KR20230151687A - Microorganism with improved lipid productivity into which tyrosine 3-monooxygenase presumed gene is introduced, and method for manufacturing lipids using the same - Google Patents
Microorganism with improved lipid productivity into which tyrosine 3-monooxygenase presumed gene is introduced, and method for manufacturing lipids using the same Download PDFInfo
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- KR20230151687A KR20230151687A KR1020220051333A KR20220051333A KR20230151687A KR 20230151687 A KR20230151687 A KR 20230151687A KR 1020220051333 A KR1020220051333 A KR 1020220051333A KR 20220051333 A KR20220051333 A KR 20220051333A KR 20230151687 A KR20230151687 A KR 20230151687A
- Authority
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- South Korea
- Prior art keywords
- monooxygenase
- tyrosine
- protein
- microorganism
- present
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Abstract
본 발명은 타이로신 3-모노옥시제네이즈 (tyrosine 3-monooxygenase) 유사 유전자, 이를 포함하는 재조합 벡터, 상기 유전자가 도입된 지질 생산성이 향상된 미생물, 상기 미생물의 제조방법 및 이를 이용한 지질 생산 방법에 관한 것이다.The present invention relates to a tyrosine 3-monooxygenase-like gene, a recombinant vector containing the same, a microorganism with improved lipid productivity into which the gene is introduced, a method for producing the microorganism, and a method for producing lipids using the same. .
Description
본 발명은 타이로신 3-모노옥시제네이즈 (tyrosine 3-monooxygenase) 유사 유전자, 이를 포함하는 재조합 벡터, 상기 유전자가 도입된 지질 생산성이 향상된 미생물, 상기 미생물의 제조방법 및 이를 이용한 지질 생산 방법에 관한 것이다.The present invention relates to a tyrosine 3-monooxygenase-like gene, a recombinant vector containing the same, a microorganism with improved lipid productivity into which the gene is introduced, a method for producing the microorganism, and a method for producing lipids using the same. .
최근 전세계적으로 화석에너지의 고갈과 지구온난화에 직면하여, 미국을 포함하는 선진국을 중심으로 환경 친화적인 바이오, 수소, 태양 등의 신 재생에너지 활용 및 확산을 위한 정책 입안과 함께 이들 신 재생에너지의 공급과 사용을 촉진하고 있다. 국내에서도 신 재생에너지 개발에 많은 노력을 하고 있지만, 현재 연구단계에 머물고 있는 실정이다. 바이오 에너지는 재생 가능하며, 이산화탄소를 고정함으로써 지구온난화 가속 현상을 감소시킬 수 있는 장점이 있어, 현재 바이오 에탄올, 바이오 부탄올, 바이오 디젤 등에 대한 연구가 활발하게 진행되고 있다.Recently, in the face of depletion of fossil energy and global warming around the world, developed countries, including the United States, have been enacting policies to utilize and expand environmentally friendly renewable energy such as bio, hydrogen, and solar energy, as well as developing new renewable energy. Promoting supply and use. Although much effort is being made in Korea to develop new renewable energy, it is currently in the research stage. Bioenergy is renewable and has the advantage of reducing the acceleration of global warming by fixing carbon dioxide, so research on bioethanol, biobutanol, biodiesel, etc. is currently being actively conducted.
바이오 에너지 중에서도, 가장 활발하게 연구가 진행되고 있는 것은 바이오 디젤이다. 바이오 디젤은 바이오 매스에 포함된 지방산 (fatty acid)의 트랜스에스터화 (transesterification) 과정에 의해 메틸 에스터 또는 에틸 에스터 형태로 전환되어 생성되는데, 150 ℃의 인화점을 가지고 있어 인화점이 64 ℃인 경유에 비해 불이 잘 붙지 않고, 낮은 온도에서 휘발성이 높은 휘발유 (45 ℃)보다 안정적이기 때문에 비가연성 액체로 분류되며, 고온에서 불이 붙기 때문에 안정성이 더 높다고 할 수 있다. 또한, 경유와 달리 바이오 디젤은 연소될 때 발암물질 및 돌연변이를 일으키는 유해 가스 방출량이 적으며 기타 배출물이 현저하게 낮은 비독성 에너지로 알려져 있다.Among bioenergy, the one on which research is most actively underway is biodiesel. Biodiesel is produced by converting fatty acids contained in biomass into methyl ester or ethyl ester form through a transesterification process. It has a flash point of 150 ℃, making it cheaper than diesel, which has a flash point of 64 ℃. It is classified as a non-flammable liquid because it does not catch fire easily and is more stable at low temperatures than highly volatile gasoline (45°C), and because it catches fire at high temperatures, it can be said to have higher stability. In addition, unlike diesel, biodiesel is known to be a non-toxic energy that emits less harmful gases that cause carcinogens and mutations when burned and has significantly lower other emissions.
이러한 바이오 디젤은 미세조류를 이용하여 생물학적으로 생산할 수 있다고 알려져 있다. 미세조류는 사료 또는 비료의 용도로 사용되고 있으며, 특히 녹조류에 속하는 스피룰리나 (Spirulina sp.), 클로렐라 (Chlorella sp.), 두날리엘라 (Dunaliella sp.), 노스톡 (Nostoc sp.) 등은 건강식품으로도 이용되고 있다. 이러한 미세조류는 태양에너지의 이용효율이 식물에 비하여 약 25배 높기 때문에, 지질의 함량이 우수한 미세조류는 바이오 디젤의 생산을 위한 바이오매스로 사용할 수 있다. It is known that such biodiesel can be produced biologically using microalgae. Microalgae are used as feed or fertilizer, and in particular, green algae such as Spirulina sp., Chlorella sp., Dunaliella sp., and Nostoc sp. are health foods. It is also used. Since the solar energy utilization efficiency of these microalgae is about 25 times higher than that of plants, microalgae with excellent lipid content can be used as biomass for the production of biodiesel.
이처럼 지질의 함량이 우수한 미세조류로는 보트리오코커스 (Botryococcus sp.)와 쉬조키트리움 (Schizochytrium sp.)이 알려져 있는데, 미세조류는 정상적인 생활환경에서는 지질의 함량이 높지 않지만, 영양공급이 중단된 상태에서 일정시간이 경과하면, 세포 내에 지질을 축적하는 특성을 나타낸다. Botryococcus sp. and Schizochytrium sp. are known as microalgae with excellent lipid content. Microalgae do not have high lipid content in normal living environments, but when nutritional supply is interrupted, microalgae do not have high lipid content. After a certain period of time passes in this state, it exhibits the characteristic of accumulating lipids within cells.
이러한 특성을 이용하여 미세조류를 바이오 디젤의 생산을 위한 바이오 매스로 사용할 수 있으나, 미세조류 내 지질함량을 증가시키기 위해서는 미세조류를 배양하여 증식하는 제1공정과, 증식된 미세조류에 영양공급을 중단하고 일정시간 동안 배양하여 세포 내 지질함량을 증대시키는 제2공정이 수행되어야 하는데, 2 단계 공정에는 과다한 시간이 요구되기 때문에 아직까지는 바이오 디젤의 산업적 생산에 사용되지 못하고 있으므로, 바이오 디젤의 생산을 위한 지질함량이 우수한 미세조류의 개발이 절실히 요구되고 있는 실정이다. Using these characteristics, microalgae can be used as biomass for the production of biodiesel. However, to increase the lipid content in microalgae, the first process of cultivating and proliferating microalgae and supplying nutrients to the proliferated microalgae are required. A second process must be performed to increase the lipid content within the cells by stopping the cultivation for a certain period of time. However, because the second-stage process requires an excessive amount of time, it has not yet been used in the industrial production of biodiesel. There is an urgent need for the development of microalgae with excellent lipid content.
