KR20230116864A - Lca 및 3-kca를 udca 및 3-kudca로 전환하기 위한 효소적방법 - Google Patents
Lca 및 3-kca를 udca 및 3-kudca로 전환하기 위한 효소적방법 Download PDFInfo
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- KR20230116864A KR20230116864A KR1020237021971A KR20237021971A KR20230116864A KR 20230116864 A KR20230116864 A KR 20230116864A KR 1020237021971 A KR1020237021971 A KR 1020237021971A KR 20237021971 A KR20237021971 A KR 20237021971A KR 20230116864 A KR20230116864 A KR 20230116864A
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Abstract
7β-하이드록실화 시스템뿐만 아니라 이 시스템으로부터 리토콜산 및 3-케토-리토콜산의 7β-하이드록시 유도체를 생산하는 방법이 제공된다. 또한 상기 효소 시스템의 생산에 유용한 재조합 유기체 및 그러한 효소를 인코딩하는 플라스미드가 제공된다.
Description
본 발명은 7β-하이드록실화 시스템, 및 이러한 시스템으로부터 리토콜산 및 3-케토-5β-콜란산의 7β-하이드록시 유도체를 생산하는 방법에 관한 것이다. 본 발명은 또한 그러한 효소 시스템의 생성에 유용한 재조합 유기체 및 그러한 효소를 인코딩하는 플라스미드에 관한 것이다.
우르소데옥시콜산(UDCA)은 케노데옥시콜산(CDCA)보다 적은 부작용으로 콜레스테롤 담석을 용해시킬 수 있기 때문에 담낭염 치료에 자주 처방되는 귀중한 담즙산이다. UDCA는 또한 항염증 특성을 가지고 있으며 낭포성 섬유증 및 원발성 담즙성 담관염과 같은 간 질환의 치료에 적용된다. UDCA의 주요 천연 공급원은 다양한 곰 종으로부터의 담즙이다.
UDCA는 또한 동물 담즙에서 얻은 콜산(CA) 또는 CDCA에서 생산될 수 있다. 에거트 등(Eggert et al. (2014))은 UDCA를 생산하기 위해 CA에서 출발하여 Wolff-Kishner 케톤 환원을 포함한 5 단계로 CDCA를 형성하는 합성 경로와 C7에서의 에피머화를 보고하였다. T. Eggert, D. Bakonyi, W. Hummel, J. Biotechnol. 2014, 191, 11-21. 젱 등(Zheng et al. (2015))은 CDCA에서 UDCA로의 생촉매 에피머화에 기반한 더 짧은 합성 경로를 보고하였다. M.-M. Zheng, R.-F. Wang, C.-X. Li, J.-H. Xu, Process Biochem. 2015, 50, 598-604.
7β-하이드록실라제 시스템과 세포막의 결합은 생촉매 시스템에 대한 특별한 도전이다. 실제로 듀라이라즈 등(Durairaj et al. (2016))은 P450nor가 지금까지 발견된 유일한 가용성 진균 CYP이며 탈질화를 수행한다고 보고했다. Durairaj et al. Microb Cell Fact (2016) 15:125. 이러한 노력은 Fusarium equiseti와 같은 전세포 진균에서 더욱 복잡해지며, 여기서 그롭 등(Grobe et al. (2020))의 보고에 따르면 여러 P450 효소의 작용으로 부산물이 형성된다. S. Grobe, C. Badenhorst, T. Bayer, et al., Angew. Chem. Int. Ed. 10.1002/anie.202012675.
이러한 장애물들을 극복하기 위해, 그롭 등(Grobe et al. (2020))은 Escherichia coli 기반 전세포 시스템에서 세포막과의 결합을 필요로 하지 않는 P450 효소인 Streptomyces antibioticus의 cyt P450 모노옥시게나제 CYP107D1(oleP)의 변이체를 사용하여 LCA로부터 UDCA의 형성에 대해 보고하였다. LCA를 6β-하이드록시 유도체인 MDCA로 전환시키는 천연 효소를 변경함으로써, 저자들은 UDCA가 MDCA보다 우선적으로 형성되도록 하이드록실화의 위치를 대부분 변경할 수 있었다. 그러나, 전환은 매우 낮은 생산성(24시간에 기껏해야 67 μM)과 불완전한 위치 선택성(기껏해야 73:27 비율의 UDCA:MDCA)으로 수행되었다.
따라서, LCA 및 3-KCA를 UDCA 및 3-KUDCA로 선택적으로 전환하기 위한 효율적이고 생산적인 방법에 대한 필요성이 존재한다. 이상적인 방법은 높은 수율을 제공하고, 확장하기 용이하며, 상업적 생산에서 구현하기 용이한 것이다. 필요한 것은 상업적 부피로 리토콜산 또는 3-KCA의 7β-하이드록실화를 위한 효율적인 효소 시스템, 과정 및 성분이다.
발명의 요약
다른 종으로부터의 천연 7β-하이드록실화 시스템으로 형질전환된 효모를 사용한 일련의 실험을 포함하여 LCA 및 3-KCA를 하이드록실화하기 위한 다양한 조작된 미생물 시스템으로 광범위한 실험을 한 후, 발명자들은 예기치 않게 LCA 및 3-KCA 및 이의 유도체로부터 UDCA 및 3-KUDCA 및 이의 유도체를 선택적으로 생산할 수 있는, 7β-하이드록실라제 활성을 발현하도록 형질전환된 효모 기반 시스템을 발견하였다. 따라서, 제1 주요 실시양태에서 본 발명은 LCA 또는 3-KCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 효모, 또는 이의 추출물 또는 용해물(lysate)의 존재 하에 7β-하이드록실라제 시스템과 접촉시키는 것을 포함하여 LCA 또는 3-KCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 UDCA 또는 3-KUDCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 이의 카복실레이트 염으로 전환시키는 방법을 제공하며, 여기서 7β-하이드록실라제 시스템은 효모 고유의 것이 아니다.
추가의 주요 실시양태는 본 발명의 유기체를 생산하기 위해 사용되는 플라스미드에 관한 것이다. 따라서, 제2 주요 실시양태에서 본 발명은 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 서열 번호 20; 서열 번호 23; 서열 번호 26; 서열 번호 29; 또는 서열 번호 32로부터 선택된 핵산 서열; 또는 전술한 임의의 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열을 포함하는 플라스미드를 제공한다.
추가 실시양태는 본 발명의 방법에 사용되는 형질전환 유기체에 관한 것이다. 따라서, 제3 주요 실시양태에서 본 발명은 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 서열 번호 20; 서열 번호 23; 서열 번호 26; 서열 번호 29; 및 서열 번호 32로부터 선택된 CYP 인코딩 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에 의해 형질전환된 유기체를 제공한다.
또 다른 실시양태는 본 발명의 전환이 일어나는 반응 혼합물에 관한 것이다. 따라서, 제4 주요 실시양태에서 본 발명은 (i) LCA 또는 3-KCA, (ii) 효모, 또는 이의 추출물 또는 용해물, (iii) 7β-하이드록실화 시스템을 포함하는 반응 혼합물을 제공한다. 제5 주요 실시양태는 효모와, P450 옥시도리덕타제("CPR") 효소 및 P450 7β-하이드록실라제("CYP") 효소를 포함하는 7β-하이드록실화 시스템을 포함하는 반응 혼합물을 제공하며, 여기서 CYP 효소는 Gibberella zeae, 바람직하게는 Gibberella zeae PH1 또는 Gibberella zeae VKM2600, 가장 바람직하게는 Gibberella zeae VKM2600 고유 효소이다.
본 발명의 추가적인 이점이 다음의 설명에 일부 기재되어 있으며, 일부는 설명으로부터 명해할 것이거나 본 발명의 실시에 의해 알 수 있을 것이다. 본 발명의 이점은 특히 이어지는 청구범위에서 제시되는 요소 및 조합에 의해 실현되고 달성될 것이다. 전술한 일반적인 설명 및 다음의 상세한 설명은 단지 예시적이고 설명을 위한 것이며 청구된 본 발명을 제한하지 않는 것으로 이해되어야 한다.
본 명세서에 통합되어 그 일부를 구성하는 첨부 도면은 본 발명의 여러 실시양태를 예시하고 설명과 함께 본 발명의 원리를 설명하기 위해 제공된다.
도 1은 실시예 17에 기술된 실험으로부터의 LCMS 크로마토그램을 도시한다. 도 1A는 추출된 브로스 샘플의 TIC 추적이다. 도 1B는 LCA 표준의 TIC 추적이다. 도 1C는 UDCA 표준의 TIC 추적이다.
도 2는 브로스 샘플로부터 추출된 UDCA(A)와 실시예 17에서 보고된 UDCA 인증(authentic) 표준(B)에 대한 MS 스펙트럼의 비교이다.
도 3은 실시예 18에 기술된 실험으로부터의 CMS 크로마토그램을 도시한다. 도 3A는 단리된 UDCA의 TIC 추적이다. 도 3B는 UDCA 표준의 TIC 추적이다.
도 4는 실시예 18에서 보고된 단리된 UDCA(A) 및 UDCA 인증 표준(B)에 대한 MS 스펙트럼의 비교이다.
도 5는 실시예 18에 기술된 실험으로부터 단리된 UDCA의 1H NMR 스펙트럼을 도시한다.
도 6은 실시예 18에 기술된 실험으로부터 단리된 UDCA의 13C NMR 스펙트럼을 도시한다.
도 7은 실시예 18에 기술된 실험으로부터의 인증 UDCA의 1H NMR 스펙트럼을 도시한다.
도 8은 실시예 18에 기술된 실험으로부터의 인증 UDCA의 13C NMR 스펙트럼을 도시한다.
도 9는 실시예 19에 기술된 실험으로부터의 LCMS 크로마토그램을 도시한다. 도 9A는 추출된 브로스 샘플의 TIC 추적이다. 도 9B는 추출된 브로스 샘플의 m/z 389.3(3-KUDCA)에 대한 추출된 이온 크로마토그램(EIC)이다. 도 9C는 3-KUDCA 표준의 TIC 추적이다. 도 9D는 3-KCA 표준의 TIC 추적이다.
도 10은 브로스 샘플(A)로부터 추출된 3-KUDCA와 실시예 19에서 보고된 3-KUDCA 인증 표준(B)에 대한 MS 스펙트럼의 비교이다.
도 11은 실시예 21에 기술된 실험으로부터의 LCMS 크로마토그램을 도시한다. 도 11A는 추출된 브로스 샘플의 TIC 추적이다. 도 11B는 추출된 브로스 샘플의 m/z 391.3(UDCA)에 대한 추출된 이온 크로마토그램(EIC)이다. 도 11C는 UDCA 표준의 TIC 추적이다.
도 12는 실시예 21에서 보고된 바와 같은, 브로스 샘플(A) 및 UDCA 인증 표준(B)으로부터 추출된 UDCA에 대한 MS 스펙트럼의 비교이다.
도 1은 실시예 17에 기술된 실험으로부터의 LCMS 크로마토그램을 도시한다. 도 1A는 추출된 브로스 샘플의 TIC 추적이다. 도 1B는 LCA 표준의 TIC 추적이다. 도 1C는 UDCA 표준의 TIC 추적이다.
도 2는 브로스 샘플로부터 추출된 UDCA(A)와 실시예 17에서 보고된 UDCA 인증(authentic) 표준(B)에 대한 MS 스펙트럼의 비교이다.
도 3은 실시예 18에 기술된 실험으로부터의 CMS 크로마토그램을 도시한다. 도 3A는 단리된 UDCA의 TIC 추적이다. 도 3B는 UDCA 표준의 TIC 추적이다.
도 4는 실시예 18에서 보고된 단리된 UDCA(A) 및 UDCA 인증 표준(B)에 대한 MS 스펙트럼의 비교이다.
도 5는 실시예 18에 기술된 실험으로부터 단리된 UDCA의 1H NMR 스펙트럼을 도시한다.
도 6은 실시예 18에 기술된 실험으로부터 단리된 UDCA의 13C NMR 스펙트럼을 도시한다.
도 7은 실시예 18에 기술된 실험으로부터의 인증 UDCA의 1H NMR 스펙트럼을 도시한다.
도 8은 실시예 18에 기술된 실험으로부터의 인증 UDCA의 13C NMR 스펙트럼을 도시한다.
도 9는 실시예 19에 기술된 실험으로부터의 LCMS 크로마토그램을 도시한다. 도 9A는 추출된 브로스 샘플의 TIC 추적이다. 도 9B는 추출된 브로스 샘플의 m/z 389.3(3-KUDCA)에 대한 추출된 이온 크로마토그램(EIC)이다. 도 9C는 3-KUDCA 표준의 TIC 추적이다. 도 9D는 3-KCA 표준의 TIC 추적이다.
도 10은 브로스 샘플(A)로부터 추출된 3-KUDCA와 실시예 19에서 보고된 3-KUDCA 인증 표준(B)에 대한 MS 스펙트럼의 비교이다.
도 11은 실시예 21에 기술된 실험으로부터의 LCMS 크로마토그램을 도시한다. 도 11A는 추출된 브로스 샘플의 TIC 추적이다. 도 11B는 추출된 브로스 샘플의 m/z 391.3(UDCA)에 대한 추출된 이온 크로마토그램(EIC)이다. 도 11C는 UDCA 표준의 TIC 추적이다.
도 12는 실시예 21에서 보고된 바와 같은, 브로스 샘플(A) 및 UDCA 인증 표준(B)으로부터 추출된 UDCA에 대한 MS 스펙트럼의 비교이다.
상세한 설명
용어의 정의 및 사용
본 명세서 및 이어지는 청구범위에서 사용되는 바와 같이, 단수 형태 "a", "an" 및 "the"는 문맥상 명백하게 달리 지시하지 않는 한 복수 지시대상을 포함한다.
본 명세서 및 이어지는 청구범위에서 사용되는 바와 같이, "포함한다"라는 단어 및 "포함하는" 및 "포함하다"와 같은 단어의 변형은 "~를 포함하지만 이에 제한되지 않는"을 의미하며, 예를 들어, 기타 첨가제, 구성요소, 정수 또는 단계를 배제하려는 의도는 아니다. 요소가 복수의 구성요소, 단계 또는 조건을 포함하는 것으로 기술될 때, 요소는 또한 그러한 복수의 임의의 조합을 포함하거나 구성요소, 단계 또는 조건의 복수 또는 조합으로 "이루어진" 또는 "본질적으로 이로 이루어진" 것으로 기술될 수 있음이 이해될 것이다.
범위의 상한과 별도로 하한을 지정하거나 특정 수치를 지정하여 범위를 부여하는 경우에는 하한 변수, 상한 변수 및 수학적으로 가능한 특정 숫자 값 중 어느 하나를 선택적으로 조합하여 범위를 정의할 수 있음을 알 수 있을 것이다. 마찬가지로, 범위가 하나의 끝점에서 다른 끝점에 걸쳐 있는 것으로 정의될 때 범위는 두 끝점 사이의 범위를 포함하고 두 끝점을 제외하는 것으로도 이해될 것이다.
본원에서 사용되는 경우 "약"이라는 용어는 화학 산업에서 허용되고 이 산업에서 제품에 내재된 변동성, 예를 들어 제조 변동으로 인한 제품 강도의 차이 및 시간에 따른 제품 열화를 보완할 것이다. 일 실시양태에서, 상기 용어는 ±5% 변동성 또는 ±10% 변동성을 허용한다.
본 발명의 조성물과 관련하여 사용되는 "허용되는"이라는 어구는 생리학적으로 허용 가능하고 전형적으로 대상체(예를 들어, 인간과 같은 포유동물)에게 투여될 때 바람직하지 않은 반응을 일으키지 않는 이러한 조성물의 분자 실체 및 기타 성분을 지칭한다.
"코딩 서열"은 단백질의 아미노산 서열을 인코딩하는 핵산(예를 들어, 유전자)의 해당 부분을 지칭한다.
"자연 발생" 또는 "야생형" 또는 "고유"는 "비자연 발생", "비야생형", "비고유" 또는 "외래"와 달리 자연에서 발견된 형태를 지칭한다. 예를 들어, 천연 발생 또는 야생형 폴리펩티드 또는 폴리뉴클레오티드 서열은 자연의 공급원으로부터 단리될 수 있고 인간 조작에 의해 의도적으로 변형되지 않은 유기체에 존재하는 서열이다.
예를 들어, 세포, 핵산 또는 폴리펩티드와 관련하여 사용될 때 "재조합"은 자연에 달리 존재하지 않을 방식으로 변형된 물질, 또는 물질의 자연 또는 천연 형태에 상응하는 물질을 지칭한다. 비제한적 예는 무엇보다도 세포의 천연(비재조합) 형태 내에서 발견되지 않는 유전자를 발현하거나, 달리 상이한 수준에서 발현되는 천연 유전자를 발현하는 재조합 세포를 포함한다.
"서열 동일성 백분율" 및 "상동성 백분율"은 폴리뉴클레오티드 및 폴리펩티드 사이의 비교를 지칭하기 위해 본원에서 상호교환적으로 사용되며, 비교 창에 걸쳐 최적으로 정렬된 2개의 서열을 비교함으로써 결정되며, 비교 창에서 폴리뉴클레오티드 또는 폴리펩티드 서열의 일부는 두 서열의 최적 정렬을 위해 참조 서열(부가 또는 결실을 포함하지 않음)과 비교하여 부가 또는 결실(즉, 갭)을 포함할 것이다. 백분율은 동일한 핵산 염기 또는 아미노산 잔기가 두 서열에서 발생하는 위치의 수를 결정하여 일치하는 위치의 수를 산출하고 일치하는 위치의 수를 비교 창의 총 위치 수로 나누고 결과에 100을 곱하여 서열 동일성의 백분율을 산출함으로써 계산된다.
당업자는 2개의 서열을 정렬하는데 이용가능한 많은 확립된 알고리즘이 있음을 인식할 것이다. 비교를 위한 서열의 최적 정렬은 예를 들어 Smith and Waterman, 1981, Adv. Appl. Math. 2:482의 국소 상동성 알고리즘, Needleman and Wunsch, 1970, J. Mol. Biol. 48:443의 상동성 정렬 알고리즘, Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444의 유사성 검색 방법, 이들 알고리즘의 컴퓨터 구현(GCG Wisconsin Software Package의 GAP, BESTFIT, FASTA, 및 TFASTA), 또는 시각적 검사(일반적으로, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, Greene Publishing Associates, Inc.와 John Wiley & Sons, Inc.의 조인트 벤처(1995 Supplement) (Ausubel) 참조)에 의해 수행될 수 있다. 퍼센트 서열 동일성 및 서열 유사성을 결정하는데 적합한 알고리즘의 예는 각각 [Altschul et al., 1990, J. Mol. Biol. 215: 403-410] 및 [Altschul et al., 1977, Nucleic Acids Res. 3389-3402]에 기술된 BLAST 및 BLAST 2.0 알고리즘이다.
"참조 서열"은 서열 비교를 위한 기초로서 사용되는 정의된 서열을 지칭한다. 참조 서열은 더 큰 서열의 하위 집합, 예를 들어 전장 유전자 또는 폴리펩티드 서열의 분절일 수 있다. 일반적으로, 참조 서열은 적어도 20개 뉴클레오티드 또는 아미노산 잔기 길이, 적어도 25개 잔기 길이, 적어도 50개 잔기 길이 또는 전장의 핵산 또는 폴리펩티드이다. 2개의 폴리뉴클레오티드 또는 폴리펩티드는 각각 (1) 2개의 서열 간에 유사한 서열(즉, 완전한 서열의 일부)을 포함할 수 있고, (2) 2개의 서열 간에 분기되는 서열을 추가로 포함할 수 있기 때문에, 2개(또는 그 이상)의 폴리뉴클레오티드 또는 폴리펩티드 사이의 서열 비교는 전형적으로 서열 유사성의 국소 영역을 식별하고 비교하기 위해 "비교 창"을 통해 2개의 폴리뉴클레오티드의 서열을 비교함으로써 수행된다.
"비교 창"은 서열이 적어도 20개의 연속 뉴클레오티드 또는 아미노산의 참조 서열과 비교될 수 있는 적어도 약 20개의 연속 뉴클레오티드 위치 또는 아미노산 잔기의 개념적 분절을 지칭하며, 여기서 비교 창에서 서열의 일부는 2개의 서열의 최적 정렬을 위해 참조 서열(추가 또는 결실을 포함하지 않음)과 비교하여 20% 이하의 추가 또는 결실(즉, 갭)을 포함할 수 있다. 비교 창은 20개 연속 잔기보다 길 수 있으며 선택적으로 30, 40, 50, 100, 150 또는 200개 이상의 창을 포함한다.
"실질적 동일성"은 참조 서열의 적어도 90%, 95%, 98%, 또는 99%를 포함하는 비교 창에 걸쳐 참조 서열과 비교하여 적어도 80%의 서열 동일성, 적어도 85%의 서열 동일성, 적어도 90%의 서열 동일성, 또는 적어도 95%의 서열 동일성, 보다 일반적으로 적어도 98% 또는 99%의 서열 동일성을 갖는 폴리뉴클레오티드 또는 폴리펩티드 서열을 지칭한다. 폴리펩티드에 적용되는 특정 실시양태에서, 용어 "실질적 동일성"은 기본 갭 가중치를 사용하는 프로그램 GAP 또는 BESTFIT와 같이 최적으로 정렬될 때 2개의 폴리펩티드 서열이 적어도 80% 서열 동일성, 바람직하게는 적어도 89% 서열 동일성, 적어도 95% 서열 동일성 또는 그 이상(예를 들어, 99% 서열 동일성)을 공유함을 의미한다. 바람직하게는, 동일하지 않은 잔기 위치는 보존적 아미노산 치환에 따라 상이하다.
본원에서 세포 유기체에 대한 언급이 주어지는 경우, 이는 야생형 상태 및 변형된 유기체로서의 둘 다의 유기체를 지칭하는 것으로 이해될 것이다. 따라서, 효모라는 용어는 재조합 기술을 사용하여 생산된 임의의 인공 효모 외에 자연에 자연적으로 존재하는 모든 야생형 효모를 포함한다.
용어 "효모"는 사카로마이세테스 강(Saccaromycetes class), 바람직하게는 사카로마이세탈레스 목(Saccharomycetales order), 바람직하게는 사카로마이세타세아 과(Saccharomycetaceae family)의 자낭균류를 지칭한다. 특히 바람직한 효모는 Pichia 및 Saccharomyces 속, 특히 Pichia pastoris 및 Saccharomyces cerevisiae에 속한다.
3-KCA 또는 3-케토-5β-콜란산은 다음 화학 구조로 표시된다:
LCA 또는 리토콜산은 다음 화학 구조로 표시된다:
3-KUDCA 또는 7β-하이드록시-3-케토-5β-콜란산은 다음 화학 구조로 표시된다:
UDCA 또는 우르소데옥시콜산은 다음 화학 구조로 표시된다:
본원에서 사용되는 카복실레이트 "염"은 개시된 화합물의 유도체를 지칭하며, 여기서 모 화합물은 존재하는 산 모이어티를 이의 염 형태로 전환시킴으로써 변형된다. 적합한 염의 예는 카복실산의 산성 잔기의 알칼리 또는 유기 염을 포함하지만 이에 제한되지 않는다. 본 발명의 염은 예를 들어 비독성 무기 또는 유기 염기로부터 형성된 모 화합물의 통상적인 무독성 염 또는 4차 암모늄 염을 포함한다. 본 발명의 염은 산성 모이어티를 함유하는 모 화합물로부터 통상적인 화학적 방법에 의해 합성될 수 있다. 일반적으로, 이러한 염은 이들 화합물의 유리산 형태를 물 또는 유기 용매 또는 이 둘의 혼합물에서 화학량론적 양의 적절한 염기와 반응시켜 제조할 수 있다.
본원에서 사용되는 "에스테르"는 바람직하게는 -COOR 모이어티를 지칭하며, 여기서 R은 임의로 치환된 C1-20 알킬 또는 임의로 치환된 아릴이다.
본원에서 사용되는 용어 "알킬"은 직쇄 또는 분지쇄인 포화 탄화수소 그룹을 지칭한다. 알킬 그룹의 예는 메틸(Me), 에틸(Et), 프로필(예: n-프로필 및 이소프로필), 부틸(예: n-부틸, 이소부틸, t-부틸), 펜틸(예: n-펜틸, 이소펜틸, 네오펜틸) 등이 있다. 본 발명의 임의의 실시양태 또는 하위 실시양태에서, 알킬 그룹은 1 내지 약 20, 2 내지 약 20, 1 내지 약 10, 1 내지 약 8, 1 내지 약 6, 1 내지 약 약 4개 또는 1 내지 약 3개의 탄소 원자를 함유한다.
본원에 사용된 "아릴"은 예를 들어 페닐, 나프틸, 안트라세닐, 페난트레닐, 인다닐, 인데닐 및 같은 모노사이클릭 또는 폴리사이클릭(예를 들어, 2, 3 또는 4개의 융합된 고리를 가짐) 방향족 탄화수소(헤테로방향족 탄화수소 포함)를 지칭한다. 일부 실시양태에서, 아릴기는 6 내지 약 20개의 탄소 원자를 갖는다.
본 발명의 실시양태 또는 하위 실시양태 중 임의의 것에서, 임의로 치환된 모이어티는 대안적으로 할로, OH, 아민, C1-6 알킬, C1-6 알콕시, C1-6 하이드록시알킬, CO(C1-6 알킬), CHO, CO2H, CO2(C1-6 알킬) 및 C1-6 할로알킬 중에서 독립적으로 선택된 0, 1, 2, 또는 3개의 치환체로 치환된 것으로 정의될 수 있다.
본원에 사용된 아미드는 바람직하게는 -C(O)N(R')(R") 모이어티를 지칭하며, 여기서 R' 및 R"는 독립적으로 임의로 치환된 C1-20 알킬 또는 임의로 치환된 아릴이다. 대안적으로, UDCA의 카복실 아미드는 타우로우르소데옥시콜산("TUDCA")일 수 있다.
본 발명의 "P450 7β-하이드록실라제 시스템"은 LCA 또는 K-LCA의 7-H 위치를 하이드록실화할 수 있는 클래스 II CYP 효소 시스템을 지칭한다. [Durairaj et al. Microb Cell Fact (2016) 15:125]에서 검토된 바와 같이, Class II CYP 효소 시스템은 2개의 통합 막 단백질: P450 7β-하이드록실라제(본원에서 때때로 "CYP"라고 함) 및 NAD(P)H에서 헴 모이어티로 2개의 전자를 전달하는 보철 보조인자 FAD 및 FMN을 함유하는 사이토크롬 P450 리덕타제(본원에서 때때로 "CRP"이라고 함)를 포함한다. 이 시스템은 또한 제3의 단백질 성분인 Cyt b5를 포함할 수 있으며, 이는 제2의 전자를 옥시철 CYP로 전달한다.
주요 실시양태의 논의
본 발명의 제1 주요 실시양태는 LCA 또는 3-KCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 효모, 또는 이의 추출물 또는 용해물의 존재 하에 7β-하이드록실라제 시스템과 접촉시키는 것을 포함하여 LCA 또는 3-KCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 UDCA 또는 3-KUDCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 이의 카복실레이트 염으로 전환시키는 방법을 제공하며, 여기서 7β-하이드록실라제 시스템은 효모 고유의 것이 아니다.
제2 주요 실시양태는 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 서열 번호 20; 서열 번호 23; 서열 번호 26; 서열 번호 29; 또는 서열 번호 32로부터 선택된 핵산 서열; 또는 전술한 임의의 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열을 포함하는 플라스미드를 제공한다.
제3 주요 실시양태는 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 서열 번호 20; 서열 번호 23; 서열 번호 26; 서열 번호 29; 및 서열 번호 32로부터 선택된 CYP 인코딩 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에 의해 형질전환된 유기체를 제공한다.
제4 주요 실시양태는 (i) LCA 또는 3-KCA, (ii) 효모, 또는 이의 추출물 또는 용해물, (iii) 7β-하이드록실화 시스템을 포함하는 반응 혼합물을 제공한다.
제5 주요 실시양태는 효모와, P450 옥시도리덕타제("CPR") 효소 및 P450 7β-하이드록실라제("CYP") 효소를 포함하는 7β-하이드록실화 시스템을 포함하는 반응 혼합물을 제공하며, 여기서 CYP 효소는 Gibberella zeae, 바람직하게는 Gibberella zeae PH1 또는 Gibberella zeae VKM2600, 가장 바람직하게는 Gibberella zeae VKM2600 고유 효소이다.
