KR100786885B1 - Fusarium oxysporum FB1501 producing enniatin H I and MK1688 - Google Patents

Fusarium oxysporum FB1501 producing enniatin H I and MK1688 Download PDF

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KR100786885B1
KR100786885B1 KR1020060048756A KR20060048756A KR100786885B1 KR 100786885 B1 KR100786885 B1 KR 100786885B1 KR 1020060048756 A KR1020060048756 A KR 1020060048756A KR 20060048756 A KR20060048756 A KR 20060048756A KR 100786885 B1 KR100786885 B1 KR 100786885B1
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송혁환
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Abstract

본 발명은 엔니아틴 H, I 및 MK1688을 생산하는 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501[KFCC-11363P] 및 상기 균주를 배양하여 엔니아틴 H, I 및 MK1688을 생산하는 방법, 또는 엔니아틴 H, I 및 MK1688과 부버리신을 동시에 생산하는 방법에 관한 것이다. 본 발명의 균주는 엔니아틴 H, I 및 MK1688를 대량생산 할 수 있는 기회를 제공하고, 또한 상기 엔니아틴은 물론 부버리신을 동시에 생산하므로써 저 생산비용으로 상기 생리활성물질의 분리가 가능하게 된다.The invention yen Fusa producing California tin H, I and MK1688 Solarium oxy's Forum (Fusarium oxysporum ) FB1501 [KFCC-11363P] and a method for culturing the strain to produce enanthine H, I and MK1688, or a method for simultaneously producing enanthine H, I and MK1688 and burberrysin. The strain of the present invention provides an opportunity to mass-produce enanthine H, I and MK1688, and also enables the isolation of the bioactive substance at low production cost by simultaneously producing the enanthine as well as butycin. do.

엔니아틴, 푸사리움 옥시스포럼(Fusarium oxysporum), 부버리신 Enniatin, Fusarium oxysporum, Burberry

Description

엔니아틴 Ｈ,Ｉ, 및 ＭＫ１６８８을 생산하는 푸사리움 옥시스포럼 ＦＢ１５０１{Fusarium oxysporum FB1501 producing enniatin H, I and MK1688}Fusarium oxysporum FB1501 producing enniatin H, I and MK1688}

도 1은 푸사리움 옥시스포럼 FB1501의 400 배 확대한 현미경 사진이다. a) 및 b)는 마이크로콘니디아(microconidia)이고, c) 및 d)는 매크로콘니디아(macroconidia)이고, e)는 돈니디오포아(donidiophore), f)는 클라미도스포아(chlamydospore)이다.1 is a 400 times magnification photomicrograph of the Fusarium Oxy Forum FB1501. a) and b) are microconidia, c) and d) are macroroconidia, e) donidiophore and f) chlamydospore.

도 2는 균주에서 DNA를 추출한 후 증폭시킨 후 아가로스 겔 전기영동을 실시한 후의 겔 사진이다. 상기 A 부분은 푸사리움 속 특이적인 프라이머(P28SL 및 P58SL)로 증폭시킨 것이고, B 부분은 푸사리움 옥시스포럼 특이적인 프라이머(FoF 및 FoR)로 증폭시킨 것이며, M 레인은 100 bp 단위의 DNA 분자량 마커이고, S-1 레인은 푸사리움 모닐리포르메 NRRL 13569이고, S-2 레인은 푸사리움 옥시스포럼 KCCT 16909이고, 레인 1501은 푸사리움 옥시스포럼 FB-1501이다.2 is a gel photograph after agarose gel electrophoresis after extracting and amplifying DNA from the strain. The A part is amplified with specific primers in the Fusarium (P28SL and P58SL), the B part is amplified with the Fusarium oxysporum specific primers (FoF and FoR), the M lane is a DNA molecular weight of 100 bp unit Marker, S-1 lane is Fusarium monoliforme NRRL 13569, S-2 lane is Fusarium oxysforum KCCT 16909 and lane 1501 is Fusarium oxysforum FB-1501.

도 3은 푸사리움 옥시스포럼 FB-1501 세포 클로로포름 추출물의 HPLC 크로마토그램이다.Figure 3 is an HPLC chromatogram of Fusarium OxyForum FB-1501 Cell Chloroform Extract.

도 4는 화합물 1의 전자분사 이온화 질량분석 스펙트럼이다.4 is an electrospray ionization mass spectrometry of compound 1. FIG.

도 5는 화합물 1의 IR 스펙트럼이다.5 is an IR spectrum of Compound 1. FIG.

도 6은 화합물 1의 HMBC와 COSY의 상관관계를 나타낸 그림이다.6 is a graph showing the correlation between HMBC and COSY of compound 1.

도 7은 화합물 1의 부분적 구조를 나타낸 그래프이다. 상기 (I)은 N-MeVal이고, (II)은 Hmp이고, (III)은 Hiv이다.7 is a graph showing the partial structure of Compound 1. (I) is N-MeVal, (II) is Hmp and (III) is Hiv.

도 8은 화합물 1(엔니아틴 H)의 화학 구조이다.8 is the chemical structure of Compound 1 (Enyatin H).

도 9는 화합물 2의 전자분사 이온화 질량분석 스펙트럼이다.9 is an electrospray ionization mass spectrometry of compound 2. FIG.

도 10은 화합물 2의 IR 스펙트럼이다.10 is an IR spectrum of Compound 2. FIG.

도 11은 화합물 2의 HMBC와 COSY의 상관관계를 나타낸 그림이다.11 is a graph showing the correlation between HMBC and COSY of compound 2.

도 12는 화합물 2의 부분적 구조를 나타낸 그래프이다.12 is a graph showing the partial structure of compound 2.

도 13은 화합물 2(부버리신)의 화학 구조이다.13 is a chemical structure of Compound 2 (buburysin).

도 14는 화합물 3의 전자분사 이온화 질량분석 스펙트럼이다.14 is an electrospray ionization mass spectrometry of compound 3. FIG.

도 15는 화합물 3의 IR 스펙트럼이다.15 is an IR spectrum of Compound 3. FIG.

도 16은 화합물 3(엔니아틴 I)의 화학 구조이다.16 is the chemical structure of Compound 3 (Enyatin I).

도 17은 화합물 4의 전자분사 이온화 질량분석 스펙트럼이다.17 is an electrospray ionization mass spectrometry of compound 4. FIG.

도 18는 화합물 4의 IR 스펙트럼이다.18 is an IR spectrum of Compound 4. FIG.

도 19는 화합물 4의 HMBC와 COSY의 상관관계를 나타낸 그림이다.19 is a graph showing the correlation between HMBC and COSY of compound 4.

도 20은 화합물 4의 부분적 구조를 나타낸 그래프이다.20 is a graph showing the partial structure of compound 4.

도 21은 화합물 4(엔니아틴 MK1688)의 화학 구조이다.21 is a chemical structure of Compound 4 (Enyatin MK1688).

본 발명은 엔니아틴 H, I 및 MK1688을 생산하는 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501[KFCC-11363P] 및 상기 균주를 배양하여 엔니아틴 H, I 및 MK1688을 생산하는 방법, 또는 엔니아틴 H, I 및 MK1688과 부버리신을 동시에 생산하는 방법에 관한 것이다. 본 발명의 균주는 엔니아틴 H, I 및 MK1688를 대량생산 할 수 있는 기회를 제공하고, 또한 상기 엔니아틴은 물론 부버리신을 동시에 생산하므로써 저 생산비용으로 상기 생리활성물질의 분리가 가능하게 된다.The invention yen Fusa producing California tin H, I and MK1688 Solarium oxy's Forum (Fusarium oxysporum ) FB1501 [KFCC-11363P] and a method for culturing the strain to produce enanthine H, I and MK1688, or a method for simultaneously producing enanthine H, I and MK1688 and burberrysin. The strain of the present invention provides an opportunity to mass-produce enanthine H, I and MK1688, and also enables the isolation of the bioactive substance at low production cost by simultaneously producing the enanthine as well as butycin. do.

