KR101226644B1 - Transformed Escherichia coli for Over-expression of Fatty Acid Biosynthesis Pathway and Method of Preparing the Same - Google Patents

Transformed Escherichia coli for Over-expression of Fatty Acid Biosynthesis Pathway and Method of Preparing the Same Download PDF

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KR101226644B1
KR101226644B1 KR1020100072465A KR20100072465A KR101226644B1 KR 101226644 B1 KR101226644 B1 KR 101226644B1 KR 1020100072465 A KR1020100072465 A KR 1020100072465A KR 20100072465 A KR20100072465 A KR 20100072465A KR 101226644 B1 KR101226644 B1 KR 101226644B1
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Abstract

본 발명은 지방산 생합성 경로에 관여하는 효소들을 암호화하는 유전자로 형질전환된 지방산 과발현용 대장균 및 그 제조방법에 관한 것이다.
본 발명의 지방산 과발현용 대장균은 유전적 안정성이 높을 뿐만 아니라 기초물질이 두 가지 경로를 통하여 지방산 회로로 유도되게 해주므로 지방산 과발현 효과가 매우 뛰어나다.The present invention relates to a fatty acid overexpression Escherichia coli transformed with a gene encoding enzymes involved in the fatty acid biosynthetic pathway and a method for producing the same.
E. coli for fatty acid overexpression of the present invention is not only high genetic stability, but also because the base material is induced to the fatty acid circuit through two pathways, the fatty acid overexpression effect is very excellent.

Description

지방산 생합성 경로의 과발현용 형질전환 대장균 및 그의 제조방법{Transformed Escherichia coli for Over-expression of Fatty Acid Biosynthesis Pathway and Method of Preparing the Same}Transformed Escherichia coli for Over-expression of Fatty Acid Biosynthesis Pathway and Method of Preparing the Same}

본 발명은 지방산 생합성 경로의 과발현용 형질전환 대장균 및 그의 제조방법에 관한 것이다.
The present invention relates to a transformant Escherichia coli for overexpression of a fatty acid biosynthetic pathway and a method for producing the same.

화석연료로부터 생산되어지는 화학제품들은 제조공정시 지구온난화 가스 및 폐기물을 대량 생산하여 인류에게 심각한 환경적인 위기를 초래하고 있다. 따라서 화석연료로부터 생산되어지는 화학공업을 대체할 수 있는, 즉 인류에게 유해한 폐기물의 생산 및 에너지 소비를 최소화할 수 있는 바이오매스를 원료로 사용하는 환경 친화적인 새로운 생물 공정의 개발이 필요하다. 바이오에너지로 대표되어지는 바이오에탄올(Bioethanol), 바이오디젤(Biodiesel), 바이오가스(Biohydrogen), 부탄올에 대한 관심이 증가하고 있으며, 언급된 종류의 바이오에너지 모두 전력생산이나 수송용 연료로 사용될 수 있으나, 실적용 및 생산 방법의 몇몇 단점으로 인해 새로운 신재생에너지 자원인 하이드로카본 형태의 화합물에 대한 관심이 증가되고 있다. 이에 따라 장쇄 지방산을 대사산물로서 생성할 수 있는 재조합 균주에 대한 관심도 증대되고 있다.
Chemical products produced from fossil fuels produce global warming gases and wastes in the manufacturing process, causing a serious environmental crisis for mankind. Therefore, there is a need to develop new environmentally friendly biological processes using biomass as a raw material that can replace the chemical industry produced from fossil fuels, that is, minimize the production and energy consumption of wastes harmful to humankind. There is increasing interest in bioethanol, biodiesel, biohydrogen and butanol, which are represented by bioenergy. All of the mentioned types of bioenergy can be used as fuels for power generation or transportation. Due to some shortcomings in the production, production and production processes, there is a growing interest in hydrocarbons in the form of new renewable energy resources. Accordingly, there is also increasing interest in recombinant strains capable of producing long chain fatty acids as metabolites.

지방산 합성(fatty acid synthases, FASs)의 typeⅡ로 분리될 수 있는 것에 의해 지방산 생합성의 첫 번째 단계는 β-케토아실-[아실운반단백질]신타아제Ⅲ (KASⅢ, fabH)에 의해 촉매 되어지는 아실-CoA 기질과 함께 말로닐-[아실운반단백질]의 농도에 의한 카르복실을 제거(decarboxylative)하는 것이다. 차후의 지방산 대사에서 신장 단계는 β-케토아실-[아실운반단백질]신타아제Ⅲ와 아실-CoA 기질보다 이실-[아실운반단백질]의 활용에 따라 다르게 촉매 되어진다.The first step in fatty acid biosynthesis by being able to be separated into type II of fatty acid synthases (FASs) is β- ketoacyl- acyl catalyzed by [acyl carrier protein] synthase III (KASIII, fabH ). With the CoA substrate, it is decarboxylative due to the concentration of malonyl- [acyl carrier protein]. In the subsequent fatty acid metabolism, the elongation stage is catalyzed differently depending on the application of Isil- [acyl carrier proteins] rather than β-ketoacyl- [acyl carrier proteins] synthase III and acyl-CoA substrates.

β-케토아실-[아실운반단백질]신타아제Ⅲ의 첫 반응은 말로닐-[아실운반단백질]와 아세틸-CoA의 농축이다. 이는 β-케토아실-[아실운반단백질]신타아제Ⅲ 효소 중에 독특한 것으로 아세틸-CoA가 공여자로 사용되고 아세틸-CoA[아실운반단백질]트랜스아실레이즈 활성을 가지게 해준다. 다음 그림에서 보여지듯이 β-케토아실-[아실운반단백질]신타아제Ⅲ, fabH 유전자는 아세틸-CoA에서 CO2와 H2O가 제외되면서 아세토아실-[아실운반단백질]로 지방산이 합성되어지며 또한 말로닐-[아실운반단백질]로부터 β-케토아실-[아실운반단백질]로 만들어져 지방산 회로로 유도된다. The first reaction of β-ketoacyl- [acyl carrier proteins] synthase III is the concentration of malonyl- [acyl carrier proteins] and acetyl-CoA. This is unique among the β-ketoacyl- [acyl carrier protein] synthase III enzymes, allowing acetyl-CoA to be used as a donor and having acetyl-CoA [acyl carrier protein] transacylase activity. As shown in the following figure, the β- ketoacyl- [acyl carrier protein] synthase III and fabH genes are synthesized as acetoacyl- [acyl carrier protein] by excluding CO 2 and H 2 O from acetyl-CoA. It is made from malonyl- [acyl carrier protein] to [beta] -ketoacyl- [acyl carrier protein] and induced into the fatty acid cycle.

Figure 112010048551716-pat00001
Figure 112010048551716-pat00001

β-케토아실-[아실운반단백질]신타아제Ⅲ 효소 이외에 지방산 합성반응은 acetyl-CoA carboxylase(accA, accBC)와 말로닐-CoA를 말로닐-CoA:ACP(acyl carrier protein)로 전환하는 효소, malonyl-CoA:ACP(acyl carrier protein) acyltransferase(fabD)는 세포질에서 아세틸-CoA는 카복실화되어서, 지방산 생합성에 있어 중요한 중간체가 되는 말로닐-CoA(malonyl-CoA)를 생산하는 방향이다. 이 반응은 아세틸-CoA 카복실라아제 복합체에 의해 촉매되는데, 이 복합체는 세 개의 효소로 구성되어 있고, 효소 활성을 위해 ATP뿐만 아니라 Mn2 +과 바이오틴을 요구한다. 아세틸-CoA 카복실라아제는 세 개의 단백질인 바이오틴 카복실라아제, 바이오틴 운반 단백질, 그리고 카복실기 전달효소로 구성되어있다. In addition to β- ketoacyl- [acyl carrier protein] synthase III enzymes, fatty acid synthesis reactions include enzymes that convert acetyl-CoA carboxylase ( accA, accBC ) and malonyl-CoA to malonyl-CoA: acP (acyl carrier protein). malonyl-CoA: acyl carrier protein (ACP) acyltransferase ( fabD ) is a direction in which acetyl-CoA is carboxylated in the cytoplasm to produce malonyl-CoA, an important intermediate for fatty acid biosynthesis. There is a reaction catalyzed by acetyl -CoA carboxyl la kinase complex, the complex is composed of three enzymes, and to the enzyme activity as well as ATP requires a Mn 2 + and biotin. Acetyl-CoA carboxylase consists of three proteins, biotin carboxylase, biotin transporter protein, and carboxyl transferase.

KASⅢ는 식물과 박테리아에서 지방산 생합성의 시작에 중심적 역할을 한다. KASⅢ는 또한 typeⅡ FAS에 의해 만들어 지는 지방산 타입(type)을 결정하는 역할을 하고 식물에서는 유전자 조작으로 오일의 구성을 바꿀 수 있었다. 대장균과 같은 대부분의 박테리아와 식물들은 직쇄형 지방산(straight-chain fatty acid, SCFAs)을 생산한다.KASIII plays a central role in the initiation of fatty acid biosynthesis in plants and bacteria. KASIII also plays a role in determining the type of fatty acids produced by type II FAS, and in plants, genetic modifications can alter the composition of the oil. Most bacteria and plants, such as E. coli, produce straight-chain fatty acids (SCFAs).

대장균 KASⅢ는 아세틸-CoA와 프로피오닐-CoA를 위한 기질을 가져야 한다고 보고되었다. KASⅢ가 지방산 생합성 초기와 지방산 생산의 타입을 결정하기 위한 원인이 된다는 것은 문헌(N. Smirnova and KA Reynolds, Branched-chain fatty acid biosynthesis in Escherichia coli, Journal of Industrial Microbio Biotechnol 27:246251(2001))에서 보고되었으며 KASⅢ의 특별한 기질 생산 혹은 대량 생산을 위한 조직 내 유전자 조작이 제안되었다. 또 다른 문헌(Christopher T. N, K. Taguchi, S. Taguchi and Y. Doi, Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III (fabH) and Polyhydroxyalka-noate Synthase (phaC) Genes Leads to Short-Chain-Length Medium-hain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in Escherichia coli JM109, Appl Inviron Microbio , 9991007(2004); NATALYA S. and K. A. REYNOLDS. Engineered Fatty Acid Biosynthesis in Streptomyces by Altered Catalytic Function of b-Ketoacyl-Acyl Carrier Protein Synthase III. JOURNAL OF BACTERIOLOGY, 23352342(2001))에서는 KASⅢ가 지방산 구성을 변화시킬 수 있다는 결과가 보고되었다.
E. coli KASIII has been reported to have substrates for acetyl-CoA and propionyl-CoA. It has been suggested that KASIII is responsible for determining the initial fatty acid biosynthesis and the type of fatty acid production (N. Smirnova and KA Reynolds, Branched-chain fatty acid biosynthesis in Escherichia). coli , Journal of Industrial Microbio Biotechnol 27: 246251 (2001), and genetic manipulation in tissues for the production or mass production of specific substrates of KASIII has been proposed. Another article (Christopher T. N, K. Taguchi, S. Taguchi and Y. Doi, Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III ( fabH ) and Polyhydroxyalka -noate Synthase ( phaC ) Genes Leads to Short-Chain- Length Medium-hain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in Escherichia coli JM109, Appl Inviron Microbio , 9991007 (2004); NATALYA S. and KA REYNOLDS. Engineered Fatty Acid Biosynthesis in Streptomyces by Altered Catalytic Function of b-Ketoacyl-Acyl Carrier Protein Synthase III. JOURNAL OF BACTERIOLOGY , 23352342 (2001), reported that KASIII could alter fatty acid composition.

본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

본 발명자들은 유전자 조작을 통해 지방산 생합성 대사산물을 안정적이고 효과적으로 생산하는 균주의 개발을 위하여 예의 연구 노력하였고 그 결과 fabH 유전자를 지방산 생합성 초기 유도 유전자들과 함께 대장균을 공동형질전환 시키는 경우 기초물질이 두 가지 경로를 통하여 지방산 회로로 유도되게 해주므로 매우 효과적으로 지방산 생합성 경로가 과발현됨을 확인하여, 본 발명을 완성하게 되었다.The present inventors have intensively studied effort to the development of strains that stable and efficient production of fatty acid biosynthesis metabolites through genetic modification that results fabH When the gene co-transforms E. coli with fatty acid biosynthesis early induction genes, it was confirmed that the fatty acid biosynthesis pathway was overexpressed very effectively since the base material was induced into the fatty acid circuit through two pathways, thus completing the present invention.

따라서 본 발명의 목적은 지방산 생합성 경로의 과발현용 형질전환 대장균 을 제공하는 데 있다.Therefore, an object of the present invention is to provide a transformant Escherichia coli for overexpression of fatty acid biosynthetic pathway.

본 발명의 다른 목적은 상기 형질전환 대장균의 제조방법을 제공하는 데 있다.Another object of the present invention to provide a method for producing the transformed E. coli.

본 발명의 또 다른 목적은 상기 형질전환 대장균을 이용하여 지방산을 생합성하는 방법을 제공하는 데 있다.
Still another object of the present invention is to provide a method for biosynthesizing fatty acids using the transformed Escherichia coli.

본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

본 발명의 일 양태에 따르면, 본 발명은 (a) β-케토아실-[아실운반단백질] 신타아제 III(β-Ketoacyl-Acyl Carrier Protein Synthase III)를 코딩하는 뉴클레오타이드 서열; 및 (b) 아세틸-CoA 카복실라제 카복시트랜스퍼라제 서브유닛 알파(acetyl-CoA carboxylase, carboxytransferase, alpha subunit)를 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복시 운반단백질 서브유닛(acetyl CoA carboxylase, biotin carboxyl carrier protein(BCCP) subunit)을 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복실라제 서브유닛(acetyl-CoA carboxylase, biotin carboxylase subunit)을 코딩하는 뉴클레오타이드 서열 및 말로닐-CoA-[아실운반단백질] 트랜스아실라제(malonyl-CoA-[acyl-carrier-protein] transacylase)를 코딩하는 뉴클레오타이드 서열로 이루어진 군으로부터 선택되는 일 이상의 뉴클레오타이드 서열을 포함하는 발현벡터로 공동형질전환된(cotransformed) 지방산 과발현용 대장균을 제공한다.According to one aspect of the present invention, the present invention provides a kit comprising: (a) a nucleotide sequence encoding β-ketoacyl- [acyl carrier protein] synthase III (β-Ketoacyl-Acyl Carrier Protein Synthase III); And (b) a nucleotide sequence encoding acetyl-CoA carboxylase carboxycitase transferase subunit alpha (acetyl-CoA carboxylase, carboxytransferase, alpha subunit), acetyl-CoA carboxylase biotin carboxy transporter subunit (acetyl CoA carboxylase, biotin carboxyl) nucleotide sequence encoding the carrier protein (BCCP) subunit), nucleotide sequence encoding the acetyl-CoA carboxylase subunit (acetyl-CoA carboxylase, biotin carboxylase subunit) and malonyl-CoA- [acyl carrier protein] trans Providing Escherichia coli co-transformed with an expression vector comprising at least one nucleotide sequence selected from the group consisting of nucleotide sequences encoding an acylase (malonyl-CoA- [acyl-carrier-protein] transacylase) do.

본 발명의 일 구현예에서, 상기 발현벡터는 (a) β-케토아실-[아실운반단백질] 신타아제 III를 코딩하는 뉴클레오타이드 서열; 및 (b) 아세틸-CoA 카복실라제 카복시트랜스퍼라제 서브유닛 알파를 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복시 운반단백질 서브유닛을 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복실라제 서브유닛을 코딩하는 뉴클레오타이드 서열 및 말로닐-CoA-[아실운반단백질] 트랜스아실라제를 코딩하는 뉴클레오타이드 서열을 모두 포함한다.
In one embodiment of the invention, the expression vector is (a) a nucleotide sequence encoding β-ketoacyl- [acyl carrier protein] synthase III; And (b) a nucleotide sequence encoding acetyl-CoA carboxylase carboxycitase transferase subunit alpha, a nucleotide sequence encoding acetyl-CoA carboxylase biotin carboxy transporter subunit, an acetyl-CoA carboxylase biotin carboxylase subunit A nucleotide sequence encoding a nucleotide sequence and a malonyl-CoA- [acyl carrier protein] transacylase.

