KR101567308B1 - Recombinant vector for increasing biomass and lipid productivity of microalgae and uses thereof - Google Patents
Recombinant vector for increasing biomass and lipid productivity of microalgae and uses thereof Download PDFInfo
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- KR101567308B1 KR101567308B1 KR1020140003840A KR20140003840A KR101567308B1 KR 101567308 B1 KR101567308 B1 KR 101567308B1 KR 1020140003840 A KR1020140003840 A KR 1020140003840A KR 20140003840 A KR20140003840 A KR 20140003840A KR 101567308 B1 KR101567308 B1 KR 101567308B1
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- KR
- South Korea
- Prior art keywords
- microalgae
- recombinant vector
- lipid
- biomass
- val
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Abstract
본 발명은 암모늄이 없는 조건 또는 질산염이 있는 조건에서 유도되는 유도성 프로모터 및 상기 프로모터 하위에 GAPDH(glyceraldehyde-3-phosphate dehydrogenase) 단백질 코딩 유전자를 포함하는 것을 특징으로 하는 재조합 벡터, 상기 재조합 벡터로 미세조류 세포를 형질전환하여 바이오매스와 지질 생산성이 증가된 형질전환 미세조류의 제조 방법, 상기 방법에 의해 제조된 형질전환 미세조류, 상기 재조합 벡터를 유효성분으로 포함하는, 미세조류의 바이오매스와 지질 생산성을 증가시키기 위한 조성물, 상기 형질전환 미세조류를 배양하여 지질을 생산하는 방법 및 상기 형질전환 미세조류를 이용한 바이오디젤의 제조 방법에 관한 것이다The present invention relates to a recombinant vector characterized by comprising an inducible promoter derived under ammonium-free conditions or nitrate-containing conditions and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein coding gene under the promoter, A method for producing a transformed microalga having an increased biomass and lipid productivity by transforming avian cells, a method for producing a microalgae of the microalgae containing the recombinant vector as an active ingredient, biomass and lipid A composition for increasing productivity, a method for producing lipid by culturing the transformed microalgae, and a method for producing biodiesel using the transformed microalgae
Description
본 발명은 미세조류의 바이오매스와 지질 생산성을 증가시키기 위한 재조합 벡터 및 이의 용도에 관한 것으로, 더욱 상세하게는 암모늄이 없는 조건 또는 질산염이 있는 조건에서 유도되는 유도성 프로모터 및 상기 프로모터 하위에 GAPDH(glyceraldehyde-3-phosphate dehydrogenase) 단백질 코딩 유전자를 포함하는 것을 특징으로 하는 재조합 벡터, 상기 재조합 벡터로 미세조류 세포를 형질전환하여 바이오매스와 지질 생산성이 증가된 형질전환 미세조류의 제조 방법, 상기 방법에 의해 제조된 형질전환 미세조류, 상기 재조합 벡터를 유효성분으로 포함하는, 미세조류의 바이오매스와 지질 생산성을 증가시키기 위한 조성물, 상기 형질전환 미세조류를 배양하여 지질을 생산하는 방법 및 상기 형질전환 미세조류를 이용한 바이오디젤의 제조 방법에 관한 것이다.The present invention relates to a recombinant vector and its use for increasing biomass and lipid productivity of microalgae, and more particularly, to an inductive promoter which is induced under ammonium-free or nitrate-containing conditions and GAPDH glyceraldehyde-3-phosphate dehydrogenase) protein coding gene, a method of transforming microalgae cells with the recombinant vector, and a method of producing transformed microalgae with increased biomass and lipid productivity, , A composition for increasing biomass and lipid productivity of microalgae containing the recombinant vector as an active ingredient, a method for producing lipids by culturing the transformed microalgae, and a method for producing the transformed microalgae And a method for producing biodiesel using algae.
남조류(cyanobacteria)를 포함하는 미세조류는 각종 지질, 탄화수소, 지방 알코올 및 기타 복합 오일들을 생산하는 것으로 보고되어 왔다. 모든 미세조류가 바이오디젤 생산에 적합하지는 않다 하더라도, 일부 미세조류 균주들이 바이오디젤 연료로서의 이용 가능성을 보여주고 있다. 클라미도모나스 레인하르티(Chlamydomonas reinhardtii)는, 가장 널리 보급되어 있는 미세조류 모델 시스템들 중 하나로, 이 미세조류는 바이오연료 공급원으로서 바람직한 특징들을 갖는다. 또한, 지질 생산 경로들의 대사 엔지니어링도 클라미도모나스 레인하르티의 모델 시스템 균주들을 이용하여 널리 연구되어 왔다.Microalgae, including cyanobacteria, have been reported to produce various lipids, hydrocarbons, fatty alcohols and other complex oils. Although not all microalgae are suitable for biodiesel production, some microalgae strains show potential for use as biodiesel fuel. Chlamydomonas reinhardtii ) is one of the most widely deployed microalgae model systems, and these microalgae have desirable characteristics as a source of biofuels. Metabolic engineering of lipid production pathways has also been extensively studied using model systems strains of C. elegans.
광합성을 하는 생물에서 일어나는 캘빈회로(calvin cycle)를 통해서 얻어진 3-포스포글리세린산(PGA, phosphoglycerate)은 세포질이나 엽록체에서 해당과정(glycolysis)이 진행되어 아세틸 조효소A(acetyl-CoA)로 전환되며, 아세틸 조효소A는 지방산(fatty acid) 생산에 크게 기여한다. 본 발명은 GAPDH(glyceraldehyde-3-phosphate dehydrogenase) 효소가 3-포스포글리세린산 합성에 긍정적인 영향을 미쳐 궁극적으로 지방산 생산량 증대에 기여할 수 있을 것으로 예상하여, 유도성 프로모터와 GAPDH 단백질 코딩 유전자를 포함하는 재조합 벡터를 제작하여 미세조류에 형질전환하고, 형질전환 미세조류의 생장 변화 및 지질함량 변화를 관찰하였다.3-phosphoglycerate (PGA) obtained through a calvin cycle in a photosynthetic organism undergoes glycolysis in the cytoplasm or chloroplast and is converted to acetyl-CoA , And acetyl coenzyme A contributes greatly to fatty acid production. The present invention contemplates that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme has a positive effect on the synthesis of 3-phosphoglycerate and ultimately contributes to an increase in the fatty acid production, and thus includes an inducible promoter and a GAPDH protein coding gene The transformed microalgae were transformed into microalgae, and the changes in the growth and lipid contents of transgenic microalgae were observed.
한편, 한국공개특허 제2013-0095517호에는 '신규한 미세조류 클라미도모나스 레인하르티 KNUA021 균주 및 이로부터의 지방 알코올 및 지방산 생산 방법'이 개시되어 있고, 한국등록특허 제1107980호에는 'glgA1 유전자와 이를 포함하는 재조합벡터와 이로부터 형질전환된 숙주세포와 이의 생합성 제어를 통한 남세균 총지질 함량 조절 방법'이 개시되어 있으나, 본 발명의 미세조류의 바이오매스와 지질 생산성을 증가시키기 위한 재조합 벡터 및 이의 용도에 대해서는 기재된 바가 없다.Korean Patent Laid-Open Publication No. 2013-0095517 discloses a novel microalgae Clamidomonas reinhardtii KNUA021 strain and a method for producing fatty alcohols and fatty acids therefrom. In Korean Patent No. 1107980, the 'glgA1 gene A method for regulating the total lipid content of a bacterium by controlling the biosynthesis of the recombinant vector and a transformed host cell from the recombinant vector is disclosed. However, in order to increase the biomass and lipid productivity of the microalgae of the present invention, There is no description about use.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명에서는 유도성 프로모터 Nia1 프로모터와 GAPDH 단백질 코딩 유전자를 포함하는 재조합 벡터로 미세조류를 형질전환하여, 암모늄이 없는 배지 조성물에서 상기 형질전환 미세조류를 배양하여 미세조류의 바이오매스 및 지질 생산량이 비형질전환체에 비해 증가된 것을 확인함으로써, 본 발명을 완성하였다.The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method for transforming microalgae with a recombinant vector comprising an inducible promoter Nia1 promoter and a GAPDH protein coding gene, Was cultured to confirm that the biomass and lipid production of microalgae were increased as compared with the non-transformant, thereby completing the present invention.
