KR102694932B1 - Protein for excretion of mycosporine-like amino acids - Google Patents
Protein for excretion of mycosporine-like amino acids Download PDFInfo
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- KR102694932B1 KR102694932B1 KR1020240034753A KR20240034753A KR102694932B1 KR 102694932 B1 KR102694932 B1 KR 102694932B1 KR 1020240034753 A KR1020240034753 A KR 1020240034753A KR 20240034753 A KR20240034753 A KR 20240034753A KR 102694932 B1 KR102694932 B1 KR 102694932B1
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- South Korea
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- mycosporine
- microorganism
- protein
- amino acids
- yor1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
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Abstract
본 발명은 마이코스포린 유사 아미노산의 배출용 단백질에 관한 것으로, 보다 상세하게는 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(oligomycin resistance ATP-dependent permease YOR1 protein)을 유효성분으로 포함하는 마이코스포린 유사 아미노산(mycosporine-like amino acids) 배출용 조성물, 상기 단백질을 발현하는 마이코스포린 유사 아미노산 생산용 미생물 및 상기 미생물을 유효성분으로 포함하는 마이코스포린 유사 아미노산 생산용 조성물에 관한 것이다. 본 발명에 따른 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질은 마이코스코린 유사 아미노산 생산 균주가 생산한 마이코스포린 유사 아미노산을 균주 외부로 배출하는 효과가 뛰어나므로, 해당 마이코스포린 유사 아미노산을 대량으로 생산하는데 유용하게 이용될 수 있다.The present invention relates to a protein for excretion of mycosporine-like amino acids, and more specifically, to a composition for excretion of mycosporine-like amino acids comprising an oligomycin resistance ATP-dependent permease YOR1 protein as an active ingredient, a microorganism for producing mycosporine-like amino acids expressing the protein, and a composition for producing mycosporine-like amino acids comprising the microorganism as an active ingredient. The oligomycin resistance ATP-dependent permease YOR1 protein according to the present invention is excellent in the effect of excreting mycosporine-like amino acids produced by a mycosporine-like amino acid producing strain to the outside of the strain, and therefore can be usefully utilized for mass production of the corresponding mycosporine-like amino acids.
Description
본 발명은 마이코스포린 유사 아미노산의 배출용 단백질에 관한 것으로, 보다 상세하게는 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(oligomycin resistance ATP-dependent permease YOR1 protein)을 유효성분으로 포함하는 마이코스포린 유사 아미노산(mycosporine-like amino acids)의 세포외 배출용 조성물, 상기 단백질을 발현하는 마이코스포린 유사 아미노산 생산용 미생물 및 상기 미생물을 유효성분으로 포함하는 마이코스포린 유사 아미노산 생산용 조성물에 관한 것이다.The present invention relates to a protein for excretion of mycosporine-like amino acids, and more specifically, to a composition for extracellular excretion of mycosporine-like amino acids comprising oligomycin resistance ATP-dependent permease YOR1 protein as an active ingredient, a microorganism for producing mycosporine-like amino acids expressing the protein, and a composition for producing mycosporine-like amino acids comprising the microorganism as an active ingredient.
자외선은 UV-A(약 320~400 nm), UV-B(약 290~320 nm) 및 UV-C(100~280 nm)로 구성되어 있다. UV-A는 진피층까지 침입해 주로 색소 침착 및 피부 노화를 유발하고, UV B는 고에너지 광선으로서 표피와 진피의 상부로 침입해 색소 침착 및 피부암의 발생으로 관여하는 것이 알려져 있다.Ultraviolet rays are composed of UV-A (approximately 320–400 nm), UV-B (approximately 290–320 nm), and UV-C (100–280 nm). UV-A penetrates the dermis layer and mainly causes pigmentation and skin aging, and UV-B is a high-energy ray that penetrates the epidermis and upper part of the dermis and is known to be involved in pigmentation and the development of skin cancer.
이러한 자외선으로부터 피부를 보호하기 위하여 다양한 화학적, 물리적 자외선 차단제들이 사용되고 있다. 그러나, 이들 자외선 차단제는 접촉성 피부염을 유발하거나, 피부의 광과민성 반응을 유발하기도 하는 등의 문제가 있어 보다 안전한 바이오 기반의 자외선 차단 물질의 개발이 요구되고 있다.To protect the skin from these ultraviolet rays, various chemical and physical sunscreens are used. However, these sunscreens have problems such as causing contact dermatitis or photosensitivity reactions in the skin, so the development of safer bio-based sunscreen materials is required.
한편, 해조류는 자외선의 직접적인 영향을 받으므로, 자외선으로부터 보호하기 위한 물질을 많이 함유하고 있다. 그 중 마이코스포린(mycosporine) 및 마이코스포린 유사 아미노산(mycosporine-like amino acids; MAAs)은 미세조류, 균계, 조류 등의 원핵 및 진핵 미생물이 생산하는 천연 자외선 차단 소재로 알려져있다. 특히, 마이코스포린 유사 아미노산은 사이클로헥센이민 코어(cyclohexenimine core) 구조에 질소 화합물이 결합한 형태로서, 결합된 화합물의 종류에 따라 30여개 이상의 다양한 마이코스포린 유사 아미노산이 보고되었다. 마이코스포린 유사 아미노산의 대표적인 예인 시노린(shinorine) 은 글라이신(glycine)과 세린(serine) 치환기를 가지고 있는 화합물로, 효과적인 자외선 차단제로 각광 받고 있다. 시노린은 매우 높은 흡광 계수(ε=28,100-50,000 M-1cm-1)를 갖는데, 이는 효과적인 자외선 차단 소재로 알려진 옥시벤존의 흡광 계수(ε=14,295) 보다 세배 가량 높은 수치이다. 따라서, 시노린은 매우 고효율의 자외선 차단물질이라 할 수 있다. 뿐만 아니라, 시노린은 지구상에 가장 많이 도달하는 자외선인 UV-A에 특이적으로 자외선 차단이 가능한 효과적인 소재이다.Meanwhile, since seaweeds are directly affected by ultraviolet rays, they contain many substances for protecting against ultraviolet rays. Among them, mycosporine and mycosporine-like amino acids (MAAs) are known as natural ultraviolet ray blocking materials produced by prokaryotic and eukaryotic microorganisms such as microalgae, fungi, and algae. In particular, mycosporine-like amino acids are in the form of a nitrogen compound bonded to a cyclohexenimine core structure, and more than 30 different mycosporine-like amino acids have been reported depending on the type of bonded compound. Shinorine, a representative example of mycosporine-like amino acids, is a compound with glycine and serine substituents, and is attracting attention as an effective ultraviolet ray blocking agent. Shinorine has a very high absorption coefficient (ε=28,100-50,000 M -1 cm -1 ), which is about three times higher than the absorption coefficient of oxybenzone (ε=14,295), which is known as an effective UV-blocking material. Therefore, Shinorine can be said to be a very efficient UV-blocking material. In addition, Shinorine is an effective material that can specifically block UV-A, the UV-ray that reaches the earth the most.
이들 마이코스포린 유사 아미노산은 상술한 미생물에 의하여 자연적으로 생산되기는 하나, 수요를 충족시키기 위한 대량 생산을 위하여 마이코스포린 유사 아미노산 생산 효율이 우수한 재조합 균주가 다양하게 개발되고 있다(등록특허 제10-2351059호 및 등록특허 제10-2548752호 등). 그러나 이러한 균주를 이용하여 마이코스포린 유사 아미노산을 생산하는 것과, 그 생산 균주로부터 생산된 마이코스포린 유사 아미노산을 추출하는 것은 또 다른 문제로서, 마이코스포린 유사 아미노산 생산능을 갖는 대부분의 균주로부터 마이코스포린 유사 아미노산을 효과적으로 추출하기 위한 새로운 방법이 필요하다.Although these mycosporine-like amino acids are naturally produced by the above-mentioned microorganisms, various recombinant strains with excellent mycosporine-like amino acid production efficiency are being developed for mass production to meet the demand (Patent Registration No. 10-2351059 and Patent Registration No. 10-2548752, etc.). However, producing mycosporine-like amino acids using these strains and extracting the mycosporine-like amino acids produced from the producing strains are two different problems, and therefore, a new method is needed to effectively extract mycosporine-like amino acids from most strains having mycosporine-like amino acid production ability.