하위 실시양태의 논의
전술한 바와 같이, 본 발명은 바람직하게는 비천연 7β-하이드록실화 시스템을 발현하도록 형질전환된 효모의 존재하에 수행된다. 효모는 바람직하게는 Saccharomyces 및 Pichia에서 선택되며, 가장 바람직하게는 Saccharomyces cerevisiae 및 Pichia pastoris에서 선택된다.
본 발명의 방법에 사용되는 유기체는 비-천연 P450 7-베타-하이드록실라제("CYP") 효소 및 임의로 비-천연 P450 옥시도리덕타제("CPR") 효소를 포함하는 비천연 7β-하이드록실라제 시스템에 의해 형질전환된 것이다. CPR 효소는 7β-하이드록실라제 시스템에 중요하지만, CPR 효소가 유기체에 외래인 것이 절대적으로 필요한 것은 아니며, 효모에 고유한 고유 효소로도 충분할 수 있다.
본 발명을 실행하기 위한 바람직한 CYP 효소는 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 서열 번호 20; 서열 번호 23; 서열 번호 26; 서열 번호 29; 및 서열 번호 32로부터 선택된 CYP 인코딩 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에 의해 인코딩된다.
CYP 인코딩 핵산은 상기 서열 번호 중 어느 하나 또는 조합으로부터 선택될 수 있고, 본 발명의 임의의 CPR 효소와 조합될 수 있다. 일 실시양태에서 인코딩 핵산 서열은 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 및 서열 번호 20; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열로부터 선택된다. 또 다른 실시양태에서 핵산은 서열 번호 23; 서열 번호 26; 또는 서열 번호 29; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열로부터 선택된다. 또 다른 실시양태에서 핵산 서열은 서열 번호 32; 또는 서열 번호 32와 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산으로부터 선택된다.
CYP 효소는 바람직하게는 서열 번호 9; 서열 번호 12; 서열 번호 15; 서열 번호 18; 서열 번호 21; 서열 번호 24; 서열 번호 27; 서열 번호 30; 또는 서열 번호 33으로부터 선택된 CYP 아미노산 서열; 또는 전술한 임의의 아미노산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 아미노산 서열로부터 선택된다.
CYP 효소는 상기 서열 번호 중 어느 하나 또는 조합으로부터 선택될 수 있고, 본 발명의 임의의 CPR 효소와 조합될 수 있다. 일 실시양태에서 CYP 효소는 서열 번호 9; 서열 번호 12; 서열 번호 15; 서열 번호 18; 및 서열 번호 21로부터 선택된 CYP 아미노산 서열; 또는 전술한 임의의 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 아미노산 서열을 포함한다. 또 다른 실시양태에서 CYP 효소는 서열 번호 24; 서열 번호 27; 또는 서열 번호 30; 또는 전술한 임의의 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 아미노산 서열을 포함한다. 또 다른 실시양태에서 CYP 효소는 서열 번호 33; 또는 서열 번호 33과 적어도 85%, 90%, 95%, 98% 또는 99% 동일성을 갖는 아미노산 서열을 포함한다.
본 발명의 CYP 효소를 인코딩하는 바람직한 플라스미드는 바람직하게는 서열 번호 7; 서열 번호 10; 서열 번호 13; 서열 번호 16; 서열 번호 19; 서열 번호 22; 서열 번호 25; 서열 번호 28; 또는 서열 번호 31로부터 선택된 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열을 포함한다.
일 실시양태에서 CYP 효소를 인코딩하는 플라스미드는 서열 번호 7; 서열 번호 10; 서열 번호 13; 서열 번호 16; 또는 서열 번호 19; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열을 포함한다. 다른 실시양태에서 CYP 효소를 인코딩하는 플라스미드는 서열 번호 22; 서열 번호 25; 또는 서열 번호 28; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열을 포함한다. 또 다른 실시양태에서 CYP 효소를 인코딩하는 플라스미드는 서열 번호 31; 또는 서열 번호 31과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열을 포함한다.
일 실시양태에서, CYP 효소는 Gibberella zeae, 바람직하게는 Gibberella zeae PH1 또는 Gibberella zeae VKM2600, 가장 바람직하게는 Gibberella zeae VKM2600 고유의 단백질이고, 유기체는 이러한 단백질을 발현하도록 형질전환된다.
7β-하이드록실화 시스템의 CPR 효소는 7β-하이드록실라제 활성이 발현되는 유기체에 고유하거나 서열 번호 2 및 서열 번호 5로부터 선택된 핵산 서열, 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에서 선택된 CPR 인코딩 핵산 서열에 의해 인코딩될 수 있다. CPR 효소는 바람직하게 서열 번호 3 및 서열 번호 6으로부터 선택된 CPR 아미노산 서열, 또는 전술한 임의의 아미노산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 아미노산 서열을 포함한다.
일 실시양태에서, 본 발명의 방법은 LCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 7β-하이드록실라제 시스템과 접촉시켜 UDCA 또는 이의 카복실산 에스테르, 카복실 아미드, 또는 카복실레이트 염을 생산하도록 실시된다. 다른 실시양태에서, 본 발명의 방법은 3-KCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 7β-하이드록실라제 시스템과 접촉시켜 3-KUDCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 생산하도록 실시된다. 3-KUDCA 또는 이의 카복실산 에스테르, 카복실 아미드, 또는 카복실레이트 염이 생산되는 경우, 본 발명의 방법은 임의로 3-KUDCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 UDCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염으로 환원시키는 단계를 추가로 포함할 것이다.
바람직한 실시양태에서, 본 발명의 방법은 7β-하이드록실라제 시스템으로부터 UDCA 또는 3-KUDCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 단리하는 것을 추가로 포함한다. 단리라는 것은 UDCA 또는 3-KUDCA가 7β-하이드록실라제 시스템 및 UDCA 또는 3-KUDCA가 생산된 반응 혼합물로부터 실질적으로 순수함을 의미한다. 따라서, UDCA 또는 3-KUDCA는 잔류 반응 혼합물의 중량을 고려할 때 적어도 90%, 적어도 95% 또는 적어도 98% 순수하다. 특히 바람직한 실시양태에서, UDCA 또는 3-KUDCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염은 실질적으로 순수한 부분입체이성질체로서 생성된다. "실질적으로 순수한 부분입체이성질체"는 이의 7α-부분입체이성질체를 고려할 때 적어도 90% 순수, 적어도 95% 순수, 또는 적어도 98% 순수한 부분입체이성질체를 지칭한다.
조작된 CYP 및 CPR 효소
본원에 개시된 효소 서열과 상이한 특성을 갖는 CYP 및 CPR 효소는 CYP 또는 CPR 효소를 인코딩하는 유전 물질을 돌연변이시키고 원하는 특성을 갖는 조작된 효소를 발현하는 폴리뉴클레오티드를 확인함으로써 얻을 수 있다. 이러한 자연 발생적이지 않은 CYP 및 CPR 효소는 시험관 내 돌연변이 또는 유도 진화와 같은 잘 알려진 다양한 기술에 의해 생성될 수 있다. 일부 실시양태에서, 유도 진화는 폴리펩티드를 코딩하는 유전자 전체에 걸쳐 돌연변이를 생산하는 것이 비교적 용이할 뿐만 아니라 이전에 돌연변이된 폴리뉴클레오티드를 취하여 이들을 선택된 효소 특성의 추가 개선을 얻기 위한 돌연변이유발 및/또는 재조합의 추가 주기에 적용시키는 능력을 제공하기 때문에 조작된 효소를 생산하기 위한 매력적인 방법이다. 전체 유전자에 돌연변이유발을 적용하면 유전자의 제한된 영역에 대한 변화를 제한함으로써 발생할 수 있는 편차를 줄일 수 있다. 그것은 또한 효소의 멀리 떨어져 있는 부분이 효소 기능의 다양한 측면에서 역할을 할 수 있기 때문에 다른 효소 특성에 영향을 받는 효소의 생산을 향상시킬 수 있다.
돌연변이유발 및 유도 진화에서, 천연 발생 또는 야생형 CYP 또는 CPR 효소를 인코딩하는 부모 또는 참조 폴리뉴클레오티드를 돌연변이 과정, 예를 들어 무작위 돌연변이유발 및 재조합에 적용하여 폴리뉴클레오티드에 돌연변이를 도입한다. 돌연변이된 폴리뉴클레오티드가 발현되고 번역되어 폴리펩티드에 변형된 조작된 CYP 또는 CPR 효소를 생성한다. 본원에서 사용되는 "변형"은 아미노산 치환, 결실 및 삽입을 포함한다. 변형 중 어느 하나 또는 조합이 자연 발생 효소적 활성 폴리펩티드에 도입되어 조작된 효소를 생성할 수 있으며, 이는 이후 특정 효소 특성에서 원하는 개선을 갖는 폴리펩티드 및 상응하는 폴리뉴클레오티드를 확인하기 위해 다양한 방법에 의해 스크리닝된다.
7-베타 하이드록실라제 환경
CYP 및 CPR 효소는 세포 내, 세포 배지, 고정 기질, 또는 효소를 발현하도록 재조합 설계된 세포의 용해물 및 추출물 또는 단리된 제제와 같은 다른 형태로 존재할 수 있다. 용어 "단리된 폴리펩티드"는 이에 자연적으로 수반되는 다른 오염물, 예를 들어 단백질, 지질 및 폴리뉴클레오티드가 실질적으로 분리된 폴리펩티드를 지칭한다. 이 용어는 자연 발생 환경 또는 발현 시스템(예: 숙주 세포 또는 시험관 내 합성)에서 제거되거나 정제된 폴리펩티드를 포함한다.
일부 실시양태에서, 단리된 CYP 및 CPR 효소는 실질적으로 순수한 폴리펩티드 조성물에 존재한다. 용어 "실질적으로 순수한 폴리펩티드"는 폴리펩티드 종이 존재하는 우세한 종(즉, 몰 또는 중량 기준으로 조성물 내의 임의의 다른 개별 거대분자 종보다 더 풍부함)인 조성물을 지칭하며, 일반적으로 대상 종이 몰 또는 중량%로 존재하는 거대분자 종의 적어도 약 50%를 포함하는 경우 실질적으로 정제된 조성물이다. 일반적으로, 실질적으로 순수한 CYP 및 CPR 효소 조성물은 조성물에 존재하는 몰 또는 중량%로 모든 거대분자 종의 약 60% 이상, 약 70% 이상, 약 80% 이상, 약 90% 이상, 약 95% 이상, 및 약 98% 이상을 포함할 것이다. 일부 실시양태에서, 대상 종은 본질적인 균질성으로 정제되며(즉, 오염 종이 통상적인 검출 방법에 의해 조성물에서 검출될 수 없음), 여기서 조성물은 본질적으로 단일 CYP 및 CPR 거대분자 종으로 이루어진다. 용매 종, 소분자(<500 달톤) 및 원소 이온 종은 거대분자 종으로 간주되지 않는다.
인코딩 폴리뉴클레오티드
CYP 및 CPR 효소를 인코딩하는 단리된 폴리뉴클레오티드는 효소의 발현을 제공하기 위해 다양한 방식으로 조작될 수 있다. 벡터에 삽입하기 전에 단리된 폴리뉴클레오티드의 조작이 발현 벡터에 따라 바람직하거나 필요할 수 있다. 재조합 DNA 방법을 이용하여 폴리뉴클레오티드 및 핵산 서열을 변형시키는 기술은 당업계에 잘 알려져 있다. 지침은 [Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press; 및 Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, 2006년에 업데이트]에서 제공된다.
따라서, 또 다른 측면에서, 본 개시내용은 또한 CYP 및 CPR 효소 폴리펩티드 또는 이의 변이체를 인코딩하는 폴리뉴클레오티드, 및 도입할 호스트의 유형에 따라 프로모터 및 종결자, 복제 기점 등과 같은 하나 이상의 발현 조절 영역을 포함하는 재조합 발현 벡터에 관한 것이다. 다양한 핵산 및 조절 서열을 함께 연결하여 하나 이상의 편리한 제한 부위를 포함할 수 있는 재조합 발현 벡터를 생성하여 이러한 부위에서 폴리펩티드를 인코딩하는 핵산 서열의 삽입 또는 치환을 허용할 수 있다. 재조합 발현 벡터를 생성함에 있어서, 코딩 서열을 벡터에 위치시켜 코딩 서열이 발현에 적절한 조절 서열과 작동가능하도록 연결한다.
재조합 발현 벡터는 임의의 벡터(예를 들어, 플라스미드 또는 바이러스)일 수 있으며, 이는 편리하게 재조합 DNA 절차에 적용될 수 있고 폴리뉴클레오티드 서열의 발현을 가져올 수 있다. 벡터의 선택은 전형적으로 벡터가 도입될 숙주 세포와 벡터의 호환성에 따라 달라질 것이다. 벡터는 선형 또는 폐쇄형 원형 플라스미드일 수 있다.
발현 벡터는 자율적으로 복제하는 벡터, 즉 복제가 염색체 복제와 독립적인 염색체외 독립체로서 존재하는 벡터, 예를 들어 플라스미드, 염색체외 요소, 미니-염색체 또는 인공 염색체일 수 있다. 벡터는 자가 복제를 보장하는 모든 수단을 포함할 수 있다. 대안적으로, 벡터는 숙주 세포에 도입될 때 게놈에 통합되고 통합된 염색체(들)와 함께 복제되는 것일 수 있다. 또한, 숙주 세포의 게놈에 도입될 전체 DNA를 함께 포함하는 단일 벡터 또는 플라스미드 또는 2 이상의 벡터 또는 플라스미드가 사용될 수 있다. 특히 바람직한 실시양태에서, 본 발명의 플라스미드 또는 벡터는 AOX1 프로모터 및 AOX1 종결자 서열의 조절 하에 있다.
용어 "조절 서열"은 본원에서 본 개시내용의 폴리펩티드의 발현에 필요하거나 유리한 모든 구성요소를 포함하는 것으로 정의된다. 각각의 조절 서열은 폴리펩티드를 인코딩하는 핵산 서열에 고유하거나 외래일 수 있다. 이러한 조절 서열은 리더, 폴리아데닐화 서열, 프로펩티드 서열, 프로모터, 신호 펩티드 서열 및 전사 종결자를 포함하지만 이에 제한되지 않는다. 조절 서열에는 최소한 프로모터, 전사 및 번역 중지 신호, 리보솜 결합 부위(번역 중지)가 포함된다. 조절 서열은 폴리펩티드를 인코딩하는 핵산 서열의 코딩 영역과 조절 서열의 결찰을 용이하게 하는 특정 제한 부위를 도입할 목적으로 링커와 함께 제공될 수 있다.
본원에서 용어 "작동 가능하게 연결된"은 조절 서열이 폴리뉴클레오티드 및/또는 폴리펩티드의 발현을 지시하도록 DNA 서열의 코딩 서열에 대한 위치에 조절 서열이 적절하게 배치된 구성으로 정의된다. 조절 서열은 적절한 프로모터 서열일 수 있다. "프로모터 서열"은 코딩 영역의 발현을 위해 숙주 세포에 의해 인식되는 핵산 서열이다. 프로모터 서열은 폴리펩티드의 발현을 매개하는 전사 조절 서열을 포함한다. 프로모터는 돌연변이, 절두 및 혼성 프로모터를 포함하여 선택된 숙주 세포에서 전사 활성을 나타내는 임의의 핵산 서열일 수 있고, 숙주 세포에 동종 또는 이종인 세포외 또는 세포내 폴리펩티드를 인코딩하는 유전자로부터 수득될 수 있다.
조절 서열은 또한 전사를 종결시키기 위해 숙주 세포에 의해 인식되는 서열인 적합한 전사 종결자 서열일 수 있다. 종결자 서열은 폴리펩티드를 인코딩하는 핵산 서열의 3' 말단에 작동가능하게 연결된다. 선택된 숙주 세포에서 기능적인 임의의 종결자가 본 발명에 사용될 수 있다.
CYP 및 CPR 폴리펩티드의 발현을 위한 숙주 세포
또 다른 측면에서, 본 개시내용은 본 개시내용의 CYP 및 CPR 효소를 인코딩하는 폴리뉴클레오티드를 포함하는 숙주 세포를 제공하며, 상기 폴리뉴클레오티드는 숙주 세포에서 CYP 및 CPR 효소의 발현을 위한 하나 이상의 조절 서열에 작동가능하게 연결된다. 본 발명의 발현 벡터에 의해 인코딩되는 CYP 및 CPR 효소를 발현하는 데 사용하기 위한 숙주 세포는 당업계에 잘 알려져 있으며 특히 본 발명의 효모 세포(예를 들어, Saccharomyces cerevisiae 또는 Pichia pastoris)를 포함한다. 하나의 특정 실시양태에서, 본 발명의 방법은 CYP 및 CPR 효소를 발현하는 전세포, 또는 이러한 세포의 추출물 또는 용해물로 수행되며, 여기서 전세포 또는 이러한 전세포의 추출물 또는 용해물은 Pichia pastoris 및 Saccharomyces cerevisiae로부터 선택된다. 전술한 숙주 세포에 대한 적절한 배양 배지 및 성장 조건은 당업계에 잘 알려져 있다.
CYP 및 CPR 효소의 발현을 위한 폴리뉴클레오티드는 당업계에 공지된 다양한 방법에 의해 세포 내로 도입될 수 있다. 본원에 기술된 효모의 경우, 전형적인 과정은 형질전환(예: 전기천공법 또는 염화칼슘 매개) 또는 접합, 또는 때때로 원형질체 융합에 의한 것이다. 폴리뉴클레오티드를 세포에 도입하기 위한 다양한 방법은 당업자에게 자명할 것이다.
반응 조건
본원에 기재된 입체선택적 하이드록실화를 수행함에 있어서, CYP 및 CPR 효소는 정제된 효소(고정화된 변이체 포함), 효소를 인코딩하는 유전자(들)로 형질전환된 전세포 및/또는 세포 추출물 및/또는 그러한 세포의 용해물의 형태로 반응 혼합물에 첨가될 수 있다. 조작된 CYP 및 CPR 효소를 인코딩하는 유전자(들)는 숙주 세포로 개별적으로 또는 함께 동일한 숙주 세포로 형질전환될 수 있다.
예를 들어, 일부 실시양태에서 한 세트의 숙주 세포가 CYP 효소를 인코딩하는 유전자(들)로 형질전환될 수 있고 또 다른 세트는 CPR 효소를 인코딩하는 유전자(들)로 형질전환될 수 있다. 양 세트의 형질전환된 세포는 전세포의 형태로, 또는 그로부터 유도된 용해물 또는 추출물의 형태로 반응 혼합물에서 함께 사용될 수 있다. 다른 실시양태에서, 숙주 세포는 조작된 CYP 및 CPR 효소 둘 모두를 인코딩하는 유전자(들)로 형질전환될 수 있다.
CYP 및 CPR 효소를 인코딩하는 유전자(들)로 형질전환된 전세포, 또는 세포 추출물 및/또는 이의 용해물은 고체(예를 들어, 동결건조, 분무 건조, 고정화 등) 또는 반고체(예: 미정제 페이스트)를 포함하는 다양한 상이한 형태로 사용될 수 있다. 세포 추출물 또는 세포 용해물은 침전(황산암모늄, 폴리에틸렌이민, 열 처리 등)에 이어 동결건조 전에 탈염 절차(예: 한외여과, 투석 등)에 의해 부분적으로 정제될 수 있다.
하이드록실화 반응에 사용되는 반응물의 양은 일반적으로 사용되는 CYP 및 CPR 효소 기질의 양에 따라 달라질 것이다. 다음 지침을 사용하여 사용할 CYP 및 CPR 효소의 양을 결정할 수 있다. 일반적으로, 스테롤 기질은 약 50 mg/리터 내지 약 5 g/리터의 하이드록실라제 시스템을 사용하여 약 1 내지 20 g/리터의 농도로 사용된다. 반응 혼합물에서 스테롤 대 하이드록실라제 시스템의 중량비는 일반적으로 약 10:1 내지 200:1이다. 당업자는 원하는 수준의 생산성 및 생산 규모에 맞추기 위해 이러한 양을 변경하는 방법을 쉽게 알 수 있을 것이다.
반응물의 첨가 순서는 중요하지 않다. 반응물은 동시에 용매(예를 들어, 단상 용매, 2상 수성 공용매 시스템 등)에 함께 첨가될 수 있거나, 대안적으로 반응물의 일부가 별도로 첨가될 수 있고 일부는 상이한 시점에 함께 첨가될 수 있다. 예를 들어, 하이드록실라제 시스템이 먼저 용매에 첨가될 수 있다. 그러나, 바람직하게는 효소 제제는 마지막에 첨가된다.
본원에 기술된 CYP 및 CPR 효소-촉매 반응을 수행하기 위한 적합한 조건은 실험 pH 및 온도에서 CYP 및 CPR 효소 및 스테롤 기질을 접촉시키고, 예를 들어 본원에 제공된 실시예에 기술된 방법을 사용하여 생성물을 검출하는 것을 포함해 다양한 조건을 포함한다.
본원에 기재된 하이드록실라제-촉매화 반응은 일반적으로 용매 중에서 수행된다. 물이 가장 바람직하지만, 에틸 아세테이트, 부틸 아세테이트, 1-옥탄올, 헵탄, 옥탄, 메틸 t-부틸 에테르(MTBE), 톨루엔 등과 같은 유기 용매 및 1-에틸 4-메틸이미다졸륨 테트라플루오로보레이트, 1-부틸-3-메틸이미다졸륨 테트라플루오로보레이트, 1-부틸-3-메틸이미다졸륨 헥사플루오로포스페이트 등과 같은 이온성 액체가 특정 상황에서 단독으로 또는 물과 함께 사용될 수 있다. 바람직한 실시양태에서, 물 및 수성 공용매 시스템을 포함하는 수성 용매가 사용된다. 용매 시스템은 바람직하게는 50%, 75%, 90%, 95% 또는 98% 초과의 물이고, 일 실시양태에서는 물 100%이다.
하이드록실화 과정 동안 반응 혼합물의 pH는 변할 수 있다. 반응 혼합물의 pH는 반응 과정 동안 산 또는 염기의 첨가에 의해 원하는 pH로 또는 원하는 pH 범위 내에서 유지될 수 있다. 대안적으로, pH는 완충액을 포함하는 용매를 사용하여 조절될 수 있다. 원하는 pH 범위를 유지하기 위한 적합한 완충액은 당업계에 공지되어 있고, 예를 들어 인산염 완충액, 트리에탄올아민 완충액 등을 포함한다. 완충 및 산 또는 염기 첨가의 조합이 또한 사용될 수 있다.
하이드록실화는 전형적으로 약 15 ℃ 내지 약 75 ℃ 범위의 온도에서 수행된다. 일부 실시양태에서, 반응은 약 20 ℃ 내지 약 55 ℃ 범위의 온도에서 수행된다. 또 다른 실시양태에서, 반응은 약 20 ℃ 내지 약 45 ℃ 범위의 온도에서 수행된다. 반응은 또한 주변 조건 하에서 수행될 수 있다.
반응은 일반적으로 기질의 하이드록실화가 본질적으로 완료되거나 거의 완료될 때까지 진행된다. 기질에서 생성물로의 하이드록실화는 기질 및/또는 생성물을 검출함으로써 공지된 방법을 사용하여 모니터링할 수 있다. 적합한 방법은 기체 크로마토그래피, HPLC 등을 포함한다. 반응 혼합물에서 생성된 스테롤 하이드록실화 생성물의 전환 수율은 일반적으로 약 50% 초과, 약 60% 초과, 약 70% 초과, 약 80% 초과, 90% 초과, 심지어는 약 97% 초과일 수 있다.
하이드록실화 생성물을 반응 혼합물로부터 회수할 수 있고 임의로 당업자에게 공지된 방법을 사용하여 추가로 정제할 수 있다. 하이드록실라제 시스템으로부터의 단리를 위한 크로마토그래피 기술에는 특히 역상 크로마토그래피 고성능 액체 크로마토그래피, 이온 교환 크로마토그래피, 겔 전기영동 및 친화성 크로마토그래피가 포함된다. 특정 스테롤을 정제하기 위한 조건은 부분적으로 순전하, 소수성, 친수성, 분자량, 분자 형태 등과 같은 인자에 의존할 것이며, 당업자에게 자명할 것이다. 생성물 정제를 위한 바람직한 방법은 유기 용매로의 추출 및 후속 결정화를 포함한다.
실시예
다음 실시예에서는 수치(예: 양, 온도 등)에 대한 정확성을 보장하기 위해 노력했지만 일부 오차 및 편차가 고려되어야 한다. 하기 실시예는 본원에 청구된 방법이 어떻게 제조되고 평가되는지에 대한 완전한 개시 및 설명을 당업자에게 제공하기 위해 제시되며, 순전히 본 발명을 예시하기 위한 것이지 발명가가 자신의 발명으로 간주하는 범위를 제한하려는 의도는 아니다.
실시예 1 내지 15에 대한 일반적인 방법
DNA의 단리, 취급 및 조작은 표준 방법(Green and Sambrook, 2012)을 사용하여 수행하였으며, 여기에는 제한 효소로의 소화, PCR, 클로닝 기술 및 박테리아 세포의 형질전환이 포함된다. 예를 들어, [Green, M.R., Sambrook, J., 2012. Molecular Cloning: A Laboratory Manual, Fourth Edition, 4 Lab edition. ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.]가 참조된다.
합성 DNA는 Eurofins Scientific SE(벨기에 브뤼셀), Integrated DNA Technologies(아이오와 코랄빌), Genewiz(Brooks Life Sciences Company)(뉴저지 사우스 플레인필드) 또는 Twist Bioscience(캘리포니아 샌프란시스코)와 같은 상업적 공급업체에서 주문하였다. 유전자는 실시예에 설명된 대로 맞춤형 벡터로 제공받았다.
배지
2TY 배지는 16 g/L 박토-트립톤(bacto-tryptone), 10 g/L 효모 추출물 및 5 g/L NaCl을 포함하며, 오토클레이빙으로 멸균시켰다. 2TY 한천은 15 g/L 한천을 추가로 포함하였다.
YPD 배지는 10 g/L 효모 추출물, 10 g/L 박토-트립톤을 포함하고 오토클레이빙으로 멸균시켰다. 멸균 40% 글루코스 스톡 용액 50 ml/L를 사용 직전에 첨가하였다. YPD 한천 플레이트는 15 g/L 한천을 추가로 포함하였다.
BMG는 100 mM 인산칼륨, pH 7.5, 13.4 g/L YNB, 0.4 mg/L 비오틴 및 1% 글리세롤을 포함하였다.
BMM은 100 mM 인산칼륨, pH 7.5, 13.4g/L YNB, 0.4 mg/L 비오틴 및 1% 메탄올을 포함하였다.
10 g 효모 추출물 및 10 g 박토-트립톤을 dH2O 700 ml에 용해시키고 오토클레이빙으로 멸균하여 BMMY를 제조하였다. 사용 직전에 YNB 스톡 용액 100 ml, 비오틴 스톡 용액 2 ml 및 100 mM 인산칼륨 완충액(pH 6.0) 100 ml를 첨가하였다.
YNB 스톡 용액은 황산 암모늄을 포함하고 아미노산이 없는 134 g/L 효모 질소 염기로 구성되며 오토클레이빙으로 멸균시켰다.
비오틴 스톡 용액은 200 mg/L 비오틴으로 구성되며 0.2 μm 필터를 사용하여 여과 멸균시켰다.
재료
제한 효소는 New England Biolabs(매사추세츠 입스위치) 또는 Promega Corporation(위스콘신 매디슨)에서 구입하였다. 배지 구성요소, 화학물질 및 PCR 프라이머는 MilliporeSigma(미주리 세인트 루이스)에서 구입하였다. 제오신은 Thermo Fisher Scientific(매사추세츠 월쌈)에서 공급받았다.
