KR101957060B1 - Transformed synechococcus elongates having capability of acetone from carbon dioxide and method for producing acetone using the same - Google Patents

Transformed synechococcus elongates having capability of acetone from carbon dioxide and method for producing acetone using the same Download PDF

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KR101957060B1
KR101957060B1 KR1020170172856A KR20170172856A KR101957060B1 KR 101957060 B1 KR101957060 B1 KR 101957060B1 KR 1020170172856 A KR1020170172856 A KR 1020170172856A KR 20170172856 A KR20170172856 A KR 20170172856A KR 101957060 B1 KR101957060 B1 KR 101957060B1
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우한민
이현정
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성균관대학교산학협력단
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Abstract

The present invention relates to a transformed Synechococcus elongatus strain capable of mass-producing acetone from carbon dioxide through a by-pass route, and a method of producing acetone using the same. The transformed Synechococcus elongatus strain of the present invention is economical by using light and carbon dioxide present in the atmosphere as a carbon source; is eco-friendly being used to remove or reduce carbon dioxide in the atmosphere using microorganisms; and can be a basic model for increasing acetone production for industrialization. A method of producing an acetone-producing strain using the by-pass of the present invention can be applied to other strains, and acetone is also useful in a method for producing isopropanol materials as a precursor of isopropanol. The Synechococcus elongatus strain comprises a pyruvate decarboxylase enzyme (pdc) gene and aldehyde dehydrogenase enzyme (ald6) gene.

Description

이산화탄소로부터의 아세톤 생산능을 갖는 형질전환된 시네코코커스 일롱게투스 균주 및 이를 이용한 아세톤 생산방법{TRANSFORMED SYNECHOCOCCUS ELONGATES HAVING CAPABILITY OF ACETONE FROM CARBON DIOXIDE AND METHOD FOR PRODUCING ACETONE USING THE SAME}TECHNICAL FIELD The present invention relates to a transformed Cynekocus Iylongutus strain having an ability to produce acetone from carbon dioxide and a method for producing acetone using the transformed Cynecococcus Iylonggetus strain.

본 발명은 우회경로를 통해 이산화탄소로부터 아세톤을 대량 생산할 수 있는 형질전환된 시네코코커스 일롱게투스 균주, 이를 이용한 아세톤 생산 방법에 관한 것이다. The present invention relates to a transformed Cynekocors Iylongutus strain capable of mass production of acetone from carbon dioxide through a bypass route, and a method for producing acetone using the same.

아세톤(Acetone)은 화학식 CH3COCH3. 분자량은 58.08으로, 향기가 있는 무색의 액체이다. 물에 잘 녹으며, 유기용매로서 다른 유기물질과도 잘 섞인다. 그래서 물로 세척이 되지 않는 물질을 아세톤으로 처리하면 쉽게 세척할 수 있어 일상 생활에 많이 사용된다. Acetone has the formula CH 3 COCH 3 . The molecular weight is 58.08, a colorless liquid with a fragrance. It dissolves well in water and as an organic solvent it mixes well with other organic materials. Therefore, it can be easily cleaned by treating acetone which can not be washed with water, so it is widely used in daily life.

또한, 아세톤은 탄소가 산소와 이중결합을 하고 있고, 그 탄소가 또 다른 두 개의 결합자리를 가지고 있어 두 알킬기(alkyl group)들과 결합하고 있는 경우, 이 물질을 케톤(ketone)이라 한다. 케톤의 가장 간단한 형태로, 양쪽에 하나씩 총 두(di)개의 메틸기(methyl)와 결합하고 있기 때문에 다이메틸케톤(dimethylketone)이라고 부르기도 한다. 또 탄소가 총 세 개인 프로페인(propane)에서 변형된 케톤(ketone)이라는 의미에서 프로판온(propanone)이라고 부르기도 한다.Acetone is also called a ketone when carbon has a double bond with oxygen and the carbon has two other bonding sites and is bonded to two alkyl groups. It is the simplest form of ketone and is sometimes called dimethylketone because it is combined with a total of two methyls on each side. It is also called propanone in the sense that it is a modified ketone in propane with a total of three carbons.

아세톤은 제일 작은 카르보닐기와 카르복실기 두가지 기능원자단을 가지고 있는 분자로서 일반 화합물보다 더욱 중요하고 특수한 화학성질을 나타내고 있으며 약물 합성과 유기 합성에 상용하고 있다. 의약, 농업, 일용화학에서 사용할 수 있어 광범하게 사용되고 있는 의약 화학 공업의 중간체이다. 이외에 아세톤은 케톤산 중에서 제일 간단한 분자 구조를 가지고 있고 생물체의 화학 대사 경로에서 중요한 역할을 담당하고 있다. 전 세계적으로 아세톤 제품은 생산량이 부족하여 시장 수요를 만족시키기에는 거리가 너무나도 크므로 더욱 경제성 있는 생산 프로세스를 대체할 것을 절박하게 수요하고 있다.Acetone is the molecule with the smallest carbonyl group and carboxyl group atomic functional group, which is more important than the general compound and has special chemical properties and is commonly used in drug synthesis and organic synthesis. It is an intermediate of the medicinal chemical industry which is widely used for medicines, agriculture and daily chemicals. In addition, acetone has the simplest molecular structure among ketone acids and plays an important role in the chemical metabolism pathway of organisms. Acetone products worldwide are desperately in need of replacing more economical production processes because their production is so short that they are too far to meet market demands.

현재 아세톤은 큐멘공법에 의해 페놀 생산 공정의 병산물로 발생하며, 공업과 화학분야에서 사용되는 중요한 유기용매 및 화학 중간물질로 석유화학공정에서 이산화탄소 발생과 함께 생산이 된다. 하지만 시아노박테리아는 태양광과 이산화탄소를 이용하여 아세톤을 생산하면서 또 다른 이산화탄소 배출 없이 공기 중의 이산화탄소를 줄일 수 있어 친환경적이다. 석유화학제품을 친환경 바이오화학제품으로 대체할 경우 석유자원 사용량 감축이 가능하며 이산화탄소 발생량도 저감이 가능하다. 현재 시아노박테리아를 이용해 아세톤 생산 연구가 진행되고 있지만 아세톤을 생산하기 위한 대사경로를 갖고 있지 않아 다른 균주에 있는 유전자를 도입하여 새로운 대사경로를 만드는 대사공학적 접근이 필요하며, 현재의 기술로써 생산할 수 있는 아세톤 생산량보다 더욱 많은 양의 아세톤을 생산할 수 있는 발현 시스템과 발효법의 필요성이 대두되고 있다. At present, acetone is produced by the cumene process as a bovine product of the phenol production process. It is an important organic solvent and chemical intermediate used in the industrial and chemical fields. It is produced with the generation of carbon dioxide in the petrochemical process. However, cyanobacteria are environmentally friendly because they produce acetone using sunlight and carbon dioxide and can reduce carbon dioxide in the air without another carbon dioxide emission. Replacing petrochemical products with environmentally friendly biochemicals can reduce the use of petroleum resources and reduce carbon dioxide emissions. Acetone production is currently under study using cyanobacteria but there is no metabolic pathway to produce acetone, so a metabolic engineering approach to introduce a gene in another strain to create a new metabolic pathway is needed. There is a need for an expression system and a fermentation method capable of producing a larger amount of acetone than the acetone production.

한국 등록특허 KR 10-1789521Korean Patent No. KR 10-1789521

상기 목적을 해결하기 위하여, 본 발명의 목적은 아세톤을 대량생산하기 위하여 아세톤 생산 능력이 있는 형질전환된 시네코코커스 일롱게투스(Synechococcus elongatus) 균주를 제공하는 것이다. In order to solve the above object, an object of the present invention is to provide a transformed strain of Synechococcus elongatus which is capable of producing acetone for mass production of acetone.

또한, 본 발명의 다른 목적은 상기 형질전환된 균주를 배양하는 단계를 포함하는 아세톤의 생산 방법을 제공하는 것이다. Another object of the present invention is to provide a method for producing acetone comprising culturing the transformed strain.

상기 목적을 달성하기 위하여, 본 발명은 서열번호 1의 서열로 표시되는 피루부산 탈탄산효소를 코딩하는 유전자 pdc(pyruvate decarboxylase enzyme); 및In order to accomplish the above object, the present invention provides a gene encoding a pyruvate decarboxylase enzyme (pdc) encoding a pyruvate decarboxylase expressed by the sequence of SEQ ID NO: 1; And

서열번호 2의 서열로 표시되는 알데히드 탈수소효소를 코딩하는 유전자 ald6(aldehyde dehydrogenase enzyme)를 포함하는, 시네코코커스 일롱게투스(Synechococcus elongatus) 균주를 제공한다. There is provided a strain of Synechococcus elongatus comprising an aldehyde dehydrogenase enzyme (ald6) encoding an aldehyde dehydrogenase represented by the sequence of SEQ ID NO: 2.

또한, 본 발명은 상기 균주를 배양하는 단계를 포함하는 아세톤의 생산 방법을 제공한다. In addition, the present invention provides a method for producing acetone comprising culturing the strain.

본 발명의 형질전환된 시네코코커스 일롱게투스 균주는 이산화탄소를 탄소원으로 사용하여 아세톤을 대량생산 할 수 있다. 시네코코커스 일롱게투스 균주는 빛과 대기중에 존재하는 이산화탄소를 탄소원으로 사용하여 경제적이며, 미생물을 사용하여 대기중의 이산화탄소를 제거 또는 저감하는데 활용될 수 있으므로 친환경 친화적이며, 산업화를 위한 아세톤 생산량 증대에 있어서 기본 모델이 될 수 있다. 본 발명에 따라 by-pass를 활용하여 아세톤 생산균주를 제작하는 방법은 또 다른 균주에도 적용될 수 있고, 아세톤은 이소프로판올의 전구체로서 이소프로판올 물질 생산을 위한 방법으로도 제시될 수 있다. The transformed CynoC.ylonggetus strain of the present invention can mass produce acetone using carbon dioxide as a carbon source. Cynicococcus Iylongus strains are economical because they use carbon dioxide present in light and air as a carbon source. They can be used to remove or reduce atmospheric carbon dioxide by using microorganisms. Therefore, they are environmentally friendly and increase acetone production for industrialization. It can be a basic model. The method for producing an acetone-producing strain using by-pass according to the present invention can be applied to another strain, and acetone can also be suggested as a method for producing isopropanol material as a precursor of isopropanol.

도 1은 형질전환된 시네코코커스 일롱게투스 균주의 아세톤 생산 경로를 나타낸 모식도이다.
도 2는 이산화탄소로부터의 아세톤 생산능을 갖는 형질전환된 시네코코커스 일롱게투스 균주를 제조하기 위하여 사용한 재조합 벡터의 모식도이다:
A: 제 1 벡터(NSI 아세톤 생산 경로), B: 제 2 벡터(NSII 우회 경로), C: 제3 벡터(NSIII 증가된 아세틸-CoA 및 말로닐-CoA).
도 3은 본 발명의 제 1 벡터(pSe1Bb1s-atoB, atoDA, adc)의 벡터 맵을 나타낸 모식도이다.
도 4는 본 발명의 제 2 벡터(pSe2Bb1k-pdc, ald6)의 벡터 맵을 나타낸 모식도이다.
도 5는 본 발명의 제 3 벡터(pSe3Bb1c-acs(L641P), accBC, accD1)의 벡터 맵을 나타낸 모식도이다.
도 6은 본 발명의 제 1 벡터 및 제 2 벡터로 형질전환된 균주의 세포 성장 및 아세톤과 아세트산 생성량을 나타낸 그래프이다:
(A) By-pass 유전자 (pdc, ald6 유전자) 포함 제 2 벡터로 형질전환된 균주,
(B) 아세톤 생산 대사경로 유전자 (atoB, atoDA, adc 유전자) 포함 제 1 벡터와 by-pass 유전자 (pdc, ald6 유전자) 포함 제 2 벡터로 형질전환된 균주,
(C) 아세톤 생산 대사경로 유전자 (atoB, ctfAB, adc 유전자) 포함 제 1 벡터와 by-pass 유전자 (pdc, ald6 유전자) 포함 제 2 벡터로 형질전환된 균주,
(D) malonyl-CoA 이용 아세톤 생산 대사경로 유전자 (nphT7, atoDA, adc 유전자) 포함 제 1 벡터와 by-pass 유전자 (pdc,ald6 유전자) 포함 제 2 벡터로 형질전환된 균주.
(E) malonyl-CoA 이용 아세톤 생산 대사경로 유전자 (nphT7, ctfAB, adc 유전자) 포함 제 1 벡터와 by-pass 유전자 (pdc,ald6 유전자) 포함 제 2 벡터로 형질전환된 균주.
도 7은 본 발명의 제 1 벡터 제 2 벡터 및 제 3 벡터로 형질전환된 균주의 세포 성장 및 아세톤과 아세트산 생성량을 나타낸 그래프이다:
(A) 아세톤 생산 대사경로 유전자 (atoB, atoDA, adc 유전자) 포함 제 1 벡터, by-pass 유전자 (pdc, ald6 유전자) 포함 제 2 벡터, acetate로부터 acetyl-CoA 전환 유전자 (acs(L641P) 유전자) 포함 제 3 벡터로 형질전환된 균주,
(B) 아세톤 생산 대사경로 유전자 (atoB, ctfAB, adc 유전자) 포함 제 1 벡터, by-pass 유전자 (pdc, ald6 유전자) 포함 제 2 벡터, acetate로부터 acetyl-CoA 전환 유전자 (acs(L641P) 유전자) 포함 제 3 벡터로 형질전환된 균주,
(C) 아세톤 생산 대사경로 유전자 (atoB, atoDA, adc 유전자) 포함 제 1 벡터, by-pass 유전자 (pdc, ald6 유전자) 포함 제 2 벡터, acetate로부터 acetyl-CoA 전환 유전자 및 acetyl-coA로부터 malonyl-CoA 전환 유전자 (acs(L641P), accBC, accD1 유전자) 포함 제 3 벡터로 형질전환된 균주의 세포성장, 아세톤 생산, 아세트산을 확인한 그래프이다.
도 8은 metabolic engineering을 통하여 아세테이트 첨가 없이 이산화탄소로부터 아세톤이 생산되는 과정을 나타낸 모식도이다.1 is a schematic diagram showing an acetone production pathway of a transformed Cynekocors lilonggetus strain.
2 is a schematic diagram of a recombinant vector used for producing a transformed Cynekocors Iylongutus strain having acetone producing ability from carbon dioxide:
A: first vector (NSI acetone production path), B: second vector (NSII bypass pathway), C: third vector (NSIII increased acetyl-CoA and malonyl-CoA).
3 is a schematic diagram showing a vector map of the first vector (pSe1Bb1s-atoB, atoDA, adc) of the present invention.
4 is a schematic diagram showing a vector map of the second vector (pSe2Bb1k-pdc, ald6) of the present invention.
5 is a schematic diagram showing a vector map of the third vector (pSe3Bb1c-acs (L641P), accBC, accD1) of the present invention.
6 is a graph showing cell growth and acetone and acetic acid production of strains transformed with the first and second vectors of the present invention;
(A) a strain transformed with a second vector containing a by-pass gene (pdc, ald6 gene)
(B) a strain transformed with a first vector containing a acetone production metabolic pathway gene (atoB, atoDA, adc gene) and a second vector containing a by-pass gene (pdc, ald6 gene)
(C) a strain transformed with a first vector containing a acetone production metabolic pathway gene (atoB, ctfAB, adc gene) and a second vector containing a by-pass gene (pdc, ald6 gene)
(D) A strain transformed with a second vector containing a first vector and a by-pass gene (pdc, ald6 gene) containing an acetone-producing metabolic pathway gene (nphT7, atoDA, adc gene) using malonyl-CoA.
(E) A strain transformed with a second vector containing a first vector and a by-pass gene (pdc, ald6 gene) containing an acetone-producing metabolic pathway gene (nphT7, ctfAB, adc gene) using malonyl-CoA.
7 is a graph showing cell growth and acetone and acetic acid production of strains transformed with the first vector second vector and the third vector of the present invention;
(A) Acetone-CoA-converting gene (acs (L641P) gene) from acetate, a vector containing acetone-producing metabolic pathway genes (atoB, atoDA and adc gene), a first vector, a by-pass gene (pdc, ald6 gene) Lt; RTI ID = 0.0 > 3 < / RTI > vector,
(B) Acetone-CoA-converting gene (acs (L641P) gene) from acetone-producing metabolic pathway gene (atoB, ctfAB, adc gene) first vector, by-pass gene (pdc, ald6 gene) Lt; RTI ID = 0.0 > 3 < / RTI > vector,
(C) A second vector containing acetone-producing metabolic pathway genes (atoB, atoDA, adc gene), a second vector containing a by-pass gene (pdc, ald6 gene), a malonyl- Acetone production, and acetic acid of a strain transformed with a third vector containing CoA-converted genes (acs (L641P), accBC, accD1 gene).
FIG. 8 is a schematic view showing a process in which acetone is produced from carbon dioxide without adding acetate through metabolic engineering.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