본 발명자들은 유전자 조작을 통해 지방산 생합성 대사산물을 안정적이고 효과적으로 생산하는 균주의 개발을 위하여 예의 연구 노력하였고, 그 결과 fabH 유전자를 지방산 생합성 초기 유도 유전자들과 함께 대장균을 공동형질전환 시키는 경우 기초물질이 두 가지 경로를 통하여 지방산 회로로 유도되게 해주므로 매우 효과적으로 지방산 생합성 경로가 과발현됨을 확인하여, 본 발명을 완성하게 되었다.The present inventors have made diligent research efforts to develop strains that stably and effectively produce fatty acid biosynthetic metabolites through genetic engineering, and as a result fabH When the gene co-transforms E. coli with fatty acid biosynthesis early induction genes, it was confirmed that the fatty acid biosynthesis pathway was overexpressed very effectively since the base material was induced into the fatty acid circuit through two pathways, thus completing the present invention.

본 발명의 β-케토아실-[아실운반단백질] 신타아제 Ⅲ(β-ketoacyl acyl carrier protein synthase Ⅲ)는 아세틸-CoA에서 아세토아실-[아실운반단백질]로 변환하는 과정 및 말로닐-[아실운반단백질]에서 β-케토아실-[아실운반단백질]로 변환하는 과정에 관여하는 촉매이며, fabH 유전자가 이를 암호화하는 유전자이다.Β-ketoacyl- [acyl carrier protein] synthase III of the present invention is a process of converting acetyl-CoA to acetoacyl- [acyl carrier protein] and malonyl- [acyl carrier] Protein] is a catalyst involved in the conversion of β-ketoacyl- [acyl carrier protein] The fabH gene is the gene that encodes it.

본 발명의 발현벡터에서 발현되는 β-케토아실-[아실운반단백질] 신타아제 Ⅲ는 대장균(Escherichia coli)으로부터 유래한 것을 사용하는 것이 바람직하다. 더욱 바람직하게는 서열목록 제1서열로 표시되는 E. coli K-12 MG1655의 β-케토아실-[아실운반단백질] 신타아제 Ⅲ를 발현하도록 할 수 있으며, 상기 β-케토아실-[아실운반단백질] 신타아제 Ⅲ를 코딩하는 뉴클레오타이드 서열은 바람직하게는 서열목록 제2서열로 표시될 수 있다.Β-ketoacyl- [acyl carrier protein] synthase III expressed in the expression vector of the present invention is Escherichia coli. Preference is given to using those derived from coli ). More preferably, it is possible to express β-ketoacyl- [acyl transport protein] synthase III of E. coli K-12 MG1655 represented by SEQ ID NO: 1, and the β-ketoacyl- [acyl transport protein The nucleotide sequence encoding synthase III may preferably be represented by the sequence listing second sequence.

또한, 본 발명의 상기 아세틸-CoA 카복실라제 카복실트랜스퍼라제(acetyl-CoA carboxylase carboxytransferase)는 아세틸-CoA를 말로닐-CoA로 변환하는 과정에 관여하는 촉매이며, 두 개의 알파 서브유닛과 두 개의 베타 서브유닛으로 구성된 테트라머이다. 본 발명의 아세틸-CoA 카복실라제 카복실트랜스퍼라제 서브유닛 알파는 상기 서브유닛 중 하나이며, accA 유전자가 이를 코딩하는 유전자이다.In addition, the acetyl-CoA carboxylase carboxytransferase (acetyl-CoA carboxylase carboxytransferase) of the present invention is a catalyst involved in the process of converting acetyl-CoA to malonyl-CoA, two alpha subunits and two beta subunits It is a tetramer composed of units. Acetyl-CoA carboxylase carboxyltransferase subunit alpha of the present invention is one of the above subunits, and the accA gene is the gene encoding it.

본 발명의 발현벡터에서 발현되는 상기 아세틸-CoA 카복실라제 카복실트랜스퍼라제 서브유닛 알파는 대장균(Escherichia coli)으로부터 유래한 것을 사용하는 것이 바람직하다. 더욱 바람직하게는 서열목록 제3서열로 표시되는 E. coli K-12 MG1655의 아세틸-CoA 카복실라제 카복실트랜스퍼라제 서브유닛 알파를 발현하도록 할 수 있으며, 상기 아세틸-CoA 카복실라제 카복실트랜스퍼라제 서브유닛 알파를 코딩하는 뉴클레오타이드 서열은 바람직하게는 서열목록 제4서열로 표시될 수 있다.The acetyl-CoA carboxylase carboxyltransferase subunit alpha expressed in the expression vector of the present invention is Escherichia coli. Preference is given to using those derived from coli ). More preferably, the acetyl-CoA carboxylase carboxytransferase subunit alpha of E. coli K-12 MG1655, which is represented by SEQ ID NO: 3, may be expressed, and the acetyl-CoA carboxylase carboxyl transferase subunit alpha Nucleotide sequence encoding the can be preferably represented by SEQ ID NO: 4 sequence.

상기 아세틸-CoA 카복실라제 바이오틴 카복시 운반단백질 서브유닛; 아세틸-CoA 카복실라제 바이오틴 카복실라제 서브유닛; 및 말로닐-CoA-[아실운반단백질] 트랜스아실라제도 각각 대장균(Escherichia coli)으로부터 유래한 것을 사용하는 것이 바람직하다. 더욱 바람직하게는 각각 서열목록 제5서열, 제7서열 및 제9서열로 표시되는 E. coli K-12 MG1655의 효소를 발현하도록 할 수 있으며, 상기 단백질을 코딩하는 뉴클레오타이드 서열은 바람직하게는 각각 서열목록 제6서열, 제8서열 및 제10서열로 표시될 수 있다.
Said acetyl-CoA carboxylase biotin carboxy transport protein subunit; Acetyl-CoA carboxylase biotin carboxylase subunits; And malonyl-CoA- [acyl carrier protein] transacilases, respectively, Escherichia Preference is given to using those derived from coli ). More preferably, it is possible to express the enzyme of E. coli K-12 MG1655 represented by SEQ ID NO: 5, 7 and 9, respectively, wherein the nucleotide sequence encoding the protein is preferably each sequence The list may be displayed as the sixth, eighth, and tenth sequences.

상기에서 기술한, (a) 내지 (b)의 지방산 생합성 과정에 관여하는 효소들을 코딩하는 각각의 뉴클레오타이드 서열들은 발현 조절서열에 작동가능하게 연결되어 발현 벡터 내에 삽입될 수 있다. 상기에서 '작동 가능하게 연결된다(operably linked to)'는 것은 하나의 핵산 단편이 다른 핵산 단편과 결합되어 그의 기능 또는 발현이 다른 핵산 단편에 의해 영향을 받는 것을 말한다. 또한, '발현 조절 서열(expression control sequence)'이란 특정한 숙주 세포에서 작동 가능하게 연결된 핵산 서열의 발현을 조절하는 DNA 서열을 의미한다. 그러한 조절 서열은 전사를 개시하기 위한 프로모터, 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열 및 전사 및 해독의 종결을 조절하는 서열을 포함한다.As described above, each of the nucleotide sequences encoding the enzymes involved in the fatty acid biosynthesis process of (a) to (b) may be operably linked to an expression control sequence and inserted into the expression vector. 'Operably linked to' as used herein means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by another nucleic acid fragment. Also, an " expression control sequence " means a DNA sequence that regulates the expression of a nucleic acid sequence operably linked to a particular host cell. Such regulatory sequences include promoters for initiating transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation.

본 발명에 있어서, 상기 발현벡터에 포함되는 프로모터는 강력한 발현능력을 가지는 것을 사용하는 것이 바람직하다.In the present invention, the promoter contained in the expression vector is preferably used having a strong expression ability.

일 구현예에서, 본 발명의 발현 벡터는 프로모터서열, 발현 대상 유전자(구조 유전자)의 뉴클레오타이드 서열 및 터미네이터 서열을 포함하며, 상기 서열들이 5'-3' 순서대로 연결되는 것이 바람직하다. In one embodiment, the expression vector of the present invention comprises a promoter sequence, the nucleotide sequence of the gene to be expressed (structural gene) and the terminator sequence, it is preferred that the sequences are linked in 5'-3 'order.

다른 구현예에서, 상기 발현벡터는 lacI 유전자를 추가적으로 포함한다. lacI 유전자는 숙주 세포가 Lac 리프레서를 코딩하는 유전자를 포함하고 있는지 불문하고, 프로모터를 조절할 수 있도록 한다. 이 경우 구조 유전자를 암호화하는뉴클레오타이드의 발현을 유도하기 위하여, IPTG(Isopropyl-β-D-thiogalactopyranoside)를 사용할 수 있다.In another embodiment, the expression vector further comprises a lac I gene. The lac I gene allows the promoter cell to be regulated regardless of whether the host cell contains a gene encoding a Lac repressor. In this case, in order to induce the expression of a nucleotide encoding a structural gene, IPTG (Isopropyl-β-D-thiogalactopyranoside) can be used.

본 발명의 일 구현예에서, 상기 발현벡터는 fabH 유전자; 및 accA , accB , accC 및 fabD로 이루어진 군으로부터 선택되는 일 이상의 유전자 시퀀스 전체를 포함할 수도 있다. 그러나, 본 발명의 발현벡터에는 상기 유전자들이 효소의 과발현 기능을 포함하여 최소한의 길이를 가지도록 RBS(ribosomal binding site) 및 효소발현에 꼭 필요한 부분을 포함하는 염기서열만을 서열로 정하여 삽입시키는 것이 숙주세포의 대사부담(metabolic burden)을 줄이는 측면에서 바람직하다. 발명의 실시예에서는 pJS12와 pJS13 발현벡터가 사용되었다.
In one embodiment of the invention, the expression vector is fabH gene; And accA , accB , accC And fabD may include all of one or more gene sequences selected from the group consisting of. However, in the expression vector of the present invention, the genes are inserted into a sequence including only a base sequence including a ribosomal binding site (RBS) and an essential part of the enzyme expression so that the genes have a minimum length including an overexpression function of the enzyme. It is desirable in terms of reducing the metabolic burden of the cells. In an embodiment of the present invention, pJS12 and pJS13 expression vectors were used.

본 발명에서 사용되는 발현벡터란 구조유전자를 암호화하는 핵산이 삽입될 수 있고, 숙주 세포 내에서 상기 핵산을 발현할 수 있는 당 분야에 공지된 플라스미드, 바이러스 벡터 또는 기타 매개체를 의미하며 바람직하게는 플라스미드 벡터일 수 있다.The expression vector used in the present invention means a plasmid, viral vector or other medium known in the art capable of inserting a nucleic acid encoding a structural gene and capable of expressing the nucleic acid in a host cell, preferably a plasmid It may be a vector.

상기 플라스미드로는 대장균에서 복제 및 발현이 가능한 운반체라면 당업계에 공지된 그 어떠한 것이라도 사용할 수 있으며, 바람직하게는 서열목록 제11서열로 표시되는 플라스미드 벡터 pTrc99A를 사용할 수 있다.
As the plasmid, any carrier known in the art may be used as long as it is a carrier capable of replication and expression in E. coli, and preferably, the plasmid vector pTrc99A represented by SEQ ID NO: 11 may be used.

본 발명의 다른 양태에 따르면, 본 발명은 (a) β-케토아실-[아실운반단백질] 신타아제 III를 코딩하는 뉴클레오타이드 서열; 및 아세틸-CoA 카복실라제 카복시트랜스퍼라제 서브유닛 알파를 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복시 운반단백질 서브유닛을 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복실라제 서브유닛을 코딩하는 뉴클레오타이드 서열 및 말로닐-CoA-[아실운반단백질] 트랜스아실라제를 코딩하는 뉴클레오타이드 서열로 이루어진 군으로부터 선택되는 일 이상의 뉴클레오타이드 서열을 발현벡터에 삽입시키는 단계; 및 (b) 상기 뉴클레오타이드 서열이 삽입된 발현벡터로 대장균을 형질전환시키는 단계를 포함하는 지방산 과발현용 형질전환 대장균의 제조방법을 제공한다.According to another aspect of the present invention, the present invention provides an antibody comprising (a) a nucleotide sequence encoding β-ketoacyl- [acyl carrier protein] synthase III; And a nucleotide sequence encoding acetyl-CoA carboxylase carboxytransferase subunit alpha, a nucleotide sequence encoding acetyl-CoA carboxylase biotin carboxy carrier protein subunit, a nucleotide sequence encoding acetyl-CoA carboxylase biotin carboxylase subunit And inserting into the expression vector one or more nucleotide sequences selected from the group consisting of nucleotide sequences encoding malonyl-CoA- [acylcarrier protein] transacylase; And (b) transforming Escherichia coli with the expression vector into which the nucleotide sequence is inserted.

본 발명의 일 구현예에서 상기 발현벡터는 (a) β-케토아실-[아실운반단백질] 신타아제 III를 코딩하는 뉴클레오타이드 서열; 및 (b) 아세틸-CoA 카복실라제 카복시트랜스퍼라제 서브유닛 알파를 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복시 운반단백질 서브유닛을 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복실라제 서브유닛을 코딩하는 뉴클레오타이드 서열 및 말로닐-CoA-[아실운반단백질] 트랜스아실라제를 코딩하는 뉴클레오타이드 서열을 모두 포함한다.In one embodiment of the present invention, the expression vector comprises: (a) a nucleotide sequence encoding β-ketoacyl- [acyl carrier protein] synthase III; And (b) a nucleotide sequence encoding acetyl-CoA carboxylase carboxycitase transferase subunit alpha, a nucleotide sequence encoding acetyl-CoA carboxylase biotin carboxy transporter subunit, an acetyl-CoA carboxylase biotin carboxylase subunit A nucleotide sequence encoding a nucleotide sequence and a malonyl-CoA- [acyl carrier protein] transacylase.

본 발명의 지방산 생합성 경로의 과발현용 형질전환 대장균의 제조방법은 상술한 형질전환 대장균을 생산하는 방법에 관한 것이므로 상술한 내용과 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다. Since the method for producing transformed Escherichia coli for overexpression of the fatty acid biosynthetic pathway of the present invention relates to the method for producing the transformed Escherichia coli, the descriptions common to the above are omitted in order to avoid excessive complexity of the present specification.

상기에서 상술하게 기술한 본 발명의 발현 벡터는 당업계에 공지된 방법, 예를 들어 이에 한정되지는 않으나, CaCl2 완충액을 사용한 컴피턴트 세포를 만든 후 열충격(42℃) 방법에 의해 발현벡터를 숙주세포 내로 넣는 방법, 전기 충격에 의한 형질전환 방법, 일시적 형질감염(transient transfection), 미세주사, 세포융합, 칼슘 포스페이트 침전법, 전기침공법(electroporation) 등에 의해 숙주 세포 내로 도입할 수 있다. 형질전환체의 안정적 제조와 효율을 높이기 위해서는 전기 충격에 의한 형질전환 방법을 사용하는 것이 바람직하다.
The expression vector of the present invention described above in detail is not limited to this method, such as, but not limited to, the expression vector by thermal shock (42 ℃) method after producing competent cells using CaCl 2 buffer. It can be introduced into the host cell by a method of putting it into a host cell, a transformation method by electric shock, transient transfection, microinjection, cell fusion, calcium phosphate precipitation, electroporation, or the like. In order to stably manufacture and improve the efficiency of the transformant, it is preferable to use a transformation method by electric shock.

본 발명의 또 다른 양태에 따르면, 본 발명은 (a) 상기에서 상술한 형질전환 대장균을 배양하여 지방산을 생합성 하는 단계; 및 (b) 상기 생합성된 지방산을 수득하는 단계를 포함하는 지방산 생합성 방법을 제공한다.According to another aspect of the present invention, the present invention comprises the steps of (a) culturing the above-described transformed E. coli biosynthesizing fatty acids; And (b) provides a fatty acid biosynthesis method comprising the step of obtaining the biosynthetic fatty acid.

본 발명의 지방산 과발현용 형질전환 대장균의 배양은 당업계에 공지된 통상의 대장균 배양 방법에 의해 배양될 수 있다. The culture of the transformed Escherichia coli for fatty acid overexpression of the present invention can be cultured by a common Escherichia coli culture method known in the art.

바람직하게는, 단계 (a)는 발현 유도제인 IPTG의 존재 하에서 실시된다. Preferably, step (a) is carried out in the presence of IPTG, which is an expression inducer.

