KR101522961B1 - Novel compound quercetin-3-O-N-acetyl-quinovosamine and method for producing the same - Google Patents
Novel compound quercetin-3-O-N-acetyl-quinovosamine and method for producing the same Download PDFInfo
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Abstract
본 발명은 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자와 바실러스 세레우스(Bacillus cereus) 유래 UDP -GlcNAc4,6-DH(UDP-N-acetylglucosamine 4,6-dehydratase) 유전자를 대사조절된 대장균에 발현시켜 퀘세틴에서 신규한 화합물인 퀘세틴-3-O-N-아세틸-퀴노보사민(quercetin-3-O-N-acetyl-quinovosamine)을 합성하는 방법에 관한 것이다.The present invention relates to Arabidopsis thaliana transforming enzyme AtUGT78D2 ( Arabidopsis thaliana UDP-dependent glycosyltransferase) genes of Bacillus cereus (Bacillus cereus) derived UDP -GlcNAc4,6-DH (UDP- N -acetylglucosamine 4,6-dehydratase) by expressing the gene in the E. coli metabolic regulation of a novel compound in Quebec Quebec paroxetine paroxetine -3- O - N-acetyl-quinolyl 3- O - N- acetyl-quinovosamine. ≪ / RTI >
Description
본 발명은 신규한 퀘세틴-3-O-N-아세틸-퀴노보사민 화합물 및 이의 제조방법에 관한 것으로, 더욱 상세하게는 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자와 바실러스 세레우스(Bacillus cereus)에서 유래한 UDP - GlcNAc4 ,6-DH(UDP-N-acetylglucosamine 4,6-dehydratase) 유전자를 대사조절된 대장균에 발현시켜 기질인 퀘세틴에서 퀘세틴-3-O-N-아세틸-퀴노보사민(quercetin-3-O-N-acetyl-quinovosamine)의 신규 화합물을 합성하는 방법에 관한 것이다.The present invention relates to novel Quebec paroxetine -3- O - N-acetyl-quinolyl Novo samin compounds and relates to a production method thereof, and more particularly, Arabidopsis thaliana-derived sugar transferase AtUGT78D2 (Arabidopsis thaliana UDP-dependent glycosyltransferase ) genes and Bacillus The expression of UDP - GlcNAc4 , 6 - DH (UDP - N -
일반적으로 배양방법과 유전자 조작기술이 잘 확립되어 있는 대장균과 같은 미생물에 유용한 유전자를 도입함으로써 천연물을 효소학적으로 변형할 수 있는데, 이러한 방법을 생물전환법(biotransformation)이라 한다. 생물전환법을 이용하면 반응 중간에 들어가는 값비싼 보조인자(cofactor)를 절약할 수 있는 장점을 가지고 있다.In general, natural products can be enzymatically modified by introducing genes useful for microorganisms such as E. coli, which are well established in culture methods and genetic engineering techniques. This method is called biotransformation. Bioconversion has the advantage of saving costly cofactors in the middle of the reaction.
새로운 의약 후보물질로 많은 관심을 불러일으키고 있는 플라보노이드(flavonoid)는 식물이 만들어 내는 많은 이차 대사산물 중 하나이다. 플라보노이드는 일상적으로 섭취하는 식품 중에 다종/다양한 형태로 포함되어 있으며, 강한 항산화활성을 갖는 것으로 알려져 있다. 그러나 그 항산화활성은 생체 외에서는 유효하지만, 경구흡수성이 낮기 때문에 생체 내에서는 활성 산소나 자유 라디칼을 소거하기에는 충분하지 않다. 따라서, 플라보노이드(헤스페리딘(hesperidine), 디오스민(diosmin), 나린진(naringin), 네오헤스페리딘(neohesperidine))에 당을 결합시킴으로써 흡수성을 높이는 방법이 제안되고 있다(일본 특허공개공보 제2000-78956호).Flavonoid, which has attracted much interest as a new drug candidate, is one of the many secondary metabolites produced by plants. Flavonoids are known to have a strong antioxidant activity and are contained in many types / diverse forms of foods that are ingested routinely. However, its antioxidant activity is effective in vitro, but it is not sufficient to eradicate active oxygen or free radicals in vivo because of its low oral absorbability. Accordingly, there has been proposed a method of increasing the absorbency by binding sugars to flavonoids (hesperidine, diosmin, naringin, neohesperidine) (Japanese Patent Application Laid-Open No. 2000-78956) .
퀘세틴(Quercetin, 3,3',4',5,7-펜타하이드록시플라본(3,3',4',5,7-pentahydroxyflavone))은 강력한 항산화활성(Middlton et al., 2000, Pharmacol. Rev. 52:673-751) 외에, 혈소판의 응집 억제 및 접착 억제 작용, 혈관 확장 작용, 항암 작용 등 다양한 생리기능을 갖는 것이 알려져 있다. 특히, 양파에 많이 함유된 퀘세틴 배당체(퀘세틴-4'-β-D-글루코시드(Quercetin-4'-β-D-glucoside) 및 퀘세틴-3,4'-β-D-글루코시드(Quercetin-3,4'-β-D-glucoside)) 쪽이 흡수성이 우수한 것으로 보고되어 있다(Hollman et al., 1998, Arch. Toxicol. Suppl. 20:237-248). 또한, 유사하게 퀘세틴의 3번 탄소 위치에 글루코오스가 β결합한 이소퀘세틴(퀘세틴-3-β-D-글루코사이드(Quercetin-3-β-D-glucoside))은 퀘세틴이나 루틴보다도 흡수성이 높은 것으로 보고되어 있다(Morand et al., 2000, Free Raf. Res. 33:667-676).Quercetin (3,3 ', 4', 5,7-pentahydroxyflavone) has a strong antioxidant activity (Middlton et al., 2000, Pharmacol . Rev. 52: 673-751), it is known that it has various physiological functions such as inhibition of aggregation of platelets and adhesion inhibition, vasodilation, and anti-cancer action. In particular, quercetin glycosides (quercetin-4'-β-D-glucoside and quercetin-3,4'-β-D-glucoside (Quercetin-3,4'-β-D-glucoside) has been reported to be superior in absorbability (Hollman et al., 1998, Arch. Toxicol. Suppl. 20: 237-248). Similarly, quercetin-3-β-D-glucoside (quercetin-3-β-D-glucoside) in which glucose is β-linked to the 3-carbon position of quercetin is more absorbent than quercetin or (Morand et al., 2000, Free Raf. Res. 33: 667-676).
당전이효소는 핵산당(nucleotide sugar)을 당 공여체(donor)로 이용하는데, 본 발명은 새로운 구조의 핵산당을 대장균에서 만들기 위해 대장균의 핵산당 대사경로를 분석하고 핵산당 합성경로에 있는 물질들을 기질로 사용할 수 있는 바실러스 세레우스 ATCC 14579(Bacillus cereus ATCC 14579) 유래 UDP - GlcNAc4 ,6-DH(UDP-N-acetylglucosamine 6-dehydrogenase) 유전자를 대장균에 도입하였다. UDP -GlcNAc4,6-DH 유전자는 UDP-N-아세틸글루코사민(acetylglucosamine)을 이용하여 UDP-N-아세틸-퀴노보사민(UDP-N-acetyl-quinovosamine)을 만들며, 이렇게 만들어진 UDP-N-아세틸-퀴노보사민은 당전이효소에 의해 기질에 당화된다. 본 발명은 기질로 퀘세틴을 사용하였고, 상기와 같은 반응을 통해 신규 화합물인 퀘세틴-3-O-N-아세틸-퀴노보사민(quercetin 3-O-N-acetyl-quinovosamine)을 합성하였다.The transglycosylase utilizes a nucleotide sugar as a donor. The present invention analyzes the nucleic acid sugar metabolism pathway of E. coli to make a new structure of the nucleic acid sugar in E. coli, Bacillus cereus ATCC 14579 ( Bacillus < RTI ID = 0.0 > cereus It was introduced GlcNAc4, 6-DH (UDP- N -acetylglucosamine 6-dehydrogenase) gene into the E. coli - ATCC 14579) derived from UDP. UDP -GlcNAc4,6-DH gene is UDP- N -acetyl-acetyl UDP- N with Glucosamine (acetylglucosamine) quinolyl Novo samin creates a (UDP- N -acetyl-quinovosamine), so made UDP- N -acetyl- Quinobosamine is glycosylated to the substrate by a glycosyltransferase. The present invention was used as a substrate Quebec paroxetine, the reaction, such as the novel compounds of Quebec paroxetine -3- O through-N-acetyl-quinolyl Novo samin (quercetin 3- O - N -acetyl- quinovosamine) was synthesized.