상기 시네코코커스 일롱게투스는 시아노박테리아의 일종으로 원핵세포인 시아노박테리아는 유전자 조작이 용이하여 대사경로를 바꾸거나 대사물질을 인위적으로 조절하는데 유리하다. 시아노박테리아의 이러한 특성과 합성생물학/대사공학적 기법을 도입하여 기존에는 없는 대사경로를 이용하여 다양한 바이오연료 대체물 또는 화학제품을 생산하고 있다. The Cynecococcus ilongotus is a kind of cyanobacteria. Cyanobacteria, which is a prokaryotic cell, is advantageous for easy manipulation of genetic code to change metabolic pathway or artificially control metabolism. These characteristics of cyanobacteria and synthetic biology / metabolism engineering techniques have been introduced to produce a variety of biofuel alternatives or chemical products using existing metabolic pathways.

이에 본 발명자들은 시아노박테리아의 한 종류인 시네코코커스 일롱게투스 균주에 다양한 유전자들은 형질전환 하여 아세톤을 대량으로 생산할 수 있는 형질전환 균주를 제작하였으며, 제작된 형질전환된 균주를 배양하여 아세톤을 대량생산할 수 있음을 확인하였으므로 본 발명을 완성하였다. Therefore, the present inventors produced a transformant strain capable of producing acetone in a large amount by transforming various genes into a strain of Cyno bacteria, Cynococcus Iylonggetus, and cultured the transformant to prepare acetone The present invention has been completed.

또한, 본 발명은 기존의 nphT7, atoDA, adc를 형질전환한 균주가 기재된 등록 특허(KR 10-1750293)는 152 mg/L의 아세톤을 생산하고, 기존의 nphT7, atoDA, adc-xpkA, pta를 형질전환한 균주가 기재된 논문(Plant Biotechnol J. 2016 Aug;14(8):1768-76. doi: 10.1111/pbi.12536. Epub 2016 Feb 16.)는 22.48 mg/L의 아세톤을 생산하였으나, 본 발명의 atoB, atoDA, adc, pdc, ald6, acs(L641P), accBC 및 accD1 유전자를 형질전환한 시네코코커스 일롱게투스 균주는 최대 280 mg/L를 생산하는 것을 확인하였습니다. 이러한 결과는 기존 아세톤 생산 특허 균주의 최고농도와 비교하여 본 발명에서 아세톤 생산이 1.8배 증가하였으며, 이전에 출원된 아세톤 생산 균주의 경우 10 mM 아세트산칼륨(potassium acetate)를 추가로 첨가하여 아세톤이 생산한 것이나, 본 발명은 metabolic engineering을 통하여 아세트산의 첨가 없이 이산화탄소로부터 아세톤이 생산되므로 아세톤 생산이 더 간편하고 효과가 뛰어나다.In addition, the present invention is based on the finding that the existing patent (KR 10-1750293) which describes a strain transformed with nphT7, atoDA and adc produces acetone of 152 mg / L and the existing nphT7, atoDA, adc-xpkA and pta (Plant Biotechnol J. 2016 Aug; 14 (8): 1768-76. Doi: 10.1111 / pbi.12536. Epub 2016 Feb. 16) produced 22.48 mg / L acetone, It was confirmed that the Cycocorcus Iylongutus strain transformed with the genes atoB, atoDA, adc, pdc, ald6, acs (L641P), accBC and accD1 of the invention produced up to 280 mg / L. These results show that the acetone production in the present invention is increased 1.8 times as compared with the highest concentration of the conventional acetone production patent strain, and in the case of the acetone-producing strains previously filed, 10 mM potassium acetate is further added to produce acetone However, since acetone is produced from carbon dioxide without adding acetic acid through metabolic engineering, the present invention is easier and more effective in producing acetone.

추가적으로 상기 논문에 발표된 아세톤 생산 균주와 최고 농도와 비교하여도 본 발명에서 아세톤 생산이 12.4배 증가한 것을 확인하였으며, 논문에 발표된 균주의 경우 1.8 L photobioreactor에서 배양하여 이산화탄소로부터 아세톤이 생산되므로 본 발명의 atoB, atoDA, adc, pdc, ald6, acs(L641P), accBC, accD1 유전자를 형질전환한 시네코코커스 일롱게투스 균주가 아세톤 생산능이 훨씬 우수하다. In addition, acetone production in the present invention was increased by 12.4-fold compared to the acetone-producing strains and highest concentrations reported in the above-mentioned papers. In the case of the strains disclosed in the present invention, since acetone was produced from carbon dioxide by culturing in 1.8 L photobioreactor, Cyclooxacillus longongus strains transformed with atoB, atoDA, adc, pdc, ald6, acs (L641P), accBC and accD1 genes are superior to acetone.

본 발명은 아세톤 생산 대사경로가 삽입되어있는 시네코코커스 일롱게투스 균주에 by-pass 및 CCM (carbon concentrating mechanism) enforced pathway를 도입하여 이산화탄소로부터 아세톤을 직접 생산할 수 있는 새로운 돌연변이 균주를 제작하였다. 선행 논문에서 발표된 4종의 아세톤 생산가능 시아노박테리아는 acetyl-CoA로부터 acetoacetyl-CoA를 생산하는 효소를 코딩하는 유전자; malonyl-CoA로부터 acetoacetyl-CoA를 생산하는 효소를 코딩하는 유전자; acetoacetyl-CoA로부터 acetoacetate를 생산하는 효소를 코딩하는 유전자; acetoacetate로부터 아세톤을 생산하는 효소를 코딩하는 유전자가 삽입되어 있나, 본 발명에서는 피루부산 탈탄산효소를 코딩하는 유전자 pdc(pyruvate decarboxylase enzyme); 및 알데히드 탈수소효소를 코딩하는 유전자 ald6(aldehyde dehydrogenase enzyme)를 포함하는 시네코코커스 일롱게투스 균주이다. The present invention introduces a by-pass and a carbon concentrating mechanism (CCM) enforced pathway into a strain of C. cinerea with an acetone production metabolic pathway to produce a new mutant strain capable of directly producing acetone from carbon dioxide. The four acetone-producing cyanobacteria reported in the preceding article are genes encoding an enzyme that produces acetoacetyl-CoA from acetyl-CoA; a gene encoding an enzyme that produces acetoacetyl-CoA from malonyl-CoA; a gene encoding an enzyme that produces acetoacetate from acetoacetyl-CoA; a gene coding for an enzyme that produces acetone from acetoacetate is inserted. In the present invention, a gene pdc (pyruvate decarboxylase enzyme) encoding a pyruvate decarboxylase is used. And a gene encoding aldehyde dehydrogenase (aldhyde dehydrogenase enzyme) ald6.

또한, 상기 균주에 acetyl-CoA로부터 acetoacetyl-CoA를 생산하는 효소를 코딩하는 유전자 AtoB(Acetyl-CoA acetyltransferase); 서열번호 4의 서열로 표시되는 acetoacetyl-CoA로부터 acetoacetate를 생산하는 효소를 코딩하는 유전자 AtoDA(acetotacetate CoA transferase); 및 서열번호 5의 서열로 표시되는 acetoacetate로부터 아세톤을 생산하는 효소를 코딩하는 유전자 Adc(Acetoacetate decarboxylase)를 더 포함할 수 있고, acetate-coA 리가아제 효소을 코딩하는 유전자 acsL641P(Salmonella enterica acetyl-CoA synthetase variant) 및 Acetyl-CoA 카르복실화 효소를 코딩하는 유전자 accBC(Acetyl-CoA carboxylase) 및 accD1(Acetyl-CoA carboxylase beta-subunit)을 추가적으로 더 포함할 수 있다. Also, a gene encoding AtoB (Acetyl-CoA acetyltransferase) encoding an enzyme that produces acetoacetyl-CoA from acetyl-CoA in the above strain; AtoDA (acetotacetate CoA transferase) gene encoding an enzyme that produces acetoacetate from acetoacetyl-CoA represented by the sequence of SEQ ID NO: 4; And acetoacetate decarboxylase (Adc) encoding an enzyme producing acetone from the acetoacetate represented by the sequence of SEQ ID NO: 5, and may further comprise a gene acs L641P (Salmonella enterica acetyl-CoA synthetase variant) and the genes accBC (Acetyl-CoA carboxylase) and accDl (Acetyl-CoA carboxylase beta-subunit) encoding the Acetyl-CoA carboxylase.

pyruvate로 부터 acetaldehyde 생산하는 효소는 Z. mobilis로부터 유래되었고, acetaldehyde로부터 acetate를 생산하는 효소는 S. cerevisiae로부터 유래한 것이다. acetate로부터 acetyl-CoA를 생산하는 효소는 S. enterica로부터 유래되었고, acetyl-CoA로부터 malonyl-CoA를 생산하는 효소는 C. glutamicum으로부터 유래한 것이다. The enzyme that acetaldehyde produces from pyruvate is derived from Z. mobilis , and the enzyme that produces acetate from acetaldehyde is derived from S. cerevisiae . The enzyme producing acetyl-CoA from acetate is derived from S. enterica , and the enzyme producing malonyl-CoA from acetyl-CoA is derived from C. glutamicum .

본 발명은 참고문헌을 통해 알려진 아세톤 생산이 가능한 1) atoB, atoDA, adc, 2) atoB, ctfAB, adc, 3) nphT7, atoDA, adc, 4) nphT7, ctfAB, adc 유전자가 삽입된 돌연변이 균주에 각각 5) pyruvate로부터 acetaldehyde를 생산하는 효소를 코딩하는 유전자 및 acetaldehyde로부터 acetate를 생산하는 효소를 코딩하는 유전자를 포함하는 균주; 6) acetate로부터 acetyl-CoA를 생산하는 효소를 코딩하는 유전자를 함께 포함하는 균주; 7) acetyl-CoA로부터 malonyl-CoA를 생산하는 효소를 코딩하는 유전자를 함께 포함하는 균주 7종이 개발되었다.The present invention relates to a mutant strain in which at least one of the following acetone production is possible: atoB, atoDA, adc, 2) atoB, ctfAB, adc, 3) nphT7, atoDA, adc, 4) nphT7, ctfAB, 5) a strain containing an enzyme encoding an enzyme producing acetaldehyde from pyruvate and a gene encoding an enzyme producing acetate from acetaldehyde; 6) a strain containing a gene encoding an enzyme that produces acetyl-CoA from acetate; 7) A total of 7 strains containing the genes encoding malonyl-CoA-producing enzymes from acetyl-CoA were developed.

본 발명의 일실시예에서, 상기 균주는 제 2 벡터로 형진전환 된 것으로서, In one embodiment of the invention, the strain is transformed into a second vector,

상기 제 2 벡터는 서열번호 1의 서열로 표시되는 피루부산 탈탄산효소를 코딩하는 유전자 pdc; 및 서열번호 2의 서열로 표시되는 알데히드 탈수소효소를 코딩하는 유전자 ald6(aldehyde dehydrogenase enzyme)를 포함할 수 있다. The second vector comprises the gene pdc encoding the fusobacterium decarboxylase expressed by the sequence of SEQ ID NO: 1; And an aldehyde dehydrogenase enzyme (ald6) encoding an aldehyde dehydrogenase represented by the sequence of SEQ ID NO: 2.

상기 균주는 제 2 벡터 외에 제 1 벡터 및/또는 제 3 벡터로 추가적으로 형질전환 된것으로, Wherein the strain is additionally transformed into a first vector and / or a third vector in addition to the second vector,

상기 제 1 벡터는 서열번호 3의 서열로 표시되는 acetyl-CoA로부터 acetoacetyl-CoA를 생산하는 효소를 코딩하는 유전자 AtoB; 서열번호 4의 서열로 표시되는 acetoacetyl-CoA로부터 acetoacetate를 생산하는 효소를 코딩하는 유전자 AtoDA; 및 서열번호 5의 서열로 표시되는 acetoacetate로부터 아세톤을 생산하는 효소를 코딩하는 유전자 Adc를 포함할 수 있고, 제 3 벡터는 서열번호 6의 서열로 표시되는 acetate-coA 리가아제 효소을 코딩하는 유전자 acsL641P, 서열번호 7의 서열로 표시되는 Acetyl-CoA 카르복실화 효소를 코딩하는 유전자 accBC 및 서열번호 8의 서열로 표시되는 Acetyl-CoA 카르복실화 효소를 코딩하는 유전자 accD1을 포함할 수 있다. Wherein said first vector is selected from the group consisting of a gene encoding atoB which encodes an enzyme that produces acetoacetyl-CoA from acetyl-CoA represented by the sequence of SEQ ID NO: 3; A gene encoding an enzyme producing acetoacetate from acetoacetyl-CoA represented by the sequence of SEQ ID NO: 4; AtoDA; And a gene Adc encoding an enzyme that produces acetone from the acetoacetate represented by the sequence of SEQ ID NO: 5, and the third vector may include the gene acs L641P encoding the acetate-coA ligase enzyme represented by SEQ ID NO: 6 , The gene accBC encoding the Acetyl-CoA carboxylase represented by the sequence of SEQ ID NO: 7, and the gene accD1 encoding the Acetyl-CoA carboxylase represented by the sequence of SEQ ID NO: 8.