상기 과제를 해결하기 위해, 본 발명은 암모늄이 없는 조건 또는 질산염이 있는 조건에서 유도되는 유도성 프로모터 및 상기 프로모터 하위에 GAPDH(glyceraldehyde-3-phosphate dehydrogenase) 단백질 코딩 유전자를 포함하는 것을 특징으로 하는 재조합 벡터를 제공한다.In order to solve the above-mentioned problems, the present invention provides a recombinant microorganism which comprises an inducible promoter derived under ammonium-free conditions or nitrate-containing conditions and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Lt; / RTI >
또한, 본 발명은 상기 재조합 벡터로 미세조류 세포를 형질전환하여 GAPDH 단백질 코딩 유전자를 발현하는 단계를 포함하는 비형질전환체에 비해 바이오매스와 지질 생산성이 증가된 형질전환 미세조류의 제조 방법을 제공한다.In addition, the present invention provides a method for producing transformed microalgae having increased biomass and lipid productivity as compared to non-transformants comprising transforming microalgae cells with the recombinant vector and expressing a GAPDH protein coding gene do.
또한, 본 발명은 상기 방법에 의해 제조된 비형질전환체에 비해 바이오매스와 지질 생산성이 증가된 형질전환 미세조류를 제공한다.In addition, the present invention provides transgenic microalgae with increased biomass and lipid productivity as compared to the non-transformant produced by the method.
또한, 본 발명은 재조합 벡터를 유효성분으로 포함하는, 미세조류의 바이오매스와 지질 생산성을 증가시키기 위한 조성물을 제공한다.The present invention also provides a composition for increasing the biomass and lipid productivity of microalgae comprising a recombinant vector as an active ingredient.
또한, 본 발명은 상기 형질전환 미세조류를 배양 배지에서 배양하는 단계를 포함하는 지질을 생산하는 방법을 제공한다.In addition, the present invention provides a method for producing a lipid comprising culturing the transformed microalgae in a culture medium.
또한, 본 발명은In addition,
(a) 상기 형질전환 미세조류를 배양하는 단계;(a) culturing the transformed microalgae;
(b) 상기 (a) 단계의 미세조류 배양액으로부터 지방산을 추출하는 단계; 및(b) extracting the fatty acid from the microalgae culture liquid of step (a); And
(c) 상기 (b) 단계의 지방산을 트랜스에스테르화시켜 지방산 에스테르를 생성하는 것을 특징으로 하는 바이오디젤의 제조 방법을 제공한다.(c) transesterifying the fatty acid in the step (b) to produce a fatty acid ester.
본 발명에서는 Nia1 프로모터와 GAPDH 단백질 코딩 유전자를 포함하는 재조합벡터를 미세조류에 형질전환하여, 미세조류의 바이오매스 및 지질 합성이 증가되는 것을 확인하였다. 기존의 재조합 벡터를 이용한 형질전환 미세조류는 야생형에 비해 바이오매스가 증가하면 바이오디젤 생산량이 감소하거나 반대로 바이오디젤 생산량이 증가하면 바이오매스가 감소하는 문제가 있었는데, 본 발명의 재조합 벡터를 이용한 형질전환 미세조류는 야생형에 비해 동일한 바이오매스에서 보다 많은 양의 바이오디젤을 생산할 뿐만 아니라, 동일 배양 조건에서는 야생형에 비해 높은 바이오매스 증가량을 보여줌으로써 궁극적으로 높은 바이오디젤 생산성을 나타내기 때문에, 본 발명의 재조합 벡터는 산업적으로 매우 유용하게 이용될 수 있을 것으로 판단된다.In the present invention, it was confirmed that the recombinant vector containing the Nia1 promoter and the GAPDH protein coding gene was transformed into microalgae to increase biomass and lipid synthesis of microalgae. Transformation microalgae using the existing recombinant vector had a problem that the biomass decreased when the biomass increased compared to the wild type or the biomass decreased when the biodiesel production increased. On the contrary, when the transformed microorganism transformed with the recombinant vector of the present invention The microalgae not only produce more biodiesel in the same biomass than the wild type but also exhibit higher biodiesel productivity than the wild type in the same culture conditions and ultimately exhibit high biodiesel productivity. It is considered that the vector can be very usefully used industrially.
도 1은 본 발명의 Nia1 프로모터와 GAPDH 단백질 코딩 유전자가 삽입된 재조합 벡터의 그림이다.
도 2는 형질전환체의 검증을 위한 PCR 결과이다. PNG gDNA, 형질전환 미세조류로부터 추출한 게노믹 DNA; PNG 벡터, 형질전환에 사용한 재조합 벡터; 양성대조군, 진핵생물 18s rRNA.
도 3은 형질전환체 7개의 암모늄이 없는 배지에서의 생장 속도(a)와 지질함량(b)을 확인한 결과이다.
도 4는 선별된 형질전환 미세조류와 야생형 미세조류를 일반 배지(TAP) 및 암모늄이 없는 배지(TAP-N)에서 배양하며 흡광도 분석을 통해 생장 속도를 확인한 결과이다.
도 5는 선별된 형질전환 미세조류와 야생형 미세조류를 일반 배지(TAP) 및 암모늄이 없는 배지(TAP-N)에서 배양하며 건조 중량 분석을 통해 생장 속도를 확인한 결과이다.
도 6은 선별된 형질전환 미세조류와 야생형 미세조류를 일반 배지(TAP) 및 암모늄이 없는 배지(TAP-N)에서 배양하며 지방산 메틸에스테르(FAME, fatty acid methyl ester)의 함량을 분석하여 지질함량을 확인한 결과이다.
도 7은 선별된 형질전환 미세조류와 야생형 미세조류를 일반 배지(TAP) 및 암모늄이 없는 배지(TAP-N)에서 배양하며 지방산 메틸에스테르 수율을 분석한 결과이다.
도 8은 선별된 형질전환 미세조류와 야생형 미세조류를 일반 배지(TAP) 및 암모늄이 없는 배지(TAP-N)에서 배양하며 지방산 메틸에스테르 생산성을 분석한 결과이다.1 is a diagram of a recombinant vector into which the Nia1 promoter of the present invention and the GAPDH protein coding gene are inserted.
Figure 2 shows PCR results for the verification of transformants. PNG gDNA, genomic DNA extracted from transformed microalgae; PNG vector, the recombinant vector used for transformation; Positive control, eukaryotic 18s rRNA.
FIG. 3 shows the results of confirming the growth rate (a) and the lipid content (b) in the culture medium containing 7 ammonium-free transformants.
FIG. 4 shows the results of culturing the selected transformed microalgae and wild-type microalgae in a TAP medium and a TAP-N medium without ammonium, and observing the growth rate by absorbance analysis.
FIG. 5 shows the results of culturing selected transformed microalgae and wild-type microalgae in TAP and ammonium-free medium (TAP-N) and confirming the growth rate by dry weight analysis.
FIG. 6 shows the results of culturing selected transformed microalgae and wild-type microalgae on TAP and ammonium-free media (TAP-N) and analyzing the content of fatty acid methyl ester (FAME) .
FIG. 7 shows the results of analysis of the yield of fatty acid methyl esters by culturing selected transformed microalgae and wild-type microalgae in a medium (TAP) and an ammonium-free medium (TAP-N).
FIG. 8 shows the results of analysis of productivity of fatty acid methyl esters by culturing selected transformed microalgae and wild-type microalgae in a TAP medium and a TAP-N medium without ammonium.
본 발명의 목적을 달성하기 위하여, 본 발명은 암모늄이 없는 조건 또는 질산염이 있는 조건에서 유도되는 유도성 프로모터 및 상기 프로모터 하위에 GAPDH(glyceraldehyde-3-phosphate dehydrogenase) 단백질 코딩 유전자를 포함하는 것을 특징으로 하는 재조합 벡터를 제공한다.In order to achieve the object of the present invention, the present invention is characterized in that it comprises an inducible promoter which is induced under ammonium-free or nitrate-containing conditions and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Lt; / RTI >
본 발명의 일 구현 예에 따른 재조합 벡터에 있어서, 상기 유도성 프로모터는 Nia1(nirate reductase) 프로모터, Cytochrome c6 프로모터, CA1(carbonic anhydrase) 프로모터 등일 수 있으며, 바람직하게는 Nia1(nirate reductase) 프로모터일 수 있으나, 이에 제한되지 않는다.In the recombinant vector according to one embodiment of the invention, the inducible promoter is Nia1 (nirate reductase) promoter, Cytochrome c 6 promoter, and the like CA1 (carbonic anhydrase) promoter, preferably Nia1 (nirate reductase) promoter days But is not limited thereto.
