KR101522962B1 - Novel compound quercetin-3-O-N-acetyl-xylosamine and method for producing the same - Google Patents
Novel compound quercetin-3-O-N-acetyl-xylosamine and method for producing the same Download PDFInfo
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- KR101522962B1 KR101522962B1 KR1020130091301A KR20130091301A KR101522962B1 KR 101522962 B1 KR101522962 B1 KR 101522962B1 KR 1020130091301 A KR1020130091301 A KR 1020130091301A KR 20130091301 A KR20130091301 A KR 20130091301A KR 101522962 B1 KR101522962 B1 KR 101522962B1
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Abstract
본 발명은 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자와 바실러스 세레우스 아종(Bacillus cereus subsp .) 유래 UGlcNAcDH(UDP-N-acetylglucosamine 6-dehydratase) 및 UXNAcS(UDP-N-acetyl-xylosamine synthase) 유전자를 대사조절된 대장균에 발현시켜 퀘세틴에서 신규한 화합물인 퀘세틴-3-O-N-아세틸-자일로사민(quercetin-3-O-N-acetyl-xylosamine)을 합성하는 방법에 관한 것이다.The present invention relates to Arabidopsis thaliana transforming enzyme AtUGT78D2 ( Arabidopsis thaliana UDP-dependent glycosyltransferase) genes and Bacillus cereus subspecies (Bacillus cereus subsp.) derived UGlcNAcDH (UDP- N -acetylglucosamine 6-dehydratase ) and UXNAcS (UDP- N -acetyl-xylosamine synthase ) expressing the gene for the E. coli metabolic regulation O - N -acetyl-xylosamine, which is a novel compound in quercetin, which is a quercetin-3- O - N- acetyl-xylosamine.
Description
본 발명은 신규한 퀘세틴-3-O-N-아세틸-자일로사민 화합물 및 이의 제조방법에 관한 것으로, 더욱 상세하게는 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자, 바실러스 세레우스 아종(Bacillus cereus subsp .)에서 유래한 UGlcNAcDH(UDP-N-acetylglucosamine 6-dehydratase) 및 UXNAcS(UDP-N-acetyl-xylosamine synthase) 유전자를 대사조절된 대장균에 발현시켜 기질인 퀘세틴에서 퀘세틴-3-O-N-아세틸-자일로사민(quercetin-3-O-N-acetyl-xylosamine)의 신규 화합물을 합성하는 방법에 관한 것이다.The present invention relates to novel Quebec paroxetine -3- O - N-acetyl-xylene to samin compounds and relates to a production method thereof, and more particularly, Arabidopsis thaliana-derived sugar transferase AtUGT78D2 (Arabidopsis thaliana UDP-glycosyltransferase dependent) gene, the Bacillus Bacillus cereus subsp . ) A UGlcNAcDH (UDP- N -acetylglucosamine 6-dehydratase ) and UXNAcS (UDP- N -acetyl-xylosamine synthase ) Quebec genes in the Quebec by expressing the E. coli metabolic regulation substrate paroxetine paroxetine-3 derived from a O - N - O - N- acetyl-xylosamine, which is a novel compound of the present invention.
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 반응중간에 들어가는 값비싼 보조인자(cofactor)를 절약할 수 있는 장점을 가지고있다. 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 아종(Bacillus cereus subsp .) 유래 UGlcNAcDH(UDP-N-acetylglucosamine 6-dehydrogenase) 및 UXNAcS(UDP-N-acetyl-xylosamine synthase) 유전자를 대장균에 도입하였다. UGlcNAcDH는 UDP-N-아세틸글루코사민을 이용하여 UDP-N-아세틸-글루코사민우론산(UDP-N-acetyl-glucosaminuronic acid)을 만들고 이를 UXNAcS가 UDP-N-아세틸-자일로사민으로 만든다. 이렇게 만들어진 UDP-N-아세틸-자일로사민은 당전이효소에 의해 기질에 당화된다. 본 발명은 기질로 퀘세틴을 사용하였고, 상기와 같은 반응을 통해 신규 화합물인 퀘세틴-3-O-N-아세틸-자일로사민(quercetin-3-O-N-acetyl-xylosamine)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 sereus subspecies ( Bacillus cereus subsp.) derived UGlcNAcDH (UDP- N -acetylglucosamine 6-dehydrogenase ) and UXNAcS (UDP- N -acetyl-xylosamine synthase ) gene was introduced into E. coli. UGlcNAcDH is UDP- N - acetylglucosamine using UDP- N - Acetyl-Glucosamine creates a uronic acid (UDP- N -acetyl-glucosaminuronic acid) that it UXNAcS UDP- N - made of samin as Giles-acetyl. The UDP- N -acetyl-xylosamine thus produced is glycosylated to the substrate by a glycosyltransferase. The present invention was used as a substrate Quebec paroxetine, through a reaction as described above in Quebec paroxetine -3- O novel compounds were synthesized - samin (N -acetyl-xylosamine quercetin-3- O) in xylene - N - acetyl .
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 세 유전자(AtUGT78D2 , UGlcNAcDH 및 UXNAcS)를 가지고 대장균을 이용한 생물전환법은 퀘세틴-3-O-N-아세틸-자일로사민을 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, the three genes of the present invention ( AtUGT78D2 , UGlcNAcDH And UXNAcS ) could be a novel way to produce quercetin-3- O - N -acetyl- xylosamine .