'제1, 제2, 제3'라는 표현은, 벡터의 종류를 구별하기 위한 것일 뿐, 형질전환의 순서나 방법을 제한하는 것이 아니다.The expressions 'first, second, and third' are only for distinguishing types of vectors, and do not limit the order or method of transformation.

본 발명의 일실시예에서, 상기 제 1 벡터는, 모균주인 시네코코커스 일롱게투스의 뉴트럴 사이트I(neutral site I, NSI)에 삽입될 수 있고, 상기 제 2 벡터는 모균주인 시네코코커스 일롱게투스의 뉴트럴 사이트II(neutral site II, NSII)에 삽입될 수 있으며, 상기 제 3 벡터는 모균주인 시네코코커스 일롱게투스의 뉴트럴 사이트III(neutral site III, NSIII)에 삽입될 수 있다(도 2 참조). In one embodiment of the present invention, the first vector may be inserted into a Neutral site I (NSI) of the parent strain Cynekocors Iylonggetus, and the second vector may be inserted into the parent strain Cynecoco Can be inserted into a neutral site II (NSII) of Cassirl longtus, and the third vector can be inserted into a neutral site III (NSIII) of the parent strain Cynokococirus ilonggetus (See Fig. 2).

상기 제 2 벡터는 카나마이신(kanamycin) 저항성 유전자; lacI 리프레서; trc 프로모터; 및 서열번호 1의 서열로 표시되는 피루부산 탈탄산효소를 코딩하는 유전자 pdc; 및 서열번호 2의 서열로 표시되는 알데히드 탈수소효소를 코딩하는 유전자 ald6을 포함하는 할 수 있고(도 4 참조), 상기 제 1 벡터는 스펙티노마이신(spectinomycin) 저항성 유전자; 저항성 유전자; lacI 리프레서; trc 프로모터; 및서열번호 3의 서열로 표시되는 acetyl-CoA로부터 acetoacetyl-CoA를 생산하는 효소를 코딩하는 유전자 AtoB(; 서열번호 4의 서열로 표시되는 acetoacetyl-CoA로부터 acetoacetate를 생산하는 효소를 코딩하는 유전자 AtoDA; 및 서열번호 5의 서열로 표시되는 acetoacetate로부터 아세톤을 생산하는 효소를 코딩하는 유전자 Adc를 포함할 수 있으며(도 3 참조), 상기 제 3 벡터는 클로람페니콜(chloramphenicol) 저항성 유전자; lacI 리프레서; trc 프로모터; 및 서열번호 6의 서열로 표시되는 acetate-coA 리가아제 효소을 코딩하는 유전자 acsL641P 및 서열번호 7 및 서열번호 8의 서열로 표시되는 Acetyl-CoA 카르복실화 효소를 코딩하는 유전자 accBC 및 accD1을 포함할 수 있다(도 5 참조). Said second vector is a kanamycin resistance gene; lacI refresher; trc promoter; And the gene pdc encoding the fructose decarboxylase expressed by the sequence of SEQ ID NO: 1; And a gene ald6 encoding an aldehyde dehydrogenase represented by the sequence of SEQ ID NO: 2 (see Fig. 4). The first vector may be a spectinomycin resistance gene; Resistance gene; lacI refresher; trc promoter; And AtoB (which encodes an enzyme producing acetoacetyl-CoA from the acetyl-CoA represented by the sequence of SEQ ID NO: 3; And a gene Adc encoding an enzyme producing acetone from the acetoacetate represented by the sequence of SEQ ID NO: 5 (see Fig. 3), and the third vector is a chloramphenicol resistance gene; a lacI repressor; a trc promoter ; And acs L641P encoding the acetate-coA ligase enzyme represented by the sequence of SEQ ID NO: 6 and the genes accBC and accD1 encoding the Acetyl-CoA carboxylase represented by SEQ ID NO: 7 and SEQ ID NO: 8 (See FIG. 5).

상기 균주는 기탁번호 KCCM12179P인 것으로, 상기 균주는 제 1 벡터, 제 2 벡터 및 제 3 벡터로 형질전환된 균주이다. The strain is the accession number KCCM12179P, and the strain is a strain transformed with a first vector, a second vector and a third vector.

또한, 본 발명은 시네코코커스 일롱게투스 균주를 배양하는 단계를 포함하는 아세톤 생산 방법이다. 균주를 배양하는 단계는 이산화탄소를 공급하는 것을 포함할 수 있다. 본 발명에서는 휘발되는 아세톤을 얻기 위하여 배양병과 물이 담긴병을 연결하여 물에 녹은 아세톤을 함께 수득하는 단계가 포함된다. 본 발명의 시네코코커스 일롱게투스 균주는 생산한 아세톤이 배지 내에서 확인이 되며, 휘발된 아세톤은 배양병과 연결된 병에서 확인되는 것이다. 본 발명은 시네코코커스 일롱게투스 균주를 배양하는 단계를 포함하는 이산화탄소 제거 방법이다(도 8 참조).In addition, the present invention is a method for producing acetone, which comprises culturing a strain of Cynekococus Iylonggetus. The step of culturing the strain may include supplying carbon dioxide. In the present invention, in order to obtain acetone to be volatilized, a culture bottle and a bottle containing water are connected to each other to obtain acetone dissolved in water. The C. cinerea longongus strain of the present invention is confirmed in the medium by the produced acetone, and the volatilized acetone is identified in the bottle connected to the culture bottle. The present invention is a method for removing carbon dioxide, which comprises culturing a strain of Cynekococus Iylonggetus (see FIG. 8).

이하, 하기의 실시예를 통하여 본 발명을 보다 구체적으로 설명한다. 그러나 하기의 실시에는 본 발명에 대한 이해를 돕기 위해 예시의 목적으로만 제공된 것일 뿐, 본 발명의 범주 및 범위가 이에 한정되지 않는다.Hereinafter, the present invention will be described more specifically with reference to the following examples. However, the following embodiments are provided for illustrative purposes only in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

<< 실시예Example 1>  1> 시네코코커스Cine Kokocers 일롱게투스Il Longgetus 균주로부터 아세톤을 생산하기 위한 전략 Strategy for the production of acetone from strains

시네코코커스 일롱게투스 균주로부터 우회 경로(by-pass) 및 CCM enforced pathway를 이용하여 아세톤을 생산하기 위한 전략을 분석하였다. Strategies for producing acetone using the by-pass and the CCM enforced pathway from Cynekokocus Iulongus strains were analyzed.

구체적으로, 시네코코커스 일롱게투스에서 아세톤을 생산한 선행 논문을 참고하여 acetyl-CoA로부터 아세톤에 이르는 atoB, AtoDA/ctfAB, Adc 유전자를 이용하여 새로운 대사경로를 만들었고, malonyl-CoA로부터 아세톤에 이르는 nphT7유전자를 이용하여 대사경로를 만들었다. 따라서 atoB-atoDA/ctfB-adc 경로와 nphT7-atoDA/ctfB-adc 경로를 가지는 4종의 시아노박테리아 균주를 확보하였다 (Chwa JW, Kim UJ, Sim SJ, Um Y, Woo HM (2016) Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition. Plant Biotech J 14:1768-1776). Specifically, a new metabolic pathway was constructed using atoB, AtoDA / ctfAB, and Adc genes from acetyl-CoA to acetone, and a new metabolic pathway from malonyl-CoA to acetone The metabolic pathway was constructed using the nphT7 gene. Thus, four strains of cyanobacteria with the pathway of atoB-atoDA / ctfB-adc and nphT7-atoDA / ctfB-adc were obtained (Chwa JW, Kim UJ, Sim SJ, Um Y, Woo HM a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition Plant Biotech J 14: 1768-1776).

본 발명에서는 아세톤 생산 경로에 by-pass를 도입하고자 Z. mobilis 의 pdc유전자와 S. cerevisiae 의 ald6 유전자의 DNA 시퀀스를 코돈 최적화 과정을 거친 후 Genscriptⓡ 에서 시퀀스를 합성 주문 제작하였다. pdc는 pyruvate decarboxylase enzyme을 코딩하는 유전자이고 ald6 유전자는 aldehyde dehydrogenase enzyme을 코딩하는 유전자이다. 따라서 atoB, atoDA, adc-pdc, ald6, atoB, ctfAB, adc-pdc, ald6, nphT7, atoDA, adc- pdc, ald6, nphT7, ctfAB,adc-pdc, ald6 이렇게 4가지 대사경로를 디자인하였다.In the present invention, in order to introduce by-pass in the acetone production path, Z. mobilis DNA sequences of the pdc gene of S. cerevisiae and the ald6 gene of S. cerevisiae were codon optimized and synthesized on Genscript. pdc is a gene encoding pyruvate decarboxylase enzyme and ald6 gene is a gene encoding aldehyde dehydrogenase enzyme. Thus, four metabolic pathways were designed: atoB, atoDA, adc-pdc, ald6, atoB, ctfAB, adc-pdc, ald6, nphT7, atoDA, adc-pdc, ald6, nphT7, ctfAB, adc-pdc and ald6.

추가로 시네코코커스 일롱게투스 내에는 아세테이트를 acetyl-CoA로 전환할 수 있는 acs 유전자가 존재한다. 그러나 by-pass에 의해 생산된 아세테이트가 acetyl-CoA로 모두 전환되지 않기 때문에 전환효율을 증가시키고자 선행논문을 참고하여 acs(L641P) 유전자를 추가로 삽입하였다 (Kocharin K, Verena YC, Siewers V, Nielsen J (2012) Engineering of acetyl-CoA metabolism for the improved production of polyhydroxybutyrate in Saccharomyces cerevisiae. AMB Express 2:52). 시네코코커스 일롱게투스 내에는 acetyl-CoA를 malonyl-CoA로 전환할 수 있는 AccBC-D 유전자가 존재하지만 말로닐코에이로 전환 효율을 증가시키고자 선행논문을 참고하여 accBC 및 accD1 유전자를 추가로 삽입하였다 (Davis MS, Solbiati J, Cronan JE (2000) Overproduction of acetyl-coA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli. J Biol Chem 275:28593-28598). Acs(L641P)는 Salmonealla enterica 균주의 유전자 시퀀스를 이용하였고, accBC 및 accD1은 Corynebacterium glutamicum 균주의 유전자 시퀀스를 이용하여 코돈 최적화 과정을 거친 후 Genscriptⓡ 에서 시퀀스를 합성 주문 제작하였다. acs(L641P) 유전자는 acetate-coA ligase enzyme을 코딩하는 유전자이고, accBC 및 accD1 유전자는 Acetyl-CoA carboxylase를 코딩하는 유전자이다. 따라서 atoB, atoDA, adc-pdc, ald6-acs(L641P), atoB,ctfAB, adc-pdc, ald6-acs(L641P), atoB, atoDA,adc-pdc,ald6-acs(L641P), accBC 및 accD1 3가지 대사경로가 추가로 디자인되었다. In addition, there is an acs gene in Acinetobacter spp. That can convert acetate into acetyl-CoA. However, acac (L641P) gene was further inserted (Kocharin K, Verena YC, Siewers V, et al.) To increase the conversion efficiency because acetate produced by by-pass was not converted to acetyl- Nielsen J (2012) Engineering of acetyl-CoA metabolism for the improved production of polyhydroxybutyrate in Saccharomyces cerevisiae . AMB Express 2:52). Although the AccBC-D gene, which can convert acetyl-CoA into malonyl-CoA, is present in Cineocococcus Iylongus, the accBC and accD1 genes are additionally inserted (Davis MS, Solbiati J, Cronan JE (2000) Overproduction of acetyl-coA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli . J Biol Chem 275: 28593-28598). Acs (L641P) is Salmonealla enterica The gene sequences of the strains were used, and accBC and accD1 corresponded to Corynebacterium glutamicum After the codon optimization process using the gene sequence of the strain, the sequence was synthesized and customized in Genscript. The acs (L641P) gene is the gene encoding the acetate-coA ligase enzyme, and the accBC and accD1 genes are the genes encoding Acetyl-CoA carboxylase. Thus, atoB, atoDA, adc-pdc, ald6-acs (L641P), atoB, ctfAB, adc-pdc, ald6-acs (L641P), atoB, atoDA, adc- Additional metabolic pathways have been designed.

<< 실시예Example 2> 아세톤 생산을 위한 재조합 벡터의 제작 2> Production of recombinant vector for acetone production

SyneBrick vector인 pSe1Bb1s-gfp, pSe2Bb1k-gfp, pSe3Bb1c-none vector를 이용하여 아세톤 생산균주 제작하였다. Acetone-producing strains were constructed using the SyneBrick vectors pSe1Bb1s-gfp, pSe2Bb1k-gfp, and pSe3Bb1c-none vectors.