이에 본 발명자들은 마이코스포린 유사 아미노산 생산능을 갖는 균주가 생산한 마이코스포린 유사 아미노산을 균주 내부에서 외부로 효과적으로 배출할 수 있는 단백질을 탐색하고자 예의 노력을 한 결과, 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(Oligomycin resistance ATP-dependent permease YOR1 protein)을 발현하는 균주의 경우, 그렇지 않은 균주와 비교하여 배양 배지 내 마이코스포린 유사 아미노산의 농도가 급격히 상승하는 것을 확인하고 본 발명을 완성하였다.Accordingly, the inventors of the present invention have made extensive efforts to search for a protein that can effectively excrete mycosporine-like amino acids produced by a strain capable of producing mycosporine-like amino acids from the inside of the strain to the outside. As a result, they confirmed that the concentration of mycosporine-like amino acids in a culture medium rapidly increases in a strain expressing the oligomycin resistance ATP-dependent permease YOR1 protein compared to a strain that does not express the protein, thereby completing the present invention.
따라서, 본 발명의 목적은 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(Oligomycin resistance ATP-dependent permease YOR1 protein)을 유효성분으로 포함하는 마이코스포린 유사 아미노산(mycosporine-like amino acids)의 세포외 배출용 조성물을 제공하는 것이다.Accordingly, the purpose of the present invention is to provide a composition for extracellular excretion of mycosporine-like amino acids, which comprises oligomycin resistance ATP-dependent permease YOR1 protein as an active ingredient.
본 발명의 다른 목적은 마이코스포린 유사 아미노산(mycosporine-like amino acids) 생산용 미생물에 있어서, 상기 미생물은 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(oligomycin resistance ATP-dependent permease YOR1 protein)을 발현하는 것인 마이코스포린 유사 아미노산 생산용 미생물을 제공하는 것이다.Another object of the present invention is to provide a microorganism for producing mycosporine-like amino acids, wherein the microorganism expresses an oligomycin resistance ATP-dependent permease YOR1 protein.
본 발명의 또 다른 목적은 상술한 미생물을 유효성분으로 포함하는 마이코스포린 유사 아미노산(mycosporine-like amino acids) 생산용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for producing mycosporine-like amino acids comprising the above-described microorganism as an effective ingredient.
본 발명의 또 다른 목적은 상술한 미생물을 배양하는 단계; 및 상기 배양된 미생물 또는 배지로부터 마이코스포린 유사 아미노산을 회수하는 단계;를 포함하는, 마이코스포린 유사 아미노산의 생산 방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing a mycosporine-like amino acid, comprising the steps of culturing the above-described microorganism; and recovering a mycosporine-like amino acid from the cultured microorganism or medium.
상기와 같은 목적을 달성하기 위하여, 본 발명은 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(Oligomycin resistance ATP-dependent permease YOR1 protein)을 유효성분으로 포함하는 마이코스포린 유사 아미노산(mycosporine-like amino acids)의 세포외 배출용 조성물을 제공한다.To achieve the above-mentioned purpose, the present invention provides a composition for extracellular excretion of mycosporine-like amino acids, which contains oligomycin resistance ATP-dependent permease YOR1 protein as an active ingredient.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 마이코스포린 유사 아미노산(mycosporine-like amino acids) 생산용 미생물에 있어서, 상기 미생물은 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(oligomycin resistance ATP-dependent permease YOR1 protein)을 발현하는 것인 마이코스포린 유사 아미노산 생산용 미생물을 제공한다.In order to achieve another object of the present invention, the present invention provides a microorganism for producing mycosporine-like amino acids, wherein the microorganism expresses an oligomycin resistance ATP-dependent permease YOR1 protein.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상술한 미생물을 유효성분으로 포함하는 마이코스포린 유사 아미노산(mycosporine-like amino acids) 생산용 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a composition for producing mycosporine-like amino acids comprising the above-described microorganism as an effective ingredient.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 상술한 미생물을 배양하는 단계; 및 상기 배양된 미생물 또는 배지로부터 마이코스포린 유사 아미노산을 회수하는 단계;를 포함하는, 마이코스포린 유사 아미노산의 생산 방법을 제공한다.In order to achieve another object of the present invention, the present invention provides a method for producing a mycosporine-like amino acid, comprising: a step of culturing the above-described microorganism; and a step of recovering a mycosporine-like amino acid from the cultured microorganism or medium.
이하 본 발명을 상세히 설명한다. The present invention is described in detail below.
본 발명자는 마이코스포린 유사 아미노산 생산 균주가 생산한 마이코스포린 유사 아미노산을 효과적으로 균주 외부로 배출할 수 있는 단백질을 탐색하기 위하여, Saccharomyces cerevisiae 균주에서 유래하는 단백질 중 플라스마막에 존재하고 링 구조를 가져, 케미컬을 배출할 수 있을 것으로 판단되는 단백질 후보군을 선정하고, 이를 마이코스포린 유사 아미노산 생산 균주에 재조합하여 발현시켰다. 그 결과 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(Oligomycin resistance ATP-dependent permease YOR1 protein)을 발현하는 균주는 그렇지 않은 균주 및 다른 후보 단백질을 발현하는 균주와 비교하여 적게는 약 1.8배, 많게는 약 2.4배 가량 더 높은 배양 배지 내 마이코스포린 유사 아미노산 농도를 보였다. In order to search for a protein capable of effectively excreting mycosporine-like amino acid produced by a mycosporine-like amino acid-producing strain to the outside of the strain, the inventors of the present invention selected a group of protein candidates derived from Saccharomyces cerevisiae strains that are present in the plasma membrane, have a ring structure, and are expected to be able to excrete chemicals, and recombinantly expressed these proteins in a mycosporine-like amino acid-producing strain. As a result, the strain expressing the oligomycin resistance ATP-dependent permease YOR1 protein showed a mycosporine-like amino acid concentration in the culture medium that was about 1.8 times higher at least and about 2.4 times higher at most compared to the strains that did not express the protein and the strains expressing other candidate proteins.
따라서, 본 발명은 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(Oligomycin resistance ATP-dependent permease YOR1 protein)을 유효성분으로 포함하는 마이코스포린 유사 아미노산(mycosporine-like amino acids)의 세포외 배출용 조성물을 제공한다.Accordingly, the present invention provides a composition for extracellular excretion of mycosporine-like amino acids, comprising oligomycin resistance ATP-dependent permease YOR1 protein as an active ingredient.
본 발명에 있어서, 상기 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(Oligomycin resistance ATP-dependent permease YOR1 protein)은 상술한 바와 같이 Saccharomyces cerevisiae 에서 유래한 것으로서, 본래 세포질에서 독성 물질을 제거하기 위하여 원형질 막에서 다발성 약물 펌프의 역할을 하는 단백질이며, 광범위한 독성 유기 음이온의 해독에 관여하는 수송체로 알려져 있다. 상기 YOR1 단백질의 아미노산 서열은 다음과 같다.In the present invention, the oligomycin resistance ATP-dependent permease YOR1 protein is derived from Saccharomyces cerevisiae as described above, and is a protein that functions as a multi-drug pump in the plasma membrane to remove toxic substances from the cytoplasm, and is known as a transporter involved in the detoxification of a wide range of toxic organic anions. The amino acid sequence of the YOR1 protein is as follows.