이러한 물질은 자연계에는 존재하지 않으며, 또한 기존의 화학적인 방법을 이용해서 합성하는 것은 거의 불가능하다. 생물전환법(효소학적 합성 방법)은 화학적 합성법으로 물질을 변형하기 어려운 위치 선택성과 키랄 선택성을 가지기 때문에 화학적 합성법에 비하여 여러 가지 장점을 가지고 있다. 따라서, 본 발명의 두 유전자(AtUGT78D2 및 UDP - GlcNAc4 ,6- DH)를 가지고 대장균을 이용한 생물전환법은 퀘세틴-3-O-N-아세틸-퀴노보사민을 생산할 수 있는 새로운 방법이 될 수 있을 것이다.These substances do not exist in nature, and it is almost impossible to synthesize them using existing chemical methods. The bioconversion method (enzymatic synthesis method) has several advantages over the chemical synthesis method because it has position selectivity and chiral selectivity which are difficult to be modified by a chemical synthesis method. Therefore, a bioconversion method using E. coli with two genes of the present invention ( AtUGT78D2 and UDP - GlcNAc4 , 6- DH ) can be a new method for producing quercetin-3- O - N -acetyl- quinobosamine There will be.
한국등록특허 제0716797호에는 '당전이 효소를 이용한 당전이 화합물의 유도체 제조방법 및 이로부터 제조된 유도체'가 개시되어 있고, 한국등록특허 제1270124호에는 '신규한 퀘세틴 3-Ｏ-6-디옥시-Ｌ-탈로사이드 화합물 및 그 화합물의 제조방법'이 개시되어 있으나, 본 발명의 신규한 퀘세틴-3-O-N-아세틸-퀴노보사민 화합물 및 이의 제조방법에 대해서는 기재된 바가 없다.Korean Patent No. 0716797 discloses a process for preparing a derivative of a precursor compound using the enzyme and a derivative prepared therefrom. Korean Patent No. 1270124 discloses a novel quercetin 3-O-6- Dioxy-L-taloside compound and a process for producing the same. However, the novel quercetin-3- O - N -acetyl-quinobosane compound of the present invention and its preparation method are not disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자는 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자와 바실러스 세레우스 ATCC 14579(Bacillus cereus ATCC 14579) 유래 UDP - GlcNAc4 ,6- DH(UDP-N-acetylglucosamine 4,6-dehydratase) 유전자를 대사조절된 대장균에 발현시키고, 상기 형질전환 대장균을 이용하여 퀘세틴을 기질로 하여 신규한 화합물인 퀘세틴-3-O-N-아세틸-퀴노보사민을 합성함으로써, 본 발명을 완성하였다.The present invention is derived by the request as described above, the present inventors transferase per Arabidopsis-derived AtUGT78D2 (Arabidopsis thaliana UDP-dependent glycosyltransferase) genes of Bacillus cereus ATCC 14579 (Bacillus cereus Of a GlcNAc4, 6- DH (UDP- N -
상기 과제를 해결하기 위해, 본 발명은 신규한 퀘세틴-3-O-N-아세틸-퀴노보사민(quercetin 3-O-N-acetyl-quinovosamine) 화합물을 제공한다.In order to solve the above problems, the present invention relates to novel Quebec paroxetine -3- O - provides a compound N-acetyl-quinolyl samin Novo (N -acetyl-quinovosamine quercetin 3- O ).
또한, 본 발명은 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자 및 바실러스 세레우스(Bacillus cereus) 유래 UDP - GlcNAc4 ,6- DH(UDP-N-acetylglucosamine 4,6-dehydratase) 유전자를 포함하는 발현벡터; 애기장대 유래 당전이효소 AtUGT78D2 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP - GlcNAc4 ,6- DH 유전자를 포함하는 발현벡터; 또는 애기장대 유래 당전이효소 AtUGT78D2 유전자 및 바실러스 세레우스 유래 UDP-GlcNAc4,6-DH 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP -GlcNAc4,6-DH 유전자를 포함하는 발현벡터를 함유하는 상기 신규 화합물의 제조용 조성물을 제공한다.The present invention is derived from Arabidopsis thaliana per transferase AtUGT78D2 (Arabidopsis thaliana UDP-glycosyltransferase dependent) gene and Bacillus cereus (Bacillus cereus) derived UDP - GlcNAc4, 6- DH (UDP- N -
또한, 본 발명은In addition,
a) 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자 및 바실러스 세레우스(Bacillus cereus) 유래 UDP - GlcNAc4 ,6- DH(UDP-N-acetylglucosamine 4,6-dehydratase) 유전자를 포함하는 발현벡터를 제조하거나, 애기장대 유래 당전이효소 AtUGT78D2 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP-GlcNAc4,6-DH 유전자를 포함하는 발현벡터를 제조하거나, 또는 애기장대 유래 당전이효소 AtUGT78D2 유전자 및 바실러스 세레우스 유래 UDP - GlcNAc4 ,6- DH 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP - GlcNAc4 ,6- DH 유전자를 포함하는 발현벡터를 제조하는 단계;a) Arabidopsis transferase enzyme AtUGT78D2 ( Arabidopsis thaliana UDP-dependent glycosyltransferase) genes and Bacillus cereus (Bacillus cereus) derived UDP - GlcNAc4, 6- DH (UDP- N -
b) 상기 발현벡터들을 대장균에 형질전환시키는 단계; 및b) transforming the expression vectors into E. coli; And
c) 상기 형질전환 대장균을 배양하면서 퀘세틴을 기질로 첨가하는 단계를 포함하는 상기 신규 화합물의 제조방법을 제공한다.c) adding the quercetin to the substrate while culturing the transformed E. coli.
또한, 본 발명은 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자 및 바실러스 세레우스 (Bacillus cereus) 유래 UDP - GlcNAc4 ,6- DH(UDP-N-acetylglucosamine 4,6-dehydratase) 유전자를 포함하는 발현벡터; 애기장대 유래 당전이효소 AtUGT78D2 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP-GlcNAc4,6-DH 유전자를 포함하는 발현벡터; 또는 애기장대 유래 당전이효소 AtUGT78D2 유전자 및 바실러스 세레우스 유래 UDP-GlcNAc4,6-DH 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP -GlcNAc4,6-DH 유전자를 포함하는 발현벡터로 형질전환되어 상기 신규 화합물을 생산하는 형질전환 대장균을 제공한다.The present invention is derived from Arabidopsis thaliana per transferase AtUGT78D2 (Arabidopsis thaliana UDP-glycosyltransferase dependent) gene and Bacillus cereus (Bacillus cereus) derived UDP - GlcNAc4, 6- DH (UDP- N -
또한, 본 발명은 신규한 퀘세틴-3-O-N-아세틸-퀴노보사민 화합물을 유효성분으로 포함하는 식품 조성물을 제공한다.The present invention also provides a food composition comprising a novel quercetin-3- O - N -acetyl-quinobosenamine compound as an active ingredient.