구체적으로, 아세톤 생산 기본 균주는 SyneBrick 벡터인 pSe1Bb1s-gfp의 gfp 부분을 EcoRI-BamHI 제한효소를 이용하여 제거한 후 그 자리에 atoB, atoDA, adc 유전자의 DNA 시퀀스가 삽입된 pSe1Bb1s-atoB, atoDA, adc 벡터와 atoB, ctfAB, adc 유전자의 DNA시퀀스가 삽입된 pSe1Bb1s- atoB, ctfAB, adc 벡터, nphT7, atoDA, adc 유전자의 DNA 시퀀스가 삽입된 pSe1Bb1s-nphT7, atoDA, adc 벡터, nphT7, ctfAB, adc 유전자의 DNA 시퀀스가 삽입된 pSe1Bb1s-nphT7, ctfAB, adc 벡터 (Chwa JW, Kim UJ, Sim SJ, Um Y, Woo HM (2016) Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition. Plant Biotech J 14:1768-1776)를 이용하였다(도 2A, 도 3).Specifically, acetone-producing strains were obtained by removing the gfp portion of SyneBrick vector pSe1Bb1s-gfp using EcoRI-BamHI restriction enzyme and then introducing pSe1Bb1s-atoB, atoDA, adc PSe1Bb1s-nphT7, atoDA, adc vector, nphT7, ctfAB, and adc genes inserted with DNA sequences of pSe1Bb1s-atoB, ctfAB, adc vector, nphT7, atoDA and adc genes inserted with DNA sequences of atoB, ctfAB and adc genes of the DNA sequence is inserted pSe1Bb1s-nphT7, ctfAB, adc vector (Chwa JW, Kim UJ, Sim SJ, Um Y, Woo HM (2016) Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in S ynechococcus elongatus PCC 7942 under light and aerobic condition. Plant Biotech J 14: 1768-1776) was used (Fig. 2A, Fig. 3).

pSe2Bb1k-gfp (Chwa JW, Kim UJ, Sim SJ, Um Y, Woo HM (2016) Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition. Plant Biotech J 14:1768-1776)의 gfp 부분을 EcoRI-BamHI 제한효소를 이용하여 제거한 후 그 자리에 합성 주문한 pdc, ald6 유전자의 DNA 시퀀스를 삽입하여 pSe2Bb1k-pdc,ald6 벡터를 완성하여 아세톤 생산 증가를 위한 제 2 벡터를 준비하였다(도 2B, 도 4). pSe2Bb1k-gfp (Chwa JW, Kim UJ, Sim SJ, Um Y, Woo HM (2016) Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition Plant Biotech J 14: 1768-1776) was digested with EcoRI-BamHI restriction enzyme, and the DNA sequence of pdc and ald6 gene synthesized in the place was inserted to complete the pSe2Bb1k-pdc and ald6 vectors, 2 vector was prepared (Fig. 2B, Fig. 4).

pSe3Bb1c-GFP (Kim WJ, Lee S-M, Um Y, Sim SJ, Woo HM (2017) Development of SyneBrick vectors as a synthetic biology platform for gene expression in Synechococcus elongatus PCC 7942. Front Plant Sci 8:293)의 GFP 부분에 EcoRI-BamHI 제한효소를 이용하여 acs(L641P) 유전자의 DNA 시퀀스를 삽입하였다. 이렇게 완성된 pSe3Bb1c-Acs(L641P) 벡터는 BamHI-XhoI 제한효소 처리 후 BglII-XhoI 제한효소로 처리된 accBC 및 accD1 유전자의 DNA 시퀀스를 삽입하였다. 이로써 pSe3Bb1c- Acs(L641P) 또는 pSe3Bb1c-Acs(L641P), accBC, accD1인 제 3벡터를 완성하였다(도 2C, 도 5).Synthesis of SyneBrick vectors as a synthetic biology platform for Synechococcus elongatus PCC 7942. Front Plant Sci 8: 293) in the GFP portion of pSe3Bb1c-GFP (Kim WJ, Lee SM, Um Y, Sim SJ, Woo HM DNA sequences of the acs (L641P) gene were inserted using EcoRI-BamHI restriction enzyme. The thus completed pSe3Bb1c-Acs (L641P) vector was inserted with a DNA sequence of accBC and accD1 gene treated with BglII-XhoI restriction enzyme after treatment with BamHI-XhoI restriction enzyme. This resulted in the completion of a third vector, pSe3Bb1c-Acs (L641P) or pSe3Bb1c-Acs (L641P), accBC, accD1 (FIG. 2C, FIG.

Neutral site-I에 atoB, atoDA, adc, atoB, ctfAB, adc, nphT7, atoDA, adc, nphT7, ctfAB, adc가 도입되어 있는 균주에 pSe2Bb1k-pdc, ald6 벡터를 Neutral site-II에 각각 삽입하여 by-pass가 도입된 4가지 아세톤 생산 균주를 개발하였다. 추가로 아세톤 생산 경로가 도입되지 않고 by-pass 만 도입되어 있는 균주를 제작하였다. 형질전환 방법은 Natural transformation 방법을 이용하여 수행하였고, 형질전환 여부는 PCR (5’-> 3’ 프라이머 시퀀스 : NSI-forward : AAG CGC TCC GCA TGG ATC TG (서열번호 9)/ reverse : CAA GGC AGC TTG GAA GGG CG(서열번호 10), NSII-forward : GGC TAC GGT TCG TAA TGC CA(서열번호 11) / reverse : GAG ATC AGG GCT GTA CTT AC(서열번호 12))을 통해 확인하였다.PSe2Bb1k-pdc, and ald6 vectors were inserted into the Neutral site-II, respectively, into strains in which atoB, atoDA, adc, atoB, ctfAB, adc, nphT7, atoDA, adc, nphT7, ctfAB, 4-acetone-producing strains were introduced. In addition, strains with only by-pass without acetone production pathway were introduced. The transformation was carried out using the natural transformation method and the transformation was performed using the PCR (5 '-> 3' primer sequence: NSI-forward: AAG CGC TCC GCA TGG ATC TG (SEQ ID NO: 9) / reverse: CAA GGC AGC TTG GAA GGG CG (SEQ ID NO: 10), NSII-forward: GGC TAC GGT TCG TAA TGC CA (SEQ ID NO: 11) / reverse: GAG ATC AGG GCT GTA CTT AC (SEQ ID NO: 12)).

Neutral site-I(제 1 벡터: 아세톤 생산 경로), Neutral site-II(제 2 벡터: 우회 경로)에 atoB, atoDA, adc-pdc, ald6, atoB, ctfAB, adc-pdc, ald6이 각각 도입되어 있는 균주에 pSe3Bb1c-acs(L641P) 또는 pSe3Bb1c-acs(L641P), accBC 및 accD1인 벡터 Neutral site-III(제 3 벡터)에 각각 삽입하여 아세틸코에이, 말로닐코에이로의 전환 효율이 좋은 아세톤 생산균주를 제작하였다. Neutral site-III의 형질전환 여부는 PCR (5’-> 3’ 프라이머 시퀀스 : NSIII-forward : CCA CGC ATT CAA CCA AGG GC(서열번호 13)/ reverse : CAC ACC CTC AGA GAC AAG CC(서열번호 14))을 통해 확인하였다.AtoB, atoDA, adc-pdc, ald6, atoB, ctfAB, adc-pdc, and ald6 were introduced into Neutral site-I (first vector: acetone production pathway) and Neutral site-II Were inserted into pSe3Bb1c-acs (L641P) or pSe3Bb1c-acs (L641P), accBC and accD1 vector Neutral site-III (third vector), respectively, to the strains in which acetonucleotide, malonylcholine, . The transformation of Neutral site-III was confirmed by PCR (5'-> 3 'primer sequence: NSIII-forward: CCA CGC ATT CAA CCA AGG GC (SEQ ID NO: 13) / reverse: CAC ACC CTC AGA GAC AAG CC )).

<< 실시예Example 3> 우회 경로(by-pass)가 삽입된 형질전환된  3> transformed with inserted by-pass 시네코코스Cine Cocos 일롱게투스Il Longgetus 균주를 이용하여 이산화탄소로부터 아세톤  Using the strain, 생산능Production capacity 분석 analysis

상기 실시예 2의 벡터로 형질전환한 돌연변이 균주들을 일정시간 배양하여 직접적으로 공급해주는 5% 이산화탄소로부터 아세톤이 생산되는지 테스트해보았다. 구체적인 배양조건은 100 mL 병에 10 mM MOPS 버퍼가 포함된 BG-11 배지를 100 mL 넣고 제작한 아세톤 생산균주를 처음 배양시 OD=0.6으로 희석하여 넣어주었다. 배지에 스펙티노마이신(spectinomycin) 항생제 10 μg/mL, 카나마이신(kanamycin) 항생제 10 μg/mL, 클로람페니콜(chloramphenicol) 항생제 3 μg/mL를 넣어준 뒤 정치배양기 30℃, 100 μE·m-2·s-1, 5% CO2를 연속하는 공급조건하에 배양하였다. The mutant strains transformed with the vector of Example 2 were cultured for a certain period of time and tested for the production of acetone from 5% carbon dioxide directly supplied. For the detailed culture conditions, 100 mL of BG-11 medium containing 10 mM MOPS buffer was added to 100 mL bottle, and the acetone-producing strain prepared was diluted to OD = 0.6 for the first incubation. Add 10 μg / ml of spectinomycin antibiotics, 10 μg / ml of kanamycin antibiotics and 3 μg / ml of chloramphenicol antibiotics to the medium, and incubate at 30 ° C for 100 μE · m -2 · s -1 , 5% CO 2 under continuous feeding conditions.

휘발되는 아세톤은 500 mL 병에 500 mL 물을 채운 뒤 배양병과 연결하여 휘발되는 아세톤이 물에 녹게 하였다. 24시간 배양 후에는 유전자 발현에 필요한 인듀서 1 mM IPTG를 넣어주었고, 배양 후 10일 동안 세포의 730 nm 파장에서의 광학 밀도(optical density)를 확인하였고, 아세톤, 아세트산의 생산량을 측정하였다. The volatilized acetone was filled with 500 mL of water into a 500 mL bottle and then connected to the culture bottle to dissolve volatilized acetone in the water. After 24 hours incubation, inducer 1 mM IPTG required for gene expression was added. The optical density at 730 nm wavelength of the cells was observed for 10 days after the incubation, and the yield of acetone and acetic acid was measured.

우회 경로에 관련된 pdc, ald6 유전자가 삽입된 형질전환 균주에서는 아세트산이 741 mg/L 생산이 되었다. atoB, atoDA, adc-pdc, ald6 유전자가 삽입된 형질전환 균주에서는 179.9 mg/L 아세톤이 생산되었고, 375.5 mg/L 아세트산이 생산되었다. atoB, ctfAB, adc-pdc, ald6 유전자가 삽입된 형질전환 균주에서는 최대 127.2 mg/L 아세톤이 생산되었고, 이때 373.0 mg/L 아세트산이 생산되었다. nphT7, atoDA, adc-pdc,ald6 유전자가 삽입된 형질전환 균주에서는 최대 94.5 mg/L 아세톤이 생산되었고, 이때 427.0 mg/L 아세트산이 생산되었다. nphT7, ctfAB, adc-pdc, ald6 유전자가 삽입된 형질전환 균주에서는 최대 61.9 mg/L 아세톤이 생산되었고, 이때 524.5 mg/L 아세트산이 생산되었다(도 6). In the transduction pathway in which the pdc and ald6 genes were inserted in the bypass pathway, acetic acid was produced at 741 mg / L. Atorvastatin, atoDA, adc-pdc and ald6 genes, 179.9 mg / L acetone was produced and 375.5 mg / L acetic acid was produced. In the transformant strain in which atoB, ctfAB, adc-pdc, and ald6 genes were inserted, up to 127.2 mg / L acetone was produced, and 373.0 mg / L acetic acid was produced. In the transfection strain in which nphT7, atoDA, adc-pdc, and ald6 genes were inserted, up to 94.5 mg / L acetone was produced, and 427.0 mg / L acetic acid was produced. In the transformant strain in which the nphT7, ctfAB, adc-pdc, and ald6 genes were inserted, up to 61.9 mg / L acetone was produced, and 524.5 mg / L acetic acid was produced (FIG.

<< 실시예Example 4>  4> CCMCCM enforced pathway가 추가로 삽입된 형질전환된  transformed with an additional enforced pathway 시네코코스Cine Cocos 일롱게투스 균주를 이용하여 이산화탄소로부터 아세톤  Using ilonggutus strains, 생산능Production capacity 분석 analysis

아세톤 생산을 확인한 4종의 돌연변이 균주에서 생산량이 높은 2종의 돌연변이 균주 (atoB, atoDA, adc-pdc, ald6/ atoB, ctfAB, adc-pdc, ald6)에 acs(L641P) 유전자를 추가로 도입하여 위와 동일한 방법으로 배양하였다. atoB, atoDA, adc-pdc, ald6-acs(L641P) 유전자가 삽입된 형질전환 균주에서는 최대 238.9 mg/L 아세톤이 생산되었고 이때 311 mg/L 아세트산이 생산되었다. (L641P) gene was further introduced into two mutant strains (atoB, atoDA, adc-pdc, ald6 / atoB, ctfAB, adc-pdc and ald6) with high yields from four mutant strains confirming acetone production And cultured in the same manner as above. Lactobacillus strains containing atoB, atoDA, adc-pdc, and ald6-acs (L641P) genes produced up to 238.9 mg / L acetone and produced 311 mg / L acetic acid.

또한, atoB, ctfAB, adc-pdc, ald6-acs(L641P) 유전자가 삽입된 형질전환 균주에서는 최대 280 mg/L 아세톤이 생산되었고 이때 220.5 mg/L 아세트산이 생산되었다. In addition, up to 280 mg / L acetone was produced in transgenic strains in which atoB, ctfAB, adc-pdc, and ald6-acs (L641P) genes were inserted, yielding 220.5 mg / L acetic acid.

추가로 atoB, atoDA, adc-pdc, ald6-acs(L641P) 유전자가 삽입된 형질전환 균주에 accBC 및 accD1 유전자를 추가로 도입하여 위와 동일한 방법으로 배양하였다. atoB, atoDA, adc-pdc, ald6-acs(L641P), accBC 및 accD1 유전자가 삽입된 형질전환 균주에서는 최대 280.2 mg/L 아세톤이 생산되었고 이때 220.5 mg/L 아세트산이 생산되었다. 따라서 by-pass 및 CCM enforced pathway 유전자가 삽입됨에 따라 아세트산이 대량 생산되며 이를 이용해서 아세톤이 생산이 증가됨을 확인하였다(도 7). In addition, accBC and accD1 genes were further introduced into the transformants into which atoB, atoDA, adc-pdc, and ald6-acs (L641P) genes were inserted and cultured in the same manner as above. In the transfection strain in which atoB, atoDA, adc-pdc, ald6-acs (L641P), accBC and accD1 genes were inserted, up to 280.2 mg / L acetone was produced and 220.5 mg / L acetic acid was produced. Therefore, acetic acid was mass-produced as a by-pass and CCM-enforced pathway gene was inserted, and acetone production was increased using this mass (FIG. 7).