본 발명에 따른 Nia1 프로모터는 서열번호 1의 염기서열을 가질 수 있다. 또한, 상기 프로모터 서열의 상동체가 본 발명의 범위 내에 포함된다. 상동체는 염기 서열은 변화되지만, 서열번호 1의 염기 서열과 유사한 기능적 특성을 갖는 염기 서열이다. 구체적으로, 상기 프로모터 서열은 서열번호 1의 염기 서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.The Nia1 promoter according to the present invention may have the nucleotide sequence of SEQ ID NO: 1. Also, homologues of the promoter sequences are included within the scope of the present invention. The homologue is a base sequence having a functional characteristic similar to that of SEQ ID NO: 1, although the base sequence is changed. Specifically, the promoter sequence has a nucleotide sequence that has at least 70% homology, more preferably at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >
본 발명에 따른 GAPDH 단백질의 범위는 서열번호 2로 표시되는 아미노산 서열을 갖는 단백질 및 상기 단백질의 기능적 동등물을 포함한다. "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 2로 표시되는 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 2로 표시되는 단백질과 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다. "실질적으로 동질의 생리활성"이란 미세조류의 바이오매스와 지질 생산성을 증가시키는 활성을 의미한다.The GAPDH The range of the protein includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous bioactivity" means an activity that increases microbial biomass and lipid productivity.
또한, 본 발명은 상기 GAPDH 단백질을 암호화하는 유전자를 제공한다. 본 발명의 유전자는 서열번호 3으로 표시되는 GAPDH cDNA 또는 서열번호 4로 표시되는 GAPDH 게노믹 DNA 염기서열을 포함할 수 있다. 또한, 상기 염기 서열의 상동체가 본 발명의 범위 내에 포함되며 구체적인 내용은 전술한 바와 같다.The present invention also provides a gene encoding the GAPDH protein. The gene of the present invention may include the GAPDH cDNA represented by SEQ ID NO: 3 or the GAPDH genomic DNA nucleotide sequence represented by SEQ ID NO: 4. In addition, homologues of the above base sequences are included within the scope of the present invention, and the details are as described above.
용어 "유도성 프로모터(inducible promoter)"는 외부 자극 시에 프로모터의 활성이 증가 또는 감소될 수 있는 프로모터를 나타낸다. 자극은 천연에서의 물리적 또는 화학적 자극, 예컨대 온도, 광, 화학물질 등일 수 있다. 프로모터 하위의 유전자의 유도는 자극의 직접적인 또는 간접적인 전달을 통해 수득될 수 있다.The term " inducible promoter "refers to a promoter capable of increasing or decreasing the activity of the promoter upon external stimulation. The stimulation may be physical or chemical stimulation in nature, such as temperature, light, chemicals, and the like. Induction of the gene under the promoter can be obtained through direct or indirect delivery of the stimulus.
용어 "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩티드, 이종의 펩티드 또는 이종의 핵산에 의해 암호된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 그러나 상기 유전자는 변형된 것으로써 인위적인 수단에 의해 세포 내 재도입된 것이다.The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been re-introduced into the cell by an artificial means as modified.
용어 "벡터"는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 지칭할 때 사용된다. 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재생산될 수 있다. 용어 "전달체"는 흔히 "벡터"와 호환하여 사용된다.The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ".
또한, 본 발명은 상기의 재조합 벡터로 미세조류 세포를 형질전환하여 GAPDH 단백질 코딩 유전자를 발현하는 단계를 포함하는 비형질전환체에 비해 바이오매스와 지질 생산성이 증가된 형질전환 미세조류의 제조 방법 및 상기의 방법에 의해 제조된 형질전환 미세조류를 제공한다.In addition, the present invention provides a method for producing transformed microalgae having enhanced biomass and lipid productivity as compared with the non-transformant comprising the step of transforming microalgae cells with the above recombinant vector and expressing the GAPDH protein coding gene, The transformed microalgae produced by the above method are provided.
본 발명의 일 구현 예에 따른 형질전환 미세조류의 제조방법에 있어서, 재조합 벡터로 미세조류 세포를 형질전환하는 방법은 유리 구슬(glass bead), 유전자 총(gene gun), 전기천공법(electroporation), 탄화규소 휘스커(silicon carbide whisker) 등의 방법일 수 있고, 바람직하게는 유리 구슬 방법일 수 있으나, 이에 제한되지 않는다.In the method of transforming microalgae according to one embodiment of the present invention, a method of transforming microalgae cells with a recombinant vector may be a glass bead, a gene gun, an electroporation method, , A silicon carbide whisker, and the like, preferably a glass bead method, but is not limited thereto.
또한, 본 발명에 따른 미세조류는 클라미도모나스(Chlamydomonas), 에틀리아(Ettlia), 두날리엘라(Dunaliella), 클로렐라(Chlorella), 난노클로롭시스(Nannochloropsis), 스피룰리나(Spirulina), 크루코커스(Chroococcus), 채토세로스(Chaetoceros), 아크난테스(Achnanthes), 엠포라(Amphora) 종 등일 수 있으며, 바람직하게는 클라미도모나스 레인하르티(Chlamydomonas reinhardtii), 클라미도모나스 카우다타 윌레(Chlamydomonas caudata Wille), 클라미도모나스 모에우시(Chlamydomonas moewusii), 클라미도모나스 니발리스(Chlamydomonas nivalis), 클라미도모나스 페리그라눌라타(Chlamydomonas perigranulata), 에틀리아 올레오아분단스(Ettlia oleoabundans), 난노클로롭시스 살리나(Nannochloropsis salina), 난노클로롭시스 가디타나(Nannochloropsis gaditana), 두날리엘라 바르다윌(Dunaliella bardawil), 두날리엘라 살리나(Dunaliella salina), 두날리엘라 프리모렉타(Dunaliella primolecta), 클로렐라 불가리스(Chlorella vulgaris), 클로렐라 에모르소니(Chlorella emorsonii), 클로렐라 미누티시마(Chlorella minutissima), 클로렐라 소로키니아나(Chlorella sorokiniana), 스피룰리나 플라텐시스(Spirulina platensis), 사이클로텔라 크립티카(Cyclotella cryptica), 테트라셀미스 수에시카(Tetraselmis suecica), 모노라피디엄(Monoraphidium), 보트리오코커스 브라우니(Botryococcus braunii), 스티코쿠스(Stichococcus), 해마토코커스 플루비알리스(Haematococcus pluvialis), 패오닥틸룸 트리코뮤텀(Phaeodactylum tricomutum), 이소크리시스 갈바나(Isochrysis galbana), 니츠쉬아 클로스테리움(Nitzschia closterium), 오란티오키트리움(Aurantiochytrium) 등일 수 있으며, 더욱 바람직하게는 클라미도모나스 레인하르티(Chlamydomonas reinhardtii)일 수 있으나, 이에 제한되지 않는다.In addition, micro-algae according to the present invention Chlamydomonas (Chlamydomonas), the wrong ah (Ettlia), two flying it Ella (Dunaliella), Chlorella (Chlorella), nanno claw drop system (Nannochloropsis), Spirulina (Spirulina), crew Lactococcus Such as Chroococcus , Chaetoceros , Achnanthes , Amphora species and the like, preferably Chlamydomonas reinhardtii ), Chlamydomonas ( Chlamydomonas caudata Wille , Chlamydomonas moewusii , Chlamydomonas nivalis , Chlamydomonas, perigranulata , Ettlia oleoabundans ), Nannochloropsis salin a), nanno claw Rob cis Gardiner appear (Nannochloropsis gaditana), two flying it Ella right Wiltshire (Dunaliella bardawil , Dunaliella salina , Dunaliella < RTI ID = 0.0 > primolecta , Chlorella vulgaris , chlorella eosinophil emorsonii), Chlorella minu Tea Island (Chlorella minutissima), Chlorella Thoreau Kearney Ana (Chlorella sorokiniana , Spirulina platensis , Cyclotella cryptica , Tetraselmis < RTI ID = 0.0 > suecica , Monoraphidium , Botryococcus, braunii , Stichococcus , Haematococcus, pluvialis , < RTI ID = 0.0 > Phaeodactylum tricomutum ), Isochrysis galbana), Chemnitz Ushuaia Claus teddy Leeum (Nitzschia closterium , Aurantiochytrium , and the like, more preferably, but not limited to, Chlamydomonas reinhardtii .