YOR1 단백질의 아미노산 서열(서열번호 1)Amino acid sequence of YOR1 protein (SEQ ID NO: 1)
MTITVGDAVSETELENKSQNVVLSPKASASSDISTDVDKDTSSSWDDKSLLPTGEYIVDRNKPQTYLNSDDIEKVTESDIFPQKRLFSFLHSKKIPEVPQTDDERKIYPLFHTNIISNMFFWWVLPILRVGYKRTIQPNDLFKMDPRMSIETLYDDFEKNMIYYFEKTRKKYRKRHPEATEEEVMENAKLPKHTVLRALLFTFKKQYFMSIVFAILANCTSGFNPMITKRLIEFVEEKAIFHSMHVNKGIGYAIGACLMMFVNGLTFNHFFHTSQLTGVQAKSILTKAAMKKMFNASNYARHCFPNGKVTSFVTTDLARIEFALSFQPFLAGFPAILAICIVLLIVNLGPIALVGIGIFFGGFFISLFAFKLILGFRIAANIFTDARVTMMREVLNNIKMIKYYTWEDAYEKNIQDIRTKEISKVRKMQLSRNFLIAMAMSLPSIASLVTFLAMYKVNKGGRQPGNIFASLSLFQVLSLQMFFLPIAIGTGIDMIIGLGRLQSLLEAPEDDPNQMIEMKPSPGFDPKLALKMTHCSFEWEDYELNDAIEEAKGEAKDEGKKNKKKRKDTWGKPSASTNKAKRLDNMLKDRDGPEDLEKTSFRGFKDLNFDIKKGEFIMITGPIGTGKSSLLNAMAGSMRKTDGKVEVNGDLLMCGYPWIQNASVRDNIIFGSPFNKEKYDEVVRVCSLKADLDILPAGDMTEIGERGITLSGGQKARINLARSVYKKKDIYLFDDVLSAVDSRVGKHIMDECLTGMLANKTRILATHQLSLIERASRVIVLGTDGQVDIGTVDELKARNQTLINLLQFSSQNSEKEDEEQEAVVAGELGQLKYESEVKELTELKKKATEMSQTANSGKIVADGHTSSKEERAVNSISLKIYREYIKAAVGKWGFIALPLYAILVVGTTFCSLFSSVWLSYWTENKFKNRPPSFYMGLYSFFVFAAFIFMNGQFTILCAMGIMASKWLNLRAVKRILHTPMSYIDTTPLGRILNRFTKDTDSLDNELTESLRLMTSQFANIVGVCVMCIVYLPWFAIAIPFLLVIFVLIADHYQSSGREIKRLEAVQRSFVYNNLNEVLGGMDTIKAYRSQERFLAKSDFLINKMNEAGYLVVVLQRWVGIFLDMVAIAFALIITLLCVTRAFPISAASVGVLLTYVLQLPGLLNTILRAMTQTENDMNSAERLVTYATELPLEASYRKPEMTPPESWPSMGEIIFENVDFAYRPGLPIVLKNLNLNIKSGEKIGICGRTGAGKSTIMSALYRLNELTAGKILIDNVDISQLGLFDLRRKLAIIPQDPVLFRGTIRKNLDPFNERTDDELWDALVRGGAIAKDDLPEVKLQKPDENGTHGKMHKFHLDQAVEEEGSNFSLGERQLLALTRALVRQSKILILDEATSSVDYETDGKIQTRIVEEFGDCTILCIAHRLKTIVNYDRILVLEKGEVAEFDTPWTLFSQEDSIFRSMCSRSGIVENDFENRS*MTITVGDAVSETELENKSQNVVLSPKASASSDISTDVDKDTSSSWDDKSLLPTGEYIVDRNKPQTYLNSDDIEKVTESDIFPQKRLFSFLHSKKIPEVPQTDDERKIYPLFHTNIISNMFFWWVLPILRVGYKRTIQPNDLFKMDPRMSIETLYDDFEKNMIYYFEKTRKKYRKRHPEATEEEVMENAKLPKHTVLRALLFTFKKQ YFMSIVFAILANCTSGFNPMITKRLIEFVEEKAIFHSMHVNKGIGYAIGACLMMFVNGLTFNHFFHTSQLTGVQAKSILTKAAMKKMFNASNYARHCFPNGKVTSFVTTDLARIEFALSFQPFLAGFPAILAICIVLLIVNLGPIALVGIGIFFGGFFISLFA FKLILGFRIAANIFTDARVTMMREVLNNIKMIKYYTWEDAYEKNIQDIRTKEISKVRKMQLSRNFLIAMAMSLPSIASLVTFLAMYKVNKGGRQPGNIFASLSLFQVLSLQMFFLPIAIGTGIDMIIGLGRLQSLLEAPEDDPNQMIEMKPSPGFDPKLALKMTHCSFEWEDYELNDAIEEAKGEAKDEGKKNKKKRKDTWGKPSASTNKA KRLDNMLKDRDGPEDLEKTSFRGFKDLNFDIKKGEFIMITGPIGTGKSSLLNAMAGSMRKTDGKVEVNGDLLMCGYPWIQNASVRDNIIFGSPFNKEKYDEVVRVCSLKADLDILPAGDMTEIGERGITLSGGQKARINLARSVYKKKDIYLFDDVLSA VDSRVGKHIMDECLTGMLANKTRILATHQLSLIERASRVIVLGTDGQVDIGTVDELKARNQTLINLLQFSSQNSEKEDEEQEAVVAGELGQLKYESEVKELTELKKKATEMSQTANSGKIVADGHTSSKEERAVNSISLKIYREYIKAAVGKWGFIALPLYAILVVGTTFCSLFSSVWLSYWTENKFKNRPPSFYMGLYSFFVFAAFIF MNGQFTILCAMGIMASKWLNLRAVKRILHTPMSYIDTTPLGRILNRFTKDTDSLDNELTESLRLMTSQFANIVGVCVMCIVYLPWFAIAIPFLLVIFVLIADHYQSSGREIKRLEAVQRSFVYNNLNEVLGGMDTIKAYRSQERFLAKSDFLINKMNEAG YLVVVLQRWVGIFLDMVAIAFALIITLLCVTRAFPISAASVGVLLTYVLQLPGLLNTILRAMTQTENDMNSAERLVTYATELPLEASYRKPEMTPPESWPSMGEIIFENVDFAYRPGLPIVLKNLNLNIKSGEKIGICGRTGAGKSTIMSALYRLNELTAGKILIDNVDISQLGLFDLRRKLAIIPQDPVLFRGTIRKNLDPFNERTDDELWDALVRRGGAIA KDDLPEVKLQKPDENGTHGKMHKFHLDQAVEEEGSNFSLGERQLLALTRALVRQSKILILDEATSSVDYETDGKIQTRIVEEFGDCTILCIAHRLKTIVNYDRILVLEKGEVAEFDTPWTLFSQEDSIFRSMCSRSGIVENDFENRS*
본 발명에서 상기 YOR1 단백질은 그와 동일하거나 전술한 본 발명 단백질의 기능적 동등물 및 그들의 염을 포함한다. 상기 '기능적 동등물'이란 전술한 본 발명의 폴리펩티드와 적어도 80％ 이상의, 바람직하게는 90％, 더욱 바람직하게는 95％ 이상의 아미노산기 서열 상동성(즉, 동일성)을 갖는 것으로 예를 들면, 80％, 81％, 82％, 83％, 84％, 85％, 86％, 87％, 88％, 89％, 90％, 91％, 92％, 93％, 94％, 95％, 96％, 97％, 98％, 99％, 100％의 서열 상동성을 갖는 것을 포함하며, 본 발명에 따른 상기 단백질과 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다. 상기에서 '실질적으로 동질의 생리활성'이란, 일례로 마이코스포린 유사 아미노산을 세포 외로 배출하는 능력을 의미한다. In the present invention, the YOR1 protein includes functional equivalents of the protein of the present invention described above and salts thereof. The term "functional equivalent" refers to a protein having at least 80%, preferably 90%, and more preferably 95% or more amino acid sequence homology (i.e., identity) with the polypeptide of the present invention described above, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology, and refers to a protein exhibiting substantially the same physiological activity as the protein according to the present invention. In the above, ‘substantially identical physiological activity’ means, for example, the ability to secrete mycosporine-like amino acids out of cells.