고리형 뎁시펩티드인 엔니아틴은 고이만 등에 의해 푸사리움 종에 의해 생산되는 식물 독소(phytotoxin)로 처음 보고되었다[G?umann, E., St. Naef-Roth, and H. Kern, Zur phytotoxischen wirksamkeit der enniatine. Phytopathologische Zeitschrift, 40: 45-51. 1960]. 엔니아틴의 종류는 1960 년대 엔니아틴 A, A1, B, B1 등이 보고된 후, 1990 년대 토모다 등에 의해 엔니아틴 D, E, F 등이 보고되었고[Tomoda, H., H. Nishida, X.X. Huang, R. Masuma, Y.K. Kim and S. Omura. New cyclodpsipeptides, enniatins D, E, and F produced by Fusarium sp. FO-1305. The Journal of Antibiotics, 45: 1207-1215. 1992], 그와 같이 비스콘티 등(1992)에 의해 엔니아틴 B2, B3, B4가 보고되었다[Visconti, A., L.A. Blais, J.W. ApSimon, E. Greenhalgh and J.D. Miller. Production of enniatins by Fusarium acuminatum and Fusarium compactum in liquid culture: isolation and characterization of three new enniatins B2, B3 and B4. Journal of Agricultural and Food Chemistry, 40, 1076-1082. 1992]. 그 이후 닐란논타 등에 의해 곤충병원성 미생물에 의해 엔니아틴 H, I 및 MK1688이 생산된다는 보고가 있었다[Nilanonta, C., M. Isaka, P. Kittakoop, S. Trakulnaleamsai, M. Tanticharoen, and Y. Thebtaranonth. Unusual enniatins produeced by the insect pathogenic fungus Verticillium hemipterigenum : isolation and studies on precursor- directed biosynthesis. Tetrahedron, 59: 1015-1020. 2003]. 이러한 엔니아틴의 기본구조는 질소에 메틸기가 붙어 있는 루신, 이소루신 또는 발린과 아이소하이드록시발레릭산(D-2- hydroxyisovaleric acid)이 펩타이드 결합과 에스터 결합으로 연결된 고리형 뎁시펩티드 구조인데, 상기 닐란논타 등(2003)에 의해 발견된 엔니아틴 H, I 및 MK1688의 경우 질소에 메틸화된 발린과 아이소하이드록시발레릭산 대신에 하이드록시 3-메틸펜타노익산(2-hydroxy-3-methylpentanoic acid)이 하나씩 치환되어 있는 형태로 밝혀졌다.Enniatin, a cyclic depsipeptide, was first reported as a phytotoxin produced by Fusarium species by Goiman et al. [G? Umann, E., St. Naef-Roth, and H. Kern, Zur phytotoxischen wirksamkeit der enniatine. Phytopathologische Zeitschrift , 40: 45-51. 1960]. Enniatine was reported as Enniatin A, A1, B, B1 in the 1960s, and then Entomine D, E, F, etc. by Tomoda et al. In the 1990s [Tomoda, H., H. Nishida, XX Huang, R. Masuma, YK Kim and S. Omura. New cyclodpsipeptides, enniatins D, E, and F produced by Fusarium sp. FO-1305. The Journal of Antibiotics, 45: 1207-1215. 1992] and Enniatin B2, B3, B4 have been reported by Visconti et al. (1992) [Visconti, A., LA Blais, JW ApSimon, E. Greenhalgh and JD Miller. Production of enniatins by Fusarium acuminatum and Fusarium compactum in liquid culture: isolation and characterization of three new enniatins B2, B3 and B4. Journal of Agricultural and Food Chemistry , 40, 1076-1082. 1992]. Since then, it has been reported that enanthine H, I and MK1688 are produced by entomogenic microorganisms by Nilanonta et al. [Nilanonta, C., M. Isaka, P. Kittakoop, S. Trakulnaleamsai, M. Tanticharoen, and Y. Thebtaranonth. Unusual enniatins produeced by the insect pathogenic fungus V erticillium hemipterigenum : isolation and studies on precursor- directed biosynthesis. Tetrahedron, 59: 1015-1020. 2003]. The basic structure of this enanthine is a cyclic depsipeptide structure in which leucine, isoleucine or valine and isohydroxyvaleric acid (D-2-hydroxyisovaleric acid) having a methyl group attached to nitrogen are connected by a peptide bond and an ester bond. Enniatin H, I, and MK1688, discovered by Nilanonta et al. (2003), substituted hydroxy 3-methylpentanoic acid instead of valine and isohydroxy valeric acid methylated in nitrogen. ) Is substituted one by one.

일반적으로 먼저 알려진 엔니아틴 A, A1, B, B1 등은 부버리신과 함께 다양한 활성들이 보고 되어 있다. 고이만 등(1960)과 버마이스터와 플라트너는 엔니아틴의 식물 독성 대하여 보고하였으며[Burmeister, H.R., and R.D. Plattner. Enniatin production by Fusarium tricinctum and its effect on germinating wheat seeds. Phytopathology , 77: 1483-1487. 1987], 그로브와 포플은 엔니아틴의 살충제 활성에 대한 보고를 하였고[Grove, J.F. and M. Pople. The insecticidal activity of beauvericin and the enniatin complex. Mycopathologia, 70: 103-105. 1980], 상기 토모다 등(1992)은 콜레스테롤 합성 효소의 저해제로 엔니아틴이 작용한다는 것을 보고하였다. 그 외에도 트레닌 등[Trenin, A.S., I.V. Tolstykh, L.P. Terekhova, V.A. Zenkova, M.O. Makarova, E.G. Gladkikh, O.P. Bychkova, G.S. Katrujfa, and I.V. Dudnik. The hypolipidemic action of antibiotic 86/88(enniatin B) in a hepatoblastoma G2 cell cultrue. Antibiot Khimioter. 45: 6-9. 2000], 울리그 등[Uhlig, S., A.C. Gutleb, U. Thrane, and A. Flaoyen. Identification of cytotoxic principles from Fusarium avenaceum using bioassay-guided fraction. Toxicon , 46: 150-159. 2005], 그리고 카미야 등(2004)에 의해 엔니아틴이 사람 또는 동물 유래 세포 또는 암세포에 세포독성이 있다는 것이 보고 되었다. 마키 등은 엔니아틴 화합물들이 항-HIV 활성을 가지고 있다는 것을 보고하였다[Makee, T.C., H.R. Bokesch, J.L. Mccormick, M.A. Rashid, D. Spielvogel, K.R. Gustafson, M.M. Alavanja, J.H. Cardellina, and M.R. Boyd. Isolation and characterization of new anti-HIV and cytotoxic leads from plants, marine, and microbial organisms. Journal of Natural Products, 60: 431-438. 1997].In general, the first known eniatin A, A1, B, B1, etc. have been reported with a variety of activities with the burberry. Goiman et al. (1960) and Burmeister and Platner reported on the phytotoxicity of enanthine [Burmeister, HR, and RD Plattner. Enniatin production by Fusarium tricinctum and its effect on germinating wheat seeds. Phytopathology , 77: 1483-1487. 1987], Grove and Popple reported the pesticidal activity of enanthine [Grove, JF and M. Pople. The insecticidal activity of beauvericin and the enniatin complex. Mycopathologia , 70: 103-105. 1980], Tomoda et al. (1992) reported that enanthine acts as an inhibitor of cholesterol synthase. In addition, Trenin et al. [Trenin, AS, IV Tolstykh, LP Terekhova, VA Zenkova, MO Makarova, EG Gladkikh, OP Bychkova, GS Katrujfa, and IV Dudnik. The hypolipidemic action of antibiotic 86/88 (enniatin B) in a hepatoblastoma G2 cell cultrue. Antibiot Khimioter . 45: 6-9. 2000, Ulrig et al. [Uhlig, S., AC Gutleb, U. Thrane, and A. Flaoyen. Identification of cytotoxic principles from Fusarium avenaceum using bioassay-guided fraction. Toxicon , 46: 150-159. [2005] and Kamiya et al. (2004) reported that niacin is cytotoxic to human or animal derived cells or cancer cells. Maki et al. Reported that enniatin compounds have anti-HIV activity [Makee, TC, HR Bokesch, JL Mccormick, MA Rashid, D. Spielvogel, KR Gustafson, MM Alavanja, JH Cardellina, and MR Boyd. Isolation and characterization of new anti-HIV and cytotoxic leads from plants, marine, and microbial organisms. Journal of Natural Products , 60: 431-438. 1997].

엔니아틴 중에서 엔니아틴 H, I 및 MK1688은 상기 닐란논타 등(2003)에 의해 항말라리아 활성을 가지고 있고, 곤충병원 곰팡이인 버티실륨 헤미테리제넘 (Verticillium hemipterigenum) 에 의해 기존의 엔니아틴들과 함께 생산된다는 것이 보고되었다. Among the enanthines, enanthine H, I and MK1688 have antimalarial activity by Nilanonta et al. (2003), and existing enanthines by Verticillium hemipterigenum , an insect pathogenic fungus. It has been reported to be produced together with.