본 발명은 애기장대 유래 당전이효소 AtUGT78D2 유전자와 바실러스 세레우스 ATCC 14579 유래 UDP - GlcNAc4 ,6- DH 유전자를 대장균에 발현시켜 신규한 화합물을 합성하는 것에 관한 것이다. 본 발명을 통하여 알 수 있는 바와 같이 본 발명의 대장균 시스템을 통하여 퀘세틴을 기질로 하여 신규한 화합물인 퀘세틴-3-O-N-아세틸-퀴노보사민을 만들어 내었다. 본 발명의 화합물인 퀘세틴-3-O-N-아세틸-퀴노보사민은 그 화합물을 구성하고 있는 각각의 구성성분이 가지는 기능성으로 인하여 각종 식품, 의약품 및 화장품 등에 유용하게 이용될 수 있다.The present invention relates to the synthesis of novel compounds by expressing the Arabidopsis glycosyltransferase AtUGT78D2 gene and the UDP - GlcNAc4,6 - DH gene derived from Bacillus cereus ATCC 14579 into E. coli. As can be seen from the present invention, quercetin-3- O - N -acetyl-quinobosamine, which is a novel compound, was produced using quercetin as a substrate through the E. coli system of the present invention. Quercetin-3- O - N -acetyl-quinobosamine, which is a compound of the present invention, can be usefully used in various foods, medicines and cosmetics due to the functionality of each component constituting the compound.
도 1은 기질 퀘세틴으로부터 신규 화합물인 퀘세틴-3-O-N-아세틸-퀴노보사민을 합성하는 것을 도식화한 그림이다.
도 2는 애기장대 유래 당전이효소 AtUGT78D2 유전자와 바실러스 세레우스 ATCC 14579 유래 UDP - GlcNAc4 ,6- DH 유전자를 형질전환시킨 대사조절된 대장균을 이용하여 퀘세틴을 기질로 생물전환을 실시하여 얻은 반응물을 고성능 액체 크로마토그래피(HPLC)로 분석한 결과이다.
도 3은 핵자기공명분광기(NMR, Nuclear Magnetic Resonance spectrometer) 분석 결과이다.
도 4는 애기장대 유래 당전이효소 AtUGT78D2 유전자와 바실러스 세레우스 ATCC 14579 유래 UDP - GlcNAc4 ,6- DH 유전자를 다양한 조합으로 대사조절된 대장균에 형질전환하여 신규 화합물의 생성량을 비교한 결과이다.FIG. 1 is a diagram illustrating the synthesis of a novel compound quercetin-3- O - N -acetyl-quinobosamine from a substrate quercetin.
FIG. 2 shows a reaction product obtained by bioconversion of quercetin to a substrate using a metabolic-controlled E. coli transformed with Arabidopsis thaliana glycoprotein AtUGT78D2 gene and UDP - GlcNAc4,6 - DH gene derived from Bacillus cereus ATCC 14579 It is the result of analysis by high performance liquid chromatography (HPLC).
FIG. 3 shows the result of nuclear magnetic resonance spectrometer (NMR) analysis.
FIG. 4 shows the results of comparing the amount of new compounds produced by transfection of Escherichia coli - derived glycosyltransferase AtUGT78D2 gene and UDP - GlcNAc4 , 6- DH gene derived from Bacillus cereus ATCC 14579 into various regulated Escherichia coli.
본 발명의 목적을 달성하기 위하여, 본 발명은 하기 화학식 1의 퀘세틴-3-O-N-아세틸-퀴노보사민(quercetin 3-O-N-acetyl-quinovosamine) 화합물을 제공한다.According to an aspect of the invention there is provided paroxetine Quebec -3- O of formula (1) - to provide the compound N-acetyl-quinolyl samin Novo (N -acetyl-quinovosamine quercetin 3- O ).
또한, 본 발명은 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자 및 바실러스 세레우스(Bacillus cereus) 유래 UDP - GlcNAc4 ,6- DH(UDP-N-acetylglucosamine 4,6-dehydratase) 유전자를 포함하는 발현벡터; 애기장대 유래 당전이효소 AtUGT78D2 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP-GlcNAc4,6-DH 유전자를 포함하는 발현벡터; 또는 애기장대 유래 당전이효소 AtUGT78D2 유전자 및 바실러스 세레우스 유래 UDP-GlcNAc4,6-DH 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP -GlcNAc4,6-DH 유전자를 포함하는 발현벡터를 함유하는 상기 화합물의 제조용 조성물을 제공한다.The present invention is derived from Arabidopsis thaliana per transferase AtUGT78D2 (Arabidopsis thaliana UDP-glycosyltransferase dependent) gene and Bacillus cereus (Bacillus cereus) derived UDP - GlcNAc4, 6- DH (UDP- N -
본 발명의 일 구현 예에 따른 조성물은 AtUGT78D2 유전자 및 UDP - GlcNAc4 ,6-DH 유전자가 하나의 발현 벡터에 함께 도입될 수도 있고, 별개의 발현 벡터에 각각 도입될 수도 있다. 또한, 본 발명의 일 구현 예에 따른 조성물은 AtUGT78D2 유전자 및 UDP - GlcNAc4 ,6- DH 유전자가 하나의 발현 벡터에 함께 도입된 것과 UDP - GlcNAc4 ,6- DH 유전자가 별개의 하나의 발현 벡터에 도입된 것을 동시에 포함할 수 있다.The composition according to one embodiment of the present invention may be introduced into an expression vector, or may be introduced into a separate expression vector, respectively, with the AtUGT78D2 gene and the UDP - GlcNAc4 , 6-DH gene. In addition, the composition according to one embodiment of the present invention AtUGT78D2 gene and UDP - GlcNAc4, 6- DH gene is introduced with the UDP as a single expression vector - GlcNAc4, 6- DH gene was introduced in a separate one of an expression vector Can be included at the same time.
본 발명의 일 구현 예에 따른 조성물에서, 상기 애기장대 유래 당전이 효소 AtUGT78D2 유전자는 서열번호 1의 염기서열로 이루어질 수 있다. 또한, 상기 염기 서열의 상동체가 본 발명의 범위 내에 포함된다. 구체적으로, 상기 유전자는 서열번호 1의 염기 서열과 각각 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.In the composition according to an embodiment of the present invention, the Arabidopsis thaliana transforming enzyme AtUGT78D2 gene may be composed of the nucleotide sequence of SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >
또한, 본 발명의 일 구현 예에 따른 조성물에서, 상기 바실러스 세레우스 유래 UDP - GlcNAc4 ,6- DH 유전자는 바람직하게는 바실러스 세레우스 ATCC 14579 유래 UDP - GlcNAc4 ,6- DH 유전자일 수 있으며, 더욱 바람직하게는 서열번호 2의 염기서열로 이루어질 수 있다. 또한, 상기 염기 서열의 상동체가 본 발명의 범위 내에 포함되는데 구체적인 내용은 전술한 바와 같다.Further, in the composition according to an embodiment of the present invention, the Bacillus cereus-derived UDP - GlcNAc4 , 6- DH gene can be preferably UDP - GlcNAc4 , 6- DH gene derived from Bacillus cereus ATCC 14579, The nucleotide sequence of SEQ ID NO: 2. In addition, homologues of the nucleotide sequences are included within the scope of the present invention, and the details thereof are as described above.
용어 "벡터"는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 지칭할 때 사용된다. 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재생산될 수 있다. 용어 "전달체"는 흔히 "벡터"와 호환하여 사용된다. 용어 "발현 벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 진핵세포에서 이용가능한 프로모터, 인핸서, 종결신호 및 폴리아데닐레이션 신호는 공지되어 있다.The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.