또한, 본 발명은 상기의 재조합 벡터를 유효성분으로 포함하는, 미세조류의 바이오매스와 지질 생산성을 증가시키기 위한 조성물을 제공한다. 상기 조성물은 유효성분으로 암모늄이 없는 조건 또는 질산염이 있는 조건에서 유도되는 유도성 프로모터 및 상기 프로모터 하위에 GAPDH 단백질 코딩 유전자를 포함하는 재조합 벡터를 함유하며, 상기 재조합 벡터를 미세조류 세포에 형질전환시킴으로써 미세조류의 바이오매스와 지질 생산성을 증가시킬 수 있는 것이다.The present invention also provides a composition for increasing the biomass and lipid productivity of microalgae comprising the recombinant vector as an active ingredient. The composition contains an inducible promoter derived under conditions in which ammonium is not present as an active ingredient or in the presence of nitrate, and a recombinant vector comprising a GAPDH protein coding gene under the promoter. By transforming the recombinant vector into microalgae cells It can increase biomass and lipid productivity of microalgae.
또한, 본 발명은 상기의 형질전환 미세조류를 배양 배지에서 배양하는 단계를 포함하는 지질을 생산하는 방법을 제공한다.The present invention also provides a method for producing a lipid comprising culturing the transgenic microalgae in a culture medium.
본 발명의 일 구현 예에 따른 지질을 생산하는 방법에 있어서, 상기 배양 배지는 암모늄이 없는 배양 배지일 수 있으나, 이에 제한되지 않는다.In the method of producing a lipid according to an embodiment of the present invention, the culture medium may be a culture medium without ammonium, but is not limited thereto.
또한, 본 발명은In addition,
(a) 상기 형질전환 미세조류를 배양하는 단계;(a) culturing the transformed microalgae;
(b) 상기 (a) 단계의 미세조류 배양액으로부터 지방산을 추출하는 단계; 및(b) extracting the fatty acid from the microalgae culture liquid of step (a); And
(c) 상기 (b) 단계의 지방산을 트랜스에스테르화시켜 지방산 에스테르를 생성하는 것을 특징으로 하는 바이오디젤의 제조 방법을 제공한다.
(c) transesterifying the fatty acid in the step (b) to produce a fatty acid ester.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
재료 및 방법Materials and methods
실험 재료Experimental material
클라미도모나스 레인하르티(Chlamydomonas reinhardtii) CC-124, CC-620 및 CC-621은 클라미도모나스 자원 센터(미국)로부터 준비하였고, 벡터 pCr102는 한국생명공학연구원(KRIBB)으로부터 구매하였다. 상기 미세조류들은 미국의 Gorman, D.S.와 Levine, R.P.가 개발한 TAP(Tris-Acetate-Phosphate) 배지를 사용하여 배양하였으며, TAP 배지의 조성은 하기 표 1과 같다. Chlamydomonas reinhardtii ) CC-124, CC-620 and CC-621 were prepared from Clamidomonas Resource Center (USA), and vector pCr102 was purchased from Korea Biotechnology Research Institute (KRIBB). The microalgae were cultured using a TAR (Tris-Acetate-Phosphate) medium developed by Gorman, DS and Levine, RP, USA. The composition of the TAP medium is shown in Table 1 below.
유도성 프로모터의 작동을 위한 조건으로는 TAP-N 배지를 사용하였는데, TAP-N 배지의 조성은 상기 표 1의 TAP 배지 조성표에서 염화암모늄 성분만 빠진 구성으로 이루어졌다.
The TAP-N medium was used as a condition for the induction of the promoter, and the composition of the TAP-N medium was composed of only the ammonium chloride component in the TAP medium composition table in Table 1 above.
재조합 벡터 제조Production of recombinant vector
pCr102 벡터를 가진 대장균을 100㎎/㎖ 암피실린이 함유된 LB 액체배지(BD bioscience, 미국)에 접종하고 200rpm의 교반 속도로 18시간 배양한 후, mini-prep kit(QIAGEN, 독일)를 사용하여 플라스미드 DNA를 추출하였다. 준비된 pCr102 벡터로 Nia1 프로모터와 GAPDH 단백질 코딩 유전자를 클로닝하기 위해, 상기 프로모터와 유전자의 클로닝 주형으로 사용할 클라미도모나스 레인하르티의 게노믹 DNA를 준비하였다. 게노믹 DNA는 TAP 배지에서 4일간 배양시킨 클라이도모나스 레인하르티 CC-124를 수확하여 HiGeneTM Genomic DNA Prep Kit(Biofact, 한국)를 사용하여 준비하였다.Escherichia coli having the pCr102 vector was inoculated into an LB liquid medium (BD bioscience, USA) containing 100 mg / ml ampicillin and cultured at a stirring speed of 200 rpm for 18 hours. Then, the plasmids were cultured in a mini-prep kit (QIAGEN, Germany) DNA was extracted. In order to clone the Nia1 promoter and the GAPDH protein coding gene into the prepared pCr102 vector, Genomic DNA of Clamidomonas reinhardtii to be used as a cloning template for the above promoter and gene was prepared. Genomic DNA was prepared by harvesting Cladomonas reinharti CC-124 cultured in TAP medium for 4 days and using HiGene ™ Genomic DNA Prep Kit (Biofact, Korea).
준비된 클라미도모나스 레인하르티의 게노믹 DNA를 주형으로 하여, 제한효소 자리가 들어간 Nia1 정방향 프라이머(5'-GGGGTACCAACCGACCAATCGATAG-3'; 서열번호 5, 밑줄 KpnI 자리) 및 Nia1 역방향 프라이머(5'-CCCAAGCTTCCCGGGACTAGTACTGGCAGGATTCGGC-3'; 서열번호 6, 밑줄 HindⅢ 자리)와 GAPDH 정방향 프라이머(5'-GACTAGTATGCAGAAGGTGCGCAG-3'; 서열번호 7, 밑줄 SpeI 자리) 및 GAPDH 역방향 프라이머(5'-TCCCCCCGGGTTATTACGCCACCCACTTC-3'; 서열번호 8, 밑줄 SmaI 자리)를 이용하여 중합효소 연쇄반응(PCR)을 수행하였다. PCR의 조건은 Nia1의 경우, 변성(denaturation)은 94℃ 30초, 어닐링(annealing)은 50℃ 30초, 연장(extension)은 72℃ 30초로 25회 반복 수행하였고, GAPDH의 경우는 변성 94℃ 30초, 어닐링 49.8℃ 30초, 연장 72℃ 1분 45초로 25회 반복 수행하였다. PCR 수행 후, PCR 산물들은 겔 추출 방법을 이용하여 준비하였다.Prepared by the Chlamydomonas to genomic DNA of lane hareuti as the template, the restriction position is entered Nia1 forward primer (5'-GG GGTACC AACCGACCAATCGATAG-3 '; SEQ ID NO: 5, Kpn I underlined digit) Nia1 and reverse primer (5 ' -CCC AAGCTT CCCGGGACTAGTACTGGCAGGATTCGGC-3 '; SEQ ID NO: 6, underlined Hind ⅲ digits) and GAPDH forward primer (5'-G ACTAGT ATGCAGAAGGTGCGCAG-3 '; SEQ ID NO: 7, Spe I underlined digit) and GAPDH reverse primer (5'-TCCC (CCCGGG TTATTACGCCACCCACTTC-3 '; SEQ ID NO: 8, underlined Sma I site) was used to perform PCR. The PCR conditions were as follows: denaturation at 94 ° C. for 30 seconds, annealing at 50 ° C. for 30 seconds, extension at 72 ° C. for 30 seconds, and GAPDH at 94 ° C. for
준비해두었던 플라스미드 DNA(pCr102 벡터)를 KpnI와 HindⅢ 제한효소로 처리하여, 원래의 psaD 프로모터 부위를 잘라내고, PCR로 증폭시켜 준비한 Nia1 프로모터 단편들을 ligation kit(Takara, 일본)를 사용하여 결합시켰다. 그 후, Nia1 프로모터가 들어간 재조합 벡터를 대상으로 SpeI와 SmaI 제한효소를 처리하고 같은 방법으로 GAPDH 단백질 코딩 유전자를 삽입하였다.