한국미생물보존센터(국외)Korea Microorganism Conservation Center (overseas) KCCM12179KCCM12179 2017113020171130

<110> Research Business Foundation SUNGKYUNKWAN UNIVERSITY <120> TRANSFORMED SYNECHOCOCCUS ELONGATES HAVING CAPABILITY OF ACETONE FROM CARBON DIOXIDE AND METHOD FOR PRODUCING ACETONE USING THE SAME <130> PN1711-422 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 1707 <212> DNA <213> Unknown <220> <223> pdc(pyruvate decarboxylase enzyme) <400> 1 atgagctaca ccgtgggcac ctacctggcc gaacgcctgg tgcagatcgg cctgaaacac 60 cactttgccg tggccggcga ttacaacctg gtgctgctgg ataacctgct gctgaacaaa 120 aacatggaac aggtgtactg ctgcaacgaa ctgaactgcg gctttagcgc cgaaggctac 180 gcccgcgcca aaggcgccgc cgccgccgtg gtgacctaca gcgtgggcgc cctgagcgcc 240 tttgatgcca tcggcggcgc ctacgccgaa aacctgcccg tgatcctgat cagcggcgcc 300 cccaacaaca acgatcacgc cgccggccac gtgctgcacc acgccctggg caaaaccgat 360 taccactacc agctggaaat ggccaaaaac atcaccgccg ccgccgaagc catctacacc 420 cccgaagaag cccccgccaa aatcgatcac gtgatcaaaa ccgccctgcg cgaaaaaaaa 480 cccgtgtacc tggaaatcgc ctgcaacatc gccagcatgc cctgcgccgc ccccggcccc 540 gccagcgccc tgtttaacga tgaagccagc gatgaagcca gcctgaacgc cgccgtggaa 600 gaaaccctga aatttatcgc caaccgcgat aaagtggccg tgctggtggg cagcaaactg 660 cgcgccgccg gcgccgaaga agccgccgtg aaatttgccg atgccctggg cggcgccgtg 720 gccaccatgg ccgccgccaa aagctttttt cccgaagaaa acccccacta catcggcacc 780 agctggggcg aagtgagcta ccccggcgtg gaaaaaacca tgaaagaagc cgatgccgtg 840 atcgccctgg cccccgtgtt taacgattac agcaccaccg gctggaccga tatccccgat 900 cccaaaaaac tggtgctggc cgaaccccgc agcgtggtgg tgaacggcat ccgctttccc 960 agcgtgcacc tgaaagatta cctgacccgc ctggcccaga aagtgagcaa aaaaaccggc 1020 gccctggatt tttttaaaag cctgaacgcc ggcgaactga aaaaagccgc ccccgccgat 1080 cccagcgccc ccctggtgaa cgccgaaatc gcccgccagg tggaagccct gctgaccccc 1140 aacaccaccg tgatcgccga aaccggcgat agctggttta acgcccagcg catgaaactg 1200 cccaacggcg cccgcgtgga atacgaaatg cagtggggcc acatcggctg gagcgtgccc 1260 gccgcctttg gctacgccgt gggcgccccc gaacgccgca acatcctgat ggtgggcgat 1320 ggcagctttc agctgaccgc ccaggaagtg gcccagatgg tgcgcctgaa actgcccgtg 1380 atcatctttc tgatcaacaa ctacggctac accatcgaag tgatgatcca cgatggcccc 1440 tacaacaaca tcaaaaactg ggattacgcc ggcctgatgg aagtgtttaa cggcaacggc 1500 ggctacgata gcggcgccgg caaaggcctg aaagccaaaa ccggcggcga actggccgaa 1560 gccatcaaag tggccctggc caacaccgat ggccccaccc tgatcgaatg ctttatcggc 1620 cgcgaagatt gcaccgaaga actggtgaaa tggggcaaac gcgtggccgc cgccaacagc 1680 cgcaaacccg tgaacaaact gctgtag 1707 <210> 2 <211> 1503 <212> DNA <213> Unknown <220> <223> ald6(aldehyde dehydrogenase enzyme) <400> 2 atgaccaaac tgcactttga taccgccgaa cccgtgaaaa tcaccctgcc caacggcctg 60 acctacgaac agcccaccgg cctgtttatc aacaacaaat ttatgaaagc ccaggatggc 120 aaaacctacc ccgtggaaga tcccagcacc gaaaacaccg tgtgcgaagt gagcagcgcc 180 accaccgaag atgtggaata cgccatcgaa tgcgccgatc gcgcctttca cgataccgaa 240 tgggccaccc aggacccccg cgaacgcggc cgcctgctga gcaaactggc cgatgaactg 300 gaaagccaga tcgatctggt gagcagcatc gaagccctgg ataacggcaa aaccctggcc 360 ctggcccgcg gcgatgtgac catcgccatc aactgcctgc gcgatgccgc cgcctacgcc 420 gataaagtga acggccgcac catcaacacc ggcgatggct acatgaactt taccaccctg 480 gaacccatcg gcgtgtgcgg ccagatcatc ccctggaact ttcccatcat gatgctggcc 540 tggaaaatcg cccccgccct ggccatgggc aacgtgtgca tcctgaaacc cgccgccgtg 600 acccccctga acgccctgta ctttgccagc ctgtgcaaaa aagtgggcat ccccgccggc 660 gtggtgaaca tcgtgcccgg ccccggccgc accgtgggcg ccgccctgac caacgatccc 720 cgcatccgca aactggcctt taccggcagc accgaagtgg gcaaaagcgt ggccgtggat 780 agcagcgaaa gcaacctgaa aaaaatcacc ctggaactgg gcggcaaaag cgcccacctg 840 gtgtttgatg atgccaacat caaaaaaacc ctgcccaacc tggtgaacgg catctttaaa 900 aacgccggcc agatttgcag cagcggcagc cgcatctacg tgcaggaagg catctacgat 960 gaactgctgg ccgcctttaa agcctacctg gaaaccgaaa tcaaagtggg caaccccttt 1020 gataaagcca actttcaggg cgccatcacc aaccgccagc agtttgatac catcatgaac 1080 tacatcgata tcggcaaaaa agaaggcgcc aaaatcctga ccggcggcga aaaagtgggc 1140 gataaaggct actttatccg ccccaccgtg ttttacgatg tgaacgaaga tatgcgcatc 1200 gtgaaagaag aaatctttgg ccccgtggtg accgtggcca aatttaaaac cctggaagaa 1260 ggcgtggaaa tggccaacag cagcgaattt ggcctgggca gcggcatcga aaccgaaagc 1320 ctgagcaccg gcctgaaagt ggccaaaatg ctgaaagccg gcaccgtgtg gatcaacacc 1380 tacaacgatt ttgatagccg cgtgcccttt ggcggcgtga aacagagcgg ctacggccgc 1440 gaaatgggcg aagaagtgta ccacgcctac accgaagtga aagccgtgcg catcaaactg 1500 tag 1503 <210> 3 <211> 1185 <212> DNA <213> Unknown <220> <223> AtoB(Acetyl-CoA acetyltransferase) <400> 3 atgaaaaact gcgtgatcgt gagcgccgtg cgcaccgcca tcggcagctt taacggcagc 60 ctggccagca ccagcgccat cgatctgggc gccaccgtga tcaaagccgc catcgaacgc 120 gccaaaatcg atagccagca cgtggatgaa gtgatcatgg gcaacgtgct ccaggccggc 180 ctgggccaga accccgcccg ccaggccctg ctgaaaagcg gcctggccga aaccgtgtgc 240 ggctttaccg tgaacaaagt gtgcggcagc ggcctgaaaa gcgtggccct ggccgcccag 300 gccatccagg ccggccaggc ccagagcatc gtggccggcg gcatggaaaa catgagcctg 360 gccccctacc tgctggatgc caaagcccgc agcggctacc gcctgggcga tggccaggtg 420 tacgatgtga tcctgcgcga tggcctgatg tgcgccaccc acggctacca catgggcatc 480 accgccgaaa acgtggccaa agaatacggc atcacccgcg aaatgcagga tgaactggcc 540 ctgcacagcc agcgcaaagc cgccgccgcc atcgaaagcg gcgcctttac cgccgaaatc 600 gtgcccgtga acgtggtgac ccgcaaaaaa acctttgtgt ttagccagga tgaatttccc 660 aaagccaaca gcaccgccga agccctgggc gccctgcgcc ccgcctttga taaagccggc 720 accgtgaccg ccggcaacgc cagcggcatc aacgatggcg ccgccgccct ggtgatcatg 780 gaagaaagcg ccgccctggc cgccggcctg acccccctgg cccgcatcaa aagctacgcc 840 agcggcggcg tgccccccgc cctgatgggc atgggccccg tgcccgccac ccagaaagcc 900 ctccagctgg ccggcctcca gctggccgat atcgatctga tcgaagccaa cgaagccttt 960 gccgcccagt ttctggccgt gggcaaaaac ctgggctttg atagcgaaaa agtgaacgtg 1020 aacggcggcg ccatcgccct gggccacccc atcggcgcca gcggcgcccg catcctggtg 1080 accctgctgc acgccatgca ggcccgcgat aaaaccctgg gcctggccac cctgtgcatc 1140 ggcggcggcc agggcatcgc catggtgatc gaacgcctga actag 1185 <210> 4 <211> 1333 <212> DNA <213> Unknown <220> <223> AtoDA(acetotacetate CoA transferase) <400> 4 atgaaaacca aactgatgac cctccaggat gccaccggct tttttcgcga tggcatgacc 60 atcatggtgg gcggctttat gggcatcggc acccccagcc gcctggtgga agccctgctg 120 gaaagcggcg tgcgcgatct gaccctgatc gccaacgata ccgcctttgt ggataccggc 180 atcggccccc tgatcgtgaa cggccgcgtg cgcaaagtga tcgccagcca catcggcacc 240 aaccccgaaa ccggccgccg catgatcagc ggcgaaatgg atgtggtgct ggtgccccag 300 ggcaccctga tcgaacagat ccgctgcggc ggcgccggcc tgggcggctt tctgaccccc 360 accggcgtgg gcaccgtggt ggaagaaggc aaacagaccc tgaccctgga tggcaaaacc 420 tggctgctgg aacgccccct gcgcgccgat ctggccctga tccgcgccca ccgctgcgat 480 accctgggca acctgaccta ccagctgagc gcccgcaact ttaaccccct gatcgccctg 540 gccgccgata tcaccctggt ggaacccgat gaactggtgg aaaccggcga actccagccc 600 gatcacatcg tgacccccgg cgccgtgatc gatcacatca tcgtgagcca ggaaagcaaa 660 tagttaaaga ggagaatact agatggatgc caaacagcgc atcgcccgcc gcgtggccca 720 ggaactgcgc gatggcgata tcgtgaacct gggcatcggc ctgcccacca tggtggccaa 780 ctacctgccc gaaggcatcc acatcaccct ccagagcgaa aacggctttc tgggcctggg 840 ccccgtgacc accgcccacc ccgatctggt gaacgccggc ggccagccct gcggcgtgct 900 gcccggcgcc gccatgtttg atagcgccat gagctttgcc ctgatccgcg gcggccacat 960 cgatgcctgc gtgctgggcg gcctccaggt ggatgaagaa gccaacctgg ccaactgggt 1020 ggtgcccggc aaaatggtgc ccggcatggg cggcgccatg gatctggtga ccggcagccg 1080 caaagtgatc atcgccatgg aacactgcgc caaagatggc agcgccaaaa tcctgcgccg 1140 ctgcaccatg cccctgaccg cccagcacgc cgtgcacatg ctggtgaccg aactggccgt 1200 gtttcgcttt atcgatggca aaatgtggct gaccgaaatc gccgatggct gcgatctggc 1260 caccgtgcgc gccaaaaccg aagcccgctt tgaagtggcc gccgatctga acacccagcg 1320 cggcgatctg tag 1333 <210> 5 <211> 735 <212> DNA <213> Unknown <220> <223> Adc(Acetoacetate decarboxylase) <400> 5 atgctgaaag atgaagtgat caaacagatc agcacccccc tgaccagccc cgcctttccc 60 cgcggcccct acaaatttca caaccgcgaa tactttaaca tcgtgtaccg caccgatatg 120 gatgccctgc gcaaagtggt gcccgaaccc ctggaaatcg atgaacccct ggtgcgcttt 180 gaaatcatgg ccatgcacga taccagcggc ctgggctgct acaccgaaag cggccaggcc 240 atccccgtga gctgcaacgg cgtgaaaggc gattacctgc acatgatgta cctggataac 300 gaacccgcca tcgccgtggg ccgcgaactg agcgcctacc ccaaaaaact gggctacccc 360 aaactgtttg tggatagcga taccctggtg ggcaccctgg attacggcaa actgcgcgtg 420 gccaccgcca ccatgggcta caaacacaaa gccctggatg ccaacgaagc caaagatcag 480 atttgccgcc ccaactacat gctgaaaatc atccccaact acgatggcag cccccgcatc 540 tgcgaactga tcaacgccaa aatcaccgat gtgaccgtgc acgaagcctg gaccggcccc 600 acccgcctcc agctgtttga tcacgccatg gcccccctga acgatctgcc cgtgaaagaa 660 atcgtgagca gcagccacat cctggccgat atcatcctgc cccgcgccga agtgatctac 720 gattacctga aatag 735 <210> 6 <211> 1959 <212> DNA <213> Unknown <220> <223> acsL641P(Salmonella enterica acetyl-CoA synthetase variant) <400> 6 atgagccaga cccacaaaca cgccatcccc gccaacatcg ccgatcgctg cctgatcaac 60 cccgaacagt acgaaaccaa atacaaacag agcatcaacg atcccgatac cttttggggc 120 gaacagggca aaatcctgga ttggatcacc ccctaccaga aagtgaaaaa caccagcttt 180 gcccccggca acgtgagcat caaatggtac gaagatggca ccctgaacct ggccgccaac 240 tgcctggatc gccacctgca ggaaaacggc gatcgcaccg ccatcatctg ggaaggcgat 300 gataccagcc agagcaaaca catcagctac cgcgaactgc accgcgatgt gtgccgcttt 360 gccaacaccc tgctggatct gggcatcaaa aaaggcgatg tggtggccat ctacatgccc 420 atggtgcccg aagccgccgt ggccatgctg gcctgcgccc gcatcggcgc cgtgcacagc 480 gtgatctttg gcggctttag ccccgaagcc gtggccggcc gcatcatcga tagcagcagc 540 cgcctggtga tcaccgccga tgaaggcgtg cgcgccggcc gcagcatccc cctgaaaaaa 600 aacgtggatg atgccctgaa aaaccccaac gtgaccagcg tggaacacgt gatcgtgctg 660 aaacgcaccg gcagcgatat cgattggcag gaaggccgcg atctgtggtg gcgcgatctg 720 atcgaaaaag ccagccccga acaccagccc gaagccatga acgccgaaga tcccctgttt 780 atcctgtaca ccagcggcag caccggcaaa cccaaaggcg tgctgcacac caccggcggc 840 tacctggtgt acgccgccac cacctttaaa tacgtgtttg attaccaccc cggcgatatc 900 tactggtgca ccgccgatgt gggctgggtg accggccaca gctacctgct gtacggcccc 960 ctggcctgcg gcgccaccac cctgatgttt gaaggcgtgc ccaactggcc cacccccgcc 1020 cgcatgtgcc aggtggtgga taaacaccag gtgaacatcc tgtacaccgc ccccaccgcc 1080 atccgcgccc tgatggccga aggcgataaa gccatcgaag gcaccgatcg cagcagcctg 1140 cgcatcctgg gcagcgtggg cgaacccatc aaccccgaag cctgggaatg gtactggaaa 1200 aaaatcggca aagaaaaatg ccccgtggtg gatacctggt ggcagaccga aaccggcggc 1260 tttatgatca cccccctgcc cggcgccatc gaactgaaag ccggcagcgc cacccgcccc 1320 ttttttggcg tgcagcccgc cctggtggat aacgaaggcc acccccagga aggcgccacc 1380 gaaggcaacc tggtgatcac cgatagctgg cccggccagg cccgcaccct gtttggcgat 1440 cacgaacgct ttgaacagac ctactttagc acctttaaaa acatgtactt tagcggcgat 1500 ggcgcccgcc gcgatgaaga tggctactac tggatcaccg gccgcgtgga tgatgtgctg 1560 aacgtgagcg gccaccgcct