KR101990014B1 - 2,4-디하이드록시부티르산의 제조 방법 - Google Patents
2,4-디하이드록시부티르산의 제조 방법 Download PDFInfo
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- KR101990014B1 KR101990014B1 KR1020137012492A KR20137012492A KR101990014B1 KR 101990014 B1 KR101990014 B1 KR 101990014B1 KR 1020137012492 A KR1020137012492 A KR 1020137012492A KR 20137012492 A KR20137012492 A KR 20137012492A KR 101990014 B1 KR101990014 B1 KR 101990014B1
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- leu
- ala
- val
- gly
- glu
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Abstract
본 발명은, 말레이트 키나아제에 의해 말레이트를 4-포스포-말레이트로 변환하는 단계를 포함하는 합성 경로에 의해, 2,4-디하이드록시부티르산 (2,4-DHB)을 제조하는 방법에 관한 것으로, 상기 4-포스포-말레이트는 말레이트 세미알데하이드 탈수소효소에 의해 말레이트-4-세미알데하이드로 변환되며, 상기 말레이트-4-세미알데하이드는 DHB 탈수소효소에 의해 2,4-DHB로 변환된다.
Description
본 발명은, 각각 말레이트 키나아제, 말레이트 세미알데하이드 탈수소효소, 및 2,4-디하이드록시부티레이트 탈수소효소 활성을 가진 효소를 포함하는 합성 경로의 구현에 의해, 말레이트로부터 2,4-디하이드록시부티르산을 제조하는 신규 방법에 관한 것이다.
본 출원에서 언급되는 카르복실산은 이의 염 (예를 들어, 2,4-디하이드록시부티레이트) 또는 산 형태 (예를 들어, 2,4-디하이드록시부티르산)의 명칭 하에 동일시된다.
2,4-디하이드록시부티르산 (2,4-DHB 또는 DHB와 동일시됨)은 상당히 경제적으로 이로운 화합물이다. DHB는 pH를 적절히 조정함으로써, 수성 매질에서 α-하이드록시-γ-부티로락톤으로 쉽게 전환될 수 있다. α-하이드록시-γ-부티로락톤은 메티오닌의 대체물이며, 동물 영양에서 그 시장이 큰, 2-하이드록시-4-(메틸티오)-부티레이트 (HMTB)를 제조하기 위한 주요 전구체이다 (Deck et al., 2008). 현재, α-하이드록시-γ-부티로락톤은, γ-부티로락톤을 α 위치에서 할로겐화하고, 이어서 할로겐 원자를 알칼리 매질에서 하이드록실기로 치환시키는 것을 포함하는 다단계 공정에 의해, γ-부티로락톤으로부터 유도된다 (Deck et al., 2008).
유가 상승으로 인해, 재생가능한 자원으로부터 DHB를 제조할 필요성이 형성되고 있다. 미생물은 바이오매스 유래의 원료, 예를 들어 당이나 유기산을 여러 가지 화학적 화합물로 변환시킬 수 있다 (Werpy & Petersen, 2004). 생화학적 및 게놈학적 정보의 증가로, 미생물이 천연적인 대사 중간산물을 고수율로 그리고 고 생산성으로 과다생성하도록, 미생물을 변형시킬 수 있다 (Bailey, 1991). 생성 미생물의 최적화에는, 때때로, 특히, 대상 대사물질의 생합성의 필수 효소의 과다발현, 및 생성물 피드백 저해의 경감을 보장하는, 대사성 네트워크의 합리적인 조작이 요구된다. 다른 가능성으로는, 대상 대사물질의 생성을 촉매하는 신규 효소 시스템을 구현하는 것이다.
대사 공학적 방식과 효소적 촉매 방식은, 대상 대사물질을 생성하는 대사 경로의 조절과 생화학에 대해 상세한 지식을 필요로 한다. DHB를 제조하는 경우, 이러한 정보는 이용가능하지 않다. 단지 소수의 연구에서, 숙신산 세미알데하이드 탈수소효소가 결핍된 환자에서 DHB의 생성이 보고되어 있지만, DHB 생성과 관련한 효소 반응은 규명되어 있지 않다 (Shinka et al., 2002). 따라서, 발효에 의해 또는 효소에 의해 DHB를 제조하는 방법은, (i) 이용가능한 전구체를 DHB로 변환하는, 열역학적으로 실현가능한 경로의 규명, (ii) 상기 경로에서 각각의 반응 단계를 촉매할 수 있는 효소의 동정 및 구축, (iii) 적절한 생산 유기체에서, 경로의 효소들의 기능적 발현이 요구된다.