특히 바이오 디젤 생산을 위해 사용되고 있는 미세조류의 경우 세포내의 대사 특성에 관한 연구가 부족하여 지질 생산성 증대를 위한 타겟 유전자 선별이 필요한 실정이다. 즉, 지질 함량이 증가된 재조합 균주를 개발하기 위하여 어떠한 유전자를 증폭해야 하는지에 관한 선행 연구가 부족하다.In particular, in the case of microalgae used for biodiesel production, there is a lack of research on intracellular metabolic characteristics, so target gene selection to increase lipid productivity is necessary. In other words, there is a lack of prior research on which genes should be amplified to develop recombinant strains with increased lipid content.
본 발명자는 방사무늬김 (Pyropia yezoensis) 유래 타이로신 3-모노옥시제네이즈 (tyrosine 3-monooxygenase) 유사 유전자를 미세조류에 도입하여 과발현시킬 경우, 재조합 미생물에서 지질 함량이 증가한다는 것을 확인하고 본 발명을 완성하게 되었다.The present inventor confirmed that when a tyrosine 3-monooxygenase-like gene derived from Pyropia yezoensis is introduced into microalgae and overexpressed, the lipid content in the recombinant microorganism increases, and the present invention It has been completed.
본 발명의 목적은 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드를 제공하는 것이다.An object of the present invention is to provide a polynucleotide encoding a tyrosine 3-monooxygenase-like protein.
본 발명의 다른 목적은 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드를 포함하는 재조합 벡터를 제공하는 것이다. Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a tyrosine 3-monooxygenase-like protein.
본 발명의 또 다른 목적은 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드를 포함하는 재조합 벡터로 형질전환된 지질 생산성이 향상된 미생물을 제공하는 것이다.Another object of the present invention is to provide a microorganism with improved lipid productivity transformed with a recombinant vector containing a polynucleotide encoding a tyrosine 3-monooxygenase-like protein.
본 발명의 또 다른 목적은 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드를 포함하는 재조합 벡터로 미생물을 형질전환하는 형질전환 단계를 포함하는 지질 생산성이 향상된 미생물의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing microorganisms with improved lipid productivity, including a transformation step of transforming the microorganism with a recombinant vector containing a polynucleotide encoding a tyrosine 3-monooxygenase-like protein. .
본 발명의 또 다른 목적은 다음의 단계를 포함하는 지질을 생산하는 방법을 제공하는 것이다:Another object of the present invention is to provide a method for producing lipids comprising the following steps:
타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드를 포함하는 재조합 벡터로 형질전환된 미생물을 배양하는 배양단계; 및A culturing step of culturing a microorganism transformed with a recombinant vector containing a polynucleotide encoding a tyrosine 3-monooxygenase-like protein; and
지질을 추출하는 추출단계.Extraction step to extract lipids.
본 발명은 타이로신 3-모노옥시제네이즈 (tyrosine 3-monooxygenase) 유사 유전자가 도입된 지질 생산성이 향상된 미생물 및 이를 이용한 지질 생산 방법에 관한 것으로서, 본 발명에 따라 타이로신 3-모노옥시제네이즈 유사 유전자가 도입된 클라미도모나스 라인하드티 (Chlamydomonas reinhardtii) 균주는 향상된 지질 생산성을 나타내었다.The present invention relates to a microorganism with improved lipid productivity into which a tyrosine 3-monooxygenase-like gene has been introduced and a lipid production method using the same. According to the present invention, the tyrosine 3-monooxygenase-like gene is The introduced Chlamydomonas reinhardtii strain showed improved lipid productivity.
본 발명자는 환경에 따른 방사무늬김 (Pyropia yezoensis) 유전자의 발현 변화로부터 특이적으로 높게 발현하는 유전자들 중에서 타이로신 3-모노옥시제네이즈 유사 유전자를 발견하고, 이 유전자를 미세조류 클라미도모나스 라인하드티에 형질전환하여 지질 함량을 평가하였다.The present inventor discovered a tyrosine 3-monooxygenase-like gene among genes that are specifically highly expressed from changes in the expression of genes in Pyropia yezoensis depending on the environment, and transferred this gene to the microalgae Chlamydomonas Reinhard. The lipid content was evaluated by transformation into T.
그 결과 타이로신 3-모노옥시제네이즈 유사 유전자가 형질전환된 재조합 클라미도모나스 라인하드티가 지질 생산을 위한 배양 환경에서 우수한 성장을 보이고, 야생형에 비하여 더 높은 함량의 지질을 합성한다는 것이 확인되었으므로, 이를 활용하여 지질, 전분 등과 같은 대사 물질을 대량 생산할 수 있을 것으로 기대된다.As a result, it was confirmed that recombinant Chlamydomonas reinhardtii transformed with a tyrosine 3-monooxygenase-like gene showed excellent growth in a culture environment for lipid production and synthesized a higher content of lipids compared to the wild type. It is expected that this will be able to mass produce metabolites such as lipids and starch.
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드에 관한 것이다.One aspect of the invention relates to a polynucleotide encoding a tyrosine 3-monooxygenase-like protein.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질은 서열번호 4의 아미노산 서열을 포함하는 펩타이드인 것일 수 있으며, 예를 들어, 서열번호 4의 아미노산 서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the tyrosine 3-monooxygenase-like protein may be a peptide containing the amino acid sequence of SEQ ID NO: 4, for example, may be composed of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto. no.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드는 서열번호 3의 염기서열 또는 이와 실질적 동일성을 갖는 염기서열을 포함하는 폴리뉴클레오타이드인 것일 수 있으며, 예를 들어, 서열번호 3의 염기서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the polynucleotide encoding a tyrosine 3-monooxygenase-like protein may be a polynucleotide containing the base sequence of SEQ ID NO: 3 or a base sequence substantially identical thereto, for example, SEQ ID NO: It may consist of a base sequence of 3, but is not limited to this.
본 발명에 있어서, “실질적 동일성”은 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 뉴클레오타이드 서열과 임의의 다른 뉴클레오타이드 서열을 최대한 대응되도록 정렬하고, 그 서열을 분석하여, 임의의 다른 뉴클레오타이드 서열이 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 뉴클레오타이드 서열과 70% 이상, 80% 이상, 90% 이상, 또는 98% 이상의 서열 상동성을 갖는 것을 의미한다.In the present invention, “substantial identity” means aligning the nucleotide sequence encoding a tyrosine 3-monooxygenase-like protein and any other nucleotide sequence to correspond as much as possible, and analyzing the sequence, so that any other nucleotide sequence is tyrosine. It means having at least 70%, at least 80%, at least 90%, or at least 98% sequence homology with the nucleotide sequence encoding a 3-monooxygenase-like protein.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드는 방사무늬김에서 유래한 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the polynucleotide encoding a tyrosine 3-monooxygenase-like protein may be derived from radioactive seaweed, but is not limited thereto.
본 발명의 다른 양태는 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드를 포함하는 재조합 벡터에 관한 것이다.Another aspect of the present invention relates to a recombinant vector comprising a polynucleotide encoding a tyrosine 3-monooxygenase-like protein.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질은 서열번호 4의 아미노산 서열을 포함하는 펩타이드인 것일 수 있으며, 예를 들어, 서열번호 4의 아미노산 서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the tyrosine 3-monooxygenase-like protein may be a peptide containing the amino acid sequence of SEQ ID NO: 4, for example, may be composed of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto. no.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드는 서열번호 3의 염기서열 또는 이와 실질적 동일성을 갖는 염기서열을 포함하는 폴리뉴클레오타이드인 것일 수 있으며, 예를 들어, 서열번호 3의 염기서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the polynucleotide encoding a tyrosine 3-monooxygenase-like protein may be a polynucleotide containing the base sequence of SEQ ID NO: 3 or a base sequence substantially identical thereto, for example, SEQ ID NO: It may consist of a base sequence of 3, but is not limited to this.