gggcaccgcc gaaatcgaaa gcgccctggt ggcccacccc 1620 aaaatcgccg aagccgccgt ggtgggcatc ccccacgcca tcaaaggcca ggccatctac 1680 gcctacgtga ccctgaacca cggcgaagaa cccagccccg aactgtacgc cgaagtgcgc 1740 aactgggtgc gcaaagaaat cggccccctg gccacccccg atgtgctgca ctggaccgat 1800 agcctgccca aaacccgcag cggcaaaatc atgcgccgca tcctgcgcaa aatcgccgcc 1860 ggcgatacca gcaacctggg cgataccagc accctggccg atcccggcgt ggtggaaaaa 1920 cccctggaag aaaaacaggc catcgccatg cccagctag 1959 <210> 7 <211> 1807 <212> DNA <213> Unknown <220> <223> accBC(Acetyl-CoA carboxylase) <400> 7 atctagggaa cgcgaaggag gtagaaaaga agtgagcgtg gaaacccgca aaatcaccaa 60 agtgctggtg gccaaccgcg gcgaaatcgc catccgcgtg tttcgcgccg cccgcgatga 120 aggcatcggc agcgtggccg tgtacgccga acccgatgcc gatgccccct ttgtgagcta 180 cgccgatgaa gcctttgccc tgggcggcca gaccagcgcc gaaagctacc tggtgatcga 240 taaaatcatc gatgccgccc gcaaaagcgg cgccgatgcc atccaccccg gctacggctt 300 tctggccgaa aacgccgatt ttgccgaagc cgtgatcaac gaaggcctga tctggatcgg 360 ccccagcccc gaaagcatcc gcagcctggg cgataaagtg accgcccgcc acatcgccga 420 taccgccaaa gcccccatgg cccccggcac caaagaaccc gtgaaagatg ccgccgaagt 480 ggtggccttt gccgaagaat ttggcctgcc catcgccatc aaagccgcct ttggcggcgg 540 cggccgcggc atgaaagtgg cctacaaaat ggaagaagtg gccgatctgt ttgaaagcgc 600 cacccgcgaa gccaccgccg cctttggccg cggcgaatgc tttgtggaac gctacctgga 660 taaagcccgc cacgtggaag cccaggtgat cgccgataaa cacggcaacg tggtggtggc 720 cggcacccgc gattgcagcc tgcagcgccg ctttcagaaa ctggtggaag aagcccccgc 780 cccctttctg accgatgatc agcgcgaacg cctgcacagc agcgccaaag ccatctgcaa 840 agaagccggc tactacggcg ccggcaccgt ggaatacctg gtgggcagcg atggcctgat 900 cagctttctg gaagtgaaca cccgcctgca ggtggaacac cccgtgaccg aagaaaccac 960 cggcatcgat ctggtgcgcg aaatgtttcg catcgccgaa ggccacgaac tgagcatcaa 1020 agaagatccc gccccccgcg gccacgcctt tgaatttcgc atcaacggcg aagatgccgg 1080 cagcaacttt atgcccgccc ccggcaaaat caccagctac cgcgaacccc agggccccgg 1140 cgtgcgcatg gatagcggcg tggtggaagg cagcgaaatc agcggccagt ttgatagcat 1200 gctggccaaa ctgatcgtgt ggggcgatac ccgcgaacag gccctgcagc gcagccgccg 1260 cgccctggcc gaatacgtgg tggaaggcat gcccaccgtg atcccctttc accagcacat 1320 cgtggaaaac cccgcctttg tgggcaacga tgaaggcttt gaaatctaca ccaaatggat 1380 cgaagaagtg tgggataacc ccatcgcccc ctacgtggat gccagcgaac tggatgaaga 1440 tgaagataaa acccccgccc agaaagtggt ggtggaaatc aacggccgcc gcgtggaagt 1500 ggccctgccc ggcgatctgg ccctgggcgg caccgccggc cccaaaaaaa aagccaaaaa 1560 acgccgcgcc ggcggcgcca aagccggcgt gagcggcgat gccgtggccg cccccatgca 1620 gggcaccgtg atcaaagtga acgtggaaga aggcgccgaa gtgaacgaag gcgataccgt 1680 ggtggtgctg gaagccatga aaatggaaaa ccccgtgaaa gcccacaaaa gcggcaccgt 1740 gaccggcctg accgtggccg ccggcgaagg cgtgaacaaa ggcgtggtgc tgctggaaat 1800 caaatag 1807 <210> 8 <211> 1632 <212> DNA <213> Unknown <220> <223> accD1(Acetyl-CoA carboxylase beta-subunit) <400> 8 atgaccatca gcagccccct gatcgatgtg gccaacctgc ccgatatcaa caccaccgcc 60 ggcaaaatcg ccgatctgaa agcccgccgc gccgaagccc actttcccat gggcgaaaaa 120 gccgtggaaa aagtgcacgc cgccggccgc ctgaccgccc gcgaacgcct ggattacctg 180 ctggatgaag gcagctttat cgaaaccgat cagctggccc gccaccgcac caccgccttt 240 ggcctgggcg ccaaacgccc cgccaccgat ggcatcgtga ccggctgggg caccatcgat 300 ggccgcgaag tgtgcatctt tagccaggat ggcaccgtgt ttggcggcgc cctgggcgaa 360 gtgtacggcg aaaaaatgat caaaatcatg gaactggcca tcgataccgg ccgccccctg 420 atcggcctgt acgaaggcgc cggcgcccgc atccaggatg gcgccgtgag cctggatttt 480 atcagccaga ccttttacca gaacatccag gccagcggcg tgatccccca gatcagcgtg 540 atcatgggcg cctgcgccgg cggcaacgcc tacggccccg ccctgaccga ttttgtggtg 600 atggtggata aaaccagcaa aatgtttgtg accggccccg atgtgatcaa aaccgtgacc 660 ggcgaagaaa tcacccagga agaactgggc ggcgccacca cccacatggt gaccgccggc 720 aacagccact acaccgccgc caccgatgaa gaagccctgg attgggtgca ggatctggtg 780 agctttctgc ccagcaacaa ccgcagctac gcccccatgg aagattttga tgaagaagaa 840 ggcggcgtgg aagaaaacat caccgccgat gatctgaaac tggatgaaat catccccgat 900 agcgccaccg tgccctacga tgtgcgcgat gtgatcgaat gcctgaccga tgatggcgaa 960 tacctggaaa tccaggccga tcgcgccgaa aacgtggtga tcgcctttgg ccgcatcgaa 1020 ggccagagcg tgggctttgt ggccaaccag cccacccagt ttgccggctg cctggatatc 1080 gatagcagcg aaaaagccgc ccgctttgtg cgcacctgcg atgcctttaa catccccatc 1140 gtgatgctgg tggatgtgcc cggctttctg cccggcgccg gccaggaata cggcggcatc 1200 ctgcgccgcg gcgccaaact gctgtacgcc tacggcgaag ccaccgtgcc caaaatcacc 1260 gtgaccatgc gcaaagccta cggcggcgcc tactgcgtga tgggcagcaa aggcctgggc 1320 agcgatatca acctggcctg gcccaccgcc cagatcgccg tgatgggcgc cgccggcgcc 1380 gtgggcttta tctaccgcaa agaactgatg gccgccgatg ccaaaggcct ggataccgtg 1440 gccctggcca aaagctttga acgcgaatac gaagatcaca tgctgaaccc ctaccacgcc 1500 gccgaacgcg gcctgatcga tgccgtgatc ctgcccagcg aaacccgcgg ccagatcagc 1560 cgcaacctgc gcctgctgaa acacaaaaac gtgacccgcc ccgcccgcaa acacggcaac 1620 atgcccctgt ag 1632 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSI-forward <400> 9 aagcgctccg catggatctg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSI-reverse <400> 10 caaggcagct tggaagggcg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSII-forward <400> 11 ggctacggtt cgtaatgcca 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSII-reverse <400> 12 gagatcaggg ctgtacttac 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSIII-forward <400> 13 ccacgcattc aaccaagggc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSIII-reverse <400> 14 cacaccctca gagacaagcc 20 <110> Research Business Foundation SUNGKYUNKWAN UNIVERSITY <120> TRANSFORMED SYNECHOCOCCUS ELONGATES HAVING CAPABILITY OF ACETONE          FROM CARBON DIOXIDE AND METHOD FOR PRODUCING ACETONE USING THE          SAME <130> PN1711-422 <160> 14 <170> KoPatentin 3.0 <210> 1 <211> 1707 <212> DNA <213> Unknown <220> Pdc (pyruvate decarboxylase enzyme) <400> 1 atgagctaca ccgtgggcac ctacctggcc gaacgcctgg tgcagatcgg cctgaaacac 60 cactttgccg tggccggcga ttacaacctg gtgctgctgg ataacctgct gctgaacaaa 120 aacatggaac aggtgtactg ctgcaacgaa ctgaactgcg gctttagcgc cgaaggctac 180 gcccgcgcca aaggcgccgc cgccgccgtg gtgacctaca gcgtgggcgc cctgagcgcc 240 tttgatgcca tcggcggcgc ctacgccgaa aacctgcccg tgatcctgat cagcggcgcc 300 cccaacaaca acgatcacgc cgccggccac gtgctgcacc acgccctggg caaaaccgat 360 tccactacc agctggaaat ggccaaaaac atcaccgccg ccgccgaagc catctacacc 420 cccgaagaag cccccgccaa aatcgatcac gtgatcaaaa ccgccctgcg cgaaaaaaaa 480 cccgtgtacc tggaaatcgc ctgcaacatc gccagcatgc cctgcgccgc ccccggcccc 540 gccagcgccc tgtttaacga tgaagccagc gatgaagcca gcctgaacgc cgccgtggaa 600 gaaaccctga aatttatcgc caaccgcgat aaagtggccg tgctggtggg cagcaaactg 660 cgcgccgccg gcgccgaaga agccgccgtg aaatttgccg atgccctggg cggcgccgtg 720 gccaccatgg ccgccgccaa aagctttttt cccgaagaaa acccccacta catcggcacc 780 agctggggcg aagtgagcta ccccggcgtg gaaaaaacca tgaaagaagc cgatgccgtg 840 atcgccctgg cccccgtgtt taacgattac agcaccaccg gctggaccga tatccccgat 900 cccaaaaaac tggtgctggc cgaaccccgc agcgtggtgg tgaacggcat ccgctttccc 960 agcgtgcacc tgaaagatta cctgacccgc ctggcccaga aagtgagcaa aaaaaccggc 1020 gccctggatt tttttaaaag cctgaacgcc ggcgaactga aaaaagccgc ccccgccgat 1080 cccagcgccc ccctggtgaa cgccgaaatc gcccgccagg tggaagccct gctgaccccc 1140 aacaccaccg tgatcgccga aaccggcgat agctggttta acgcccagcg catgaaactg 1200 cccaacggcg cccgcgtgga atacgaaatg cagtggggcc acatcggctg gagcgtgccc 1260 gccgcctttg gctacgccgt gggcgccccc gaacgccgca acatcctgat ggtgggcgat 1320 ggcagctttc agctgaccgc ccaggaagtg gcccagatgg tgcgcctgaa actgcccgtg 1380 atcatctttc tgatcaacaa ctacggctac accatcgaag tgatgatcca cgatggcccc 1440 tacaacaca tcaaaaactg ggattacgcc ggcctgatgg aagtgtttaa cggcaacggc 1500 ggctacgata gcggcgccgg caaaggcctg aaagccaaaa ccggcggcga actggccgaa 1560 gccatcaaag tggccctggc caacaccgat ggccccaccc tgatcgaatg ctttatcggc 1620 cgcgaagatt gcaccgaaga actggtgaaa tggggcaaac gcgtggccgc cgccaacagc 1680 cgcaaacccg tgaacaaact gctgtag 1707 <210> 2 <211> 1503 <212> DNA <213> Unknown <220> Ald6 (aldehyde dehydrogenase enzyme) <400> 2 atgaccaaac tgcactttga taccgccgaa cccgtgaaaa tcaccctgcc caacggcctg 60 acctacgaac agcccaccgg cctgtttatc aacaacaaat ttatgaaagc ccaggatggc 120 aaaacctacc ccgtggaaga tcccagcacc gaaaacaccg tgtgcgaagt gagcagcgcc 180 accaccgaag atgtggaata cgccatcgaa tgcgccgatc gcgcctttca cgataccgaa 240 tgggccaccc aggacccccg cgaacgcggc cgcctgctga gcaaactggc cgatgaactg 300 gaaagccaga tcgatctggt gagcagcatc gaagccctgg ataacggcaa aaccctggcc 360 ctggcccgcg gcgatgtgac catcgccatc aactgcctgc gcgatgccgc cgcctacgcc 420 gataaagtga acggccgcac catcaacacc ggcgatggct acatgaactt taccaccctg 480 gaacccatcg gcgtgtgcgg ccagatcatc ccctggaact ttcccatcat gatgctggcc 540 tggaaaatcg cccccgccct ggccatgggc aacgtgtgca tcctgaaacc cgccgccgtg 600 acccccctga acgccctgta ctttgccagc ctgtgcaaaa aagtgggcat ccccgccggc 660 gtggtgaaca tcgtgcccgg ccccggccgc accgtgggcg ccgccctgac caacgatccc 720 cgcatccgca aactggcctt taccggcagc accgaagtgg gcaaaagcgt ggccgtggat 780 agcagcgaaa gcaacctgaa aaaaatcacc ctggaactgg gcggcaaaag cgcccacctg 840 gtgtttgatg atgccaacat caaaaaaacc ctgcccaacc tggtgaacgg catctttaaa 900 aacgccggcc agatttgcag cagcggcagc cgcatctacg tgcaggaagg catctacgat 960 gaactgctgg ccgcctttaa agcctacctg gaaaccgaaa tcaaagtggg caaccccttt 1020 gataaagcca actttcaggg cgccatcacc aaccgccagc agtttgatac catcatgaac 1080 tacatcgata tcggcaaaaa agaaggcgcc aaaatcctga ccggcggcga aaaagtgggc 1140 gataaaggct actttatccg ccccaccgtg ttttacgatg tgaacgaaga tatgcgcatc 1200 gtgaaagaag aaatctttgg ccccgtggtg accgtggcca aatttaaaac cctggaagaa 1260 ggcgtggaaa tggccaacag cagcgaattt ggcctgggca gcggcatcga aaccgaaagc 1320 ctgagcaccg gcctgaaagt ggccaaaatg ctgaaagccg gcaccgtgtg gatcaacacc 1380 tacaacgatt ttgatagccg cgtgcccttt ggcggcgtga aacagagcgg ctacggccgc 1440 gaaatgggcg aagaagtgta ccacgcctac accgaagtga aagccgtgcg catcaaactg 1500 tag 1503 <210> 3 <211> 1185 <212> DNA <213> Unknown <220> <223> AtoB (Acetyl-CoA acetyltransferase) <400> 3 atgaaaaact gcgtgatcgt gagcgccgtg cgcaccgcca tcggcagctt taacggcagc 60 ctggccagca ccagcgccat cgatctgggc gccaccgtga tcaaagccgc catcgaacgc 120 gccaaaatcg atagccagca cgtggatgaa gtgatcatgg gcaacgtgct ccaggccggc 180 ctgggccaga accccgcccg ccaggccctg ctgaaaagcg gcctggccga aaccgtgtgc 240 ggctttaccg tgaacaaagt gtgcggcagc ggcctgaaaa gcgtggccct ggccgcccag 300 gccatccagg ccggccaggc ccagagcatc gtggccggcg gcatggaaaa catgagcctg 360 gccccctacc tgctggatgc caaagcccgc agcggctacc gcctgggcga tggccaggtg 420 tacgatgtga tcctgcgcga tggcctgatg tgcgccaccc acggctacca catgggcatc 480 accgccgaaa acgtggccaa agaatacggc atcacccgcg aaatgcagga tgaactggcc 540 ctgcacagcc agcgcaaagc cgccgccgcc atcgaaagcg gcgcctttac cgccgaaatc 600 gtgcccgtga acgtggtgac ccgcaaaaaa acctttgtgt ttagccagga tgaatttccc 660 aaagccaaca gcaccgccga agccctgggc gccctgcgcc