상기 형질전환 대장균 내에서 생합성된 지방산을 분리하는 단계는 당업계에 공지된 통상의 분리 또는 정제 방법에 따라 실시될 수 있다(참조: B. Aurousseau et al., Journal of the American Oil Chemists' Society, 57(3):1558-9331( 1980); Frank C. Magne et al., Journal of the American Oil Chemists' Society, 34(3):127-129(1957)).
The step of separating the biosynthetic fatty acid in the transformed E. coli can be carried out according to conventional separation or purification methods known in the art (see B. Aurousseau et al., Journal of the American Oil Chemists' Society , 57 (3): 1558-9331 (1980); Frank C. Magne et al., Journal of the American Oil Chemists' Society , 34 (3): 127-129 (1957).

본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:

(ⅰ) 지방산 생합성 경로에 관여하는 효소들을 암호화하는 유전자로 형질전환된 지방산 과발현용 대장균 및 그 제조방법을 제공한다. (Iii) Provided are Escherichia coli transformed with a gene encoding enzymes involved in the fatty acid biosynthesis pathway, and a method for producing the same.

(ⅱ) 본 발명의 지방산 과발현용 대장균은 유전적 안정성이 높을 뿐만 아니라 기초물질이 두 가지 경로를 통하여 지방산 회로로 유도되게 해주므로 지방산 과발현 효과가 매우 뛰어나다.
(Ii) Escherichia coli for fatty acid overexpression of the present invention not only has high genetic stability but also allows the basic material to be induced into the fatty acid circuit through two pathways, and thus, the effect of fatty acid overexpression is excellent.

도 1은 대장균 생체 내에서 포도당에서부터 지방산까지의 지방산 생합성 경로를 보여주는 도식이다.
도 2는 대장균의 염색체를 주형으로 하여 fabH 유전자를 PCR 수행 후 확인한 전기영동 사진이다(레인 1: 사이즈 마커, 레인 2,3: fabH).
도 3은 pJS12 재조합 플라스미드를 제한효소를 이용하여 확인한 전기영동 사진이다(레인 1: 사이즈마커, 레인 2-4: 플라스미드를 XbaⅠ과 BamHⅠ로 절단하여 D얻은 fabH).
도 4는 pTrc99A 벡터에 클로닝한 재조합 플라스미드 pJS13의 accABC , fabD 및 fabH 유전자를 PCR로 확인한 전기영동 사진이다(레인 1: 사이즈 마커, 레인 2,7: accA, 레인 3,8: accBC , 레인 4,9: fabH. 레인 5,10: fabHD , 레인 6: 공란)
도 5는 대장균과 같은 박테리아용 발현벡터, pTrc99A에 accABC 및 fabD 유전자가 삽입되어있는 구조의 도식화이다(lacI: repressor를 코딩, teminator: rrnB termimator(rrnB T1, rrnB T2), origin: pBR322-origin).
도 6은 대장균과 같은 박테리아용 발현벡터, pTrc99A에 accABC , fabD 및 fabH 유전자가 삽입되어있는 구조의 도식화이다(lacI: repressor를 코딩, teminator: rrnB termimator(rrnB T1, rrnB T2), origin: pBR322-origin).
도 7는 본 발명의 재조합 대장균의 생장곡선을 야생종(wild type) 대장균과 비교한 결과를 그린 그래프이다.
도 8은 본 발명의 재조합 대장균의 세포외에서 분석된 아세트산과 말론산의 양을 야생종(wild type), 다른 지방산 생산 유도체 유전자종과 대장균의 실험 결과와 비교한 결과이다.
도 9는 재조합 대장균의 세포내에서 분석된 지방산(fatty acid) 구성과 생산량의 차이를 배양 24시간 배양 후 지방산 추출방법에 의해 야생종(wild type) 대장균의 실험 결과와 비교한 결과이다.
도 10은 본 발명의 재조합대장균의 지방산에서 (A) 헥사데칸산, (B) 9-헥사데켄산, 7-헥사데칸산, (C) 헵타데칸산, (D) 9-옥타데켄산 및 (E) 옥타데칸산의 지방산함량 증진을 야생종(wild type) 대장균의 실험 결과와 비교한 결과이다. 도 10a는 (A) 내지 (E)의 피크를 함께 표시한 것이고, 도 10b는 (A)의 피크를 확대한 것이고, 도 10c 및 도 10d는 (B), (C) 및 (D), (E)를 각각 확대한 것이다.1 is a schematic showing the fatty acid biosynthetic pathway from glucose to fatty acids in E. coli in vivo.
Figure 2 shows the chromosome of E. coli as a template fabH The electrophoresis photographs confirmed after PCR were performed (lane 1: size marker, lanes 2 and 3: fabH ).
Figure 3 is an electrophoresis picture of the pJS12 recombinant plasmid using restriction enzymes (lane 1: size marker, lane 2-4: fabH obtained by cutting the plasmid with Xba I and BamH I ).
4 shows accABC , fabD, and fabH of recombinant plasmid pJS13 cloned into pTrc99A vector Electrophoresis pictures of genes identified by PCR (lane 1: size marker, lanes 2,7: accA , lanes 3,8: accBC , lanes 4,9: fabH . Lanes 5,10: fabHD , lane 6: blank)
5 is Such as E. coli Expression vector for bacteria, pTrc99A accABC and fabD Scheme of the structure in which the gene is inserted (lacI: coding repressor, teminator: rrnB termimator (rrnB T1, rrnB T2), origin: pBR322-origin).
6, such as E. coli Expression vector for bacteria, pTrc99A accABC , fabD, and fabH Scheme of the structure in which the gene is inserted (lacI: coding repressor, teminator: rrnB termimator (rrnB T1, rrnB T2), origin: pBR322-origin).
Figure 7 is a graph showing the result of comparing the growth curve of the recombinant E. coli of the present invention with wild type E. coli.
8 is a result of comparing the amount of acetic acid and malonic acid analyzed extracellularly of the recombinant E. coli of the present invention with the experimental results of wild type, other fatty acid producing derivative genotype and E. coli.
9 is a result of comparing the difference in the composition and production of fatty acid (fatty acid) analyzed in the cells of recombinant E. coli compared with the experimental results of wild type E. coli by the fatty acid extraction method after culturing for 24 hours.
10 shows (A) hexadecanoic acid, (B) 9-hexadecanoic acid, 7-hexadecanoic acid, (C) heptadecanoic acid, (D) 9-octadecanoic acid and E) The fatty acid content of octadecanoic acid is compared with the experimental results of wild type E. coli. 10A shows the peaks of (A) to (E) together, FIG. 10B shows an enlarged peak of (A), and FIGS. 10C and 10D show (B), (C) and (D), ( E) is expanded respectively.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명 하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

실시예Example

실시예 1: 중합효소연쇄반응(Polymerase Chain Reaction, PCR)을 이용한 Example 1: Polymerase Chain Reaction (PCR) accA, accB, accC, fabD accA, accB, accC, fabD 및 And fabH fabH 유전자의 증폭 및 Amplification of genes and fabH fabH 유전자를 발현하는 재조합 플라스미드의 제조Preparation of Recombinant Plasmids Expressing Genes

에스케리키아 콜라이(Escherichia coli) K-12 MG1655의 염색체(ATCC, USA)를 주형으로 하여 accA, accB, accC, fabD 및 fabH 유전자를 각각의 프라이머(primer)를 제작하여 중합효소연쇄반응(PCR, DaKaRa) 방법을 통해 클로닝 하였다. 각 유전자가 코딩하는 효소명 및 상기에서 사용한 프라이머를 각각 하기 표 1 및 표 2에 정리하였다. 도 2는 fabH 유전자가 RBS(Ribosome Binding Site)를 포함하여, 975 bp에서 증폭된 것을 나타내고 있다. Using the chromosome of Escherichia coli K-12 MG1655 (ATCC, USA) as a template, the primers of the accA, accB, accC, fabD and fabH genes were prepared to produce a polymerase chain reaction (PCR, DaKaRa) was cloned through the method. The enzyme names encoded by each gene and the primers used above are summarized in Tables 1 and 2, respectively. Figure 2 shows that the fabH gene was amplified at 975 bp, including the Ribosome Binding Site (RBS).

유전자서열
(GenBank 허가번호)Gene sequence
(GenBank clearance number) 유전자gene 생산 효소Production enzyme 서열목록 제4서열
(GeneID:944895)SEQ ID NO: 4 Sequence
(GeneID: 944895) accAaccA acetyl-CoA carboxylase,
carboxytransferase, alpha subunitacetyl-CoA carboxylase,
carboxytransferase, alpha subunit 서열목록 제6서열
(GeneID:947758)SEQ ID NO: 6 Sequence
(GeneID: 947758) accB accB acetyl CoA carboxylase, biotin carboxyl carrier protein(BCCP) subunitacetyl CoA carboxylase, a biotin carboxyl carrier protein (BCCP) subunit 서열목록 제8서열
(GeneID:947761)SEQ ID NO: 8 Sequence
(GeneID: 947761) accC  accC acetyl-CoA carboxylase,
biotin carboxylase subunitacetyl-CoA carboxylase,
biotin carboxylase subunit 서열목록 제10서열
(GeneID:945766)SEQ ID NO: 10
(GeneID: 945766) fabDfabD malonyl-CoA-[acyl-carrier-protein] transacylase   malonyl-CoA- [acyl-carrier-protein] transacylase 서열목록 제2서열
(GeneID:946003)SEQ ID NO 2
(GeneID: 946003) fabHfabH β-Ketoacyl-Acyl Carrier Protein Synthase IIIβ-Ketoacyl-Acyl Carrier Protein Synthase III

유전자gene 프라이머 서열Primer sequence 제한효소Restriction enzyme accAaccA FF 5‘-GAATTCTACAGGAATACTATGAGTCTGAAT-3’5'-GAATTCTACAGGAATACTATGAGTCTGAAT-3 ' EcoRⅠEcoRⅠ RR 5‘-GAGCTCTTADGDGTAACCGTAGCTCATC-3’5'-GAGCTCTTADGDGTAACCGTAGCTCATC-3 ' SacⅠSacⅠ accBCaccBC FF 5'-GAGCTCACGGAACCCACTCATGGATATTC-3'5'-GAGCTCACGGAACCCACTCATGGATATTC-3 ' SacⅠSacⅠ RR 5'-GGATCCTTGTCGATCCCCAGTAATAAAAAA-3'5'-GGATCCTTGTCGATCCCCAGTAATAAAAAA-3 ' BamHⅠBamHⅠ fabHDfabHD FF 5'-GGATCCCCGAAAAGTGACTGAGCGTACA-3'5'-GGATCCCCGAAAAGTGACTGAGCGTACA-3 ' BamHⅠBamHⅠ RR 5'-TCTAGATTAAAGCTCGAGCGCCGCTGC-3'5'-TCTAGATTAAAGCTCGAGCGCCGCTGC-3 ' XbaⅠXbaⅠ fabHfabH FF 5'-GGATCCCCGAAAAGTGACTGAGCGTACA-3'5'-GGATCCCCGAAAAGTGACTGAGCGTACA-3 ' BamHIBamHI RR 5'-GGATCCCCGAAAAGTGACTGAGCGTACA-3'5'-GGATCCCCGAAAAGTGACTGAGCGTACA-3 ' XbaIXbaI

각각의 중합효소연쇄반응은 통상적인 반응조건(10 mM Tris-HCl(pH 9.0), 50 mM KCI, 0.1% Triton X-100, 2 mM MgSO4 및 Taq DNA 중합효소(DaKaRa)) 아래에서 95℃/5분(denaturation), 65℃/1분(annealing) 및 72℃/1분(extension)으로 1회 수행한 후, 95℃/1분(denaturation), 65℃/30초(annealing) 및 72℃/1분(extension)으로 30회 반복 수행하였다. 마지막 단계로 안정적인 신장(extension)을 위해 95℃/1분(denaturation), 60℃/1분(annealing) 및 72℃/5분(extension) 반응하였다.Each polymerase chain reaction was performed under conventional reaction conditions (10 mM Tris-HCl, pH 9.0), 50 mM KCI, 0.1% Triton X-100, 2 mM MgSO 4 And 95 ° C / 1 min under Taq DNA polymerase (DaKaRa) at 95 ° C./5 min (denaturation), 65 ° C./1 min (annealing) and 72 ° C./1 min (extension). denaturation, 65 ° C./30 sec (annealing) and 72 ° C./1 min (extension) were performed 30 times. The final step was 95 ° C./1 min (denaturation), 60 ° C./1 min (annealing) and 72 ° C./5 min (extension) for stable extension.

중합효소연쇄반응 후 fabH의 증폭된 DNA는 0.8% 아가로즈 젤 상에서 확인 후 정제하여 T-벡터 클로닝에 이용하였다. pGEM-T easy 벡터(DaKaRa)에 라이게이션을 수행하여 재조합 플라스미드인 pGEM::fabH를 제작하였다. 상기 재조합 플라스미드 pGEM::fabH들은 대장균(E. coli AG1 competent cell, Stratagene)에서 형질 전환하여 균주를 제조하였다.
Of fabH after polymerase chain reaction Amplified DNA was identified and purified on 0.8% agarose gel and used for T-vector cloning. Ligation was performed on the pGEM-T easy vector (DaKaRa) to prepare a recombinant plasmid pGEM :: fabH . The recombinant plasmid pGEM :: fabHs are E. coli A strain was prepared by transformation in AG1 competent cell (Stratagene).

실시예Example 2: 재조합 플라스미드  2: recombinant plasmid pJS12pJS12 와 Wow pJS13pJS13 의 제조 Manufacturing

상기 실시예 1의 재조합 플라스미드인 pGEM-T::fabH와 대장균 벡터인 pTrc99A(Christopher, 2004)를 표 2의 제한효소(XbaI, BamHI)로 37℃ 항온수조(water bath)에서 약 2시간 반응하였다. 또한, accA, accBC 및 fabD의 각각의 DNA 절편들을 상기 표 2에 열거한 제한효소로 각각 절단한 뒤 pTrc99A 벡터의 다중삽입부위(multicloning site)에 도 5 및 6에 개시된 것과 같이 T4 라이게이즈(Dakara)를 이용, 16℃에서 반응, 라이게이션을 하여 재조합 플라스미드를 제조하고 이를 각각 pJS12와 pJS13라 명명하였다. 도 5 및 6은 상기 pJS12(accA , accBC 및 fabD 유전자 포함), pJS13(accA, accBC, fabD 및 fabH 유전자 포함)라 명명된 벡터의 지도(map)를 나타내는 도면이다. PGEM -T :: fabH , which is the recombinant plasmid of Example 1 E. coli vector pTrc99A (Christopher, 2004) was reacted with a restriction enzyme (XbaI, BamHI) of Table 2 in a 37 ℃ water bath for about 2 hours. Also, accA, accBC And of fabD Each DNA fragment was digested with the restriction enzymes listed in Table 2 above, followed by T4 ligase (Dakara) at 16 ° C. at the multicloning site of the pTrc99A vector as shown in FIGS. 5 and 6. Reaction and ligation were performed to prepare recombinant plasmids, which were named pJS12 and pJS13, respectively. 5 and 6 show the pJS12 ( accA , accBC And fabD Genes), pJS13 (including accA, accBC , fabD and fabH genes).

각 단계마다 삽입된 유전자의 삽입여부를 확인하기 위해 대장균(E. coli AG1 competentcell, stratagene)에 형질 전환 시킨 후 플라즈미드를 추출하여 이를 다시 원래 삽입부위의 제한효소로 처리하여 잘린 DNA 조작의 크기를 0.8% 아가로즈 젤 상에서 전기영동하여 비교하는 방법을 사용하였다. 도면 3에서는 재조합된 플라즈미드가 제한효소에 의해 절단된 결과를 보여주었다. E. coli ( E. coli) to confirm the insertion of the inserted gene at each step After transforming to AG1 competentcell, stratagene), plasmids were extracted and treated with restriction enzymes at the original insertion sites to compare the size of the cut DNA by electrophoresis on 0.8% agarose gel. 3 shows the result of cleavage of the recombinant plasmid by restriction enzyme.

실시예Example 3: 대장균 3: Escherichia coli 형질전환체 Transformant SGJS12SGJS12 및  And SGJS13SGJS13 의 제조Manufacturing

통상적으로 클로닝용으로 많이 쓰이는 대장균의 경우 CaCl2 버퍼를 사용한 컴피턴트 세포를 만든 후 열 충격(42℃) 방법에 의해 플라즈미드를 숙주세포내로 넣지만, 본 발명에서는 형질전환체의 안정적 제조와 효율을 높이기 위해 전기천공법(electroporation)에 의한 형질전환방법을 사용하였다. In general, Escherichia coli, which is widely used for cloning, prepares competent cells using CaCl 2 buffer and then inserts plasmid into host cells by a heat shock (42 ° C.) method. In order to increase the transformation method by electroporation (electroporation) was used.