Pichia Pastoris의 형질전환
Pichia pastoris(Komagataella phaffi NRRL Y-11430/ATCC 76273, 이후 Pichia pastoris SAND101로 지칭함)를 30 ℃에서 250 RPM으로 진탕시키면서 10 ml YPD에서 밤새 성장시켰다. 이 배양물을 사용하여 500 ml YPD를 OD600 0.1로 접종한 다음 30 ℃에서 250 RPM에서 진탕하면서 OD600 1.3-1.5로 인큐베이션하였다. 세포를 2000 xg에서 4 ℃에서 10분 동안 원심분리하여 수확하고 20 ml 1 M HEPES, pH 8.0 및 2.5 ml 1 M DTT가 보충된 100 ml YPD에 재현탁하였다. 세포를 15분 동안 진탕 없이 30 ℃에서 인큐베이션하였다. 차가운 dH2O를 500 ml의 최종 부피로 첨가하고 세포를 10분 동안 4 ℃에서 2000 xg로 원심분리하여 수확하였다. 세포를 250 ml의 차가운 dH2O로 세척하고 2000 xg에서 4 ℃에서 10분 동안 원심분리하여 수확하였다. 세포를 차가운 1M 소르비톨 20 ml로 세척하고 4 ℃에서 10분 동안 2000 xg에서 원심분리하여 수확하였다. 세포를 차가운 1M 소르비톨 500 μl에 재현탁하였다. 100 ng DNA를 수용성 세포 40 μl에 첨가하고 얼음 위에서 미리 냉각시킨 2 mm 간격 전기천공 큐벳으로 옮겼다. 세포를 1500 V, 200 Ω, 25 μF 설정을 사용하여 BTRX ECM 630 감쇠파 전기천공 시스템에서 전기천공하였다. 차가운 1M 소르비톨 1 ml를 즉시 첨가하고 혼합물을 멸균된 Eppendorf 튜브로 옮겼다. 세포를 적어도 30분 동안 250 RPM에서 진탕하면서 30 ℃에서 재생하였다. 그런 다음 세포를 적절한 항생제가 포함된 YPD 한천 플레이트에 스프레딩한 다음 30 ℃에서 2일 동안 또는 콜로니가 보일 때까지 인큐베이션하였다.
실시예 1: 서열 번호 2(FGSG_04903)를 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND102를 상업 공급자로부터 서열 번호 1을 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 3을 가진 P450 리덕타제를 인코딩하는 서열 번호 2를 가진 유전자, 이어 AOX1 종결자 서열을 포함한다. AOX1 프로모터는 플라스미드 pSAND102의 선형화를 허용하는 고유한 PmeI 제한 부위를 포함한다.
플라스미드 pSAND102를 제한 효소 PmeI로 선형화하였다. 선형화된 플라스미드를 예를 들어 상업적으로 이용 가능한 컬럼 정제 키트를 사용하여 반응 혼합물로부터 정제하였다. 균주 Pichia pastoris SAND101의 전기수용성(Electrocompetent) 세포를 PmeI 선형화 플라스미드 pSAND102로 형질전환하여 AOX1 프로모터에서 게놈에 통합될 수 있도록 하였다. 형질전환체를 100 μg/ml 누세오트리신(nourseothricin)을 함유한 YPD 한천에 플레이팅하고 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND102이다.
실시예 2: 서열 번호 5(FGSG_03175)를 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND103을 상업 공급자로부터 서열 번호 4를 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 6을 가진 P450 리덕타제를 인코딩하는 서열 번호 5를 가진 유전자, 이어 AOX1 종결자 서열을 포함한다. AOX1 프로모터는 플라스미드 pSAND103의 선형화를 허용하는 고유한 PmeI 제한 부위를 포함한다.
플라스미드 pSAND103을 제한 효소 PmeI로 선형화하였다. 선형화된 플라스미드를 예를 들어 상업적으로 이용 가능한 컬럼 정제 키트를 사용하여 반응 혼합물로부터 정제하였다. 균주 Pichia pastoris SAND101의 전기수용성 세포를 PmeI 선형화 플라스미드 pSAND103으로 형질전환하여 AOX1 프로모터에서 게놈에 통합될 수 있도록 하였다. 형질전환체를 100 μg/ml 누세오트리신을 함유한 YPD 한천에 플레이팅하고 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND103이다.
실시예 3: 서열 번호 8(FGSG_05333)을 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND104를 상업 공급자로부터 서열 번호 7을 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 9를 가진 P450를 인코딩하는 서열 번호 8을 가진 유전자, 이어 AOX1 종결자 서열을 포함한다.
균주 Pichia pastoris SAND102의 전기수용성 세포를 플라스미드 pSAND104로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND104이다.
균주 Pichia pastoris SAND103의 전기수용성 세포를 플라스미드 pSAND104로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND105이다.
실시예 4: 서열 번호 11(FGSG_02672)을 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND105를 상업 공급자로부터 서열 번호 10을 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 12를 가진 P450를 인코딩하는 서열 번호 11을 가진 유전자, 이어 AOX1 종결자 서열을 포함한다.
균주 Pichia pastoris SAND102의 전기수용성 세포를 플라스미드 pSAND105로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND106이다.
균주 Pichia pastoris SAND103의 전기수용성 세포를 플라스미드 pSAND105로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND107이다.
실시예 5: 서열 번호 14(FGSG_10695)를 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND106을 상업 공급자로부터 서열 번호 13을 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 15를 가진 P450를 인코딩하는 서열 번호 14를 가진 유전자, 이어 AOX1 종결자 서열을 포함한다.
균주 Pichia pastoris SAND102의 전기수용성 세포를 플라스미드 pSAND106으로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND108이다.
균주 Pichia pastoris SAND103의 전기수용성 세포를 플라스미드 pSAND106으로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND109이다.
실시예 6: 서열 번호 17(P450 51(1) - FGSG_04092)을 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND107을 상업 공급자로부터 서열 번호 16을 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 18을 가진 P450를 인코딩하는 서열 번호 17을 가진 유전자, 이어 AOX1 종결자 서열을 포함한다.
균주 Pichia pastoris SAND102의 전기수용성 세포를 플라스미드 pSAND107로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND110이다.
균주 Pichia pastoris SAND103의 전기수용성 세포를 플라스미드 pSAND107로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND111이다.
실시예 7: 서열 번호 20(P450 51(2) - FGSG_01000)을 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND108을 상업 공급자로부터 서열 번호 19를 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 21을 가진 P450를 인코딩하는 서열 번호 20을 가진 유전자, 이어 AOX1 종결자 서열을 포함한다.
균주 Pichia pastoris SAND102의 전기수용성 세포를 플라스미드 pSAND108로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND112이다.
균주 Pichia pastoris SAND103의 전기수용성 세포를 플라스미드 pSAND108로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND113이다.
실시예 8: 서열 번호 23(FGRAMPH1_01T05089)을 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND109를 상업 공급자로부터 서열 번호 22를 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 24를 가진 P450를 인코딩하는 서열 번호 23을 가진 유전자, 이어 AOX1 종결자 서열을 포함한다.
균주 Pichia pastoris SAND102의 전기수용성 세포를 플라스미드 pSAND109로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND114이다.
균주 Pichia pastoris SAND103의 전기수용성 세포를 플라스미드 pSAND109로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND115이다.
실시예 9: 서열 번호 26(FGRAMPH1_01T09325)을 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND110을 상업 공급자로부터 서열 번호 25를 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 27을 가진 P450를 인코딩하는 서열 번호 26을 가진 유전자, 이어 AOX1 종결자 서열을 포함한다.
균주 Pichia pastoris SAND102의 전기수용성 세포를 플라스미드 pSAND110으로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND116이다.
균주 Pichia pastoris SAND103의 전기수용성 세포를 플라스미드 pSAND110으로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND117이다.
실시예 10: 서열 번호 29(FGRAMPH1_01T21239)를 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND111을 상업 공급자로부터 서열 번호 28을 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 30을 가진 P450를 인코딩하는 서열 번호 29를 가진 유전자, 이어 AOX1 종결자 서열을 포함한다.
균주 Pichia pastoris SAND102의 전기수용성 세포를 플라스미드 pSAND111로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND118이다.
균주 Pichia pastoris SAND103의 전기수용성 세포를 플라스미드 pSAND111로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND119이다.
실시예 11: 서열 번호 32(FGSG_02672V2)를 발현할 수 있는 PICHIA PASTORIS 균주의 작제
플라스미드 pSAND112를 상업 공급자로부터 서열 번호 31을 가진 합성 DNA로 입수하였다. 간략히, 이는 AOX1 프로모터 서열, 이어 AOX1 프로모터의 조절 하에 서열 번호 33을 가진 P450를 인코딩하는 서열 번호 32를 가진 유전자, 이어 AOX1 종결자 서열을 포함한다.
균주 Pichia pastoris SAND102의 전기수용성 세포를 플라스미드 pSAND112로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND120이다.
균주 Pichia pastoris SAND103의 전기수용성 세포를 플라스미드 pSAND112로 형질전환시키고, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 YPD 한천에 플레이팅한 다음, 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주명은 Pichia pastoris SAND121이다.
실시예 12:
PICHIA PASTORIS 균주 PICHIA PASTORIS SAND104 - PICHIA PASTORIS SAND121에서 P450 및 P450 리덕타제 유전자의 발현
균주 Pichia pastoris SAND104, Pichia pastoris SAND105, Pichia pastoris SAND106, Pichia pastoris SAND107, Pichia pastoris SAND108, Pichia pastoris SAND109, Pichia pastoris SAND110, Pichia pastoris SAND111, Pichia pastoris SAND112, Pichia pastoris SAND113, Pichia pastoris SAND114, Pichia pastoris SAND115, Pichia pastoris SAND116, Pichia pastoris SAND117, Pichia pastoris SAND118, Pichia pastoris SAND119, Pichia pastoris SAND120, 및 Pichia pastoris SAND121에 의한 리토콜산의 우르소데옥시콜산으로의 전환을 표준 방법을 사용하여 유전자 발현 유도에 의해 시험하였다. 이 방법 중 하나에서, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유하는 YPD 배지에 균주의 신선한 단일 콜로니를 접종하고 30 ℃에서 250 RPM으로 진탕하면서 밤새 인큐베이션하였다. 2 mM 아미노레불린산, 100 μl/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 신선한 BMMY 배지에 1/10 부피의 하룻밤 배양액을 접종하고 OD600이 1.0에 도달할 때까지 250 RPM에서 진탕하면서 30 ℃에서 인큐베이션하였다. 메탄올을 최종 농도 0.5%(v/v)로 첨가하고, 리토콜산을 최종 농도 1 mM로 첨가한 뒤, 2-3일 동안 250 RPM에서 진탕하면서 30 ℃에서 인큐베이션을 재개하였다.
UDCA를 포함한 산물을 [X. Ma, and X. Cao, Bioresources and Bioprocessing volume 1, Article number: 5 (2014) 및 F. Tonin and I. Arends, Beilstein J Org Chem. 2018; 14: 470-483]에 기재된 바와 같은 표준 방법을 사용하여 브로스로부터 추출하였다. 한 방법에서, 배양물을 동 부피의 에틸 아세테이트로 추출하고 산을 첨가하여 pH를 4 미만으로 조정하고 에틸 아세테이트 상을 분리한 다음 증발에 의해 용매를 제거하고 관심 스테롤을 크로마토그래피를 사용하여 정제하였다.
실시예 13:
BMG 배지에서 성장한 PICHIA PASTORIS 균주 PICHIA PASTORIS SAND104 - PICHIA PASTORIS SAND121의 전세포를 사용한 LCA 전환
균주 Pichia pastoris SAND104, Pichia pastoris SAND105, Pichia pastoris SAND106, Pichia pastoris SAND107, Pichia pastoris SAND108, Pichia pastoris SAND109, Pichia pastoris SAND110, Pichia pastoris SAND111, Pichia pastoris SAND112, Pichia pastoris SAND113, Pichia pastoris SAND114, Pichia pastoris SAND115, Pichia pastoris SAND116, Pichia pastoris SAND117, Pichia pastoris SAND118, Pichia pastoris SAND119, Pichia pastoris SAND120, 및 Pichia pastoris SAND121에 의한 리토콜산의 우르소데옥시콜산으로의 전환을 [W. Lu, J. Feng, X. Chen, et al., 2019 Appl. Environ. Microbiol. 85, e01182-19]에 기재된 바와 같은 표준 방법을 사용하여 유전자 발현 유도에 의해 시험하였다. 이 방법 중 하나에서, 25 ml BMG 배지에 균주의 신선한 단일 콜로니를 접종하고 30 ℃에서 250 RPM으로 진탕하면서 10의 OD600으로 인큐베이션하였다. 세포를 4000 xg에서 5분 동안 원심분리하여 수확하고 1.0의 OD600으로 2 mM 아미노레불렌산을 함유한 BMM 배지에 현탁하였다. 배양물을 5일 동안 24시간마다 메탄올(1% v/v)을 첨가하면서 20 ℃에서 250 RPM으로 진탕하에 인큐베이션하였다.
세포를 4000 xg에서 5분 동안 원심분리에 의해 수확하고 2 mM 아미노레불린산 및 1 mM 리토콜산을 함유하는 pH 7.5의 50 mM 인산칼륨 완충액 30 ml에 재현탁하였다. 세포 현탁액을 3일 동안 24시간마다 메탄올(1% v/v)을 첨가하면서 30 ℃에서 200 RPM으로 진탕하에 인큐베이션하였다.
UDCA를 포함한 산물을 [X. Ma, and X. Cao, Bioresources and Bioprocessing volume 1, Article number: 5 (2014) 및 F. Tonin and I. Arends, Beilstein J Org Chem. 2018; 14: 470-483]에 기재된 바와 같은 표준 방법을 사용하여 브로스로부터 추출하였다. 한 방법에서, 배양물을 동 부피의 에틸 아세테이트로 추출하고 산을 첨가하여 pH를 4 미만으로 조정하고 에틸 아세테이트 상을 분리한 다음 증발에 의해 용매를 제거하고 관심 스테롤을 크로마토그래피를 사용하여 정제하였다.
실시예 14: YPD 배지에서 성장한 PICHIA PASTORIS 균주 PICHIA PASTORIS SAND104 - PICHIA PASTORIS SAND121의 전세포를 사용한 3-KCA 전환
균주 Pichia pastoris SAND104, Pichia pastoris SAND105, Pichia pastoris SAND106, Pichia pastoris SAND107, Pichia pastoris SAND108, Pichia pastoris SAND109, Pichia pastoris SAND110, Pichia pastoris SAND111, Pichia pastoris SAND112, Pichia pastoris SAND113, Pichia pastoris SAND114, Pichia pastoris SAND115, Pichia pastoris SAND116, Pichia pastoris SAND117, Pichia pastoris SAND118, Pichia pastoris SAND119, Pichia pastoris SAND120, 및 Pichia pastoris SAND121에 의한 3-케토-5-베타-콜란산(3-KCA)산의 3-케토-7-베타-히드록시-5-베타-콜란산(3-KUDCA)으로의 전환을 표준 방법을 사용하여 유전자 발현 유도에 의해 시험하였다. 이 방법 중 하나에서, 100 μg/ml 누세오트리신 및 100 μg/ml 제오신을 함유하는 YPD 배지에 균주의 신선한 단일 콜로니를 접종하고 30 ℃에서 250 RPM으로 진탕하면서 밤새 인큐베이션하였다. 2 mM 아미노레불린산, 100 μl/ml 누세오트리신 및 100 μg/ml 제오신을 함유한 신선한 BMMY 배지에 1/10 부피의 하룻밤 배양액을 접종하고 OD600이 1.0에 도달할 때까지 250 RPM에서 진탕하면서 30 ℃에서 인큐베이션하였다. 메탄올을 최종 농도 0.5%(v/v)로 첨가하고, 3-KCA를 최종 농도 1 mM로 첨가한 뒤, 2-3일 동안 250 RPM에서 진탕하면서 30 ℃에서 인큐베이션을 재개하였다.
3-KUDCA를 포함한 산물을 표준 방법을 사용하여 브로스로부터 추출하였다. 한 방법에서, 배양물을 동 부피의 에틸 아세테이트로 추출하고 산을 첨가하여 pH를 4 미만으로 조정하고 에틸 아세테이트 상을 분리한 다음 증발에 의해 용매를 제거하고 관심 스테롤을 크로마토그래피를 사용하여 정제하였다.
실시예 15: BMG 배지에서 성장한 PICHIA PASTORIS 균주 PICHIA PASTORIS SAND104 - PICHIA PASTORIS SAND121의 전세포를 사용한 3-KCA 전환
균주 Pichia pastoris SAND104, Pichia pastoris SAND105, Pichia pastoris SAND106, Pichia pastoris SAND107, Pichia pastoris SAND108, Pichia pastoris SAND109, Pichia pastoris SAND110, Pichia pastoris SAND111, Pichia pastoris SAND112, Pichia pastoris SAND113, Pichia pastoris SAND114, Pichia pastoris SAND115, Pichia pastoris SAND116, Pichia pastoris SAND117, Pichia pastoris SAND118, Pichia pastoris SAND119, Pichia pastoris SAND120, 및 Pichia pastoris SAND121에 의한 3-KCA의 3-KUDCA로의 전환을 [W. Lu, J. Feng, X. Chen, et al., 2019 Appl. Environ. Microbiol. 85, e01182-19]에 기재된 바와 같은 표준 방법을 사용하여 유전자 발현 유도에 의해 시험하였다. 이 방법에서, 25 ml BMG 배지에 균주의 신선한 단일 콜로니를 접종하고 30 ℃에서 250 RPM으로 진탕하면서 10의 OD600으로 인큐베이션하였다. 세포를 4000 xg에서 5분 동안 원심분리하여 수확하고 1.0의 OD600으로 2 mM 아미노레불렌산을 함유한 BMM 배지에 현탁하였다. 배양물을 5일 동안 24시간마다 메탄올(1% v/v)을 첨가하면서 20 ℃에서 250 RPM으로 진탕하에 인큐베이션하였다.
세포를 4000 xg에서 5분 동안 원심분리에 의해 수확하고 2 mM 아미노레불린산 및 1 mM 리토콜산을 함유하는 pH 7.5의 50 mM 인산칼륨 완충액 30 ml에 재현탁하였다. 세포 현탁액을 3일 동안 24시간마다 메탄올(1% v/v)을 첨가하면서 30 ℃에서 200 RPM으로 진탕하에 인큐베이션하였다.
3-KUDCA를 포함한 산물을 표준 방법을 사용하여 브로스로부터 추출하였다. 한 방법에서, 배양물을 동 부피의 에틸 아세테이트로 추출하고 산을 첨가하여 pH를 4 미만으로 조정하고 에틸 아세테이트 상을 분리한 다음 증발에 의해 용매를 제거하고 관심 스테롤을 크로마토그래피를 사용하여 정제하였다.
실시예 16-21을 위한 일반적인 방법
배양 추출물의 분석
실시예에 기술된 바와 같은 액체 배양물의 용매 추출 후, 샘플을 60 ℃에서 작동하고 Acquity in line 컬럼 필터 및 Waters VanGuard가 장착된 Waters XSelect CSH C18 컬럼(2.1 mm x 50 mM x 3.5 μm)을 갖춘 Agilent 1100 HPLC에서 UDCA 및 3-KUDCA의 생성에 대해 분석하였다. 이동상은 용매 A(0.005M 아세트산암모늄, 0.012% 포름산) 및 용매 B(95% 메탄올, 5% 물, 0.012% 포름산)로 구성되며 유속은 1.0 mL/분이다. 구배는 9.5분에 걸쳐 50% 용매 B에서 100% 용매 B로 실행되었다. 샘플을 212 nm에서 UV로, 그리고 질량 범위 m/z가 150-500인 전자분무 음이온 모드에서 실행되는 Waters ZQ 단일 사중극자 MS를 사용하여 MS로 분석하였다.
배지
2TY 배지는 16 g/L 박토-트립톤, 10 g/L 효모 추출물 및 5 g/L NaCl을 포함하며 오토클레이빙으로 멸균하였다. 2TY 한천은 15 g/L 한천을 추가로 포함하였다.
합성 덱스트로즈 최소 배지(Synthetic Dextrose Minimal Medium)는 아미노산이 없는 6.7 g/L 효모 질소 염기, 20 g/L 덱스트로즈 및 1.3 g/L 아미노산 드롭아웃 분말을 포함하며 오토클레이빙으로 멸균하였다. 합성 덱스트로즈 최소 한천 배지는 20 g/L 한천을 함유하였다.
합성 갈락토스 최소 배지는 아미노산이 없는 6.7 g/L 효모 질소 염기, 20 g/L 갈락토스 및 1.3 g/L 아미노산 드롭아웃 분말을 함유하고 오토클레이빙에 의해 멸균하였다. 합성 갈락토스 최소 한천 배지는 20 g/L 한천을 포함하였다.
Pichia pastoris
의 형질전환
Pichia pastoris(Komagataella phaffi NRRL Y-11430/ATCC 76273, 이하 Pichia pastoris SAND101로 지칭함)를 30 ℃에서 250 RPM으로 진탕시키면서 10 ml YPD에서 밤새 성장시켰다. 이 배양물을 사용하여 500 ml YPD를 OD600 0.1로 접종한 다음 30 ℃에서 250 RPM에서 진탕하면서 OD600 1.3-1.5로 인큐베이션하였다. 세포를 2000 xg에서 4 ℃에서 10분 동안 원심분리하여 수확하고 20 ml 1 M HEPES, pH 8.0 및 2.5 ml 1 M DTT가 보충된 100 ml YPD에 재현탁하였다. 세포를 15분 동안 진탕 없이 30 ℃에서 인큐베이션하였다. 차가운 dH2O를 500 ml의 최종 부피로 첨가하고 세포를 10분 동안 4 ℃에서 2000 xg로 원심분리하여 수확하였다. 세포를 250 ml의 차가운 dH2O로 세척하고 2000 xg에서 4 ℃에서 10분 동안 원심분리하여 수확하였다. 세포를 차가운 1M 소르비톨 20 ml로 세척하고 4 ℃에서 10분 동안 2000 xg에서 원심분리하여 수확하였다. 세포를 차가운 1M 소르비톨 500 μl에 재현탁하였다. 100 ng DNA를 수용성 세포 40 μl에 첨가하고 얼음 위에서 미리 냉각시킨 2 mm 간격 전기천공 큐벳으로 옮겼다. 세포를 1500 V, 200 Ω, 25 μF 설정을 사용하여 BTRX ECM 630 감쇠파 전기천공 시스템에서 전기천공하였다. 차가운 1M 소르비톨 1 ml를 즉시 첨가하고 혼합물을 멸균된 Eppendorf 튜브로 옮겼다. 세포를 적어도 30분 동안 250 RPM에서 진탕하면서 30 ℃에서 재생하였다. 그런 다음 세포를 적절한 항생제가 포함된 YPD 한천 플레이트에 스프레딩한 다음 30 ℃에서 2일 동안 또는 콜로니가 보일 때까지 인큐베이션하였다.
Saccharomyces cerevisiae
의
형질전환
Saccharomyces cerevisiae YPH499(Agilent)를 30 ℃에서 250 RPM으로 진탕하면서 10 mL YPD에서 밤새 성장시켰다. 이 배양물을 사용하여 500 mL YPD를 OD600 0.1로 접종한 다음 30 ℃에서 250 RPM에서 진탕하면서 OD600 1.3-1.5로 인큐베이션하였다. 세포를 2000 xg에서 4 ℃에서 10분 동안 원심분리하여 수확하고 20 ml 1 M HEPES, pH 8.0 및 2.5 ml 1 M DTT가 보충된 100 ml YPD에 재현탁하였다. 세포를 15분 동안 진탕 없이 30 ℃에서 인큐베이션하였다. 차가운 dH2O를 500 ml의 최종 부피로 첨가하고 세포를 10분 동안 4 ℃에서 2000 xg로 원심분리하여 수확하였다. 세포를 250 ml의 차가운 dH2O로 세척하고 2000 xg에서 4 ℃에서 10분 동안 원심분리하여 수확하였다. 세포를 차가운 1M 소르비톨 20 ml로 세척하고 4 ℃에서 10분 동안 2000 xg에서 원심분리하여 수확하였다. 세포를 차가운 1M 소르비톨 500 μl에 재현탁하였다. 100 ng DNA를 수용성 세포 40 μl에 첨가하고 얼음 위에서 미리 냉각시킨 2 mm 간격 전기천공 큐벳으로 옮겼다. 세포를 1500 V, 200 Ω, 25 μF 설정을 사용하여 BTRX ECM 630 감쇠파 전기천공 시스템에서 전기천공하였다. 차가운 1M 소르비톨 1 ml를 즉시 첨가하고 혼합물을 멸균된 Eppendorf 튜브로 옮겼다. 세포를 적어도 30분 동안 250 RPM에서 진탕하면서 30 ℃에서 재생하였다. 그런 다음 세포를 적절한 항생제가 포함된 YPD 한천 플레이트에 스프레딩한 다음 30 ℃에서 3일 동안 또는 콜로니가 보일 때까지 인큐베이션하였다.
실시예 16: 서열 번호 2 및 서열 번호 32를 발현할 수 있는 Pichia pastoris 균주의 작제.
플라스미드 pSAND101을 다음과 같이 작제하였다. 플라스미드 pPICHOLI-1(MoBiTec GmbH, 독일)을 제한 효소 BsaI 및 PciI로 절단하였다. 서열 번호 34를 합성 DNA(Integrated DNA Technologies)로 주문하고 융합 클로닝(Takara Bio)에 의해 절단된 pPICHOLI-1에 삽입한 후 표준 방법을 사용하여 대장균의 형질전환을 진행하였다. 형질전환체를 100 μg/mL 누세오트리신을 함유하는 2TY 한천에 플레이팅하였다. pSAND101의 올바른 조립을 제한 소화에 의해 확인하였다.
플라스미드 pSAND102를 다음과 같이 작제하였다. 플라스미드 pSAND101을 제한 효소 EcoRI 및 SalI로 절단하였다. 서열 번호 35를 합성 DNA(Twist Bioscience)로 주문하고 제한 효소 EcoRI 및 SalI로 절단하였다. 소화된 합성 DNA를 표준 방법에 따라 결찰시켜 절단된 pSAND101에 삽입하였다. 대장균 형질전환체를 100 μg/mL 누세오트리신을 함유하는 2TY 한천에 플레이팅하였다. pSAND102의 올바른 조립을 제한 소화에 의해 확인하였다.
플라스미드 pSAND112를 다음과 같이 작제하였다. 플라스미드 pPICHOLI-1을 제한 효소 EcoRI 및 SalI로 절단하였다. 서열 번호 36을 합성 DNA(Twist Bioscience)로 주문하고 제한 효소 EcoRI 및 SalI로 절단하였다. 소화된 합성 DNA를 표준 방법에 따라 결찰시켜 절단된 pPICHOLI-1에 삽입하였다. 대장균 형질전환체를 100 μg/mL 제오신을 함유하는 2TY 한천에 플레이팅하였다. pSAND112의 올바른 조립을 제한 소화에 의해 확인하였다.
플라스미드 pSAND102를 제한 효소 PmeI로 소화시켜 선형화하였다. 선형화된 pSAND102를 표준 방법을 사용하여 전기천공에 의해 Pichia pastoris SAND101을 형질전환하는 데 사용하였다. 생성된 균주를 Pichia pastoris SAND102로 명하였다.
플라스미드 pSAND112를 사용하여 표준 방법을 사용하여 전기천공에 의해 Pichia pastoris SAND102를 형질전환하는 데 사용하였다. 생성된 균주를 Pichia pastoris SAND121로 명하였다.
실시예 17: Pichia pastoris SAND121에 의한 LCA의 UDCA로의 생물전환
Pichia pastis SAND121을 250 mL 삼각 플라스크(Erlenmeyer flask)에서 100 μg/mL 제오신이 첨가된 25 mL BMG 배지에 접종하고 30 ℃에서 2일 동안 250 RPM으로 진탕하면서 인큐베이션하여 시드 배양물로 사용하였다.
시드 배양물로부터의 세포를 원심분리에 의해 수확하고 이를 사용하여 1L 삼각 플라스크에서 2 mM 5-아미노레불린산(5-ALA)을 함유하는 250 mL BMM을 OD595 1.0으로 접종하고 20 ℃에서 5일 동안 인큐베이션하여 발현 배양물로 사용하였다. 발현 배양물을 1일 동안 170 RPM으로 진탕한 다음 나머지 4일 동안 250 RPM으로 진탕시켰다. 메탄올을 매일 1% v/v의 농도로 발현 배양물에 공급하였다.
80 mL 발현 배양물로부터 세포를 원심분리에 의해 수확하고, pH 7.5에서 30 mL 여과 멸균된 인산칼륨 완충액에 현탁시키고, 250 mL 삼각 플라스크로 옮겼다. 80 mL 발현 배양물로부터 세포를 원심분리에 의해 수확하고, pH 9에서 30 mL 여과 멸균된 인산칼륨 완충액에 현탁시키고, 250 mL 삼각 플라스크로 옮겼다. 각 플라스크에 0.25 mL 5-ALA 수용액(200 mM) 및 38.8 mg/mL LCA를 함유하는 0.35 mL 메탄올을 첨가하였다. 생물전환 배양물로 사용되는 두 플라스크를 250 RPM에서 진탕하면서 30 ℃에서 인큐베이션하였다. 생물전환 배양물에 매일 0.35 mL의 메탄올을 공급한 후 2일 간 인큐베이션을 계속하였다. 그런 다음 생물전환 배양물에 1.0 mL 메탄올을 공급한 후 3일 동안 인큐베이션을 계속하였다.