The prepared plasmid DNA (pCr102 vector) you wrote was treated with Kpn I and Hind Ⅲ restriction enzyme, was bonded was cut out of the original psaD promoter region, using the Nia1 promoter fragment prepared by amplifying by PCR ligation kit (Takara, Japan) . Then, the recombinant vector containing the Nia1 promoter was treated with Spe I and Sma I restriction enzymes, and the GAPDH protein coding gene was inserted in the same manner.
미세조류 형질전환Microalgae transformation
클라미도모나스 레인하르티 CC-620과 CC-621 균주의 교배를 통해서 얻어진 자가분해효소(autolysin)를 CC-124 균주에 미리 처리하여, DNA가 쉽게 들어갈 수 있도록 세포벽을 분해하였다. Nia1 프로모터와 GAPDH 단백질 코딩 유전자가 삽입된 재조합 벡터를 XbaI 제한효소로 처리하여 선형 DNA 형태로 바꾸어주고, 100ng/㎕ 농도의 선형 DNA 10㎕를 상기 CC-124 세포들과 20% 폴리에틸렌글리콜(PEG)과 함께 멸균시킨 글래스 비드(glass bead)에 넣고 약 30초간 볼텍싱하였다. 그 후, 15㎍/㎖의 하이그로마이신이 들어가 있는 TAP 플레이트에 도말하고, 2~3주 배양하여 콜로니를 확인하였다.
The autolysin obtained by crossing of CC-620 and CC-621 was pre-treated with strain CC-124, and the cell wall was disassembled so that the DNA could easily enter. The recombinant vector in which the Nia1 promoter and the GAPDH protein coding gene were inserted was transformed into a linear DNA form by treating with Xba I restriction enzyme, and 10 μl of a linear DNA at a concentration of 100 ng / μl was added to the CC-124 cells and 20% polyethylene glycol ), And then vortexed for about 30 seconds in a glass bead sterilized. Thereafter, the cells were plated on a TAP plate containing 15 μg / ml of hygromycin, and cultured for 2 to 3 weeks to confirm colonies.
실시예Example 1. 형질전환 미세조류의 검증 및 선별 1. Validation and selection of transgenic microalgae
형질전환 후 항생제가 포함된 고체 배지 및 액체 배지 상태에서 각각 계대 배양하면서 형질전환체들을 가려낼 수 있었다. 일련의 계대 과정을 통해서 얻어진 형질전환체들이 Nia1 프로모터와 GAPDH 단백질 코딩 유전자를 모두 가지고 있는지 검증하기 위해서 형질전환체로부터 게노믹 DNA를 추출하고 이를 기반으로 PCR을 통해 삽입 유전자의 유무를 확인하였다. GAPDH 단백질 코딩 유전자의 경우 길이가 길어서 한번에 PCR하기 어렵기 때문에, 유전자를 부분적으로 나누어서 확인하였고, 사용한 프라이머 정보는 하기 표 2와 같다. 또한 형질전환에 사용된 재조합 벡터를 대조구로 함께 사용하였다.Transformants were screened by subculture in solid and liquid medium containing antibiotics after transfection. Genomic DNA was extracted from the transformants to confirm whether the transformants obtained through a series of passages contained both the Nia1 promoter and the GAPDH protein coding gene. Based on the extracted genomic DNA, the presence of the inserted gene was confirmed by PCR. Since the GAPDH protein coding gene has a long length and is difficult to PCR at once, the gene was partially confirmed and the primer information used is shown in Table 2 below. The recombinant vector used for transformation was used as a control.
그 결과, 형질전환체의 게노믹 DNA상에서 재조합 벡터의 선별 마커 및 GAPDH 도입 유전자가 확인되었다(도 2).As a result, a recombinant vector selection marker and GAPDH-introduced gene were confirmed on the genomic DNA of the transformant (FIG. 2).
또한, 얻어진 7개의 형질전환체 중에서 가장 높은 생장 속도와 지질함량을 가진 형질전환체를 선별하기 위해, 암모늄이 없는 TAP 배지에서 형질전환체를 배양하면서 750nm에서 흡광도를 측정하여 생장 속도를 확인하였고, 지방산 메틸에스테르(FAME, fatty acid methylester) 분석을 통해 간접적으로 지질함량을 확인하였다. 그 결과, 형질전환체 #7이 암모늄이 없는 배지에서 비형질전환 야생형에 비해서 55% 증가한 생장 속도와, 31% 높은 FAME 함량이 확인되었다(도 3). 이에 형질전환체 #7을 PNG로 명명하고 추가 연구를 수행하였다.
In order to select the transformants having the highest growth rate and lipid content among the 7 transformants obtained, the growth rate was confirmed by measuring the absorbance at 750 nm while culturing the transformant in the TAP medium without ammonium, Fatty acid methyl ester (FAME, fatty acid methylester) analysis was used to confirm the lipid content indirectly. As a result, a growth rate of 55% and a FAME content of 31% were confirmed in
실시예Example 2. 생장 속도 분석 2. Growth rate analysis
야생형 및 GAPDH 단백질 코딩 유전자가 도입된 형질전환체(PNG)를 암모늄이 있는 배지(TAP) 및 암모늄이 제거된 배지(TAP-N)에서 배양하며 흡광도 측정 및 건조 중량 측정을 통해 생장 속도를 관찰하였다.Transformants (PNG), into which wild-type and GAPDH protein coding genes were introduced, were cultured in ammonium-containing medium (TAP) and ammonium-depleted medium (TAP-N) and the growth rate was observed by absorbance measurement and dry weight measurement .
그 결과, 750nm에서 흡광도 측정을 통해 형질전환체(PNG)와 야생형 모두 암모늄이 없는 TAP-N 배지보다, 암모늄이 있는 TAP 배지에서 더 잘 자라는 것을 확인할 수 있었다. TAP 배지에서 배양한 형질전환체는 야생형과 유사하거나 조금 더 높은 흡광도 값을 유지하였다. 하지만 암모늄이 없는 TAP-N 배지에서는 두 종은 다른 양상을 보여주었는데, 야생형은 배양 2일 후부터 흡광도의 변화없이 일정한 수준을 유지하는 반면, 형질전환체는 배양 후 4일까지 꾸준히 흡광도가 증가하고 있으며, 특히 배양 4일째에는 TAP 배지에서 배양한 야생형의 흡광도와 비슷한 결과를 나타내었다(도 4). 또한, 동일한 부피로 설정한 건조 중량(dry weight) 측정을 통해 확인한 생장 속도도 흡광도 측정 결과와 유사한 형태로 확인되었다(도 5).As a result, it was confirmed that the absorbance at 750 nm allowed the TAP-N medium, which is free of both the transformant (PNG) and wild type ammonium, to grow better on the ammonium-containing TAP medium. Transformants cultured in TAP medium maintained a similar or slightly higher absorbance value to the wild type. However, in the TAP-N medium without ammonium, the two species exhibited different patterns. The wild type maintained a constant level without change of absorbance after 2 days of culture, while the transformant was continuously increased in absorbance until the 4th day after culturing , Especially on the fourth day of culture, similar to that of the wild type cultured on TAP medium (Fig. 4). In addition, the growth rate determined by measuring the dry weight set to the same volume was also confirmed to be similar to the absorbance measurement result (FIG. 5).
이상의 결과는 암모늄이 없는 TAP-N 배지에서 미세조류의 바이오매스(biomass) 생산량이 낮다는 기존의 결과들과 상이한 결과이며, 이를 통해 본 발명의 재조합 벡터를 사용하여 바이오매스가 고려된 높은 지질 생산성을 기대할 수 있었다.
These results are in contrast to previous results that the biomass yield of microalgae is low in ammonium-free TAP-N medium, which allows the biomass to be considered for high lipid productivity using the recombinant vector of the present invention .