ccgcctttga taaagccggc 720 accgtgaccg ccggcaacgc cagcggcatc aacgatggcg ccgccgccct ggtgatcatg 780 gaagaaagcg ccgccctggc cgccggcctg acccccctgg cccgcatcaa aagctacgcc 840 agcggcggcg tgccccccgc cctgatgggc atgggccccg tgcccgccac ccagaaagcc 900 ctccagctgg ccggcctcca gctggccgat atcgatctga tcgaagccaa cgaagccttt 960 gccgcccagt ttctggccgt gggcaaaaac ctgggctttg atagcgaaaa agtgaacgtg 1020 aacggcggcg ccatcgccct gggccacccc atcggcgcca gcggcgcccg catcctggtg 1080 accctgctgc acgccatgca ggcccgcgat aaaaccctgg gcctggccac cctgtgcatc 1140 ggcggcggcc agggcatcgc catggtgatc gaacgcctga actag 1185 <210> 4 <211> 1333 <212> DNA <213> Unknown <220> <223> AtoDA (acetotacetate CoA transferase) <400> 4 atgaaaacca aactgatgac cctccaggat gccaccggct tttttcgcga tggcatgacc 60 atcatggtgg gcggctttat gggcatcggc acccccagcc gcctggtgga agccctgctg 120 gaaagcggcg tgcgcgatct gaccctgatc gccaacgata ccgcctttgt ggataccggc 180 atcggccccc tgatcgtgaa cggccgcgtg cgcaaagtga tcgccagcca catcggcacc 240 aaccccgaaa ccggccgccg catgatcagc ggcgaaatgg atgtggtgct ggtgccccag 300 ggcaccctga tcgaacagat ccgctgcggc ggcgccggcc tgggcggctt tctgaccccc 360 accggcgtgg gcaccgtggt ggaagaaggc aaacagaccc tgaccctgga tggcaaaacc 420 tggctgctgg aacgccccct gcgcgccgat ctggccctga tccgcgccca ccgctgcgat 480 accctgggca acctgaccta ccagctgagc gcccgcaact ttaaccccct gatcgccctg 540 gccgccgata tcaccctggt ggaacccgat gaactggtgg aaaccggcga actccagccc 600 gatcacatcg tgacccccgg cgccgtgatc gatcacatca tcgtgagcca ggaaagcaaa 660 tagttaaaga ggagaatact agatggatgc caaacagcgc atcgcccgcc gcgtggccca 720 ggaactgcgc gatggcgata tcgtgaacct gggcatcggc ctgcccacca tggtggccaa 780 ctacctgccc gaaggcatcc acatcaccct ccagagcgaa aacggctttc tgggcctggg 840 ccccgtgacc accgcccacc ccgatctggt gaacgccggc ggccagccct gcggcgtgct 900 gcccggcgcc gccatgtttg atagcgccat gagctttgcc ctgatccgcg gcggccacat 960 cgatgcctgc gtgctgggcg gcctccaggt ggatgaagaa gccaacctgg ccaactgggt 1020 ggtgcccggc aaaatggtgc ccggcatggg cggcgccatg gatctggtga ccggcagccg 1080 caaagtgatc atcgccatgg aacactgcgc caaagatggc agcgccaaaa tcctgcgccg 1140 ctgcaccatg cccctgaccg cccagcacgc cgtgcacatg ctggtgaccg aactggccgt 1200 gtttcgcttt atcgatggca aaatgtggct gaccgaaatc gccgatggct gcgatctggc 1260 cccgtgcgc gccaaaaccg aagcccgctt tgaagtggcc gccgatctga acacccagcg 1320 cggcgatctg tag 1333 <210> 5 <211> 735 <212> DNA <213> Unknown <220> <223> Acetoacetate decarboxylase (Adc) <400> 5 atgctgaaag atgaagtgat caaacagatc agcacccccc tgaccagccc cgcctttccc 60 cgcggcccct acaaatttca caaccgcgaa tactttaaca tcgtgtaccg caccgatatg 120 gatgccctgc gcaaagtggt gcccgaaccc ctggaaatcg atgaacccct ggtgcgcttt 180 cggccggcc atccccgtga gctgcaacgg cgtgaaaggc gattacctgc acatgatgta cctggataac 300 gaacccgcca tcgccgtggg ccgcgaactg agcgcctacc ccaaaaaact gggctacccc 360 aaactgtttg tggatagcga taccctggtg ggcaccctgg attacggcaa actgcgcgtg 420 gccaccgcca ccatgggcta caaacacaaa gccctggatg ccaacgaagc caaagatcag 480 atttgccgcc ccaactacat gctgaaaatc atccccaact acgatggcag cccccgcatc 540 tgcgaactga tcaacgccaa aatcaccgat gtgaccgtgc acgaagcctg gaccggcccc 600 acccgcctcc agctgtttga tcacgccatg gcccccctga acgatctgcc cgtgaaagaa 660 atcgtgagca gcagccacat cctggccgat atcatcctgc cccgcgccga agtgatctac 720 gattacctga aatag 735 <210> 6 <211> 1959 <212> DNA <213> Unknown <220> &Lt; 223 > AcSL641P (Salmonella enterica acetyl-CoA synthetase variant) <400> 6 atgagccaga cccacaaaca cgccatcccc gccaacatcg ccgatcgctg cctgatcaac 60 cccgaacagt acgaaaccaa atacaaacag agcatcaacg atcccgatac cttttggggc 120 gaacagggca aaatcctgga ttggatcacc ccctaccaga aagtgaaaaa caccagcttt 180 gcccccggca acgtgagcat caaatggtac gaagatggca ccctgaacct ggccgccaac 240 tgcctggatc gccacctgca ggaaaacggc gatcgcaccg ccatcatctg ggaaggcgat 300 gataccagcc agagcaaaca catcagctac cgcgaactgc accgcgatgt gtgccgcttt 360 gccaacaccc tgctggatct gggcatcaaa aaaggcgatg tggtggccat ctacatgccc 420 atggtgcccg aagccgccgt ggccatgctg gcctgcgccc gcatcggcgc cgtgcacagc 480 gtgatctttg gcggctttag ccccgaagcc gtggccggcc gcatcatcga tagcagcagc 540 cgcctggtga tcaccgccga tgaaggcgtg cgcgccggcc gcagcatccc cctgaaaaaa 600 aacgtggatg atgccctgaa aaaccccaac gtgaccagcg tggaacacgt gatcgtgctg 660 aaacgcaccg gcagcgatat cgattggcag gaaggccgcg atctgtggtg gcgcgatctg 720 atcgaaaaag ccagccccga acaccagccc gaagccatga acgccgaaga tcccctgttt 780 atcctgtaca ccagcggcag caccggcaaa cccaaaggcg tgctgcacac caccggcggc 840 tacctggtgt acgccgccac cacctttaaa tacgtgtttg attaccaccc cggcgatatc 900 tactggtgca ccgccgatgt gggctgggtg accggccaca gctacctgct gtacggcccc 960 ctggcctgcg gcgccaccac cctgatgttt gaaggcgtgc ccaactggcc cacccccgcc 1020 cgcatgtgcc aggtggtgga taaacaccag gtgaacatcc tgtacaccgc ccccaccgcc 1080 atccgcgccc tgatggccga aggcgataaa gccatcgaag gcaccgatcg cagcagcctg 1140 cgcatcctgg gcagcgtggg cgaacccatc aaccccgaag cctgggaatg gtactggaaa 1200 aaaatcggca aagaaaaatg ccccgtggtg gatacctggt ggcagaccga aaccggcggc 1260 tttatgatca cccccctgcc cggcgccatc gaactgaaag ccggcagcgc cacccgcccc 1320 ttttttggcg tgcagcccgc cctggtggat aacgaaggcc acccccagga aggcgccacc 1380 gaaggcaacc tggtgatcac cgatagctgg cccggccagg cccgcaccct gtttggcgat 1440 cacgaacgct ttgaacagac ctactttagc acctttaaaa acatgtactt tagcggcgat 1500 ggcgcccgcc gcgatgaaga tggctactac tggatcaccg gccgcgtgga tgatgtgctg 1560 aacgtgagcg gccaccgcct gggcaccgcc gaaatcgaaa gcgccctggt ggcccacccc 1620 aaaatcgccg aagccgccgt ggtgggcatc ccccacgcca tcaaaggcca ggccatctac 1680 gcctacgtga ccctgaacca cggcgaagaa cccagccccg aactgtacgc cgaagtgcgc 1740 aactgggtgc gcaaagaaat cggccccctg gccacccccg atgtgctgca ctggaccgat 1800 agcctgccca aaacccgcag cggcaaaatc atgcgccgca tcctgcgcaa aatcgccgcc 1860 ggcgatacca gcaacctggg cgataccagc accctggccg atcccggcgt ggtggaaaaa 1920 cccctggaag aaaaacaggc catcgccatg cccagctag 1959 <210> 7 <211> 1807 <212> DNA <213> Unknown <220> &Lt; 223 > AccBC (Acetyl-CoA carboxylase) <400> 7 atctagggaa cgcgaaggag gtagaaaaga agtgagcgtg gaaacccgca aaatcaccaa 60 agtgctggtg gccaaccgcg gcgaaatcgc catccgcgtg tttcgcgccg cccgcgatga 120 aggcatcggc agcgtggccg tgtacgccga acccgatgcc gatgccccct ttgtgagcta 180 cgccgatgaa gcctttgccc tgggcggcca gaccagcgcc gaaagctacc tggtgatcga 240 taaaatcatc gatgccgccc gcaaaagcgg cgccgatgcc atccaccccg gctacggctt 300 tctggccgaa aacgccgatt ttgccgaagc cgtgatcaac gaaggcctga tctggatcgg 360 ccccagcccc gaaagcatcc gcagcctggg cgataaagtg accgcccgcc acatcgccga 420 taccgccaaa gcccccatgg cccccggcac caaagaaccc gtgaaagatg ccgccgaagt 480 ggtggccttt gccgaagaat ttggcctgcc catcgccatc aaagccgcct ttggcggcgg 540 cggccgcggc atgaaagtgg cctacaaaat ggaagaagtg gccgatctgt ttgaaagcgc 600 ccccgcgaa gccaccgccg cctttggccg cggcgaatgc tttgtggaac gctacctgga 660 taaagcccgc cacgtggaag cccaggtgat cgccgataaa cacggcaacg tggtggtggc 720 cggcacccgc gattgcagcc tgcagcgccg ctttcagaaa ctggtggaag aagcccccgc 780 cccctttctg accgatgatc agcgcgaacg cctgcacagc agcgccaaag ccatctgcaa 840 agaagccggc tactacggcg ccggcaccgt ggaatacctg gtgggcagcg atggcctgat 900 cagctttctg gaagtgaaca cccgcctgca ggtggaacac cccgtgaccg aagaaaccac 960 cggcatcgat ctggtgcgcg aaatgtttcg catcgccgaa ggccacgaac tgagcatcaa 1020 agaagatccc gccccccgcg gccacgcctt tgaatttcgc atcaacggcg aagatgccgg 1080 cagcaacttt atgcccgccc ccggcaaaat caccagctac cgcgaacccc agggccccgg 1140 cgtgcgcatg gatagcggcg tggtggaagg cagcgaaatc agcggccagt ttgatagcat 1200 gctggccaaa ctgatcgtgt ggggcgatac ccgcgaacag gccctgcagc gcagccgccg 1260 cgccctggcc gaatacgtgg tggaaggcat gcccaccgtg atcccctttc accagcacat 1320 cgtggaaaac cccgcctttg tgggcaacga tgaaggcttt gaaatctaca ccaaatggat 1380 cgaagaagtg tgggataacc ccatcgcccc ctacgtggat gccagcgaac tggatgaaga 1440 tgaagataaa acccccgccc agaaagtggt ggtggaaatc aacggccgcc gcgtggaagt 1500 ggccctgccc ggcgatctgg ccctgggcgg caccgccggc cccaaaaaaa aagccaaaaa 1560 acgccgcgcc ggcggcgcca aagccggcgt gagcggcgat gccgtggccg cccccatgca 1620 gggcaccgtg atcaaagtga acgtggaaga aggcgccgaa gtgaacgaag gcgataccgt 1680 ggtggtgctg gaagccatga aaatggaaaa ccccgtgaaa gcccacaaaa gcggcaccgt 1740 gccggcctg accgtggccg ccggcgaagg cgtgaacaaa ggcgtggtgc tgctggaaat 1800 caaatag 1807 <210> 8 <211> 1632 <212> DNA <213> Unknown <220> &Lt; 223 > accDl (Acetyl-CoA carboxylase beta-subunit) <400> 8 atgaccatca gcagccccct gatcgatgtg gccaacctgc ccgatatcaa caccaccgcc 60 ggcaaaatcg ccgatctgaa agcccgccgc gccgaagccc actttcccat gggcgaaaaa 120 gccgtggaaa aagtgcacgc cgccggccgc ctgaccgccc gcgaacgcct ggattacctg 180 ctggatgaag gcagctttat cgaaaccgat cagctggccc gccaccgcac caccgccttt 240 ggcctgggcg ccaaacgccc cgccaccgat ggcatcgtga ccggctgggg caccatcgat 300 ggccgcgaag tgtgcatctt tagccaggat ggcaccgtgt ttggcggcgc cctgggcgaa 360 gtgtacggcg aaaaaatgat caaaatcatg gaactggcca tcgataccgg ccgccccctg 420 atcggcctgt acgaaggcgc cggcgcccgc atccaggatg gcgccgtgag cctggatttt 480 atcagccaga ccttttacca gaacatccag gccagcggcg tgatccccca gatcagcgtg 540 atcatgggcg cctgcgccgg cggcaacgcc tacggccccg ccctgaccga ttttgtggtg 600 atggtggata aaaccagcaa aatgtttgtg accggccccg atgtgatcaa aaccgtgacc 660 ggcgaagaaa tcacccagga agaactgggc ggcgccacca cccacatggt gaccgccggc 720 aacagccact acaccgccgc caccgatgaa gaagccctgg attgggtgca ggatctggtg 780 agctttctgc ccagcaacaa ccgcagctac gcccccatgg aagattttga tgaagaagaa 840 ggcggcgtgg aagaaaacat caccgccgat gatctgaaac tggatgaaat catccccgat 900 agcgccaccg tgccctacga tgtgcgcgat gtgatcgaat gcctgaccga tgatggcgaa 960 tacctggaaa tccaggccga tcgcgccgaa aacgtggtga tcgcctttgg ccgcatcgaa 1020 ggccagagcg tgggctttgt ggccaaccag cccacccagt ttgccggctg cctggatatc 1080 gatagcagcg aaaaagccgc ccgctttgtg cgcacctgcg atgcctttaa catccccatc 1140 gtgatgctgg tggatgtgcc cggctttctg cccggcgccg gccaggaata cggcggcatc 1200 ctgcgccgcg gcgccaaact gctgtacgcc tacggcgaag ccaccgtgcc caaaatcacc 1260 gtgaccatgc gcaaagccta cggcggcgcc tactgcgtga tgggcagcaa aggcctgggc 1320 agcgatatca acctggcctg gcccaccgcc cagatcgccg tgatgggcgc cgccggcgcc 1380 gtgggcttta tctaccgcaa agaactgatg gccgccgatg ccaaaggcct ggataccgtg 1440 gccctggcca aaagctttga acgcgaatac gaagatcaca tgctgaaccc ctaccacgcc 1500 gccgaacgcg gcctgatcga tgccgtgatc ctgcccagcg aaacccgcgg ccagatcagc 1560 cgcaacctgc gcctgctgaa acacaaaaac gtgacccgcc ccgcccgcaa acacggcaac 1620 atgcccctgt ag 1632 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSI-forward <400> 9 aagcgctccg catggatctg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSI-reverse <400> 10 caaggcagct tggaagggcg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSII-forward <400> 11 ggctacggtt cgtaatgcca 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSII-reverse <400> 12 gagatcaggg ctgtacttac 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSIII-forward <400> 13 ccacgcattc aaccaagggc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NSIII-reverse <400> 14 cacaccctca gagacaagcc 20