엔니아틴과 함께 부버리신은 하밀 등(1969)과 굽타 등(1995)에 의해 부버리아(Beauveria) 종 및 푸사리움 종 등이 생산하는 것으로 알려졌다[Hamill, R.L., C.E. Higgens, H.E. Boaz, and M. Gorman. The structure of beauvericin, a new depsipeptide antibiotic toxic to Artemia salina . Tetrahedron Letters, 49: 4255-4258. 1969; Gupta, S., C. Montllor, and Y.-S. Hwang. Isolation of novel beauvericin analogues from the fungus Beauveria bassiana. Journal of Natural Products, 58: 733-738. 1995]. 부버리신의 구조는 아이소하이드록시발레릭산과 페닐알라닌이 펩타이드 결합과 에스터 결합으로 연결되어 있는 것으로 보고되었다. 부버리신 A, B, C, 알루부버리신 A, B, C 등이 유도체가 보고 되고 있으며 현재 합성효소와 합성 메카니즘이 밝혀지지 않았다. 상기 하밀 등(1969)은 부버리신의 살충제 활성에 대하여 보고하였으며, 상기 그로브와 포플은(1980) 제초제 활성에 대해 보고 하였다. 상기 토모다 등(1992)은 콜레스테롤 합성 효소의 저해제로 부버리신이 작용한다는 것을 보고하였다. 그 외에도 부버리신은 다수의 박테리아와 균류에 대한 항박테리아 및 항진균성물질로 작용한다는 것을 상기 하밀 등(1969)과 오프킨니코프 등(1971)에 의해 보고되었다[Ovchinnikov, Y.A., V.T. Ivanov, and I.I. Mikhaleva. The synthesis and some properties of beauvericin. Tetrahedron Letters, 3: 159-162. 1971]. 엔니아틴과의 구조유사성으로 인해 포유류에서 콜레스테롤 아실 트랜스퍼라아제(cholesterol acyl transferase)의 저해제로 작용한다고 알려져 있다. 또한 오이셔스 등[Ojcious, D.M., A. Zychlinsky, L.M. Zheng, and J.D.E. Young. Ionophoore-induced apoptosis: role of DNA fragmentation and calcium fluxes. Experimental Cell Research, 197: 43-49. 1991], 로그리코 등[Logrieco, A., A. Moretti, A. Ritieni, M.F. Caiffa, and L. Macchia, Beauvericin: chemistry, biology and significance. In: Advances in microbial toxin research and its biotechnological exploitation. Kluwer Academic/Plenum Publishers, New York, 23-30. 2002a]과 마치아 등[Macchia, L., R. Di Paola, F. fornelli, S. Nenna, A. Moretti, R. Napoletano, A. Logrieco, M.F. Caiaffa, and A. Bottalico. Cytotoxicity of beauvericin to mammalian cells. In Abstracts of the International Seminar on Fusarium: Mycotoxins, Taxonomy and Pathogenicity, 9 to 13 May Martina Franca, Italy. Stampasud Mottola, Italy. 72-73. 1995]에 의해 부버리신의 암세포 세포 독성이 보고 되었다. Together with enniatin, burberry is known to be produced by Beauveria species and Fusarium species by Hamil et al. (1969) and Gupta et al. (1995) [Hamill, RL, CE Higgens, HE Boaz, and M]. Gorman. The structure of beauvericin, a new depsipeptide antibiotic toxic to Artemia salina . Tetrahedron Letters , 49: 4255-4258. 1969; Gupta, S., C. Montllor, and Y.-S. Hwang. Isolation of novel beauvericin analogues from the fungus Beauveria bassiana . Journal of Natural Products , 58: 733-738. 1995]. It has been reported that the structure of burberrysin is isohydroxy valeric acid and phenylalanine are linked by peptide bonds and ester bonds. Derivatives have been reported for Burburysin A, B, C, and Alububurysin A, B, C and the like, and no synthetase and synthetic mechanisms have been identified. Hamil et al. (1969) reported on the insecticide activity of bublycine, and the groves and poples (1980) reported on herbicide activity. Tomoda et al. (1992) reported that burberrysin acts as an inhibitor of cholesterol synthase. In addition, Burburyin has been reported by Hamil et al. (1969) and Offkinnikov et al. (1971), above, to act as antibacterial and antifungal agents for many bacteria and fungi [Ovchinnikov, YA, VT Ivanov, and II]. Mikhaleva. The synthesis and some properties of beauvericin. Tetrahedron Letters, 3: 159-162. 1971]. Due to its structural similarity to Enniatin, it is known to act as an inhibitor of cholesterol acyl transferase in mammals. See also Ouscious et al., Ojcious, DM, A. Zychlinsky, LM Zheng, and JDE Young. Ionophoore-induced apoptosis: role of DNA fragmentation and calcium fluxes. Experimental Cell Research, 197: 43-49. 1991], Logrieco et al. [Logrieco, A., A. Moretti, A. Ritieni, MF Caiffa, and L. Macchia, Beauvericin: chemistry, biology and significance. In : Advances in microbial toxin research and its biotechnological exploitation. Kluwer Academic / Plenum Publishers, New York , 23-30. 2002a and Machia et al. [Macchia, L., R. Di Paola, F. fornelli, S. Nenna, A. Moretti, R. Napoletano, A. Logrieco, MF Caiaffa, and A. Bottalico. Cytotoxicity of beauvericin to mammalian cells. In Abstracts of the International Seminar on Fusarium : Mycotoxins, Taxonomy and Pathogenicity, 9 to 13 May Martina Franca, Italy. Stampasud Mottola, Italy . 72-73. 1995] reported cancer cell cytotoxicity of burberrysin.

지금까지 엔니아틴 H, I 및 MK1688을 생산하는 균주로는 곤충병원 곰팡이인 버티실륨 헤미테리제넘 (Verticillium hemipterigenum)이 알려져 있을 뿐 푸사리움 옥시스포럼에서 엔니아틴 H, I 및 MK1688을 생산한다는 보고는 없었으며, 특히 상기 엔니아틴 H, I 및 MK1688과 부버리신을 동시에 생산하는 균주에 대해서도 알져진 바가 없었다. 따라서 다양한 생리활성의 연구를 수행하기에 충분한 양의 엔니아틴 H, I 및 MK1688의 생산방법 또는 생산균주의 필요성이 증대되고 있었다.To date, strains that produce enanthine H, I and MK1688 are Verticillium , a fungal vermicillium hemiterigenum hemipterigenum ) is known, but there have been no reports of the production of Enniatin H, I and MK1688 at the Fusarium Oxygen Forum, particularly for strains producing both Enniatin H, I and MK1688 and Burberrysin at the same time. There was no bar. Therefore, there is an increasing need for production methods or strains of enanthine H, I and MK1688 in sufficient amounts to carry out various biological activities.

본 발명은 엔니아틴 H, I 및 MK1688을 생산하는 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501을 제공한다.The present invention provides the Fusarium oxysporum FB1501, which produces eniatin H, I and MK1688.

또한 본 발명은 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501을 배양하여 엔니아틴 H, I 및 MK1688을 생산하는 방법, 또는 엔니아틴 H, I 및 MK1688과 부버리신을 동시에 생산하는 방법을 제공한다.The invention also Fusarium oxy's Forum (Fusarium oxysporum ) A method of culturing FB1501 to produce eniatin H, I and MK1688, or a method of simultaneously producing niatine H, I and MK1688 and burberrysin.

본 발명은 엔니아틴 H, I 및 MK1688을 생산하는 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501[KFCC-11363P]를 특징으로 한다.The present invention features Fusarium oxysporum FB1501 [KFCC-11363P] producing niacin H, I and MK1688.

또한 본 발명은 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501[KFCC-11363P]를 배양하여 엔니아틴 H, I 및 MK1688을 생산하는 방법, 또는 엔니아틴 H, I 및 MK1688과 부버리신을 동시에 생산하는 방법을 특징으로 한다.The invention also Fusarium oxy's Forum (Fusarium oxysporum ) FB1501 [KFCC-11363P] is cultivated to produce enanthine H, I and MK1688, or to simultaneously produce enanthine H, I and MK1688 and burberrysin.

이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다.Referring to the present invention in more detail as follows.

본 발명자들은 토양 시료로부터 고리형 뎁시펩티드 생산 균주를 분리하였으며, 본원발명의 균주는 푸사리움 옥시스포럼으로 동정되었다. 상기 분리한 균주는 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501으로 명명하고, 한국미생물보존센터에 2006년 1 월 6일 자로 기탁하였다[수탁번호 : KFCC-11363P]. 상기 균주는 엔니아틴 H, I 및 MK1688을 생산하고, 또한 동시에 부버리신을 생산하는 것으로 밝혀졌다.We isolated cyclic depsipeptide producing strains from soil samples, and the strains of the present invention were identified as Fusarium oxis forum. The isolated strain was a Fusarium oxime forum ( Fusarium) oxysporum ) FB1501, which was deposited with the Korea Microorganism Conservation Center on January 6, 2006 [Accession Number: KFCC-11363P]. The strain has been found to produce eniatin H, I and MK1688, and at the same time produce burberrysin.

이하, 실시예에 의거하여 본 발명을 더욱 상세하게 설명하나, 하기 실시예는 본 발명을 예시하기 위한 것이며 본 발명을 한정하는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are intended to illustrate the present invention and do not limit the present invention.