상기 전기 충격에 의한 형질전환방법을 위해 16시간동안 전배양 된 30 ㎕ (0.1%)의 야생종 에스케리키아 콜라이(Escherichia coli) K12 MG1655(ATCC, USA) 배양액을 시험관에 들어있는 3 ㎖의 LB(10 g/L tripton, 10 g/L NaCl 및 5 g/L yeast extract)배지에 접종하여 배양액의 흡광도(absorbance)가 600nm 파장에서 0.6에 이르렀을 때, 배양액 전체에 해당하는 3 ㎖의 배양액은 원심 분리(12000 rpm, 1분)하여 상등액과 세포를 분리하였다. 모아진 세포는 10% 글리세롤 1 ㎖로 1회 세척해준 후, 다시 원심분리(12000 rpm, 1분)하여 상등액과 세포로 나누어 주었다. 세포는 10% 글리세롤 80 ㎕로 현탁하였다. 현탁 된 세포에 1-3 ㎕의 상기 실시예 2에서 제조한 재조합 플라즈미드(pJS12 및 pJS13)를 첨가시켜주었다.30 μl (0.1%) of wild species Escherichia coli ( Esherichia ) precultured for 16 hours for the electroshock transformation method coli ) K12 MG1655 (ATCC, USA) cultures were inoculated into 3 ml LB (10 g / L tripton, 10 g / L NaCl and 5 g / L yeast extract) media in test tubes to increase the absorbance of the cultures. When it reached 0.6 at a wavelength of 600 nm, 3 ml of the culture solution corresponding to the whole culture solution was centrifuged (12000 rpm, 1 minute) to separate the supernatant from the cells. The collected cells were washed once with 1 ml of 10% glycerol, and then centrifuged again (12000 rpm, 1 minute), and the supernatant and the cells were divided. The cells were suspended in 80 μl of 10% glycerol. 1-3 μl of the recombinant plasmids prepared in Example 2 (pJS12 and pJS13) were added to the suspended cells.

상기의 플라즈미드가 들어있는 분주된 80 ㎕의 액체는 전기천공법 (electroporation)용 큐벳(BIO-RAD, Gene pulser cuvette)에 담아 BIO-RAD, Gene pulser Xcell로 전기 충격(1800v, 25uF, 200Ω)을 가하였다. 미리 준비해둔 1 ㎖ LB(10 g/L tripton, 10 g/L NaCl 및 5 g/L yeast extract)를 첨가한 뒤 1시간 동안 200 rpm, 37℃에서 진탕 배양하였다. 배양된 형질전환체는 엠피실린(50 ㎍/㎖)이 첨가된 LB 아가(10 g/L tripton, 10 g/L NaCl, 5 g/L yeast extract 및 agar 20 g/L)에서 단일 균체가 생성될 때까지 37℃에서 배양하였다. 이를 통해 재조합 플라스미드인 pJS12 및 pJS13 각각을 에스케리키아 콜라이(Escherichia coli) K12 MG1655 균주에 형질 전환한 재조합 균주를 제조하고 이를 각각 SGJS12 및 SGJS13라 명명하였다(표 3).The 80 μl of the liquid containing the plasmid was placed in a cuvette (BIO-RAD, Gene pulser cuvette) for electroporation and subjected to electric shock (1800v, 25uF, 200Ω) with BIO-RAD, Gene pulser Xcell. Was added. 1 ml LB (10 g / L tripton, 10 g / L NaCl and 5 g / L yeast extract) prepared in advance was added thereto, followed by shaking culture at 200 rpm and 37 ° C. for 1 hour. Cultured transformants produced single cells in LB agar (10 g / L tripton, 10 g / L NaCl, 5 g / L yeast extract and agar 20 g / L) to which empicillin (50 μg / ml) was added. Incubated at 37 ° C. until This Escherichia coli recombinant plasmid pJS12 and pJS13 each via (Escherichia coli ) recombinant strains transformed into the K12 MG1655 strain were prepared and named SGJS12 and SGJS13, respectively (Table 3).

개발균주Development strain SGJS12SGJS12 Escherichia coli K12 MG1655::pTrc99A::accA :: accBC :: fabD Escherichia coli K12 MG1655 :: pTrc99A :: accA :: accBC :: fabD SGJS13SGJS13 Escherichia coli K12 MG1655::pTrc99A::accA :: accBC :: fabH :: fabD Escherichia coli K12 MG1655 :: pTrc99A :: accA :: accBC :: fabH :: fabD

SGJS12와 SGJS13은 여러 유전자가 함께 삽입되었다. 따라서 본 발명의 목적인 fabH 유전자가 다른 유전자들과 함께 모두 삽입이 되어 있는지 확인하기 위해서 재조합 균주 SGJS13의 플라즈미드를 추출하여 PCR을 통해 확인하였다. 이때 사용한 프라이머는 하기 표 4에 나타내었다. 표 4에 제시한 프라이머를 사용하여 반응조건(10 mM Tris-HCl(pH9.0), 50 mM KCI, 0.1% Triton X-100, 2 mM MgSO4 및 Taq DNA 중합효소(DaKaRa)) 아래에서 95℃/5분(denaturation), 63℃/1분(annealing) 및 72℃/1분(extension)으로 1회 수행한 후, 95℃/1분(denaturation), 63℃/30초(annealing) 및 72℃/1분(extension)으로 30회 반복 수행하였다. 마지막 단계로 안정적인 신장(extension)을 위해 95℃/1분(denaturation), 63℃/1분 (annealing) 및 72℃/5분(extension) 반응하였다. 증폭결과는 0.8% 아가로즈 젤 상에서 전기영동을 통해 도면 4에서와 같이 확인으로 모든 유전자가 삽입되었음을 알 수 있었다.SGJS12 and SGJS13 have several genes inserted together. Therefore, in order to confirm whether the fabH gene, which is an object of the present invention, is inserted together with other genes, the plasmid of the recombinant strain SGJS13 was extracted and confirmed by PCR. The primers used at this time are shown in Table 4 below. Reaction conditions (10 mM Tris-HCl (pH 9.0), 50 mM KCI, 0.1% Triton X-100, 2 mM MgSO 4 using the primers shown in Table 4) And 95 ° C / 1 min under Taq DNA polymerase (DaKaRa) at 95 ° C / 5 min (denaturation), 63 ° C / 1 min (annealing) and 72 ° C / 1 min (extension). denaturation, 63 ° C./30 sec (annealing) and 72 ° C./1 min (extension) were performed 30 times. The final step was 95 ° C./1 min (denaturation), 63 ° C./1 min (annealing) and 72 ° C./5 min (extension) for stable extension. The amplification result was confirmed that as shown in Figure 4 through the electrophoresis on 0.8% agarose gel all genes were inserted.

유전자gene 프라이머 서열Primer sequence accAaccA FF 5‘-GAATTCTACAGGAATACTATGAGTCTGAAT-3’5'-GAATTCTACAGGAATACTATGAGTCTGAAT-3 ' RR 5‘-GAGCTCTTADGDGTAACCGTAGCTCATC-3’5'-GAGCTCTTADGDGTAACCGTAGCTCATC-3 ' accBCaccBC FF 5'-GAGCTCACGGAACCCACTCATGGATATTC-3'5'-GAGCTCACGGAACCCACTCATGGATATTC-3 ' RR 5'-GGATCCTTGTCGATCCCCAGTAATAAAAAA-3'5'-GGATCCTTGTCGATCCCCAGTAATAAAAAA-3 ' fabHfabH FF 5'-GGATCCCCGAAAAGTGACTGAGCGTACA-3'5'-GGATCCCCGAAAAGTGACTGAGCGTACA-3 ' RR 5'-TCTAGACTAGAAACGAACCAGCGCGGAG-3'5'-TCTAGACTAGAAACGAACCAGCGCGGAG-3 ' fabHDfabHD FF 5'-GGATCCCCGAAAAGTGACTGAGCGTACA-3'5'-GGATCCCCGAAAAGTGACTGAGCGTACA-3 ' RR 5'-TCTAGATTAAAGCTCGAGCGCCGCTGC-3'5'-TCTAGATTAAAGCTCGAGCGCCGCTGC-3 '

실시예Example 4: 대장균의 배양 및 재조합 균주로부터 단백질의 생산  4: Culture of Escherichia Coli and Production of Proteins from Recombinant Strains

에스케리키아 콜라이( Escherichia coli) K12 MG1655 균주에 형질 전환한 재조합 균주들을 LB(암페실린 50 ㎍/㎖ 포함)배지에 16시간 정도 전 배양 시키고 그 배양액을 다시 암피실린이 포함된 LB 배지에 접종하여 흡광도가 600 nm에서 0.6-1.0 되었을 때 글리세롤 농도가 25%가 되게 보관용 균액으로 만든 다음 -80℃에서 배양실험 시까지 저장하였다.Escherichia coli (Escherichia coli ) Recombinant strains transformed into K12 MG1655 strain were incubated for 16 hours in LB (containing 50 μg / ml of ampicillin) medium, and the culture was inoculated in LB medium containing ampicillin and absorbance was 0.6- at 600 nm. When the concentration was 1.0, the glycerol concentration was 25%, and then stored as a culture solution and stored at -80 ℃ until the culture experiment.

상기에서 개발된 재조합 균주의 배양은 보관용 균액 30 ㎕을 10 mL 저면 튜브에 들어있는 암페실린(50 ㎍/㎖)이 첨가된 3 ㎖의 LB에 16시간동안 배양한 후, 이를 다시 500 ㎖ 플라스크에 들어있는 암페실린(50 ㎍/㎖)이 첨가된 200 ㎖의 LB (10 g/L tripton, 10 g/L NaCl 및 5 g/L yeast extract)배지에 0.5%의 전기 배양액을 접종하여 37℃, 170 rpm 상태에서 24시간동안 배양하였다. 재조합 균주와 비교하기 위한 에스케리키아 콜라이(Escherichia coli) K12 MG1655 야생종은 엠피실린이 들어있지 않은 LB에서 재조합 균주와 동일한 조건으로 실험을 실시하였다. 단백질 발현을 위해 배양액의 흡광도(absorbance)가 600 nm파장에서 0.6에 이르렀을 때 유도물질인 IPTG(isopropylthio-β-D-galactoside, sigma, USA)를 첨가(1 mM/㎖)하여 재조합 단백질의 발현을 유도하였다. 상기 예를 따라 초기 유도체의 발현으로 지방산 함량의 생산증대를 비교하기 위한 종과 본 발명의 목적으로 재조합 대장균과 야생종의 생장곡선은 도 7에서 보여주고 있다.Culture of the recombinant strain developed above was incubated for 16 hours in 30 ml of storage bacteria solution in 3 ml of LB to which ampicillin (50 µg / ml) contained in a 10 mL bottom tube was added for 16 hours, and then again a 500 ml flask. 200 ml LB (10 g / L tripton, 10 g / L NaCl and 5 g / L yeast extract) medium containing ampicillin (50 μg / ml) was inoculated with 0.5% of the electroculture solution at 37 ° C. Incubated for 24 hours at 170 rpm. For comparison with the recombinant strain Escherichia coli (Escherichia coli ) K12 MG1655 wild species were tested in the same conditions as recombinant strains in LB containing no empicillin. When protein absorbance was 0.6 at 600 nm wavelength, the protein was expressed by adding inducer IPTG (isopropylthio-β-D-galactoside, sigma, USA) (1 mM / ml). Induced. According to the above example, the growth curves of the recombinant Escherichia coli and the wild species for the purpose of the present invention and the species for comparing the production of fatty acid content by the expression of the initial derivatives are shown in FIG. 7.

본 발명에서 재조합 균주, SGJS13은 생장곡선에서 대장균 야생종과 다른 지방산 유도 재조합 균주 SGJS12와 비교하여 큰 차이가 없었다. 유전자의 삽입이나 과발현에 의한 독성이나 저해는 없었던 것으로 사료된다. 이는 유전자의 암호화부분은 효소의 과발현 기능을 포함하여 최소한의 길이를 가지도록 RBS(Ribosomal Binding Site) 및 효소발현에 꼭 필요한 부분을 포함하는 염기서열만을 서열로 정하여 유전자의 발현에 의한 숙주세포의 대사부담(metabolic burden)을 줄이고자 하였고 생산 경로에 맞춰 두 유전자의 순서가 순서대로 연결되게 개발하였기 때문이라고 예상된다.
In the present invention, the recombinant strain, SGJS13, was not significantly different in the growth curve compared with E. coli wild species and other fatty acid-derived recombinant strain SGJS12. There was no toxicity or inhibition by gene insertion or overexpression. This is because the coding part of the gene has a minimum length including the overexpression function of the enzyme, and only the base sequence including the necessary part for the RBS (Ribosomal Binding Site) and the enzyme expression is sequenced to metabolize the host cell by the expression of the gene. It is expected to reduce the burden on the metabolic burden and develop the sequence of the two genes in order according to the production route.

실시예Example 5: 재조합 대장균 5: recombinant E. coli 세포외로부터의From the outside 아세트산( Acetic acid ( aceticacetic acidacid ) 및 말론산 () And malonic acid ( malonicmalonic acidacid )의 측정)

재조합 균주의 배양액 내의 지방산 중간 유도체 물질인 아세트산과 말론산의 농도를 측정하기 위해 배양 중 일정시간마다 채취한 시료를 원심분리(12000 rpm, 10분) 후 상등액과 세포를 분리하여 -40℃에서 보관하였다. 상기 배양액으로부터 분리된 상등액은 각각 1 ㎖씩 0.2 ㎛ 필터를 이용하여 정제한 후, 하단의 조건하에서 고속액체크로마토그래피(HPLC) 정량 분석을 실시하였다. In order to measure the concentrations of acetic acid and malonic acid, which are fatty acid intermediate derivatives in the culture medium of the recombinant strain, the samples collected every certain time during the culture were centrifuged (12000 rpm, 10 minutes), and the supernatant and the cells were separated and stored at -40 ° C. It was. The supernatant separated from the culture solution was purified using a 0.2 μm filter, each 1 ml, and then subjected to high performance liquid chromatography (HPLC) quantitative analysis under the following conditions.

품 목subject 조 건Condition HPLC 모델HPLC Model (영린기기, Korea), UV730D(영린기기, Korea)  (Younglin, Korea), UV730D (Younglin, Korea) 컬럼column Aminex HPX-87H(Biorad, USA)Aminex HPX-87H (Biorad, USA) 유속Flow rate 0.6 mi/min0.6 mi / min 주입부피Injection volume 30 ㎕30 μl 이동상Mobile phase 0.005 N 황산0.005 N sulfuric acid 오븐 온도Oven temperature 50℃50 ℃ 작동 시간Working time 30분30 minutes UV 검출파장UV detection wavelength 210 nm210 nm

도 8에서 보여주듯이, 본 발명의 SGJS12 및 SGJS13은 야생종 대장균과 엑스트라셀룰라(extracellular)에서 분석되어지는 아세트산(acetic acid)과 말론산 (malonic acid)의 생산량 경향에 차이를 보였다. 배양이 시작된 후 fabH 유전자에 의해 SGJS13은 야생종과 다른 지방산 유도를 유도하는 유전자가 삽입된 종, SGJS12과 비교하였을 때 지방산 생산의 중간체인 아세트산(acetic acid) 및 말론산(malonic acid)이 모두 많이 증가된 것을 확인하였다. SGJS13은 지방산 생산증대에 영향을 미치는 여러 유도체, 유전자들이 많이 삽입되어있음에도 불구하고 야생종과 비교하여 아세트산과 말론산이 관찰 시간 동안 많이 생산되는 것을 확인 할 수 있었다. As shown in Figure 8, SGJS12 and SGJS13 of the present invention showed a difference in the production trend of acetic acid and malonic acid analyzed in wild E. coli and extracellular (extracellular). After incubation begins fabH SGJS13 was found to have significantly increased both acetic acid and malonic acid, which are intermediates of fatty acid production, compared to SGJS12. SGJS13 was found to produce a lot of acetic acid and malonic acid during the observation time, despite the fact that many derivatives and genes affecting fatty acid production were inserted.