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-자일로사민 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-xylosamine 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 아종(Bacillus cereus subsp .) 유래 UGlcNAcDH(UDP-N-acetylglucosamine 6-dehydratase) 및 UXNAcS(UDP-N-acetyl-xylosamine synthase) 유전자를 대사조절된 대장균에 발현시키고, 상기 형질전환 대장균을 이용하여 퀘세틴을 기질로 하여 신규한 화합물인 퀘세틴-3-O-N-아세틸-자일로사민을 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 and subspecies of Bacillus cereus (Bacillus cereus subsp . ) Derived UGlcNAcDH (UDP- N -acetylglucosamine 6-dehydratase ) and UXNAcS (UDP- N -acetyl-xylosamine synthase ) and expressing the gene in the metabolism of E. coli, using the transformed E. coli by the Quebec novel paroxetine as substrate O - N -acetyl-xylosamine, which is a compound, quercetin-3- O - N , to complete the present invention.
상기 과제를 해결하기 위해, 본 발명은 신규한 퀘세틴-3-O-N-아세틸-자일로사민(quercetin-3-O-N-acetyl-xylosamine) 화합물을 제공한다.In order to solve the above problems, the present invention relates to novel Quebec paroxetine -3- O - provides compounds - samin (N -acetyl-xylosamine quercetin-3- O) in xylene-N-acetyl.
또한, 본 발명은 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자를 포함하는 발현벡터 및 바실러스 세레우스 아종(Bacillus cereus subsp .) 유래 UGlcNAcDH(UDP-N-acetylglucosamine 6-dehydratase) 및 UXNAcS(UDP-N-acetyl-xylosamine synthase) 유전자를 포함하는 발현벡터를 함유하는 상기 신규 화합물의 제조용 조성물을 제공한다.The present invention is derived from Arabidopsis thaliana transferase per AtUGT78D2 (Arabidopsis thaliana UDP-glycosyltransferase dependent) The expression vector and Bacillus cereus subspecies including the gene (Bacillus cereus subsp . ) Provides a composition for preparing the novel compounds containing an expression vector comprising an origin UGlcNAcDH (UDP- N -acetylglucosamine 6-dehydratase ) and UXNAcS (UDP- N -acetyl-xylosamine synthase ) gene.
또한, 본 발명은In addition,
a) 애기장대 유래 AtUGT78D2 유전자를 포함하는 발현벡터와 바실러스 세레우스 아종 유래 UGlcNAcDH 및 UXNAcS 유전자를 포함하는 발현벡터를 제조하는 단계;a) Expression vector containing Arabidopsis-derived AtUGT78D2 gene and UGlcNAcDH and UXNAcS derived from Bacillus cereus subsp. Preparing an expression vector containing the gene;
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 유전자를 포함하는 발현벡터 및 바실러스 세레우스 아종 유래 UGlcNAcDH 및 UXNAcS 유전자를 포함하는 발현벡터로 형질전환되어 상기 신규 화합물을 생산하는 형질전환 대장균을 제공한다.The present invention also relates to an expression vector comprising the Arabidopsis thaliana glycosyltransferase AtUGT78D2 gene and UGlcNAcDH and UXNAcS derived from Bacillus cereus subsp. Which is transformed with an expression vector containing the gene to produce the novel compound.
또한, 본 발명은 신규한 퀘세틴-3-O-N-아세틸-자일로사민 화합물을 유효성분으로 포함하는 식품 조성물을 제공한다.The present invention also provides a food composition comprising a novel quercetin-3- O - N -acetyl-xylosamine compound as an active ingredient.
본 발명은 애기장대 유래 당전이효소 AtUGT78D2 유전자와 바실러스 세레우스 아종 유래 UGlcNAcDH 및 UXNAcS 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-자일로사민을 만들어 내었다. 본 발명의 화합물인 퀘세틴-3-O-N-아세틸-자일로사민은 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 UGlcNAcDH and UXNAcS genes derived from Bacillus cereus subsp . As can be seen from the present invention, quercetin-3- O - N -acetyl-xylosamine, 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-xylosamine, 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-자일로사민을 합성하는 것을 도식화한 그림이다.
도 2는 애기장대 유래 당전이효소 AtUGT78D2 유전자와 바실러스 세레우스 아종 유래 UGlcNAcDH 및 UXNAcS 유전자를 형질전환시킨 대사조절된 대장균을 이용하여 퀘세틴을 기질로 생물전환을 실시하여 얻은 반응물을 고성능 액체 크로마토그래피(HPLC)로 분석한 결과이다.
도 3은 애기장대 유래 당전이효소 AtUGT78D2 유전자와 바실러스 세레우스 아종 유래 UGlcNAcDH 및 UXNAcS 유전자를 다양한 조합으로 대사조절된 대장균에 형질전환하고, 퀘세틴을 기질로 생물전환을 실시하여 얻은 반응물을 고성능 액체 크로마토그래피로 분석한 결과(A)와 분석 결과를 정량화한 그래프(B)이다.
도 4는 신규 화합물의 최적의 생산 조건을 확립하기 위해 단백질 발현 유도 배양 조건 중 온도 조건을 변화시켜 생성량을 비교한 결과이다.
도 5는 신규 화합물의 최적의 생산 조건을 확립하기 위해 대장균의 배양 세포 농도별 생성량을 비교한 결과이다.Figure 1 is a schematic illustration of the synthesis of quercetin-3- O - N -acetyl-xylosamine, a novel compound from a substrate quercetin.
FIG. 2 is a graph showing the results of biotransformation of quercetin to a substrate using metabolic control E. coli transformed with Arabidopsis thaliana glycoprotein AtUGT78D2 gene, UGlcNAcDH and UXNAcS gene derived from Bacillus cereus subsp. HPLC).