실시예Example 3. 지질 함량 분석 3. Analysis of lipid content
지질 함량을 확인하는 방법으로 가스 크로마토그라피(GC, Gas chromatograph) 방법을 사용하였고, 지방산의 메탄올을 이용한 에스테르 교환반응 결과물인 지방산 메틸에스테르(FAME, fatty acid methyl ester)의 함량을, 프로모터 유도 직전과 유도 후 0.5일, 1일, 2일, 4일 및 14일 시점에서 분석하였다.The content of fatty acid methyl ester (FAME), which is the result of transesterification of fatty acid with methanol, was measured by using a gas chromatograph (GC) At day 0.5,
그 결과, 프로모터 유도 직전에서는 형질전환체와 야생형 모두 9% 수준의 FAME 함량을 보였으며, 개체간 차이가 크지 않음을 확인하였다. 또한, 암모늄이 있는 TAP 배지에서 배양한 야생형과 형질전환체 모두 전체 배양기간 동안 FAME 함량 수준이 8~9%를 유지하고 있으며, 야생형과 형질전환체간 차이는 확인되지 않았다. 하지만, 암모늄이 없는 TAP-N 배지에서는 시간에 따라서 지질 함량이 야생형과 형질전환체 모두에서 암모늄이 있는 TAP 배지에서보다 훨씬 더 증가하는 현상을 보여주었다. 그 중에서도 형질전환체의 경우 배양시간에 지남에 따라서 야생형에 비해 0.5일에는 20%, 1일에는 17%, 2일에는 31%, 4일에는 49%까지 FAME의 함량이 증가하였으며, 14일 장기 배양에서는 97%나 증가한 결과를 얻었다. 야생형의 경우 암모늄이 없는 TAP-N 배지에서 2일 및 4일에서 FAME 함량의 최대치를 나타냈지만 그 후로 증가되지 않고 정체된 반면, 형질전환체의 경우는 배양기간에 비례하여 계속해서 증가되는 양상을 보였다(도 6).
As a result, the FAME content of the transformant and the wild type was found to be about 9% immediately before the induction of the promoter, and it was confirmed that the individual difference was not large. In addition, the wild type and transformant cultured on TAP medium containing ammonium maintained the FAME content level of 8 ~ 9% during the entire culture period, and no difference between the wild type and the transformant was observed. However, in the TAP-N medium without ammonium, the lipid content over time showed a much greater increase than in the TAP medium with ammonium in both the wild type and the transformant. Among the transgenic plants, the content of FAME increased up to 20%, 0.5%, 17%, 31%, and 49%, respectively, over the incubation time, In culture, it increased by 97%. In the wild type, the maximum value of FAME content was shown at 2 days and 4 days in ammonium-free TAP-N medium, but it did not increase after that, but in the case of the transformant, (Fig. 6).
실시예Example 4. 지방산 4. Fatty acids 메틸에스테르Methyl ester (( FAMEFAME ) 수율 및 생산성 분석) Yield and productivity analysis
실질적으로 바이오 디젤을 생산함에 있어서 중요한 두 가지 요소는, 바이오매스 생산량과 그 바이오매스에 해당하는 지질 함량이다. 앞서 측정한 각 결과를 고려하여 최종적으로 바이오 디젤 생산량의 기대치가 높은 형질전환 균주가 야생형보다 바이오 디젤 생산에 있어서 우수한 균주임을 증명하였다.Two important factors in producing biodiesel in practice are the biomass production and the lipid content of the biomass. Considering the above results, it is proved that the transgenic strains with high expected biodiesel yields are superior to the wild type in producing biodiesel.
도 7의 수치는 암모늄이 없는 배지에서의 FAME 수율을 비교한 값으로, 형질전환체의 경우 배지 교체 직후에는 29% 정도의 수준이었지만, 배양 4일 후에는 140%, 14일까지 배양한 결과에서는 321%까지 증가한 값이 확인되었다(도 7). 또한 FAME 수율을 배양기간으로 나누어 주면서 FAME 생산성(productivity)을 계산한 결과, 매 시간마다 암모늄이 없는 TAP-N 배지 조건의 형질전환체가 다른 조건에 비해 훨씬 높은 생산성을 갖는 양상을 확인할 수 있었다(도 8).The values in Fig. 7 are the values obtained by comparing the yields of FAME in the culture medium without ammonium. In the case of the transformant, the level was about 29% immediately after the medium replacement, but 140% after 4 days of culture and 14 days after culture And a value increased to 321% (FIG. 7). FAME productivity was also calculated by dividing FAME yield by incubation period. As a result, the productivity of transformants in TAP-N medium without ammonium was found to be much higher than other conditions 8).
이상의 결과를 통해서, 유전적 변이를 통한 바이오 디젤의 생산성 향상의 잠재성을 확인할 수 있었다.From the above results, it was confirmed that the productivity of biodiesel can be improved through genetic variation.
<110> ADVANCED BIOMESS R&D CENTER <120> Recombinant vector for increasing biomass and lipid productivity of microalgae and uses thereof <130> PN13402 <160> 13 <170> KopatentIn 2.0 <210> 1 <211> 287 <212> DNA <213> Chlamydomonas reinhardtii <400> 1 aaccgaccaa tcgatagtct tgtgcgaccg tgcacgtgtg cagcaatagt taggtcgata 60 accacgttga acttgcgtct ctcttcgtgg cgcctcctgc ttggtgctcc acttcacttg 120 tcgctatata gcacagcgtt gaaagcaaag gccacactaa tacagccggg ctcgagagtc 180 cgtctgcgtt tgcattgttg gccaagggct gctttgtagc caaagccata cacgaagctt 240 cacttgatta gctttacgac cctcagccga atcctgccag tactagt 287 <210> 2 <211> 374 <212> PRT <213> Chlamydomonas reinhardtii <400> 2 Met Ala Ala Met Met Gln Lys Ser Ala Phe Thr Gly Ser Ala Val Ser 1 5 10 15 Ser Lys Ser Gly Val Arg Ala Lys Ala Ala Arg Ala Val Val Asp Val 20 25 30 Arg Ala Glu Lys Lys Ile Arg Val Ala Ile Asn Gly Phe Gly Arg Ile 35 40 45 Gly Arg Asn Phe Leu Arg Cys Trp His Gly Arg Gln Asn Thr Leu Leu 50 55 60 Asp Val Val Ala Ile Asn Asp Ser Gly Gly Val Lys Gln Ala Ser His 65 70 75 80 Leu Leu Lys Tyr Asp Ser Thr Leu Gly Thr Phe Ala Ala Asp Val Lys 85 90 95 Ile Val Asp Asp Ser His Ile Ser Val Asp Gly Lys Gln Ile Lys Ile 100 105 110 Val Ser Ser Arg Asp Pro Leu Gln Leu Pro Trp Lys Glu Met Asn Ile 115 120 125 Asp Leu Val Ile Glu Gly Thr Gly Val Phe Ile Asp Lys Val Gly Ala 130 135 140 Gly Lys His Ile Gln Ala Gly Ala Ser Lys Val Leu Ile Thr Ala Pro 145 150 155 160 Ala Lys Asp Lys Asp Ile Pro Thr Phe Val Val Gly Val Asn Glu Gly 165 170 175 Asp Tyr Lys His Glu Tyr Pro Ile Ile Ser Asn Ala Ser Cys Thr Thr 180 185 190 Asn Cys Leu Ala Pro Phe Val Lys Val Leu Glu Gln Lys Phe Gly Ile 195 200 205 Val Lys Gly Thr Met Thr Thr Thr His Ser Tyr Thr Gly Asp Gln Arg 210 215 220 Leu Leu Asp Ala Ser His Arg Asp Leu Arg Arg Ala Arg Ala Ala Ala 225 230 235 240 Leu Asn Ile Val Pro Thr Thr Thr Gly Ala Ala Lys Ala Val Ser Leu 245 250 255 Val Leu Pro Ser Leu Lys Gly Lys Leu Asn Gly Ile Ala Leu Arg Val 260 265 270 Pro Thr Pro Thr Val Ser Val Val Asp Leu Val Val Gln Val Glu Lys 275 280 285 Lys Thr Phe Ala Glu Glu Val Asn Ala Ala Phe Arg Glu Ala Ala Asn 290 295 300 Gly Pro Met Lys Gly Val Leu His Val Glu Asp Ala Pro Leu Val Ser 305 310 315 320 Ile Asp Phe Lys Cys Thr Asp Gln Ser Thr Ser Ile Asp Ala Ser Leu 325 330 335 Thr Met Val Met Gly Asp Asp Met Val Lys Val Val Ala Trp Tyr Asp 340 