이는 β-케토아실-[아실운반단백질]신타아제Ⅲ, fabH 유전자는 아세틸-CoA에서 CO2와 H2O가 제외되면서 아세토아실-[아실운반단백질]로 지방산이 합성되어지며 또한 말로닐-[아실운반단백질]로부터 β-케토아실-[아실운반단백질]로 만들어져 지방산 회로로 유도되게 해주어 두 회로로 모두 진행하게 할 수 있어 과발현되어 더욱 증진된 것으로 사료되어진다. 또한 β-케토아실-[아실운반단백질]신타아제Ⅲ 효소는 아세틸-CoA를 공여자로 사용되고 아세틸-CoA [아실운반단백질]트랜스아실레이즈 활성을 가지게 해주어서 아세트산은 관찰시간 동안 줄어들고 말론산이 증가된 것으로 사료된다. 이를 통해 fabH 유전자의 과발현으로 인해 지방산의 생산의 중간체인 말론산이 증진되었고 이로 인해 지방산 함량증진을 가져올 것이라 예상할 수 있다.
This is because the β- ketoacyl- [acyl carrier protein] synthase III and fabH genes are excluded from CO 2 and H 2 O in acetyl-CoA, and fatty acids are synthesized as acetoacyl- [acyl carrier proteins] and malonyl- [ Acyl carrier protein] is made from β-ketoacyl- [acyl carrier protein] to be induced into the fatty acid circuit and can proceed to both circuits. In addition, β-ketoacyl- [acyl carrier protein] synthase III enzyme was used as a donor and acetyl-CoA [acyl carrier protein] transacylase activity, so that acetic acid was decreased during observation time and malonic acid was increased. It is feed. because of this Overexpression of the fabH gene promoted malonic acid, an intermediate in fatty acid production, and could be expected to result in increased fatty acid content.

실시예Example 6: 재조합 대장균의  6: of recombinant E. coli 세포내로부터의From within the cell 지방산 ( Fatty acids ( fattyfatty acidacid ) 측정) Measure

실시예 5의 조건으로 배양한 배양액 중 IPTG를 첨가한 후 4시간 후와 24시간 동안 세포양이 충분해 졌을 때 세포 내에 있는 지방산(fatty acid)를 측정하기 위해서 지방산 추출을 다음과 같은 방법으로 진행하였다. 추출과정은 5 단계로 나누어지며 첫 번째 과정 배양단계로 배양액 5 ㎖를 원심분리(4500 rpm, 10분) 후 세포는 -80℃에 저장하였다. 두 번째 과정은 비누화(saponification)단계로 저장된 재조합 세포들을 용액 1(NaOH 45 g, MeOH 150 ㎖ 및 탈염수 150 ㎖) 1 ㎖을 넣고 5-10초간 볼텍스 해주었다. 100℃에서 5분 동안 반응해 준 뒤 다시 5-10초 동안 볼텍스 해준 후 다시 100℃, 25분 반응시켜 준 뒤 열을 식혀주었다. 세 번째 과정은 메틸화(methylation)단계로, 용액 2(6 N HCl 325 ㎖, MeOH 275 ㎖) 2 ㎖을 넣고 5-10초 동안 볼텍스 해준 뒤 80℃에서 10분 동안 반응시켜주었다. 반응이 끝난 후에는 빨리 열을 식혀주도록 하였다. 네 번째 단계는 추출(extration)단계로, 용액 3(헥산/메틸 터셔리부틸 에스터=1/1) 1.25 ㎖을 첨가하고 10분간 위아래로 흔들어 준 뒤 층이 분리 되면 아래층은 추출해서 버렸다. 다섯 번째 단계는 GC로 분석을 용이하게 하기위해 세척단계로 앞 단계의 남은 상층액에 용액 4(NaOH 10.8 g, 탈염수 900 ㎖) 3 ㎖을 첨가해서 5분 동안 위아래로 흔들어 준 뒤 상층액을 추출하여 GC/MS로 분석하였다.Fatty acid extraction was carried out in the following manner to measure fatty acids in cells after 4 hours and 24 hours after the addition of IPTG in the culture medium cultured in Example 5 It was. The extraction process was divided into five stages. In the first process culture stage, 5 ml of the culture solution was centrifuged (4500 rpm, 10 minutes), and the cells were stored at -80 ° C. In the second process, the recombinant cells stored in the saponification step were added with 1 ml of Solution 1 (45 g of NaOH, 150 ml of MeOH and 150 ml of demineralized water) and vortexed for 5-10 seconds. After reacting at 100 ° C. for 5 minutes, the solution was vortexed again for 5-10 seconds, then reacted again at 100 ° C. for 25 minutes, and then cooled. The third process is a methylation step. 2 ml of solution 2 (6 N HCl 325 ml, MeOH 275 ml) was added and vortexed for 5-10 seconds, followed by reaction at 80 ° C. for 10 minutes. After the reaction was completed, the heat was cooled quickly. The fourth step is an extraction step, in which 1.25 ml of solution 3 (hexane / methyl tertiary butyl ester = 1/1) was added, shaken up and down for 10 minutes, and the bottom layer was extracted and discarded. In the fifth step, 3 g of solution 4 (NaOH 10.8 g, demineralized water 900 ml) was added to the remaining supernatant of the previous step in order to facilitate the analysis by GC. After shaking for 5 minutes, the supernatant was extracted. Was analyzed by GC / MS.

본 발명에서 사용한 CG/MS는 5975 시리즈 MSD 및 Agilent 7890A이며 HP-5 컬럼(30m X 0.32 mm, 필름 두께 0.25 )을 사용하였다. 분석하기 위한 GC조건은 온도 프로그램은 40℃ 5분간 -> 분당 3℃로 220℃까지 -> 분당 3℃로 250℃까지 -> 250℃ 5분간이었으며 MS 내부온도는 160℃이었다. The CG / MS used in the present invention was 5975 series MSD and Agilent 7890A and used HP-5 column (30m × 0.32 mm, film thickness 0.25). GC conditions for the analysis were 40 ℃ for 5 minutes-> 3 ℃ per minute to 220 ℃-> 3 ℃ per minute to 250 ℃-> 250 ℃ for 5 minutes and MS internal temperature was 160 ℃.

상기 예를 따라 지방산 함량 증진을 위해 형질전환 된 재조합 대장균 2종과 야생종 대장균의 지방산 추출 분석결과를 도 9 및 도 10에 나타내었다. 이 추출 방법으로 헥사데칸산(Hexadecanoic acid, C16)과 옥타데칸산(Octadecanoic acid, C18)이 추출되고 분석됨을 알 수 있었다. 도 9는 배양 24시간 결과 후의 지방산 추출 결과이다. 이 결과, 야생종보다 초기 지방산 합성에 관여하는 유전자가 과발현된 재조합 대장균의 지방산, 헥사데칸산(C16)과 옥타데칸산(C18)이 더 생산 되는 것을 알 수 있었다. 특히 β-케토아실-[아실운반단백질]신타아제Ⅲ( fabH )가 포함된 재조합균이 아세틸-CoA 카복실라제(accA, accBC)와 말로닐-CoA:ACP(acyl carrier protein) 아실트랜스퍼라제(fabD)만 있는 재조합균주보다 월등히 더 많이 지방산이 생산된 것을 확인 할 수 있었다. 따라서 지방산 합성 경로에 관여하는 다른 유전자들과 함께 fabH 유전자로 공동형질 전환하는 경우 지방산 생산에 있어 시너지 효과가 있는 것으로 판단된다.
9 and 10 show the results of fatty acid extraction analysis of the recombinant E. coli and wild E. coli transformed to improve the fatty acid content. Hexadecanoic acid (C16) and octadecanoic acid (C18) were extracted and analyzed by this extraction method. 9 shows fatty acid extraction results after 24 hours of culture. As a result, it was found that more fatty acids, hexadecanoic acid (C16) and octadecanoic acid (C18) of recombinant E. coli overexpressed genes involved in the initial fatty acid synthesis than wild species were produced. In particular, the recombinant bacteria containing β-ketoacyl- [acyl carrier protein] synthase III ( fabH ) are acetyl-CoA carboxylase ( accA, accBC ) and malonyl-CoA: acP (acyl carrier protein) acyl transferase ( fabD). It was confirmed that the fatty acid produced much more than the recombinant strain containing only). Therefore, cotransformation into the fabH gene along with other genes involved in fatty acid synthesis pathways is thought to have a synergistic effect on fatty acid production.

도 10은 지방산 추출 방법으로 추출된 여러 지방산 종류를 확인할 수 있었는데 (A)헥사데칸산(Hexadecanoic acid), (B) 9-헥사데켄산(9-Hexadecenoic acid), 7-헥사데칸산(7-Hexadecenoic acid), (C) 헵타데칸산(Heptadecanoic acid), (D) 9-옥타데켄산(9-Octadecenoic acid) 및 (E) 옥타데칸산(Octadecanoic acid)이 확인 되었다. 52-56분 사이에서는 헥사데칸산(C16)과 관련된 지방산이 추출되었고 58-60분 사이는 옥타데칸산(C18)과 관련된 지방산이 추출된 것을 확인 할 수 있었다.
Figure 10 was able to confirm the various fatty acid types extracted by fatty acid extraction method (A) Hexadecanoic acid (Hexadecanoic acid), (B) 9- hexadecenoic acid, 7-hexadecanoic acid (7- Hexadecenoic acid), (C) heptadecanoic acid, (D) 9-octadecenoic acid and (E) octadecanoic acid. Fatty acid associated with hexadecanoic acid (C16) was extracted between 52-56 minutes and fatty acid associated with octadecanoic acid (C18) was extracted between 58-60 minutes.