본 발명에 있어서, “실질적 동일성”은 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 뉴클레오타이드 서열과 임의의 다른 뉴클레오타이드 서열을 최대한 대응되도록 정렬하고, 그 서열을 분석하여, 임의의 다른 뉴클레오타이드 서열이 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 뉴클레오타이드 서열과 70% 이상, 80% 이상, 90% 이상, 또는 98% 이상의 서열 상동성을 갖는 것을 의미한다.In the present invention, “substantial identity” means aligning the nucleotide sequence encoding a tyrosine 3-monooxygenase-like protein and any other nucleotide sequence to correspond as much as possible, and analyzing the sequence, so that any other nucleotide sequence is tyrosine. It means having at least 70%, at least 80%, at least 90%, or at least 98% sequence homology with the nucleotide sequence encoding a 3-monooxygenase-like protein.
본 명세서 상의 용어 '벡터'는 숙주 세포에서 목적 유전자를 발현시키기 위한 수단을 의미하며, 숙주 세포의 형질전환을 위한 재조합 벡터는 클로닝 벡터 (cloning vector)와 발현 벡터 (expression vector)를 모두 포함한다. 예를 들어, 플라스미드 벡터, 코즈미드 벡터 및 박테리오파아지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터 및 아데노 연관 바이러스 벡터와 같은 바이러스 벡터를 포함한다. 또한 재조합 벡터로 사용될 수 있는 벡터는 당업계에서 종종 사용되는 플라스미드 (예를 들면, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX 시리즈, pET 시리즈 및 pUC19 등), 파지 (예를 들면, λgt4λB, λ-Charon, λΔz1 및 M13 등) 또는 바이러스 (예를 들면, SV40 등) 등이 있을 수 있으나, 이에 한정되는 것은 아니다.The term 'vector' in this specification refers to a means for expressing a target gene in a host cell, and recombinant vectors for transformation of host cells include both cloning vectors and expression vectors. Examples include plasmid vectors, cosmid vectors and viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated viral vectors. In addition, vectors that can be used as recombinant vectors include plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.), or viruses (e.g., SV40, etc.), but are not limited thereto.
본 발명의 또 다른 양태는 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드를 포함하는 재조합 벡터로 형질전환된 지질 생산성이 향상된 미생물에 관한 것이다. Another aspect of the present invention relates to a microorganism with improved lipid productivity transformed with a recombinant vector containing a polynucleotide encoding a tyrosine 3-monooxygenase-like protein.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질은 서열번호 4의 아미노산 서열을 포함하는 펩타이드인 것일 수 있으며, 예를 들어, 서열번호 4의 아미노산 서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the tyrosine 3-monooxygenase-like protein may be a peptide containing the amino acid sequence of SEQ ID NO: 4, for example, may be composed of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto. no.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드는 서열번호 3의 염기서열 또는 이와 실질적 동일성을 갖는 염기서열을 포함하는 폴리뉴클레오타이드인 것일 수 있으며, 예를 들어, 서열번호 3의 염기서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the polynucleotide encoding a tyrosine 3-monooxygenase-like protein may be a polynucleotide containing the base sequence of SEQ ID NO: 3 or a base sequence substantially identical thereto, for example, SEQ ID NO: It may consist of a base sequence of 3, but is not limited to this.
본 발명에 있어서, “실질적 동일성”은 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 뉴클레오타이드 서열과 임의의 다른 뉴클레오타이드 서열을 최대한 대응되도록 정렬하고, 그 서열을 분석하여, 임의의 다른 뉴클레오타이드 서열이 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 뉴클레오타이드 서열과 70% 이상, 80% 이상, 90% 이상, 또는 98% 이상의 서열 상동성을 갖는 것을 의미한다.In the present invention, “substantial identity” means aligning the nucleotide sequence encoding a tyrosine 3-monooxygenase-like protein and any other nucleotide sequence to correspond as much as possible, and analyzing the sequence, so that any other nucleotide sequence is tyrosine. It means having at least 70%, at least 80%, at least 90%, or at least 98% sequence homology with the nucleotide sequence encoding a 3-monooxygenase-like protein.
본 발명에 있어서, 지질 (lipid)은 비극성 용매에 용해되는 생분자들을 총칭하는 표현으로, 지질에는 지방산 (fatty acid), 글리세롤 지질 (glycerolipid), 스테롤 지질, (sterol), 인지질 (phospholipid), 스핑고 지질(sphingolipid), 프레놀 지질 (prenol lipid) 등이 있을 수 있으나, 이에 한정되는 것은 아니다.In the present invention, lipid is a general term for biomolecules soluble in non-polar solvents. Lipids include fatty acids, glycerol lipids, sterol lipids, phospholipids, and lipids. There may be sphingolipid, prenol lipid, etc., but it is not limited thereto.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드는 방사무늬김에서 유래한 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the polynucleotide encoding a tyrosine 3-monooxygenase-like protein may be derived from radioactive seaweed, but is not limited thereto.
본 발명에 있어서, 미생물은 미세조류일 수 있으며, 예를 들어, 클라미도모나스 라인하드티(Chlamydomonas reinhardtii), 쉬조키트리움 맹그로베이(Schizochytrium mangrovei), 보트리오코커스 브라우니(Botryococcus braunii)로 이루어진 군 중에서 어느 하나 이상일 수 있으나, 이에 한정되는 것은 아니다. In the present invention, the microorganism may be microalgae, for example, a group consisting of Chlamydomonas reinhardtii, Schizochytrium mangrovei, and Botryococcus braunii. It may be one or more of these, but is not limited thereto.
본 발명의 또 다른 양태는 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드를 포함하는 재조합 벡터로 미생물을 형질전환하는 형질전환 단계를 포함하는 지질 생산성이 향상된 미생물의 제조방법에 관한 것이다.Another aspect of the present invention relates to a method for producing a microorganism with improved lipid productivity, comprising a transformation step of transforming the microorganism with a recombinant vector containing a polynucleotide encoding a tyrosine 3-monooxygenase-like protein.
본 발명의 일 구현예에서, 목적 단백질을 코딩하는 유전자가 삽입된 발현 벡터를 숙주세포에 삽입함으로써 지질 생산능력이 향상된 형질전환체를 만들 수 있다.In one embodiment of the present invention, a transformant with improved lipid production ability can be created by inserting an expression vector containing a gene encoding a target protein into a host cell.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질은 서열번호 4의 아미노산 서열을 포함하는 펩타이드인 것일 수 있으며, 예를 들어, 서열번호 4의 아미노산 서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the tyrosine 3-monooxygenase-like protein may be a peptide containing the amino acid sequence of SEQ ID NO: 4, for example, may be composed of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto. no.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드는 서열번호 3의 염기서열 또는 이와 실질적 동일성을 갖는 염기서열을 포함하는 폴리뉴클레오타이드인 것일 수 있으며, 예를 들어, 서열번호 3의 염기서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the polynucleotide encoding a tyrosine 3-monooxygenase-like protein may be a polynucleotide containing the base sequence of SEQ ID NO: 3 or a base sequence substantially identical thereto, for example, SEQ ID NO: It may consist of a base sequence of 3, but is not limited to this.