본 발명에서 용어 '상동성'은 주어진 아미노산 서열 또는 뉴클레오티드 서열과 일치하는 정도를 의미하며 백분율로 표시될 수 있다. 본 명세서에서, 주어진 아미노산 서열 또는 뉴클레오티드 서열과 동일하거나 유사한 활성을 가지는 그의 상동성은 '% 상동성'으로 표시된다. 예를 들면, 점수(score), 동일성(identity) 및 유사도(similarity) 등의 매개 변수(parameter)들을 계산하는 표준 소프트웨어, 구체적으로 BLAST 2.0을 이용하거나, 정의된 엄격한 조건(stringent condition)하에서 썼던 혼성화 실험에 의해 서열을 비교함으로써 확인할 수 있으며, 정의되는 적절한 혼성화 조건은 해당 기술 범위 내이고, 통상의 기술자에게 잘 알려진 방법(예컨대, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New YORk, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New YORk)으로 결정될 수 있다. 상기에서 용어 '엄격한 조건'이란 폴리뉴클레오티드 간의 특이적 혼성화를 가능하게 하는 조건을 의미한다. In the present invention, the term 'homology' means the degree of matching with a given amino acid sequence or nucleotide sequence, and can be expressed as a percentage. In the present specification, the homology thereof having the same or similar activity as a given amino acid sequence or nucleotide sequence is expressed as '% homology'. For example, the sequences can be compared using standard software that calculates parameters such as score, identity and similarity, specifically BLAST 2.0, or by a hybridization experiment performed under defined stringent conditions, and the defined appropriate hybridization conditions are within the scope of the relevant technology and can be determined by a method well known to those of ordinary skill in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). The term 'stringent conditions' as used herein means conditions that enable specific hybridization between polynucleotides.
또한, 상기 서열과 상동성을 가지는 서열로서 실질적으로 기재된 서열번호의 효소 단백질과 동일하거나 상응하는 생물학적 활성을 가지는 아미노산 서열이라면, 일부 서열이 결실, 변형, 삽입, 치환(비보존적 또는 보존적 치환), 또는 이들의 조합에 의해 생성된 것일 수 있다. 상기에서 아미노산의 치환은 바람직하게는 보존적 치환일 수 있다. 천연에 존재하는 아미노산의 보존적 치환의 예는 다음과 같다; 지방족 아미노산(Gly, Ala, Pro), 소수성 아미노산(Ile, Leu, Val), 방향족 아미노산(Phe, Tyr, Trp), 산성 아미노산(Asp, Glu), 염기성 아미노산(His, Lys, Arg, Gln, Asn) 및 황함유 아미노산(Cys, Met). 분자의 활성을 전체적으로 변경시키지 않는 아미노산 교환은 당해 분야에 공지되어 있다(H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). In addition, if it is an amino acid sequence having the same or corresponding biological activity as the enzyme protein of the sequence number substantially described as a sequence having homology with the above sequence, some of the sequence may be generated by deletion, modification, insertion, substitution (non-conservative or conservative substitution), or a combination thereof. The substitution of the amino acid in the above may preferably be a conservative substitution. Examples of conservative substitutions of amino acids existing in nature are as follows: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn), and sulfur-containing amino acids (Cys, Met). Amino acid exchanges that do not change the overall activity of the molecule are known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979).
본 발명에 있어서, 상기 마이코스포린 유사 아미노산(mycosporine-like amino acids, MAA)은 자외선을 흡수하는 고리형의 화합물을 의미한다. 상기 마이코스포린 유사 아미노산은 4-데옥시가두솔(4-Deoxygadusol: 4-DG)을 전구체로 하여 아미노시클로헥세논(aminocyclohexenone)과 아미노클로헥세이민(aminocycloheximine)의 구조를 가지며, 여기에 아미노산 등 다양한 물질이 결합된 화합물일 수 있다. 보다 구체적으로 상기 마이코스포린 유사 아미노산은 마이코스포린-2-글라이신(Mycosporine-2-glycine), 팰라이티놀(Palythinol), 팰라이텐산(Palythenic acid), 데옥시가두솔(deoxygadusol), 마이코스포린-메틸아민-트레오닌(Mycosporine-methylaminethreonine), 마이코스포린-글라이신-발린(Mycosporine-glycine-valine), 팰라이틴(Palythine), 아스테리나-330(Asterina-330), 시노린(Shinorine), 포피라-334(Porphyra-334), 유하로테스-362(Euhalothece-362), 마이코스포린-글라이신(Mycosporine-glycine), 마이코스포린-오르니틴(Mycosporine-ornithine), 마이코스포린-라이신(Mycosporine-lysine), 마이코스포린-글루탐산-글라이신(Mycosporine-glutamic acid-glycine), 마이코스포린-메틸아민-세린(Mycosporine-methylamine-serine), 마이코스포린-타우린(Mycosporine-taurine), 팰라이텐(Palythene), 팰라이텐-세린(Palythine-serine), 팰라이텐-세린-설페이트(Palythine-serine-sulfate), 팰라이티놀(Palythinol) 및 우수지렌(Usujirene)으로 이루어진 군에서 선택된 어느 하나일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the mycosporine-like amino acids (MAA) refer to a cyclic compound that absorbs ultraviolet rays. The mycosporine-like amino acids have a structure of aminocyclohexenone and aminocycloheximine using 4-deoxygadusol (4-DG) as a precursor, and may be a compound in which various substances such as amino acids are combined. More specifically, the mycosporine-like amino acids are Mycosporine-2-glycine, Palythinol, Palythenic acid, deoxygadusol, Mycosporine-methylaminethreonine, Mycosporine-glycine-valine, Palythine, Asterina-330, Shinorine, Porphyra-334, Euhalothece-362, Mycosporine-glycine, Mycosporine-ornithine, It may be any one selected from the group consisting of Mycosporine-lysine, Mycosporine-glutamic acid-glycine, Mycosporine-methylamine-serine, Mycosporine-taurine, Palythene, Palythine-serine, Palythine-serine-sulfate, Palythinol, and Usujirene, but is not limited thereto.
본 발명에 따른 상기 YOR1 단백질은 마이코스포린 유사 아미노산 생산 균주가 생산한 마이코스포린 유사 아미노산을 균주의 내부에서 외부로 배출하는 기능을 갖는 점에서, 어느 특정한 마이코스포린 유사 아미노산 생산 균주에 제한되지 아니하며, 마이코스포린 유사 아미노산을 생산할 수 있는 균주라면 제한없이 적용되어 생산된 마이코스포린 유사 아미노산을 균주 내부에서 배지로 효과적으로 배출할 수 있다.The YOR1 protein according to the present invention is not limited to any specific mycosporine-like amino acid producing strain in that it has the function of excreting mycosporine-like amino acid produced by the strain from the inside to the outside of the strain, and can be applied without limitation to any strain capable of producing mycosporine-like amino acid, thereby effectively excreting the produced mycosporine-like amino acid from the inside of the strain to the medium.
따라서, 본 발명은 마이코스포린 유사 아미노산(mycosporine-like amino acids) 생산용 미생물에 있어서, 상기 미생물은 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질(oligomycin resistance ATP-dependent permease YOR1 protein)을 발현하는 것인 마이코스포린 유사 아미노산 생산용 미생물을 제공한다.Accordingly, the present invention provides a microorganism for producing mycosporine-like amino acids, wherein the microorganism expresses an oligomycin resistance ATP-dependent permease YOR1 protein.
본 발명에 있어서, 상기 '마이코스포린 유사 아미노산 생산용 미생물'은 마이코스포린 유사 아미노산 생합성 유전자를 원래부터 가지고 있던 천연형의 미생물, 또는 이종 및/또는 동종 유래 마이코스포린 유사 아미노산 생합성 유전자가 도입된 재조합 미생물일 수 있고, 또한, 상기 미생물은 내재적 및/또는 도입된 마이코스포린 유사 아미노산 생합성 유전자가 코딩하는 효소의 활성이 강화된 미생물일 수 있으나, 이에 제한되는 것은 아니다. 따라서 본 발명에 따른 'YOR1 단백질을 발현하는 마이코스포린 유사 아미노산 생산용 미생물'이란, 상술한 YOR1 단백질을 코딩하는 유전자를 원래부터 가지고 있거나 새롭게 도입된 상기 마이코스포린 유사 아미노산 생산용 미생물일 수 있고, 또는 내재적 및/또는 도입된 YOR1 단백질의 활성이 강화된 상기 마이코스포린 유사 아미노산 생산용 미생물을 의미한다. In the present invention, the 'microorganism for producing a mycosporine-like amino acid' may be a natural microorganism that originally has a mycosporine-like amino acid biosynthetic gene, or a recombinant microorganism into which a heterologous and/or homologous mycosporine-like amino acid biosynthetic gene has been introduced, and further, the microorganism may be a microorganism in which the activity of an enzyme encoded by the endogenous and/or introduced mycosporine-like amino acid biosynthetic gene has been enhanced, but is not limited thereto. Therefore, the 'microorganism for producing a mycosporine-like amino acid expressing a YOR1 protein' according to the present invention may be a microorganism for producing a mycosporine-like amino acid which originally has a gene encoding the above-described YOR1 protein or which has been newly introduced, or means a microorganism for producing a mycosporine-like amino acid in which the activity of the endogenous and/or introduced YOR1 protein has been enhanced.