345 350 Asn Glu Trp Gly Tyr Ser Gln Arg Val Val Asp Leu Ala Glu Val Thr 355 360 365 Ala Lys Lys Trp Val Ala 370 <210> 3 <211> 1128 <212> DNA <213> Chlamydomonas reinhardtii <400> 3 atggccgcca tgatgcagaa gagcgccttc accggcagcg ccgtgtcctc caagtctggc 60 gtccgcgcca aggctgcccg cgccgtcgtc gacgtgcgcg cggagaagaa gatccgcgtg 120 gccatcaacg gcttcggtcg cattggccgc aacttcctgc gctgctggca cggtcgccag 180 aacaccctgc tggacgtggt tgccatcaac gacagcggcg gtgtcaagca ggccagccac 240 ctgctgaagt acgactccac cctgggcacg ttcgccgccg atgttaagat cgtcgacgac 300 agccacatct cggtggacgg caagcagatc aagattgtgt ccagccgcga ccccctgcag 360 ctgccctgga aggagatgaa catcgacctg gtcattgagg gcactggtgt cttcattgac 420 aaggttggcg ctggcaagca catccaggcc ggtgcctcca aggtgctgat caccgccccc 480 gccaaggaca aggacatccc caccttcgtg gtcggtgtga acgagggcga ctacaagcac 540 gagtacccca tcatctccaa cgcctcgtgc accaccaact gcctggcccc cttcgtcaag 600 gtgctggagc agaagttcgg cattgtcaag ggcacgatga ccaccaccca ctcctacacc 660 ggtgaccagc gcctgctgga cgcgtcccac cgcgacctgc gccgcgcccg cgccgccgcc 720 ctgaacattg tgcccaccac caccggtgcc gccaaggccg tgtcgctggt gctgcccagc 780 ctgaagggca agctgaacgg cattgccctg cgcgtgccca cccccaccgt gtcggtcgtc 840 gacctggtcg tccaggttga gaagaagacc ttcgccgagg aggtgaacgc cgccttccgc 900 gaggccgcca acggccccat gaagggcgtg ctgcacgtcg aggacgcccc cctggtgtcc 960 attgacttca agtgcaccga ccagtcgacc tccatcgacg cctccctgac catggtcatg 1020 ggcgacgaca tggtcaaggt cgtggcctgg tacgacaacg agtggggcta ctcccagcgc 1080 gtggtcgacc tggctgaggt caccgccaag aagtgggtgg cgtaataa 1128 <210> 4 <211> 1971 <212> DNA <213> Chlamydomonas reinhardtii <400> 4 atgcagaagg tgcgcagtgc actcaatacc gaacagcaat acatatccaa agaccagcga 60 cttgtcaccc agaacttcca gatgtgatcc cgacccaagc gcctcaggcg cctctttctt 120 ctagtcctga ccgcgcctgt ttgtttcttg cgcttgcaga gcgccttcac cggcagcgcc 180 gtgtcctcca agtctggcgt ccgcgccaag gtgggcatgg ctcggctttt gcttgcggac 240 ttgggttctt gggtcgcaca gcccagcact cgctggtctt cggcgggctt ctgctacgtg 300 tgtgatcagt tgataaataa gatagttggg ttgccaggtc aacgttcacc gatcctactc 360 tcttgcatcg cgctcgcagg ctgcccgcgc cgtcgtcgac gtgcgcgcgg agaagaagat 420 ccgcgtggcc atcaacggct tcggtcgcat tggccgcaac ttcctgcgct gctggcacgg 480 tcgccagaac accctgctgg acgtggttgc catcaacgac agcggcggtg tcaagcaggc 540 cagccacctg ctgaagtacg actccaccct gggcacgttc gccgccgatg ttaagatcgt 600 cgacgacagc cacatctcgg tggacggcaa gcagatcaag attgtgtcca gccgcgaccc 660 cctgcagctg ccctggaagg agatgaacat cgacctggtc attgagggca ctggtgtctt 720 cattgacaag gttggcgctg gcaagcacat ccaggtgagc atgccgcaat taaagcgcat 780 ctctgggccg ggaagcgacg ggggttttgg gtgtgcacgt tttgggtcgg cactgctcgc 840 tagcaagtgg gggggtgttg gcgttacgag taacgcgctc gctcgcccat tgagctcgtt 900 ccccctgctg acatgacccc cctgccttgc cccacacatc tataggccgg tgcctccaag 960 gtgctgatca ccgcccccgc caaggacaag gacatcccca ccttcgtggt cggtgtgaac 1020 gagggcgact acaagcacga gtaccccatc atctccaacg cctcgtgcac caccaactgc 1080 ctggccccct tcgtcaaggt aaggaagctt acgcatgttg cttgctgtgg ctttttcgtc 1140 aggaataaat gcggggggcg gcaagagcgt ccacggtgtt gcgggtccga gtgccctggg 1200 atatcttgtc ttgtcagctc acctgtctct gacccggccc ttcccctttc ccctctccct 1260 tgcctacccc aggtgctgga gcagaagttc ggcattgtca agggcacgat gaccaccacc 1320 cactcctaca ccggtgacca gcgcctgctg gacgcgtccc accgcgacct gcgccgcgcc 1380 cgcgccgccg ccctgaacat tgtgcccacc accaccggtg ccgccaaggc cgtgtcgctg 1440 gtgctgccca gcctgaaggg caagctgaac ggcattgccc tgcgcgtgcc cacccccacc 1500 gtgtcggtcg tcgacctggt cgtccaggtg agactgcctc cccttgcgct gtaacaacta 1560 cacctggaca gtgggcgccg cttaccgaat tcttgtatat ttcgtgttgc cggtggatgc 1620 tgcacgcctc agtaacttgt cttgaggctg tcactgaccc accctcctgt cctgtcctgc 1680 gctcccgctc aaccacaggt tgagaagaag accttcgccg aggaggtgaa cgccgccttc 1740 cgcgaggccg ccaacggccc catgaagggc gtgctgcacg tcgaggacgc ccccctggtg 1800 tccattgact tcaagtgcac cgaccagtcg acctccatcg acgcctccct gaccatggtc 1860 atgggcgacg acatggtcaa ggtcgtggcc tggtacgaca acgagtgggg ctactcccag 1920 cgcgtggtcg acctggctga ggtcaccgcc aagaagtggg tggcgtaata a 1971 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggggtaccaa ccgaccaatc gatag 25 <210> 6 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cccaagcttc ccgggactag tactggcagg attcggc 37 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gactagtatg cagaaggtgc gcag 24 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tccccccggg ttattacgcc acccacttc 29 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 agcgagctac caaagccata 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 taccgcttca gcacttgaga 20 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ggggtaccaa ccgaccaatc gatag 25 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cccgggttat tacgccaccc acttc 25 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggaggacacg gcgctgccgg t 21 <110> ADVANCED BIOMESS R & D CENTER <120> Recombinant vector for increasing biomass and lipid productivity of microalgae and uses thereof <130> PN13402 <160> 13 <170> Kopatentin 2.0 <210> 1 <211> 287 <212> DNA <213> Chlamydomonas reinhardtii <400> 1 aaccgaccaa tcgatagtct tgtgcgaccg tgcacgtgtg cagcaatagt taggtcgata 60 accacgttga acttgcgtct ctcttcgtgg cgcctcctgc ttggtgctcc acttcacttg 120 tcgctatata gcacagcgtt gaaagcaaag gccacactaa tacagccggg ctcgagagtc 180 cgtctgcgtt tgcattgttg gccaagggct gctttgtagc caaagccata cacgaagctt 240 cacttgatta gctttacgac cctcagccga atcctgccag tactagt 287 <210> 2 <211> 374 <212> PRT <213> Chlamydomonas reinhardtii <400> 2 Met Ala Met Met Gln Lys Ser Ala Phe Thr Gly Ser Ala Val Ser 1 5 10 15 Ser Lys Ser Gly Val Arg Ala Lys Ala Ala Arg Ala Val Val Asp Val 20 25 30 Arg Ala Glu Lys Lys Ile Arg Val Ala Ile Asn Gly Phe Gly Arg Ile 35 40 45 Gly Arg Asn Phe Leu Arg Cys Trp His Gly Arg Gln Asn Thr Leu Leu 50 55 60 Asp Val Val Ala Ile Asn Asp Ser Gly Gly Val Lys Gln Ala Ser His 65 70 75 80 Leu Leu Lys Tyr Asp Ser Thr Leu Gly Thr Phe Ala Ala Asp Val Lys 85 90 95 Ile Val Asp Asp Ser His Ile Ser Val Asp Gly Lys Gln Ile Lys