본 발명에 있어서, “실질적 동일성”은 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 뉴클레오타이드 서열과 임의의 다른 뉴클레오타이드 서열을 최대한 대응되도록 정렬하고, 그 서열을 분석하여, 임의의 다른 뉴클레오타이드 서열이 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 뉴클레오타이드 서열과 70% 이상, 80% 이상, 90% 이상, 또는 98% 이상의 서열 상동성을 갖는 것을 의미한다.In the present invention, “substantial identity” means aligning the nucleotide sequence encoding a tyrosine 3-monooxygenase-like protein and any other nucleotide sequence to correspond as much as possible, and analyzing the sequence, so that any other nucleotide sequence is tyrosine. It means having at least 70%, at least 80%, at least 90%, or at least 98% sequence homology with the nucleotide sequence encoding a 3-monooxygenase-like protein.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드는 방사무늬김에서 유래한 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the polynucleotide encoding a tyrosine 3-monooxygenase-like protein may be derived from radioactive seaweed, but is not limited thereto.
본 발명에 있어서 숙주세포는 삽입된 발현 벡터를 안정되면서 연속적으로 클로닝 또는 발현시킬 수 있는 세포로서 당업계에 공지된 어떠한 숙주 세포도 이용할 수 있고, 형질전환체는 재조합 벡터를 적절한 숙주 세포에 도입시킴으로써 얻어진 것일 수 있다. In the present invention, the host cell is a cell capable of stably and continuously cloning or expressing the inserted expression vector, and any host cell known in the art can be used, and the transformant is created by introducing the recombinant vector into an appropriate host cell. It may have been obtained.
본 발명에 있어서, 미생물은 미세조류일 수 있으며, 예를 들어, 클라미도모나스 라인하드티(Chlamydomonas reinhardtii), 쉬조키트리움 맹그로베이(Schizochytrium mangrovei), 보트리오코커스 브라우니(Botryococcus braunii)로 이루어진 군 중에서 어느 하나 이상일 수 있으나, 이에 한정되는 것은 아니다. In the present invention, the microorganism may be microalgae, for example, a group consisting of Chlamydomonas reinhardtii, Schizochytrium mangrovei, and Botryococcus braunii. It may be one or more of these, but is not limited thereto.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 유전자 또는 이를 포함하는 재조합 벡터의 숙주 세포 내로의 운반(도입)은, 당업계에 널리 알려진 운반 방법을 사용할 수 있다. 재조합 벡터의 운반 방법은 예를 들어, 숙주 세포가 원핵 세포인 경우, CaCl2 방법 또는 전기 천공 방법 등을 사용할 수 있고, 숙주 세포가 진핵 세포인 경우, 미세 주입법, 칼슘 포스페이트 침전법, 전기 천공법, 리포좀매개 형질감염법 및 유전자 밤바드먼트 등을 사용할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, a tyrosine 3-monooxygenase-like gene or a recombinant vector containing the same can be transported (introduced) into a host cell using a transport method widely known in the art. For example, if the host cell is a prokaryotic cell, the CaCl 2 method or electroporation method can be used as a transport method for the recombinant vector, and if the host cell is a eukaryotic cell, microinjection method, calcium phosphate precipitation method, or electroporation method can be used. , liposome-mediated transfection, and gene bombardment can be used, but are not limited to these.
본 발명에 있어서, 형질 전환된 숙주 세포의 선별은 선택 표지에 의해 발현되는 표현형을 이용하여, 당업계에 널리 알려진 방법에 따라 용이하게 이루어질 수 있다. 예를 들어, 선택 표지가 특정 항생제 내성 유전자인 경우에는, 특정 항생제가 함유된 배지에서 형질전환체를 배양함으로써 형질전환체를 용이하게 선별할 수 있다.In the present invention, selection of transformed host cells can be easily performed using a phenotype expressed by a selection marker, according to methods widely known in the art. For example, if the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the specific antibiotic.
본 발명의 또 다른 양태는 다음의 단계를 포함하는 지질을 생산하는 방법에 관한 것이다:Another aspect of the invention relates to a method for producing lipids comprising the following steps:
타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드를 포함하는 재조합 벡터로 형질전환된 미생물을 배양하는 배양단계; 및A culturing step of culturing a microorganism transformed with a recombinant vector containing a polynucleotide encoding a tyrosine 3-monooxygenase-like protein; and
지질을 추출하는 추출단계.Extraction step to extract lipids.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질은 서열번호 4의 아미노산 서열을 포함하는 펩타이드인 것일 수 있으며, 예를 들어, 서열번호 4의 아미노산 서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the tyrosine 3-monooxygenase-like protein may be a peptide containing the amino acid sequence of SEQ ID NO: 4, for example, may be composed of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto. no.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드는 서열번호 3의 염기서열 또는 이와 실질적 동일성을 갖는 염기서열을 포함하는 폴리뉴클레오타이드인 것일 수 있으며, 예를 들어, 서열번호 3의 염기서열로 이루어진 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the polynucleotide encoding a tyrosine 3-monooxygenase-like protein may be a polynucleotide containing the base sequence of SEQ ID NO: 3 or a base sequence substantially identical thereto, for example, SEQ ID NO: It may consist of a base sequence of 3, but is not limited to this.
본 발명에 있어서, “실질적 동일성”은 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 뉴클레오타이드 서열과 임의의 다른 뉴클레오타이드 서열을 최대한 대응되도록 정렬하고, 그 서열을 분석하여, 임의의 다른 뉴클레오타이드 서열이 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 뉴클레오타이드 서열과 70% 이상, 80% 이상, 90% 이상, 또는 98% 이상의 서열 상동성을 갖는 것을 의미한다.In the present invention, “substantial identity” means aligning the nucleotide sequence encoding a tyrosine 3-monooxygenase-like protein and any other nucleotide sequence to correspond as much as possible, and analyzing the sequence, so that any other nucleotide sequence is tyrosine. It means having at least 70%, at least 80%, at least 90%, or at least 98% sequence homology with the nucleotide sequence encoding a 3-monooxygenase-like protein.
본 발명에 있어서, 추출되는 지질은 지방산의 트랜스에스터화 과정에 의해 에스터 (ester)의 형태로 추출되는 것일 수 있다.In the present invention, the extracted lipid may be extracted in the form of an ester through a transesterification process of fatty acids.
본 발명에 있어서, 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드는 방사무늬김에서 유래한 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the polynucleotide encoding a tyrosine 3-monooxygenase-like protein may be derived from radioactive seaweed, but is not limited thereto.
본 발명에 있어서, 미생물은 미세조류일 수 있으며, 예를 들어, 클라미도모나스 라인하드티(Chlamydomonas reinhardtii), 쉬조키트리움 맹그로베이(Schizochytrium mangrovei), 보트리오코커스 브라우니(Botryococcus braunii)로 이루어진 군 중에서 어느 하나 이상일 수 있으나, 이에 한정되는 것은 아니다. In the present invention, the microorganism may be microalgae, for example, a group consisting of Chlamydomonas reinhardtii, Schizochytrium mangrovei, and Botryococcus braunii. It may be one or more of these, but is not limited thereto.
본 발명의 일 구현예에서, 재조합 클라미도모나스 라인하드티는 타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드가 도입되지 않은 야생형 대조군에 비하여 지질의 함량이 높아져 지질의 함량이 높아져 바이오디젤 생산량이 현저히 증가되는 효과를 나타내었고, 이는 향후 바이오디젤 생산에 활용될 수 있다.In one embodiment of the present invention, the recombinant Chlamydomonas reinhardtii has an increased lipid content compared to the wild-type control group in which a polynucleotide encoding a tyrosine 3-monooxygenase-like protein is not introduced, and the lipid content is increased to produce biodiesel. It showed the effect of significantly increasing production, which can be used for biodiesel production in the future.