또한, 상기 미생물은 효모(yeast), 코리네박테리움 속(Corynebacterium sp.) 미생물, 또는 에스케리키아 속(Escherichia sp.) 미생물일 수 있다. 여기에서 상기 효모는 사카로마이세스(Saccharomyces), 캔디다(Candida), 디베리오마이세스(Debaryomyces), 한세눌라(Hansenula), 클루이베로마이세스 (Kluyveromyces), 피키아(Pichia), 스키조사카로마이세스(Schizosaccharomyces), 야로이야(Yarrowia), 슈완니오마이세스(Schwanniomyces), 아르술라(Arxula), 말라세지아(Malassezia) 속 등에 해당하는 효모일 수 있으나, 이에 제한되는 것은 아니다. 보다 구체적으로 상기 효모는 사카로마이세스 세레비지애(Saccharomyces cerevisiae), 캔디다 트로피칼리스(Candida tropicalis), 캔디다 유틸리스(Candida utilis), 캔디다 보이디니(Candida boidinii), 캔디다 알비칸스(Candida albicans), 클루이베로마이세스 락티스(Kluyveromyces lactis), 피키아 파스토리스(Pichia pastoris), 피키아 스티피티스(Pichia stipitis), 스키조카로마이세스 폼베(Schizosaccharomyces pombe), 한세눌라 폴리모르파(Hansenula polymorpha), 야로이야 리폴리티카(Yarrowia lipolytica), 슈완니오마이세스 옥시덴탈리스(Schwanniomyces occidentalis), 아르술라 아데니니모란스(Arxula adeninivorans), 말라세지아 리스트릭타(Malassezia restricta), 말라세지아 퍼퍼(Malassezai furfur) 등일 수 있다.In addition, the microorganism may be a yeast, a microorganism of the genus Corynebacterium sp., or a microorganism of the genus Escherichia sp. Here, the yeast may be a yeast corresponding to the genus Saccharomyces , Candida , Debaryomyces , Hansenula , Kluyveromyces , Pichia , Schizosaccharomyces , Yarrowia , Schwanniomyces , Arxula , Malassezia , or the like, but is not limited thereto. More specifically, the yeasts are Saccharomyces cerevisiae , Candida tropicalis , Candida utilis , Candida boidinii , Candida albicans , Kluyveromyces lactis , Pichia pastoris , Pichia stipitis , Schizosaccharomyces pombe , Hansenula polymorpha, Yarrowia lipolytica , Schwanniomyces occidentalis , Arxula adeninivorans , and Malassezia restricta. These may include Malassezia restricta , Malassezia furfur , etc.
본 발명에 있어서 용어, '활성의 강화'는 효소 단백질의 활성이 도입되거나, 미생물이 가진 내재적 활성 또는 변형 전 활성에 비하여 활성이 향상된 것을 의미한다. 상기 활성의 '도입'은, 미생물이 본래 가지고 있지 않았던 특정 단백질의 활성이 자연적 혹은 인위적으로 나타나게 되는 것을 의미한다. 보다 구체적으로, 본 발명에서 활성 강화의 방법으로는, 1) 상기 효소들을 암호화하는 폴리뉴클레오티드의 카피수 증가, 2) 상기 폴리뉴클레오티드의 발현이 증가하도록 발현조절 서열의 변형, 3) 상기 효소들의 활성이 강화되도록 염색체 상의 폴리뉴클레오티드 서열의 변형, 또는 4) 이의 조합에 의해 변형하는 방법 등에 의하여 수행할 수 있으나, 이에 제한되지 않는다.In the present invention, the term 'enhancement of activity' means that the activity of an enzyme protein is introduced, or that the activity is enhanced compared to the inherent activity of a microorganism or the activity before modification. The 'introduction' of the activity means that the activity of a specific protein that the microorganism did not originally have appears naturally or artificially. More specifically, the method of enhancing the activity in the present invention can be performed by, but is not limited to, 1) increasing the copy number of a polynucleotide encoding the enzymes, 2) modifying an expression regulatory sequence so that the expression of the polynucleotide increases, 3) modifying a polynucleotide sequence on a chromosome so that the activity of the enzymes is enhanced, or 4) modifying by a combination thereof.
또한, 본 발명은 상술한 YOR1 단백질을 발현하는 마이코스포린 유사 아미노산 생산용 미생물을 유효성분으로 포함하는 마이코스포린 유사 아미노산(mycosporine-like amino acids) 생산용 조성물을 제공한다.In addition, the present invention provides a composition for producing mycosporine-like amino acids, which comprises a microorganism for producing mycosporine-like amino acids expressing the above-described YOR1 protein as an effective ingredient.
또한, 본 발명은 상술한 YOR1 단백질을 발현하는 마이코스포린 유사 아미노산 생산용 미생물을 배양하는 단계; 및 상기 배양된 미생물 또는 배지로부터 마이코스포린 유사 아미노산을 회수하는 단계;를 포함하는, 마이코스포린 유사 아미노산의 생산 방법을 제공한다.In addition, the present invention provides a method for producing a mycosporine-like amino acid, comprising: a step of culturing a microorganism for producing a mycosporine-like amino acid expressing the above-described YOR1 protein; and a step of recovering the mycosporine-like amino acid from the cultured microorganism or medium.
본 발명에 있어서, 상기 미생물 및 마이코스포린 유사 아미노산은 상술한 바와 같다. In the present invention, the microorganism and mycosporine-like amino acid are as described above.
본 발명에서, 상기 '배양'은 상기 미생물을 적당히 조절된 환경 조건에서 생육시키는 것을 의미한다. 배양과정은 당업계에 알려진 적당한 배지와 배양조건에 따라 이루어질 수 있다. 이러한 배양 과정은 선택되는 미생물에 따라 통상의 기술자가 용이하게 조정하여 사용할 수 있다. 구체적으로 상기 배양은 회분식, 연속식 및/또는 유가식일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the 'cultivation' means growing the microorganism under appropriately controlled environmental conditions. The culturing process can be performed according to appropriate media and culturing conditions known in the art. This culturing process can be easily adjusted and used by a person skilled in the art according to the selected microorganism. Specifically, the culturing may be batch, continuous, and/or fed-batch, but is not limited thereto.
상기 '배양'에 있어서 배양 온도는 20℃ 내지 45℃, 구체적으로는 25℃ 내지 40℃를 유지하는 것일 수 있고, 배양 시간은 약 10 내지 160 시간 동안 배양하는 것일 수 있으나, 이에 제한되는 것은 아니다.In the above 'cultivation', the culture temperature may be maintained at 20℃ to 45℃, specifically 25℃ to 40℃, and the culture time may be cultured for about 10 to 160 hours, but is not limited thereto.
본 발명에서, 상기 '배지' 또는 '배양 배지'는 상기 미생물을 배양하기 위해 필요로 하는 영양물질을 주성분으로 혼합한 물질을 의미하며, 생존 및 발육에 불가결한 물을 비롯하여 영양물질 및 발육인자 등을 공급한다. 구체적으로, 상기 미생물의 배양에 사용되는 배지 및 기타 배양 조건은 통상의 미생물의 배양에 사용되는 배지라면 특별한 제한 없이 어느 것이나 사용할 수 있으나, 상기 미생물을 적당한 탄소원, 질소원, 인원, 무기화합물, 아미노산 및/또는 비타민 등을 함유한 통상의 배지 내에서 호기성 조건 하에서 온도, pH 등을 조절하면서 배양할 수 있다.In the present invention, the 'medium' or 'culture medium' refers to a material containing nutrients necessary for culturing the microorganism as a main component, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism may be any medium used for culturing general microorganisms without particular limitation, but the microorganism may be cultured in a general medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid, and/or vitamin, etc., under aerobic conditions while controlling temperature, pH, etc.