Ile 100 105 110 Val Ser Ser Arg Asp Pro Leu Gln Leu Pro Trp Lys Glu Met Asn Ile 115 120 125 Asp Leu Val Ile Glu Gly Thr Gly Val Phe Ile Asp Lys Val Gly Ala 130 135 140 Gly Lys His Ile Gln Ala Gly Ala Ser Lys Val Leu Ile Thr Ala Pro 145 150 155 160 Ala Lys Asp Lys Asp Ile Pro Thr Phe Val Val Gly Val Asn Glu Gly 165 170 175 Asp Tyr Lys His Glu Tyr Pro Ile Ile Ser Asn Ala Ser Cys Thr Thr 180 185 190 Asn Cys Leu Ala Pro Phe Val Lys Val Leu Glu Gln Lys Phe Gly Ile 195 200 205 Val Lys Gly Thr Met Thr Thr Thr His Ser Tyr Thr Gly Asp Gln Arg 210 215 220 Leu Leu Asp Ala Ser His Arg Asp Leu Arg Arg Ala Arg Ala Ala Ala 225 230 235 240 Leu Asn Ile Val Pro Thr Thr Thr Gly Ala Ala Lys Ala Val Ser Leu 245 250 255 Val Leu Pro Ser Leu Lys Gly Lys Leu Asn Gly Ile Ala Leu Arg Val 260 265 270 Pro Thr Pro Thr Val Ser Val Val Asp Leu Val Val Gln Val Glu Lys 275 280 285 Lys Thr Phe Ala Glu Glu Val Asn Ala Ala Phe Arg Glu Ala Ala Asn 290 295 300 Gly Pro Met Lys Gly Val Leu His Val Glu Asp Ala Pro Leu Val Ser 305 310 315 320 Ile Asp Phe Lys Cys Thr Asp Gln Ser Thr Ser Ile Asp Ala Ser Leu 325 330 335 Thr Met Val Met Gly Asp Met Val Lys Val Val Ala Trp Tyr Asp 340 345 350 Asn Glu Trp Gly Tyr Ser Gln Arg Val Val Asp Leu Ala Glu Val Thr 355 360 365 Ala Lys Lys Trp Val Ala 370 <210> 3 <211> 1128 <212> DNA <213> Chlamydomonas reinhardtii <400> 3 atggccgcca tgatgcagaa gagcgccttc accggcagcg ccgtgtcctc caagtctggc 60 gtccgcgcca aggctgcccg cgccgtcgtc gacgtgcgcg cggagaagaa gatccgcgtg 120 gccatcaacg gcttcggtcg cattggccgc aacttcctgc gctgctggca cggtcgccag 180 aacaccctgc tggacgtggt tgccatcaac gacagcggcg gtgtcaagca ggccagccac 240 ctgctgaagt acgactccac cctgggcacg ttcgccgccg atgttaagat cgtcgacgac 300 ccccctgcag 360 ctgccctgga aggagatgaa catcgacctg gtcattgagg gcactggtgt cttcattgac 420 aaggttggcg ctggcaagca catccaggcc ggtgcctcca aggtgctgat caccgccccc 480 gccaaggaca aggacatccc caccttcgtg gtcggtgtga acgagggcga ctacaagcac 540 gagtacccca tcatctccaa cgcctcgtgc accaccaact gcctggcccc cttcgtcaag 600 gtgctggagc agaagttcgg cattgtcaag ggcacgatga ccaccaccca ctcctacacc 660 ggtgaccagc gcctgctgga cgcgtcccac cgcgacctgc gccgcgcccg cgccgccgcc 720 ctgaacattg tgcccaccac caccggtgcc gccaaggccg tgtcgctggt gctgcccagc 780 ctgaagggca agctgaacgg cattgccctg cgcgtgccca cccccaccgt gtcggtcgtc 840 gacctggtcg tccaggttga gaagaagacc ttcgccgagg aggtgaacgc cgccttccgc 900 gaggccgcca acggccccat gaagggcgtg ctgcacgtcg aggacgcccc cctggtgtcc 960 attgacttca agtgcaccga ccagtcgacc tccatcgacg cctccctgac catggtcatg 1020 ggcgacgaca tggtcaaggt cgtggcctgg tacgacaacg agtggggcta ctcccagcgc 1080 gtggtcgacc tggctgaggt caccgccaag aagtgggtgg cgtaataa 1128 <210> 4 <211> 1971 <212> DNA <213> Chlamydomonas reinhardtii <400> 4 atgcagaagg tgcgcagtgc actcaatacc gaacagcaat acatatccaa agaccagcga 60 cttgtcaccc agaacttcca gatgtgatcc cgacccaagc gcctcaggcg cctctttctt 120 ctagtcctga ccgcgcctgt ttgtttcttg cgcttgcaga gcgccttcac cggcagcgcc 180 gtgtcctcca agtctggcgt ccgcgccaag gtgggcatgg ctcggctttt gcttgcggac 240 ttgggttctt gggtcgcaca gcccagcact cgctggtctt cggcgggctt ctgctacgtg 300 tgtgatcagt tgataaataa gatagttggg ttgccaggtc aacgttcacc gatcctactc 360 tcttgcatcg cgctcgcagg ctgcccgcgc cgtcgtcgac gtgcgcgcgg agaagaagat 420 ccgcgtggcc atcaacggct tcggtcgcat tggccgcaac ttcctgcgct gctggcacgg 480 tcgccagaac accctgctgg acgtggttgc catcaacgac agcggcggtg tcaagcaggc 540 cagccacctg ctgaagtacg actccaccct gggcacgttc gccgccgatg ttaagatcgt 600 cgacgacagc cacatctcgg tggacggcaa gcagatcaag attgtgtcca gccgcgaccc 660 cctgcagctg ccctggaagg agatgaacat cgacctggtc attgagggca ctggtgtctt 720 cattgacaag gttggcgctg gcaagcacat ccaggtgagc atgccgcaat taaagcgcat 780 ctctgggccg ggaagcgacg ggggttttgg gtgtgcacgt tttgggtcgg cactgctcgc 840 tagcaagtgg gggggtgttg gcgttacgag taacgcgctc gctcgcccat tgagctcgtt 900 ccccctgctg acatgacccc cctgccttgc cccacacatc tataggccgg tgcctccaag 960 gtgctgatca ccgcccccgc caaggacaag gacatcccca ccttcgtggt cggtgtgaac 1020 gagggcgact acaagcacga gtaccccatc atctccaacg cctcgtgcac caccaactgc 1080 ctggccccct tcgtcaaggt aaggaagctt acgcatgttg cttgctgtgg ctttttcgtc 1140 aggaataaat gcggggggcg gcaagagcgt ccacggtgtt gcgggtccga gtgccctggg 1200 atatcttgtc ttgtcagctc acctgtctct gacccggccc ttcccctttc ccctctccct 1260 tgcctacccc aggtgctgga gcagaagttc ggcattgtca agggcacgat gaccaccacc 1320 cactcctaca ccggtgacca gcgcctgctg gacgcgtccc accgcgacct gcgccgcgcc 1380 cgcgccgccg ccctgaacat tgtgcccacc accaccggtg ccgccaaggc cgtgtcgctg 1440 gtgctgccca gcctgaaggg caagctgaac ggcattgccc tgcgcgtgcc cacccccacc 1500 gtgtcggtcg tcgacctggt cgtccaggtg agactgcctc cccttgcgct gtaacaacta 1560 cacctggaca gtgggcgccg cttaccgaat tcttgtatat ttcgtgttgc cggtggatgc 1620 tgcacgcctc agtaacttgt cttgaggctg tcactgaccc accctcctgt cctgtcctgc 1680 gctcccgctc aaccacaggt tgagaagaag accttcgccg aggaggtgaa cgccgccttc 1740 cgcgaggccg ccaacggccc catgaagggc gtgctgcacg tcgaggacgc ccccctggtg 1800 tccattgact tcaagtgcac cgaccagtcg acctccatcg acgcctccct gaccatggtc 1860 atgggcgacg acatggtcaa ggtcgtggcc tggtacgaca acgagtgggg ctactcccag 1920 cgcgtggtcg acctggctga ggtcaccgcc aagaagtggg tggcgtaata a 1971 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggggtaccaa ccgaccaatc gatag 25 <210> 6 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cccaagcttc ccgggactag tactggcagg attcggc 37 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gactagtatg cagaaggtgc gcag 24 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tccccccggg ttattacgcc acccacttc 29 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 agcgagctac caaagccata 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 taccgcttca gcacttgaga 20 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ggggtaccaa ccgaccaatc gatag 25 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cccgggttat tacgccaccc acttc 25 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggaggacacg gcgctgccgg t 21
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