본 발명에 있어서, 바이오 디젤은 지방산 (fatty acid)의 트랜스에스터화 (transesterification) 과정에 의해 메틸 에스터 또는 에틸 에스터 형태로 전환되어 생성되는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, biodiesel may be produced by converting fatty acids into methyl ester or ethyl ester form through a transesterification process, but is not limited thereto.
본 발명은 방사무늬김 (Pyropia yezoensi)에서 유래한 타이로신 3-모노옥시제네이즈 (tyrosine 3-monooxygenase) 유사 유전자를 암호화하는 단리된 폴리뉴클레오타이드를 포함하는 재조합 벡터로 형질전환된 클라미도모나스 라인하드티 (Chlamydomonas reinhardtii) 및 이를 이용한 지질 생산 방법에 관한 것으로서, 본 발명에 따라 제조된 클라미도모나스 라인하드티는 지질 생산을 위한 배양 환경에서 우수한 성장을 보이고, 이를 통해 지질, 전분 등과 같은 대사 물질을 대량 생산할 수 있을 것으로 기대된다.The present invention relates to Chlamydomonas reinhardtii transformed with a recombinant vector containing an isolated polynucleotide encoding a tyrosine 3-monooxygenase-like gene derived from Pyropia yezoensi . ( Chlamydomonas reinhardtii ) and a method for producing lipids using the same. Chlamydomonas reinhardtii produced according to the present invention shows excellent growth in a culture environment for lipid production, thereby producing large amounts of metabolites such as lipids and starch. It is expected that production will be possible.
도 1은 본 발명의 일 실시예에 따라 타이로신 3-모노옥시제네이즈(tyrosine 3-monooxygenase) 유사 유전자가 도입된 재조합 클라미도모나스 라인하드티 (Chlamydomonas reinhardtii)의 야생형 대비 지질 함량을 보여주는 그래프이다.Figure 1 is a graph showing the lipid content of recombinant Chlamydomonas reinhardtii into which a tyrosine 3-monooxygenase-like gene has been introduced according to an embodiment of the present invention compared to the wild type.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
실시예 1: 방사무늬김으로부터 RNA 추출 및 cDNA 합성Example 1: RNA extraction and cDNA synthesis from spiny-patterned seaweed
방사무늬김 (Pyropia yezoensi)은 국립수산과학원 해조류연구센터로부터 분양 받아 사용하였다. White seaweed ( Pyropia yezoensi ) was used after receiving it from the Seaweed Research Center of the National Institute of Fisheries Science.
구체적으로, MGM (Modified Grund Media) 배지를 C10H14O6N2Na2·2H2O (Na2EDTA) 3.72 g, FeSO4·7H2O 0.28 g, Na2HPO4·12H2O 10.74 g, NaNO3 42.5 g, MnCl2·7H2O 0.019197 g 및 비타민 B12 (Cyanocobalamin) 1 mg를 포함한 멸균된 해수 1리터 (ℓ)를 사용하여 제조하였다. Specifically, MGM (Modified Grund Media) medium was composed of 3.72 g of C 10 H 14 O 6 N 2 Na 2· 2H 2 O (Na 2 EDTA), 0.28 g of FeSO 4· 7H 2 O, and Na 2 HPO 4· 12H 2 O. It was prepared using 1 liter (ℓ) of sterilized seawater containing 10.74 g, NaNO 3 42.5 g, MnCl 2· 7H 2 O 0.019197 g, and vitamin B 12 (Cyanocobalamin) 1 mg.
그 다음, 방사무늬김을 MGM 배지에 넣고, 방사무늬김이 담긴 MGM 배지를 식물 배양 접시 (Plant culture dish; 100 x 40 mm)에 100 mL의 부피로 첨가한 후, 광도 >20 umol photon m-2s-1, 12:12 Light:Dark 사이클, 온도 12 ℃를 유지하여 1 내지 10주 동안 배양하였다.Next, put the spiny-patterned seaweed into the MGM medium, add the MGM medium containing the spiny-patterned seaweed to a plant culture dish (100 The culture was maintained for 1 to 10 weeks at 2 s -1 , 12:12 Light:Dark cycle, and temperature of 12°C.
배양된 방사무늬김으로부터 RNA를 얻기 위해, RNA 정제 키트 [QIAGEN RNeasy®Mini kit]를 사용하였다. To obtain RNA from cultured spiny seaweed, an RNA purification kit [QIAGEN RNeasy ® Mini kit] was used.
구체적으로, 방사무늬김 엽상체 100 mg을 액체질소로 냉동시켜 멸균된 막자 사발로 잘게 분쇄하였다. Specifically, 100 mg of spiny seaweed fronds were frozen in liquid nitrogen and finely ground in a sterilized mortar and pestle.
가루가 된 조직 100 mg을 1.5 mL 마이크로 센트리퓨즈 (micro centrifuge) 튜브에 넣고 450 uL RLT 버퍼를 넣고 교반한 후, QIAshredder 스핀 컬럼 (spin column)에 옮겨 2분간 15,000 rpm으로 원심분리 하였다.100 mg of powdered tissue was placed in a 1.5 mL micro centrifuge tube, added with 450 uL RLT buffer, stirred, then transferred to a QIAshredder spin column and centrifuged at 15,000 rpm for 2 minutes.
그 다음, 분리된 버퍼에 에탄올 (ethanol) 200 uL 볼륨을 섞어주고, RNeasy 스핀 컬럼에 걸어 15초 >8,000 g로 원심분리 하였다. 떨어진 버퍼를 제거하고 컬럼에 700 uL RW1 버퍼로 15초 >8,000 g로 세척하였다. 그 다음, 떨어진 버퍼를 제거하고 500 uL RPE 버퍼로 15초, 2분 >8,000 g로 2번 세척하였다. Next, a 200 uL volume of ethanol was mixed with the separated buffer, placed on an RNeasy spin column, and centrifuged at >8,000 g for 15 seconds. The buffer that fell off was removed and the column was washed with 700 uL RW1 buffer for 15 seconds at >8,000 g. Next, the dropped buffer was removed and washed twice with 500 uL RPE buffer for 15 seconds, 2 minutes at >8,000 g.
컬럼을 새로운 1.5 mL 마이크로 센트리퓨즈 튜브에 옮겨 30 내지 50 uL의 RNA 제거수 (RNase-free water)를 첨가하여 1분 >8,000 g로 원심분리 후 각각의 RNA 시료를 수득하였다.The column was transferred to a new 1.5 mL micro centrifuge tube, 30 to 50 uL of RNA removal water (RNase-free water) was added, and each RNA sample was obtained after centrifugation at >8,000 g for 1 minute.
분광광도계 [Biodrop(Biochrom, German)]를 흡광도 230 nm와 흡광도 280 nm의 파장 범위로 설정하고, RNA 제거수 2 uL를 스팟에 넣고 베이스 (base)를 잡았다. 앞서 수득한 RNA 시료 2 uL를 스팟에 넣고 흡광도 260 nm를 통해 RNA 정량 값을 확인하였다.The spectrophotometer [Biodrop (Biochrom, German)] was set to a wavelength range of 230 nm and 280 nm, and 2 uL of RNA removal water was added to the spot to set the base. 2 uL of the previously obtained RNA sample was added to the spot, and the RNA quantification value was confirmed through absorbance at 260 nm.
cDNA 합성 키트 [Transcriptor Fiest Strand cDNA Synthesis Kit(Roche, Germany)]를 사용하여 정량한 2 uL RNA 시료에 대한 cDNA를 합성하였다. cDNA was synthesized for the quantified 2 uL RNA sample using a cDNA synthesis kit [Transcriptor Fiest Strand cDNA Synthesis Kit (Roche, Germany)].