상기 탄소원으로는 글루코오스, 사카로오스, 락토오스, 프룩토오스, 수크로오스, 말토오스 등과 같은 탄수화물; 만니톨, 소르비톨 등과 같은 당 알코올, 피루브산, 락트산, 시트르산 등과 같은 유기산; 글루탐산, 메티오닌, 리신 등과 같은 아미노산 등이 포함될 수 있다. 또한, 전분 가수분해물, 당밀, 블랙스트랩 당밀, 쌀겨울, 카사버, 사탕수수 찌꺼기 및 옥수수 침지액 같은 천연의 유기 영양원을 사용할 수 있으며, 구체적으로는 글루코오스 및 살균된 전처리 당밀(즉, 환원당으로 전환된 당밀) 등과 같은 탄수화물이 사용될 수 있으며, 그 외의 적정량의 탄소원을 제한없이 다양하게 이용할 수 있다. 이들 탄소원은 단독으로 사용되거나 2 종 이상이 조합되어 사용될 수 있으나, 이에 제한되는 것은 아니다.The carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane residue, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.
상기 질소원으로는 암모니아, 황산암모늄, 염화암모늄, 초산암모늄, 인산암모늄, 탄산안모늄, 질산암모늄 등과 같은 무기질소원; 글루탐산, 메티오닌, 글루타민 등과 같은 아미노산, 펩톤, NZ-아민, 육류 추출물, 효모 추출물, 맥아 추출물, 옥수수 침지액, 카세인 가수분해물, 어류 또는 그의 분해생성물, 탈지 대두 케이크 또는 그의 분해 생성물 등과 같은 유기 질소원이 사용될 수 있다. 이들 질소원은 단독으로 사용되거나 2 종 이상이 조합되어 사용될 수 있으나, 이에 제한되는 것은 아니다.The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.
상기 인원으로는 인산 제1칼륨, 인산 제2칼륨, 또는 이에 대응되는 소디움-함유 염 등이 포함될 수 있다. 무기 화합물로는 염화나트륨, 염화칼슘, 염화철, 황산마그네슘, 황산철, 황산망간, 탄산칼슘 등이 사용될 수 있으며, 그 외에 아미노산, 비타민 및/또는 적절한 전구체 등이 포함될 수 있다. 이들 구성성분 또는 전구체는 배지에 회분식 또는 연속식으로 첨가될 수 있다. 그러나, 이에 제한되는 것은 아니다.The above-mentioned personnel may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and/or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner. However, the present invention is not limited thereto.
또한, 상기 배양 중에 수산화암모늄, 수산화칼륨, 암모니아, 인산, 황산 등과 같은 화합물을 배지에 적절한 방식으로 첨가하여, 배지의 pH를 조정할 수 있다. 또한, 배양 중에는 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 또한, 배지의 호기 상태를 유지하기 위하여, 배지 내로 산소 또는 산소 함유 기체를 주입하거나 혐기 및 미호기 상태를 유지하기 위해 기체의 주입 없이 혹은 질소, 수소 또는 이산화탄소 가스를 주입할 수 있으나, 이에 제한되는 것은 아니다.In addition, during the above culturing, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or in order to maintain the anaerobic and microaerobic state, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.
상기 배양에 따른 배지(배양이 수행된 배지) 또는 상기 미생물로부터 마이코스포린 유사 아미노산을 회수하는 단계에서, 상기 회수는 상기 미생물의 배양 방법, 예를 들어 회분식, 연속식 또는 유가식 배양 방법 등에 따라 당해 기술 분야에 공지된 적합한 방법을 이용하여 목적하는 마이코스포린 유사 아미노산을 수집(collect)하는 것일 수 있다. 예를 들어, 원심 분리, 여과, 결정화 단백질 침전제에 의한 처리(염석법), 추출, 초음파 파쇄, 한외여과, 투석법, 분자체 크로마토그래피(겔여과), 흡착크로마토그래피, 이온교환 크로마토그래피, 친화도 크로마토그래피 등의 각종 크로마토그래피, HPLC 또는 이들의 방법을 조합하여 사용될 수 있으며, 통상의 기술자는 당해 분야에 공지된 적합한 방법을 이용하여 배지 또는 미생물로부터 목적하는 마이코스포린 유사 아미노산을 회수할 수 있다.In the step of recovering the mycosporine-like amino acid from the medium according to the above culture (the medium in which the culture is performed) or the microorganism, the recovery may be to collect the target mycosporine-like amino acid using a suitable method known in the art according to the culture method of the microorganism, for example, a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination of these methods may be used, and a person skilled in the art can recover the target mycosporine-like amino acid from the medium or the microorganism using a suitable method known in the art.
또한, 상기 생산방법은 추가적으로 정제 단계를 포함할 수 있다. 상기 정제는 당해 기술분야에 공지된 적합한 방법을 이용하여 수행할 수 있다. 일 예에서, 상기 생산방법이 회수 단계와 정제 단계를 모두 포함하는 경우, 상기 회수 단계와 정제 단계는 순서에 상관없이 연속적 또는 비연속적으로 수행되거나, 동시에 또는 하나의 단계로 통합되어 수행될 수 있으나, 이에 제한되는 것은 아니다.In addition, the above production method may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, when the above production method includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.
본 발명에 따른 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질은 마이코스코린 유사 아미노산 생산 균주가 생산한 마이코스포린 유사 아미노산을 균주 외부로 배출하는 효과가 뛰어나므로, 해당 마이코스포린 유사 아미노산을 대량으로 생산하는데 유용하게 이용될 수 있다.The oligomycin-resistant ATP-dependent permease YOR1 protein according to the present invention is excellent in the effect of excreting mycosporine-like amino acids produced by a mycosporine-like amino acid producing strain to the outside of the strain, and therefore can be usefully used to produce the mycosporine-like amino acids in large quantities.
도 1은 마이코스포린 유사 아미노산 배출자 후보군 단백질인 YOR1, PDR5, PDR10 및 PDR15를 각각 발현하는 마이코스포린 유사 아미노산 생산 균주를 배양한 후, 48시간 및 72시간 경과 시점에서 배지 내 시노린 농도를 도시한 것이다.
도 2는 마이코스포린 유사 아미노산 배출자 후보군 단백질인 YOR1, PDR5, PDR10 및 PDR15를 각각 발현하는 마이코스포린 유사 아미노산 생산 균주를 배양한 후, 72시간 경과 시점에서 배지 내 포피라-334 농도를 도시한 것이다.Figure 1 shows the concentration of shinorine in the medium at 48 and 72 hours after culturing mycosporine-like amino acid producing strains expressing mycosporine-like amino acid excretor candidate proteins YOR1, PDR5, PDR10, and PDR15, respectively.
Figure 2 shows the concentration of Porphyra-334 in the medium at 72 hours after culturing mycosporine-like amino acid producing strains expressing mycosporine-like amino acid excretor candidate proteins YOR1, PDR5, PDR10, and PDR15, respectively.
이하 본 발명을 상세히 설명한다.The present invention is described in detail below.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are only intended to illustrate the present invention, and the content of the present invention is not limited to the following examples.
실시예 1. 마이코스포린 유사 아미노산 배출자 후보유전자 과발현용 벡터의 제작Example 1. Construction of a vector for overexpression of a candidate gene for mycosporine-like amino acid excretor
마이코스포린 유사 아미노산(mycosporine-like amino acid, MAA)의 배출자 탐색을 위해 Saccharomyces cerevisiae 유래 배출자 중 플라스마 막에 존재하고 링 구조를 가져, 케미컬을 배출할 수 있을 것으로 판단되는 후보들을 선택하였다. 선택된 후보 단백질 및 이들의 아미노산 서열은 다음과 같다.To search for mycosporine-like amino acid (MAA) excretors, candidates that exist in the plasma membrane and have a ring structure and are expected to be able to excrete chemicals were selected from among excretors derived from Saccharomyces cerevisiae . The selected candidate proteins and their amino acid sequences are as follows.