본 발명의 벡터는 전형적으로 클로닝 또는 발현을 위한 벡터로서 구축될 수 있다. 또한, 본 발명의 벡터는 원핵세포를 숙주로 하여 구축될 수 있다. 예를 들어, 본 발명의 벡터가 발현 벡터이고, 원핵 세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터(예: pLλ프로모터, trp 프로모터, lac 프로모터, T7 프로모터, tac 프로모터 등), 해독의 개시를 위한 리보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 숙주 세포로서 대장균(E. coli)이 이용되는 경우, 대장균 트립토판 생합성 경로의 프로모터 및 오퍼레이터 부위, 그리고 파아지 λ의 좌향 프로모터(pLλ프로모터)가 조절 부위로서 이용될 수 있다.The vector of the present invention can typically be constructed as a vector for cloning or expression. In addition, the vector of the present invention can be constructed using prokaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pL? Promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) It is common to include a ribosome binding site and a transcription / translation termination sequence for initiation of translation. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthesis pathway and the left promoter of the phage lambda (pL 貫 promoter) can be used as a regulatory region.
한편, 본 발명에 이용될 수 있는 벡터는 당업계에서 종종 사용되는 플라스미드(예: pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX 시리즈, pET 시리즈 및 pUC19 등), 파지(예: λgt4·λB, λ-Charon, λΔz1 및 M13 등) 또는 바이러스(예: SV40 등)를 조작하여 제작될 수 있다.The vectors that can be used in the present invention include plasmids such as pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19 which are frequently used in the art, phages such as λgt4 · λB ,? -charon,?? z1, and M13), or a virus (e.g., SV40, etc.).
본 발명의 벡터는 선택표지로서, 당업계에서 통상적으로 이용되는 항생제 내성 유전자를 포함할 수 있으며, 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 게네티신, 네오마이신 및 테트라사이클린에 대한 내성 유전자가 있다.The vector of the present invention may be a selection marker and may include an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.
또한, 본 발명의 일 구현 예에 따른 조성물은 대장균을 추가로 포함하는 것이 바람직하고, 더욱 바람직하게는 글루코스-1-포스페이트 우리딜일트랜스퍼라제(glucose-1-phosphate uridylyltransferase)가 결손된 대장균을 포함하는 것일 수 있으나, 이에 제한되지 않는다.In addition, the composition according to an embodiment of the present invention preferably further comprises E. coli, more preferably, E. coli containing glucose-1-phosphate uridyl lransferase deficient But is not limited thereto.
또한, 본 발명은In addition,
애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자 및 바실러스 세레우스(Bacillus cereus) 유래 UDP -GlcNAc4,6-DH(UDP-N-acetylglucosamine 4,6-dehydratase) 유전자를 포함하는 발현벡터를 제조하거나, 애기장대 유래 당전이효소 AtUGT78D2 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP-GlcNAc4,6-DH 유전자를 포함하는 발현벡터를 제조하거나, 또는 애기장대 유래 당전이효소 AtUGT78D2 유전자 및 바실러스 세레우스 유래 UDP - GlcNAc4 ,6- DH 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP - GlcNAc4 ,6- DH 유전자를 포함하는 발현벡터를 제조하는 단계;Arabidopsis thaliana-derived sugar transferase AtUGT78D2 (Arabidopsis thaliana UDP-glycosyltransferase dependent) gene and Bacillus cereus (Bacillus cereus) derived UDP -GlcNAc4,6-DH (UDP- N -
상기 발현벡터들을 대장균에 형질전환시키는 단계; 및Transforming the expression vectors into Escherichia coli; And
상기 형질전환 대장균을 배양하면서 퀘세틴을 기질로 첨가하는 단계를 포함하는 퀘세틴-3-O-N-아세틸-퀴노보사민의 제조방법을 제공한다. O - N -acetyl-quinobosamine comprising culturing the transformed Escherichia coli and adding quercetin to the substrate.
본 발명의 일 구현 예에 따른 방법에서, 상기 애기장대 유래 당전이 효소 AtUGT78D2 유전자는 서열번호 1의 염기서열로, 상기 바실러스 세레우스 유래 UDP -GlcNAc4,6-DH 유전자는 서열번호 2의 염기서열로 이루어질 수 있다. 또한, 상기 염기 서열의 상동체가 본 발명의 범위 내에 포함되는데 구체적인 내용은 전술한 바와 같다.In the method according to one embodiment of the present invention, the Arabidopsis thaliana derived ATUGT78D2 gene is the nucleotide sequence of SEQ ID NO: 1, and the Bacillus cereus - derived UDP- GlcNAc4,6-DH gene is the nucleotide sequence of SEQ ID NO: 2 Lt; / RTI > In addition, homologues of the nucleotide sequences are included within the scope of the present invention, and the details thereof are as described above.
본 발명의 벡터를 숙주세포 내로 운반하는 방법은, 숙주 세포가 원핵 세포인 경우, CaCl2 방법, 하나한 방법(Hanahan, 1983, J. Mol. Biol. 166:557-580) 및 전기천공 방법 등에 의해 실시될 수 있다.The method of delivering the vector of the present invention into a host cell can be carried out by a CaCl 2 method, a Han method (Hanahan, 1983, J. Mol. Biol. 166: 557-580) and an electroporation method when the host cell is a prokaryotic cell . ≪ / RTI >
본 발명의 일 구현 예에 따른 방법에서, 상기 발현벡터들로 형질전환된 대장균은 바람직하게는 글루코스-1-포스페이트 우리딜일트랜스퍼라제(glucose-1-phosphate uridylyltransferase)가 결손된 대장균일 수 있으나, 이에 제한되지 않는다.In the method according to an embodiment of the present invention, the E. coli transformed with the expression vectors may be E. coli preferably lacking glucose-1-phosphate uridyl lyransferase, It is not limited.
본 발명은 또한, 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자 및 바실러스 세레우스(Bacillus cereus) 유래 UDP - GlcNAc4 ,6- DH(UDP-N-acetylglucosamine 4,6-dehydratase) 유전자를 포함하는 발현벡터; 애기장대 유래 당전이효소 AtUGT78D2 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP-GlcNAc4,6-DH 유전자를 포함하는 발현벡터; 또는 애기장대 유래 당전이효소 AtUGT78D2 유전자 및 바실러스 세레우스 유래 UDP-GlcNAc4,6-DH 유전자를 포함하는 발현벡터 및 바실러스 세레우스 유래 UDP -GlcNAc4,6-DH 유전자를 포함하는 발현벡터로 형질전환되어 퀘세틴-3-O-N-아세틸-퀴노보사민을 생산하는 대장균을 제공한다.The invention also Arabidopsis thaliana-derived sugar transferase AtUGT78D2 (Arabidopsis thaliana UDP-glycosyltransferase dependent) gene and Bacillus cereus (Bacillus cereus) derived UDP - GlcNAc4, 6- DH (UDP- N -
본 발명의 일 구현 예에 있어서, 상기 대장균은 바람직하게는 글루코스-1-포스페이트 우리딜일트랜스퍼라제(glucose-1-phosphate uridylyltransferase)가 결손된 대장균일 수 있으나, 이에 제한되지 않는다.In one embodiment of the present invention, the E. coli is preferably E. coli deficient in glucose-1-phosphate uridyl lyransferase, but is not limited thereto.