실시예Example 1: 균주의 분리 및 동정 1: Isolation and Identification of Strains

(1) 분리 및 형태학적 동정(1) Separation and Morphological Identification

고리형 뎁시펩티드 생산 균주는 제주의 흙에서 분리하였고, 분리된 균주는 400배의 현미경으로 소형분생자, 대형분생자, 후막포자와 분생자병을 확인하였다. 소형분생자는 다량으로 보이며, 모양 및 크기는 여러 가지고 난형, 타원형 또는 원통형을 하고 있었다. 또한, 직선 혹은 약간 곡선 모양을 이루고 있었다. 대형분생자는 드문드문 생기고, 그 안데 격벽을 형성하고 있는 것을 볼 수 있었다. 이에 반해, 후막포자는 단일 혹은 쌍을 이루고 있었으며, 분생자병의 경우는 단경자가 가지치기 형태로 되어 있었다(도 1). 이러한 형태학적 특징으로 이 균주는 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501로 명명하고, 한국미생물보존센터에 2006년 1월 6일자로 기탁을 완료하고 수탁번호 KFCC 11363P를 부여받았다. Cyclic depsipeptide-producing strains were isolated from the soil of Jeju, and the isolated strains were identified as small conidia, large conidia, thick film spores and conidia disease under a microscope of 400 times. Small conidia appeared in large quantities and had various shapes and sizes with ovoid, oval or cylindrical shape. In addition, it was straight or slightly curved. Large conidia were sparse, forming an inner bulkhead. On the contrary, the thick film spores were single or paired, and in the case of conidia, the short stinger was pruned (FIG. 1). These morphological features make this strain the Fusarium Fusarium. oxysporum ) FB1501, the Korea Microorganisms Conservation Center completed the deposit on January 6, 2006 and was given accession number KFCC 11363P.

(2) 분자생물학적 특징에 의한 균주 확인(2) Identification of strains by molecular biological characteristics

가. DNA 추출end. DNA extraction

포테이토 덱스트로즈 아가 배지에 상기 균주 1501을 접종하여 10 일 동안 키운 후 균사가 덮어 있는 배지를 함께 떼어내어 멸균 튜브에 넣고 액체질소에 첨가하여 세포를 깬다. 액체 질소가 증발한 후 한번 더 넣어 세포를 깬다. 액체 질소가 모두 증발된 후에 65℃ 라이시스 용액 [50mM Tris pH 8.0, 50mM ethylenediamine tetraacetic acid(EDTA), 3% sodium dodecylsulfate (SDS), 1% 2-mercaptoethanol 과 0.1 m/ml proteinase K]을 첨가 한 후 65 ℃에서 한 시간 반응시킨다. 반응 후 0.5 ml 페놀 용액을 넣고 조심스럽게 흔든 후 8,000 rpm의 속 도로 5 분 동안 원심분리하여 페놀층과 수용액층을 분리한다. 수용액층을 조심스럽게 다른 튜브로 옮긴 후 수용액층에 남아 있는 페놀층을 제거하기 위해 클로로포름과 아소아밀알콜이 24 : 1의 비율로 섞여 있는 용액 0.4 ml을 넣고 흔들 후 원심분리하여 수용액층을 새 튜브로 옮긴다. 여기에 0.05 ml의 7.5M 암모늄 아세테이트 용액을 넣어 조심스럽게 섞는다. 이 후 -20 ℃의 95 % 에탄올을 0.88 ml을 넣은 후 13,000 rpm의 속도로 20 분 동안 원심분리하여 DNA를 침전시킨다. 상등액을 버리고 70 % 에탄올을 넣어 DNA를 씻어주고 다시 원심분리하여 DNA를 침전시켜 70 % 에탄올을 버리고 TE 용액(10 mM Tris, 1 mM EDTA, pH 8.0)을 넣어 보관한다. After inoculating the strain 1501 on the potato dextrose agar medium and growing for 10 days, the medium covered with the mycelium is removed, placed in a sterile tube, and added to the liquid nitrogen to break the cells. After the liquid nitrogen evaporates, add it once more to break the cells. After all the liquid nitrogen had been evaporated, a 65 ° C Lysis solution [50 mM Tris pH 8.0, 50 mM ethylenediamine tetraacetic acid (EDTA), 3% sodium dodecylsulfate (SDS), 1% 2-mercaptoethanol and 0.1 m / ml proteinase K] was added. After the reaction at 65 ℃ for one hour. After the reaction, add 0.5 ml phenol solution, shake carefully, and centrifuge for 5 minutes at a speed of 8,000 rpm to separate the phenol layer and the aqueous layer. After carefully transferring the aqueous layer to another tube, in order to remove the remaining phenol layer in the aqueous layer, add 0.4 ml of a solution containing chloroform and asoamyl alcohol in a ratio of 24: 1, shake, and centrifuge to shake the aqueous layer. Move to. Add 0.05 ml of 7.5M ammonium acetate solution and mix carefully. Thereafter, 0.88 ml of 95% ethanol at -20 ° C. was added, followed by centrifugation at 13,000 rpm for 20 minutes to precipitate DNA. Discard the supernatant, wash the DNA with 70% ethanol, and centrifuge again to precipitate the DNA. Discard 70% ethanol and store TE solution (10 mM Tris, 1 mM EDTA, pH 8.0).

나. DNA I. DNA 상동성을Homology 이용한 동정 Sympathy

푸사리움 종을 확인하기 위하여 휴 등이 제안한 푸사리움 확인 프라이머를 제작하여 사용하였다[Hue, F.X., M. Huerre, M.A. Rouffault, and C.D. Bievre. Specified detection of Fusarium species in blood and tissues by a PCR technique. Journal of Clinical Microbiology, 37: 2434-2438. 1999]. In order to identify the Fusarium species, the Fusarium identification primer proposed by Hugh et al. Was used and used [Hue, FX, M. Huerre, MA Rouffault, and CD Bievre. Specified detection of Fusarium species in blood and tissues by a PCR technique. Journal of Clinical Microbiology, 37: 2434-2438. 1999].

상기 가.에서 추출한 1 ng의 DNA를 상기 서열번호 1의 P28SL(5'-ACA AAT TAC AAC TCG GGC CCG AGA-3')와 서열번호 2의 P58SL(5'-AGT ATT CTG GCG GGC ATG CCT GT-3')프라이머 및 프로메가에서 구입한 PCR 프리-믹스쳐(pre-mixture)를 사용하여 증폭하였다. PCR 조건은 최초 변성(denaturation)은 94 ℃에서 10 분 반응 시켰다. 그 후 94 ℃, 1 분 동안 변성(denaturation), 60 ℃에서 1 분 동안 어닐 링(annealing), 72 ℃에서 1 분 동안 신장(extention) 반응을 40 번 반복하여 DNA 단편을 증폭하였다. 증폭 후 마지막 신장(extention)을 72 ℃에서 10 분 동안 반응시킨 후 서열분석기(Applied Biosystems model 310 automatic DNA sequencer)로 서열을 분석하였고 이를 서열번호 3에 나타내었다.1 ng of DNA extracted in the above A. P28SL (5'-ACA AAT TAC AAC TCG GGC CCG AGA-3 ') of SEQ ID NO: 1 and P58SL (5'-AGT ATT CTG GCG GGC ATG CCT GT) of SEQ ID NO: 2 -3 ') amplified using PCR pre-mixture purchased from Primer and Promega. PCR conditions were initially denatured for 10 minutes at 94 ℃. Thereafter, DNA fragments were amplified by repeating denaturation at 94 ° C. for 1 minute, annealing at 60 ° C. for 1 minute, and extension reaction at 72 ° C. for 1 minute. After amplification, the last extension was reacted at 72 ° C. for 10 minutes, and the sequence was analyzed by an Applied Biosystems model 310 automatic DNA sequencer, which is shown in SEQ ID NO: 3.

DNA 상동성 확인 결과 본 발명의 1501 균주는 푸사리움 옥시스포럼과 100 % 일치하였고, 푸사리움 모닐리포미스와 98 % 상동성을 나타내었다. 따라서 본 발명의 균주가 푸사리움 옥시스포럼에 해당하는 것인지를 재확인하기 위해서 옥시스포럼 특이적인 프라이머를 이용하여 다음의 실험을 진행하였다.As a result of DNA homology, the 1501 strain of the present invention was 100% identical to the Fusarium oxis forum and showed 98% homology with the Fusarium moniliformis. Therefore, in order to reconfirm whether the strain of the present invention corresponds to the Fusarium oxis forum, the following experiment was carried out using an oxis forum specific primer.

다. DNA 전기영동을 이용한 동정All. Identification using DNA electrophoresis

상기 나.의 서열번호 1 및 2의 프라이머를 이용해 증폭시킨 DNA를 이용하여 아가로스 전기영동을 통해 본 발명의 1501 균주가 푸사리움 속임을 확인하였고(도 2의 A), 또한 푸사리움 옥시스포럼 종을 확인하기 위하여 미쉬라 등이 제안한 서열번호 4의 FoF (5'-ACA TAC CAC TTG TTG CCT CG-3')와 서열번호 5의 FoR (5'-CGC CAA TCA ATT TGA GGA ACG-3')의 푸사리움 옥시스포럼 확인 프라머를 제작하여 사용하였다.It was confirmed that 1501 strain of the present invention was a Fusarium decease through agarose electrophoresis using DNA amplified by using the primers of SEQ ID NOs. 1 and 2 of B. (A in FIG. 2), and also the Fusarium Oxy Forum FoF of SEQ ID NO: 4 (5'-ACA TAC CAC TTG TTG CCT CG-3 ') and FoR of SEQ ID NO: 5 (5'-CGC CAA TCA ATT TGA GGA ACG-3') Fusarium Oxysis Forum confirmation primer was used.