상기 후보군 중 마이코스포린 유사 아미노산의 배출에 효과가 있는 단백질을 확인하기 위해, 이들 유전자를 개별적으로 발현하는 플라스미드를 제작하였다. 각각의 유전자 단편은 S. cerevisiae의 genomic DNA로부터 PCR을 통해 확보하였고, 각 유전자는 GAP promoter의 조절 하에 발현하였다. 프로모터-유전자-터미네이터 카세트를 Star41-Kan 플라스미드에 삽입하여, Star41-Kan-PGAP-YOR1, Star41-Kan-PGAP-PDR5, Star41-Kan-PGAP-PDR10, 및 Star41-Kan-PGAP-PDR15 플라스미드를 각각 제작하였다. 이렇게 제작된 플라스미드는 하기의 표 2과 같다.In order to identify proteins that are effective in excreting mycosporine-like amino acids among the above candidates, plasmids expressing these genes individually were constructed. Each gene fragment was obtained from the genomic DNA of S. cerevisiae by PCR, and each gene was expressed under the control of the GAP promoter. The promoter-gene-terminator cassette was inserted into the Star41-Kan plasmid, and Star41-Kan-PGAP-YOR1, Star41-Kan-PGAP-PDR5, Star41-Kan-PGAP-PDR10, and Star41-Kan-PGAP-PDR15 plasmids were constructed, respectively. The plasmids constructed in this way are shown in Table 2 below.
실시예 2. 배출자 후보유전자의 과발현을 통한 마이코스포린 유사 아미노산 배출능 평가 1Example 2. Evaluation of mycosporine-like amino acid excretion ability through overexpression of an excretor candidate gene 1
JHSM132 균주에 상기 실시예 1에서 제작된 Star41-Kan-PGAP-TPI1, Star41-Kan-PGAP-YOR1-TPI1, Star41-Kan-PGAP-PDR5-TPI1, Star41-Kan-PGAP-PDR10-TPI1, 및 Star41-Kan-PGAP-PDR15-TPI1 플라스미드를 각각 LiAc/SS carrier DNA/PEG 방법을 사용하여 YPD-G418 평판배지(Yeast extract 10 g/L, peptone 20 g/L, dextrose 20 g/L, G418 100 ug/L)에 형질전환시켰다. The Star41-Kan-PGAP-TPI1, Star41-Kan-PGAP-YOR1-TPI1, Star41-Kan-PGAP-PDR5-TPI1, Star41-Kan-PGAP-PDR10-TPI1, and Star41-Kan-PGAP-PDR15-TPI1 plasmids constructed in Example 1 were each transformed into YPD-G418 plate medium (yeast extract 10 g/L, peptone 20 g/L, dextrose 20 g/L, G418 100 ug/L) using the LiAc/SS carrier DNA/PEG method.
본 실시예에서 사용된 형질전환 균주는 야생형의 Saccharomyces cerevisiae 효모 균주인 CEN.PK2-1C (EUROSCARF)에 마이코스포린 유사 아미노산 생산을 위한 유전자를 재조합한 균주로서, 그 정보는 표 3에 기재된 바와 같으며, 보다 상세한 정보는 Metabolic Engineering 78 (2023) 137-147 논문을 참고할 수 있다.The transformed strain used in this example is a strain in which a gene for producing mycosporine-like amino acids is recombined into the wild-type Saccharomyces cerevisiae yeast strain CEN.PK2-1C (EUROSCARF). The information is as described in Table 3, and more detailed information can be found in the paper Metabolic Engineering 78 (2023) 137-147.
이렇게 얻어진 형질전환 효모 균주를 YPD-G418 배지 (Yeast extract 10 g/L, peptone 20 g/L, dextrose 10 g/L, xylose 10 g/L, G418 100 ug/L)에 O.D.600 = 0.2로 접종하여 30℃ 및 220 rpm 조건 하에서 72시간 동안 배양하였다. 배지 내 마이코스포린 유사 아미노산을 정량하기 위하여 배양액 1 mL을 원심분리한 후 상등액을 얻고, 이를 0.22 ㎛ 필터로 여과하여 HPLC 분석을 수행하였다. Agilent Eclipse XDB-C18 컬럼(5 ㎛, 4.6×250 mm)을 갖춘 1260 infinity HPLC 시스템(Agilent)를 이용하였고, 컬럼 온도는 40℃로 유지하며 0.5 mL/분의 유속으로 용매(0.25% Trifluoroacetic acid가 포함된 물 : 0.25% Trifluoroacetic acid가 포함된 아세토니트릴 = 97.5 : 2.5)를 흘려주었다. 파장 334 nm에서 UV-vis 검출기로 시노린(shinorine) 및 포피라-334(porphyra-334)를 검출하였다. 이렇게 HPLC를 이용하여 마이코스포린 유사 아미노산(시노린 및 포피라-334)을 정량한 결과를 하기의 표 4 및 도 1에 표시하였다. The transformed yeast strain thus obtained was inoculated into YPD-G418 medium (yeast extract 10 g/L, peptone 20 g/L, dextrose 10 g/L, xylose 10 g/L, G418 100 ug/L) at an O.D.600 of 0.2 and cultured for 72 hours under conditions of 30°C and 220 rpm. In order to quantify mycosporine-like amino acids in the medium, 1 mL of the culture solution was centrifuged to obtain the supernatant, which was filtered through a 0.22 ㎛ filter and subjected to HPLC analysis. A 1260 infinity HPLC system (Agilent) equipped with an Agilent Eclipse XDB-C18 column (5 μm, 4.6 × 250 mm) was used, the column temperature was maintained at 40°C, and the solvent (water containing 0.25% Trifluoroacetic acid: acetonitrile containing 0.25% Trifluoroacetic acid = 97.5:2.5) was flowed at a flow rate of 0.5 mL/min. Shinorine and porphyra-334 were detected with a UV-vis detector at a wavelength of 334 nm. The results of quantifying mycosporine-like amino acids (shinorine and porphyra-334) using HPLC are shown in Table 4 and Fig. 1 below.
상기 표 4 및 도 1에서 확인할 수 있는 바와 같이, YOR1 유전자를 강화한 경우에만 배지 내 시노린의 농도가 크게 증가하는 것이 확인되었다. 공벡터를 포함하는 JHSM132/Star41 균주와 비교하여 JHSM132/YOR1 균주의 시노린 배출양은 48시간과 72시간에 각각 약 2.4배, 2.0배 증가한 것을 확인할 수 있었다.As can be confirmed in Table 4 and Fig. 1 above, it was confirmed that the concentration of shinorine in the medium significantly increased only when the YOR1 gene was strengthened. Compared to the JHSM132/Star41 strain containing the empty vector, it was confirmed that the amount of shinorine secreted by the JHSM132/YOR1 strain increased approximately 2.4 times and 2.0 times at 48 hours and 72 hours, respectively.
실시예 3. 배출자 후보유전자의 과발현을 통한 마이코스포린 유사 아미노산 배출능 평가 2Example 3. Evaluation of mycosporine-like amino acid excretion ability through overexpression of an excretor candidate gene 2
JHSM133 균주에 상기 실시예 1에서 제작된 Star41-Kan-PGAP-TPI1, Star41-Kan-PGAP-YOR1-TPI1, Star41-Kan-PGAP-PDR5-TPI1, Star41-Kan-PGAP-PDR10-TPI1, 및 Star41-Kan-PGAP-PDR15-TPI1 플라스미드를 각각 LiAc/SS carrier DNA/PEG 방법을 사용하여 YPD-G418 평판배지(Yeast extract 10 g/L, peptone 20 g/L, dextrose 20 g/L, G418 100 ug/L)에 형질전환시켰다. The Star41-Kan-PGAP-TPI1, Star41-Kan-PGAP-YOR1-TPI1, Star41-Kan-PGAP-PDR5-TPI1, Star41-Kan-PGAP-PDR10-TPI1, and Star41-Kan-PGAP-PDR15-TPI1 plasmids constructed in Example 1 were each transformed into YPD-G418 plate medium (yeast extract 10 g/L, peptone 20 g/L, dextrose 20 g/L, G418 100 ug/L) using the LiAc/SS carrier DNA/PEG method.