3 is Arabidopsis-derived sugar transferase AtUGT78D2 gene and Bacillus cereus subspecies derived UGlcNAcDH and UXNAcS gene the transformed to the metabolic control of E. coli in a variety of combinations and, Quebec paroxetine a substrate a high-performance liquid chromatography and the reaction is obtained by performing the biotransformation in (A) and the graph (B) which quantifies the analysis results.
FIG. 4 shows the result of comparing the amount of production by changing the temperature condition in the protein expression inducing culture condition in order to establish optimal production conditions of the novel compound.
Fig. 5 shows the results of comparing the amount of production of E. coli per culture cell concentration in order to establish optimal production conditions of a novel compound.
본 발명의 목적을 달성하기 위하여, 본 발명은 하기 화학식 1의 퀘세틴-3-O-N-아세틸-자일로사민(quercetin-3-O-N-acetyl-xylosamine) 화합물을 제공한다.According to an aspect of the present invention, the present invention relates to paroxetine Quebec -3- O of formula (1) provides a compound-samin (N -acetyl-xylosamine quercetin-3- O) in xylene-N-acetyl.
또한, 본 발명은 애기장대 유래 당전이효소 AtUGT78D2(Arabidopsis thaliana UDP-dependent glycosyltransferase) 유전자를 포함하는 발현벡터 및 바실러스 세레우스 아종(Bacillus cereus subsp.) 유래 UGlcNAcDH(UDP-N-acetylglucosamine 6-dehydratase) 및 UXNAcS(UDP-N-acetyl-xylosamine synthase) 유전자를 포함하는 발현벡터를 함유하는 상기 화합물의 제조용 조성물을 제공한다.In addition, the present invention relates to a Arabidopsis thaliana transforming enzyme, AtUGT78D2 ( Arabidopsis thaliana UDP-glycosyltransferase dependent) The expression vector and Bacillus cereus subspecies including the gene (Bacillus N- acetylglucosamine 6-dehydratase (UDP- N- acetylglucosamine 6-dehydratase) and UNA - Cs (UDP- N- acetyl-xylosamine synthase) gene derived from S. cereus subsp .
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 아종 유래 UGlcNAcDH 및 UXNAcS 유전자는 서열번호 2와 서열번호 3의 염기서열로 이루어질 수 있다. 또한, 상기 각 염기 서열의 상동체가 본 발명의 범위 내에 포함되는데 구체적인 내용은 전술한 바와 같다.In addition, in the composition according to an embodiment of the present invention, UGlcNAcDH and UXNAcS derived from Bacillus cereus subsp. The gene may consist of the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3. In addition, the homologues of the respective nucleotide sequences are included within the scope of the present invention, and the details 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 포스포글루코뮤타제(phsophoglucomutase) 또는 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 a phosphoglucurcomutase or glucose-1-phosphate uridylyltransferase) is defective, but the present invention is not limited thereto.
또한, 본 발명은In addition,
애기장대 유래 AtUGT78D2 유전자를 포함하는 발현벡터와 바실러스 세레우스 아종 유래 UGlcNAcDH 및 UXNAcS 유전자를 포함하는 발현벡터를 제조하는 단계;Expression vector containing Arabidopsis-derived AtUGT78D2 gene and UGlcNAcDH and UXNAcS derived from Bacillus cereus subsp. Preparing an expression vector containing the gene;
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-자일로사민의 제조방법을 제공한다.3- O- N-acetyl-xylosamine comprising the step of adding quercetin to a substrate while culturing the transformed E. coli.
본 발명의 일 구현 예에 따른 방법에서, 상기 애기장대 유래 당전이 효소 AtUGT78D2 유전자는 서열번호 1의 염기서열로, 상기 바실러스 세레우스 아종 유래 UGlcNAcDH 및 UXNAcS 유전자는 각각 서열번호 2 및 서열번호 3의 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 transforming enzyme AtUGT78D2 gene is the nucleotide sequence of SEQ ID NO: 1, and the UGlcNAcDH and UXNAcS The gene may consist of the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3, respectively. 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 포스포글루코뮤타제(phsophoglucomutase) 또는 글루코스-1-포스페이트 우리딜일트랜스퍼라제(glucose-1-phosphate uridylyltransferase)가 결손된 대장균일 수 있으나, 이에 제한되지 않는다.In the method according to one embodiment of the present invention, the E. coli transformed with the above expression vectors are preferably selected from the group consisting of phosophoglucumutase or glucose-1-phosphate uridyl lyransferase, But the present invention is not limited thereto.
본 발명의 또 다른 구현 예에 따른 방법에서, 퀘세틴-3-O-N-아세틸-자일로사민 생산을 위한 상기 형질전환 대장균의 단백질 발현 유도 배양 조건은 15~30℃의 온도 및 세포농도 OD600=2~10일 수 있고, 바람직하게는 17~19℃의 온도 및 세포농도 OD600=6~8일 수 있고, 가장 바람직하게는 18℃의 온도 및 세포농도 OD600=8일 수 있으나, 이에 제한되지 않는다.In the method according to another embodiment of the present invention, the protein expression inducing culture conditions of the transformed E. coli for production of quesetin-3- O - N -acetyl-xylosamine are at a temperature of 15 to 30 DEG C and a cell concentration OD 600 = 2 to 10, preferably 17 to 19 ° C and a cell concentration OD 600 = 6 to 8, most preferably a temperature of 18 ° C and a cell concentration OD 600 = 8, But is not limited thereto.