본 발명은 이들 요구를 충족시키는 것을 과제로 한다.
따라서, 본 발명의 한 가지 과제는, 말레이트 키나아제에 의해 말레이트를 4-포스포-말레이트로 변환하는 제1 단계, 말레이트 세미알데하이드 탈수소효소에 의해 4-포스포-말레이트를 말레이트-4-세미알데하이드로 변환하는 제2 단계, 및 DHB 탈수소효소에 의해 말레이트-4-세미알데하이드를 2,4-DHB로 변환하는 제3 단계를 포함하는, 2,4-DHB의 제조 방법이다.
제1 반응에서 (도 1 (i) 참고), 말레이트 (1)는, 말레이트 키나아제 활성을 가지는 효소의 작용에 의해 4-포스포-말레이트 (2)로 전환된다 (A). 제2 반응 (B)에서, 4-포스포-말레이트는 말레이트 세미알데하이드 탈수소효소 활성을 가지는 효소의 작용에 의해 말레이트-4-세미알데하이드 (3)로 전환된다. 더욱 정확하게는,반응 (B)는 경로의 생합성 측면에서, 4-포스포-말레이트를 탈인산화하는 환원효소 활성을 가진 효소에 의해 촉매된다. 제3 반응 (C)에서, 말레이트-4-세미알데하이드는 DHB 탈수소효소 활성을 가진 효소의 작용에 의해 DHB (4)로 전환된다. 더욱 정확하게는, 반응 (C)은 경로의 생합성 측면에서, 말레이트-4-세미알데하이드 환원효소 활성을 가진 효소에 의해 촉매된다.
전술한 효소와 중간체 중 어느 것도, 지금까지 살아 있는 세포에서 확인되거나 동정된 바 없었다. 말레이트 키나아제와 마찬가지로, 말레이트 세미알데하이드 탈수소효소, DHB 탈수소효소 및 4-포스포-말레이트는 본 발명의 다른 과제들이다.
본 발명의 다른 측면에서, 2,4-DHB를 제조하는 방법에서 제1 단계는, 말레이트를 4-포스포-말레이트로 변환하는 것을 특징으로 하는, 말레이트 키나아제를 수반한다. 상기 효소는, 효소의 하나 이상의 돌연변이화에 의해 수득될 수 있으며, 상기 돌연변이(들)는 말레이트에 대한, 돌연변이 효소의 활성 및/또는 기질 친화성을 개선한다.
본 발명에서, "활성 및/또는 기질 친화성을 개선한다"라는 표현은, 돌연변이 전의 효소가:
- 기질 (말레이트, 4-포스포-말레이트 또는 말레이트-4-세미알데하이드)을 이용할 수 없으며, 및/또는
- 반응 산물 (4-포스포-말레이트 또는 말레이트-4-세미알데하이드 또는 DHB)을 적어도 3배 더 낮은 최대 비속도 (specific rate)로 합성하며, 및/또는
- 말레이트, 4-포스포-말레이트 또는 말레이트-4- 세미알데하이드에 대한 친화성이 적어도 3배 더 낮으며, 및/또는
- 천연 기질 (아스파르테이트, 4-포스포-아스파르테이트, 아스파르테이트-4-세미알데하이드)에 대한 친화성이 적어도 3배 더 높다는 의미이다.
본 발명은 또 다른 측면에서, 말레이트를 4-포스포-말레이트로 변환하는 말레이트 키나아제의 용도에 관한 것이다.
말레이트 키나아제 활성은 실시예 1에서 기술된 효소 테스트 ("효소 분석" 참고)에 의해 측정될 수 있다.
본 발명의 다른 측면에 따르면, 말레이트 키나아제는 아스파르테이트 키나아제의 돌연변이에 의해 수득될 수 있다.
도 2는, 여러 가지 생물 기원의 아스파르테이트 키나아제의 아미노산 서열을 정렬한 것이다. 아미노산 위치는 모두, E. coli의 LysC 유전자에 의해 코딩되는 아스파르테이트 키나아제의 아미노산 서열 (서열 번호 4로 표시됨)을 근거로 참고한다. 다른 유기체 유래의, 또 다른 아스파르테이트 키나아제에 있는 상응하는 보존 영역의 상대적인 위치는, 하기에서 열거한 효소와 함께 도 2에서 표시된 단순 서열 정렬을 통해, 당해 분야의 당업자가 쉽게 파악할 수 있다:
- AKIII - E. coli 유래의 아스파르테이트 키나아제 III (서열 번호 4),
- AKI (서열 번호 87) - E. coli 유래의 아스파르테이트 키나아제 I,
- AKII (서열 번호 88) - E. coli 유래의 아스파르테이트 키나아제 II,
- MJ - 메타노코커스 얀나스치이 (Methanococcus jannaschii) (서열 번호 89),
- TT - 써무스 써모필러스 (Thermus thermophilus) (서열 번호 90),
- CG - 코리네박테리움 글루타미쿰 (서열 번호 91),
- AT - 아라비돕시스 탈리아나 (Arabidopsis thaliana) (서열 번호 92),
- SC - 사카로마이세스 세레비지애 (서열 번호 93).