이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> Industry-University Cooperation Foundation Sogang University <120> Transformed Escherichia coli for Over-expression of Fatty Acid Biosynthesis Pathway and Method of Preparing the Same <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 317 <212> PRT <213> Escherichia coli strain K-12 MG1655, fabH <400> 1 Met Tyr Thr Lys Ile Ile Gly Thr Gly Ser Tyr Leu Pro Glu Gln Val 1 5 10 15 Arg Thr Asn Ala Asp Leu Glu Lys Met Val Asp Thr Ser Asp Glu Trp 20 25 30 Ile Val Thr Arg Thr Gly Ile Arg Glu Arg His Ile Ala Ala Pro Asn 35 40 45 Glu Thr Val Ser Thr Met Gly Phe Glu Ala Ala Thr Arg Ala Ile Glu 50 55 60 Met Ala Gly Ile Glu Lys Asp Gln Ile Gly Leu Ile Val Val Ala Thr 65 70 75 80 Thr Ser Ala Thr His Ala Phe Pro Ser Ala Ala Cys Gln Ile Gln Ser 85 90 95 Met Leu Gly Ile Lys Gly Cys Pro Ala Phe Asp Val Ala Ala Ala Cys 100 105 110 Ala Gly Phe Thr Tyr Ala Leu Ser Val Ala Asp Gln Tyr Val Lys Ser 115 120 125 Gly Ala Val Lys Tyr Ala Leu Val Val Gly Ser Asp Val Leu Ala Arg 130 135 140 Thr Cys Asp Pro Thr Asp Arg Gly Thr Ile Ile Ile Phe Gly Asp Gly 145 150 155 160 Ala Gly Ala Ala Val Leu Ala Ala Ser Glu Glu Pro Gly Ile Ile Ser 165 170 175 Thr His Leu His Ala Asp Gly Ser Tyr Gly Glu Leu Leu Thr Leu Pro 180 185 190 Asn Ala Asp Arg Val Asn Pro Glu Asn Ser Ile His Leu Thr Met Ala 195 200 205 Gly Asn Glu Val Phe Lys Val Ala Val Thr Glu Leu Ala His Ile Val 210 215 220 Asp Glu Thr Leu Ala Ala Asn Asn Leu Asp Arg Ser Gln Leu Asp Trp 225 230 235 240 Leu Val Pro His Gln Ala Asn Leu Arg Ile Ile Ser Ala Thr Ala Lys 245 250 255 Lys Leu Gly Met Ser Met Asp Asn Val Val Val Thr Leu Asp Arg His 260 265 270 Gly Asn Thr Ser Ala Ala Ser Val Pro Cys Ala Leu Asp Glu Ala Val 275 280 285 Arg Asp Gly Arg Ile Lys Pro Gly Gln Leu Val Leu Leu Glu Ala Phe 290 295 300 Gly Gly Gly Phe Thr Trp Gly Ser Ala Leu Val Arg Phe 305 310 315 <210> 2 <211> 954 <212> DNA <213> Escherichia coli strain K-12 MG1655, fabH cDNA <400> 2 atgtatacga agattattgg tactggcagc tatctgcccg aacaagtgcg gacaaacgcc 60 gatttggaaa aaatggtgga cacctctgac gagtggattg tcactcgtac cggtatccgc 120 gaacgccaca ttgccgcgcc aaacgaaacc gtttcaacca tgggctttga agcggcgaca 180 cgcgcaattg agatggcggg cattgagaaa gaccagattg gcctgatcgt tgtggcaacg 240 acttctgcta cgcacgcttt cccgagcgca gcttgtcaga ttcaaagcat gttgggcatt 300 aaaggttgcc cggcatttga cgttgcagca gcctgcgcag gtttcaccta tgcattaagc 360 gtagccgatc aatacgtgaa atctggggcg gtgaagtatg ctctggtcgt cggttccgat 420 gtactggcgc gcacctgcga tccaaccgat cgtgggacta ttattatttt tggcgatggc 480 gcgggcgctg cggtgctggc tgcctctgaa gagccgggaa tcatttccac ccatctgcat 540 gccgacggta gttatggtga attgctgacg ctgccaaacg ccgaccgcgt gaatccagag 600 aattcaattc atctgacgat ggcgggcaac gaagtcttca aggttgcggt aacggaactg 660 gcgcacatcg ttgatgagac gctggcggcg aataatcttg accgttctca actggactgg 720 ctggttccgc atcaggctaa cctgcgtatt atcagtgcaa cggcgaaaaa actcggtatg 780 tctatggata atgtcgtggt gacgctggat cgccacggta atacctctgc ggcctctgtc 840 ccgtgcgcgc tggatgaagc tgtacgcgac gggcgcatta agccggggca gttggttctg 900 cttgaagcct ttggcggtgg attcacctgg ggctccgcgc tggttcgttt ctag 954 <210> 3 <211> 319 <212> PRT <213> Escherichia coli strain K-12 MG1655, accA <400> 3 Met Ser Leu Asn Phe Leu Asp Phe Glu Gln Pro Ile Ala Glu Leu Glu 1 5 10 15 Ala Lys Ile Asp Ser Leu Thr Ala Val Ser Arg Gln Asp Glu Lys Leu 20 25 30 Asp Ile Asn Ile Asp Glu Glu Val His Arg Leu Arg Glu Lys Ser Val 35 40 45 Glu Leu Thr Arg Lys Ile Phe Ala Asp Leu Gly Ala Trp Gln Ile Ala 50 55 60 Gln Leu Ala Arg His Pro Gln Arg Pro Tyr Thr Leu Asp Tyr Val Arg 65 70 75 80 Leu Ala Phe Asp Glu Phe Asp Glu Leu Ala Gly Asp Arg Ala Tyr Ala 85 90 95 Asp Asp Lys Ala Ile Val Gly Gly Ile Ala Arg Leu Asp Gly Arg Pro 100 105 110 Val Met Ile Ile Gly His Gln Lys Gly Arg Glu Thr Lys Glu Lys Ile 115 120 125 Arg Arg Asn Phe Gly Met Pro Ala Pro Glu Gly Tyr Arg Lys Ala Leu 130 135 140 Arg Leu Met Gln Met Ala Glu Arg Phe Lys Met Pro Ile Ile Thr Phe 145 150 155 160 Ile Asp Thr Pro Gly Ala Tyr Pro Gly Val Gly Ala Glu Glu Arg Gly 165 170 175 Gln Ser Glu Ala Ile Ala Arg Asn Leu Arg Glu Met Ser Arg Leu Gly 180 185 190 Val Pro Val Val Cys Thr Val Ile Gly Glu Gly Gly Ser Gly Gly Ala 195 200 205 Leu Ala Ile Gly Val Gly Asp Lys Val Asn Met Leu Gln Tyr Ser Thr 210 215 220 Tyr Ser Val Ile Ser Pro Glu Gly Cys Ala Ser Ile Leu Trp Lys Ser 225 230 235 240 Ala Asp Lys Ala Pro Leu Ala Ala Glu Ala Met Gly Ile Ile Ala Pro 245 250 255 Arg Leu Lys Glu Leu Lys Leu Ile Asp Ser Ile Ile Pro Glu Pro Leu 260 265 270 Gly Gly Ala His Arg Asn Pro Glu Ala Met Ala Ala Ser Leu Lys Ala 275 280 285 Gln Leu Leu Ala Asp Leu Ala Asp Leu Asp Val Leu Ser Thr Glu Asp 290 295 300 Leu Lys Asn Arg Arg Tyr Gln Arg Leu Met Ser Tyr Gly Tyr Ala 305 310 315 <210> 4 <211> 960 <212> DNA <213> Escherichia coli strain K-12 MG1655, accA cDNA <400> 4 atgagtctga atttccttga ttttgaacag ccgattgcag agctggaagc gaaaatcgat 60 tctctgactg cggttagccg tcaggatgag aaactggata ttaacatcga tgaagaagtg 120 catcgtctgc gtgaaaaaag cgtagaactg acacgtaaaa tcttcgccga tctcggtgca 180 tggcagattg cgcaactggc acgccatcca cagcgtcctt ataccctgga ttacgttcgc 240 ctggcatttg atgaatttga cgaactggct ggcgaccgcg cgtatgcaga cgataaagct 300 atcgtcggtg gtatcgcccg tctcgatggt cgtccggtga tgatcattgg tcatcaaaaa 360 ggtcgtgaaa ccaaagaaaa aattcgccgt aactttggta tgccagcgcc agaaggttac 420 cgcaaagcac tgcgtctgat gcaaatggct gaacgcttta agatgcctat catcaccttt 480 atcgacaccc cgggggctta tcctggcgtg ggcgcagaag agcgtggtca gtctgaagcc 540 attgcacgca acctgcgtga aatgtctcgc ctcggcgtac cggtagtttg tacggttatc 600 ggtgaaggtg gttctggcgg tgcgctggcg attggcgtgg gcgataaagt gaatatgctg 660 caatacagca cctattccgt tatctcgccg gaaggttgtg cgtccattct gtggaagagc 720 gccgacaaag cgccgctggc ggctgaagcg atgggtatca ttgctccgcg tctgaaagaa 780 ctgaaactga tcgactccat catcccggaa ccactgggtg gtgctcaccg taacccggaa 840 gcgatggcgg catcgttgaa agcgcaactg ctggcggatc tggccgatct cgacgtgtta 900 agcactgaag atttaaaaaa tcgtcgttat cagcgcctga tgagctacgg ttacgcgtaa 960 960 <210> 5 <211> 156 <212> PRT <213> Escherichia coli strain K-12 MG1655, accB <400> 5 Met Asp Ile Arg Lys Ile Lys Lys Leu Ile Glu Leu Val Glu Glu Ser 1 5 10 15 Gly Ile Ser Glu Leu Glu Ile Ser Glu Gly Glu Glu Ser Val Arg Ile 20 25 30 Ser Arg Ala Ala Pro Ala Ala Ser Phe Pro Val Met Gln Gln Ala Tyr 35 40 45 Ala Ala Pro Met Met Gln Gln Pro Ala Gln Ser Asn Ala Ala Ala Pro 50 55 60 Ala Thr Val Pro Ser Met Glu Ala Pro Ala Ala Ala Glu Ile Ser Gly 65 70 75 80 His Ile Val Arg Ser Pro Met Val Gly Thr Phe Tyr Arg Thr Pro Ser 85 90 95 Pro Asp Ala Lys Ala Phe Ile Glu Val Gly Gln Lys Val Asn Val Gly 100 105 110 Asp Thr Leu Cys Ile Val Glu Ala Met Lys Met Met Asn Gln Ile Glu 115 120 125 Ala Asp Lys Ser Gly Thr Val Lys Ala Ile Leu Val Glu Ser Gly Gln 130 135 140 Pro Val Glu Phe Asp Glu Pro Leu Val Val Ile Glu 145 150 155 <210> 6 <211> 471 <212> DNA <213> Escherichia coli strain K-12 MG1655, accB cDNA <400> 6 atggatattc gtaagattaa aaaactgatc gagctggttg aagaatcagg catctccgaa 60 ctggaaattt ctgaaggcga agagtcagta cgcattagcc gtgcagctcc tgccgcaagt 120 ttccctgtga tgcaacaagc ttacgctgca ccaatgatgc agcagccagc tcaatctaac 180 gcagccgctc cggcgaccgt tccttccatg gaagcgccag cagcagcgga aatcagtggt 240 cacatcgtac gttccccgat ggttggtact ttctaccgca ccccaagccc ggacgcaaaa 300 gcgttcatcg aagtgggtca gaaagtcaac gtgggcgata ccctgtgcat cgttgaagcc 360 atgaaaatga tgaaccagat cgaagcggac aaatccggta ccgtgaaagc aattctggtc 420 gaaagtggac aaccggtaga atttgacgag ccgctggtcg tcatcgagta a 471 <210> 7 <211> 449 <212> PRT <213> Escherichia coli strain K-12 MG1655, accC <400> 7 Met Leu Asp Lys Ile Val Ile Ala Asn Arg Gly Glu Ile Ala Leu Arg 1 5 10 15 Ile Leu Arg Ala Cys Lys Glu Leu Gly Ile Lys Thr Val Ala Val His 20 25 30 Ser Ser Ala Asp Arg Asp Leu Lys His Val Leu Leu Ala Asp Glu Thr 35 40 45 Val Cys Ile Gly Pro Ala Pro Ser Val Lys Ser Tyr Leu Asn Ile Pro 50 55 60 Ala Ile Ile Ser Ala Ala Glu Ile Thr Gly Ala Val Ala Ile His Pro 65 70 75 80 Gly Tyr Gly Phe Leu Ser Glu Asn Ala Asn Phe Ala Glu Gln Val Glu 85 90 95 Arg Ser Gly Phe Ile Phe Ile Gly Pro Lys Ala Glu Thr Ile Arg Leu 100 105 110 Met Gly Asp Lys Val Ser Ala Ile Ala Ala Met Lys Lys Ala Gly Val 115 120 125 Pro Cys Val Pro Gly Ser Asp Gly Pro Leu Gly Asp Asp Met Asp Lys 130 135 140 Asn Arg Ala Ile Ala Lys Arg Ile Gly Tyr Pro Val Ile Ile Lys Ala 145 150 155 160 Ser Gly Gly Gly Gly Gly Arg Gly Met Arg Val Val Arg Gly Asp Ala 165 170 175 Glu Leu Ala Gln Ser Ile Ser Met Thr Arg Ala Glu Ala Lys Ala Ala 180 185 190 Phe Ser Asn Asp Met Val Tyr Met Glu Lys Tyr Leu Glu Asn Pro Arg 195 200 205 His Val Glu Ile Gln Val Leu Ala Asp Gly Gln Gly Asn Ala Ile Tyr 210 215 220 Leu Ala Glu Arg Asp Cys Ser Met Gln Arg Arg His Gln Lys Val Val 225 230 235 240 Glu Glu Ala Pro Ala Pro Gly Ile Thr Pro Glu Leu Arg Arg Tyr Ile 245 250 255 Gly Glu Arg Cys Ala Lys Ala Cys Val Asp Ile Gly Tyr Arg Gly Ala 260 265 270 Gly Thr Phe Glu Phe Leu Phe Glu Asn Gly Glu Phe Tyr Phe Ile Glu 275 280 285 Met Asn Thr Arg Ile Gln Val Glu His Pro Val Thr Glu Met Ile Thr 290 295 300 Gly Val Asp Leu Ile Lys Glu Gln Leu Arg Ile Ala Ala Gly Gln Pro 305 310 315 320 Leu Ser Ile Lys Gln Glu Glu Val His Val Arg Gly His Ala Val Glu 325 330 335 Cys Arg Ile Asn Ala Glu Asp Pro Asn Thr Phe Leu Pro Ser Pro Gly 340 345 350 Lys Ile Thr Arg Phe His Ala Pro Gly Gly Phe Gly Val Arg Trp Glu 355 360 365 Ser His Ile Tyr Ala Gly Tyr Thr Val Pro Pro Tyr Tyr Asp Ser Met 370 375 380 Ile Gly Lys Leu Ile Cys Tyr Gly Glu Asn Arg Asp Val Ala Ile Ala 385 390 395 400 Arg Met Lys Asn Ala Leu Gln Glu Leu Ile Ile Asp Gly Ile Lys Thr 405 410 415 Asn Val Asp Leu Gln Ile Arg Ile Met Asn Asp Glu Asn Phe Gln His 420 425 430 Gly Gly Thr Asn Ile His Tyr Leu Glu Lys Lys Leu Gly Leu Gln Glu 435 440 445 Lys <210> 8 <211> 1350 <212> DNA <213> Escherichia coli strain K-12 MG1655, accC cDNA <400> 8 atgctggata aaattgttat tgccaaccgc ggcgagattg cattgcgtat tcttcgtgcc 60 tgtaaagaac tgggcatcaa gactgtcgct gtgcactcca gcgcggatcg cgatctaaaa 120 cacgtattac tggcagatga aacggtctgt attggccctg ctccgtcagt aaaaagttat 180 ctgaacatcc cggcaatcat cagcgccgct gaaatcaccg gcgcagtagc aatccatccg 240 ggttacggct tcctctccga gaacgccaac tttgccgagc aggttgaacg ctccggcttt 300 atcttcattg gcccgaaagc agaaaccatt cgcctgatgg gcgacaaagt atccgcaatc 360 gcggcgatga aaaaagcggg cgtcccttgc gtaccgggtt ctgacggccc gctgggcgac 420 gatatggata aaaaccgtgc cattgctaaa cgcattggtt atccggtgat tatcaaagcc 480 tccggcggcg gcggcggtcg cggtatgcgc gtagtgcgcg gcgacgctga actggcacaa 540 tccatctcca tgacccgtgc ggaagcgaaa gctgctttca gcaacgatat ggtttacatg 600 gagaaatacc tggaaaatcc tcgccacgtc gagattcagg tactggctga cggtcagggc 660 aacgctatct atctggcgga acgtgactgc tccatgcaac gccgccacca gaaagtggtc 720 gaagaagcgc cagcaccggg cattaccccg gaactgcgtc gctacatcgg cgaacgttgc 780 gctaaagcgt gtgttgatat cggctatcgc ggtgcaggta ctttcgagtt cctgttcgaa 840 aacggcgagt tctatttcat cgaaatgaac acccgtattc aggtagaaca cccggttaca 900 gaaatgatca ccggcgttga cctgatcaaa gaacagctgc gtatcgctgc cggtcaaccg 960 ctgtcgatca agcaagaaga agttcacgtt cgcggccatg cggtggaatg tcgtatcaac 1020 gccgaagatc cgaacacctt cctgccaagt ccgggcaaaa tcacccgttt ccacgcacct 1080 ggcggttttg gcgtacgttg ggagtctcat atctacgcgg gctacaccgt accgccgtac 1140 tatgactcaa tgatcggtaa gctgatttgc tacggtgaaa accgtgacgt ggcgattgcc 1200 cgcatgaaga atgcgctgca ggagctgatc atcgacggta tcaaaaccaa cgttgatctg 1260 cagatccgca tcatgaatga cgagaacttc cagcatggtg gcactaacat ccactatctg 1320 gagaaaaaac tcggtcttca ggaaaaataa 1350 <210> 9 <211> 309 <212> PRT <213> Escherichia coli strain K-12 MG1655, fabD <400> 9 Met Thr Gln Phe Ala Phe Val Phe Pro Gly Gln Gly Ser Gln Thr Val 1 5 10 15 Gly Met Leu Ala Asp Met Ala Ala Ser Tyr Pro Ile Val Glu Glu Thr 20 25 30 Phe Ala Glu Ala Ser Ala Ala Leu Gly Tyr Asp Leu Trp Ala Leu Thr 35 40 45 Gln Gln Gly Pro Ala Glu Glu Leu Asn Lys Thr Trp Gln Thr Gln Pro 50 55 60 Ala Leu Leu Thr Ala Ser Val Ala Leu Tyr Arg Val Trp Gln Gln Gln 65 70 75 80 Gly Gly Lys Ala Pro Ala Met Met Ala Gly His Ser Leu Gly Glu Tyr 85 90 95 Ser Ala Leu Val Cys Ala Gly Val Ile Asp Phe Ala Asp Ala Val Arg 100 105 110 Leu Val Glu Met Arg Gly Lys Phe Met Gln Glu Ala Val Pro Glu Gly 115 120 125 Thr Gly Ala Met Ala Ala Ile Ile Gly Leu Asp Asp Ala Ser Ile Ala 130 135 140 Lys Ala Cys Glu Glu Ala Ala Glu Gly Gln Val Val Ser Pro Val Asn 145 150 155 160 Phe Asn Ser Pro Gly Gln Val Val Ile Ala Gly His Lys Glu Ala Val 165 170 175 Glu Arg Ala Gly Ala Ala Cys Lys Ala Ala Gly Ala Lys Arg Ala Leu 180 185 190 Pro Leu Pro Val Ser Val Pro Ser His Cys Ala Leu Met Lys Pro Ala 195 200 205 Ala Asp Lys Leu Ala Val Glu Leu Ala Lys Ile Thr Phe Asn Ala Pro 210 215 220 Thr Val Pro Val Val Asn Asn Val Asp Val Lys Cys Glu Thr Asn Gly 225 230 235 240 Asp Ala Ile Arg Asp Ala Leu Val Arg Gln Leu Tyr Asn Pro Val Gln 245 250 255 Trp Thr Lys Ser Val Glu Tyr Met Ala Ala Gln Gly Val Glu His Leu 260 265 270 Tyr Glu Val Gly Pro Gly Lys Val Leu Thr Gly Leu Thr Lys Arg Ile 275 280 285 Val Asp Thr Leu Thr Ala Ser Ala Leu Asn Glu Pro Ser Ala Met Ala 290 295 300 Ala Ala Leu Glu Leu 305 <210> 10 <211> 930 <212> DNA <213> Escherichia coli strain K-12 MG1655, fabD cDNA <400> 10 atgacgcaat ttgcatttgt gttccctgga cagggttctc aaaccgttgg aatgctggct 60 gatatggcgg cgagctatcc aattgtcgaa gaaacgtttg ctgaagcttc tgcggcgctg 120 ggctacgacc tgtgggcgct gacccagcag gggccagctg aagaactgaa taaaacctgg 180 caaactcagc ctgcgctgtt gactgcatct gttgcgctgt atcgcgtatg gcagcagcag 240 ggcggtaaag caccggcaat gatggccggt cacagcctgg gggaatactc cgcgctggtt 300 tgcgctggtg tgattgattt cgctgatgcg gtgcgtctgg ttgagatgcg cggcaagttc 360 atgcaagaag ccgtaccgga aggcacgggc gctatggcgg caatcatcgg tctggatgat 420 gcgtctattg cgaaagcgtg tgaagaagct gcagaaggtc aggtcgtttc tccggtaaac 480 tttaactctc cgggacaggt ggttattgcc ggtcataaag aagcggttga gcgtgctggc 540 gctgcctgta aagcggcggg cgcaaaacgc gcgctgccgt taccagtgag cgtaccgtct 600 cactgtgcgc tgatgaaacc agcagccgac aaactggcag tagaattagc gaaaatcacc 660 tttaacgcac caacagttcc tgttgtgaat aacgttgatg tgaaatgcga aaccaatggt 720 gatgccatcc gtgacgcact ggtacgtcag ttgtataacc cggttcagtg gacgaagtct 780 gttgagtaca tggcagcgca aggcgtagaa catctctatg aagtcggccc gggcaaagtg 840 cttactggcc tgacgaaacg cattgtcgac accctgaccg cctcggcgct gaacgaacct 900 tcagcgatgg cagcggcgct cgagctttaa 930 <210> 11 <211> 4176 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence vector <400> 11 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc 60 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc 120 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc 180 tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga 240 taacaatttc acacaggaaa cagaccatgg aattcgagct cggtacccgg ggatcctcta 300 gagtcgacct gcaggcatgc aagcttggct gttttggcgg atgagagaag attttcagcc 360 tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca 420 gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg 480 atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga 540 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 600 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg 660 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg 720 acggatggcc tttttgcgtt tctacaaact ctttttgttt atttttctaa atacattcaa 780 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 840 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 900 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 960 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 1020 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 1080 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 1140 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 1200 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 1260 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 1320 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 1380 cgatgcctac agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 1440 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 1500 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 1560 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 1620 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 1680 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1740 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 1800 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 1860 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 1920 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1980 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 2040 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 2100 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 2160 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 2220 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 2280 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 2340 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 2400 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 2460 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 2520 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 2580 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 2640 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 2700 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc 2760 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc 2820 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 2880 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa 2940 ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca ccatcgaatg gtgcaaaacc 3000 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa 3060 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc 3120 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg 3180 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg 3240 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg 3300 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc 3360 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg 3420 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat 3480 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc 3540 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc 3600 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat 3660 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc 3720 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg 3780 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg 3840 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat 3900 atcccgccgt caaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac 3960 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca 4020 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 4080 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg 4140 caacgcaatt aatgtgagtt agcgcgaatt gatctg 4176 <110> Industry-University Cooperation Foundation Sogang University <120> Transformed Escherichia coli for Over-expression of Fatty Acid          Biosynthesis Pathway and Method of Preparing the Same <160> 11 <170> Kopatentin 1.71 <210> 1 <211> 317 <212> PRT <213> Escherichia coli strain K-12 MG1655, fabH <400> 1 Met Tyr Thr Lys Ile Ile Gly Thr Gly Ser Tyr Leu Pro Glu Gln Val   1 5 10 15 