또한, 본 발명은 신규한 퀘세틴-3-O-N-아세틸-퀴노보사민 화합물을 유효성분으로 포함하는 식품 조성물을 제공한다. 본 발명의 화합물을 포함하는 식품으로서는 제한은 없으나, 아이스크림, 샤베트, 빙과 등의 냉과류; 유음료, 유산균 음료, 청량음료(과즙이 함유된 것을 포함), 탄산음료, 야채/과실 음료, 스포츠음료, 분말음료 등의 음료류; 리큐르 등의 알코올음료; 커피음료, 홍차 음료 등의 차 음료류; 콘소메 스프, 포타지 스프 등의 스프류; 카스타드 푸딩, 밀크 푸딩, 과즙 함유 푸딩 등의 푸딩류, 젤리, 바발로아(babaloa) 및 요구르트 등의 디저트류; 츄잉검이나 풍선검 등의 검류(스틱검, 당코팅된 검볼); 마블 쵸콜렛 등의 코팅된 쵸콜렛 외에 딸기 쵸콜렛, 블루베리 쵸콜렛 및 멜론 쵸콜렛 등의 풍미를 부가한 쵸콜렛 등의 쵸콜렛류; 경질 사탕(hard candy)(봉봉, 버터볼, 마블 등을 포함), 연질 사탕(softcandy)(캬라멜, 누가(nougat), 구미캔디(gummy candy), 마쉬멜로우(marshmallow) 등을 포함), 드로프(drop), 태피(taffy) 등의 캬라멜류; 하드 비스켓, 쿠키, 오카키(okaki, 쌀 크래커), 전병(sembei, 쌀 크래커) 등의 구운 과자류; 절임류, 김치, 세퍼레이트 드레싱, 오일 프리 드레싱, 케첩, 딥, 소스 등의 소스류; 딸기잼, 블루베리잼, 마말레이드, 사과잼, 살구잼, 설탕조림과일(preserves) 등의 잼류; 적포도주 등의 과실주; 시럽에 조린 체리, 살구, 사과, 딸기, 배 등의 가공용 과실; 햄, 소시지, 로스트 포크 등의 육류 가공품; 어육햄, 어육소시지, 어육 필레(fillet), 어묵; 치즈 등의 낙농 제품류; 우동, 냉국수, 소면, 메밀국수, 중국식 메밀국수, 스파게티, 마카로니, 쌀국수, 당면 및 만두국 등의 면류; 이외 각종 부식 등의 다양한 가공 식품을 들 수 있다.
The present invention also provides a food composition comprising a novel quercetin-3- O - N -acetyl-quinobosenamine compound as an active ingredient. Foods containing the compound of the present invention are not limited, but include cold confectionery products such as ice cream, sherbet and ice cream; Beverages such as milk drinks, lactic acid beverages, soft drinks (including juice), carbonated drinks, vegetable / fruit drinks, sports drinks, powdered drinks; Alcoholic drinks such as liqueur; Coffee beverages such as coffee beverages and tea beverages; Soups such as consommé and soup; Puddings such as custard pudding, milk pudding, juice-containing pudding, desserts such as jelly, babaloa and yogurt; Chewing gum or balloon gum (stick gum, sugar coated gum ball); Chocolate such as chocolate coated with berries such as strawberry chocolate, blueberry chocolate and melon chocolate in addition to coated chocolate such as marble chocolate; Such as hard candy (including sweets, butter balls, marbles, etc.), soft candy (including caramel, nougat, gummy candy, marshmallow, etc.) caramels such as drop and taffy; Baked confectionery such as hard biscuits, cookies, okaki (rice crackers), sembei (rice crackers); Sauces such as pickles, kimchi, separate dressings, oil-free dressings, ketchup, dips and sauces; Jams such as strawberry jam, blueberry jam, marmalade, apple jam, apricot jam, and preserves; Fruit wine such as red wine; Fruit for processing syrup, cherry, apricot, apple, strawberry, and pear; Meat products such as ham, sausage, and roast pork; Fish meat ham, fish sausage, fish fillet, fish cake; Dairy products such as cheese; Noodles such as udon noodles, cold noodles, somen noodles, buckwheat noodles, Chinese buckwheat noodles, spaghetti, macaroni, rice noodles, vermicelli and mandarin; And various kinds of processed foods such as various kinds of corrosion.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
실시예Example 1. One. UDPUDP -- GlcNAc4GlcNAc4 ,6-, 6- DHDH 유전자의 합성 및 대장균 발현벡터 제작Synthesis of genes and Escherichia coli expression vector production
UDP - GlcNAc4 ,6- DH(UDP-N-acetylglucosamine 4,6-dehydratase) 유전자는 바실러스 세레우스 ATCC 14579(Bacillus cereus ATCC 14579)에서 밝혀진 염기 서열(Genbank AE016877.1)을 토대로 PCR을 실시하여 클로닝하였다. 이때, 주형으로 바실러스 세레우스 ATCC 14579의 게노믹 DNA를 이용하였고, 제한효소 자리가 들어간 정방향 프라이머(5'-CGGCATATGTTAAATAAAATAATTTTAATT-3'; 서열번호 3, 밑줄; NdeI 자리) 및 역방향 프라이머(5'-CATCTCGAGTCATCGCAAAAACCCTCCTTT-3'; 서열번호 4, 밑줄; XhoI 자리)를 사용하여 PCR을 수행하였다.
UDP - GlcNAc4,6 - DH (UDP- N-
UDP - GlcNAc4 ,6- DH 유전자를 발현시키기 위한 벡터 제작 UDP - GlcNAc4 , 6- DH Vector generation for gene expression
합성한 UDP - GlcNAc4 ,6- DH 유전자를 발현시키기 위하여 상기 PCR 산물을 제한효소로 처리하고, 복제수가 각기 다른 대장균 발현벡터인 pACYCDuet(10-12 카피), pCDFDuet(20-40 카피) 및 pETDuet(40 카피 이상, Novagen, 독일) 벡터에 클로닝하고 각각 pA-UDP-GlcNAc4,6-DH, pC-UDP-GlcNAc4,6-DH 및 pE-UDP-GlcNAc4,6-DH라 명명하였다.
In order to express the synthesized UDP - GlcNAc4,6 - DH gene, the PCR products were treated with restriction enzymes, and pACYCDuet (10-12 copies), pCDFDuet (20-40 copies) and pETDuet UC-UDP-GlcNAc4,6-DH and pE-UDP-GlcNAc4,6-DH, respectively.
실시예Example 2. 애기장대 2. The Arabian Pole 당전이효소Transglycosylase 유전자의 Gene 클로닝Cloning 및 대장균 발현벡터 제작 And Escherichia coli Expression Vector Production
애기장대로부터 분리한 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자를 주형으로 하고, 제한효소 자리를 포함한 정방향 프라이머(5'-ATGAATTCGATGACCAAACCCTCCGACCC-3'; 서열번호 5, 밑줄; EcoRI 자리) 및 역방향 프라이머(5'-AAGCGGCCGCTTAAGTTTCTTAGCATTACA-3'; 서열번호 6, 밑줄; NotI 자리)를 이용하여 PCR을 수행하였다. PCR 조건은 전변성(pre-denaturaton) 94℃ 14분, 변성(denaturation) 94℃ 1분, 어닐링(annealing) 55℃ 1분 및 신장(extension) 72℃ 1분 30초 조건으로 수행하였다. 이렇게 얻어진 산물을 제한효소로 처리하여, 앞서 구축한 pA-UDP-GlcNAc4,6-DH, pC-UDP-GlcNAc4,6-DH 및 pE-UDP-GlcNAc4,6-DH 벡터에 클로닝하였다.
AtUGT78D2 isolated from Arabidopsis ( Arabidopsis -ATA GAATTC GATGACCAAACCCTCCGACCC-3 '(SEQ ID NO: 5, underlined, EcoRI digest ) and reverse primer (5'-AA GCGGCCGC TTAAGTTTCTTAGCATTACA-3') containing a restriction enzyme site and a thaliana UDP-dependent glycosyltransferase gene as a template, 3 ', SEQ ID NO: 6, underline, NotI position). PCR conditions were 94 ° C for 14 minutes, denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and extension at 72 ° C for 1 minute and 30 seconds. The resulting product was treated with restriction enzymes and cloned into the pA-UDP-GlcNAc4,6-DH, pC-UDP-GlcNAc4,6-DH and pE-UDP-GlcNAc4,6-DH vectors constructed above.