PCR 조건은 상기 나.와 동일하게 하여 DNA를 증폭시켰고, 2 % 아가로스 겔을 제조하여 PCR 반응 산물을 10 ㎕와 염색약(loading dye)을 (5:1)의 비율로 섞어서 아가로스 겔에 로딩하였다. 겔에 트리스-아세테이트-이디티에이(Tris-acetate-EDTA) 용액과 함께 전기를 걸어주었다. 전기영동이 끝난 후 에티듐 브로마이드 (ethidium bromide) 용액에 넣어 염색하였다. 염색된 겔을 자외선에 쬐어 주면 증폭된 밴드를 확인할 수 있고, 그 결과를 도 2의 B에 나타내었다. 상기 전기영동에서 양성 대조군으로 푸사리움 옥시스포럼(Fusarium oxysporum) KCCT 16909를 사용하였고 음성 대조군으로 푸사리움 모닐리포르메(Fusarium moniliforme) NRRL 13569 균주를 사용하였다. PCR conditions were the same as above B. DNA was amplified. A 2% agarose gel was prepared, and 10 μl of the PCR reaction product was mixed with a loading dye (5: 1) and loaded on the agarose gel. It was. The gel was energized with Tris-acetate-EDTA solution. After electrophoresis, the dye was added to an ethidium bromide solution. Amplified band can be confirmed by exposing the stained gel to ultraviolet rays, and the result is shown in B of FIG. 2. In the electrophoresis, Fusarium oxime forum ( Fusarium) as a positive control oxysporum ) KCCT 16909 was used as the negative control Fusarium moniliforme ( Fusarium) moniliforme ) NRRL 13569 strain was used.

상기 전기영동 결과 1501 균주는 350 bp 크기의 밴드를 가진 푸사리움이었고, 그중에서도 푸사리움 옥시스포럼 임을 확인할 수 있었다.As a result of the electrophoresis, the 1501 strain was a Fusarium with a 350 bp band, and among them, it was confirmed that the Fusarium Oxis Forum.

실시예Example 2: 균주의 배양 및 고리형  2: Cultivation and Cyclic Strains 뎁시펩티드의Depsipeptide 분리 detach

1 ×105 spores/ml의 상기 균주 1501의 포자를 FDM 배지 (25 g of sucrose, 4.25 g of NaNO3, 5 g of NaCl, 2.5 g of MgSO47H2O, 1.36 g of KH2PO4, 0.01 g of FeSO47H2O, and 0.0029 g of ZnSO47H2O per liter) 250 mL에 접종하여 25 ℃, 암소에서 10 일간 120 rpm의 조건으로 배양하였다.Spores of the strain 1501 at 1 × 10 5 spores / ml were collected in FDM medium (25 g of sucrose, 4.25 g of NaNO 3, 5 g of NaCl, 2.5 g of MgSO 47 H 2 O, 1.36 g of KH 2 PO 4, 0.01 g of FeSO 47 H 2 O, and 0.0029 g of ZnSO47H2O per liter) was inoculated into 250 mL and incubated at 25 ° C. in the dark at 120 rpm for 10 days.

균사체를 포함하고 있는 배양액에 2 배에 해당하는 클로로포름을 넣고 격렬하게 흔들어준 후 분획 깔대기를 이용하여 클로로포름층을 취하고 한번 더 클로로포름 추출과정을 거친다. 분리된 클로로포름을 건조하여 메탄올로 다시 녹인 후 0.45 ㎛ 필터로 필터한 후 HPLC를 이용하여 고리형 뎁시펩티드를 정제하였다. Crom-sil pack ODS preparative column (1.0 ×25 cm)을 이용하여 아세토니트릴:물을 60:40 부피비로 혼합한 용액을 분당 4 mL의 유속으로 하여 4 개의 화합물을 분 리하였다. 상기 4 개의 화합물은 다시 Shiseido pack C18 column을 이용하여 아세토니트릴:물을 70:30 부피비로 혼합한 용액을 분당 1 mL의 유속으로 정제하였다(도 3).Add 2 times the chloroform to the culture solution containing the mycelium, shake it vigorously, take a chloroform layer using a fraction funnel, and go through the chloroform extraction process once more. The separated chloroform was dried, re-dissolved in methanol, filtered with a 0.45 μm filter, and then purified using cyclic depsipeptide using HPLC. Four compounds were separated using a Crom-sil pack ODS preparative column (1.0 × 25 cm) with acetonitrile: water in a 60:40 volume ratio at a flow rate of 4 mL per minute. The four compounds were again purified using a Shiseido pack C18 column with a solution of acetonitrile: water in a 70:30 volume ratio at a flow rate of 1 mL per minute (FIG. 3).

실시예Example 3: 고리형  3: annular 뎁시펩티드의Depsipeptide 확인 Confirm

실시예 2의 도 3의 각 화합물들의 구조분석을 위해 전자분사 이온화 질량분석기(elecrospray ionization mass spectrometry, ESI-MS)를 이용하여 분자량을 측정하였고, 적외선 분석(IR)을 실시하였다. 도 3의 화합물 1, 3 및 4의 1D-NMR (1H NMR, 13C NMR, 과 DEPT-135) 측정은 Bruker Avance 500 spectrometer system을 이용하였고, 2D-NMR (COSY, HMQC, and HMBC) 은 Bruker DMX 600 spectrometer system을 사용하여 측정하였다. 또한 도 3의 화합물 2의 모든 NMR 분석은 Bruker Avance 400 spectrometer system을 이용하여 분석하였다. For the structural analysis of each compound of FIG. 3 of Example 2, molecular weight was measured using an electrospray ionization mass spectrometer (ESI-MS), and infrared analysis (IR) was performed. 1D-NMR ( 1 H NMR, 13 C NMR, and DEPT-135) of Compounds 1, 3 and 4 of FIG. 3 were measured using a Bruker Avance 500 spectrometer system, and 2D-NMR (COSY, HMQC, and HMBC) was Measurements were made using a Bruker DMX 600 spectrometer system. In addition, all NMR analysis of the compound 2 of Figure 3 was analyzed using a Bruker Avance 400 spectrometer system.

(1) 화합물 1(1) Compound 1

화합물 1은 ESI-MS 측정에 의해 분자량이 654.5로 측정되었고(도 4), IR 스펙트럼에서에서 υ 1650.95 cm-1 과 υ 1733.89 cm-1 에 아미드 결합과 에스터 결합이 존재하는 것으로 확인되었다(도 5). 13C 및 1H NMR 스펙트럼(in CDCl3)의 화학 이동(chemical shifts)에서 13C 및 1H 의 연결은 13C-1H HMQC 스펙트럼에 의해 분 석되었고, 이를 표 1에 나타내었다. 13C-1H HMBC 측정으로 13C-1H의 긴 범위의 커플링(long range couplings) 을 확인하고 1H-1H COSY 측정으로 분석된 구조내의 수소와 수소의 관계를 도 6에 나타내었다. 모든 NMR 분석을 통해 피크 1은 3개의 부분구조를 가지고 있는 것으로 확인되었다(도 7). 이 3개의 부분구조는 N-methyl valine (N-MeVal)과 D-hydroxyisovaleic acid (Hiv) 그리고 3-methylpentanoic acid (Hmp)로 확인되었다. 그리고 ESI-MS 분석으로 확인된 분자량으로 화합물 1은 3 개의 N-MeVal과 2 개의 Hiv 그리고 1 개의 Hmp로 구성되어 있음을 알 수 있었다. 그리고 IR 분석에서 확인된 아미드 결합과 에스터 결합으로 화합물 1이 (N-MeVal)-(Hiv)-(N-Meval)-(Hiv)-(N-MeVal)-(Hmp)로 구성된 고리형 뎁시펩티드 임을 확인하였으며, 이물질은 닐란논타 등(2003)에 의해 구조가 확인된 엔니아틴 H로 확인되었다(도 8). Compound 1 was determined to have a molecular weight of 654.5 by ESI-MS measurement (FIG. 4), and it was found that amide and ester bonds exist at υ 1650.95 cm −1 and υ 1733.89 cm −1 in the IR spectrum (FIG. 5). ). The linkage of 13 C and 1 H in the chemical shifts of the 13 C and 1 H NMR spectra (in CDCl 3 ) was analyzed by the 13 C- 1 H HMQC spectrum, which is shown in Table 1. 13 shows the relationship between the hydrogen and the hydrogen in the check C- 1 H HMBC measured by coupling 13 (long range couplings) of the long range of the C- and 1 H analysis of 1 H- 1 H COSY measurement structure in Figure 6 . All NMR analyzes confirmed that peak 1 had three substructures (FIG. 7). These three substructures were identified as N-methyl valine (N-MeVal), D-hydroxyisovaleic acid (Hiv) and 3-methylpentanoic acid (Hmp). And the molecular weight confirmed by ESI-MS analysis it can be seen that Compound 1 is composed of three N-MeVal, two Hiv and one Hmp. And the amide and ester bonds identified in the IR analysis were used to determine that Compound 1 was composed of (N-MeVal)-(Hiv)-(N-Meval)-(Hiv)-(N-MeVal)-(Hmp). It was confirmed that, the foreign material was confirmed as enanthine H structure confirmed by Nilanonta et al. (2003) (Fig. 8).