구체적으로, 정량된 각각의 2 uL RNA 시료에 올리고-dT 1 uL와 DEPC 처리수 (DEPC-water)를 이용하여 총 13 uL로 맞춰 65 ℃에서 10분간 인큐베이션 (incubation)하였다. Specifically, each quantified 2 uL RNA sample was mixed with 1 uL of oligo-dT and DEPC-treated water (DEPC-water) to make a total of 13 uL and incubated at 65°C for 10 minutes.
처리 후 RT reaction (5x) 4 uL, 역전사효소 (Reverse transcriptase) 1 uL, RNase 억제제 (RNase inhibitor) 0.5 uL, DNTP 2 uL를 첨가하여 50 ℃에서 60분, 85 ℃에서 5분간 인큐베이션하여 각각의 cDNA를 합성하였다.After treatment, 4 uL of RT reaction (5x), 1 uL of reverse transcriptase, 0.5 uL of RNase inhibitor, and 2 uL of DNTP were added and incubated at 50 ℃ for 60 minutes and 85 ℃ for 5 minutes to produce each cDNA. was synthesized.
이후 합성된 cDNA에서 타이로신 3-모노옥시제네이즈 (tyrosine 3-monooxygenase) 유사 유전자를 하기 표 1의 프라이머를 사용하여 PCR 증폭하였다. Afterwards, the tyrosine 3-monooxygenase-like gene was PCR amplified from the synthesized cDNA using the primers shown in Table 1 below.
구체적으로, 매 사이클별로, cDNA의 변성단계 (denaturation)는 94 ℃에서 5분간 이루어졌고, 프라이머 결합단계 (annealing)는 61 ℃에서 1분간 이루어졌다. DNA 합성단계 (extension)는 Pfu 중합효소 (Pfu DNA Polymerase)를 이용하여 72 ℃에서 2분간 이루어졌다. Specifically, for each cycle, denaturation of cDNA was performed at 94°C for 5 minutes, and primer binding step (annealing) was performed at 61°C for 1 minute. The DNA synthesis step (extension) was performed at 72°C for 2 minutes using Pfu DNA Polymerase.
PCR은 총 30 사이클 이루어졌으며, 마지막 사이클에는 DNA 합성단계를 72 ℃에서 10분간 진행하였다. PCR was performed for a total of 30 cycles, and in the last cycle, the DNA synthesis step was performed at 72°C for 10 minutes.
증폭된 타이로신 3-모노옥시제네이즈 유사 유전자의 염기서열 및 아미노산 서열은 하기 표 1과 같다.The base sequence and amino acid sequence of the amplified tyrosine 3-monooxygenase-like gene are shown in Table 1 below.
실시예 2: 클라미도모나스 형질전환체 수득Example 2: Obtaining Chlamydomonas transformants
증폭된 타이로신 3-모노옥시제네이즈 유사 유전자가 클라미도모나스 라인하드티 (Chlamydomonas reinhardtii)에서 지질 생산에 영향을 미치는지 확인하였다.It was confirmed whether the amplified tyrosine 3-monooxygenase-like gene affects lipid production in Chlamydomonas reinhardtii .
구체적으로, 하기 표 2의 프라이머를 사용하여 타이로신 3-모노옥시제네이즈 유사 유전자를 PCR 증폭한 후, 재조합 단백질 발현 키트 [GeneArt®Chlamydomonas TOPO®Engineering Kits (A14264)]의 벡터 pChlamy_3/D-TOPO를 이용하여 유전자를 발현시켰다. Specifically, the tyrosine 3-monooxygenase-like gene was PCR amplified using the primers in Table 2 below, and then the vector pChlamy_3/D-TOPO from the recombinant protein expression kit [GeneArt ® Chlamydomonas TOPO ® Engineering Kits (A14264)] was used. The gene was expressed using
이때, PCR 증폭은 프라이머 결합단계의 온도를 61℃로 설정한 것을 제외하면 상술한 실시예 1의 PCR 증폭 과정과 동일하게 진행되었다.At this time, the PCR amplification was carried out in the same manner as the PCR amplification process in Example 1 described above, except that the temperature of the primer binding step was set to 61°C.
벡터 pChlamy_1/D-TOPO가 히그로마이신 (hygromycin) 내성유전자를 가지므로, 야생형인 클라미도모나스 라인하드티 C137에 Gene Pulser (Bio-Rad, US)를 이용하여 형질전환 하였으며 2 내지 3주간 배양한 후, 15 mg/L 히그로마이신이 첨가된 배지에서 선별하여 형질전환체를 수득하였다.Since the vector pChlamy_1/D-TOPO has a hygromycin resistance gene, wild type Chlamydomonas Rheinhardti C137 was transformed using Gene Pulser (Bio-Rad, US) and cultured for 2 to 3 weeks. Afterwards, transformants were obtained by selection in a medium supplemented with 15 mg/L hygromycin.
실시예 3: 재조합 클라미도모나스의 지질 함량 비교Example 3: Comparison of lipid content of recombinant Chlamydomonas
방사무늬김 (Pyropia yezoensi)에서 유래한 타이로신 3-모노옥시제네이즈 (tyrosine 3-monooxygenase) 유사 유전자가 발현된 재조합 클라미도모나스 라인하드티 (Chlamydomonas reinhardtii)와 야생형 클라미도모나스 라인하드를 MBBM (Modified Bold`s Basal medium) 배지에 넣은 다음, 광도 20 umol photon m-2s-1, 12:12 Light:Dark cycle, 온도 20 ℃를 유지하여 7일 동안 배양하였다.Recombinant Chlamydomonas reinhardtii, which expressed a tyrosine 3-monooxygenase-like gene derived from Pyropia yezoensi, and wild-type Chlamydomonas reinhardtii were mixed with MBBM (Modified). Bold's Basal medium) and cultured for 7 days at a light intensity of 20 umol photon m -2 s -1 , a 12:12 Light:Dark cycle, and a temperature of 20°C.
배양이 끝난 균주들의 지질 함량을 분석하였으며, 지질 함량 분석은 공지된 기술에 따라 지방산 메틸 에스터 (FA methyl esters; FAMEs)의 형태에 대해 이루어졌다.The lipid content of the cultivated strains was analyzed, and the lipid content analysis was performed in the form of fatty acid methyl esters (FA methyl esters; FAMEs) according to known techniques.
구체적으로, 5 mg의 동결 건조된 균주 세포를 1 mL 2 M NaOH-CH3OH 용액에 현탁시키고 실온 (room temperature; RT)에서 1시간동안 흔들고 (110 rpm) 75 ℃에서 15분 동안 인큐베이션 (incubation)하였다. Specifically, 5 mg of freeze-dried strain cells were suspended in 1 mL 2 M NaOH-CH 3 OH solution, shaken (110 rpm) for 1 hour at room temperature (RT), and incubated at 75°C for 15 minutes. ) was done.
냉각 후, 혼합액에 1 mL 4 M HCl-CH3OH 용액을 첨가하고 HCl로 pH를 2.0 미만으로 조절한 뒤, 75 ℃에서 15분 동안 인큐베이션하였다. After cooling, 1 mL 4 M HCl-CH 3 OH solution was added to the mixture, the pH was adjusted to less than 2.0 with HCl, and the mixture was incubated at 75°C for 15 minutes.