본 발명은 또한, 애기장대 유래 당전이효소 AtUGT78D2 유전자를 포함하는 발현벡터 및 바실러스 세레우스 아종 유래 UGlcNAcDH 및 UXNAcS 유전자를 포함하는 발현벡터로 형질전환되어 퀘세틴-3-O-N-아세틸-자일로사민을 생산하는 대장균을 제공한다.The present invention also relates to an expression vector comprising the Arabidopsis thaliana glycosyltransferase AtUGT78D2 gene and an expression vector comprising UGlcNAcDH and UXNAcS derived from Bacillus cereus subsp. O - N -acetyl-xylosamine, which is transformed with an expression vector containing the gene.
본 발명의 일 구현 예에 있어서, 상기 대장균은 바람직하게는 포스포글루코뮤타제(phsophoglucomutase) 또는 글루코스-1-포스페이트 우리딜일트랜스퍼라제(glucose-1-phosphate uridylyltransferase)가 결손된 대장균일 수 있으나, 이에 제한되지 않는다.In one embodiment of the present invention, the Escherichia coli is preferably Escherichia coli lacking a phosphoglucormutase or glucose-1-phosphate uridyl lransferase, It is not limited.
또한, 본 발명은 신규한 퀘세틴-3-O-N-아세틸-자일로사민 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-xylosamine 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. 애기장대 1. 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 삽입하여 만든 정방향 프라이머(5'-ATGAATTCGATGACCAAACCCTCCGACCC-3'; 서열번호 4, 밑줄; EcoRI 자리) 및 역방향 프라이머(5'-AACTCGAGTTAAGTTTCTTAGCATTACA-3'; 서열번호 5, 밑줄; XhoI 자리)와 pfu Taq 폴리머라제를 이용하여 PCR을 수행하였다. PCR 산물은 제한효소로 처리하여 pGEX 벡터에 클로닝하였다.
AtUGT78D2 isolated from Arabidopsis ( Arabidopsis thaliana UDP-glycosyltransferase dependent) and the gene as the template, created by inserting the restriction position the forward primer (5'-AT GAATTC GATGACCAAACCCTCCGACCC-3 '; SEQ ID NO: 4, underlined; EcoRI digits) and reverse primers (5'-AA CTCGAG TTAAGTTTCTTAGCATTACA-3 '; SEQ ID NO: 5, underline; XhoI site) and pfu Taq polymerase. The PCR products were cloned into pGEX vector by treatment with restriction enzymes.
실시예Example 2. 2. UGlcNAcDHUGlcNAcDH 및 And UXNAcSUXNAcS 유전자의 합성 및 대장균 발현벡터 제작Synthesis of genes and Escherichia coli expression vector production
바실러스 세레우스 아종 cytotoxis NVH391-98(Bacillus cereus subsp. cytotoxis NVH391-98) 유래 UGlcNAcDH 및 UXNAcS 유전자는 이전에 발표된 논문(Gu et al., 2010, J. Biol. Chem. 285:24825-24833)의 염기서열을 바탕으로 바이오니아(한국)에서 합성하였고, 코돈 최적화를 통해 대장균에서 단백질이 더욱 잘 발현되도록 하였다. 또한 UGlcNAcDH 유전자는 합성시 5' 말단에 EcoRI 자리 및 3' 말단에 NotI 자리를, 그리고 UXNAcS 유전자는 합성시 5' 말단에 NdeI 자리 및 3' 말단에 XhoI 자리를 넣어서 합성을 진행하였다.Bacillus cereus subsp. Cytotoxis NVH391-98 ( Bacillus cereus subsp. cytotoxis NVH391-98) derived UGlcNAcDH And UXNAcS The gene was synthesized in biona (Korea) based on the nucleotide sequence of a previously published paper (Gu et al., 2010, J. Biol. Chem. 285: 24825-24833) Lt; / RTI > Also UGlcNAcDH The gene has EcoRI site at the 5 'end and NotI site at the 3' end in synthesis, and UXNAcS The gene was synthesized by incorporating NdeI site at the 5 'end and XhoI site at the 3' end in the synthesis.
합성한 UGlcNAcDH 및 UXNAcS 유전자를 발현시키기 위하여 UGlcNAcDH 유전자는 EcoRI과 NotI 제한효소로, UXNAcS 유전자는 NdeI과 XhoI 제한효소로 각각 처리하고, 대장균 발현벡터인 pACYCDuet(Novagen, 독일) 벡터에 삽입하였다.
The synthesized UGlcNAcDH and UXNAcS In order to express the gene, UGlcNAcDH gene was EcoRI and NotI restriction enzyme, and UXNAcS The genes are NdeI and XhoI Restriction enzymes, and inserted into the vector pACYCDuet (Novagen, Germany), an E. coli expression vector.