위치location 화합물 1 (Enniatin H) Compound 1 (Enniatin H) 화합물 3 (Enniatin I) Compound 3 (Enniatin I) 화합물 4 (Enniatin MK1688) Compound 4 (Enniatin MK1688) 13C (ppm) 13 C (ppm) 1H (ppm) 1 H (ppm) 13C (ppm) 13 C (ppm) 1H (ppm) 1 H (ppm) 13C (ppm) 13 C (ppm) 1H (ppm) 1 H (ppm) N-MeValN-MeVal 3 units3 units 3 units3 units 3 units3 units 1One 170.541170.541 170.613170.613 170.644170.644 22 63.030, 63.42963.030, 63.429 4.564 (1H, m)4.564 (1 H, m) 63.31963.319 4.569(1H, m)4.569 (1 H, m) 63.31163.311 4.599 (1H, d, J =8.75 Hz)4.599 (1H, d, J = 8.75 Hz) 33 28.063, 28.11628.063, 28.116 2.293(1H, m)a 2.293 (1H, m) a 28.030, 28.11928.030, 28.119 2.289 (1H, m)a 2.289 (1H, m) a 28.06828.068 2.281 (1H, m)2.281 (1H, m) 44 20.55520.555 1.067 (3H, m)1.067 (3H, m) 20.56820.568 1.065 (3H, m)1.065 (3H, m) 20.49720.497 1.060 (3H d, J =6.11 Hz)1.060 (3H d, J = 6.11 Hz) 55 19.481, 19.579, 19.69119.481, 19.579, 19.691 0.916 (3H, m)0.916 (3H, m) 19.510, 19.600, 19.71519.510, 19.600, 19.715 0.919 (3H, m)0.919 (3H, m) 19.57119.571 0.914 (3H q, J =14.8, 7.18 Hz)0.914 (3H q, J = 14.8, 7.18 Hz) 66 33.21833.218 3.129, 3.128, 3.146 (3H, s)3.129, 3.128, 3.146 (3H, s) 32.999, 33.16932.999, 33.169 3.102, 3.117, 3.133 (3H, s)3.102, 3.117, 3.133 (3H, s) 32.92132.921 3.105 (1H, s)3.105 (1H, s) HmpHmp 1 unit1 unit 2 units2 units 3 units3 units 77 169.536169.536 169.529169.529 169.490169.490 88 74.52674.526 5.271 (1H, d, J =6.75 Hz)5.271 (1H, d, J = 6.75 Hz) 74.454, 74.60074.454, 74.600 5.269 (1H, t, J =7.74 Hz)5.269 (1H, t, J = 7.74 Hz) 74.56274.562 5.286 (1H, d, J =6.02 Hz)5.286 (1H, doublet, J = 6.02 Hz) 99 36.29736.297 2.013 (1H, m)2.013 (1 H, m) 36.35236.352 2.012 (1H, m)2.012 (1 H, m) 36.41836.418 2.011 (1H, m)2.011 (1 H, m) 1010 25.60025.600 1.184 (1H, m), 1.438 (1H, m)1.184 (1 H, m), 1.438 (1 H, m) 25.59425.594 1.162 (1H, m), 1.459 (1H, m)1.162 (1 H, m), 1.459 (1 H, m) 25.54925.549 1.189 (1H, m), 1.459 (1H, m)1.189 (1H, m), 1.459 (1H, m) 1111 11.51411.514 0.916 (3H, m)0.916 (3H, m) 11.54911.549 0.919 (3H, m)0.919 (3H, m) 11.56411.564 0.914 (3H, t, J =7.18 Hz)0.914 (3H, t, J = 7.18 Hz) 1212 14.80314.803 0.963 (3H, m)a 0.963 (3H, m) a 14.80014.800 0.962 (3H, m)0.962 (3H, m) 14.81714.817 0.962 (3H, d, J =6.11 Hz)0.962 (3H, d, J = 6.11 Hz) HivHiv 2 units2 units 1 unit1 unit 1313 169.536169.536 169.529169.529 1414 75.853, 76.02275.853, 76.022 5.144 (1H, m)5.144 (1H, m) 75.94275.942 5.149 (1H, d, J =8.09 Hz)5.149 (1H, doublet, J = 8.09 Hz) 1515 29.881, 30.15829.881, 30.158 2.293 (1H, m)a 2.293 (1H, m) a 30.19230.192 2.289 (1H, m)a 2.289 (1H, m) a 1616 18.722b 18.722 b 0.963 (3H, d, J =6.70 Hz)a0.963 (3H, d, J = 6.70 Hz) a 18.710b 18.710 b 0.962 (3H, d, J=9.32 Hz)a 0.962 (3H, d, J = 9.32 Hz) a 1717 18.881b 18.881 b 0.992 (3H, d, J =6.70 Hz)a 0.992 (3H, d, J = 6.70 Hz) a 18.881b 18.881 b 0.988 (3H, d, J =6.83 Hz)a 0.988 (3H, d, J = 6.83 Hz) a

상기 위첨자 a는 1H 신호가 겹친 경우이고, 상기 위첨자 b의 대입(assignments)은 변경될 수 있습니다. The superscript a is the case where the 1 H signals overlap, and the assignments of the superscript b can be changed.

(2) 화합물 2(2) compound 2

화합물 2의 분자량은 ESI-MS 측정에 의해 784.5로 측정되었고(도 9) IR 스펙트럼에서 υ 1650.95 cm-1 과 υ 1733.89 cm-1에 아미드 결합과 에스터 결합이 존재하는 것으로 확인되었다(도 10). 13C 및 1H NMR 스펙트럼(in CDCl3)의 화학 이동에서 13C 및 1H 의 연결은 13C-1H HMQC 스펙트럼에 의해 분석되어 표 2에 나타내었다. 13C-1H HMBC 측정으로 13C-1H의 긴 범위의 커플링(long range couplings) 을 확인하고 1H-1H COSY 측정으로 분석된 구조내의 수소와 수소의 관계를 도 11에 나타내었다. 모든 NMR 분석을 통해 화합물 2는 2 개의 부분구조를 가지고 있는 것으로 확인되었다(도 12). 이 화합물의 부분구조는 N-methyl phenylalanine (N-MePhe)과 D-hydroxyisovaleic acid (Hiv)로 확인되었다. 그리고 ESI-MS 측정에 의한 분자량 분석으로 화합물 2는 3 개의 N-MePhe과 3 개의 Hiv 로 구성되어 있고, IR 분석에서 확인된 아미드 결합과 에스터 결합으로 인해 (N-MePhe)-(Hiv)-(N-MePhe)-(Hiv)- (N-MePhe)-(Hiv)로 구성된 고리형 뎁시펩티드 임을 확인하였다. 상기 분석된 화합물 2는 하밀 등(1969)에 의해 구조가 확인된 부버리신으로 밝혀졌다(도 13).The molecular weight of compound 2 was determined to be 784.5 by ESI-MS measurement (FIG. 9) and it was found that amide bonds and ester bonds exist at υ 1650.95 cm −1 and υ 1733.89 cm −1 in the IR spectrum (FIG. 10). 13 C and 1 H NMR spectrum (in CDCl 3) chemical shift at 13 C and 1 H of connection are analyzed by a 13 C- 1 H HMQC spectra are shown in Table 2 below. 13 shows the relationship between the hydrogen and the hydrogen in the check C- 1 H HMBC measured by coupling 13 (long range couplings) of the long range of the C- and 1 H analysis of 1 H- 1 H COSY measurement structure in Figure 11 . All NMR analyzes confirmed that Compound 2 had two substructures (FIG. 12). The partial structure of this compound was identified as N-methyl phenylalanine (N-MePhe) and D-hydroxyisovaleic acid (Hiv). In the molecular weight analysis by ESI-MS measurement, Compound 2 was composed of three N-MePhe and three Hiv, and due to the amide bond and the ester bond identified in the IR analysis, (N-MePhe)-(Hiv)-( It was confirmed that it is a cyclic depsipeptide consisting of N-MePhe)-(Hiv)-(N-MePhe)-(Hiv). The analyzed compound 2 was found to be bublycine whose structure was confirmed by Hamil et al. (1969) (FIG. 13).

