyalkanoate-co-lactate] and Method for Preparing the Same | |
KR100957774B1 (en) | Novel Copolymer of [polyMCL 3-hydroxyalkanoate-co-lactate] and Method for Preparing the Same | |
KR100926489B1 (en) | Novel Copolymer of [Poly3-hydroxyvutyrate-co-lactate] and Method for Preparing the Same | |
KR101293904B1 (en) | Recombinant microorganism capable of producing polylactate or polylactate copolymer from xylose and method for producing polylactate or polylactate copolymer from xylose using the same | |
KR100957776B1 (en) | Novel Copolymer of [poly3-hydroxybutyrate-co-hydroxypropionate-co-lactate] and Method for Preparing the Same | |
KR101260187B1 (en) | Recombinant microorganism able to produce polylactate or polylactate copolymer from glycerol and method for producing polylactate or polylactate copolymer from glycerol using the same | |
KR100948777B1 (en) | Novel Copolymer of [poly3-hydroxybutyrate-co-3-hydroxyvalerate-co-lactate] and Method for Preparing the Same | |
KR100926491B1 (en) | Novel Copolymer of [poly3-hydroxybutyrate-co-3-hydroxypropionate-co-4-hydroxybutyrate-co-lactate] and Method for Preparing the Same | |
KR100926488B1 (en) | Novel Copolymer of [poly4-hydroxybutyrate-co-3-hydroxypropionate-co-lactate] and Method for Preparing the Same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A201 | Request for examination | ||
E902 | Notification of reason for refusal | ||
E701 | Decision to grant or registration of patent right | ||
GRNT | Written decision to grant | ||
FPAY | Annual fee payment |
Payment date: 20141017 Year of fee payment: 4 |
|
FPAY | Annual fee payment |
Payment date: 20150923 Year of fee payment: 5 |
|
FPAY | Annual fee payment |
Payment date: 20160928 Year of fee payment: 6 |
|
FPAY | Annual fee payment |
Payment date: 20170919 Year of fee payment: 7 |
|
FPAY | Annual fee payment |
Payment date: 20181016 Year of fee payment: 8 |
|
FPAY | Annual fee payment |
Payment date: 20191016 Year of fee payment: 9 |