Arg Thr Asn Ala Asp Leu Glu Lys Met Val Asp Thr Ser Asp Glu Trp              20 25 30 Ile Val Thr Arg Thr Gly Ile Arg Glu Arg His Ile Ala Ala Pro Asn          35 40 45 Glu Thr Val Ser Thr Met Gly Phe Glu Ala Ala Thr Arg Ala Ile Glu      50 55 60 Met Ala Gly Ile Glu Lys Asp Gln Ile Gly Leu Ile Val Val Ala Thr  65 70 75 80 Thr Ser Ala Thr His Ala Phe Pro Ser Ala Ala Cys Gln Ile Gln Ser                  85 90 95 Met Leu Gly Ile Lys Gly Cys Pro Ala Phe Asp Val Ala Ala Ala Cys             100 105 110 Ala Gly Phe Thr Tyr Ala Leu Ser Val Ala Asp Gln Tyr Val Lys Ser         115 120 125 Gly Ala Val Lys Tyr Ala Leu Val Val Gly Ser Asp Val Leu Ala Arg     130 135 140 Thr Cys Asp Pro Thr Asp Arg Gly Thr Ile Ile Ile Phe Gly Asp Gly 145 150 155 160 Ala Gly Ala Ala Val Leu Ala Ala Ser Glu Glu Pro Gly Ile Ile Ser                 165 170 175 Thr His Leu His Ala Asp Gly Ser Tyr Gly Glu Leu Leu Thr Leu Pro             180 185 190 Asn Ala Asp Arg Val Asn Pro Glu Asn Ser Ile His Leu Thr Met Ala         195 200 205 Gly Asn Glu Val Phe Lys Val Ala Val Thr Glu Leu Ala His Ile Val     210 215 220 Asp Glu Thr Leu Ala Ala Asn Asn Leu Asp Arg Ser Gln Leu Asp Trp 225 230 235 240 Leu Val Pro His Gln Ala Asn Leu Arg Ile Ile Ser Ala Thr Ala Lys                 245 250 255 Lys Leu Gly Met Ser Met Asp Asn Val Val Val Thr Leu Asp Arg His             260 265 270 Gly Asn Thr Ser Ala Ala Ser Val Pro Cys Ala Leu Asp Glu Ala Val         275 280 285 Arg Asp Gly Arg Ile Lys Pro Gly Gln Leu Val Leu Leu Glu Ala Phe     290 295 300 Gly Gly Gly Phe Thr Trp Gly Ser Ala Leu Val Arg Phe 305 310 315 <210> 2 <211> 954 <212> DNA <213> Escherichia coli strain K-12 MG1655, fabH cDNA <400> 2 atgtatacga agattattgg tactggcagc tatctgcccg aacaagtgcg gacaaacgcc 60 gatttggaaa aaatggtgga cacctctgac gagtggattg tcactcgtac cggtatccgc 120 gaacgccaca ttgccgcgcc aaacgaaacc gtttcaacca tgggctttga agcggcgaca 180 cgcgcaattg agatggcggg cattgagaaa gaccagattg gcctgatcgt tgtggcaacg 240 acttctgcta cgcacgcttt cccgagcgca gcttgtcaga ttcaaagcat gttgggcatt 300 aaaggttgcc cggcatttga cgttgcagca gcctgcgcag gtttcaccta tgcattaagc 360 gtagccgatc aatacgtgaa atctggggcg gtgaagtatg ctctggtcgt cggttccgat 420 gtactggcgc gcacctgcga tccaaccgat cgtgggacta ttattatttt tggcgatggc 480 gcgggcgctg cggtgctggc tgcctctgaa gagccgggaa tcatttccac ccatctgcat 540 gccgacggta gttatggtga attgctgacg ctgccaaacg ccgaccgcgt gaatccagag 600 aattcaattc atctgacgat ggcgggcaac gaagtcttca aggttgcggt aacggaactg 660 gcgcacatcg ttgatgagac gctggcggcg aataatcttg accgttctca actggactgg 720 ctggttccgc atcaggctaa cctgcgtatt atcagtgcaa cggcgaaaaa actcggtatg 780 tctatggata atgtcgtggt gacgctggat cgccacggta atacctctgc ggcctctgtc 840 ccgtgcgcgc tggatgaagc tgtacgcgac gggcgcatta agccggggca gttggttctg 900 cttgaagcct ttggcggtgg attcacctgg ggctccgcgc tggttcgttt ctag 954 <210> 3 <211> 319 <212> PRT <213> Escherichia coli strain K-12 MG1655, accA <400> 3 Met Ser Leu Asn Phe Leu Asp Phe Glu Gln Pro Ile Ala Glu Leu Glu   1 5 10 15 Ala Lys Ile Asp Ser Leu Thr Ala Val Ser Arg Gln Asp Glu Lys Leu              20 25 30 Asp Ile Asn Ile Asp Glu Glu Val His Arg Leu Arg Glu Lys Ser Val          35 40 45 Glu Leu Thr Arg Lys Ile Phe Ala Asp Leu Gly Ala Trp Gln Ile Ala      50 55 60 Gln Leu Ala Arg His Pro Gln Arg Pro Tyr Thr Leu Asp Tyr Val Arg  65 70 75 80 Leu Ala Phe Asp Glu Phe Asp Glu Leu Ala Gly Asp Arg Ala Tyr Ala                  85 90 95 Asp Asp Lys Ala Ile Val Gly Gly Ile Ala Arg Leu Asp Gly Arg Pro             100 105 110 Val Met Ile Ile Gly His Gln Lys Gly Arg Glu Thr Lys Glu Lys Ile         115 120 125 Arg Arg Asn Phe Gly Met Pro Ala Pro Glu Gly Tyr Arg Lys Ala Leu     130 135 140 Arg Leu Met Gln Met Ala Glu Arg Phe Lys Met Pro Ile Ile Thr Phe 145 150 155 160 Ile Asp Thr Pro Gly Ala Tyr Pro Gly Val Gly Ala Glu Glu Arg Gly                 165 170 175 Gln Ser Glu Ala Ile Ala Arg Asn Leu Arg Glu Met Ser Arg Leu Gly             180 185 190 Val Pro Val Val Cys Thr Val Ile Gly Glu Gly Gly Ser Gly Gly Ala         195 200 205 Leu Ala Ile Gly Val Gly Asp Lys Val Asn Met Leu Gln Tyr Ser Thr     210 215 220 Tyr Ser Val Ile Ser Pro Glu Gly Cys Ala Ser Ile Leu Trp Lys Ser 225 230 235 240 Ala Asp Lys Ala Pro Leu Ala Ala Glu Ala Met Gly Ile Ile Ala Pro                 245 250 255 Arg Leu Lys Glu Leu Lys Leu Ile Asp Ser Ile Ile Pro Glu Pro Leu             260 265 270 Gly Gly Ala His Arg Asn Pro Glu Ala Met Ala Ala Ser Leu Lys Ala         275 280 285 Gln Leu Leu Ala Asp Leu Ala Asp Leu Asp Val Leu Ser Thr Glu Asp     290 295 300 Leu Lys Asn Arg Arg Tyr Gln Arg Leu Met Ser Tyr Gly Tyr Ala 305 310 315 <210> 4 <211> 960 <212> DNA <213> Escherichia coli strain K-12 MG1655, accA cDNA <400> 4 atgagtctga atttccttga ttttgaacag ccgattgcag agctggaagc gaaaatcgat 60 tctctgactg cggttagccg tcaggatgag aaactggata ttaacatcga tgaagaagtg 120 catcgtctgc gtgaaaaaag cgtagaactg acacgtaaaa tcttcgccga tctcggtgca 180 tggcagattg cgcaactggc acgccatcca cagcgtcctt ataccctgga ttacgttcgc 240 ctggcatttg atgaatttga cgaactggct ggcgaccgcg cgtatgcaga cgataaagct 300 atcgtcggtg gtatcgcccg tctcgatggt cgtccggtga tgatcattgg tcatcaaaaa 360 ggtcgtgaaa ccaaagaaaa aattcgccgt aactttggta tgccagcgcc agaaggttac 420 cgcaaagcac tgcgtctgat gcaaatggct gaacgcttta agatgcctat catcaccttt 480 atcgacaccc cgggggctta tcctggcgtg ggcgcagaag agcgtggtca gtctgaagcc 540 attgcacgca acctgcgtga aatgtctcgc ctcggcgtac cggtagtttg tacggttatc 600 ggtgaaggtg gttctggcgg tgcgctggcg attggcgtgg gcgataaagt gaatatgctg 660 caatacagca cctattccgt tatctcgccg gaaggttgtg cgtccattct gtggaagagc 720 gccgacaaag cgccgctggc ggctgaagcg atgggtatca ttgctccgcg tctgaaagaa 780 ctgaaactga tcgactccat catcccggaa ccactgggtg gtgctcaccg taacccggaa 840 gcgatggcgg catcgttgaa agcgcaactg ctggcggatc tggccgatct cgacgtgtta 900 agcactgaag atttaaaaaa tcgtcgttat cagcgcctga tgagctacgg ttacgcgtaa 960                                                                          960 <210> 5 <211> 156 <212> PRT <213> Escherichia coli strain K-12 MG1655, accB <400> 5 Met Asp Ile Arg Lys Ile Lys Lys Leu Ile Glu Leu Val Glu Glu Ser   1 5 10 15 Gly Ile Ser Glu Leu Glu Ile Ser Glu Gly Glu Glu Ser Val Arg Ile              20 25 30 Ser Arg Ala Ala Pro Ala Ala Ser Phe Pro Val Met Gln Gln Ala Tyr          35 40 45 Ala Ala Pro Met Met Gln Gln Pro Ala Gln Ser Asn Ala Ala Ala Pro      50 55 60 Ala Thr Val Pro Ser Met Glu Ala Pro Ala Ala Ala Glu Ile Ser Gly  65 70 75 80 His Ile Val Arg Ser Pro Met Val Gly Thr Phe Tyr Arg Thr Pro Ser                  85 90 95 Pro Asp Ala Lys Ala Phe Ile Glu Val Gly Gln Lys Val Asn Val Gly             100 105 110 Asp Thr Leu Cys Ile Val Glu Ala Met Lys Met Met Asn Gln Ile Glu         115 120 125 Ala Asp Lys Ser Gly Thr Val Lys Ala Ile Leu Val Glu Ser Gly Gln     130 135 140 Pro Val Glu Phe Asp Glu Pro Leu Val Val Ile Glu 145 150 155 <210> 6 <211> 471 <212> DNA <213> Escherichia coli strain K-12 MG1655, accB cDNA <400> 6 atggatattc gtaagattaa aaaactgatc gagctggttg aagaatcagg catctccgaa 60 ctggaaattt ctgaaggcga agagtcagta cgcattagcc gtgcagctcc tgccgcaagt 120 ttccctgtga tgcaacaagc ttacgctgca ccaatgatgc agcagccagc tcaatctaac 180 gcagccgctc cggcgaccgt tccttccatg gaagcgccag cagcagcgga aatcagtggt 240 cacatcgtac gttccccgat ggttggtact ttctaccgca ccccaagccc ggacgcaaaa 300 gcgttcatcg aagtgggtca gaaagtcaac gtgggcgata ccctgtgcat cgttgaagcc 360 atgaaaatga tgaaccagat cgaagcggac aaatccggta ccgtgaaagc aattctggtc 420 gaaagtggac aaccggtaga atttgacgag ccgctggtcg tcatcgagta a 471 <210> 7 <211> 449 <212> PRT <213> Escherichia coli strain K-12 MG1655, accC <400> 7 Met Leu Asp Lys Ile Val Ile Ala Asn Arg Gly Glu Ile Ala Leu Arg   1 5 10 15 Ile Leu Arg Ala Cys Lys Glu Leu Gly Ile Lys Thr Val Ala Val His              20 25 30 Ser Ser Ala Asp Arg Asp Leu Lys His Val Leu Leu Ala Asp Glu Thr          35 40 45 Val Cys Ile Gly Pro Ala Pro Ser Val Lys Ser Tyr Leu Asn Ile Pro      50 55 60 Ala Ile Ile Ser Ala Ala Glu Ile Thr Gly Ala Val Ala Ile His Pro  65 70 75 80 Gly Tyr Gly Phe Leu Ser Glu Asn Ala Asn Phe Ala Glu Gln Val Glu                  85 90 95 Arg Ser Gly Phe Ile Phe Ile Gly Pro Lys Ala Glu Thr Ile Arg Leu             100 105 110 Met Gly Asp Lys Val Ser Ala Ile Ala Ala Met Lys Lys Ala Gly Val         115 120 125 Pro Cys Val Pro Gly Ser Asp Gly Pro Leu Gly Asp Asp Met Asp Lys     130 135 140 Asn Arg Ala Ile Ala Lys Arg Ile Gly Tyr Pro Val Ile Ile Lys Ala 145 150 155 160 Ser Gly Gly Gly Gly Gly Arg Gly Met Arg Val Val Arg Gly Asp Ala                 165 170 175 Glu Leu Ala Gln Ser Ile Ser Met Thr Arg Ala Glu Ala Lys Ala Ala             180 185 190 Phe Ser Asn Asp Met Val Tyr Met Glu Lys Tyr Leu Glu Asn Pro Arg         195 200 205 His Val Glu Ile Gln Val Leu Ala Asp Gly Gln Gly Asn Ala Ile Tyr     210 215 220 Leu Ala Glu Arg Asp Cys Ser Met Gln Arg Arg His Gln Lys Val Val 225 230 235 240 Glu Glu Ala Pro Ala Pro Gly Ile Thr Pro Glu Leu Arg Arg Tyr Ile                 245 250 255 Gly Glu Arg Cys Ala Lys Ala Cys Val Asp Ile Gly Tyr Arg Gly Ala             260 265 270 Gly Thr Phe Glu Phe Leu Phe Glu Asn Gly Glu Phe Tyr Phe Ile Glu         275 280 285 Met Asn Thr Arg Ile Gln Val Glu His Pro Val Thr Glu Met Ile Thr     290 295 300 Gly Val Asp Leu Ile Lys Glu Gln Leu Arg Ile Ala Ala Gly Gln Pro 305 310 315 320 Leu Ser Ile Lys Gln Glu Glu Val His Val Arg Gly His Ala Val Glu                 325 330 335 Cys Arg Ile Asn Ala Glu Asp Pro Asn Thr Phe Leu Pro Ser Pro Gly             340 345 350 Lys Ile Thr Arg Phe His Ala Pro Gly Gly Phe Gly Val Arg Trp Glu         355 360 365 Ser His Ile Tyr Ala Gly Tyr Thr Val Pro Pro Tyr Tyr Asp Ser Met     370 375 380 Ile Gly Lys Leu Ile Cys Tyr Gly Glu Asn Arg Asp Val Ala Ile Ala 385 390 395 400 Arg Met Lys Asn Ala Leu Gln Glu Leu Ile Ile Asp Gly Ile Lys Thr                 405 410 415 Asn Val Asp Leu Gln Ile Arg Ile Met Asn Asp Glu Asn Phe Gln His             420 425 430 Gly Gly Thr Asn Ile His Tyr Leu Glu Lys Lys Leu Gly Leu Gln Glu         435 440 445 Lys     <210> 8 <211> 1350 <212> DNA <213> Escherichia coli strain K-12 MG1655, accC cDNA <400> 8 atgctggata aaattgttat tgccaaccgc ggcgagattg cattgcgtat tcttcgtgcc 60 tgtaaagaac tgggcatcaa gactgtcgct gtgcactcca gcgcggatcg cgatctaaaa 120 cacgtattac tggcagatga aacggtctgt attggccctg ctccgtcagt aaaaagttat 180 ctgaacatcc cggcaatcat cagcgccgct gaaatcaccg gcgcagtagc aatccatccg 240 ggttacggct tcctctccga gaacgccaac tttgccgagc aggttgaacg ctccggcttt 300 atcttcattg gcccgaaagc agaaaccatt cgcctgatgg gcgacaaagt atccgcaatc 360 gcggcgatga aaaaagcggg cgtcccttgc gtaccgggtt ctgacggccc gctgggcgac 420 gatatggata aaaaccgtgc cattgctaaa cgcattggtt atccggtgat tatcaaagcc 480 tccggcggcg gcggcggtcg cggtatgcgc gtagtgcgcg gcgacgctga actggcacaa 540 tccatctcca tgacccgtgc ggaagcgaaa gctgctttca gcaacgatat ggtttacatg 600 gagaaatacc tggaaaatcc tcgccacgtc gagattcagg tactggctga cggtcagggc 660 aacgctatct atctggcgga acgtgactgc tccatgcaac gccgccacca gaaagtggtc 720 gaagaagcgc cagcaccggg cattaccccg gaactgcgtc gctacatcgg cgaacgttgc 780 gctaaagcgt gtgttgatat cggctatcgc ggtgcaggta ctttcgagtt cctgttcgaa 840 aacggcgagt tctatttcat cgaaatgaac acccgtattc aggtagaaca cccggttaca 900 gaaatgatca ccggcgttga cctgatcaaa gaacagctgc gtatcgctgc cggtcaaccg 960 ctgtcgatca agcaagaaga agttcacgtt cgcggccatg cggtggaatg tcgtatcaac 1020 gccgaagatc cgaacacctt cctgccaagt ccgggcaaaa tcacccgttt ccacgcacct 1080 ggcggttttg gcgtacgttg ggagtctcat atctacgcgg gctacaccgt accgccgtac 1140 tatgactcaa tgatcggtaa gctgatttgc tacggtgaaa accgtgacgt ggcgattgcc 1200 cgcatgaaga atgcgctgca ggagctgatc atcgacggta tcaaaaccaa cgttgatctg 1260 cagatccgca tcatgaatga cgagaacttc cagcatggtg gcactaacat ccactatctg 1320 gagaaaaaac tcggtcttca ggaaaaataa 1350 <210> 9 <211> 309 <212> PRT <213> Escherichia coli strain K-12 MG1655, fabD <400> 9 Met Thr Gln Phe Ala Phe Val Phe Pro Gly Gln Gly Ser Gln Thr Val   1 5 10 15 Gly Met Leu Ala Asp Met Ala Ala Ser Tyr Pro Ile Val Glu Glu Thr              20 25 30 Phe Ala Glu Ala Ser Ala Ala Leu Gly Tyr Asp Leu Trp Ala Leu Thr          35 40 45 Gln Gln Gly Pro Ala Glu Glu Leu Asn Lys Thr Trp Gln Thr Gln Pro      50 55 60 Ala Leu Leu Thr Ala Ser Val Ala Leu Tyr Arg Val Trp Gln Gln Gln  65 70 75 80 Gly Gly Lys Ala Pro Ala Met Met Ala Gly His Ser Leu Gly Glu Tyr                  85 90 95 Ser Ala Leu Val Cys Ala Gly Val Ile Asp Phe Ala Asp Ala Val Arg             100 105 110 Leu Val Glu Met Arg Gly Lys Phe Met Gln Glu Ala Val Pro Glu Gly         115 120 125 Thr Gly Ala Met Ala Ala Ile Ile Gly Leu Asp Asp Ala Ser Ile Ala     130 135 140 Lys Ala Cys Glu Glu Ala Ala Glu Gly Gln Val Val Ser Pro Val Asn 145 150 155 160 Phe Asn Ser Pro Gly Gln Val Val Ile Ala Gly His Lys Glu Ala Val                 165 170 175 Glu Arg Ala Gly Ala Ala Cys Lys Ala Ala Gly Ala Lys Arg Ala Leu             180 185 190 Pro Leu Pro Val Ser Val Pro Ser His Cys Ala Leu Met Lys Pro Ala         195 200 205 Ala Asp Lys Leu Ala Val Glu Leu Ala Lys Ile Thr Phe Asn Ala Pro     210 215 220 Thr Val Pro Val Val Asn Asn Val Asp Val Lys Cys Glu Thr Asn Gly 225 230 235 240 Asp Ala Ile Arg Asp Ala Leu Val Arg Gln Leu Tyr Asn Pro Val Gln                 245 250 255 Trp Thr Lys Ser Val Glu Tyr Met Ala Ala Gln Gly Val Glu His Leu             260 265 270 Tyr Glu Val Gly Pro Gly Lys Val Leu Thr Gly Leu Thr Lys Arg Ile         275 280 285 Val Asp Thr Leu Thr Ala Ser Ala Leu Asn Glu Pro Ser Ala Met Ala     290 295 300 Ala Ala Leu Glu Leu 305 <210> 10 <211> 930 <212> DNA <213> Escherichia coli strain K-12 MG1655, fabD cDNA <400> 10 atgacgcaat ttgcatttgt gttccctgga cagggttctc aaaccgttgg aatgctggct 60 gatatggcgg cgagctatcc aattgtcgaa gaaacgtttg ctgaagcttc tgcggcgctg 120 ggctacgacc tgtgggcgct gacccagcag gggccagctg aagaactgaa taaaacctgg 180 caaactcagc ctgcgctgtt gactgcatct gttgcgctgt atcgcgtatg gcagcagcag 240 ggcggtaaag caccggcaat gatggccggt cacagcctgg gggaatactc cgcgctggtt 300 tgcgctggtg tgattgattt cgctgatgcg gtgcgtctgg ttgagatgcg cggcaagttc 360 atgcaagaag ccgtaccgga aggcacgggc gctatggcgg caatcatcgg tctggatgat 420 gcgtctattg cgaaagcgtg tgaagaagct gcagaaggtc aggtcgtttc tccggtaaac 480 tttaactctc cgggacaggt ggttattgcc ggtcataaag aagcggttga gcgtgctggc 540 gctgcctgta aagcggcggg cgcaaaacgc gcgctgccgt taccagtgag cgtaccgtct 600 cactgtgcgc tgatgaaacc agcagccgac aaactggcag tagaattagc gaaaatcacc 660 tttaacgcac caacagttcc tgttgtgaat aacgttgatg tgaaatgcga aaccaatggt 720 gatgccatcc gtgacgcact ggtacgtcag ttgtataacc cggttcagtg gacgaagtct 780 gttgagtaca tggcagcgca aggcgtagaa catctctatg aagtcggccc gggcaaagtg 840 cttactggcc tgacgaaacg cattgtcgac accctgaccg cctcggcgct gaacgaacct 900 tcagcgatgg cagcggcgct cgagctttaa 930 <210> 11 <211> 4176 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence vector <400> 11 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc 60 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc 120 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc 180 tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga 240 taacaatttc acacaggaaa cagaccatgg aattcgagct cggtacccgg ggatcctcta 300 gagtcgacct gcaggcatgc aagcttggct gttttggcgg atgagagaag attttcagcc 360 tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca 420 gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg 480 atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga 540 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 600 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg 660 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg 720 acggatggcc tttttgcgtt tctacaaact ctttttgttt atttttctaa atacattcaa 780 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 840 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 900 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 960 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 1020 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 1080 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 1140 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 1200 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 1260 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 1320 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 1380 cgatgcctac agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 1440 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 1500 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 1560 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 1620 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 1680 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1740 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 1800 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 1860 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 1920 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1980 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 2040 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 2100 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 2160 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 2220 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 2280 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 2340 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 2400 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 2460 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 2520 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 2580 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 2640 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 2700 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc 2760 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc 2820 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 2880 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa 2940 ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca ccatcgaatg gtgcaaaacc 3000 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa 3060 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc 3120 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg 3180 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg 3240 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg 3300 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc 3360 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg 3420 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat 3480 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc 3540 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc 3600 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat 3660 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc 3720 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg 3780 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg 3840 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat 3900 atcccgccgt caaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac 3960 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca 4020 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 4080 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg 4140 caacgcaatt aatgtgagtt agcgcgaatt gatctg 4176