Claims (12)

서열번호 1의 서열로 표시되는 피루부산 탈탄산효소를 코딩하는 유전자 pdc(pyruvate decarboxylase enzyme); 및 서열번호 2의 서열로 표시되는 알데히드 탈수소효소를 코딩하는 유전자 ald6(aldehyde dehydrogenase enzyme),
서열번호 3의 서열로 표시되는 acetyl-CoA로부터 acetoacetyl-CoA를 생산하는 효소를 코딩하는 유전자 AtoB(Acetyl-CoA acetyltransferase); 서열번호 4의 서열로 표시되는 acetoacetyl-CoA로부터 acetoacetate를 생산하는 효소를 코딩하는 유전자 AtoDA(acetotacetate CoA transferase); 및 서열번호 5의 서열로 표시되는 acetoacetate로부터 아세톤을 생산하는 효소를 코딩하는 유전자 Adc(Acetoacetate decarboxylase),
및 서열번호 6의 서열로 표시되는 acetate-coA 리가아제 효소을 코딩하는 유전자 acsL641P(Salmonella enterica acetyl-CoA synthetase variant); 서열번호 7의 서열로 표시되는 Acetyl-CoA 카르복실화 효소를 코딩하는 유전자 accBC(Acetyl-CoA carboxylase); 및 서열번호 8의 서열로 표시되는 Acetyl-CoA 카르복실화 효소를 코딩하는 유전자 accD1(Acetyl-CoA carboxylase beta-subunit)를 포함하는, 시네코코커스 일롱게투스(Synechococcus elongatus) 균주.A gene pdc (pyruvate decarboxylase enzyme) encoding a pyruvate decarboxylase expressed by the sequence of SEQ ID NO: 1; And aldehyde dehydrogenase enzyme (ald6) gene encoding an aldehyde dehydrogenase represented by the sequence of SEQ ID NO: 2,
AtoB (Acetyl-CoA acetyltransferase) gene encoding an enzyme that produces acetoacetyl-CoA from acetyl-CoA represented by the sequence of SEQ ID NO: 3; AtoDA (acetotacetate CoA transferase) gene encoding an enzyme that produces acetoacetate from acetoacetyl-CoA represented by the sequence of SEQ ID NO: 4; And Adc (Acetoacetate decarboxylase), which encodes an enzyme that produces acetone from acetoacetate represented by the sequence of SEQ ID NO: 5,
And the gene acsL641P (Salmonella enterica acetyl-CoA synthetase variant) encoding the acetate-coA ligase enzyme represented by the sequence of SEQ ID NO: 6; The gene accBC (Acetyl-CoA carboxylase) encoding the Acetyl-CoA carboxylating enzyme represented by the sequence of SEQ ID NO: 7; And Synechococcus elongatus strain comprising the gene accDl (Acetyl-CoA carboxylase beta-subunit) encoding the Acetyl-CoA carboxylatase represented by the sequence of SEQ ID NO: 8. 삭제delete 삭제delete 제 1항에 있어서,
상기 균주는 제 2 벡터로 형질전환 된 것으로서,
상기 제 2 벡터는 서열번호 1의 서열로 표시되는 피루부산 탈탄산효소를 코딩하는 유전자 pdc(pyruvate decarboxylase enzyme); 및
서열번호 2의 서열로 표시되는 알데히드 탈수소효소를 코딩하는 유전자 ald6(aldehyde dehydrogenase enzyme)를 포함하는 시네코코커스 일롱게투스 균주.The method according to claim 1,
The strain was transformed with a second vector,
The second vector comprises a gene encoding pyruvate decarboxylase enzyme pdc (pyruvate decarboxylase enzyme) represented by the sequence of SEQ ID NO: 1; And
A Cynekococus Iylongus strain comprising the aldehyde dehydrogenase enzyme gene encoding the aldehyde dehydrogenase represented by the sequence of SEQ ID NO: 2. 재 1항에 있어서,
상기 균주는 제 1 벡터로 형질전환 된 것으로서,
상기 제 1 벡터는 서열번호 3의 서열로 표시되는 acetyl-CoA로부터 acetoacetyl-CoA를 생산하는 효소를 코딩하는 유전자 AtoB(Acetyl-CoA acetyltransferase); 서열번호 4의 서열로 표시되는 acetoacetyl-CoA로부터 acetoacetate를 생산하는 효소를 코딩하는 유전자 AtoDA(acetotacetate CoA transferase); 및 서열번호 5의 서열로 표시되는 acetoacetate로부터 아세톤을 생산하는 효소를 코딩하는 유전자 Adc(Acetoacetate decarboxylase)를 포함하는 시네코코커스 일롱게투스 균주.In the first aspect,
The strain was transformed with a first vector,
The first vector comprises a gene (Acoyl-CoA acetyltransferase) encoding an enzyme that produces acetoacetyl-CoA from acetyl-CoA represented by the sequence of SEQ ID NO: 3; AtoDA (acetotacetate CoA transferase) gene encoding an enzyme that produces acetoacetate from acetoacetyl-CoA represented by the sequence of SEQ ID NO: 4; And a gene encoding Adc (Acetoacetate decarboxylase) encoding an enzyme that produces acetone from acetoacetate represented by the sequence of SEQ ID NO: 5. 제 1항에 있어서,
상기 균주는 제 3 벡터로 형질전환 된 것으로서,
상기 제 3 벡터는 서열번호 6의 서열로 표시되는 acetate-coA 리가아제 효소을 코딩하는 유전자 acsL641P(Salmonella enterica acetyl-CoA synthetase variant); 서열번호 7의 서열로 표시되는 Acetyl-CoA 카르복실화 효소를 코딩하는 유전자 accBC(Acetyl-CoA carboxylase); 및 서열번호 8의 서열로 표시되는 Acetyl-CoA 카르복실화 효소를 코딩하는 유전자 accD1(Acetyl-CoA carboxylase beta-subunit)을 포함하는 시네코코커스 일롱게투스 균주.The method according to claim 1,
The strain was transformed with a third vector,
The third vector is a gene acs L641P (Salmonella enterica acetyl-CoA synthetase variant) encoding an acetate-coA ligase enzyme represented by the sequence of SEQ ID NO: 6; The gene accBC (Acetyl-CoA carboxylase) encoding the Acetyl-CoA carboxylating enzyme represented by the sequence of SEQ ID NO: 7; And a gene accDl (Acetyl-CoA carboxylase beta-subunit) encoding the Acetyl-CoA carboxylating enzyme represented by the sequence of SEQ ID NO: 8. 제 4항에 있어서,
상기 제 2 벡터는
카나마이신(kanamycin) 저항성 유전자; lacI 리프레서; trc 프로모터; 및
서열번호 1의 서열로 표시되는 피루부산 탈탄산효소를 코딩하는 유전자 pdc(pyruvate decarboxylase enzyme); 및
서열번호 2의 서열로 표시되는 알데히드 탈수소효소를 코딩하는 유전자 ald6(aldehyde dehydrogenase enzyme)을 포함하는 시네코코커스 일롱게투스 균주.5. The method of claim 4,
The second vector
Kanamycin resistance gene; lacI refresher; trc promoter; And
A gene pdc (pyruvate decarboxylase enzyme) encoding a pyruvate decarboxylase expressed by the sequence of SEQ ID NO: 1; And
A Cynekocus Iylongutus strain comprising the aldehyde dehydrogenase enzyme gene encoding an aldehyde dehydrogenase represented by the sequence of SEQ ID NO: 2. 제 5항에 있어서,
상기 제 1 벡터는
스펙티노마이신(spectinomycin) 저항성 유전자; lacI 리프레서; trc 프로모터; 및
서열번호 3의 서열로 표시되는 acetyl-CoA로부터 acetoacetyl-CoA를 생산하는 효소를 코딩하는 유전자 AtoB(Acetyl-CoA acetyltransferase); 서열번호 4의 서열로 표시되는 acetoacetyl-CoA로부터 acetoacetate를 생산하는 효소를 코딩하는 유전자 AtoDA(acetotacetate CoA transferase); 및 서열번호 5의 서열로 표시되는 acetoacetate로부터 아세톤을 생산하는 효소를 코딩하는 유전자 Adc(Acetoacetate decarboxylase)를 포함하는 시네코코커스 일롱게투스 균주.6. The method of claim 5,
The first vector
A spectinomycin resistance gene; lacI refresher; trc promoter; And
AtoB (Acetyl-CoA acetyltransferase) gene encoding an enzyme that produces acetoacetyl-CoA from acetyl-CoA represented by the sequence of SEQ ID NO: 3; AtoDA (acetotacetate CoA transferase) gene encoding an enzyme that produces acetoacetate from acetoacetyl-CoA represented by the sequence of SEQ ID NO: 4; And a gene encoding Adc (Acetoacetate decarboxylase) encoding an enzyme that produces acetone from acetoacetate represented by the sequence of SEQ ID NO: 5. 제 6항에 있어서,
상기 제 3 벡터는
클로람페니콜(Chloramphenicol) 저항성 유전자; lacI 리프레서; trc 프로모터; 및
서열번호 6의 서열로 표시되는 acetate-coA 리가아제 효소을 코딩하는 유전자 acsL641P(Salmonella enterica acetyl-CoA synthetase variant); 서열번호 7의 서열로 표시되는 Acetyl-CoA 카르복실화 효소를 코딩하는 유전자 accBC(Acetyl-CoA carboxylase); 및 서열번호 8의 서열로 표시되는 Acetyl-CoA 카르복실화 효소를 코딩하는 유전자 accD1(Acetyl-CoA carboxylase beta-subunit)을 포함하는 시네코코커스 일롱게투스 균주. The method according to claim 6,
The third vector
Chloramphenicol resistance gene; lacI refresher; trc promoter; And
The gene acs L641P (Salmonella enterica acetyl-CoA synthetase variant) encoding the acetate-coA ligase enzyme represented by the sequence of SEQ ID NO: 6; The gene accBC (Acetyl-CoA carboxylase) encoding the Acetyl-CoA carboxylating enzyme represented by the sequence of SEQ ID NO: 7; And a gene accDl (Acetyl-CoA carboxylase beta-subunit) encoding the Acetyl-CoA carboxylating enzyme represented by the sequence of SEQ ID NO: 8. 제 4항에 있어서, 상기 균주는
상기 제2 벡터가, 모균주인 시네코코커스 일롱게투스 PCC7942(기탁번호: ATCC 33912)에 형질전환된 것인, 시네코코커스 일롱게투스 균주.5. The method according to claim 4, wherein the strain
Wherein the second vector is transformed into the parent strain Cynokocors lllngotus PCC7942 (Accession No .: ATCC 33912). 제 1항에 있어서,
상기 균주는 기탁번호가 KCCM12179P인 것인, 시네코코커스 일롱게투스 균주.The method according to claim 1,
Wherein the strain is the KCCM 12179P accession number. 제 1항의 균주를 배양하는 단계를 포함하는 아세톤의 생산 방법.A method for producing acetone comprising culturing the strain of claim 1.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
KR20160107633A (en) * 2015-03-04 2016-09-19 한국과학기술연구원 Transformed synechococcus elongatus having capability of producing acetone
KR101789521B1 (en) 2016-03-29 2017-10-26 한국과학기술연구원 Transformed synechococcus elongates having capability of amorphadiene from carbon dioxide and method for producing amorphadiene using the same

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160107633A (en) * 2015-03-04 2016-09-19 한국과학기술연구원 Transformed synechococcus elongatus having capability of producing acetone
KR101789521B1 (en) 2016-03-29 2017-10-26 한국과학기술연구원 Transformed synechococcus elongates having capability of amorphadiene from carbon dioxide and method for producing amorphadiene using the same

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