실시예Example 3. 3. AtUGT78D2AtUGT78D2 유전자 및 Gene and UDPUDP -- GlcNAc4GlcNAc4 ,6-, 6- DHDH 유전자 형질전환 대장균 제조 및 생물전환방법을 이용한 새로운 물질 생산 Generation of new substances using recombinant E. coli and bioconversion
글루코스-1-포스페이트 우리딜일트랜스퍼라제(galU, glucose-1-phosphate uridylyltransferase)가 결손된 것을 특징으로 하는 대장균 BL21(△galU)에 상기 AtUGT78D2 유전자와 UDP - GlcNAc4 ,6- DH 유전자를 포함하는 pCDFDuet 발현벡터를 형질전환하였다. AtUGT78D2 유전자와 UDP - GlcNAc4 ,6- DH 유전자를 발현하는 형질전환 대장균을 50㎍/㎖ 스펙티노마이신의 항생제를 첨가한 LB 배지에 종균배양 하였다. 밤샘 배양한 종균배양 대장균 균주를 새로운 LB 배지에 접종하고, 600nm에서 흡광도 값 0.8까지 되게 배양하였다. 배양된 대장균에 IPTG를 최종농도 1mM로 첨가해 주고, 30℃에서 20시간 동안 단백질을 발현시킨다. 배양된 대장균은 원심분리를 하여 상등액은 버리고, 회수된 대장균은 50㎍/㎖ 스펙티노마이신 및 2% 글루코스가 첨가된 M9 배지에 흡광도 600nm에서 값이 2.0가 되게 맞추어 주었다. 여기에 최종 농도가 100μM이 되게 기질인 퀘세틴을 첨가해주고 30℃에서 48시간 동안 진탕 배양하였다. 배양액은 100℃의 물에서 3분 동안 끓인 후, 13,000rpm의 속도로 3분간 원심분리하여 상등액만을 취하여, 고성능 액체 크로마토그래피(HPLC) 분석에 이용하였다.Expression of pCDFDuet containing the AtUGT78D2 gene and UDP - GlcNAc4 , 6- DH gene in Escherichia coli BL21 (? GalU), which is characterized in that glucose-1-phosphate wollidyltransferase Vector. Transformed Escherichia coli expressing AtUGT78D2 gene and UDP - GlcNAc4 , 6 - DH gene were cultured on LB medium supplemented with 50 .mu.g / ml of antibiotic of spectinomycin. The overnight cultured Escherichia coli strain was inoculated into fresh LB medium and cultured at 600 nm to an absorbance value of 0.8. IPTG was added to the cultured Escherichia coli at a final concentration of 1 mM, and the protein was expressed at 30 DEG C for 20 hours. The cultured Escherichia coli was centrifuged and the supernatant was discarded. The recovered Escherichia coli was adjusted to 2.0 at an absorbance of 600 nm on M9 medium supplemented with 50 μg / ml of spectinomycin and 2% glucose. The substrate quercetin was added thereto to a final concentration of 100 μM and incubated at 30 ° C for 48 hours with shaking. The culture broth was boiled in water at 100 ° C for 3 minutes, centrifuged at 13,000 rpm for 3 minutes, and the supernatant was taken for high performance liquid chromatography (HPLC) analysis.
그 결과, 퀘세틴으로부터 새로운 화합물의 생성이 확인되었고(도 2), 이 물질의 분자량을 질량분석으로 확인해본 결과 489-Da 크기의 물질임이 확인되었다. 또한 NMR 분석 결과 생성물의 구조는 예측하였던 퀘세틴-3-O-N-아세틸-퀴노보사민으로 밝혀졌다(도 3).
As a result, the generation of a new compound from quercetin was confirmed (FIG. 2), and the molecular weight of this substance was confirmed by mass spectrometry and found to be 489-Da. Further, NMR analysis revealed that the structure of the product was quercetin-3- O - N -acetyl-quinobosamine which was predicted (Fig. 3).
실시예Example 4: 대사조절 대장균 및 발현 벡터 종류별 4: Metabolism control E. coli and expression vector type 퀘세틴Quercetin -3--3- OO -- NN -아세틸--Acetyl- 퀴노보사민의Quinobosamine 생산량 비교 Comparison of production
UDP-N-아세틸-퀴노보사민(acetyl-quinovosamine)의 합성경로는 UDP-N-아세틸글루코사민을 기질로 하여 UDP - GlcNAc4 ,6- DH라는 유전자에 의해 합성된다. 대장균에서 UDP-N-아세틸-퀴노보사민의 함량을 증가시키기 위해 이 물질의 생합성 경로에서 전구체 물질인 글루코스-6-포스페이트(glucose-6-phosphate)를 생합성에 이용해 글루코스-1-포스페이트를 만드는 유전자 포스포글루코뮤타제(pgm, phsophoglucomutase)를 결손시켰다. 또한, 본 발명의 AtUGT78D2 유전자는 UDP-글루코스와 UDP-N-아세틸글루코사민을 당 공여체(sugar donor)로 경쟁한다. 이에 글루코스-1-포스페이트(glucose-1-phosphate)에서 UDP-글루코스로 전환시키는 글루코스-1-포스페이트 우리딜일트랜스퍼라제(galU, glucose-1-phosphate uridylyltransferase) 유전자를 대장균에서 결손시켰다. 상기 유전자들의 결손은 Quick and Easy Conditional Knockout kit(Gene Bridges, 독일)를 사용하였다.UDP- N-acetyl-quinolyl novo synthesis path of samin (acetyl-quinovosamine) is UDP- N-synthesized by a gene called GlcNAc4, 6- DH-UDP to a acetylglucosamine as a substrate. In order to increase the content of UDP-N-acetyl-quinobosamine in Escherichia coli, glucose-6-phosphate, which is a precursor substance in the biosynthesis pathway of this substance, is used for biosynthesis to produce glucose- (Pgm, phsophoglucomutase). In addition, the AtUGT78D2 gene of the present invention competes with UDP-glucose and UDP-N-acetylglucosamine as a sugar donor. Thus, a glucose-1-phosphate glucose-1-phosphate uridyl lransferase gene which converts glucose-1-phosphate into UDP-glucose was deleted in E. coli. The deletion of these genes was performed using Quick and Easy Conditional Knockout kit (Gene Bridges, Germany).