그 후, FAME을 핵세인 1 mL로 추출하고, 손으로 30초 흔들어준 후 3500 g에서 2분간 원심분리하였다. Afterwards, FAME was extracted with 1 mL of hexane, shaken by hand for 30 seconds, and then centrifuged at 3500 g for 2 minutes.
최종적으로, 혼합액 중 FAME의 양을 측정하여, 그 결과를 도 1 및 표 3에 나타내었다.Finally, the amount of FAME in the mixed solution was measured, and the results are shown in Figure 1 and Table 3.
(mg/L)lipid content
(mg/L)
도 1 및 표 3에서 확인할 수 있듯이, 방사무늬김에서 유래한 타이로신 3-모노옥시제네이즈 유사 유전자가 발현된 재조합 클라미도모나스 라인하드에서는 67.55 mg/L의 지질량이 얻어졌으며, 야생형 클라미도모나스 라인하드에서 측정된 51.21 mg/L에 비하여 31.9% 더 높은 지질량을 갖는 것이 확인되었다.As can be seen in Figure 1 and Table 3, a lipid amount of 67.55 mg/L was obtained in the recombinant Chlamydomonas Rheinhard expressing a tyrosine 3-monooxygenase-like gene derived from the wild-type Chlamydomonas line. It was confirmed that the lipid content was 31.9% higher than the 51.21 mg/L measured on the hard disk.
<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Microorganism with improved lipid productivity into which tyrosine 3-monooxygenase presumed gene is introduced, and method for manufacturing lipids using the same <130> PN220074 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 atgcctcagc cagcagagtt a 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 cgatctgccc attagtgcgc cc 22 <210> 3 <211> 1314 <212> DNA <213> Unknown <220> <223> tyrosine 3-monooxygenase (DNA) <400> 3 atgtcaactt tggcaatatt agttttttcg tccgtcaact caaagcctca aaaccaaaaa 60 ttaccattaa taattaaaat tcattcccaa aatcactttt ttgtttcact tttaccactt 120 accaattcca ccatgagcca cactgctact actgatgcgt cagccatcac ctttacagca 180 aagacagacg acattgggtc gatctttgcc tgtgtcaagg aggcagagcc tacctccttt 240 cgatttgagg gtacctccaa gaatgaagcc aaagaggttt ccgtcacttg cgagggtact 300 accaagactg cgctggaaaa ggctttcaac gatcattcag tggacgcgtc aaaaatcatc 360 aagtttacga tggaagagtt tgactttccc cgccacaaat ctgatcttga cgcctttgca 420 aacaagactc tggcgcttgg cgaaggtctg acgctcacag acgaccatcc gggtgccaag 480 gatgaaaagt acatcgaaag tcgcaacttg cacgctgaaa cagccatcaa ttaccgtcac 540 ggcgaggcta ttccagacgc ccactacaca ctagtggaga ctgaaacctg gaagactgtg 600 tacgaaaacc tttttggcat gtactccaca catgcttgct ccgagtatca ggtcgccatg 660 aagtcatttc aggacgagtg caacttttcg gccgaggcca ttccgcaaca ggcggtgatt 720 tcgagatttc tcatgagccg cacagggttt actctgcgcc cagtcgctgg tctcctgtcc 780 gctcgcaact tcctgaacgg tcttgccttc cgcgtatttc acgcaaccca gtacatccgc 840 caccactcta agcccctcta cacacctgag cccgacgtct gccatgagct ccttggccac 900 gtgcctctct ttgccgaccc aacttttgcc gatttctccc acgaaattgg tcttctgtca 960 atcggtgcgt cggactggga cattgaacgc ctgtccactc tgtactggtt caccattgag 1020 tttggtctga ccaaggaggc caacggcgaa atgcgcgcgt acggtgcggg acttctctct 1080 agctttggtg agctggagta ctgcctgtcc gacaagccca aggtgcttcc ctttgacccc 1140 gaggtcgcgt cgacaaccga ataccccatc acgtgcttcc aacccaccta cttctgcgct 1200 gagtcatttg ccgatgccct tgcgaaagtc cgtgcatttg ccaagggcat gaatcgcacc 1260 ttctcggtga cctacgatgc cgagactgag tctctcaagg ttcacaaagt gtga 1314 <210> 4 <211> 437 <212> PRT <213> Unknown <220> <223> tyrosine 3-monooxygenase (protein) <400> 4 Met Ser Thr Leu Ala Ile Leu Val Phe Ser Ser Val Asn Ser Lys Pro 1 5 10 15 Gln Asn Gln Lys Leu Pro Leu Ile Ile Lys Ile His Ser Gln Asn His 20 25 30 Phe Phe Val Ser Leu Leu Pro Leu Thr Asn Ser Thr Met Ser His Thr 35 40 45 Ala Thr Thr Asp Ala Ser Ala Ile Thr Phe Thr Ala Lys Thr Asp Asp 50 55 60 Ile Gly Ser Ile Phe Ala Cys Val Lys Glu Ala Glu Pro Thr Ser Phe 65 70 75 80 Arg Phe Glu Gly Thr Ser Lys Asn Glu Ala Lys Glu Val Ser Val Thr 85 90 95 Cys Glu Gly Thr Thr Lys Thr Ala Leu Glu Lys Ala Phe Asn Asp His 100 105 110 Ser Val Asp Ala Ser Lys Ile Ile Lys Phe Thr Met Glu Glu Phe Asp 115 120 125 Phe Pro Arg His Lys Ser Asp Leu Asp Ala Phe Ala Asn Lys Thr Leu 130 135 140 Ala Leu Gly Glu Gly Leu Thr Leu Thr Asp Asp His Pro Gly Ala Lys 145 150 155 160 Asp Glu Lys Tyr Ile Glu Ser Arg Asn Leu His Ala Glu Thr Ala Ile 165 170 175 Asn Tyr Arg His Gly Glu Ala Ile Pro Asp Ala His Tyr Thr Leu Val 180 185 190 Glu Thr Glu Thr Trp Lys Thr Val Tyr Glu Asn Leu Phe Gly Met Tyr 195 200 205 Ser Thr His Ala Cys Ser Glu Tyr Gln Val Ala Met Lys Ser Phe Gln 210 215 220 Asp Glu Cys Asn Phe Ser Ala Glu Ala Ile Pro Gln Gln Ala Val Ile 225 230 235 240 Ser Arg Phe Leu Met Ser Arg Thr Gly Phe Thr Leu Arg Pro Val Ala 245 250 255 Gly Leu Leu Ser Ala Arg Asn Phe Leu Asn Gly Leu Ala Phe Arg Val 260 265 270 Phe His Ala Thr Gln Tyr Ile Arg His His Ser Lys Pro Leu Tyr Thr 275 280 285 Pro Glu Pro Asp Val Cys His Glu Leu Leu Gly His Val Pro Leu Phe 290 295 300 Ala Asp Pro Thr Phe Ala Asp Phe Ser His Glu Ile Gly Leu Leu Ser 305 310 315 320 Ile Gly Ala Ser Asp Trp Asp Ile Glu Arg Leu Ser Thr Leu Tyr Trp 325 330 335 Phe Thr Ile Glu Phe Gly Leu Thr Lys Glu Ala Asn Gly Glu Met Arg 340 345 350 Ala Tyr Gly Ala Gly Leu Leu Ser Ser Phe Gly Glu Leu Glu Tyr Cys 355 360 365 Leu Ser Asp Lys Pro Lys Val Leu Pro Phe Asp Pro Glu Val Ala Ser 370 375 380 Thr Thr Glu Tyr Pro Ile Thr Cys Phe Gln Pro Thr Tyr Phe Cys Ala 385 390 395 400 Glu Ser Phe Ala Asp Ala Leu Ala Lys Val