500 μL 샘플을 생물전환 배양물에서 꺼내고 0.1% 포름산을 포함하는 동 부피의 에틸 아세테이트와 45분 동안 진탕하여 추출하였다. 원심분리로 상을 분리하고 용매상 20 μL를 깨끗한 튜브에 옮겨 증발시켰다. 펠렛을 20 μL의 메탄올에 용해시키고 50% 이동상 용액 A와 50% 이동상 용액 B의 혼합물에서 10배 희석하고 HPLC-MS로 분석하였다(일반적인 방법 참조). 나란히 실행된 UDCA 표준에서 볼 수 있는 바와 같이 동일한 체류 시간 및 질량 스펙트럼 프로파일을 가진 피크가 나타났다(도 1 및 도 2 참조).
나머지 생물전환 배양물을 50-mL Falcon 튜브로 옮기고 나중의 UDCA 분리를 위해 -20 ℃에서 보관하였다(실시예 18 참조).
실시예 18: UDCA의 단리 및 인증 표준과의 비교
실시예 17에 기재된 바와 같이 -20 ℃에서 보관된 생물전환 배양물을 해동하고 4500 RPM에서 15분 동안 원심분리하였다. 상청액 100 mL를 따라내고 0.1% 포름산을 포함하는 동 부피의 에틸 아세테이트와 45분 동안 교반하면서 3회 추출하였다. 유기상을 모으고 진공하에서 증발시켜 179 mg의 미정제물을 얻었다.
미정제물을 80 mL의 에틸 아세테이트에 용해시키고 진공에서 용매를 제거하여 1.5 g의 실리카겔(Merck 등급 9385, 200-400 메쉬 입자 크기)에 건조 로딩하였다. 건조된 실리카를 25 g Biotage KP-Sil Snap 카트리지(Biotage)의 사전 팩킹된 실리카 위에 부었다. 컬럼을 10 컬럼 부피에 걸쳐 10% 에틸 아세테이트에서 100% 에틸 아세테이트의 에틸 아세테이트-헥산 구배로 용출시켰다. 분획을 수집하고 LCMS로 분석하였다. 선택된 분획을 합치고 회전식 증발기에서 용매를 증발시켜 11.3 mg 중량의 추출물을 얻었다.
그런 다음 이 추출물을 아세토니트릴(0.3 mL)과 DMSO(0.7 mL)에 용해시키고 25% 아세토니트릴과 75% 물의 혼합물로 사전 평형화한 12 g Snap Ultra 카트리지(Biotage)에 주입하였다. 컬럼을 10 컬럼 부피에 걸쳐 25% 아세토니트릴에서 80% 아세토니트릴의 아세토니트릴-물 구배로 용출하였다. 분획을 수집한 다음 LC-MS로 분석하였다. 선택된 분획을 모으고 LCMS로 분석(도 3 및 도 4 참조)한 다음 동결 건조하여 3.8 mg 중량의 흰색 분말을 얻었다.
이 샘플에 대해 d4-메탄올에서 NMR 분광법을 수행하고 동시에 실행된 상업적으로 입수한 UDCA(Sigma-Aldrich) 샘플과 비교하였다. NMR 스펙트럼을 1H 및 13C에 대해 각각 500.05 MHz 및 125.75 MHz에서 작동하는 298K에서의 Bruker 500MHz DCH Cryoprobe Spectrometer에서 기록하였다. 상업적으로 이용 가능한 UDCA 표준 NMR 스펙트럼은 샘플 NMR 스펙트럼과 일치하였다(도 5, 도 6, 도 7 및 도 8 참조).
실시예 19: Pichia pastoris SAND121에 의한 3-KCA의 3-KUDCA로의 생물전환
Pichia pastoris SAND121을 사용하여 250 mL 삼각 플라스크에서 100 μg/mL 누세오트리신 및 100 μg/mL 제오신이 보충된 25 mL BMG 배지를 접종하고 250 RPM에서 3일 동안 진탕하면서 30 ℃에서 인큐베이션하였다. 0.25 mL 5-ALA 수용액(200 mM) 및 37.6 mg/mL 3-케톨리토콜산(3-KCA)을 함유하는 0.25 mL 메탄올을 배양물에 첨가한 다음, 전과 같이 1일 동안 인큐베이션을 계속하였다. 0.25 mL 메탄올을 배양물에 첨가한 다음, 전과 같이 1일 동안 인큐베이션을 계속하였다. 배양물에서 800 μL의 브로스를 꺼내고 0.1% 포름산을 함유한 동 부피의 에틸 아세테이트와 45분 동안 진탕하여 추출하였다. 원심분리로 상을 분리하고 용매상 400 μL를 깨끗한 튜브에 옮겨 증발시켰다. 펠릿을 400 μL의 메탄올에서 10분 동안 혼합하여 용해시키고 12000 xg에서 1분 동안 원심분리하였다. 15 μL의 메탄올 용액을 50% 이동상 용액 A와 50% 이동상 용액 B의 혼합물에서 10배 희석하고 HPLC-MS로 분석하였다(일반적인 방법 참조). 나란히 실행된 3-KUDCA 표준에서 볼 수 있는 바와 같이 동일한 체류 시간 및 질량 스펙트럼 프로필을 가진 피크가 나타났다(도 9 및 도 10 참조).
실시예 20: 서열 번호 2 및 서열 번호 32를 발현할 수 있는 Saccharomyces cerevisiae 균주의 작제
Gal1 프로모터의 조절 하에 서열 번호 33의 서열을 가진 P450를 인코딩하는 유전자, 및 Gal10 프로모터의 조절 하에 서열 번호 3의 서열을 가진 P450를 인코딩하는 유전자를 발현하기 위한 플라스미드 pSAND113을 다음과 같이 작제하였다.
플라스미드 pESC-URA(Agilent)를 제한 효소 EcoRI 및 SpeI로 절단하였다. 서열 번호 37 및 서열 번호 38의 프라이머를 사용하여 플라스미드 pSAND102로부터 837bp 단편을 증폭시켰다. 이 837 bp 단편을 SLiCE 클로닝 방법(Zhang et al., 2014)을 사용하여 EcoRI-SpeI 소화된 pESC-URA에 삽입하여 중간 플라스미드를 형성하였다. 삽입 및 삽입물의 정체를 제한 소화에 의해 확인하였다.
중간 플라스미드를 제한 효소 HindIII 및 SalI로 절단하였다. 서열 번호 39 및 서열 번호 40의 프라이머를 사용하여 플라스미드 pSAND110로부터 1584 bp 단편을 증폭시켰다. 이 1584 bp 단편을 SLiCE 클로닝 방법(Zhang et al., 2014)을 사용하여 HindIII-SalI 소화된 중간 플라스미드에 삽입하여 플라스미드 pSAND113을 형성하였다. 삽입 및 삽입물의 정체를 제한 소화에 의해 확인하였다.
Saccharomyces cerevisiae 균주 YPH499(Agilent)를 표준 방법을 사용하여 전기천공법에 의해 플라스미드 pSAND113으로 형질전환한 후 세포 현탁액을 우라실이 없는 합성 글루코스 최소 한천 배지에 플레이팅하고 콜로니가 보일 때까지 30 ℃에서 인큐베이션하였다. 생성된 균주를 Saccharomyces cerevisiae SAND122로 명명하였다.
실시예 21: Saccharomyces cerevisiae SAND122에 의한 LCA의 UDCA로의 생물전환
50 mL Falcon 튜브 중의 우라실이 결여된 7 mL 합성 덱스트로즈 최소 배지에 Saccharomyces cerevisiae SAND122를 접종하고 30 ℃에서 250 RPM으로 24시간 동안 진탕 배양하여 시드 배양물로 사용하였다.
시드 배양물 1 mL를 짧게 원심분리하여 세포를 수확하였다. 상청액을 버리고 나머지 세포 펠릿을 폼 마개로 막은 50 mL Falcon 튜브 중의 우라실이 없는 5 mL 합성 갈락토스 최소 배지에 현탁하였다. 이 배양물을 24시간 동안 250 RPM으로 진탕하면서 30 ℃에서 배양하여 발현 배양물로 사용하였다.
발현 배양물 4 mL를 짧게 원심분리하여 세포를 수확하였다. 상청액을 버리고 나머지 세포 펠릿을 폼 마개로 막은 50-mL Falcon 튜브 중의 5 mL 생물전환 완충액(0.1 M 인산칼륨 완충액(pH 10), 1% 갈락토스 및 650 mg/L LCA)에 현탁하였다. 이 현탁액을 250 RPM에서 72시간 동안 진탕시키면서 30 ℃에서 인큐베이션하여 생물전환 배양물로 사용하였다.
생물전환 배양물에서 500 μL 샘플을 꺼내고 0.1% 포름산을 포함하는 동 부피의 에틸 아세테이트와 45분 동안 진탕하여 추출하였다. 원심분리에 의해 상을 분리하고 용매상 20 μL를 깨끗한 튜브에 옮겨 증발시켰다. 펠렛을 20 μL의 메탄올에 용해시키고 50% 이동상 용액 A와 50% 이동상 용액 B의 혼합물에서 10배 희석한 뒤 HPLC-MS로 분석하였다(일반적인 방법 참조). 함게 실행된 UDCA 표준과 같이 동일한 체류 시간 및 질량 스펙트럼 프로필을 가진 피크가 관찰되었다(도 11 및 도 12 참조).
인용된 참고문헌
Zhang, Y., Werling, U., Ederlmann, W. (2014). Seamless Ligation Cloning Extract (SLiCE) Cloning Method. Methods in Molecular Biology 1116, 235―244.
본 출원 전반에 걸쳐 다양한 간행물이 참조되었다. 이들 공보의 간행물의 내용은 본 발명이 속하는 최신 기술을 보다 완전하게 설명하기 위해 본 출원에 참조로 포함되었다. 본 발명의 범위 또는 사상을 벗어나지 않고 본 발명에 다양한 수정 및 변형이 이루어질 수 있음은 당업자에게 자명할 것이다. 본 발명의 다른 실시양태는 상기 명세서 및 본원에 개시된 본 발명의 실시를 검토 시 당업자에게 명백할 것이다. 본 명세서 및 실시예는 단지 예시로서 간주되며, 본 발명의 실제 범위 및 사상은 하기 청구범위에 의해 나타내어지는 것으로 의도된다.
SEQUENCE LISTING
<110> Sandhill One
<120> Title Of Invention TBC
<130> Application File Reference TBC
<160> 40
<170> PatentIn version 3.5
<210> 1
<211> 4157
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 1
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg gcccttcgaa cgtccctatc acgacccgta ccgcttctgg 1080
ctacacttac tgccagcgca atcggagtat ccatattgtc taaaatgatg ttttcaacag 1140
caagtgcaga gagtccatct ccgcaaaaaa ttttttccgg tgcttttgct tccgtaaaac 1200
tcccgctgca ttcaagtgaa tacgagtccc atgacacaaa gaggcttcgt ttcaaacttc 1260
cgcaagagac tgcagtaacg ggtttaccgt tagcttactt ggttcacatt ccaccgtccc 1320
accatcaaag ggacttgact acgccggatg aacctggata catggacctg ttggtaaaga 1380
aataccccaa aggccagggc tcgacatatc tacactccct ccagcccggt gatacgttat 1440
ccttcacatc tctacccctc aaaccagctt ggaaaacaaa caattttcct cacatcactc 1500
ttatagctgg agggtgtggg atcacgccat tattcaactt ggctcaaggg atacttagag 1560
atccggccga aaaaactagg atgaccttta tttttggtgc acgatcagac gaggacgtat 1620
tactgaaaaa ggagttagat ggctttgcaa aagagttccc ggaaagattc gaggtgaaat 1680
atacagcact tttggaagag gtcctagggg gcgtgggtcg tgatactaag gtctttgtct 1740
gtgggccgaa ggagatggaa aaggcacttg taggaggccg tggcgtatta aaggaaatag 1800
gcttcgaaaa gtctcagatc catacttttt gagtcgacct gcaagatctg cggccgcgaa 1860
ttaattcgcc ttagacatga ctgttcctca gttcaagttg ggcacttacg agaagaccgg 1920
tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tgcctgagag atgcaggctt 1980
catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt gtcattttgt 2040
ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agctgatgaa tatcttgtgg 2100
taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc actcctcttc 2160
agagtacaga agattaaggc gcgccgcaag ccaagcctgc gaagaatgta gtcgagaatt 2220
gagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca gaataccctc 2280
cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag 2340
cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga 2400
agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc cctcacagac 2460
gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca 2520
ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag ttctcacatc 2580
acatccgaac ataaacaaaa atgaccactt tggatgatac tgcttacaga tacagaactt 2640
ctgttccagg tgatgctgaa gctattgaag ctttggatgg atctttcacc actgatactg 2700
ttttcagagt cactgctact ggtgatggat tcactttgag agaagttcct gttgatcctc 2760
ctttgaccaa agtttttcct gatgatgaat ctgatgatga atctgatgct ggtgaagatg 2820
gtgatccaga ttctagaact tttgttgctt atggtgatga tggtgatttg gctggatttg 2880
ttgttgtttc ttattctgga tggaacagaa gattgactgt tgaagatatt gaagttgctc 2940
cagaacatag aggtcatggt gttggaagag ctttgatggg attggcaact gagtttgcca 3000
gagaaagagg tgctggtcat ctttggttgg aagtcaccaa tgtcaatgct ccagctattc 3060
atgcttacag aagaatggga ttcactcttt gtggattgga tactgctttg tatgatggaa 3120
ctgcttctga tggagaacaa gctttgtaca tgtccatgcc atgtccttaa agtaactgac 3180
aataaaaaga ttcttgtttt caagaacttg tcatttgtat agttttttta tattgtagtt 3240
gttctatttt aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga catcatctgc 3300
ccagatgcga agttaagtgc gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg 3360
gtcgctatac tgctgtcgat tcgatactaa cgccgccatc cagtgtcgga tctgtgagca 3420
aacccgggca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 3480
ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 3540
cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 3600
ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 3660
tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 3720
gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 3780
tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 3840
gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 3900
tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 3960
ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 4020
agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 4080
gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 4140
attttggtca tgagatc 4157
<210> 2
<211> 795
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic DNA
<400> 2
atggcccttc gaacgtccct atcacgaccc gtaccgcttc tggctacact tactgccagc 60
gcaatcggag tatccatatt gtctaaaatg atgttttcaa cagcaagtgc agagagtcca 120
tctccgcaaa aaattttttc cggtgctttt gcttccgtaa aactcccgct gcattcaagt 180
gaatacgagt cccatgacac aaagaggctt cgtttcaaac ttccgcaaga gactgcagta 240
acgggtttac cgttagctta cttggttcac attccaccgt cccaccatca aagggacttg 300
actacgccgg atgaacctgg atacatggac ctgttggtaa agaaataccc caaaggccag 360
ggctcgacat atctacactc cctccagccc ggtgatacgt tatccttcac atctctaccc 420
ctcaaaccag cttggaaaac aaacaatttt cctcacatca ctcttatagc tggagggtgt 480
gggatcacgc cattattcaa cttggctcaa gggatactta gagatccggc cgaaaaaact 540
aggatgacct ttatttttgg tgcacgatca gacgaggacg tattactgaa aaaggagtta 600
gatggctttg caaaagagtt cccggaaaga ttcgaggtga aatatacagc acttttggaa 660
gaggtcctag ggggcgtggg tcgtgatact aaggtctttg tctgtgggcc gaaggagatg 720
gaaaaggcac ttgtaggagg ccgtggcgta ttaaaggaaa taggcttcga aaagtctcag 780
atccatactt tttga 795
<210> 3
<211> 264
<212> PRT
<213> Fusarium graminearum
<400> 3
Met Ala Leu Arg Thr Ser Leu Ser Arg Pro Val Pro Leu Leu Ala Thr
1 5 10 15
Leu Thr Ala Ser Ala Ile Gly Val Ser Ile Leu Ser Lys Met Met Phe
20 25 30
Ser Thr Ala Ser Ala Glu Ser Pro Ser Pro Gln Lys Ile Phe Ser Gly
35 40 45
Ala Phe Ala Ser Val Lys Leu Pro Leu His Ser Ser Glu Tyr Glu Ser
50 55 60
His Asp Thr Lys Arg Leu Arg Phe Lys Leu Pro Gln Glu Thr Ala Val
65 70 75 80
Thr Gly Leu Pro Leu Ala Tyr Leu Val His Ile Pro Pro Ser His His
85 90 95
Gln Arg Asp Leu Thr Thr Pro Asp Glu Pro Gly Tyr Met Asp Leu Leu
100 105 110
Val Lys Lys Tyr Pro Lys Gly Gln Gly Ser Thr Tyr Leu His Ser Leu
115 120 125
Gln Pro Gly Asp Thr Leu Ser Phe Thr Ser Leu Pro Leu Lys Pro Ala
130 135 140
Trp Lys Thr Asn Asn Phe Pro His Ile Thr Leu Ile Ala Gly Gly Cys
145 150 155 160
Gly Ile Thr Pro Leu Phe Asn Leu Ala Gln Gly Ile Leu Arg Asp Pro
165 170 175
Ala Glu Lys Thr Arg Met Thr Phe Ile Phe Gly Ala Arg Ser Asp Glu
180 185 190
Asp Val Leu Leu Lys Lys Glu Leu Asp Gly Phe Ala Lys Glu Phe Pro
195 200 205
Glu Arg Phe Glu Val Lys Tyr Thr Ala Leu Leu Glu Glu Val Leu Gly
210 215 220
Gly Val Gly Arg Asp Thr Lys Val Phe Val Cys Gly Pro Lys Glu Met
225 230 235 240
Glu Lys Ala Leu Val Gly Gly Arg Gly Val Leu Lys Glu Ile Gly Phe
245 250 255
Glu Lys Ser Gln Ile His Thr Phe
260
<210> 4
<211> 4400
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 4
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg aaggaggcta tcgttaagaa agatgcaagt gttgaggtag 1080
tggacagtcc aataccgaaa cctgggacga atcctaaaga ttggaaaata ccagcctttt 1140
atggaacgga gtctaattct ggagatgaca ttgccgggtt ggttgaggca gtcggggaaa 1200
atgttgtagg tttccataaa ggagacaggg tggcagcttt tcacgaaatg ctgactcccc 1260
atggagcctt tgctgaatat gcaattgcac actattacac tacgttccat attccagaca 1320
gcatatccta cgaagaggct gccacgatac ctttggctgc ctatacttcc gtatgcgcct 1380
tgtttcaaga gctacagtta ccagatcctt ggagtcccct cgccaagtta gacgagaaaa 1440
gaccgttgct cgtatacgga gcatcaacgg ctacggctgc cttcgcaata aaactggccg 1500
ctgccgcaaa cgtacaccca atcatagccg tgggctctca aagaagcgaa tttgtaaaac 1560
catttctaga tgagtcaaag ggcgacctat tagtcgatta cacgctgcac gatacagaag 1620
ataaactggt ggcagccatc caagacgcaa ttaaaaagtc aggtgcaccc gacggtaggt 1680
gttgggtcgc atacgattca gtgtcagagg acagcaccgt ccgtctggtg accaaagcaa 1740
tcgctggccc gccagatgca aatggtcgaa aacctcgaat gacaaattta ctcatgaaat 1800
ccaacgtgga aggtgtggat ccctctgtcg aaatagtaca taccaaagta tctcaggtac 1860
acgaaaaaaa cgaaaaagat cagatgttgg gcctgacgtg ggctgccgca tttagtaggg 1920
gcctaagaga gggatggctt actgctcacc cctatatcgt gggaaagaac ggactacagg 1980
gactcagtga gggtctagtg gccctgcgtg atggtaagac aaaagcaaat aagttcctca 2040
ctatactgtc tgaaactcct ggggctactg cttgagtcga cctgcaagat ctgcggccgc 2100
gaattaattc gccttagaca tgactgttcc tcagttcaag ttgggcactt acgagaagac 2160
cggtcttgct agattctaat caagaggatg tcagaatgcc atttgcctga gagatgcagg 2220
cttcattttt gatacttttt tatttgtaac ctatatagta taggattttt tttgtcattt 2280
tgtttcttct cgtacgagct tgctcctgat cagcctatct cgcagctgat gaatatcttg 2340
tggtaggggt ttgggaaaat cattcgagtt tgatgttttt cttggtattt cccactcctc 2400
ttcagagtac agaagattaa ggcgcgccgc aagccaagcc tgcgaagaat gtagtcgaga 2460
attgagcttg cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc 2520
ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt 2580
tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg 2640
cgaagcaaaa attacggctc ctcgctgcag acctgcgagc agggaaacgc tcccctcaca 2700
gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg 2760
ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata cagttctcac 2820
atcacatccg aacataaaca aaaatgacca ctttggatga tactgcttac agatacagaa 2880
cttctgttcc aggtgatgct gaagctattg aagctttgga tggatctttc accactgata 2940
ctgttttcag agtcactgct actggtgatg gattcacttt gagagaagtt cctgttgatc 3000
ctcctttgac caaagttttt cctgatgatg aatctgatga tgaatctgat gctggtgaag 3060
atggtgatcc agattctaga acttttgttg cttatggtga tgatggtgat ttggctggat 3120
ttgttgttgt ttcttattct ggatggaaca gaagattgac tgttgaagat attgaagttg 3180
ctccagaaca tagaggtcat ggtgttggaa gagctttgat gggattggca actgagtttg 3240
ccagagaaag aggtgctggt catctttggt tggaagtcac caatgtcaat gctccagcta 3300
ttcatgctta cagaagaatg ggattcactc tttgtggatt ggatactgct ttgtatgatg 3360
gaactgcttc tgatggagaa caagctttgt acatgtccat gccatgtcct taaagtaact 3420
gacaataaaa agattcttgt tttcaagaac ttgtcatttg tatagttttt ttatattgta 3480
gttgttctat tttaatcaaa tgttagcgtg atttatattt tttttcgcct cgacatcatc 3540
tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg 3600
ctggtcgcta tactgctgtc gattcgatac taacgccgcc atccagtgtc ggatctgtga 3660
gcaaacccgg gcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 3720
ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 3780
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 3840
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 3900
ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag 3960
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 4020
ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 4080
gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 4140
aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 4200
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 4260
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 4320
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 4380
gggattttgg tcatgagatc 4400
<210> 5
<211> 1038
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 5
atgaaggagg ctatcgttaa gaaagatgca agtgttgagg tagtggacag tccaataccg 60
aaacctggga cgaatcctaa agattggaaa ataccagcct tttatggaac ggagtctaat 120
tctggagatg acattgccgg gttggttgag gcagtcgggg aaaatgttgt aggtttccat 180
aaaggagaca gggtggcagc ttttcacgaa atgctgactc cccatggagc ctttgctgaa 240
tatgcaattg cacactatta cactacgttc catattccag acagcatatc ctacgaagag 300
gctgccacga tacctttggc tgcctatact tccgtatgcg ccttgtttca agagctacag 360
ttaccagatc cttggagtcc cctcgccaag ttagacgaga aaagaccgtt gctcgtatac 420
ggagcatcaa cggctacggc tgccttcgca ataaaactgg ccgctgccgc aaacgtacac 480
ccaatcatag ccgtgggctc tcaaagaagc gaatttgtaa aaccatttct agatgagtca 540
aagggcgacc tattagtcga ttacacgctg cacgatacag aagataaact ggtggcagcc 600
atccaagacg caattaaaaa gtcaggtgca cccgacggta ggtgttgggt cgcatacgat 660
tcagtgtcag aggacagcac cgtccgtctg gtgaccaaag caatcgctgg cccgccagat 720
gcaaatggtc gaaaacctcg aatgacaaat ttactcatga aatccaacgt ggaaggtgtg 780
gatccctctg tcgaaatagt acataccaaa gtatctcagg tacacgaaaa aaacgaaaaa 840
gatcagatgt tgggcctgac gtgggctgcc gcatttagta ggggcctaag agagggatgg 900
cttactgctc acccctatat cgtgggaaag aacggactac agggactcag tgagggtcta 960
gtggccctgc gtgatggtaa gacaaaagca aataagttcc tcactatact gtctgaaact 1020
cctggggcta ctgcttga 1038
<210> 6
<211> 345
<212> PRT
<213> Fusarium graminearum
<400> 6
Met Lys Glu Ala Ile Val Lys Lys Asp Ala Ser Val Glu Val Val Asp
1 5 10 15
Ser Pro Ile Pro Lys Pro Gly Thr Asn Pro Lys Asp Trp Lys Ile Pro
20 25 30
Ala Phe Tyr Gly Thr Glu Ser Asn Ser Gly Asp Asp Ile Ala Gly Leu
35 40 45
Val Glu Ala Val Gly Glu Asn Val Val Gly Phe His Lys Gly Asp Arg
50 55 60
Val Ala Ala Phe His Glu Met Leu Thr Pro His Gly Ala Phe Ala Glu
65 70 75 80
Tyr Ala Ile Ala His Tyr Tyr Thr Thr Phe His Ile Pro Asp Ser Ile
85 90 95
Ser Tyr Glu Glu Ala Ala Thr Ile Pro Leu Ala Ala Tyr Thr Ser Val
100 105 110
Cys Ala Leu Phe Gln Glu Leu Gln Leu Pro Asp Pro Trp Ser Pro Leu
115 120 125
Ala Lys Leu Asp Glu Lys Arg Pro Leu Leu Val Tyr Gly Ala Ser Thr
130 135 140
Ala Thr Ala Ala Phe Ala Ile Lys Leu Ala Ala Ala Ala Asn Val His
145 150 155 160
Pro Ile Ile Ala Val Gly Ser Gln Arg Ser Glu Phe Val Lys Pro Phe
165 170 175
Leu Asp Glu Ser Lys Gly Asp Leu Leu Val Asp Tyr Thr Leu His Asp
180 185 190
Thr Glu Asp Lys Leu Val Ala Ala Ile Gln Asp Ala Ile Lys Lys Ser
195 200 205
Gly Ala Pro Asp Gly Arg Cys Trp Val Ala Tyr Asp Ser Val Ser Glu
210 215 220
Asp Ser Thr Val Arg Leu Val Thr Lys Ala Ile Ala Gly Pro Pro Asp
225 230 235 240
Ala Asn Gly Arg Lys Pro Arg Met Thr Asn Leu Leu Met Lys Ser Asn
245 250 255
Val Glu Gly Val Asp Pro Ser Val Glu Ile Val His Thr Lys Val Ser
260 265 270
Gln Val His Glu Lys Asn Glu Lys Asp Gln Met Leu Gly Leu Thr Trp
275 280 285
Ala Ala Ala Phe Ser Arg Gly Leu Arg Glu Gly Trp Leu Thr Ala His
290 295 300
Pro Tyr Ile Val Gly Lys Asn Gly Leu Gln Gly Leu Ser Glu Gly Leu
305 310 315 320
Val Ala Leu Arg Asp Gly Lys Thr Lys Ala Asn Lys Phe Leu Thr Ile
325 330 335
Leu Ser Glu Thr Pro Gly Ala Thr Ala
340 345
<210> 7
<211> 4418
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 7
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg gactgtaacc ccgactatga aaatgccacc tgggcttttt 1080
atagatttgt ccccagtaaa gaagccaata ttgtttttgt ggtattgttc gccataacca 1140
cattgcttca tgtgctgcaa ctttggagaa cacgaacgtg gtacctaatt ccactcgtag 1200
tcgggggcgt aagtgccagt ggcgaggtca taggatacat aggccgagta ttaaacacga 1260
atgaagagcc cggttgttgg accatgggcc catacataat gcagtccgtg ttgatattaa 1320
ttgctcctgc tctatttgca gcttctattt acatgatact gggccgtatt atcattctta 1380
ccgaaggcga acatcacagc ctgatccctt taaagtggtt aacgaagctt ttcgtttttg 1440
gggatgtcgc ttcatttatg ctacaatcaa gtgggggtgg cctgatggca atacaggatt 1500
taaataagat gggagagaaa attatcgttg gcggtttatt tgtgcagctt ttctttttcg 1560
gttgttttat tatagtctca gctgtgttcc atatacgaat gcttagagct ccgacgccta 1620
acagttcgca aactagggta cgatggcaaa catatttagc aactttgtac gtcactggtg 1680
tgcttatctg ggtgcgatct ttgttcagag tcattgagtt catagagggt aatgatggac 1740
acttgatgcg ttcagaggtt tgggttttcg ttttcgatgg catgttaatg ttattggtac 1800
tcgtgtggat gaactggttc catcccggtg aaatcggcct tctgataaga ggagaagagt 1860
ccataaccaa cggattggaa cttatgaaac ttggtggcag tggtcgtagg tcccgagtgg 1920
atacgatgga gtcactgggc agcggcagac accttgagga aaataccgaa agataagtcg 1980
acctgcaaga tctgcggccg cgaattaatt cgccttagac atgactgttc ctcagttcaa 2040
gttgggcact tacgagaaga ccggtcttgc tagattctaa tcaagaggat gtcagaatgc 2100
catttgcctg agagatgcag gcttcatttt tgatactttt ttatttgtaa cctatatagt 2160
ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc ttgctcctga tcagcctatc 2220
tcgcagctga tgaatatctt gtggtagggg tttgggaaaa tcattcgagt ttgatgtttt 2280
tcttggtatt tcccactcct cttcagagta cagaagatta agtgagacct tcgtttgtgc 2340
ggatccaatt aatatttact tattttggtc aaccccaaat aggttgattt catacttggt 2400
tcattcaaaa ataagtagtc ttttgagatc tttcaatatt ataataaata tactataaca 2460
gccgacttgt ttcattttcg cgaatgttcc cccagcttat cggatccccc acacaccata 2520
gcttcaaaat gtttctactc cttttttact cttccagatt ttctcggact ccgcgcatcg 2580
ccgtaccact tcaaaacacc caagcacagc atactaaatt tcccctcttt cttcctctag 2640
ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg cctcgtttct 2700
ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct tgaaattttt 2760
ttttttagtt tttttctctt tcagtgacct ccattgatat ttaagttaat aaacggtctt 2820
caatttctca agtttcagtt tcatttttct tgttctatta caactttttt tacttcttgt 2880
tcattagaaa gaaagcatag caatctaatc taaggggcgg tgttgacaat taatcatcgg 2940
catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga 3000
ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg 3060
accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg 3120
acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct 3180
gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga 3240
acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg 3300
agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact 3360
gacacgtccg acggcggccc acgggtccca ggcctcggag atccgtcccc cttttccttt 3420
gtcgatatca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct 3480
ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata 3540
gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga 3600
cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg 3660
aaggctttaa tttgcaagct ggagaccaac atgtgagcaa aaggccagca aaaggccagg 3720
aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 3780
cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 3840
gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 3900
tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg 3960
tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 4020
cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 4080
gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 4140
ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 4200
ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 4260
ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 4320
agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 4380
aacgaaaact cacgttaagg gattttggtc atgagatc 4418
<210> 8
<211> 939
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 8
atggactgta accccgacta tgaaaatgcc acctgggctt tttatagatt tgtccccagt 60
aaagaagcca atattgtttt tgtggtattg ttcgccataa ccacattgct tcatgtgctg 120
caactttgga gaacacgaac gtggtaccta attccactcg tagtcggggg cgtaagtgcc 180
agtggcgagg tcataggata cataggccga gtattaaaca cgaatgaaga gcccggttgt 240
tggaccatgg gcccatacat aatgcagtcc gtgttgatat taattgctcc tgctctattt 300
gcagcttcta tttacatgat actgggccgt attatcattc ttaccgaagg cgaacatcac 360
agcctgatcc ctttaaagtg gttaacgaag cttttcgttt ttggggatgt cgcttcattt 420
atgctacaat caagtggggg tggcctgatg gcaatacagg atttaaataa gatgggagag 480
aaaattatcg ttggcggttt atttgtgcag cttttctttt tcggttgttt tattatagtc 540
tcagctgtgt tccatatacg aatgcttaga gctccgacgc ctaacagttc gcaaactagg 600
gtacgatggc aaacatattt agcaactttg tacgtcactg gtgtgcttat ctgggtgcga 660
tctttgttca gagtcattga gttcatagag ggtaatgatg gacacttgat gcgttcagag 720
gtttgggttt tcgttttcga tggcatgtta atgttattgg tactcgtgtg gatgaactgg 780
ttccatcccg gtgaaatcgg ccttctgata agaggagaag agtccataac caacggattg 840
gaacttatga aacttggtgg cagtggtcgt aggtcccgag tggatacgat ggagtcactg 900
ggcagcggca gacaccttga ggaaaatacc gaaagataa 939
<210> 9
<211> 312
<212> PRT
<213> Fusarium graminearum
<400> 9
Met Asp Cys Asn Pro Asp Tyr Glu Asn Ala Thr Trp Ala Phe Tyr Arg
1 5 10 15
Phe Val Pro Ser Lys Glu Ala Asn Ile Val Phe Val Val Leu Phe Ala
20 25 30
Ile Thr Thr Leu Leu His Val Leu Gln Leu Trp Arg Thr Arg Thr Trp
35 40 45
Tyr Leu Ile Pro Leu Val Val Gly Gly Val Ser Ala Ser Gly Glu Val
50 55 60
Ile Gly Tyr Ile Gly Arg Val Leu Asn Thr Asn Glu Glu Pro Gly Cys
65 70 75 80
Trp Thr Met Gly Pro Tyr Ile Met Gln Ser Val Leu Ile Leu Ile Ala
85 90 95
Pro Ala Leu Phe Ala Ala Ser Ile Tyr Met Ile Leu Gly Arg Ile Ile
100 105 110
Ile Leu Thr Glu Gly Glu His His Ser Leu Ile Pro Leu Lys Trp Leu
115 120 125
Thr Lys Leu Phe Val Phe Gly Asp Val Ala Ser Phe Met Leu Gln Ser
130 135 140
Ser Gly Gly Gly Leu Met Ala Ile Gln Asp Leu Asn Lys Met Gly Glu
145 150 155 160
Lys Ile Ile Val Gly Gly Leu Phe Val Gln Leu Phe Phe Phe Gly Cys
165 170 175
Phe Ile Ile Val Ser Ala Val Phe His Ile Arg Met Leu Arg Ala Pro
180 185 190
Thr Pro Asn Ser Ser Gln Thr Arg Val Arg Trp Gln Thr Tyr Leu Ala
195 200 205
Thr Leu Tyr Val Thr Gly Val Leu Ile Trp Val Arg Ser Leu Phe Arg
210 215 220
Val Ile Glu Phe Ile Glu Gly Asn Asp Gly His Leu Met Arg Ser Glu
225 230 235 240
Val Trp Val Phe Val Phe Asp Gly Met Leu Met Leu Leu Val Leu Val
245 250 255
Trp Met Asn Trp Phe His Pro Gly Glu Ile Gly Leu Leu Ile Arg Gly
260 265 270
Glu Glu Ser Ile Thr Asn Gly Leu Glu Leu Met Lys Leu Gly Gly Ser
275 280 285
Gly Arg Arg Ser Arg Val Asp Thr Met Glu Ser Leu Gly Ser Gly Arg
290 295 300
His Leu Glu Glu Asn Thr Glu Arg
305 310
<210> 10
<211> 5072
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 10
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg gaggccgtac acgccgacgt ttcacaatac gaatatgcct 1080
tagacgtaga agtgggtaaa accgcacgac tactgccact agaccttgac tattgggtca 1140
gtggacagta cgcagctagg cttatgcact tgccgtatag tttacttggg aacgggggta 1200
agcagtaccc atacattaac cccaaaaagc cattcgaact tagcaatcag cgtgttgtac 1260
aggattttat agagaatgct cgagacatac tgactaaagg aaggtcgtta tacaaagata 1320
caccttataa agcacatacc gacctggggg atgttctggt tatacctcca gaatttgccg 1380
atgctttaaa gagcgaacga caattagatt ttacagaggt agcaagagac gatacacacg 1440
ggtacatacc gggtttcgaa ccgattggtt ctcctttcga tttagtaccc ttggtgaaca 1500
aatacctaac tagggccctg gccaaactga ccaagccgct gtgggccgaa gcctctctgg 1560
gagtcaacca tgtgttgggt acttcaacag aatggcatcc gatcaatcca ggagaggaca 1620
taatgcgaat cgtctcccgt atgtcgtcaa gaatatttat gggcgaggaa ctctgcaagg 1680
atgacgattg gctcaaggtt tctatagagt acactgtgca gttgttccaa acggcagacg 1740
agctaaggaa ctatccgaga tggacacgtc cgtacattca ttggtttctc cctagttgcc 1800
aaggagtcag gcgtaaacta caggaggcca gagatctgtt gcaaccccat atagacaggc 1860
gtaacgccgt aaaaaaggaa gcaatagctg aaggacgtcc ttcccccttc gacgatagca 1920
tcgaatggtt tgaaaatgag tacgaaggaa agtctgaccc ggcaactgaa caaattaagc 1980
tcagccttgt cgccatacac acaactacag acctgttgtc tgaaactatg tttaatattg 2040
cactgcagcc tgagttgcta ggtcctcttc gtgaggaaat agttaccgta ctatcgaccg 2100
agggtctaaa gaaaacatct ttctacaatt tgaaacttat ggactcggtt ataaaggaga 2160
gccagaggct tcgaccggtc ctattgggtg ccttcagacg aatggcactt gctgatgtaa 2220
cactgcctaa tggcgacgta attaagaaag ggacaaaaat tatctgtgat acaacccacc 2280
aatggaatcc ggagtactat ccagacgcaa gcaaattcaa tgcctacagg tttcttcaga 2340
tgaggcaaac accagggcaa gataaacgag cccacttagt ctccacatca cacgatcaaa 2400
tgggttttgg ccacggcctc cacgcttgtc cgggtagatt ctttgctgca aacgaaatta 2460
aaatagccct atgccacatg ttgttaaagt acgactggaa gctacctgag ggtgtcgttc 2520
cgaaaagtaa ggctctcggt atgtcactcc tgggagacag agaggcaaaa ttgatggtca 2580
agagaagggc tgccgagatc gatatagaca ctattggtag tgacgaatag gtcgacctgc 2640
aagatctgcg gccgcgaatt aattcgcctt agacatgact gttcctcagt tcaagttggg 2700
cacttacgag aagaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg 2760
cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga 2820
ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc tatctcgcag 2880
ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg tttttcttgg 2940
tatttcccac tcctcttcag agtacagaag attaagtgag accttcgttt gtgcggatcc 3000
aattaatatt tacttatttt ggtcaacccc aaataggttg atttcatact tggttcattc 3060
aaaaataagt agtcttttga gatctttcaa tattataata aatatactat aacagccgac 3120
ttgtttcatt ttcgcgaatg ttcccccagc ttatcggatc ccccacacac catagcttca 3180
aaatgtttct actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac 3240
cacttcaaaa cacccaagca cagcatacta aatttcccct ctttcttcct ctagggtgtc 3300
gttaattacc cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct 3360
tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaat tttttttttt 3420
agtttttttc tctttcagtg acctccattg atatttaagt taataaacgg tcttcaattt 3480
ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc ttgttcatta 3540
gaaagaaagc atagcaatct aatctaaggg gcggtgttga caattaatca tcggcatagt 3600
atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg 3660
ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc 3720
tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga 3780
ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt 3840
gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc 3900
gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg 3960
ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg 4020
tccgacggcg gcccacgggt cccaggcctc ggagatccgt cccccttttc ctttgtcgat 4080
atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc 4140
gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 4200
ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg 4260
tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct 4320
ttaatttgca agctggagac caacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 4380
aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 4440
aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4500
ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 4560
tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc 4620
agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 4680
gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 4740
tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 4800
acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 4860
tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 4920
caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 4980
aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 5040
aactcacgtt aagggatttt ggtcatgaga tc 5072
<210> 11
<211> 1593
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 11
atggaggccg tacacgccga cgtttcacaa tacgaatatg ccttagacgt agaagtgggt 60
aaaaccgcac gactactgcc actagacctt gactattggg tcagtggaca gtacgcagct 120
aggcttatgc acttgccgta tagtttactt gggaacgggg gtaagcagta cccatacatt 180
aaccccaaaa agccattcga acttagcaat cagcgtgttg tacaggattt tatagagaat 240
gctcgagaca tactgactaa aggaaggtcg ttatacaaag atacacctta taaagcacat 300
accgacctgg gggatgttct ggttatacct ccagaatttg ccgatgcttt aaagagcgaa 360
cgacaattag attttacaga ggtagcaaga gacgatacac acgggtacat accgggtttc 420
gaaccgattg gttctccttt cgatttagta cccttggtga acaaatacct aactagggcc 480
ctggccaaac tgaccaagcc gctgtgggcc gaagcctctc tgggagtcaa ccatgtgttg 540
ggtacttcaa cagaatggca tccgatcaat ccaggagagg acataatgcg aatcgtctcc 600
cgtatgtcgt caagaatatt tatgggcgag gaactctgca aggatgacga ttggctcaag 660
gtttctatag agtacactgt gcagttgttc caaacggcag acgagctaag gaactatccg 720
agatggacac gtccgtacat tcattggttt ctccctagtt gccaaggagt caggcgtaaa 780
ctacaggagg ccagagatct gttgcaaccc catatagaca ggcgtaacgc cgtaaaaaag 840
gaagcaatag ctgaaggacg tccttccccc ttcgacgata gcatcgaatg gtttgaaaat 900
gagtacgaag gaaagtctga cccggcaact gaacaaatta agctcagcct tgtcgccata 960
cacacaacta cagacctgtt gtctgaaact atgtttaata ttgcactgca gcctgagttg 1020
ctaggtcctc ttcgtgagga aatagttacc gtactatcga ccgagggtct aaagaaaaca 1080
tctttctaca atttgaaact tatggactcg gttataaagg agagccagag gcttcgaccg 1140
gtcctattgg gtgccttcag acgaatggca cttgctgatg taacactgcc taatggcgac 1200
gtaattaaga aagggacaaa aattatctgt gatacaaccc accaatggaa tccggagtac 1260
tatccagacg caagcaaatt caatgcctac aggtttcttc agatgaggca aacaccaggg 1320
caagataaac gagcccactt agtctccaca tcacacgatc aaatgggttt tggccacggc 1380
ctccacgctt gtccgggtag attctttgct gcaaacgaaa ttaaaatagc cctatgccac 1440
atgttgttaa agtacgactg gaagctacct gagggtgtcg ttccgaaaag taaggctctc 1500
ggtatgtcac tcctgggaga cagagaggca aaattgatgg tcaagagaag ggctgccgag 1560
atcgatatag acactattgg tagtgacgaa tag 1593
<210> 12
<211> 530
<212> PRT
<213> Fusarium graminearum
<400> 12
Met Glu Ala Val His Ala Asp Val Ser Gln Tyr Glu Tyr Ala Leu Asp
1 5 10 15
Val Glu Val Gly Lys Thr Ala Arg Leu Leu Pro Leu Asp Leu Asp Tyr
20 25 30
Trp Val Ser Gly Gln Tyr Ala Ala Arg Leu Met His Leu Pro Tyr Ser
35 40 45
Leu Leu Gly Asn Gly Gly Lys Gln Tyr Pro Tyr Ile Asn Pro Lys Lys
50 55 60
Pro Phe Glu Leu Ser Asn Gln Arg Val Val Gln Asp Phe Ile Glu Asn
65 70 75 80
Ala Arg Asp Ile Leu Thr Lys Gly Arg Ser Leu Tyr Lys Asp Thr Pro
85 90 95
Tyr Lys Ala His Thr Asp Leu Gly Asp Val Leu Val Ile Pro Pro Glu
100 105 110
Phe Ala Asp Ala Leu Lys Ser Glu Arg Gln Leu Asp Phe Thr Glu Val
115 120 125
Ala Arg Asp Asp Thr His Gly Tyr Ile Pro Gly Phe Glu Pro Ile Gly
130 135 140
Ser Pro Phe Asp Leu Val Pro Leu Val Asn Lys Tyr Leu Thr Arg Ala
145 150 155 160
Leu Ala Lys Leu Thr Lys Pro Leu Trp Ala Glu Ala Ser Leu Gly Val
165 170 175
Asn His Val Leu Gly Thr Ser Thr Glu Trp His Pro Ile Asn Pro Gly
180 185 190
Glu Asp Ile Met Arg Ile Val Ser Arg Met Ser Ser Arg Ile Phe Met
195 200 205
Gly Glu Glu Leu Cys Lys Asp Asp Asp Trp Leu Lys Val Ser Ile Glu
210 215 220
Tyr Thr Val Gln Leu Phe Gln Thr Ala Asp Glu Leu Arg Asn Tyr Pro
225 230 235 240
Arg Trp Thr Arg Pro Tyr Ile His Trp Phe Leu Pro Ser Cys Gln Gly
245 250 255
Val Arg Arg Lys Leu Gln Glu Ala Arg Asp Leu Leu Gln Pro His Ile
260 265 270
Asp Arg Arg Asn Ala Val Lys Lys Glu Ala Ile Ala Glu Gly Arg Pro
275 280 285
Ser Pro Phe Asp Asp Ser Ile Glu Trp Phe Glu Asn Glu Tyr Glu Gly
290 295 300
Lys Ser Asp Pro Ala Thr Glu Gln Ile Lys Leu Ser Leu Val Ala Ile
305 310 315 320
His Thr Thr Thr Asp Leu Leu Ser Glu Thr Met Phe Asn Ile Ala Leu
325 330 335
Gln Pro Glu Leu Leu Gly Pro Leu Arg Glu Glu Ile Val Thr Val Leu
340 345 350
Ser Thr Glu Gly Leu Lys Lys Thr Ser Phe Tyr Asn Leu Lys Leu Met
355 360 365
Asp Ser Val Ile Lys Glu Ser Gln Arg Leu Arg Pro Val Leu Leu Gly
370 375 380
Ala Phe Arg Arg Met Ala Leu Ala Asp Val Thr Leu Pro Asn Gly Asp
385 390 395 400
Val Ile Lys Lys Gly Thr Lys Ile Ile Cys Asp Thr Thr His Gln Trp
405 410 415
Asn Pro Glu Tyr Tyr Pro Asp Ala Ser Lys Phe Asn Ala Tyr Arg Phe
420 425 430
Leu Gln Met Arg Gln Thr Pro Gly Gln Asp Lys Arg Ala His Leu Val
435 440 445
Ser Thr Ser His Asp Gln Met Gly Phe Gly His Gly Leu His Ala Cys
450 455 460
Pro Gly Arg Phe Phe Ala Ala Asn Glu Ile Lys Ile Ala Leu Cys His
465 470 475 480
Met Leu Leu Lys Tyr Asp Trp Lys Leu Pro Glu Gly Val Val Pro Lys
485 490 495
Ser Lys Ala Leu Gly Met Ser Leu Leu Gly Asp Arg Glu Ala Lys Leu
500 505 510
Met Val Lys Arg Arg Ala Ala Glu Ile Asp Ile Asp Thr Ile Gly Ser
515 520 525
Asp Glu
530
<210> 13
<211> 4802
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 13
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg gcagctacgc taattgtgtt cgggggtttg ctgctcttgg 1080
cctggcttgt caacatcgct tatcgatcgt tgtttcaccc cttagctaaa tttccgggcc 1140
ctaaactagc cgcagtctct gacatttggt atgctattaa gtggacatct ggtagatatc 1200
cttttataat ggaagagact catcgtaagt acggggatgt cgttagaata gcccccaatg 1260
aactatcatt cgcaacagtt caagcctatc aagacatcta cggacacgca ctaaaaggaa 1320
agaaaaagtt tgtaaaatcc aactggtatg atacagctgg tgatcaccct ggaatagttt 1380
cagtgcgtga ccctaaagag cactctcgac aaagaaagta tctatcacac gccttctctg 1440
caaagagcct gagagggcaa gaagtgctgg ttcatgggta tgtcaacttg ttcctggacc 1500
agttaaggga ccttgcattt ggggaatcgt tcgatgcagt tgctaacgga aaaactcact 1560
tttgggttag catcattata gacgccacat acactagcat gctatctgct cttaggaagc 1620
gagtaccgct agtcaacttg tacctgccat tcgtcgtgcc taaagatgct aaggccacat 1680
accaaaaaca tcgtgcactt acccgtgaaa aaatgctaaa gaggcttgat atgcctaatt 1740
ccgaggacag aggtgatttt ttcgccagtt tgctaaggaa gggtggaaac gaagtgcccg 1800
agccagagct actgcagcaa tctaacaccc tgatagtagc aggttccgaa actacagcca 1860
catgtttgac cggcatagta ttctgtctat tgtccaaccc cagctgcctt gaagccttat 1920
ctaacgaagt aaggtctaga tttcagtcgg atagtgaaat cacgggcgac gctacagctg 1980
atatgaaata cctgtctgca gttatagaag aggggttgag aatcttcccg cctgccccat 2040
ttggcctgcc cagaatttct ccaggcgccg tgattgacgg tcactatgtg ccacctggtg 2100
tgacggtgag tgtcgatcat tggaccacga aacatgaccg tcgatactgg aaagaccctt 2160
atagttttat tcccgagcga tggatcgatg aagggtttgg cgacacaaag caggcttcac 2220
aaccattttc tctaggaccc agagcatgct tggggatcaa ccttgcttac ctagaaatgc 2280
gaattatcat tgcaaaaatg gtatattgct tcgattggga actcccacga ttaatggtca 2340
gattccatcc ccataattag gtcgacctgc aagatctgcg gccgcgaatt aattcgcctt 2400
agacatgact gttcctcagt tcaagttggg cacttacgag aagaccggtc ttgctagatt 2460
ctaatcaaga ggatgtcaga atgccatttg cctgagagat gcaggcttca tttttgatac 2520
ttttttattt gtaacctata tagtatagga ttttttttgt cattttgttt cttctcgtac 2580
gagcttgctc ctgatcagcc tatctcgcag ctgatgaata tcttgtggta ggggtttggg 2640
aaaatcattc gagtttgatg tttttcttgg tatttcccac tcctcttcag agtacagaag 2700
attaagtgag accttcgttt gtgcggatcc aattaatatt tacttatttt ggtcaacccc 2760
aaataggttg atttcatact tggttcattc aaaaataagt agtcttttga gatctttcaa 2820
tattataata aatatactat aacagccgac ttgtttcatt ttcgcgaatg ttcccccagc 2880
ttatcggatc ccccacacac catagcttca aaatgtttct actccttttt tactcttcca 2940
gattttctcg gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta 3000
aatttcccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa 3060
gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc 3120
acgtttcttt ttcttgaaat tttttttttt agtttttttc tctttcagtg acctccattg 3180
atatttaagt taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct 3240
attacaactt tttttacttc ttgttcatta gaaagaaagc atagcaatct aatctaaggg 3300
gcggtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga 3360
ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg 3420
ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg 3480
acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg 3540
tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg 3600
agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga 3660
tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc 3720
acttcgtggc cgaggagcag gactgacacg tccgacggcg gcccacgggt cccaggcctc 3780
ggagatccgt cccccttttc ctttgtcgat atcatgtaat tagttatgtc acgcttacat 3840
tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc 3900
taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa 3960
atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg 4020
cttgagaagg ttttgggacg ctcgaaggct ttaatttgca agctggagac caacatgtga 4080
gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 4140
aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 4200
ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 4260
gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 4320
ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4380
ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 4440
cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 4500
attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 4560
ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 4620
aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 4680
gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 4740
tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 4800
tc 4802
<210> 14
<211> 1323
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 14
atggcagcta cgctaattgt gttcgggggt ttgctgctct tggcctggct tgtcaacatc 60
gcttatcgat cgttgtttca ccccttagct aaatttccgg gccctaaact agccgcagtc 120
tctgacattt ggtatgctat taagtggaca tctggtagat atccttttat aatggaagag 180
actcatcgta agtacgggga tgtcgttaga atagccccca atgaactatc attcgcaaca 240
gttcaagcct atcaagacat ctacggacac gcactaaaag gaaagaaaaa gtttgtaaaa 300
tccaactggt atgatacagc tggtgatcac cctggaatag tttcagtgcg tgaccctaaa 360
gagcactctc gacaaagaaa gtatctatca cacgccttct ctgcaaagag cctgagaggg 420
caagaagtgc tggttcatgg gtatgtcaac ttgttcctgg accagttaag ggaccttgca 480
tttggggaat cgttcgatgc agttgctaac ggaaaaactc acttttgggt tagcatcatt 540
atagacgcca catacactag catgctatct gctcttagga agcgagtacc gctagtcaac 600
ttgtacctgc cattcgtcgt gcctaaagat gctaaggcca cataccaaaa acatcgtgca 660
cttacccgtg aaaaaatgct aaagaggctt gatatgccta attccgagga cagaggtgat 720
tttttcgcca gtttgctaag gaagggtgga aacgaagtgc ccgagccaga gctactgcag 780
caatctaaca ccctgatagt agcaggttcc gaaactacag ccacatgttt gaccggcata 840
gtattctgtc tattgtccaa ccccagctgc cttgaagcct tatctaacga agtaaggtct 900
agatttcagt cggatagtga aatcacgggc gacgctacag ctgatatgaa atacctgtct 960
gcagttatag aagaggggtt gagaatcttc ccgcctgccc catttggcct gcccagaatt 1020
tctccaggcg ccgtgattga cggtcactat gtgccacctg gtgtgacggt gagtgtcgat 1080
cattggacca cgaaacatga ccgtcgatac tggaaagacc cttatagttt tattcccgag 1140
cgatggatcg atgaagggtt tggcgacaca aagcaggctt cacaaccatt ttctctagga 1200
cccagagcat gcttggggat caaccttgct tacctagaaa tgcgaattat cattgcaaaa 1260
atggtatatt gcttcgattg ggaactccca cgattaatgg tcagattcca tccccataat 1320
tag 1323
<210> 15
<211> 440
<212> PRT
<213> Fusarium graminearum
<400> 15
Met Ala Ala Thr Leu Ile Val Phe Gly Gly Leu Leu Leu Leu Ala Trp
1 5 10 15
Leu Val Asn Ile Ala Tyr Arg Ser Leu Phe His Pro Leu Ala Lys Phe
20 25 30
Pro Gly Pro Lys Leu Ala Ala Val Ser Asp Ile Trp Tyr Ala Ile Lys
35 40 45
Trp Thr Ser Gly Arg Tyr Pro Phe Ile Met Glu Glu Thr His Arg Lys
50 55 60
Tyr Gly Asp Val Val Arg Ile Ala Pro Asn Glu Leu Ser Phe Ala Thr
65 70 75 80
Val Gln Ala Tyr Gln Asp Ile Tyr Gly His Ala Leu Lys Gly Lys Lys
85 90 95
Lys Phe Val Lys Ser Asn Trp Tyr Asp Thr Ala Gly Asp His Pro Gly
100 105 110
Ile Val Ser Val Arg Asp Pro Lys Glu His Ser Arg Gln Arg Lys Tyr
115 120 125
Leu Ser His Ala Phe Ser Ala Lys Ser Leu Arg Gly Gln Glu Val Leu
130 135 140
Val His Gly Tyr Val Asn Leu Phe Leu Asp Gln Leu Arg Asp Leu Ala
145 150 155 160
Phe Gly Glu Ser Phe Asp Ala Val Ala Asn Gly Lys Thr His Phe Trp
165 170 175
Val Ser Ile Ile Ile Asp Ala Thr Tyr Thr Ser Met Leu Ser Ala Leu
180 185 190
Arg Lys Arg Val Pro Leu Val Asn Leu Tyr Leu Pro Phe Val Val Pro
195 200 205
Lys Asp Ala Lys Ala Thr Tyr Gln Lys His Arg Ala Leu Thr Arg Glu
210 215 220
Lys Met Leu Lys Arg Leu Asp Met Pro Asn Ser Glu Asp Arg Gly Asp
225 230 235 240
Phe Phe Ala Ser Leu Leu Arg Lys Gly Gly Asn Glu Val Pro Glu Pro
245 250 255
Glu Leu Leu Gln Gln Ser Asn Thr Leu Ile Val Ala Gly Ser Glu Thr
260 265 270
Thr Ala Thr Cys Leu Thr Gly Ile Val Phe Cys Leu Leu Ser Asn Pro
275 280 285
Ser Cys Leu Glu Ala Leu Ser Asn Glu Val Arg Ser Arg Phe Gln Ser
290 295 300
Asp Ser Glu Ile Thr Gly Asp Ala Thr Ala Asp Met Lys Tyr Leu Ser
305 310 315 320
Ala Val Ile Glu Glu Gly Leu Arg Ile Phe Pro Pro Ala Pro Phe Gly
325 330 335
Leu Pro Arg Ile Ser Pro Gly Ala Val Ile Asp Gly His Tyr Val Pro
340 345 350
Pro Gly Val Thr Val Ser Val Asp His Trp Thr Thr Lys His Asp Arg
355 360 365
Arg Tyr Trp Lys Asp Pro Tyr Ser Phe Ile Pro Glu Arg Trp Ile Asp
370 375 380
Glu Gly Phe Gly Asp Thr Lys Gln Ala Ser Gln Pro Phe Ser Leu Gly
385 390 395 400
Pro Arg Ala Cys Leu Gly Ile Asn Leu Ala Tyr Leu Glu Met Arg Ile
405 410 415
Ile Ile Ala Lys Met Val Tyr Cys Phe Asp Trp Glu Leu Pro Arg Leu
420 425 430
Met Val Arg Phe His Pro His Asn
435 440
<210> 16
<211> 5003
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 16
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg ttccaccttc tgatatatcc actatgggtc ttggtggcat 1080
tattcgccgt cattatcgca aacctgctat atcaacagct gccaagacgt cctgatgaac 1140
ccccattagt ctttcactgg ttcccatttt tcggtaatgc agtcgcctat ggattggatc 1200
cctgtggctt tttcgagaaa tgcagggaga agcacgggga cgtattcaca ttcattttat 1260
ttggtcgaaa aattgtagcc tgcctgggcg tggacggaaa cgatttcgtt ctgaactcta 1320
ggctccaaga cgccaacgct gaagaggtct acgggccact caccattcct gtatttggca 1380
gcgacgttgt ctatgactgc cctaattcga agctaatgga acaaaagaaa ttcgtcaaat 1440
tcggtttaac gcagaaggct ttggagagtc atgtgcaact tatcgagagg gaggtgttgg 1500
attacgtcga gacagacccc tcattcagtg ggagaacatc aacaatagat gttccgaaag 1560
ccatggctga gatcacaatc ttcactgcta gtcgtagttt gcagggcgag gaagtcagga 1620
gaaagctgac tgcagagttc gcagccctct accatgatct cgacctgggc tttaggccgg 1680
ttaactttct gttcccttgg ttgccgctgc cccataacag gaagcgtgac gctgcccaca 1740