AtUGT78D2 유전자와 UDP - GlcNAc4 ,6- DH 유전자를 포함하는 발현벡터들을 BL21 DE3 야생형, BL21(△pgm) 및 BL21(△galU) 변이체에 형질전환시켜 기질인 퀘세틴으로부터 신규 화합물의 생성량을 비교해 본 결과, 글루코스-1-포스페이트 우리딜일트랜스퍼라제(galU) 유전자를 결손시킨 변이체 대장균에서 퀘세틴-3-O-N-아세틸-퀴노보사민(quercetin-3-O-N-acetyl-quinovosamine)의 함량이 야생형 대장균에 비해 약 1.5배 정도 증가하였다. 그리고 UDP - GlcNAc4 ,6- DH 유전자를 복제수가 다른 pACYCDuet, pCDFDuet 및 pETDuet 벡터에 클로닝하여 추가로 상기 대장균주들에 도입시켜 신규 화합물인 퀘세틴-3-O-N-아세틸-퀴노보사민의 생성량을 비교해 본 결과, AtUGT78D2 유전자와 UDP - GlcNAc4 ,6- DH 두 유전자를 포함하는 pACYCDuet 벡터로 형질전환시킨 BL21(△galU) 변이체 대장균에 UDP-GlcNAc4,6-DH 유전자를 pCDFDuet 벡터에 클로닝하여 추가로 형질전환한 경우에 추가 형질전환 전보다 신규 화합물의 생성량이 약 2배 증가하였고, AtUGT78D2 유전자와 UDP - GlcNAc4 ,6- DH 두 유전자를 포함하는 pCDFDuet 벡터로 형질전환시킨 BL21(△galU) 변이체 대장균에 UDP - GlcNAc4 ,6- DH 유전자를 pETDuet 벡터에 클로닝하여 추가로 형질전환한 경우에도 추가 형질전환 전보다 신규 화합물의 생성량이 약 2배 증가함을 확인할 수 있었다(도 4).Expression vectors containing AtUGT78D2 gene and UDP - GlcNAc4 , 6- DH gene were transformed into BL21 DE3 wild type, BL21 (Δpgm) and BL21 (Δgal U) mutants and the amount of new compounds produced from quercetin , glucose-1-phosphate transferase we dilil (galU) -3- O Quebec paroxetine in mutant E. coli which the gene defect - the content of N-acetyl-quinolyl samin Novo (N -acetyl-quinovosamine quercetin-3- O) Which was about 1.5 times higher than that of wild-type E. coli. The UDP - GlcNAc4 , 6- DH gene was further cloned into the pACYCDuet, pCDFDuet and pETDuet vectors in which the number of replicons was different, and the resultant was further introduced into the Escherichia coli strains to produce the amount of quercetin-3- O - N -acetyl-quinobosamine the present results, AtUGT78D2 gene compared with UDP-to GlcNAc4, 6- DH UDP-GlcNAc4,6- DH gene in the two genes pACYCDuet vector was transformed BL21 (△ galU) mutant E. coli containing additionally cloned in vector pCDFDuet transformants than transgenic added when switching the production of the novel compounds was increased about twice, AtUGT78D2 gene and UDP - GlcNAc4, 6- DH transformed with the vector containing the two genes was pCDFDuet BL21 (△ galU) UDP in mutant E. coli - GlcNAc4, 6- than the DH gene transfection more even when transformed with pETDuet added by cloning the vector and confirmed that the increase about twice amount of the novel compounds (Fig. 4).
<110> Konkuk University Industrial Cooperation Corp <120> Novel compound quercetin-3-O-N-acetyl-quinovosamine and method for producing the same <130> PN13211 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 1295 <212> DNA <213> Arabidopsis thaliana <400> 1 atgaccaaac cctccgaccc aaccagagac tcccacgtgg cagttctcgc ttttcctttc 60 ggcactcatg cagctcctct cctcaccgtc acgcgccgcc tcgcctccgc ctctccttcc 120 accgtcttct ctttcttcaa caccgcacaa tccaactctt cgttattttc ctccggtgac 180 gaagcagatc gtccggcgaa catcagagta tacgatattg ccgacggtgt tccggaggga 240 tacgtgttta gcgggagacc acaggaggcg atcgagctgt ttcttcaagc tgcgccggag 300 aatttccgga gagaaatcgc gaaggcggag acggaggttg gtacggaagt gaaatgtttg 360 atgactgatg cgttcttctg gttcgcggct gatatggcga cggagataaa tgcgtcgtgg 420 attgcgtttt ggaccgccgg agcaaactca ctctctgctc atctctacac agatctcatc 480 agagaaacca tcggtgtcaa agaagtaggt gagcgtatgg aggagacaat aggggttatc 540 tcaggaatgg agaagatcag agtcaaagat acaccagaag gagttgtgtt tgggaattta 600 gactctgttt tctcaaagat gcttcatcaa atgggtcttg ctttgcctcg tgccactgct 660 gttttcatca attcttttga agatttggat cctacattga cgaataacct cagatcgaga 720 tttaaacgat atctgaacat cggtcctctc gggttattat cttctacatt gcaacaacta 780 gtgcaagatc ctcacggttg tttggcttgg atggagaaga gatcttctgg ttctgtggcg 840 tacattagct ttggtacggt catgacaccg cctcctggag agcttgcggc gatagcagaa 900 gggttggaat cgagtaaagt gccgtttgtt tggtcgctta aggagaagag cttggttcag 960 ttaccaaaag ggtttttgga taggacaaga gagcaaggga tagtggttcc atgggcaccg 1020 caagtggaac tgctgaaaca cgaagcaacg ggtgtgtttg tgacgcattg tggatggaac 1080 tcggtgttgg agagtgtatc gggtggtgta ccgatgattt gcaggccatt ttttggggat 1140 cagagattga acggaagagc ggtggaggtt gtgtgggaga ttggaatgac gattatcaat 1200 ggagtcttca cgaaagatgg gtttgagaag tgtttggata aagttttagt tcaagatgat 1260 ggtaagaaga tgaaatgtaa tgctaagaaa cttaa 1295 <210> 2 <211> 1782 <212> DNA <213> Bacillus cereus <400> 2 ttgattttat tagattcatt tattgtatta actgccgtgt atttaagtta ttggtttata 60 catccaaatg tattaaacaa aattcctatg acagtagtta ttagttctat tacattattg 120 tgtagtcatc atgtttttgc agctatttat aagctttata acaaggcgtg ggaatatgca 180 agtattggag agttaaagca aatatttaag gcgattacgc tatcaatttt agtaacagca 240 attgttcaac aaattattaa tcacgatatt tatgttcgaa ttttagcaat cgcatggatg 300 ctacatttat tattaatcgg cggttctcgt tttgtatggc gtatgttccg tgatacatat 360 attacaaaag ctactgataa aaaacgaaca ttaattattg gtgctggttc agcaggaacg 420 atggtagtgc gtcaattaca acataataaa gaagcagatt tatatccaat tgcgtttgtc 480 gatgatgata gaaataagca aaaattggag atttataatg tgccggttat tggtacaaca 540 aatcatattc aagaaattgt agaagataat gatatagaac atatcattat tgcaatccct 600 tcattaaata gagggcaaat aaatgagatt tttgagaagt gtagaaaaac gaaggcgaaa 660 acgcaaattg tgccaatgtt agaagactta ttagatggaa aagtgtctgt aaatgaattt 720 cgtgatgtgc aagtggaaga tttactaggg agagagcctg ttcaattaga tgataaggga 780 ataggtgaga agattcaaga taagacggtt ttaataacgg gtgctggggg ctctattgga 840 tcagagattt gccgtcagat tttaaaatat aaaccagcaa aaatggttct tttaggacat 900 ggagagaata gtatttatca tatcgagatg gaattgagaa ctaagtataa agagcaagca 960 gaatttgtaa cagaaattgc tgacatacaa gatcgaaata aaatatttga aataatgaaa 1020 aaacacaagc cttttgttgt ataccatgca gctgcacata aacacgttcc attgatggaa 1080 agaaatcctg aagaagctgt aaagaataat ataattggta ccaaaaatgt tgcagaagcc 1140 gcagatacat ttggaataaa tacatttgtc atggtttcaa cagataaagc ggtaaaccca 1200 acaaatgtaa tgggagcaac aaaacgtgtt gcagaaatgg ttgttcaaca catggctatg 1260 atcagtcaaa caagatttgt tgctgttagg tttggaaatg tattaggtag tcgcggaagt 1320 gtaattccat tgtttaaaaa acaaattcaa agtgggggac cagttacagt tacccaccca 1380 gatattaccc gttactttat gaccattcca gaggcatcac gacttgtaat ccaagcagga 1440 tctttggcaa gaggtggaga gctatttgta ttggatatgg gcgaacctgt taaaattgca 1500 gatttggcaa agaatttaat tcagctttcg ggttattcta ttgaggaaat tggaattgaa 1560 tatagtggac taagaccggg agagaagatg tacgaagagt tgttaaatga taatgaaatt 1620 cataaggaac aagtatttcc taaaatccat ataggaaagg ctgttttaaa