Arg Ala Phe Ala Lys Gly 405 410 415 Met Asn Arg Thr Phe Ser Val Thr Tyr Asp Ala Glu Thr Glu Ser Leu 420 425 430 Lys Val His Lys Val 435 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> foward primer <400> 5 caccatgcct cagccagcag agtta 25 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Microorganism with improved lipid productivity into which tyrosine 3-monooxygenase presumed gene is introduced, and method for manufacturing lipids using the same <130> PN220074 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 atgcctcagc cagcagagtt a 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 cgatctgccc attagtgcgc cc 22 <210> 3 <211> 1314 <212> DNA <213> Unknown <220> <223> tyrosine 3-monooxygenase (DNA) <400> 3 atgtcaactt tggcaatatt agttttttcg tccgtcaact caaagcctca aaaccaaaaa 60 ttaccattaa taattaaaat tcattcccaa aatcactttt ttgtttcact tttaccactt 120 accaattcca ccatgagcca cactgctact actgatgcgt cagccatcac ctttacagca 180 aagacagacg acattgggtc gatctttgcc tgtgtcaagg aggcagagcc tacctccttt 240 cgatttgagg gtacctccaa gaatgaagcc aaagaggttt ccgtcacttg cgagggtact 300 accaagactg cgctggaaaa ggctttcaac gatcattcag tggacgcgtc aaaaatcatc 360 aagtttacga tggaagagtt tgactttccc cgccacaaat ctgatcttga cgcctttgca 420 aacaagactc tggcgcttgg cgaaggtctg acgctcacag acgaccatcc gggtgccaag 480 gatgaaaagt acatcgaaag tcgcaacttg cacgctgaaa cagccatcaa ttaccgtcac 540 ggcgaggcta ttccagacgc ccactacaca ctagtggaga ctgaaacctg gaagactgtg 600 tacgaaaacc tttttggcat gtactccaca catgcttgct ccgagtatca ggtcgccatg 660 aagtcatttc aggacgagtg caacttttcg gccgaggcca ttccgcaaca ggcggtgatt 720 tcgagatttc tcatgagccg cacagggttt actctgcgcc cagtcgctgg tctcctgtcc 780 gctcgcaact tcctgaacgg tcttgccttc cgcgtatttc acgcaaccca gtacatccgc 840 caccactcta agcccctcta cacacctgag cccgacgtct gccatgagct ccttggccac 900 gtgcctctct ttgccgaccc aacttttgcc gatttctccc acgaaattgg tcttctgtca 960 atcggtgcgt cggactggga cattgaacgc ctgtccactc tgtactggtt caccattgag 1020 tttggtctga ccaaggaggc caacggcgaa atgcgcgcgt acggtgcggg acttctctct 1080 agctttggtg agctggagta ctgcctgtcc gacaagccca aggtgcttcc ctttgacccc 1140 gaggtcgcgt cgacaaccga ataccccatc acgtgcttcc aacccaccta cttctgcgct 1200 gagtcatttg ccgatgccct tgcgaaagtc cgtgcatttg ccaagggcat gaatcgcacc 1260 ttctcggtga cctacgatgc cgagactgag tctctcaagg ttcacaaagt gtga 1314 <210> 4 <211> 437 <212> PRT <213> Unknown <220> <223> tyrosine 3-monooxygenase (protein) <400> 4 Met Ser Thr Leu Ala Ile Leu Val Phe Ser Ser Val Asn Ser Lys Pro 1 5 10 15 Gln Asn Gln Lys Leu Pro Leu Ile Ile Lys Ile His Ser Gln Asn His 20 25 30 Phe Phe Val Ser Leu Leu Pro Leu Thr Asn Ser Thr Met Ser His Thr 35 40 45 Ala Thr Thr Asp Ala Ser Ala Ile Thr Phe Thr Ala Lys Thr Asp Asp 50 55 60 Ile Gly Ser Ile Phe Ala Cys Val Lys Glu Ala Glu Pro Thr Ser Phe 65 70 75 80 Arg Phe Glu Gly Thr Ser Lys Asn Glu Ala Lys Glu Val Ser Val Thr 85 90 95 Cys Glu Gly Thr Thr Lys Thr Ala Leu Glu Lys Ala Phe Asn Asp His 100 105 110 Ser Val Asp Ala Ser Lys Ile Ile Lys Phe Thr Met Glu Glu Phe Asp 115 120 125 Phe Pro Arg His Lys Ser Asp Leu Asp Ala Phe Ala Asn Lys Thr Leu 130 135 140 Ala Leu Gly Glu Gly Leu Thr Leu Thr Asp Asp His Pro Gly Ala Lys 145 150 155 160 Asp Glu Lys Tyr Ile Glu Ser Arg Asn Leu His Ala Glu Thr Ala Ile 165 170 175 Asn Tyr Arg His Gly Glu Ala Ile Pro Asp Ala His Tyr Thr Leu Val 180 185 190 Glu Thr Glu Thr Trp Lys Thr Val Tyr Glu Asn Leu Phe Gly Met Tyr 195 200 205 Ser Thr His Ala Cys Ser Glu Tyr Gln Val Ala Met Lys Ser Phe Gln 210 215 220 Asp Glu Cys Asn Phe Ser Ala Glu Ala Ile Pro Gln Gln Ala Val Ile 225 230 235 240 Ser Arg Phe Leu Met Ser Arg Thr Gly Phe Thr Leu Arg Pro Val Ala 245 250 255 Gly Leu Leu Ser Ala Arg Asn Phe Leu Asn Gly Leu Ala Phe Arg Val 260 265 270 Phe His Ala Thr Gln Tyr Ile Arg His His Ser Lys Pro Leu Tyr Thr 275 280 285 Pro Glu Pro Asp Val Cys His Glu Leu Leu Gly His Val Pro Leu Phe 290 295 300 Ala Asp Pro Thr Phe Ala Asp Phe Ser His Glu Ile Gly Leu Leu Ser 305 310 315 320 Ile Gly Ala Ser Asp Trp Asp Ile Glu Arg Leu Ser Thr Leu Tyr Trp 325 330 335 Phe Thr Ile Glu Phe Gly Leu Thr Lys Glu Ala Asn Gly Glu Met Arg 340 345 350 Ala Tyr Gly Ala Gly Leu Leu Ser Ser Phe Gly Glu Leu Glu Tyr Cys 355 360 365 Leu Ser Asp Lys Pro Lys Val Leu Pro Phe Asp Pro Glu Val Ala Ser 370 375 380 Thr Thr Glu Tyr Pro Ile Thr Cys Phe Gln Pro Thr Tyr Phe Cys Ala 385 390 395 400 Glu Ser Phe Ala Asp Ala Leu Ala Lys Val Arg Ala Phe Ala Lys Gly 405 410 415 Met Asn Arg Thr Phe Ser Val Thr Tyr Asp Ala Glu Thr Glu Ser Leu 420 425 430 Lys Val His Lys Val 435 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 caccatgcct cagccagcag agtta 25
Claims (11)
타이로신 3-모노옥시제네이즈 유사 단백질을 코딩하는 폴리뉴클레오타이드를 포함하는 재조합 벡터로 형질전환된 미생물을 배양하는 배양단계; 및
지질을 추출하는 추출단계.Method for producing lipids, comprising the following steps:
A culturing step of culturing a microorganism transformed with a recombinant vector containing a polynucleotide encoding a tyrosine 3-monooxygenase-like protein; and
Extraction step to extract lipids.
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