실시예Example 3. 3. AtUGT78D2AtUGT78D2 ,, UGlcNAcDHUGlcNAcDH 및 And UXNAcSUXNAcS 유전자 형질전환 대장균 제조 및 생물전환방법을 이용한 새로운 물질 생산Generation of new substances using recombinant E. coli and bioconversion
AtUGT78D2 유전자를 포함하는 발현벡터와 UGlcNAcDH 및 UXNAcS 유전자를 포함하고 있는 발현벡터를 대장균 BL21(DE3)에 형질전환하였다. 상기 대장균 BL21(DE3)를 50㎍/㎖ 엠피실린과 50㎍/㎖ 클로람페니콜을 첨가한 LB 배지에 종균 배양하였다. 밤샘 배양한 종균 배양 대장균을 새로운 LB 배지 3㎖에 접종하고, 흡광도 600nm에서 값이 0.8까지 되도록 배양하였다. 배양된 대장균에 IPTG를 1mM로 첨가해 주고, 18℃에서 18시간 동안 단백질을 발현시켰다. 배양된 대장균은 원심분리를 하여 상등액을 버리고, 회수된 대장균은 50㎍/㎖ 엠피실린, 50㎍/㎖ 클로람페니콜 및 2% 글루코스가 첨가된 M9 배지에 흡광도 600nm에서 값이 2.0이 되게 맞추어 주었다. 여기에 최종 농도가 100μM이 되도록 기질인 퀘세틴을 첨가해주고 30℃에서 6시간 동안 진탕 배양하였다. 상등액은 에틸아세테이트를 이용하여 추출하고, 추출된 반응물은 진공농축 건조기(speed vacuum dryer)를 이용하여 건조시켰다. 건조된 반응물을 DMSO(Dimethyl Sulfoxide)에 녹여 고성능 액체 크로마토그래피 분석에 이용하였다.An expression vector containing the AtUGT78D2 gene and an expression vector containing the UGlcNAcDH and UXNAcS genes were transformed into E. coli BL21 (DE3). Escherichia coli BL21 (DE3) was inoculated on LB medium supplemented with 50 mu g / ml ampicillin and 50 mu g / ml chloramphenicol. E. coli cultured overnight were inoculated in 3 ml of fresh LB medium and cultured at an absorbance of 600 nm to a value of 0.8. IPTG was added to the cultured Escherichia coli at 1 mM, and the protein was expressed at 18 DEG C for 18 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 in M9 medium supplemented with 50 μg / ml of ampicillin, 50 μg / ml of chloramphenicol and 2% of glucose. The substrate quercetin was added to the suspension to a final concentration of 100 μM and incubated at 30 ° C. for 6 hours with shaking. The supernatant was extracted with ethyl acetate, and the extracted reactants were dried using a speed vacuum dryer. The dried reaction product was dissolved in DMSO (dimethyl sulfoxide) and used for high performance liquid chromatography analysis.
그 결과, 퀘세틴으로부터 새로운 화합물 퀘세틴-3-O-N-아세틸-자일로사민의 생성이 확인되었고(도 2), 이 물질의 분자량을 확인해본 결과 475-Da 크기의 물질임이 확인되었다.
As a result, the formation of a new compound quercetin-3- O - N -acetyl-xylosamine from quercetin was confirmed (Fig. 2) and the molecular weight of this substance was confirmed to be 475-Da.
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-자일로사민의 합성경로는 UDP-N-아세틸글루코사민을 기질로 하여 UGlcNAcDH라는 유전자에 의해 UDP-N-아세틸-글루코사민우론산(UDP-N-acetyl-glucosaminuronic acid)이 합성되고, 이 UDP-N-아세틸-글루코사민우론산을 기질로 하여 UXNAcS 유전자에 의해 UDP-N-아세틸-자일로사민이 합성된다. 대장균에서 UDP-N-아세틸-자일로사민의 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 발명에 사용한 당전이 효소 AtUGT78D2 유전자는 UDP-글루코스와 UDP-N-아세틸-자일로사민을 당 공여체(sugar donor)로 경쟁한다. 이에 본 발명에서는 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.The route of synthesis of samin in xylene is UDP- N - - UDP- N-acetyl-glucosamine as a substrate by the acetylation by genetic UGlcNAcDH called UDP- N-acetyl-glucosamine-uronic acid (UDP- N -acetyl-glucosaminuronic acid) is synthesized , the UDP- N -acetyl - is synthesized in a samin xylene-acetyl-UDP- N by UXNAcS gene glucosamine uronic acid as a substrate. In order to increase the content of UDP- N -acetyl-xylosamine in E. coli, glucose-6-phosphate, which is a precursor substance in the biosynthesis pathway of this substance, is used for biosynthesis to produce glucose-1-phosphate (Pgm, phsophoglucomutase). In addition, the sugar- transferase AtUGT78D2 gene used in the present invention competes with UDP-glucose and UDP- N -acetyl- xylosamine as a sugar donor. In the present invention, a gene for glucose-1-phosphate glucose-1-phosphate uridyl lransferase (Glu-1-phosphate uridyl lransferase) 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).
UGlcNAcDH 및 UXNAcS 유전자를 카피 수가 다른 발현벡터별로 BL21(DE3) 야생형, BL21(△pgm) 및 BL21(△galU) 변이체에 형질전환시키고 AtUGT78D2 유전자를 포함하는 벡터 역시 형질전환시킨 후에, 기질인 퀘세틴으로부터 신규 화합물의 생성량을 고성능 액체 크로마토그래피로 분석하여 비교해 본 결과(도 3A), UGlcNAcDH 및 UXNAcS 유전자를 포함하는 발현벡터 중 pCDFDuet 벡터가 pACYCDuet 벡터보다 약 1.5배, 그리고 야생형 대장균과 변이체 대장균을 비교해 본 경우에는 포스포글루코뮤타제 유전자가 결손된 대장균 변이체(△pgm)가 야생형 대장균에 비해 약 2.7배 신규 화합물의 생성량이 증가된 것을 확인하였다(도 3B).
The UGlcNAcDH and UXNAcS genes were transformed into BL21 (DE3) wild type, BL21 (Δpgm) and BL21 (Δgal U) mutants by different expression vectors and AtUGT78D2 (Fig. 3A), and the pCDFDuet vector among the expression vectors containing the UGlcNAcDH and UXNAcS genes was analyzed by high performance liquid chromatography using the pACYCDuet And the wild type Escherichia coli and the mutant Escherichia coli were compared with each other by about 1.5 times, and it was confirmed that the Escherichia coli mutant (Δpgm) lacking the phosphoglucuronimutase gene increased the amount of the new compound about 2.7 times as compared with the wild type Escherichia coli (Fig. 3B).