agattttgaa 1680 tccattcaac aatttataga tgagtttgag cagatgagtc aagagagtat taggaaaacc 1740 ttattagatt ttgcgaataa taaagttgaa gtgaatagct aa 1782 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cggcatatgt taaataaaat aattttaatt 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 catctcgagt catcgcaaaa accctccttt 30 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atgaattcga tgaccaaacc ctccgaccc 29 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aagcggccgc ttaagtttct tagcattaca 30 <110> Konkuk University Industrial Cooperation Corp <120> Novel compound quercetin-3-O-N-acetyl-quinovosamine and method for producing the same <130> PN13211 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 1295 <212> DNA <213> Arabidopsis thaliana <400> 1 atgaccaaac cctccgaccc aaccagagac tcccacgtgg cagttctcgc ttttcctttc 60 ggcactcatg cagctcctct cctcaccgtc acgcgccgcc tcgcctccgc ctctccttcc 120 accgtcttct ctttcttcaa caccgcacaa tccaactctt cgttattttc ctccggtgac 180 gaagcagatc gtccggcgaa catcagagta tacgatattg ccgacggtgt tccggaggga 240 tacgtgttta gcgggagacc acaggaggcg atcgagctgt ttcttcaagc tgcgccggag 300 aatttccgga gagaaatcgc gaaggcggag acggaggttg gtacggaagt gaaatgtttg 360 atgactgatg cgttcttctg gttcgcggct gatatggcga cggagataaa tgcgtcgtgg 420 attgcgtttt ggaccgccgg agcaaactca ctctctgctc atctctacac agatctcatc 480 agagaaacca tcggtgtcaa agaagtaggt gagcgtatgg aggagacaat aggggttatc 540 tcaggaatgg agaagatcag agtcaaagat acaccagaag gagttgtgtt tgggaattta 600 gactctgttt tctcaaagat gcttcatcaa atgggtcttg ctttgcctcg tgccactgct 660 gttttcatca attcttttga agatttggat cctacattga cgaataacct cagatcgaga 720 tttaaacgat atctgaacat cggtcctctc gggttattat cttctacatt gcaacaacta 780 gtgcaagatc ctcacggttg tttggcttgg atggagaaga gatcttctgg ttctgtggcg 840 tacattagct ttggtacggt catgacaccg cctcctggag agcttgcggc gatagcagaa 900 gggttggaat cgagtaaagt gccgtttgtt tggtcgctta aggagaagag cttggttcag 960 ttaccaaaag ggtttttgga taggacaaga gagcaaggga tagtggttcc atgggcaccg 1020 caagtggaac tgctgaaaca cgaagcaacg ggtgtgtttg tgacgcattg tggatggaac 1080 tcggtgttgg agagtgtatc gggtggtgta ccgatgattt gcaggccatt ttttggggat 1140 cagagattga acggaagagc ggtggaggtt gtgtgggaga ttggaatgac gattatcaat 1200 ggagtcttca cgaaagatgg gtttgagaag tgtttggata aagttttagt tcaagatgat 1260 ggtaagaaga tgaaatgtaa tgctaagaaa cttaa 1295 <210> 2 <211> 1782 <212> DNA <213> Bacillus cereus <400> 2 ttgattttat tagattcatt tattgtatta actgccgtgt atttaagtta ttggtttata 60 catccaaatg tattaaacaa aattcctatg acagtagtta ttagttctat tacattattg 120 tgtagtcatc atgtttttgc agctatttat aagctttata acaaggcgtg ggaatatgca 180 agtattggag agttaaagca aatatttaag gcgattacgc tatcaatttt agtaacagca 240 attgttcaac aaattattaa tcacgatatt tatgttcgaa ttttagcaat cgcatggatg 300 ctacatttat tattaatcgg cggttctcgt tttgtatggc gtatgttccg tgatacatat 360 attacaaaag ctactgataa aaaacgaaca ttaattattg gtgctggttc agcaggaacg 420 atggtagtgc gtcaattaca acataataaa gaagcagatt tatatccaat tgcgtttgtc 480 gatgatgata gaaataagca aaaattggag atttataatg tgccggttat tggtacaaca 540 aatcatattc aagaaattgt agaagataat gatatagaac atatcattat tgcaatccct 600 tcattaaata gagggcaaat aaatgagatt tttgagaagt gtagaaaaac gaaggcgaaa 660 acgcaaattg tgccaatgtt agaagactta ttagatggaa aagtgtctgt aaatgaattt 720 cgtgatgtgc aagtggaaga tttactaggg agagagcctg ttcaattaga tgataaggga 780 ataggtgaga agattcaaga taagacggtt ttaataacgg gtgctggggg ctctattgga 840 tcagagattt gccgtcagat tttaaaatat aaaccagcaa aaatggttct tttaggacat 900 ggagagaata gtatttatca tatcgagatg gaattgagaa ctaagtataa agagcaagca 960 gaatttgtaa cagaaattgc tgacatacaa gatcgaaata aaatatttga aataatgaaa 1020 aaacacaagc cttttgttgt ataccatgca gctgcacata aacacgttcc attgatggaa 1080 agaaatcctg aagaagctgt aaagaataat ataattggta ccaaaaatgt tgcagaagcc 1140 gcagatacat ttggaataaa tacatttgtc atggtttcaa cagataaagc ggtaaaccca 1200 acaaatgtaa tgggagcaac aaaacgtgtt gcagaaatgg ttgttcaaca catggctatg 1260 atcagtcaaa caagatttgt tgctgttagg tttggaaatg tattaggtag tcgcggaagt 1320 gtaattccat tgtttaaaaa acaaattcaa agtgggggac cagttacagt tacccaccca 1380 gatattaccc gttactttat gaccattcca gaggcatcac gacttgtaat ccaagcagga 1440 tctttggcaa gaggtggaga gctatttgta ttggatatgg gcgaacctgt taaaattgca 1500 gatttggcaa agaatttaat tcagctttcg ggttattcta ttgaggaaat tggaattgaa 1560 tatagtggac taagaccggg agagaagatg tacgaagagt tgttaaatga taatgaaatt 1620 cataaggaac aagtatttcc taaaatccat ataggaaagg ctgttttaaa agattttgaa 1680 tccattcaac aatttataga tgagtttgag cagatgagtc aagagagtat taggaaaacc 1740 ttattagatt ttgcgaataa taaagttgaa gtgaatagct aa 1782 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cggcatatgt taaataaaat aattttaatt 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 catctcgagt catcgcaaaa accctccttt 30 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atgaattcga tgaccaaacc ctccgaccc 29 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aagcggccgc ttaagtttct tagcattaca 30
Claims (12)
[화학식 1]
[Chemical Formula 1]
b) 상기 발현벡터들을 대장균에 형질전환시키는 단계; 및
c) 상기 형질전환 대장균을 배양하면서 퀘세틴을 기질로 첨가하는 단계를 포함하는 하기 화학식 1의 퀘세틴-3-O-N-아세틸-퀴노보사민(quercetin 3-O-N-acetyl-quinovosamine) 화합물의 제조방법.
[화학식 1]
b) transforming the expression vectors into E. coli; And
c) the transformed E. coli to the culture and a step of adding a substrate to the Quebec paroxetine paroxetine Quebec -3- O of Formula 1-N-acetyl-quinolyl Novo samin (quercetin 3- O - N -acetyl- quinovosamine) ≪ / RTI >
[Chemical Formula 1]
[화학식 1]
[Chemical Formula 1]
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| KR20120049684A (en) * | 2010-11-09 | 2012-05-17 | 건국대학교 산학협력단 | A novel compound, quercetin 3-o-n-acetylglucosamine, gene for producing the compound, and method for producing the compound |
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| KR20120049684A (en) * | 2010-11-09 | 2012-05-17 | 건국대학교 산학협력단 | A novel compound, quercetin 3-o-n-acetylglucosamine, gene for producing the compound, and method for producing the compound |
Non-Patent Citations (2)
| Title |
|---|
| B. G. KIM et al, Appl Microbiol Biotechnol, 2012, 93, pp. 2447-2453 * |
| B. G. KIM et al, Appl Microbiol Biotechnol, 2012, 93, pp. 2447-2453* |
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