본 실시예에서 사용된 형질전환 균주는 야생형의 Saccharomyces cerevisiae 효모 균주인 CEN.PK2-1C (EUROSCARF)에 마이코스포린 유사 아미노산 생산을 위한 유전자를 재조합한 균주로서, 그 정보는 표 5에 기재된 바와 같으며, 보다 상세한 정보는 Metabolic Engineering 78 (2023) 137-147 논문을 참고할 수 있다.The transformed strain used in this example is a strain in which a gene for producing mycosporine-like amino acids is recombined into the wild-type Saccharomyces cerevisiae yeast strain CEN.PK2-1C (EUROSCARF). The information is as described in Table 5, and more detailed information can be found in the paper Metabolic Engineering 78 (2023) 137-147.
이렇게 얻어진 형질전환 효모 균주를 YPD-G418 배지 (Yeast extract 10 g/L, peptone 20 g/L, dextrose 10 g/L, xylose 10 g/L, G418 100 ug/L)에 O.D.600 = 0.2로 접종하여 30℃ 및 220 rpm 조건 하에서 72시간 동안 배양하였다. 배지 내 마이코스포린 유사 아미노산을 정량하기 위하여 배양액 1 mL을 원심분리한 후 상등액을 얻고, 이를 0.22 ㎛ 필터로 여과하여 HPLC 분석을 수행하였다. Agilent Eclipse XDB-C18 컬럼(5 ㎛, 4.6×250 mm)을 갖춘 1260 infinity HPLC 시스템(Agilent)를 이용하였고, 컬럼 온도는 40℃로 유지하며 0.5 mL/분의 유속으로 용매(0.25% Trifluoroacetic acid가 포함된 물 : 0.25% Trifluoroacetic acid가 포함된 아세토니트릴 = 97.5 : 2.5)를 흘려주었다. 파장 334 nm에서 UV-vis 검출기로 시노린(shinorine) 및 포피라-334(porphyra-334)를 검출하였다. 이렇게 HPLC를 이용하여 마이코스포린 유사 아미노산(시노린 및 포피라-334)을 정량한 결과를 하기의 표 6 및 도 2에 표시하였다. The transformed yeast strain thus obtained was inoculated into YPD-G418 medium (yeast extract 10 g/L, peptone 20 g/L, dextrose 10 g/L, xylose 10 g/L, G418 100 ug/L) at an O.D.600 of 0.2 and cultured for 72 hours under conditions of 30°C and 220 rpm. In order to quantify mycosporine-like amino acids in the medium, 1 mL of the culture solution was centrifuged to obtain the supernatant, which was filtered through a 0.22 ㎛ filter and subjected to HPLC analysis. A 1260 infinity HPLC system (Agilent) equipped with an Agilent Eclipse XDB-C18 column (5 μm, 4.6 × 250 mm) was used, the column temperature was maintained at 40°C, and the solvent (water containing 0.25% Trifluoroacetic acid: acetonitrile containing 0.25% Trifluoroacetic acid = 97.5:2.5) was flowed at a flow rate of 0.5 mL/min. Shinorine and porphyra-334 were detected with a UV-vis detector at a wavelength of 334 nm. The results of quantifying mycosporine-like amino acids (shinorine and porphyra-334) using HPLC are shown in Table 6 and Fig. 2 below.
상기 표 6 및 도 2에서 확인할 수 있는 바와 같이, YOR1 유전자를 강화한 경우에만 배지 내 포피라-334의 농도가 크게 증가하는 것이 확인되었다. 공벡터를 포함하는 JHSM133/Star41 균주와 비교하여 JHSM133/YOR1 균주의 시노린 배출양은 72시간에 각각 약 2.0배 증가한 것을 확인할 수 있었다.As can be confirmed in Table 6 and Fig. 2 above, the concentration of Porphyra-334 in the medium was confirmed to significantly increase only when the YOR1 gene was strengthened. Compared to the JHSM133/Star41 strain containing the empty vector, the amount of shinorine secreted by the JHSM133/YOR1 strain was confirmed to increase by approximately 2.0 times at 72 hours.
이상 살펴본 바와 같이, 본 발명에 따른 올리고마이신 저항 ATP 의존성 투과효소 YOR1 단백질은 마이코스코린 유사 아미노산 생산 균주가 생산한 마이코스포린 유사 아미노산을 균주 외부로 배출하는 효과가 뛰어나므로, 해당 마이코스포린 유사 아미노산을 대량으로 생산하는데 유용하게 이용될 수 있어 산업상 이용가능성이 높다.As described above, the oligomycin-resistant ATP-dependent permease YOR1 protein according to the present invention is excellent in the effect of excreting mycosporine-like amino acids produced by a mycosporine-like amino acid producing strain out of the strain, and therefore can be usefully used to mass-produce the mycosporine-like amino acids, and thus has high industrial applicability.
서열목록 전자파일 첨부Attach electronic file of sequence list
Claims (11)
A composition for extracellular excretion of Shinorine or Porphyra-334, comprising oligomycin resistance ATP-dependent permease YOR1 protein as an active ingredient.
A composition for extracellular excretion of shinorine or porphyra-334, characterized in that in claim 1, the oligomycin-resistant ATP-dependent permease YOR1 protein is derived from Saccharomyces cerevisiae.
A composition for extracellular excretion of shinorine or porphyra-334, characterized in that in claim 1, the oligomycin-resistant ATP-dependent permease YOR1 protein consists of an amino acid sequence represented by sequence number 1.
A microorganism for producing shinorine or Porphyra-334, wherein the microorganism is transformed with a nucleic acid encoding an oligomycin resistance ATP-dependent permease YOR1 protein.
In claim 5, a microorganism for producing shinorine or porphyra-334, characterized in that the oligomycin-resistant ATP-dependent permease YOR1 protein is derived from Saccharomyces cerevisiae.
In claim 5, a microorganism for producing shinorine or porphyra-334, characterized in that the oligomycin-resistant ATP-dependent permease YOR1 protein consists of an amino acid sequence represented by sequence number 1.
A microorganism for producing shinorine or porphyra-334, characterized in that in claim 5, the microorganism is a yeast, a Corynebacterium sp. microorganism, or an Escherichia sp. microorganism.
A composition for producing shinorine or porphyra-334, comprising a microorganism according to any one of claims 5 to 7 and 9 as an effective ingredient.
상기 배양된 미생물 또는 배지로부터 시노린(Shinorine) 또는 포피라-334(Porphyra-334)를 회수하는 단계;
를 포함하는, 시노린 또는 포피라-334의 생산 방법.A step of culturing a microorganism according to any one of claims 5 to 7 and 9; and
A step of recovering Shinorine or Porphyra-334 from the cultured microorganism or medium;
A method for producing shinorine or porphyra-334, comprising:
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| KR102003911B1 (en) * | 2018-02-23 | 2019-07-25 | 씨제이제일제당 주식회사 | A microorganism for producing a Mycosporine-like amino acid and a method for preparing a Mycosporine-like amino acid using the same |
| KR20210082021A (en) * | 2019-12-24 | 2021-07-02 | 씨제이제일제당 (주) | Microorganism for Producing Mycosporine-like Amino Acid and Method for Preparing Mycosporine-like Amino Acid Using the Same |
| KR102548752B1 (en) | 2022-09-28 | 2023-07-03 | 큐티스바이오 주식회사 | Microorganisms having mycosporine-like amino acid production ability and method for producing mycosporin-like amino acids using the same |
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| KR20210082021A (en) * | 2019-12-24 | 2021-07-02 | 씨제이제일제당 (주) | Microorganism for Producing Mycosporine-like Amino Acid and Method for Preparing Mycosporine-like Amino Acid Using the Same |
| KR102351059B1 (en) | 2019-12-24 | 2022-01-12 | 씨제이제일제당 (주) | Microorganism for Producing Mycosporine-like Amino Acid and Method for Preparing Mycosporine-like Amino Acid Using the Same |
| KR102548752B1 (en) | 2022-09-28 | 2023-07-03 | 큐티스바이오 주식회사 | Microorganisms having mycosporine-like amino acid production ability and method for producing mycosporin-like amino acids using the same |
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