실시예Example 5: 형질전환 대장균의 단백질 발현 유도 조건 최적화를 통한 신규 화합물의 생산량 비교 5: Comparison of the yield of new compounds by optimizing protein expression induction conditions of transformed E. coli
AtUGT78D2 유전자를 포함하는 발현벡터(pGEX)와 UGlcNAcDH 및 UXNAcS 유전자를 포함하고 있는 발현벡터(pCDFDuet)를 포스포글루코뮤타제 유전자가 결손된 대장균 변이체(△pgm)에 형질전환하고, 단백질 발현 유도 배양 조건 중 온도조건을 다르게 하여 신규 화합물의 생성량을 비교해보았다.Transformed with an expression vector (pGEX) and the expression vector (pCDFDuet) containing UGlcNAcDH and UXNAcS genes including AtUGT78D2 gene in phosphoglucoisomerase mu hydratase E. coli mutant (△ pgm) a gene defect, and protein expression induced culture conditions And the amount of the new compound was compared by varying the medium temperature condition.
2㎖ LB 배지에 50㎍/㎖의 엠피실린과 50㎍/㎖ 클로람페니콜을 첨가하여 상기 형질전환 대장균을 접종한 후 밤새도록 종균 배양하였다. 종균 배양된 대장균을 새로운 3㎖ LB 배지에 100㎕ 접종한 다음 37℃ 교반 배양기(shaking incubator)에서 배양하였다. 대장균을 흡광도 600nm에서 값이 0.6이 될 때까지 배양한 후, IPTG를 최종농도 1mM이 되도록 첨가해 주고, 18℃, 25℃ 및 30℃에서 20시간 단백질을 발현시켰다. 배양된 대장균은 원심분리를 하여 상등액은 버리고, 회수된 대장균은 50㎍/㎖의 엠피실린과 50㎍/㎖ 클로람페니콜 항생제와 2% 글루코스가 첨가된 M9 배지에 흡광도 600nm에서 값이 2.0이 되도록 맞추어 주었다. 여기에 농도가 100μM이 되도록 기질을 첨가해주고, 30℃에서 2시간 동안 진탕 배양하였다. 300㎕의 반응물을 800㎕의 에틸아세테이트를 첨가하여 잘 섞은 다음 700㎕ 추출하고, 추출된 반응물은 진공농축 건조기를 이용하여 완전히 건조시켰다. 건조된 반응물은 다시 70μM의 DMSO에 녹여 고성능 액체 크로마토그래피 분석에 이용하였다. 그 결과, 18℃의 온도조건에서 20시간 단백질 발현 유도한 배양 조건에서, 19.93㎎/ℓ의 가장 많은 양의 신규 화합물이 생성되는 것을 확인하였다(도 4).
50 mu g / ml of ampicillin and 50 mu g / ml of chloramphenicol were added to 2 ml of LB medium, and the transformed E. coli was inoculated and cultured overnight. Escherichia coli Cultured Escherichia coli was inoculated in a fresh 3 ml LB medium at 100 배 and cultured in a 37 째 C shaking incubator. Escherichia coli was cultured at an absorbance of 600 nm to a value of 0.6, IPTG was added to a final concentration of 1 mM, and proteins were expressed at 18 ° C, 25 ° C and 30 ° 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 in 50 ml of ampicillin and 50 ml / ml of M9 medium supplemented with chloramphenicol antibiotic and 2% glucose . Substrates were added to the solution to give a concentration of 100 μM, and cultured at 30 ° C for 2 hours with shaking. 300 [mu] l of the reaction mixture was mixed with 800 [mu] l of ethyl acetate, and 700 [mu] l of the reaction mixture was extracted, and the extracted reaction product was completely dried using a vacuum condenser dryer. The dried reactants were again dissolved in 70 μM DMSO and used for high performance liquid chromatography analysis. As a result, it was confirmed that the highest amount of new compound of 19.93 mg / L was produced under culture conditions induced by protein expression for 20 hours at a temperature of 18 캜 (FIG. 4).
실시예Example 6: 형질전환 대장균의 세포 농도 조건 최적화를 통한 신규 화합물의 생산량 비교 6: Comparison of the yield of new compounds by optimizing cell concentration conditions of transformed E. coli
AtUGT78D2 유전자를 포함하는 발현벡터(pGEX)와 UGlcNAcDH 및 UXNAcS 유전자를 포함하고 있는 발현벡터(pCDFDuet)를 포스포글루코뮤타제 유전자가 결손된 대장균 변이체(△pgm)에 형질전환하고, 단백질 발현 유도 배양 조건은 18℃의 온도에서 20시간으로 고정하고, 세포농도를 다르게 하여 신규 화합물의 생성량을 비교해보았다.Transformed with an expression vector (pGEX) and the expression vector (pCDFDuet) containing UGlcNAcDH and UXNAcS genes including AtUGT78D2 gene in phosphoglucoisomerase mu hydratase E. coli mutant (△ pgm) a gene defect, and protein expression induced culture conditions Was fixed at a temperature of 18 캜 for 20 hours, and the amount of the new compound was compared by varying the cell concentration.