13C 화학 이동 (ppm) 13 C chemical shift (ppm) 1H 화학 이동 (ppm) 1 H chemical shift (ppm) N-MePheN-MePhe C-1C-1 169.70169.70 3 units3 units C-2C-2 57.1657.16 5.52 (1H, dd, J = 11.9, 4.9)5.52 (1H, doublet of doublets, J = 11.9, 4.9) C-3C-3 34.7534.75 3.38 (2H, dd, J = 14.6, 4.9 Hz)3.38 (2H, doublet of doublets, J = 14.6, 4.9 Hz) C-4C-4 136.58136.58 C-5C-5 128.83128.83 7.18-7.35 (1H, m)7.18-7.35 (1H, m) C-6C-6 128.57128.57 7.18-7.35 (1H, m)7.18-7.35 (1H, m) C-7C-7 126.82126.82 7.18-7.35 (1H, m)7.18-7.35 (1H, m) C-8C-8 128.57128.57 7.18-7.35 (1H, m)7.18-7.35 (1H, m) C-9C-9 128.83128.83 7.18-7.35 (1H, m)7.18-7.35 (1H, m) C-10C-10 32.2232.22 3.01 (1H, s)3.01 (1H, s) HivHiv C-11C-11 169.95169.95 3 units3 units C-12C-12 75.6075.60 4.90 (1H, d, J = 8.5)4.90 (1H, doublet, J = 8.5) C-13C-13 29.7529.75 2.00 (1H, m)2.00 (1H, m) C-14C-14 18.7518.75 0.42 (3H, d, J = 6.9 Hz)0.42 (3H, d, J = 6.9 Hz) C-15C-15 17.3817.38 0.80 (3H, d, J = 6.6 Hz)0.80 (3H, d, J = 6.6 Hz)

(3) 화합물 3(3) compound 3

화합물 3은 ESI-MS 측정에 의해 분자량이 668.5로 측정되었고(도 14) IR 스펙트럼에서 υ 1656.74 cm-1 과 υ 1733.89 cm-1 에 아미드 결합과 에스터 결합이 존재하는 것으로 확인되었다(도 15). 13C 및 1H NMR 스펙트럼(in CDCl3)의 화학 이동에서 13C 및 1H 의 연결은 13C-1H HMQC 스펙트럼에 의해 분석되어 상기 표 1에서 나타내었다. 13C-1H HMBC 측정으로 13C-1H의 긴 범위의 커플링(long range couplings) 을 확인하고 1H-1H COSY 측정으로 분석된 구조내의 수소와 수소의 관계들은 화합물 1에 나타난 관계들과 비슷한 결과를 나타내었다. 모든 NMR 분석을 통해 화합물 3은 화합물 1과 같은 N-methyl valine (N-MeVal)과 D-hydroxyisovaleic acid (Hiv) 그리고 3-methylpentanoic acid (Hmp)로 이루어진 3개의 부분구조를 가지고 있는 것으로 확인되었다(도 7). 그리고 ESI-MS분석으로 확인된 분자량으로 화합물 3은 3 개의 N-MeVal과 1 개의 Hiv 그리고 2 개의 Hmp로 구성되어 있음을 알 수 있었다. 그리고 IR 분석에서 확인된 아미드 결합과 에스터 결합으로 화합물 3이 (N-MeVal)-(Hiv)- (N-Meval)- (Hmp)-(N-MeVal)-(Hmp)로 구성된 고리형 뎁시펩티드 임을 확인하였으며, 이물질은 닐란논타 등(2003)에 의해 구조가 확인된 엔니아틴 I로 확인되었다(도 16). Compound 3 was determined to have a molecular weight of 668.5 by ESI-MS measurement (FIG. 14) and it was found that amide bonds and ester bonds exist at υ 1656.74 cm −1 and υ 1733.89 cm −1 in the IR spectrum (FIG. 15). 13 C and 1 H NMR spectrum (in CDCl 3) chemical shift at 13 C and 1 H of connections is analyzed by a 13 C- 1 H HMQC spectra are shown in Table 1 above. 13 make the coupling (long range couplings) of the long range of 13 C- 1 H by C- 1 H HMBC measurement and relationship between the hydrogen and the hydrogen in the structure analysis of 1 H- 1 H COSY measurements relationship shown in compound 1 Similar results were obtained. All NMR analyzes confirmed that Compound 3 had three substructures, the same as Compound 1, consisting of N-methyl valine (N-MeVal), D-hydroxyisovaleic acid (Hiv), and 3-methylpentanoic acid (Hmp). 7). And the molecular weight confirmed by ESI-MS analysis, it can be seen that Compound 3 consists of three N-MeVal, one Hiv and two Hmp. And the amide and ester bonds identified in the IR analysis showed that the compound 3 consisted of (N-MeVal)-(Hiv)-(N-Meval)-(Hmp)-(N-MeVal)-(Hmp). It was confirmed that, and the foreign material was confirmed as Enyatin I confirmed the structure by Nilanonta et al. (2003) (Fig. 16).

(4) 화합물 4(4) compound 4

화합물 4는 ESI-MS 측정에 의해 분자량이 682.6로 측정되었고(도 17) IR 스펙트럼에서 υ 1654.81 cm-1 과 υ 1739.67 cm-1 에 아미드 결합과 에스터 결합이 존재하는 것으로 확인되었다(도 18). 13C 및 1H NMR 스펙트럼(in CDCl3)의 화학 이동에서 13C 및 1H 의 연결은 13C-1H HMQC 스펙트럼에 의해 분석되어 상기 표 1에서 나타내었다. 13C-1H HMBC 측정으로 13C-1H의 긴 범위의 커플링(long range couplings) 을 확인하고 1H-1H COSY 측정으로 분석된 구조내의 수소와 수소의 관계를 도 19에 나타내었다. 모든 NMR 분석과 ESI-MS을 통한 분자량으로 화합물 4는 N-methyl valine (N-MeVal)과 3-methylpentanoic acid (Hmp)가 연결되어 있고 이 부분구조가 3 번 반복되어 있는 것으로 확인되었다(도 20). 그리고 IR 분석에서 확인된 아미드 결합과 에스터 결합으로 화합물 4가 (N-MeVal)-(Hmp)-(N-Meval)-(Hmp)- (N-MeVal)- (Hmp)로 구성된 고리형 뎁시펩티드 임을 확인하였으며, 이물질은 미카와 등(1991)과 닐란논타 등(2003)에 의해 구조가 확인된 엔니아틴 MK1688로 확인되었다(도 21). Compound 4 was determined to have a molecular weight of 682.6 by ESI-MS measurement (FIG. 17) and was found to have amide and ester bonds at υ 1654.81 cm −1 and υ 1739.67 cm −1 in the IR spectrum (FIG. 18). 13 C and 1 H NMR spectrum (in CDCl 3) chemical shift at 13 C and 1 H of connections is analyzed by a 13 C- 1 H HMQC spectra are shown in Table 1 above. 13 shows the relationship between the hydrogen and the hydrogen in the check C- 1 H HMBC measured by coupling 13 (long range couplings) of the long range of the C- and 1 H analysis of 1 H- 1 H COSY measurement structure 19 . Molecular weight through all NMR analysis and ESI-MS, Compound 4 is connected to N-methyl valine (N-MeVal) and 3-methylpentanoic acid (Hmp) and confirmed that this substructure is repeated three times (Fig. 20). ). In addition, the amide and ester bonds identified in the IR analysis showed that the compound 4 is a cyclic dipeptipeptide composed of (N-MeVal)-(Hmp)-(N-Meval)-(Hmp)-(N-MeVal)-(Hmp). It was confirmed that, the foreign material was confirmed as Enyatin MK1688 whose structure was confirmed by Mikawa et al. (1991) and Nilanonta et al. (2003) (Fig. 21).

실시예Example 4: 배지의 선택 4: selection of badge

엔니아틴 H, I 및 MK1688의 최대생산에 적합한 액체배지를 조사하기 위해 완전배지 5종류(표 3)와 화학적 선택배지 5종류(표 4)를 선행 연구를 참고로 선별하였다. 선별된 배지 각각에 본 발명의 1501 균주를 포테이토 덱스트로스 아가(PDA)에서 5 일간 배양 시킨 뒤, 마드리(Madry) 등에 의해 고안된 FDM에 옮겨져 7 일간 25 ℃에서 120 rpm의 조건에서 전 배양 한 뒤 약 1 ×105 spores/ml에 해당하는 포자를 접종하여 10 일간 25 ℃에서 120 rpm의 조건에서 배양하였다. Five complete media (Table 3) and five chemically selected media (Table 4) were selected by reference to previous studies to investigate liquid media suitable for the maximum production of Enniatin H, I and MK1688. After incubating the 1501 strain of the present invention in potato dextrose agar (PDA) for 5 days in each of the selected media, it was transferred to FDM designed by Madry et al., And pre-incubated at 25 rpm for 7 days. Spores corresponding to approximately 1 × 10 5 spores / ml were inoculated and incubated at 25 rpm for 10 days at 120 rpm.