tcaaaatgag ggaggtctat atggacatta taaatgacag acgaaaaggg ggaatacgta 1800
ccgaggacgg tacggatatg attgccaatt taatgggatg cacatataag aatggccagc 1860
cagttcctga taaggagatt gcacacatga tgattacgct gctcatggca ggtcaacact 1920
catccagctc ggcttcttca tggattgtcc tgcatttagc ctcgagtcct gacattacgg 1980
aagagttgta ccaagagcaa ctcgtcaatt tatcagtcaa cggggccctt cccccgcttc 2040
agtactctga cctagacaaa ttgccgttgt tacagaatgt tgtaaaggaa acgctccgag 2100
ttcattctag tattcatagt attcttagga aagttaagcg tccgatgcaa gtccccaact 2160
caccatatac tattaccacg gataaggtca tcatggcctc ccccacggtg acagcaatgt 2220
cagaagagta cttcgagaat gctaaaacgt ggaaccctca cagatgggac aacagggcta 2280
aagaggaagt ggataccgag gatgtaatag actatggata cggagctgtc agtaaaggaa 2340
caaagtctcc ttatctaccg tttggggcag ggagacatcg atgcatcggc gaaaagttcg 2400
catacgtgaa tttgggggtc atagttgcta cgcttgtgag aaacttcagg ttatcgacaa 2460
tagacggccg acctggtgtt cctgaaaccg actatacatc cctattctcc cgaccggctc 2520
agccggcctt cattcgatgg gaacgaagga aaaagattta ggtcgacctg caagatctgc 2580
ggccgcgaat taattcgcct tagacatgac tgttcctcag ttcaagttgg gcacttacga 2640
gaagaccggt cttgctagat tctaatcaag aggatgtcag aatgccattt gcctgagaga 2700
tgcaggcttc atttttgata cttttttatt tgtaacctat atagtatagg attttttttg 2760
tcattttgtt tcttctcgta cgagcttgct cctgatcagc ctatctcgca gctgatgaat 2820
atcttgtggt aggggtttgg gaaaatcatt cgagtttgat gtttttcttg gtatttccca 2880
ctcctcttca gagtacagaa gattaagtga gaccttcgtt tgtgcggatc caattaatat 2940
ttacttattt tggtcaaccc caaataggtt gatttcatac ttggttcatt caaaaataag 3000
tagtcttttg agatctttca atattataat aaatatacta taacagccga cttgtttcat 3060
tttcgcgaat gttcccccag cttatcggat cccccacaca ccatagcttc aaaatgtttc 3120
tactcctttt ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa 3180
acacccaagc acagcatact aaatttcccc tctttcttcc tctagggtgt cgttaattac 3240
ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa 3300
aaggcaataa aaatttttat cacgtttctt tttcttgaaa tttttttttt tagttttttt 3360
ctctttcagt gacctccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt 3420
cagtttcatt tttcttgttc tattacaact ttttttactt cttgttcatt agaaagaaag 3480
catagcaatc taatctaagg ggcggtgttg acaattaatc atcggcatag tatatcggca 3540
tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg 3600
tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct 3660
cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca 3720
tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg 3780
gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct 3840
ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg 3900
acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtccgacggc 3960
ggcccacggg tcccaggcct cggagatccg tccccctttt cctttgtcga tatcatgtaa 4020
ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa 4080
ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta 4140
agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt 4200
aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc 4260
aagctggaga ccaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 4320
gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 4380
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 4440
agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 4500
ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg 4560
taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 4620
gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4680
gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 4740
ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 4800
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 4860
gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 4920
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 4980
taagggattt tggtcatgag atc 5003
<210> 17
<211> 1524
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 17
atgttccacc ttctgatata tccactatgg gtcttggtgg cattattcgc cgtcattatc 60
gcaaacctgc tatatcaaca gctgccaaga cgtcctgatg aacccccatt agtctttcac 120
tggttcccat ttttcggtaa tgcagtcgcc tatggattgg atccctgtgg ctttttcgag 180
aaatgcaggg agaagcacgg ggacgtattc acattcattt tatttggtcg aaaaattgta 240
gcctgcctgg gcgtggacgg aaacgatttc gttctgaact ctaggctcca agacgccaac 300
gctgaagagg tctacgggcc actcaccatt cctgtatttg gcagcgacgt tgtctatgac 360
tgccctaatt cgaagctaat ggaacaaaag aaattcgtca aattcggttt aacgcagaag 420
gctttggaga gtcatgtgca acttatcgag agggaggtgt tggattacgt cgagacagac 480
ccctcattca gtgggagaac atcaacaata gatgttccga aagccatggc tgagatcaca 540
atcttcactg ctagtcgtag tttgcagggc gaggaagtca ggagaaagct gactgcagag 600
ttcgcagccc tctaccatga tctcgacctg ggctttaggc cggttaactt tctgttccct 660
tggttgccgc tgccccataa caggaagcgt gacgctgccc acatcaaaat gagggaggtc 720
tatatggaca ttataaatga cagacgaaaa gggggaatac gtaccgagga cggtacggat 780
atgattgcca atttaatggg atgcacatat aagaatggcc agccagttcc tgataaggag 840
attgcacaca tgatgattac gctgctcatg gcaggtcaac actcatccag ctcggcttct 900
tcatggattg tcctgcattt agcctcgagt cctgacatta cggaagagtt gtaccaagag 960
caactcgtca atttatcagt caacggggcc cttcccccgc ttcagtactc tgacctagac 1020
aaattgccgt tgttacagaa tgttgtaaag gaaacgctcc gagttcattc tagtattcat 1080
agtattctta ggaaagttaa gcgtccgatg caagtcccca actcaccata tactattacc 1140
acggataagg tcatcatggc ctcccccacg gtgacagcaa tgtcagaaga gtacttcgag 1200
aatgctaaaa cgtggaaccc tcacagatgg gacaacaggg ctaaagagga agtggatacc 1260
gaggatgtaa tagactatgg atacggagct gtcagtaaag gaacaaagtc tccttatcta 1320
ccgtttgggg cagggagaca tcgatgcatc ggcgaaaagt tcgcatacgt gaatttgggg 1380
gtcatagttg ctacgcttgt gagaaacttc aggttatcga caatagacgg ccgacctggt 1440
gttcctgaaa ccgactatac atccctattc tcccgaccgg ctcagccggc cttcattcga 1500
tgggaacgaa ggaaaaagat ttag 1524
<210> 18
<211> 507
<212> PRT
<213> Fusarium graminearum
<400> 18
Met Phe His Leu Leu Ile Tyr Pro Leu Trp Val Leu Val Ala Leu Phe
1 5 10 15
Ala Val Ile Ile Ala Asn Leu Leu Tyr Gln Gln Leu Pro Arg Arg Pro
20 25 30
Asp Glu Pro Pro Leu Val Phe His Trp Phe Pro Phe Phe Gly Asn Ala
35 40 45
Val Ala Tyr Gly Leu Asp Pro Cys Gly Phe Phe Glu Lys Cys Arg Glu
50 55 60
Lys His Gly Asp Val Phe Thr Phe Ile Leu Phe Gly Arg Lys Ile Val
65 70 75 80
Ala Cys Leu Gly Val Asp Gly Asn Asp Phe Val Leu Asn Ser Arg Leu
85 90 95
Gln Asp Ala Asn Ala Glu Glu Val Tyr Gly Pro Leu Thr Ile Pro Val
100 105 110
Phe Gly Ser Asp Val Val Tyr Asp Cys Pro Asn Ser Lys Leu Met Glu
115 120 125
Gln Lys Lys Phe Val Lys Phe Gly Leu Thr Gln Lys Ala Leu Glu Ser
130 135 140
His Val Gln Leu Ile Glu Arg Glu Val Leu Asp Tyr Val Glu Thr Asp
145 150 155 160
Pro Ser Phe Ser Gly Arg Thr Ser Thr Ile Asp Val Pro Lys Ala Met
165 170 175
Ala Glu Ile Thr Ile Phe Thr Ala Ser Arg Ser Leu Gln Gly Glu Glu
180 185 190
Val Arg Arg Lys Leu Thr Ala Glu Phe Ala Ala Leu Tyr His Asp Leu
195 200 205
Asp Leu Gly Phe Arg Pro Val Asn Phe Leu Phe Pro Trp Leu Pro Leu
210 215 220
Pro His Asn Arg Lys Arg Asp Ala Ala His Ile Lys Met Arg Glu Val
225 230 235 240
Tyr Met Asp Ile Ile Asn Asp Arg Arg Lys Gly Gly Ile Arg Thr Glu
245 250 255
Asp Gly Thr Asp Met Ile Ala Asn Leu Met Gly Cys Thr Tyr Lys Asn
260 265 270
Gly Gln Pro Val Pro Asp Lys Glu Ile Ala His Met Met Ile Thr Leu
275 280 285
Leu Met Ala Gly Gln His Ser Ser Ser Ser Ala Ser Ser Trp Ile Val
290 295 300
Leu His Leu Ala Ser Ser Pro Asp Ile Thr Glu Glu Leu Tyr Gln Glu
305 310 315 320
Gln Leu Val Asn Leu Ser Val Asn Gly Ala Leu Pro Pro Leu Gln Tyr
325 330 335
Ser Asp Leu Asp Lys Leu Pro Leu Leu Gln Asn Val Val Lys Glu Thr
340 345 350
Leu Arg Val His Ser Ser Ile His Ser Ile Leu Arg Lys Val Lys Arg
355 360 365
Pro Met Gln Val Pro Asn Ser Pro Tyr Thr Ile Thr Thr Asp Lys Val
370 375 380
Ile Met Ala Ser Pro Thr Val Thr Ala Met Ser Glu Glu Tyr Phe Glu
385 390 395 400
Asn Ala Lys Thr Trp Asn Pro His Arg Trp Asp Asn Arg Ala Lys Glu
405 410 415
Glu Val Asp Thr Glu Asp Val Ile Asp Tyr Gly Tyr Gly Ala Val Ser
420 425 430
Lys Gly Thr Lys Ser Pro Tyr Leu Pro Phe Gly Ala Gly Arg His Arg
435 440 445
Cys Ile Gly Glu Lys Phe Ala Tyr Val Asn Leu Gly Val Ile Val Ala
450 455 460
Thr Leu Val Arg Asn Phe Arg Leu Ser Thr Ile Asp Gly Arg Pro Gly
465 470 475 480
Val Pro Glu Thr Asp Tyr Thr Ser Leu Phe Ser Arg Pro Ala Gln Pro
485 490 495
Ala Phe Ile Arg Trp Glu Arg Arg Lys Lys Ile
500 505
<210> 19
<211> 5060
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 19
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg ggacttttgc aagaacttgc cgggcacccc ctagcacagc 1080
aattccagga acttcctttg ggtcaacagg ttggaattgg ctttgccgtt tttttggtcc 1140
tctcggtagt ccttaatgtt ctaaaccagc ttttattcag gaatccaaat gaaccgccaa 1200
tggtctttca ttggttccct tttgtaggga gcacaatcac gtacggtatg gatcccccta 1260
catttttcag agaaaacaga gctaaacatg gcgacgtatt cacctttatt ctcttgggaa 1320
agaaaactac ggttgctgtc ggcccggcag gaaatgactt cattttaaac ggtaagctta 1380
aggacgtatg tgctgaagag atctacacgg ttctcacaac tccagtattc ggcaaagatg 1440
tcgtttatga ttgtccaaac gctaagttaa tggaacaaaa aaagttcatg aaaattgctc 1500
tcacgacaga ggcatttaga tcttatgtgc ccataatcag ttcagaagtc agagactact 1560
ttaagagaag tccagacttc aagggaaagt ccggtattgc agatatacca aaaaagatgg 1620
ctgagattac aatattcact gcttcccacg ccctccaagg ttcggctata agaagtaagt 1680
ttgatgagag cttggcagct ttgtatcacg atctagacat gggctttaca ccgattaact 1740
ttatgttaca ctgggcaccg ctgccttgga acaggaagcg agatcacgct caaagaacgg 1800
tcgcaaaaat atatatggat acgattaaag agcgacgtgc aaaaggtaac aatgaatcag 1860
aacatgatat gatgaagcat ctgatgaact cgacgtacaa aaatggaata cgagttcccg 1920
atcacgaggt tgcacacatg atgatcgcac tccttatggc tggacagcat agttcttcaa 1980
gtactagctc gtggataatg ctgcgtttgg ctcagtatcc ccatatcatg gaggaattat 2040
atcaggagca ggtaaagaat ttaggggcag atctgcctcc attgacatat gaggatctag 2100
ccaaacttcc gttgaatcaa gctatcgtaa aagaaacttt acgtttacat gctccaatcc 2160
actctattat gagggctgtc aaatccccaa tgcccgtacc tggcaccaaa tatgtgatac 2220
cgacatcaca cacacttcta gctgcacccg gtgtctcggc tacggactct gcatttttcc 2280
caaatcctga tgaatgggac cctcacagat gggaggctga ttcccctaac tttcccagga 2340
tggcttcgaa aggagaggac gaggaaaaaa tagattatgg gtatggttta gtctcaaaag 2400
gctccgcttc gccgtatctg ccctttggag ctggtaggca ccgatgcatt ggggaacact 2460
ttgctaatgc tcaattacag acaatcgtag ctgaagtcgt gagggaattt aaatttcgta 2520
atgtcgatgg aggtcacacg ttaattgata ctgattacgc ctcattgttc tcgcgaccct 2580
tggaacccgc taacatccat tgggaacgta gacaataggt cgacctgcaa gatctgcggc 2640
cgcgaattaa ttcgccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 2700
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 2760
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 2820
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 2880
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 2940
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatccaa ttaatattta 3000
cttattttgg tcaaccccaa ataggttgat ttcatacttg gttcattcaa aaataagtag 3060
tcttttgaga tctttcaata ttataataaa tatactataa cagccgactt gtttcatttt 3120
cgcgaatgtt cccccagctt atcggatccc ccacacacca tagcttcaaa atgtttctac 3180
tcctttttta ctcttccaga ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca 3240
cccaagcaca gcatactaaa tttcccctct ttcttcctct agggtgtcgt taattacccg 3300
tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag 3360
gcaataaaaa tttttatcac gtttcttttt cttgaaattt ttttttttag tttttttctc 3420
tttcagtgac ctccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag 3480
tttcattttt cttgttctat tacaactttt tttacttctt gttcattaga aagaaagcat 3540
agcaatctaa tctaaggggc ggtgttgaca attaatcatc ggcatagtat atcggcatag 3600
tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc 3660
tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc 3720
gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca 3780
gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc 3840
tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg 3900
ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc 3960
cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtc cgacggcggc 4020
ccacgggtcc caggcctcgg agatccgtcc cccttttcct ttgtcgatat catgtaatta 4080
gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga aaaggaagga 4140
gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga 4200
acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac 4260
attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag 4320
ctggagacca acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4380
ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 4440
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4500
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4560
ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag 4620
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 4680
ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 4740
gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 4800
aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 4860
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 4920
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 4980
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 5040
gggattttgg tcatgagatc 5060
<210> 20
<211> 1581
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 20
atgggacttt tgcaagaact tgccgggcac cccctagcac agcaattcca ggaacttcct 60
ttgggtcaac aggttggaat tggctttgcc gtttttttgg tcctctcggt agtccttaat 120
gttctaaacc agcttttatt caggaatcca aatgaaccgc caatggtctt tcattggttc 180
ccttttgtag ggagcacaat cacgtacggt atggatcccc ctacattttt cagagaaaac 240
agagctaaac atggcgacgt attcaccttt attctcttgg gaaagaaaac tacggttgct 300
gtcggcccgg caggaaatga cttcatttta aacggtaagc ttaaggacgt atgtgctgaa 360
gagatctaca cggttctcac aactccagta ttcggcaaag atgtcgttta tgattgtcca 420
aacgctaagt taatggaaca aaaaaagttc atgaaaattg ctctcacgac agaggcattt 480
agatcttatg tgcccataat cagttcagaa gtcagagact actttaagag aagtccagac 540
ttcaagggaa agtccggtat tgcagatata ccaaaaaaga tggctgagat tacaatattc 600
actgcttccc acgccctcca aggttcggct ataagaagta agtttgatga gagcttggca 660
gctttgtatc acgatctaga catgggcttt acaccgatta actttatgtt acactgggca 720
ccgctgcctt ggaacaggaa gcgagatcac gctcaaagaa cggtcgcaaa aatatatatg 780
gatacgatta aagagcgacg tgcaaaaggt aacaatgaat cagaacatga tatgatgaag 840
catctgatga actcgacgta caaaaatgga atacgagttc ccgatcacga ggttgcacac 900
atgatgatcg cactccttat ggctggacag catagttctt caagtactag ctcgtggata 960
atgctgcgtt tggctcagta tccccatatc atggaggaat tatatcagga gcaggtaaag 1020
aatttagggg cagatctgcc tccattgaca tatgaggatc tagccaaact tccgttgaat 1080
caagctatcg taaaagaaac tttacgttta catgctccaa tccactctat tatgagggct 1140
gtcaaatccc caatgcccgt acctggcacc aaatatgtga taccgacatc acacacactt 1200
ctagctgcac ccggtgtctc ggctacggac tctgcatttt tcccaaatcc tgatgaatgg 1260
gaccctcaca gatgggaggc tgattcccct aactttccca ggatggcttc gaaaggagag 1320
gacgaggaaa aaatagatta tgggtatggt ttagtctcaa aaggctccgc ttcgccgtat 1380
ctgccctttg gagctggtag gcaccgatgc attggggaac actttgctaa tgctcaatta 1440
cagacaatcg tagctgaagt cgtgagggaa tttaaatttc gtaatgtcga tggaggtcac 1500
acgttaattg atactgatta cgcctcattg ttctcgcgac ccttggaacc cgctaacatc 1560
cattgggaac gtagacaata g 1581
<210> 21
<211> 526
<212> PRT
<213> Fusarium graminearum
<400> 21
Met Gly Leu Leu Gln Glu Leu Ala Gly His Pro Leu Ala Gln Gln Phe
1 5 10 15
Gln Glu Leu Pro Leu Gly Gln Gln Val Gly Ile Gly Phe Ala Val Phe
20 25 30
Leu Val Leu Ser Val Val Leu Asn Val Leu Asn Gln Leu Leu Phe Arg
35 40 45
Asn Pro Asn Glu Pro Pro Met Val Phe His Trp Phe Pro Phe Val Gly
50 55 60
Ser Thr Ile Thr Tyr Gly Met Asp Pro Pro Thr Phe Phe Arg Glu Asn
65 70 75 80
Arg Ala Lys His Gly Asp Val Phe Thr Phe Ile Leu Leu Gly Lys Lys
85 90 95
Thr Thr Val Ala Val Gly Pro Ala Gly Asn Asp Phe Ile Leu Asn Gly
100 105 110
Lys Leu Lys Asp Val Cys Ala Glu Glu Ile Tyr Thr Val Leu Thr Thr
115 120 125
Pro Val Phe Gly Lys Asp Val Val Tyr Asp Cys Pro Asn Ala Lys Leu
130 135 140
Met Glu Gln Lys Lys Phe Met Lys Ile Ala Leu Thr Thr Glu Ala Phe
145 150 155 160
Arg Ser Tyr Val Pro Ile Ile Ser Ser Glu Val Arg Asp Tyr Phe Lys
165 170 175
Arg Ser Pro Asp Phe Lys Gly Lys Ser Gly Ile Ala Asp Ile Pro Lys
180 185 190
Lys Met Ala Glu Ile Thr Ile Phe Thr Ala Ser His Ala Leu Gln Gly
195 200 205
Ser Ala Ile Arg Ser Lys Phe Asp Glu Ser Leu Ala Ala Leu Tyr His
210 215 220
Asp Leu Asp Met Gly Phe Thr Pro Ile Asn Phe Met Leu His Trp Ala
225 230 235 240
Pro Leu Pro Trp Asn Arg Lys Arg Asp His Ala Gln Arg Thr Val Ala
245 250 255
Lys Ile Tyr Met Asp Thr Ile Lys Glu Arg Arg Ala Lys Gly Asn Asn
260 265 270
Glu Ser Glu His Asp Met Met Lys His Leu Met Asn Ser Thr Tyr Lys
275 280 285
Asn Gly Ile Arg Val Pro Asp His Glu Val Ala His Met Met Ile Ala
290 295 300
Leu Leu Met Ala Gly Gln His Ser Ser Ser Ser Thr Ser Ser Trp Ile
305 310 315 320
Met Leu Arg Leu Ala Gln Tyr Pro His Ile Met Glu Glu Leu Tyr Gln
325 330 335
Glu Gln Val Lys Asn Leu Gly Ala Asp Leu Pro Pro Leu Thr Tyr Glu
340 345 350
Asp Leu Ala Lys Leu Pro Leu Asn Gln Ala Ile Val Lys Glu Thr Leu
355 360 365
Arg Leu His Ala Pro Ile His Ser Ile Met Arg Ala Val Lys Ser Pro
370 375 380
Met Pro Val Pro Gly Thr Lys Tyr Val Ile Pro Thr Ser His Thr Leu
385 390 395 400
Leu Ala Ala Pro Gly Val Ser Ala Thr Asp Ser Ala Phe Phe Pro Asn
405 410 415
Pro Asp Glu Trp Asp Pro His Arg Trp Glu Ala Asp Ser Pro Asn Phe
420 425 430
Pro Arg Met Ala Ser Lys Gly Glu Asp Glu Glu Lys Ile Asp Tyr Gly
435 440 445
Tyr Gly Leu Val Ser Lys Gly Ser Ala Ser Pro Tyr Leu Pro Phe Gly
450 455 460
Ala Gly Arg His Arg Cys Ile Gly Glu His Phe Ala Asn Ala Gln Leu
465 470 475 480
Gln Thr Ile Val Ala Glu Val Val Arg Glu Phe Lys Phe Arg Asn Val
485 490 495
Asp Gly Gly His Thr Leu Ile Asp Thr Asp Tyr Ala Ser Leu Phe Ser
500 505 510
Arg Pro Leu Glu Pro Ala Asn Ile His Trp Glu Arg Arg Gln
515 520 525
<210> 22
<211> 4994
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 22
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg ggagtcaata acgcgacttt gggcttggta tgctgtgtta 1080
tcgtcgcggt ggttgcttta gcgacgcgaa aggggcctga ctcaagagag cccccgtatg 1140
ttaaggaaag ggtcccctac ttcagtcaca tctacggact tttgaagcat ggcttacgtt 1200
attttgatgt tgtcagtgct cagcaacccc accccatatt tacgatagat atgtcgggcc 1260
agaagaacta tatagtaact tctcctgaac tggttcaagc ggtgcaacgc aacacaacgt 1320
cgttgagctt ctccccggca atgattcccg cttttcgacg catgatgggg tttgatgaag 1380
ctgggatcga gctgattttt cgggatgcac atacagaaaa aggcatgtac ggggaaattc 1440
acagggtcca gaaggcgtct ttacttccgg gaactgagtc gttggacgaa ctttgcacca 1500
ttatacgagg taagttgtta acaattgtga atgacatgcc ctcctctcaa acaatcgatc 1560
tgtacgcgtg ggtccaggac ctttacatga ggacaaataa ctctgcttgc tttggcgcaa 1620
aggatccttt tactttaaac ccgtccctga tttcgacctt ctggttgtgg gaggcgaata 1680
ttaaggtatt gttactgggg attccatggt tcctatcccc ctcaaaatat tcaactgctc 1740
agcgaactag aaacgattta gtgaacgcgt tcacgcaata cttgggtaat gatgggcttg 1800
aaactgcttg tagctttatc aaagaactat ctaatttggg gattcgtaga ggccttagta 1860
ccgaaaataa cgcgagggcg ctggtcggca gcatcctggc aatcgtgggg aatacaattc 1920
cgacaacctt ttggcttctc attcagatct tctccaggcc agacctgctc aaggagatac 1980
gttctgagct tgaggcaacg ctggaagatc catctagtcg atcagaaata tcactcaact 2040
atactgtgat cagagaaaag tgtccagttc ttatgtctac atatgaggaa attctcagga 2100
tgacgagcgg tatcgcaaca gtcaggtaca cgaatgagga tacgttaatc caggaccgct 2160
ggttgttaaa gaaaggcgca caagtgcaaa tgcccactgc cttcatacat gccgacccaa 2220
ccacgtgggg cgcagacgcg gaggtctttg atcacactag gttcttgaaa tctaaggttc 2280
tgacaaaaga gcaaaaagcg cgcagagccg ctgccttccg gccttttggg ggtggcaaca 2340
ccctgtgccc gggacggcac ttcgcgtctt atgaggtgct taccttcgcc gggagcatcc 2400
tgctcggttt tgatatgaca cccacaactg aagctttcaa cctccccgag atggataggt 2460
ctaagcttcc tctgacctcc ctgaaaccag ctggggatat caaagtcaac ctaacccgcc 2520
gttccgggtg ggagaaggtg caattcaagt gagtcgacct gcaagatctg cggccgcgaa 2580
ttaattcgcc ttagacatga ctgttcctca gttcaagttg ggcacttacg agaagaccgg 2640
tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tgcctgagag atgcaggctt 2700
catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt gtcattttgt 2760
ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agctgatgaa tatcttgtgg 2820
taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc actcctcttc 2880
agagtacaga agattaagtg agaccttcgt ttgtgcggat ccaattaata tttacttatt 2940
ttggtcaacc ccaaataggt tgatttcata cttggttcat tcaaaaataa gtagtctttt 3000
gagatctttc aatattataa taaatatact ataacagccg acttgtttca ttttcgcgaa 3060
tgttccccca gcttatcgga tcccccacac accatagctt caaaatgttt ctactccttt 3120
tttactcttc cagattttct cggactccgc gcatcgccgt accacttcaa aacacccaag 3180
cacagcatac taaatttccc ctctttcttc ctctagggtg tcgttaatta cccgtactaa 3240
aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata 3300
aaaattttta tcacgtttct ttttcttgaa attttttttt ttagtttttt tctctttcag 3360
tgacctccat tgatatttaa gttaataaac ggtcttcaat ttctcaagtt tcagtttcat 3420
ttttcttgtt ctattacaac tttttttact tcttgttcat tagaaagaaa gcatagcaat 3480
ctaatctaag gggcggtgtt gacaattaat catcggcata gtatatcggc atagtataat 3540
acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg 3600
cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact 3660
tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg 3720
tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg 3780
agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg 3840
ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg 3900
gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtccgacgg cggcccacgg 3960
gtcccaggcc tcggagatcc gtcccccttt tcctttgtcg atatcatgta attagttatg 4020
tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga 4080
caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta 4140
tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata 4200
ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg caagctggag 4260
accaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4320
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4380
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4440
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4500
ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4560
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4620
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 4680
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4740
tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 4800
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4860
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 4920
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4980
ttggtcatga gatc 4994
<210> 23
<211> 1515
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 23
atgggagtca ataacgcgac tttgggcttg gtatgctgtg ttatcgtcgc ggtggttgct 60
ttagcgacgc gaaaggggcc tgactcaaga gagcccccgt atgttaagga aagggtcccc 120
tacttcagtc acatctacgg acttttgaag catggcttac gttattttga tgttgtcagt 180
gctcagcaac cccaccccat atttacgata gatatgtcgg gccagaagaa ctatatagta 240
acttctcctg aactggttca agcggtgcaa cgcaacacaa cgtcgttgag cttctccccg 300
gcaatgattc ccgcttttcg acgcatgatg gggtttgatg aagctgggat cgagctgatt 360
tttcgggatg cacatacaga aaaaggcatg tacggggaaa ttcacagggt ccagaaggcg 420
tctttacttc cgggaactga gtcgttggac gaactttgca ccattatacg aggtaagttg 480
ttaacaattg tgaatgacat gccctcctct caaacaatcg atctgtacgc gtgggtccag 540
gacctttaca tgaggacaaa taactctgct tgctttggcg caaaggatcc ttttacttta 600
aacccgtccc tgatttcgac cttctggttg tgggaggcga atattaaggt attgttactg 660
gggattccat ggttcctatc cccctcaaaa tattcaactg ctcagcgaac tagaaacgat 720
ttagtgaacg cgttcacgca atacttgggt aatgatgggc ttgaaactgc ttgtagcttt 780
atcaaagaac tatctaattt ggggattcgt agaggcctta gtaccgaaaa taacgcgagg 840
gcgctggtcg gcagcatcct ggcaatcgtg gggaatacaa ttccgacaac cttttggctt 900
ctcattcaga tcttctccag gccagacctg ctcaaggaga tacgttctga gcttgaggca 960
acgctggaag atccatctag tcgatcagaa atatcactca actatactgt gatcagagaa 1020
aagtgtccag ttcttatgtc tacatatgag gaaattctca ggatgacgag cggtatcgca 1080
acagtcaggt acacgaatga ggatacgtta atccaggacc gctggttgtt aaagaaaggc 1140
gcacaagtgc aaatgcccac tgccttcata catgccgacc caaccacgtg gggcgcagac 1200
gcggaggtct ttgatcacac taggttcttg aaatctaagg ttctgacaaa agagcaaaaa 1260
gcgcgcagag ccgctgcctt ccggcctttt gggggtggca acaccctgtg cccgggacgg 1320
cacttcgcgt cttatgaggt gcttaccttc gccgggagca tcctgctcgg ttttgatatg 1380
acacccacaa ctgaagcttt caacctcccc gagatggata ggtctaagct tcctctgacc 1440
tccctgaaac cagctgggga tatcaaagtc aacctaaccc gccgttccgg gtgggagaag 1500
gtgcaattca agtga 1515
<210> 24
<211> 504
<212> PRT
<213> Fusarium graminearum
<400> 24
Met Gly Val Asn Asn Ala Thr Leu Gly Leu Val Cys Cys Val Ile Val
1 5 10 15
Ala Val Val Ala Leu Ala Thr Arg Lys Gly Pro Asp Ser Arg Glu Pro
20 25 30
Pro Tyr Val Lys Glu Arg Val Pro Tyr Phe Ser His Ile Tyr Gly Leu
35 40 45
Leu Lys His Gly Leu Arg Tyr Phe Asp Val Val Ser Ala Gln Gln Pro
50 55 60
His Pro Ile Phe Thr Ile Asp Met Ser Gly Gln Lys Asn Tyr Ile Val
65 70 75 80
Thr Ser Pro Glu Leu Val Gln Ala Val Gln Arg Asn Thr Thr Ser Leu
85 90 95
Ser Phe Ser Pro Ala Met Ile Pro Ala Phe Arg Arg Met Met Gly Phe
100 105 110
Asp Glu Ala Gly Ile Glu Leu Ile Phe Arg Asp Ala His Thr Glu Lys
115 120 125
Gly Met Tyr Gly Glu Ile His Arg Val Gln Lys Ala Ser Leu Leu Pro
130 135 140
Gly Thr Glu Ser Leu Asp Glu Leu Cys Thr Ile Ile Arg Gly Lys Leu
145 150 155 160
Leu Thr Ile Val Asn Asp Met Pro Ser Ser Gln Thr Ile Asp Leu Tyr
165 170 175
Ala Trp Val Gln Asp Leu Tyr Met Arg Thr Asn Asn Ser Ala Cys Phe
180 185 190
Gly Ala Lys Asp Pro Phe Thr Leu Asn Pro Ser Leu Ile Ser Thr Phe
195 200 205
Trp Leu Trp Glu Ala Asn Ile Lys Val Leu Leu Leu Gly Ile Pro Trp
210 215 220
Phe Leu Ser Pro Ser Lys Tyr Ser Thr Ala Gln Arg Thr Arg Asn Asp
225 230 235 240
Leu Val Asn Ala Phe Thr Gln Tyr Leu Gly Asn Asp Gly Leu Glu Thr
245 250 255
Ala Cys Ser Phe Ile Lys Glu Leu Ser Asn Leu Gly Ile Arg Arg Gly
260 265 270
Leu Ser Thr Glu Asn Asn Ala Arg Ala Leu Val Gly Ser Ile Leu Ala
275 280 285
Ile Val Gly Asn Thr Ile Pro Thr Thr Phe Trp Leu Leu Ile Gln Ile
290 295 300
Phe Ser Arg Pro Asp Leu Leu Lys Glu Ile Arg Ser Glu Leu Glu Ala
305 310 315 320
Thr Leu Glu Asp Pro Ser Ser Arg Ser Glu Ile Ser Leu Asn Tyr Thr
325 330 335
Val Ile Arg Glu Lys Cys Pro Val Leu Met Ser Thr Tyr Glu Glu Ile
340 345 350
Leu Arg Met Thr Ser Gly Ile Ala Thr Val Arg Tyr Thr Asn Glu Asp
355 360 365
Thr Leu Ile Gln Asp Arg Trp Leu Leu Lys Lys Gly Ala Gln Val Gln
370 375 380
Met Pro Thr Ala Phe Ile His Ala Asp Pro Thr Thr Trp Gly Ala Asp
385 390 395 400
Ala Glu Val Phe Asp His Thr Arg Phe Leu Lys Ser Lys Val Leu Thr
405 410 415
Lys Glu Gln Lys Ala Arg Arg Ala Ala Ala Phe Arg Pro Phe Gly Gly
420 425 430
Gly Asn Thr Leu Cys Pro Gly Arg His Phe Ala Ser Tyr Glu Val Leu
435 440 445
Thr Phe Ala Gly Ser Ile Leu Leu Gly Phe Asp Met Thr Pro Thr Thr
450 455 460
Glu Ala Phe Asn Leu Pro Glu Met Asp Arg Ser Lys Leu Pro Leu Thr
465 470 475 480
Ser Leu Lys Pro Ala Gly Asp Ile Lys Val Asn Leu Thr Arg Arg Ser
485 490 495
Gly Trp Glu Lys Val Gln Phe Lys
500
<210> 25
<211> 5051
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 25
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg atattcgaca acttgtcgct cagcaacacg tgggttgtgt 1080
tagtacttag cgcggtattt cttgtgcttt cccgttttat tgctccgaca atctcagaga 1140
acgagcctcc catcgtcaag ccaagggccc ccttcattgg acacattatc tccatgttga 1200
gggacggctc cgatatctac gttaatttgt ttaagcaaag aaaggaacca atagttactt 1260
tacccatgtt aaatggaaaa ttatacgtga taaattctcc agacctcata caggccgcat 1320
tgcgtaacaa tgacatctct ttcacaccgt tcattcttga gtcgtcaaaa gcaatgtggg 1380
ggttatctga taatgcgatg gcgagcatat ctgaccttgc caacttgaaa ggcggtatgc 1440
agattatcca ctcaaccctc ggaggggagt cgcttcataa attgaacata tcgtctctga 1500
gtaggttcat gacttatttg aatcgcgtta aacccggcga aaatattggt atagccgaca 1560
cttatatttg gctgagagac atgctcaccg acgctagcgc gaccgcggtc tatggtccta 1620
agaatccaat aaccgtcgat aaaatgcacc tagtatggta ctcgttacta caatccattt 1680
actctacttg ttccaacagt ggtcgagatt acgataaaca agcgttactt gtcgcaatag 1740
gcctcccttc cttcgtgaca aaagccgcga taaatgctcg tctaaaggtt aataacttgc 1800
ttctgtcgta ctataaaaat ggtggcaacc atgaaaaagg ggcgtctgaa atcatacaac 1860
agcgggcaac gtatctgcga aagacagggt tcacagatga cgatttgtcc cacatggagt 1920
tcatgatact atgggtagga gtgactaata ctgcacccgt tctattctgg ttgtttgtcc 1980
acgttcttac gtctgctggc tatacgagcc gcgtgcgggc tgagatagag gcgataacaa 2040
taatcaccaa gacgccagag ggcagaaaag caaccttcga tacccgttta ctcgagaaat 2100
cctgcccatt cctcaacgcg tgttaccagg aatgccttcg acattactct cactcgatcg 2160
gtaatcgtcg agtcatgcag gatactgaga tccaagattc tcagggccga aagtaccttc 2220
taaagaaagg cgttaacgtt caatggccgc ctccggtcac acatttcaat acggaagttt 2280
ggggccagga cgcggatgta tttcgtccag aaagatttat ggacgtcact cctcaggacg 2340
aaaaaaagag gagaggcgcc ctgttatcct tcggaggtgg caaacacctt tgcccgggta 2400
gaaagttcgc gtacacagaa ttgctagggc ttgtgggggt tgtggctctt ggcttcgaag 2460
ttaagggtct ggagctaccc gaaagtaaat acgcaggaat cggcatagga ggcaagatgc 2520
ctgattggga gaatatggaa aaaggcttcg gtctaagacg tcgagagggg tgggaggatg 2580
ttacctgggt ctttgatgga gataattgag tcgacctgca agatctgcgg ccgcgaatta 2640
attcgcctta gacatgactg ttcctcagtt caagttgggc acttacgaga agaccggtct 2700
tgctagattc taatcaagag gatgtcagaa tgccatttgc ctgagagatg caggcttcat 2760
ttttgatact tttttatttg taacctatat agtataggat tttttttgtc attttgtttc 2820
ttctcgtacg agcttgctcc tgatcagcct atctcgcagc tgatgaatat cttgtggtag 2880
gggtttggga aaatcattcg agtttgatgt ttttcttggt atttcccact cctcttcaga 2940
gtacagaaga ttaagtgaga ccttcgtttg tgcggatcca attaatattt acttattttg 3000
gtcaacccca aataggttga tttcatactt ggttcattca aaaataagta gtcttttgag 3060
atctttcaat attataataa atatactata acagccgact tgtttcattt tcgcgaatgt 3120
tcccccagct tatcggatcc cccacacacc atagcttcaa aatgtttcta ctcctttttt 3180
actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac 3240
agcatactaa atttcccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg 3300
tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa 3360
atttttatca cgtttctttt tcttgaaatt ttttttttta gtttttttct ctttcagtga 3420
cctccattga tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt 3480
tcttgttcta ttacaacttt ttttacttct tgttcattag aaagaaagca tagcaatcta 3540
atctaagggg cggtgttgac aattaatcat cggcatagta tatcggcata gtataatacg 3600
acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc 3660
gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg 3720
tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc 3780
aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc 3840
tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca 3900
tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca 3960
actgcgtgca cttcgtggcc gaggagcagg actgacacgt ccgacggcgg cccacgggtc 4020
ccaggcctcg gagatccgtc ccccttttcc tttgtcgata tcatgtaatt agttatgtca 4080
cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa 4140
cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt 4200
atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg 4260
aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa gctggagacc 4320
aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 4380
tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 4440
tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 4500
cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 4560
agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 4620
tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 4680
aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 4740
ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 4800
cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 4860
accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 4920
ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 4980
ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 5040
gtcatgagat c 5051
<210> 26
<211> 1572
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 26
atgatattcg acaacttgtc gctcagcaac acgtgggttg tgttagtact tagcgcggta 60
tttcttgtgc tttcccgttt tattgctccg acaatctcag agaacgagcc tcccatcgtc 120
aagccaaggg cccccttcat tggacacatt atctccatgt tgagggacgg ctccgatatc 180
tacgttaatt tgtttaagca aagaaaggaa ccaatagtta ctttacccat gttaaatgga 240
aaattatacg tgataaattc tccagacctc atacaggccg cattgcgtaa caatgacatc 300
tctttcacac cgttcattct tgagtcgtca aaagcaatgt gggggttatc tgataatgcg 360
atggcgagca tatctgacct tgccaacttg aaaggcggta tgcagattat ccactcaacc 420
ctcggagggg agtcgcttca taaattgaac atatcgtctc tgagtaggtt catgacttat 480
ttgaatcgcg ttaaacccgg cgaaaatatt ggtatagccg acacttatat ttggctgaga 540
gacatgctca ccgacgctag cgcgaccgcg gtctatggtc ctaagaatcc aataaccgtc 600
gataaaatgc acctagtatg gtactcgtta ctacaatcca tttactctac ttgttccaac 660
agtggtcgag attacgataa acaagcgtta cttgtcgcaa taggcctccc ttccttcgtg 720
acaaaagccg cgataaatgc tcgtctaaag gttaataact tgcttctgtc gtactataaa 780
aatggtggca accatgaaaa aggggcgtct gaaatcatac aacagcgggc aacgtatctg 840
cgaaagacag ggttcacaga tgacgatttg tcccacatgg agttcatgat actatgggta 900
ggagtgacta atactgcacc cgttctattc tggttgtttg tccacgttct tacgtctgct 960
ggctatacga gccgcgtgcg ggctgagata gaggcgataa caataatcac caagacgcca 1020
gagggcagaa aagcaacctt cgatacccgt ttactcgaga aatcctgccc attcctcaac 1080
gcgtgttacc aggaatgcct tcgacattac tctcactcga tcggtaatcg tcgagtcatg 1140
caggatactg agatccaaga ttctcagggc cgaaagtacc ttctaaagaa aggcgttaac 1200
gttcaatggc cgcctccggt cacacatttc aatacggaag tttggggcca ggacgcggat 1260
gtatttcgtc cagaaagatt tatggacgtc actcctcagg acgaaaaaaa gaggagaggc 1320
gccctgttat ccttcggagg tggcaaacac ctttgcccgg gtagaaagtt cgcgtacaca 1380
gaattgctag ggcttgtggg ggttgtggct cttggcttcg aagttaaggg tctggagcta 1440
cccgaaagta aatacgcagg aatcggcata ggaggcaaga tgcctgattg ggagaatatg 1500
gaaaaaggct tcggtctaag acgtcgagag gggtgggagg atgttacctg ggtctttgat 1560
ggagataatt ga 1572
<210> 27
<211> 523
<212> PRT
<213> Fusarium graminearum
<400> 27
Met Ile Phe Asp Asn Leu Ser Leu Ser Asn Thr Trp Val Val Leu Val
1 5 10 15
Leu Ser Ala Val Phe Leu Val Leu Ser Arg Phe Ile Ala Pro Thr Ile
20 25 30
Ser Glu Asn Glu Pro Pro Ile Val Lys Pro Arg Ala Pro Phe Ile Gly
35 40 45
His Ile Ile Ser Met Leu Arg Asp Gly Ser Asp Ile Tyr Val Asn Leu
50 55 60
Phe Lys Gln Arg Lys Glu Pro Ile Val Thr Leu Pro Met Leu Asn Gly
65 70 75 80
Lys Leu Tyr Val Ile Asn Ser Pro Asp Leu Ile Gln Ala Ala Leu Arg
85 90 95
Asn Asn Asp Ile Ser Phe Thr Pro Phe Ile Leu Glu Ser Ser Lys Ala
100 105 110
Met Trp Gly Leu Ser Asp Asn Ala Met Ala Ser Ile Ser Asp Leu Ala
115 120 125
Asn Leu Lys Gly Gly Met Gln Ile Ile His Ser Thr Leu Gly Gly Glu
130 135 140
Ser Leu His Lys Leu Asn Ile Ser Ser Leu Ser Arg Phe Met Thr Tyr
145 150 155 160
Leu Asn Arg Val Lys Pro Gly Glu Asn Ile Gly Ile Ala Asp Thr Tyr
165 170 175
Ile Trp Leu Arg Asp Met Leu Thr Asp Ala Ser Ala Thr Ala Val Tyr
180 185 190
Gly Pro Lys Asn Pro Ile Thr Val Asp Lys Met His Leu Val Trp Tyr
195 200 205
Ser Leu Leu Gln Ser Ile Tyr Ser Thr Cys Ser Asn Ser Gly Arg Asp
210 215 220
Tyr Asp Lys Gln Ala Leu Leu Val Ala Ile Gly Leu Pro Ser Phe Val
225 230 235 240
Thr Lys Ala Ala Ile Asn Ala Arg Leu Lys Val Asn Asn Leu Leu Leu
245 250 255
Ser Tyr Tyr Lys Asn Gly Gly Asn His Glu Lys Gly Ala Ser Glu Ile
260 265 270
Ile Gln Gln Arg Ala Thr Tyr Leu Arg Lys Thr Gly Phe Thr Asp Asp
275 280 285
Asp Leu Ser His Met Glu Phe Met Ile Leu Trp Val Gly Val Thr Asn
290 295 300
Thr Ala Pro Val Leu Phe Trp Leu Phe Val His Val Leu Thr Ser Ala
305 310 315 320
Gly Tyr Thr Ser Arg Val Arg Ala Glu Ile Glu Ala Ile Thr Ile Ile
325 330 335
Thr Lys Thr Pro Glu Gly Arg Lys Ala Thr Phe Asp Thr Arg Leu Leu
340 345 350
Glu Lys Ser Cys Pro Phe Leu Asn Ala Cys Tyr Gln Glu Cys Leu Arg
355 360 365
His Tyr Ser His Ser Ile Gly Asn Arg Arg Val Met Gln Asp Thr Glu
370 375 380
Ile Gln Asp Ser Gln Gly Arg Lys Tyr Leu Leu Lys Lys Gly Val Asn
385 390 395 400
Val Gln Trp Pro Pro Pro Val Thr His Phe Asn Thr Glu Val Trp Gly
405 410 415
Gln Asp Ala Asp Val Phe Arg Pro Glu Arg Phe Met Asp Val Thr Pro
420 425 430
Gln Asp Glu Lys Lys Arg Arg Gly Ala Leu Leu Ser Phe Gly Gly Gly
435 440 445
Lys His Leu Cys Pro Gly Arg Lys Phe Ala Tyr Thr Glu Leu Leu Gly
450 455 460
Leu Val Gly Val Val Ala Leu Gly Phe Glu Val Lys Gly Leu Glu Leu
465 470 475 480
Pro Glu Ser Lys Tyr Ala Gly Ile Gly Ile Gly Gly Lys Met Pro Asp
485 490 495
Trp Glu Asn Met Glu Lys Gly Phe Gly Leu Arg Arg Arg Glu Gly Trp
500 505 510
Glu Asp Val Thr Trp Val Phe Asp Gly Asp Asn
515 520
<210> 28
<211> 5048
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 28
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg gagtccatga taattactcc tgagatgaac tcaactttaa 1080
agatcgcgga tgtccaagcc cacgacttac ctttgcaaca caactttctg tcatacttgt 1140
ttggattgct aatcgccaca tatatagtat ggcagtattt cctgcgaact ggagtcacgg 1200
agtcagcttg ctccgagcct ccaatgctac cctattggat ccccgtggta ggtcatacct 1260
tcagtttctt gactaatact cataatacga taatgtcggg ccggagtcac ttcaaatcta 1320
taacacatcc cttctctctg ttgattggag gtagaaggac ttacgtagtc cttgacccgc 1380
actatattgg aaaggtgtac aagaaaacga aagatttggt tcatgagccg tttatagatc 1440
acttaatgat gtgcatcggg acaactcaaa aaacgaggga cataatgtgg aacacaatga 1500
tcggggactc cagtctaacc gattcggctc tcgattggct tagggaggaa gtctcccaat 1560
cgccttctag ccaaccattt ttcgacagat tcatgatgga attggatcat ggcctccagc 1620
aaggcgaccc gcttactacg gggcgacttc gggaacataa catgcttaag tttgttgaaa 1680
caattataat caccgtatca actaatagct tctttgggaa ggtgcttcta aaacaatctc 1740
cagaaattct tgactcgttt ccaatttttg accgacacgt ctggaagatg gtattccgcg 1800
caccaaaatt tactttcatg acggcacaca acgcgaaggg ttctgtcatc gacggtctta 1860
ctaaatattt tgatttacca caaagtgaga gacaggacgc cgcttctttt atccttaaaa 1920
gtgaggacgc aatgcgtgag aatggaatct gctcacggga gattgcggcc ctgctcttta 1980
aattcttttg gggcataaat ggcatgcccg cgacactggc cttctggttt cttgccagga 2040
ctgtctacac accacacctt tgggaggata tacgtgcaga ggtcgcaccg gcctttagga 2100
atggtattca ttcaccccca gacatagggt atttgaaaaa gtgcccaaaa ttaaacgcca 2160
ccttccacga aacgttacgc atccacggtg ggacggctgg atttaggcaa gtcgcgagtg 2220
ataccgtcat aggtggattt accttcaagg ccgggtccga cgttataatg ccgtaccggc 2280
aaatgcacct agatgagggg atctgggggc aggacgctaa gacttttgat attgatcgct 2340
ttattcataa cccgaaacta gctaccgcaa agacatttaa gccttttgga ggcggtgtaa 2400
cattgtgtcc aggacgcttc catgcgcacc gaactgctct gagctttatt gcgattgtta 2460
taacccgata cgacatccac gttgtgggcg gttgcgaatc gcgacccttc ccacatatga 2520
atacacgcgg accagaggtt ggtgttatat tcccagtctt ggagcaggtg ccacaaatta 2580
tagtaaaaaa tgttgacatt gaatgagtcg acctgcaaga tctgcggccg cgaattaatt 2640
cgccttagac atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc 2700
tagattctaa tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt 2760
tgatactttt ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc 2820
tcgtacgagc ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg 2880
tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta 2940
cagaagatta agtgagacct tcgtttgtgc ggatccaatt aatatttact tattttggtc 3000
aaccccaaat aggttgattt catacttggt tcattcaaaa ataagtagtc ttttgagatc 3060
tttcaatatt ataataaata tactataaca gccgacttgt ttcattttcg cgaatgttcc 3120
cccagcttat cggatccccc acacaccata gcttcaaaat gtttctactc cttttttact 3180
cttccagatt ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc 3240
atactaaatt tcccctcttt cttcctctag ggtgtcgtta attacccgta ctaaaggttt 3300
ggaaaagaaa aaagagaccg cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt 3360
tttatcacgt ttctttttct tgaaattttt ttttttagtt tttttctctt tcagtgacct 3420
ccattgatat ttaagttaat aaacggtctt caatttctca agtttcagtt tcatttttct 3480
tgttctatta caactttttt tacttcttgt tcattagaaa gaaagcatag caatctaatc 3540
taaggggcgg tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca 3600
aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg 3660
acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg 3720
aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg 3780
accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt 3840
acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga 3900
ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact 3960
gcgtgcactt cgtggccgag gagcaggact gacacgtccg acggcggccc acgggtccca 4020
ggcctcggag atccgtcccc cttttccttt gtcgatatca tgtaattagt tatgtcacgc 4080
ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct 4140
gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata 4200
tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa 4260
accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcaagct ggagaccaac 4320
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 4380
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 4440
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 4500
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 4560
gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 4620
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 4680
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 4740
aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 4800
aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 4860
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 4920
ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 4980
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 5040
atgagatc 5048
<210> 29
<211> 1569
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 29
atggagtcca tgataattac tcctgagatg aactcaactt taaagatcgc ggatgtccaa 60
gcccacgact tacctttgca acacaacttt ctgtcatact tgtttggatt gctaatcgcc 120
acatatatag tatggcagta tttcctgcga actggagtca cggagtcagc ttgctccgag 180
cctccaatgc taccctattg gatccccgtg gtaggtcata ccttcagttt cttgactaat 240
actcataata cgataatgtc gggccggagt cacttcaaat ctataacaca tcccttctct 300
ctgttgattg gaggtagaag gacttacgta gtccttgacc cgcactatat tggaaaggtg 360
tacaagaaaa cgaaagattt ggttcatgag ccgtttatag atcacttaat gatgtgcatc 420
gggacaactc aaaaaacgag ggacataatg tggaacacaa tgatcgggga ctccagtcta 480
accgattcgg ctctcgattg gcttagggag gaagtctccc aatcgccttc tagccaacca 540
tttttcgaca gattcatgat ggaattggat catggcctcc agcaaggcga cccgcttact 600
acggggcgac ttcgggaaca taacatgctt aagtttgttg aaacaattat aatcaccgta 660
tcaactaata gcttctttgg gaaggtgctt ctaaaacaat ctccagaaat tcttgactcg 720
tttccaattt ttgaccgaca cgtctggaag atggtattcc gcgcaccaaa atttactttc 780
atgacggcac acaacgcgaa gggttctgtc atcgacggtc ttactaaata ttttgattta 840
ccacaaagtg agagacagga cgccgcttct tttatcctta aaagtgagga cgcaatgcgt 900
gagaatggaa tctgctcacg ggagattgcg gccctgctct ttaaattctt ttggggcata 960
aatggcatgc ccgcgacact ggccttctgg tttcttgcca ggactgtcta cacaccacac 1020
ctttgggagg atatacgtgc agaggtcgca ccggccttta ggaatggtat tcattcaccc 1080
ccagacatag ggtatttgaa aaagtgccca aaattaaacg ccaccttcca cgaaacgtta 1140
cgcatccacg gtgggacggc tggatttagg caagtcgcga gtgataccgt cataggtgga 1200
tttaccttca aggccgggtc cgacgttata atgccgtacc ggcaaatgca cctagatgag 1260
gggatctggg ggcaggacgc taagactttt gatattgatc gctttattca taacccgaaa 1320
ctagctaccg caaagacatt taagcctttt ggaggcggtg taacattgtg tccaggacgc 1380
ttccatgcgc accgaactgc tctgagcttt attgcgattg ttataacccg atacgacatc 1440
cacgttgtgg gcggttgcga atcgcgaccc ttcccacata tgaatacacg cggaccagag 1500
gttggtgtta tattcccagt cttggagcag gtgccacaaa ttatagtaaa aaatgttgac 1560
attgaatga 1569
<210> 30
<211> 522
<212> PRT
<213> Fusarium graminearum
<400> 30
Met Glu Ser Met Ile Ile Thr Pro Glu Met Asn Ser Thr Leu Lys Ile
1 5 10 15
Ala Asp Val Gln Ala His Asp Leu Pro Leu Gln His Asn Phe Leu Ser
20 25 30
Tyr Leu Phe Gly Leu Leu Ile Ala Thr Tyr Ile Val Trp Gln Tyr Phe
35 40 45
Leu Arg Thr Gly Val Thr Glu Ser Ala Cys Ser Glu Pro Pro Met Leu
50 55 60
Pro Tyr Trp Ile Pro Val Val Gly His Thr Phe Ser Phe Leu Thr Asn
65 70 75 80
Thr His Asn Thr Ile Met Ser Gly Arg Ser His Phe Lys Ser Ile Thr
85 90 95
His Pro Phe Ser Leu Leu Ile Gly Gly Arg Arg Thr Tyr Val Val Leu
100 105 110
Asp Pro His Tyr Ile Gly Lys Val Tyr Lys Lys Thr Lys Asp Leu Val
115 120 125
His Glu Pro Phe Ile Asp His Leu Met Met Cys Ile Gly Thr Thr Gln