방법은 종균배양 및 단백질 발현 유도 과정까지는 실시예 5와 같으며, 다만 세포농도를 흡광도 600nm에서 값이 1, 2, 3, 4, 5, 6, 7, 8, 9 및 10으로 맞추어 100μM의 기질을 넣어주고, 30℃에서 배양하다가, 1.5, 3.0, 4.5 및 6.0 시간째에 기질을 추가로 넣어준 후, 8시간 후에 최종 추출하였다. 추출한 반응물을 고성능 액체 크로마토그래피로 분석한 결과, 세포농도 OD600=8.0에서 160㎎/ℓ의 가장 많은 양의 신규 화합물이 생성되는 것을 확인하였다(도 5).The method is the same as in Example 5 until the seed culture and the induction of protein expression are carried out except that the cell concentration is adjusted to 100, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 at an absorbance of 600 nm , And incubated at 30 ° C. Substrates were further added at 1.5, 3.0, 4.5, and 6.0 hours, followed by final extraction at 8 hours. The extracted reaction product was analyzed by high performance liquid chromatography. As a result, it was confirmed that the largest amount of new compound with a cell concentration OD 600 = 8.0 to 160 mg / l was produced (FIG. 5).
<110> Konkuk University Industrial Cooperation Corp <120> Novel compound quercetin-3-O-N-acetyl-xylosamine and method for producing the same <130> PN13212 <160> 5 <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> 1287 <212> DNA <213> Bacillus cereus subsp. <400> 2 gaattcgatg gaaaaagaga aaggtgaaga aaaaatgaac gattctcgca aaatcactgt 60 tatcggattg ggctatgttg gcctgccgct ggcgatccat tttgcggaac ggggctataa 120 cgtcgtcggc ttagataagg acaaacgcaa aattgaacgc attgaaaaag gagattctta 180 cataccagac gtttcctcga acgttttaaa agatttggta caaaacaaac agttggtagt 240 ttatacacct cataccggga tagaagagtt tcagaatagt gaatatgtga tcgtgacggt 300 cccgacgcca atcaataaac agaaagaacc tgatttatcg gctctcattg cagcgtcaca 360 ctatataaaa cagaatcttc aggcaggaca gaccttcatt tttgaaagtt ccacatatcc 420 gggtacactt gaagaggtca tcattccgat tattgcccaa tcaggtaagg aagttggaaa 480 agattattat attggctaca gccctgagcg tattgatccg gccaatcaac agtacacagt 540 tcagaccatt ccaaaagtga tttcgggtca aacagagaga tgtaagcagc aagtccagaa 600 attgtatagc accatttttg acaccgtcgt tccagtgagt tccccaaaag tagcagagat 660 gtgtaaactg ttcgaaaata tacagcgtct ggttaacata tccctggtaa atgagttaaa 720 catcctttgc gaaaagctgg ggattgactt tcgggaggct cttgaagccg cggccaccaa 780 accattcggt tttacgccat actggccggg cccaggtata gggggacatt gtattccggt 840 tgatccctta tactttcagt ggaaagcccg tcagctaggg caatcatcac agctgatcga 900 agtggctcac atgattaatg agaagatgcc gcaacaaatc gtaacgcagg tgaaggagct 960 gagtgctccc ccagggactg tcttcctgat cggcattgcg tacaaaaaag acgtgaatga 1020 tttacgagag tctcctgcac tcccgattat cgagctgctc gtgaacgagg gttataaagt 1080 gcaatatcac gatccctaca ttagctctgc gaaaatcggt gacaaaatat acgattctat 1140 cccccttaaa aagaaaaccc tggaaaaggc agattgcatt ctaattgtaa cggaccatag 1200 caatatagac tggaatatat gcaagggaat gaagcatgta atcgatactc gcggtgtatt 1260 gaagaaggtt agcgcataag cggccgc 1287 <210> 3 <211> 975 <212> DNA <213> Bacillus cereus subsp. <400> 3 catatgaaga aacgttgttt gataaccgga ggtgcaggct ttatcggttc acatctggcg 60 gaagaactcg tcaaacgggg tcatccggtt acgatcgttg ataattttta caaaggcaaa 120 agcaaatatc acgaggagtt aacaggtaat atcccgataa ttccaatcag tatactggat 180 aaaaactcaa tgcatgaact ggtaaatcag cacgatgttg tgtttcatct tgccgctatt 240 ttaggtgtga agactacaat ggagaagagc attgaactga tagaaactaa tttcgatggc 300 acgagaaaca ttctgcaagc agccttaaag gggaaaaaaa aagtgatttt cgcctccact 360 tctgaggtgt acggaaaagg gacgccgccc ttctcggaag atgatgatcg gctgtacggg 420 gctacttcga agattcgttg gagctatgcc atttgcaaga ccttggaaga gacactgtgt 480 ttaggatatg ctctacaggg tctgcccgta acaattgtcc gatattttaa tatctatggc 540 ccgagggcaa aagacggtcc ttacgctggc gtcatcccgc gcttcatacg tgccgcactg 600 cagggtgatg atcttcttgt gtatggcgat ggaaagcaga cccgctgttt tacgtatgta 660 agtgatgcgg ttgaggcgac cattgccgcg atggacgaaa aagtcaacgg agagattatt 720 aacatagggt ctgaggacga aaaaagcatc caggaggtag cgcaagacat tcaccagttg 780 acccatagtt cttccaagat tgttcatgtg ccatttgaaa aggtttatcc acatgggttt 840 gaggaaatcc cgaaccgcaa acctgacgtt accaagctga aagaaatgtg ccaattccac 900 cctaatgtgt catgggaaca aggcctcaaa gaaacaatcc agtggtttcg tgaaatcgag 960 aatgactaac tcgag 975 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atgaattcga