배지badge 원재료Raw materials 사용량 (g/L)Usage (g / L) PD PD Potato infusion formPotato infusion form 200.0200.0 DextroseDexrose 20.020.0 Malt extract Malt extract Malt extract baseMalt extract base 6.06.0 MaltoseMaltose 1.81.8 DextroseDexrose 6.06.0 Yeast extractYeast extract 1.21.2 YM YM Malt extractMalt extract 3.03.0 Yeast extractYeast extract 3.03.0 PeptoneEptone 5.05.0 DextroseDexrose 10.010.0 FCM FCM MolassesMolasses 3.03.0 Corn steep liquorCorn steep liquor 3.03.0 FNM FNM GlucoseGlucose 20.020.0 PeptoneEptone 5.05.0 Yeast extractYeast extract 10.010.0 Corn steep liquorCorn steep liquor 10.010.0

배지badge 원재료Raw materials 사용량 (g/L)Usage (g / L) FDM FDM SucroseSucrose 25.025.0 NaNO3 NaNO 3 4.254.25 NaClNaCl 5.05.0 MgSO4.7H2OMgSO 4 .7H 2 O 2.52.5 KH2PO4 KH 2 PO 4 1.361.36 FeSO4.7H2OFeSO 4 .7H 2 O 0.010.01 ZnSO4.7H2O ZnSO 4 .7H 2 O 0.00290.0029 Nash & Snyder Nash & Snyder PeptoneEptone 15.015.0 KH2PO4 KH 2 PO 4 1.01.0 MgSO4.7H2OMgSO 4 .7H 2 O 0.50.5 FBM FBM D-GalactoseD-Galactose 20.020.0 L-AsparagineL-Asparagine 2.02.0 KH2PO4 KH 2 PO 4 1.01.0 KClKCl 0.50.5 MgSO4.7H2OMgSO 4 .7H 2 O 0.50.5 Czapek-Dox Czapek-Dox SucroseSucrose 30.030.0 NaNO3 NaNO 3 3.03.0 K2HPO4 K 2 HPO 4 1.01.0 MgSO4.7H2OMgSO 4 .7H 2 O 0.50.5 KClKCl 0.50.5 FeSO4.7H2OFeSO 4 .7H 2 O 0.010.01 MCzD MCzD DextroseDexrose 20.020.0 KH2PO4 KH 2 PO 4 0.50.5 NaNO3 NaNO 3 2.02.0 MgSO4.7H2OMgSO 4 .7H 2 O 0.50.5 Yeast extractYeast extract 1.01.0 1% ferrous sulfate solution1% ferrous sulfate solution 1.0 ml1.0 ml

완전배지 5가지(PD, malt extract, FCM, FNM, YM)와 화학적 선택배지 5종류(Nash & Snyder, FBM, Czapek-dox, FDM, MCzD)에 따른 엔니아틴 H, I 및 MK1688의 생산량을 조사한 결과는 표 5에 나타내었다.Production of Enniatin H, I and MK1688 according to 5 complete media (PD, malt extract, FCM, FNM, YM) and 5 chemical selective media (Nash & Snyder, FBM, Czapek-dox, FDM, MCzD) The results of the investigation are shown in Table 5.

최대 균체량을 보인 배지는 에프씨엠 배지(FCM)에서 나타났으며 나쉬앤스나이더 배지(Nash & Snyder)에서 최소의 균체량을 생산하였다. 그에 반에 엔니아틴 H, I 및 MK1688의 총 생산량은 화학적 선택배지 중에 하나인 프란키아 디파인드 최소 배지(Frankia Defined Minimal Medium, FDM)에서 엔니아닌 H이 0.16 g/L, I가 0.55 g/L 그리고 MK1688가 0.81 g/L 생산되어, 총 생산량 1.5 g/L로 최대 생산량을 보였으며, 부버리신도 0.17 g/L 생산되었고, 에프씨엠 배지(FCM)와 나쉬앤스나이더 배지(Nash & Snyder)에서는 엔니아틴이 생산되지 않았다. Medium showing the maximum cell weight was shown in the FCM medium (FCM) and produced the minimum cell mass in Nash & Snyder (Nash & Snyder). In contrast, the total production of Enniatin H, I, and MK1688 was 0.16 g / L for Ennianin H and 0.55 g / L for Frankia Defined Minimal Medium (FDM), one of the chemical selective media. L and MK1688 produced 0.81 g / L, yielding a maximum yield of 1.5 g / L total, with 0.17 g / L of burberry-sin, FCM medium and Nash & Snyder medium. Enanthine was not produced.

배지badge 건조 세포무게(g/L)Dry cell weight (g / L) pHpH EN H (g/L)EN H (g / L) EN I (g/L)EN I (g / L) MK1688 (g/L)MK1688 (g / L) 엔니아틴총량 (g/L)Enanthine total amount (g / L) 비 생산량 (g/gDCW)Specific yield (g / gDCW) PD brothPD broth 3.43.4 7.17.1 0.040.04 0.040.04 0.020.02 0.10.1 0.030.03 ME brothME broth 1.61.6 6.76.7 0.030.03 0.030.03 0.020.02 0.080.08 0.050.05 FCMFCM 10.810.8 8.88.8 -- -- -- -- FNMFNM 9.09.0 9.19.1 0.040.04 0.060.06 0.040.04 0.140.14 0.010.01 YM brothYM broth 10.210.2 9.29.2 0.120.12 0.170.17 0.110.11 0.40.4 0.040.04 Nash & SnyderNash & Snyder 1.51.5 8.88.8 -- -- -- -- FBMFBM 8.38.3 3.73.7 0.0030.003 0.070.07 0.040.04 0.10.1 0.010.01 Czappek-doxCzappek-dox 6.06.0 6.26.2 0.050.05 0.210.21 0.130.13 0.40.4 0.070.07 FDMFDM 8.38.3 9.09.0 0.160.16 0.550.55 0.810.81 1.51.5 0.20.2 MCzDMCzD 4.64.6 3.23.2 -- -- -- -- --

이상에서 상세히 설명한 바와 같이, 엔니아틴 H, I 및 MK1688 그리고 부버리 신은 식물 독소(phytotoxin)로 알려져 있었으나, 최근 항암 활성 또는 항말라리아 활성 등의 생리활성이 밝혀지기 시작하면서 많은 연구자들의 주목을 받기 시작하였으나, 실험에 사용할 수 있을 만큼 충분한 엔니아틴 H, I 및 MK1688의 확보가 어렵다는 문제가 있었다. 본 발명의 엔니아틴 H, I 및 MK1688을 생산하는 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501는 엔니아틴 H, I 및 MK1688를 대량생산 할 수 있는 기회를 제공하고, 또한 상기 엔니아틴은 물론 부버리신을 동시에 생산하므로써 저 생산비용으로 상기 생리활성물질의 분리가 가능하게 된다.As described in detail above, Enniatin H, I and MK1688 and Burberry Sin was known as a plant toxin (phytotoxin), but recently the physiological activity such as anti-cancer activity or anti-malarial activity has attracted the attention of many researchers It started, but there was a problem that it is difficult to secure enough eniatin H, I and MK1688 to be used in the experiment. Fusarium oxysporum FB1501, which produces eniatin H, I and MK1688 of the present invention, provides an opportunity to mass-produce eniatin H, I and MK1688. Of course, it is possible to separate the physiologically active substance at a low production cost by simultaneously producing burberrysin.

서열목록 전자파일 첨부 Attach sequence list electronic file

Claims (5)

엔니아틴 H, I 및 MK1688을 생산하는 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501[KFCC-11363P].Yen California tin H, Fusarium oxy's forum to produce I and MK1688 (Fusarium oxysporum ) FB1501 [KFCC-11363P]. 청구항 1에 있어서,The method according to claim 1, 상기 엔니아틴 H, I 및 MK1688와 부버리신을 동시에 생산하는 것을 특징으로 하는 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501[KFCC-11363P].Fusarium oxis Forum ( Fusarium) characterized in that the production of the niacin H, I and MK1688 and Burberry at the same time oxysporum ) FB1501 [KFCC-11363P]. 푸사리움 옥시스포럼(Fusarium oxysporum) FB1501[KFCC-11363P]를 배양하여 엔니아틴 H, I 및 MK1688을 생산하는 방법.Fusarium Fusarium oxysporum ) A method of producing Enniatin H, I and MK1688 by culturing FB1501 [KFCC-11363P]. 청구항 3에 있어서, The method according to claim 3, 상기 배양으로 엔니아틴 H, I 및 MK1688과 동시에 부버리신을 생산하는 방법.The culture is a method for producing buryinsin at the same time with the enanthine H, I and MK1688. 청구항 3 또는 청구항 4에 있어서,The method according to claim 3 or 4, 상기 배양에서 배지 1L당 설탕 25 g, 질산나트륨 4.25 g, 염화나트륨 5 g, 황산마그네슘 7수화물 2.5 g, 인산이수소칼륨 1.36 g, 황산철 7수화물 0.01 g 및 황산아연 7수화물 0.0029 g을 포함하여 이루어진 프란키아 디파인드 최소 배지(Frankia Defined Minimal Medium)를 사용하는 것을 특징으로 하는 방법.The culture medium comprises 25 g of sugar per 1 L of medium, sodium nitrate 4.25 g, sodium chloride 5 g, magnesium sulfate heptahydrate 2.5 g, potassium dihydrogen phosphate 1.36 g, iron sulfate heptahydrate 0.01 g and zinc sulfate heptahydrate 0.0029 g. The method characterized by using Frankia Defined Minimal Medium.
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