Claims (10)

다음을 포함하는 발현벡터로 공동형질전환된(cotransformed) 지방산 과발현용 대장균:
(a) β-케토아실-[아실운반단백질] 신타아제 III(β-Ketoacyl-Acyl Carrier Protein Synthase III)를 코딩하는 뉴클레오타이드 서열; 및
(b) 아세틸-CoA 카복실라제 카복시트랜스퍼라제 서브유닛 알파(acetyl-CoA carboxylase, carboxytransferase, alpha subunit)를 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복시 운반단백질 서브유닛(acetyl CoA carboxylase, biotin carboxyl carrier protein(BCCP) subunit)을 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복실라제 서브유닛(acetyl-CoA carboxylase, biotin carboxylase subunit)을 코딩하는 뉴클레오타이드 서열 및 말로닐-CoA-[아실운반단백질] 트랜스아실라제(malonyl-CoA-[acyl-carrier-protein] transacylase)를 코딩하는 뉴클레오타이드 서열.
Escherichia coli coexpressed with an expression vector comprising:
(a) a nucleotide sequence encoding a β-ketoacyl- [acyl carrier protein] synthase III (β-Ketoacyl-Acyl Carrier Protein Synthase III); And
(b) nucleotide sequence encoding acetyl-CoA carboxylase carboxycitase transferase subunit alpha (acetyl-CoA carboxylase, carboxytransferase, alpha subunit), acetyl-CoA carboxylase biotin carboxy carrier subunit (acetyl CoA carboxylase, biotin carboxyl carrier nucleotide sequence encoding protein (BCCP) subunit), nucleotide sequence encoding acetyl-CoA carboxylase subunit (acetyl-CoA carboxylase, biotin carboxylase subunit) and malonyl-CoA- [acyl carrier protein] transacyl Nucleotide sequence encoding a lase (malonyl-CoA- [acyl-carrier-protein] transacylase).
삭제delete 제 1 항에 있어서, 상기 β-케토아실-[아실운반단백질] 신타아제 III는 서열목록 제1서열로 표시되는 아미노산 서열로 이루어진 것을 특징으로 하는 지방산 과발현용 대장균.
The E. coli for fatty acid overexpression according to claim 1, wherein the β-ketoacyl- [acyl carrier protein] synthase III is composed of an amino acid sequence represented by SEQ ID NO: 1.
제 1 항에 있어서, 상기 아세틸-CoA 카복실라제 카복시트랜스퍼라제 서브유닛 알파는 서열목록 제3서열로 표시되는 아미노산 서열로 이루어지고, 아세틸-CoA 카복실라제 바이오틴 카복시 운반단백질 서브유닛은 서열목록 제5서열로 표시되는 아미노산 서열로 이루어지고, 아세틸-CoA 카복실라제 바이오틴 카복실라제 서브유닛은 서열목록 제7서열로 표시되는 아미노산 서열로 이루어지고, 말로닐-CoA-[아실운반단백질] 트랜스아실라제는 서열목록 제9서열로 표시되는 아미노산 서열로 이루어진 것을 특징으로 하는 지방산 과발현용 대장균.
According to claim 1, wherein the acetyl-CoA carboxylase carboxysheet transferase subunit alpha consists of an amino acid sequence represented by SEQ ID NO: 3 sequence, acetyl-CoA carboxylase biotin carboxy carrier protein subunit is SEQ ID NO: 5 An acetyl-CoA carboxylase biotin carboxylase subunit, consisting of an amino acid sequence represented by SEQ ID NO: 7, and a malonyl-CoA- [acyl carrier protein] transacylase E. coli for fatty acid overexpression, characterized in that consisting of the amino acid sequence represented by the ninth sequence.
제 1 항에 있어서, 상기 β-케토아실-[아실운반단백질] 신타아제 III를 코딩하는 뉴클레오타이드 서열(a)는 서열목록 제2서열로 표시되는 것을 특징으로 하는 지방산 과발현용 대장균.
[Claim 2] The Escherichia coli for fatty acid overexpression according to claim 1, wherein the nucleotide sequence (a) encoding β-ketoacyl- [acyl carrier protein] synthase III is represented by SEQ ID NO: 2.
제 1 항에 있어서, 상기 발현 벡터는 서열목록 제11서열로 표시되는 뉴클레오타이드 서열을 포함하는 백본벡터에 상기 뉴클레오타이드 서열 (a) 및 상기 뉴클레오타이드 서열 (b)를 도입한 것을 특징으로 하는 지방산 과발현용 대장균.
According to claim 1, wherein the expression vector E. coli for fatty acid overexpression, characterized in that the nucleotide sequence (a) and the nucleotide sequence (b) introduced into the backbone vector comprising the nucleotide sequence represented by SEQ ID NO: 11 .
다음 단계를 포함하는 지방산 과발현용 형질전환 대장균의 제조방법:
(a) β-케토아실-[아실운반단백질] 신타아제 III를 코딩하는 뉴클레오타이드 서열; 및
아세틸-CoA 카복실라제 카복시트랜스퍼라제 서브유닛 알파를 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복시 운반단백질 서브유닛을 코딩하는 뉴클레오타이드 서열, 아세틸-CoA 카복실라제 바이오틴 카복실라제 서브유닛을 코딩하는 뉴클레오타이드 서열 및 말로닐-CoA-[아실운반단백질] 트랜스아실라제를 코딩하는 뉴클레오타이드 서열
을 발현벡터에 삽입시키는 단계; 및
(b) 상기 뉴클레오타이드 서열이 삽입된 발현벡터로 대장균을 형질전환시키는 단계.
Method for producing a transforming Escherichia coli for fatty acid overexpression comprising the following steps:
(a) a nucleotide sequence encoding β-ketoacyl- [acyl carrier protein] synthase III; And
Nucleotide sequence encoding acetyl-CoA carboxylase carboxytransferase subunit alpha, nucleotide sequence encoding acetyl-CoA carboxylase biotin carboxy carrier protein subunit, nucleotide sequence encoding acetyl-CoA carboxylase biotin carboxylase subunit Nucleotide sequence encoding a malonyl-CoA- [acyl carrier protein] transacylase
Inserting into the expression vector; And
(b) transforming Escherichia coli with the expression vector into which the nucleotide sequence is inserted.
삭제delete 제 7 항에 있어서, 상기 대장균을 형질전환시키는 단계(b)는 전기충격 방법으로 수행되는 것을 특징으로 하는 지방산 과발현용 형질전환 대장균의 제조방법.
According to claim 7, wherein the step of transforming Escherichia coli (b) is a method for producing a transformed Escherichia coli for fatty acid overexpression, characterized in that carried out by the electroshock method.
(a) 제 1 항 및 제 3 항 내지 제 6 항 중 어느 한 항의 대장균을 배양하여 지방산을 생합성 하는 단계; 및 (b) 상기 생합성된 지방산을 수득하는 단계를 포함하는 지방산 생합성 방법.(a) culturing the E. coli of any one of claims 1 and 3 to 6 to biosynthesize fatty acids; And (b) obtaining the biosynthesized fatty acid.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150139310A (en) * 2014-06-03 2015-12-11 서강대학교산학협력단 Novel strain secreting fatty acids by phospholipase and method for producing fatty acids using it

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Appl. Environ. Microbiol. Vol.67(1):426-433 (2001. 1.) *
Appl. Environ. Microbiol. Vol.67(1):426-433 (2001. 1.)*
J. Bacteriol. Vol.175(21):6881-6889 (1993. 11.) *
J. Bacteriol. Vol.177(23):7003-7006 (1995. 12.) *

Cited By (2)

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KR101598348B1 (en) 2014-06-03 2016-02-29 서강대학교산학협력단 Novel strain secreting fatty acids by phospholipase and method for producing fatty acids using it

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