tgaccaaacc ctccgaccc 29 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 aactcgagtt aagtttctta gcattaca 28 <110> Konkuk University Industrial Cooperation Corp <120> Novel compound quercetin-3-O-N-acetyl-xylosamine and method for producing the same <130> PN13212 <160> 5 <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> 1287 <212> DNA <213> Bacillus cereus subsp. <400> 2 gaattcgatg gaaaaagaga aaggtgaaga aaaaatgaac gattctcgca aaatcactgt 60 tatcggattg ggctatgttg gcctgccgct ggcgatccat tttgcggaac ggggctataa 120 cgtcgtcggc ttagataagg acaaacgcaa aattgaacgc attgaaaaag gagattctta 180 cataccagac gtttcctcga acgttttaaa agatttggta caaaacaaac agttggtagt 240 ttatacacct cataccggga tagaagagtt tcagaatagt gaatatgtga tcgtgacggt 300 cccgacgcca atcaataaac agaaagaacc tgatttatcg gctctcattg cagcgtcaca 360 ctatataaaa cagaatcttc aggcaggaca gaccttcatt tttgaaagtt ccacatatcc 420 gggtacactt gaagaggtca tcattccgat tattgcccaa tcaggtaagg aagttggaaa 480 agattattat attggctaca gccctgagcg tattgatccg gccaatcaac agtacacagt 540 tcagaccatt ccaaaagtga tttcgggtca aacagagaga tgtaagcagc aagtccagaa 600 attgtatagc accatttttg acaccgtcgt tccagtgagt tccccaaaag tagcagagat 660 gtgtaaactg ttcgaaaata tacagcgtct ggttaacata tccctggtaa atgagttaaa 720 catcctttgc gaaaagctgg ggattgactt tcgggaggct cttgaagccg cggccaccaa 780 accattcggt tttacgccat actggccggg cccaggtata gggggacatt gtattccggt 840 tgatccctta tactttcagt ggaaagcccg tcagctaggg caatcatcac agctgatcga 900 agtggctcac atgattaatg agaagatgcc gcaacaaatc gtaacgcagg tgaaggagct 960 gagtgctccc ccagggactg tcttcctgat cggcattgcg tacaaaaaag acgtgaatga 1020 tttacgagag tctcctgcac tcccgattat cgagctgctc gtgaacgagg gttataaagt 1080 gcaatatcac gatccctaca ttagctctgc gaaaatcggt gacaaaatat acgattctat 1140 cccccttaaa aagaaaaccc tggaaaaggc agattgcatt ctaattgtaa cggaccatag 1200 caatatagac tggaatatat gcaagggaat gaagcatgta atcgatactc gcggtgtatt 1260 gaagaaggtt agcgcataag cggccgc 1287 <210> 3 <211> 975 <212> DNA <213> Bacillus cereus subsp. <400> 3 catatgaaga aacgttgttt gataaccgga ggtgcaggct ttatcggttc acatctggcg 60 gaagaactcg tcaaacgggg tcatccggtt acgatcgttg ataattttta caaaggcaaa 120 agcaaatatc acgaggagtt aacaggtaat atcccgataa ttccaatcag tatactggat 180 aaaaactcaa tgcatgaact ggtaaatcag cacgatgttg tgtttcatct tgccgctatt 240 ttaggtgtga agactacaat ggagaagagc attgaactga tagaaactaa tttcgatggc 300 acgagaaaca ttctgcaagc agccttaaag gggaaaaaaa aagtgatttt cgcctccact 360 tctgaggtgt acggaaaagg gacgccgccc ttctcggaag atgatgatcg gctgtacggg 420 gctacttcga agattcgttg gagctatgcc atttgcaaga ccttggaaga gacactgtgt 480 ttaggatatg ctctacaggg tctgcccgta acaattgtcc gatattttaa tatctatggc 540 ccgagggcaa aagacggtcc ttacgctggc gtcatcccgc gcttcatacg tgccgcactg 600 cagggtgatg atcttcttgt gtatggcgat ggaaagcaga cccgctgttt tacgtatgta 660 agtgatgcgg ttgaggcgac cattgccgcg atggacgaaa aagtcaacgg agagattatt 720 aacatagggt ctgaggacga aaaaagcatc caggaggtag cgcaagacat tcaccagttg 780 acccatagtt cttccaagat tgttcatgtg ccatttgaaa aggtttatcc acatgggttt 840 gaggaaatcc cgaaccgcaa acctgacgtt accaagctga aagaaatgtg ccaattccac 900 cctaatgtgt catgggaaca aggcctcaaa gaaacaatcc agtggtttcg tgaaatcgag 960 aatgactaac tcgag 975 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atgaattcga tgaccaaacc ctccgaccc 29 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 aactcgagtt aagtttctta gcattaca 28
Claims (13)
[화학식 1]
[Chemical Formula 1]
b) 상기 발현벡터들을 대장균에 형질전환시키는 단계; 및
c) 상기 형질전환 대장균을 배양하면서 퀘세틴을 기질로 첨가하는 단계;를 포함하는 제1항의 화합물의 제조방법.a) an expression vector containing the Arabidopsis derived AtUGT78D2 gene having the nucleotide sequence of SEQ ID NO: 1 and an expression vector comprising the UGlcNAcDH and UXNAcS gene derived from Bacillus subtilis subspecies having the nucleotide sequences of SEQ ID NOS: 2 and 3, respectively ;
b) transforming the expression vectors into E. coli; And
c) adding quercetin as a substrate while culturing the transformed E. coli.
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