130 135 140
Lys Thr Arg Asp Ile Met Trp Asn Thr Met Ile Gly Asp Ser Ser Leu
145 150 155 160
Thr Asp Ser Ala Leu Asp Trp Leu Arg Glu Glu Val Ser Gln Ser Pro
165 170 175
Ser Ser Gln Pro Phe Phe Asp Arg Phe Met Met Glu Leu Asp His Gly
180 185 190
Leu Gln Gln Gly Asp Pro Leu Thr Thr Gly Arg Leu Arg Glu His Asn
195 200 205
Met Leu Lys Phe Val Glu Thr Ile Ile Ile Thr Val Ser Thr Asn Ser
210 215 220
Phe Phe Gly Lys Val Leu Leu Lys Gln Ser Pro Glu Ile Leu Asp Ser
225 230 235 240
Phe Pro Ile Phe Asp Arg His Val Trp Lys Met Val Phe Arg Ala Pro
245 250 255
Lys Phe Thr Phe Met Thr Ala His Asn Ala Lys Gly Ser Val Ile Asp
260 265 270
Gly Leu Thr Lys Tyr Phe Asp Leu Pro Gln Ser Glu Arg Gln Asp Ala
275 280 285
Ala Ser Phe Ile Leu Lys Ser Glu Asp Ala Met Arg Glu Asn Gly Ile
290 295 300
Cys Ser Arg Glu Ile Ala Ala Leu Leu Phe Lys Phe Phe Trp Gly Ile
305 310 315 320
Asn Gly Met Pro Ala Thr Leu Ala Phe Trp Phe Leu Ala Arg Thr Val
325 330 335
Tyr Thr Pro His Leu Trp Glu Asp Ile Arg Ala Glu Val Ala Pro Ala
340 345 350
Phe Arg Asn Gly Ile His Ser Pro Pro Asp Ile Gly Tyr Leu Lys Lys
355 360 365
Cys Pro Lys Leu Asn Ala Thr Phe His Glu Thr Leu Arg Ile His Gly
370 375 380
Gly Thr Ala Gly Phe Arg Gln Val Ala Ser Asp Thr Val Ile Gly Gly
385 390 395 400
Phe Thr Phe Lys Ala Gly Ser Asp Val Ile Met Pro Tyr Arg Gln Met
405 410 415
His Leu Asp Glu Gly Ile Trp Gly Gln Asp Ala Lys Thr Phe Asp Ile
420 425 430
Asp Arg Phe Ile His Asn Pro Lys Leu Ala Thr Ala Lys Thr Phe Lys
435 440 445
Pro Phe Gly Gly Gly Val Thr Leu Cys Pro Gly Arg Phe His Ala His
450 455 460
Arg Thr Ala Leu Ser Phe Ile Ala Ile Val Ile Thr Arg Tyr Asp Ile
465 470 475 480
His Val Val Gly Gly Cys Glu Ser Arg Pro Phe Pro His Met Asn Thr
485 490 495
Arg Gly Pro Glu Val Gly Val Ile Phe Pro Val Leu Glu Gln Val Pro
500 505 510
Gln Ile Ile Val Lys Asn Val Asp Ile Glu
515 520
<210> 31
<211> 5021
<212> DNA
<213> Artificial Sequence
<220>
<223> plasmid
<400> 31
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tcctacgtat taatacgact 960
cactatattt gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac 1020
aatttcacac agaattcatg gccacggatc ttgacctcgt gctgggaaaa agtcagtacg 1080
cattattttg tggcataact ttatttagct ttttcatact aaagtattcc cttctcggaa 1140
acgggggcaa gcaataccct tatatcaacc ccaagaaacc ctttgagctg tcgaaccagc 1200
gagtagtcca ggatttcatc gagaacgcac gagacattct tactaagggt cgctcacttt 1260
acaaggatac gccctacaag gcgcataccg atttagggga cgtcctcgta atcccgcccg 1320
agtttgccga cgctctcaag tccgaaagac agcttgactt taccgaggtc gcgagagacg 1380
atactcacgg ttacattcct ggattcgagc ccataggttc cccgttcgat ctggtgccgc 1440
tcgtcaacaa gtatcttaca agggcgttgg caaaactaac aaagccactg tgggccgaag 1500
cctcgttagg tgtaaaccat gttctgggca cgtctacgga gtggcatccc attaacccag 1560
gcgaagatat catgaggata gtctccagaa tgtcatccag aatattcatg ggtgaggaac 1620
tttgtaaaga tgacgattgg ctgaaagtgt cgattgagta cactgtgcag ctgtttcaaa 1680
ccgcagacga attacgtaac tatccacgtt ggacgcggcc ctatattcac tggttcttgc 1740
cttcctgtca gggggttcgt cgcaagttgc aggaggcgcg tgatttattg caaccccata 1800
ttgataggag aaatgcagtg aagaaagaag cgatcgctga aggtagaccc tcaccattcg 1860
acgattcaat agagtggttt gaaaatgagt acgagggcaa atctgatccc gccactgaac 1920
aaattaaact atcactggtg gcgattcaca caaccacgga cctcctgtct gaaaccatgt 1980
tcaatatagc tttgcagcca gaactccttg gtcccctacg tgaagagata gttacggttc 2040
tttccacgga aggtctaaaa aagacgtcgt tttacaattt gaagttgatg gattcggtca 2100
taaaggagtc acagcgactt cgacccgttc ttctcggtgc gttccgaaga atggcactcg 2160
ctgacgtaac cttgcccaat ggcgacgtaa taaagaaagg gaccaagatc atttgcgaca 2220
ctacacatca gtggaaccca gaatactatc ccgatgccag caagttcaat gcatatcggt 2280
ttctccaaat gagacagacg cccggtcagg acaaaagagc acaccttgtc agcacaagcc 2340
acgatcaaat ggggttcgga cacggcttgc acgcgtgccc aggccggttt ttcgcagcca 2400
atgagataaa gatagcgctg tgtcacatgc tattgaagta tgactggaag cttccagaag 2460
gtgttgtacc taagtctaag gccctcggca tgtccttact gggggaccgg gaagccaaac 2520
tgatggtcaa gaggagagca gccgaaatcg atatagacac tattgggagc gatgaatgag 2580
tcgacctgca agatctgcgg ccgcgaatta attcgcctta gacatgactg ttcctcagtt 2640
caagttgggc acttacgaga agaccggtct tgctagattc taatcaagag gatgtcagaa 2700
tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg taacctatat 2760
agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc tgatcagcct 2820
atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg agtttgatgt 2880
ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga ccttcgtttg 2940
tgcggatcca attaatattt acttattttg gtcaacccca aataggttga tttcatactt 3000
ggttcattca aaaataagta gtcttttgag atctttcaat attataataa atatactata 3060
acagccgact tgtttcattt tcgcgaatgt tcccccagct tatcggatcc cccacacacc 3120
atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca 3180
tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc 3240
tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt 3300
tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaatt 3360
ttttttttta gtttttttct ctttcagtga cctccattga tatttaagtt aataaacggt 3420
cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt ttttacttct 3480
tgttcattag aaagaaagca tagcaatcta atctaagggg cggtgttgac aattaatcat 3540
cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt 3600
tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga 3660
ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg 3720
acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg 3780
cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca 3840
cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc 3900
gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg 3960
actgacacgt ccgacggcgg cccacgggtc ccaggcctcg gagatccgtc ccccttttcc 4020
tttgtcgata tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc 4080
gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt 4140
atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac 4200
agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc 4260
tcgaaggctt taatttgcaa gctggagacc aacatgtgag caaaaggcca gcaaaaggcc 4320
aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 4380
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 4440
caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 4500
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt 4560
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 4620
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 4680
cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 4740
ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 4800
tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 4860
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 4920
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 4980
tggaacgaaa actcacgtta agggattttg gtcatgagat c 5021
<210> 32
<211> 1542
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 32
atggccacgg atcttgacct cgtgctggga aaaagtcagt acgcattatt ttgtggcata 60
actttattta gctttttcat actaaagtat tcccttctcg gaaacggggg caagcaatac 120
ccttatatca accccaagaa accctttgag ctgtcgaacc agcgagtagt ccaggatttc 180
atcgagaacg cacgagacat tcttactaag ggtcgctcac tttacaagga tacgccctac 240
aaggcgcata ccgatttagg ggacgtcctc gtaatcccgc ccgagtttgc cgacgctctc 300
aagtccgaaa gacagcttga ctttaccgag gtcgcgagag acgatactca cggttacatt 360
cctggattcg agcccatagg ttccccgttc gatctggtgc cgctcgtcaa caagtatctt 420
acaagggcgt tggcaaaact aacaaagcca ctgtgggccg aagcctcgtt aggtgtaaac 480
catgttctgg gcacgtctac ggagtggcat cccattaacc caggcgaaga tatcatgagg 540
atagtctcca gaatgtcatc cagaatattc atgggtgagg aactttgtaa agatgacgat 600
tggctgaaag tgtcgattga gtacactgtg cagctgtttc aaaccgcaga cgaattacgt 660
aactatccac gttggacgcg gccctatatt cactggttct tgccttcctg tcagggggtt 720
cgtcgcaagt tgcaggaggc gcgtgattta ttgcaacccc atattgatag gagaaatgca 780
gtgaagaaag aagcgatcgc tgaaggtaga ccctcaccat tcgacgattc aatagagtgg 840
tttgaaaatg agtacgaggg caaatctgat cccgccactg aacaaattaa actatcactg 900
gtggcgattc acacaaccac ggacctcctg tctgaaacca tgttcaatat agctttgcag 960
ccagaactcc ttggtcccct acgtgaagag atagttacgg ttctttccac ggaaggtcta 1020
aaaaagacgt cgttttacaa tttgaagttg atggattcgg tcataaagga gtcacagcga 1080
cttcgacccg ttcttctcgg tgcgttccga agaatggcac tcgctgacgt aaccttgccc 1140
aatggcgacg taataaagaa agggaccaag atcatttgcg acactacaca tcagtggaac 1200
ccagaatact atcccgatgc cagcaagttc aatgcatatc ggtttctcca aatgagacag 1260
acgcccggtc aggacaaaag agcacacctt gtcagcacaa gccacgatca aatggggttc 1320
ggacacggct tgcacgcgtg cccaggccgg tttttcgcag ccaatgagat aaagatagcg 1380
ctgtgtcaca tgctattgaa gtatgactgg aagcttccag aaggtgttgt acctaagtct 1440
aaggccctcg gcatgtcctt actgggggac cgggaagcca aactgatggt caagaggaga 1500
gcagccgaaa tcgatataga cactattggg agcgatgaat ga 1542
<210> 33
<211> 513
<212> PRT
<213> Fusarium graminearum
<400> 33
Met Ala Thr Asp Leu Asp Leu Val Leu Gly Lys Ser Gln Tyr Ala Leu
1 5 10 15
Phe Cys Gly Ile Thr Leu Phe Ser Phe Phe Ile Leu Lys Tyr Ser Leu
20 25 30
Leu Gly Asn Gly Gly Lys Gln Tyr Pro Tyr Ile Asn Pro Lys Lys Pro
35 40 45
Phe Glu Leu Ser Asn Gln Arg Val Val Gln Asp Phe Ile Glu Asn Ala
50 55 60
Arg Asp Ile Leu Thr Lys Gly Arg Ser Leu Tyr Lys Asp Thr Pro Tyr
65 70 75 80
Lys Ala His Thr Asp Leu Gly Asp Val Leu Val Ile Pro Pro Glu Phe
85 90 95
Ala Asp Ala Leu Lys Ser Glu Arg Gln Leu Asp Phe Thr Glu Val Ala
100 105 110
Arg Asp Asp Thr His Gly Tyr Ile Pro Gly Phe Glu Pro Ile Gly Ser
115 120 125
Pro Phe Asp Leu Val Pro Leu Val Asn Lys Tyr Leu Thr Arg Ala Leu
130 135 140
Ala Lys Leu Thr Lys Pro Leu Trp Ala Glu Ala Ser Leu Gly Val Asn
145 150 155 160
His Val Leu Gly Thr Ser Thr Glu Trp His Pro Ile Asn Pro Gly Glu
165 170 175
Asp Ile Met Arg Ile Val Ser Arg Met Ser Ser Arg Ile Phe Met Gly
180 185 190
Glu Glu Leu Cys Lys Asp Asp Asp Trp Leu Lys Val Ser Ile Glu Tyr
195 200 205
Thr Val Gln Leu Phe Gln Thr Ala Asp Glu Leu Arg Asn Tyr Pro Arg
210 215 220
Trp Thr Arg Pro Tyr Ile His Trp Phe Leu Pro Ser Cys Gln Gly Val
225 230 235 240
Arg Arg Lys Leu Gln Glu Ala Arg Asp Leu Leu Gln Pro His Ile Asp
245 250 255
Arg Arg Asn Ala Val Lys Lys Glu Ala Ile Ala Glu Gly Arg Pro Ser
260 265 270
Pro Phe Asp Asp Ser Ile Glu Trp Phe Glu Asn Glu Tyr Glu Gly Lys
275 280 285
Ser Asp Pro Ala Thr Glu Gln Ile Lys Leu Ser Leu Val Ala Ile His
290 295 300
Thr Thr Thr Asp Leu Leu Ser Glu Thr Met Phe Asn Ile Ala Leu Gln
305 310 315 320
Pro Glu Leu Leu Gly Pro Leu Arg Glu Glu Ile Val Thr Val Leu Ser
325 330 335
Thr Glu Gly Leu Lys Lys Thr Ser Phe Tyr Asn Leu Lys Leu Met Asp
340 345 350
Ser Val Ile Lys Glu Ser Gln Arg Leu Arg Pro Val Leu Leu Gly Ala
355 360 365
Phe Arg Arg Met Ala Leu Ala Asp Val Thr Leu Pro Asn Gly Asp Val
370 375 380
Ile Lys Lys Gly Thr Lys Ile Ile Cys Asp Thr Thr His Gln Trp Asn
385 390 395 400
Pro Glu Tyr Tyr Pro Asp Ala Ser Lys Phe Asn Ala Tyr Arg Phe Leu
405 410 415
Gln Met Arg Gln Thr Pro Gly Gln Asp Lys Arg Ala His Leu Val Ser
420 425 430
Thr Ser His Asp Gln Met Gly Phe Gly His Gly Leu His Ala Cys Pro
435 440 445
Gly Arg Phe Phe Ala Ala Asn Glu Ile Lys Ile Ala Leu Cys His Met
450 455 460
Leu Leu Lys Tyr Asp Trp Lys Leu Pro Glu Gly Val Val Pro Lys Ser
465 470 475 480
Lys Ala Leu Gly Met Ser Leu Leu Gly Asp Arg Glu Ala Lys Leu Met
485 490 495
Val Lys Arg Arg Ala Ala Glu Ile Asp Ile Asp Thr Ile Gly Ser Asp
500 505 510
Glu
<210> 34
<211> 1280
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 34
gtacagaaga ttaaggcgcg ccgcaagcca agcctgcgaa gaatgtagtc gagaattgag 60
cttgcctcgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa taccctcctt 120
gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta catttagccc 180
atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac ggcgcgaagc 240
aaaaattacg gctcctcgct gcagacctgc gagcagggaa acgctcccct cacagacgcg 300
ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga tttgccactg 360
aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc tcacatcaca 420
tccgaacata aacaaaaatg accactttgg atgatactgc ttacagatac agaacttctg 480
ttccaggtga tgctgaagct attgaagctt tggatggatc tttcaccact gatactgttt 540
tcagagtcac tgctactggt gatggattca ctttgagaga agttcctgtt gatcctcctt 600
tgaccaaagt ttttcctgat gatgaatctg atgatgaatc tgatgctggt gaagatggtg 660
atccagattc tagaactttt gttgcttatg gtgatgatgg tgatttggct ggatttgttg 720
ttgtttctta ttctggatgg aacagaagat tgactgttga agatattgaa gttgctccag 780
aacatagagg tcatggtgtt ggaagagctt tgatgggatt ggcaactgag tttgccagag 840
aaagaggtgc tggtcatctt tggttggaag tcaccaatgt caatgctcca gctattcatg 900
cttacagaag aatgggattc actctttgtg gattggatac tgctttgtat gatggaactg 960
cttctgatgg agaacaagct ttgtacatgt ccatgccatg tccttaaagt aactgacaat 1020
aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt 1080
ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca 1140
gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc 1200
gctatactgc tgtcgattcg atactaacgc cgccatccag tgtcggatct gtgagcaaac 1260
ccgggcatgt gagcaaaagg 1280
<210> 35
<211> 807
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 35
gaattcatgg cccttcgaac gtccctatca cgacccgtac cgcttctggc tacacttact 60
gccagcgcaa tcggagtatc catattgtct aaaatgatgt tttcaacagc aagtgcagag 120
agtccatctc cgcaaaaaat tttttccggt gcttttgctt ccgtaaaact cccgctgcat 180
tcaagtgaat acgagtccca tgacacaaag aggcttcgtt tcaaacttcc gcaagagact 240
gcagtaacgg gtttaccgtt agcttacttg gttcacattc caccgtccca ccatcaaagg 300
gacttgacta cgccggatga acctggatac atggacctgt tggtaaagaa ataccccaaa 360
ggccagggct cgacatatct acactccctc cagcccggtg atacgttatc cttcacatct 420
ctacccctca aaccagcttg gaaaacaaac aattttcctc acatcactct tatagctgga 480
gggtgtggga tcacgccatt attcaacttg gctcaaggga tacttagaga tccggccgaa 540
aaaactagga tgacctttat ttttggtgca cgatcagacg aggacgtatt actgaaaaag 600
gagttagatg gctttgcaaa agagttcccg gaaagattcg aggtgaaata tacagcactt 660
ttggaagagg tcctaggggg cgtgggtcgt gatactaagg tctttgtctg tgggccgaag 720
gagatggaaa aggcacttgt aggaggccgt ggcgtattaa aggaaatagg cttcgaaaag 780
tctcagatcc atactttttg agtcgac 807
<210> 36
<211> 1554
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic DNA
<400> 36
gaattcatgg ccacggatct tgacctcgtg ctgggaaaaa gtcagtacgc attattttgt 60
ggcataactt tatttagctt tttcatacta aagtattccc ttctcggaaa cgggggcaag 120
caataccctt atatcaaccc caagaaaccc tttgagctgt cgaaccagcg agtagtccag 180
gatttcatcg agaacgcacg agacattctt actaagggtc gctcacttta caaggatacg 240
ccctacaagg cgcataccga tttaggggac gtcctcgtaa tcccgcccga gtttgccgac 300
gctctcaagt ccgaaagaca gcttgacttt accgaggtcg cgagagacga tactcacggt 360
tacattcctg gattcgagcc cataggttcc ccgttcgatc tggtgccgct cgtcaacaag 420
tatcttacaa gggcgttggc aaaactaaca aagccactgt gggccgaagc ctcgttaggt 480
gtaaaccatg ttctgggcac gtctacggag tggcatccca ttaacccagg cgaagatatc 540
atgaggatag tctccagaat gtcatccaga atattcatgg gtgaggaact ttgtaaagat 600
gacgattggc tgaaagtgtc gattgagtac actgtgcagc tgtttcaaac cgcagacgaa 660
ttacgtaact atccacgttg gacgcggccc tatattcact ggttcttgcc ttcctgtcag 720
ggggttcgtc gcaagttgca ggaggcgcgt gatttattgc aaccccatat tgataggaga 780
aatgcagtga agaaagaagc gatcgctgaa ggtagaccct caccattcga cgattcaata 840
gagtggtttg aaaatgagta cgagggcaaa tctgatcccg ccactgaaca aattaaacta 900
tcactggtgg cgattcacac aaccacggac ctcctgtctg aaaccatgtt caatatagct 960
ttgcagccag aactccttgg tcccctacgt gaagagatag ttacggttct ttccacggaa 1020
ggtctaaaaa agacgtcgtt ttacaatttg aagttgatgg attcggtcat aaaggagtca 1080
cagcgacttc gacccgttct tctcggtgcg ttccgaagaa tggcactcgc tgacgtaacc 1140
ttgcccaatg gcgacgtaat aaagaaaggg accaagatca tttgcgacac tacacatcag 1200
tggaacccag aatactatcc cgatgccagc aagttcaatg catatcggtt tctccaaatg 1260
agacagacgc ccggtcagga caaaagagca caccttgtca gcacaagcca cgatcaaatg 1320
gggttcggac acggcttgca cgcgtgccca ggccggtttt tcgcagccaa tgagataaag 1380
atagcgctgt gtcacatgct attgaagtat gactggaagc ttccagaagg tgttgtacct 1440
aagtctaagg ccctcggcat gtccttactg ggggaccggg aagccaaact gatggtcaag 1500
aggagagcag ccgaaatcga tatagacact attgggagcg atgaatgagt cgac 1554
<210> 37
<211> 38
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 37
aatttttgaa aattcgaatt catggccctt cgaacgtc 38
<210> 38
<211> 53
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 38
ttgtaatcca tcgatactag ttcaaaaagt atggatctga gacttttcga agc 53
<210> 39
<211> 39
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 39
ctatagggcc cgggcgtcga catggccacg gatcttgac 39
<210> 40
<211> 59
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 40
gctagccgcg gtaccaagct ttcattcatc gctcccaata gtgtctatat cgatttcgg 59
Claims (35)
- LCA 또는 3-KCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 UDCA 또는 3-KUDCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염으로 전환시키는 방법으로서, LCA 또는 3-KCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 효모, 또는 이의 추출물 또는 용해물(lysate)의 존재 하에 7β-하이드록실라제 시스템과 접촉시키는 단계를 포함하며, 여기서 7β-하이드록실라제 시스템은 효모 고유의 것이 아닌, 방법.
- 제1항에 있어서, 효모는 사카로마이세스(Saccharomyces) 및 피치아(Pichia) 로부터 선택되는, 방법.
- 제1항에 있어서, 효모는 사카로마이세스 세레비지애(Saccharomyces cerevisiae) 및 피치아 파스토리스(Pichia pastoris)로부터 선택되는, 방법.
- 제1항에 있어서, 효모, 또는 이의 추출물 또는 용해물은 유기체 외래인 7β-하이드록실라제 시스템에 의해 형질전환되는, 방법.
- 제4항에 있어서, 7β-하이드록실화 시스템은 P450 옥시도리덕타제("CPR") 효소 및 P450 7-베타-하이드록실라제("CYP") 효소를 포함하고, CYP 효소는 효모 고유의 것이 아니며, CPR 효소는 효모에 고유한 것이거나 고유한 것이 아닐 수 있는, 방법.
- 제5항에 있어서, CYP 효소는 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 서열 번호 20; 서열 번호 23; 서열 번호 26; 서열 번호 29; 및 서열 번호 32로부터 선택된 CYP 인코딩 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에 의해 인코딩되는, 방법.
- 제5항 또는 제6항에 있어서, CPR 효소는 서열 번호 2 및 서열 번호 5로부터 선택된 CPR 인코딩 핵산 서열, 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에 의해 인코딩되는, 방법.
- 제5항에 있어서, CYP 효소는 서열 번호 9; 서열 번호 12; 서열 번호 15; 서열 번호 18; 서열 번호 21; 서열 번호 24; 서열 번호 27; 서열 번호 30; 또는 서열 번호 33으로부터 선택되는 CYP 아미노산 서열, 또는 전술한 임의의 아미노산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 아미노산 서열을 포함하는, 방법.
- 제5항 또는 제8항에 있어서, CPR 효소는 서열 번호 3 및 서열 번호 6으로부터 선택되는 CPR 아미노산 서열, 또는 전술한 임의의 아미노산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 아미노산 서열을 포함하는, 방법.
- 제1항에 있어서, 7β-하이드록실라제 시스템은 푸사리움 그라미네아룸(F. graminearum) 또는 지베렐라 제아(Gibberella zeae), 바람직하게는 지베렐라 제아(Gibberella zeae) PH1 또는 지베렐라 제아(Gibberella zeae) VKM2600, 가장 바람직하게는 지베렐라 제아(Gibberella zeae) VKM2600 고유의 P450 7-베타-하이드록실라제("CYP") 효소를 포함하는, 방법.
- 제8항에 있어서, LCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 7β-하이드록실라제 시스템과 접촉시켜 UDCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 생성하는 단계를 포함하는 방법.
- 제8항에 있어서, 3-KCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 7β-하이드록실라제 시스템과 접촉시켜 3-KUDCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 생성하는 단계를 포함하는 방법.
- 제12항에 있어서, 3-KUDCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 UDCA 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염으로 환원시키는 단계를 추가로 포함하는 방법.
- 제11항 내지 제13항 중 어느 한 항에 있어서, 7β-하이드록실라제 시스템으로부터 UDCA 또는 3-KUDCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염을 단리하는 단계를 추가로 포함하는 방법.
- 제11항 내지 제13항 중 어느 한 항에 있어서, UDCA 또는 3-KUDCA, 또는 이의 카복실산 에스테르, 카복실 아미드 또는 카복실레이트 염은 실질적으로 순수한 부분입체이성질체로서 생성되는, 방법.
- 제11항 내지 제13항 중 어느 한 항에 있어서, 약 15 ℃ 내지 약 75 ℃의 온도에서 수행되는 방법.
- 제11항 내지 제13항 중 어느 한 항에 있어서, 약 pH 5 내지 약 pH 9의 pH에서 수행되는 방법.
- 제1항 내지 제17항 중 어느 한 항에 있어서, LCA 또는 3-KCA 대 7β-하이드록실라제 시스템의 중량비가 약 10:1 내지 200:1인, 방법.
- 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 서열 번호 20; 서열 번호 23; 서열 번호 26; 서열 번호 29; 또는 서열 번호 32으로부터 선택된 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열을 포함하는 플라스미드.
- 제19항에 있어서, 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 및 서열 번호 20으로부터 선택된 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열을 포함하는 플라스미드.
- 제19항에 있어서, 서열 번호 23; 서열 번호 26; 또는 서열 번호 29로부터 선택된 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열을 포함하는 플라스미드.
- 제19항에 있어서, 서열 번호 32로부터 선택된 핵산 서열; 또는 서열 번호 32와 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열을 포함하는 플라스미드.
- 제19항 내지 제22항 중 어느 한 항에 있어서, AOX1 프로모터 및 AOX1 종결자 서열의 조절 하에 있는 플라스미드.
- 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 서열 번호 20; 서열 번호 23; 서열 번호 26; 서열 번호 29; 및 서열 번호 32로부터 선택된 CYP 인코딩 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에 의해 형질전환된 유기체.
- 제24항에 있어서, 서열 번호 8; 서열 번호 11; 서열 번호 14; 서열 번호 17; 및 서열 번호 20으로부터 선택된 CYP 인코딩 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에 의해 형질전환된 유기체.
- 제24항에 있어서, 서열 번호 23; 서열 번호 26; 및 서열 번호 29로부터 선택된 CYP 인코딩 핵산 서열; 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에 의해 형질전환된 유기체.
- 제24항에 있어서, 서열 번호 32로부터 선택된 CYP 인코딩 핵산 서열; 또는 서열 번호 32와 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에 의해 형질전환된 유기체.
- 제24항 내지 제27항 중 어느 한 항에 있어서, 서열 번호 2 또는 서열 번호 5를 포함하는 CPR 인코딩 핵산 서열, 또는 전술한 임의의 핵산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 핵산 서열에 의해 형질전환된 유기체.
- 제24항 내지 제27항 중 어느 한 항에 있어서, 유기체는 효모, 바람직하게는 사카로마이세스(Saccharomyces) 또는 피치아(Pichia), 및 보다 바람직하게는 사카로마이세스 세레비지애(Saccharomyces cerevisiae) 또는 피치아 파스토리스(Pichia pastoris)인, 유기체.
- (i) LCA 또는 3-KCA, (ii) 효모, 또는 이의 추출물 또는 용해물, (iii) 7β-하이드록실화 시스템을 포함하는 반응 혼합물.
- 제30항에 있어서, 7β-하이드록실화 시스템은 P450 옥시도리덕타제("CPR") 효소 및 P450 7β-하이드록실라제("CYP") 효소를 포함하고, 여기서 CYP 효소는 서열 번호 9; 서열 번호 12; 서열 번호 15; 서열 번호 18; 서열 번호 21; 서열 번호 24; 서열 번호 27; 서열 번호 30; 또는 서열 번호 33으로부터 선택된 아미노산 서열; 또는 전술한 임의의 아미노산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 아미노산 서열을 포함하는, 반응 혼합물.
- 제30항 또는 제31항에 있어서, CPR 효소는 서열 번호 3 및 서열 번호 6으로부터 선택된 아미노산 서열; 또는 전술한 임의의 아미노산 서열과 적어도 85%, 90%, 95%, 98%, 또는 99% 동일성을 갖는 아미노산 서열을 포함하는, 반응 혼합물.
- 제30항 또는 제31항에 있어서, 효모는 사카로마이세스(Saccharomyces) 또는 피치아(Pichia), 보다 바람직하게는 사카로마이세스 세레비지애(Saccharomyces cerevisiae) 또는 피치아 파스토리스(Pichia pastoris)인, 반응 혼합물.
- 효모와 P450 옥시도리덕타제("CPR") 효소 및 P450 7β-하이드록실라제("CYP") 효소를 포함하는 7β-하이드록실화 시스템을 포함하는 반응 혼합물로서, 여기서 CYP 효소는 지베렐라 제아(Gibberella zeae), 바람직하게는 지베렐라 제아(Gibberella zeae) PH1 또는 지베렐라 제아(Gibberella zeae) VKM2600, 가장 바람직하게는 지베렐라 제아(Gibberella zeae) VKM2600 고유의 효소인, 반응 혼합물.
- 제34항에 있어서, LCA 또는 3-KCA를 추가로 포함하는 반응 혼합물.
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