상기 정렬은 ClustalW2 소프트웨어로 수행될 수 있다. 예를 들어, 서열 번호 4로 표시되는 아스파르테이트 키나아제의 E119 잔기는 아라비돕시스 탈리아나의 아스파르테이트 키나아제의 E207 잔기 (서열 번호 50)에 상응하거나, 사카로마이세스 세레비지애의 아스파르테이트 키나아제의 E147 잔기 (서열 번호 51)에 상응한다.
본 발명에 따른 아스파르테이트 키나아제 돌연변이체는 야생형 효소와 비교해, S39, T45, V115, E119, F184 및/또는 S201의 위치 중 하나 이상에, 하나 이상의 돌연변이를 포함하며, 상기 위치에서 천연 아미노산이, 다른 19개의 천연 단백질성 (proteinogenic) 아미노산인 알라닌, 아르기닌, 아스파라긴, 아스파르트산, 시스테인, 글루타민산, 글루타민, 글리신, 히스티딘, 이소루신, 루신, 라이신, 메티오닌, 페닐알라닌, 프롤린, 세린, 트레오닌, 트립토판, 티로신, 또는 발린 중 어느 하나로 치환된다.
비-배타적인 실시예에, 에스케리키아 콜라이의 아스파르테이트 키나아제 Lys C를 주형으로 사용한 부위 특이적인 돌연변이유발에 의한 말레이트 키나아제의 구축이 기술되어 있다. 본 발명의 한 측면에 따르면, 위치 119의 글루타민산을 아스파라긴, 글루타민, 시스테인, 프롤린, 세린, 트레오닌, 발린 또는 글리신으로 치환하여, 말레이트에 대한 LysC의 기질 특이성을 변화시켰다.
본 발명의 다른 측면에서, 말레이트 키나아제는 서열 번호 9로 표시되며, 더욱 구체적으로는 서열 번호 12, 서열 번호 14, 서열 번호 16, 서열 번호 18, 서열 번호 20, 서열 번호 22, 서열 번호 24 또는 서열 번호 26으로 표시된다.
아스파르테이트 키나아제는 전형적으로 메티오닌, 트레오닌 또는 라이신에 의해 저해된다. 따라서, 아스파르테이트 키나아제의 무작위 또는 부위 특이적인 돌연변이유발에 의해 제작된 말레이트 키나아제는 상기 아미노산에 의해 저해될 수도 있다. 본 발명의 다른 측면에서, 메티오닌, 라이신 또는 트레오닌에 의한 말레이트 키나아제의 저해는, 말레이트 키나아제의 돌연변이화에 의해 감소한다.
본 발명의 구체적인 측면에서, 전술한 돌연변이 LysC (말레이트 키나아제)는, 하기 아미노산인 E250, M318, S321, V339, S338, F324, L325, V339, S345, E346, D340, T344 및/또는 T352 중 하나 이상에 돌연변이가 생김으로써, 라이신 저해에 둔감해지게 된다 (실시예 3 참고).
본 발명은 또한, 상기 개질된 효소, 보다 구체적으로는 서열 번호 39, 서열 번호 41, 서열 번호 43 또는 서열 번호 45로 표시되는 효소들을 포함한다.
더욱 다른 측면에서, 본 발명에 따라 2,4-DHB를 제조하는 방법에서 제2 단계는, 4-포스포-말레이트를 말레이트-4-세미알데하이드로 변환하는 것을 특징으로 하는 말레이트 세미알데하이드 탈수소효소를 포함하며, 상기 효소는 경로의 생합성 측면에서, 4-포스포-말레이트를 탈인산화하는 환원효소 활성을 가진다.
말레이트 세미알데하이드 탈수소효소 활성은 실시예 4에서 기술된 효소 테스트에 의해 측정될 수 있다 ("효소 분석" 참고).
이러한 효소는, 효소에 생긴 하나 이상의 돌연변이에 의해 수득될 수 있으며, 상기 돌연변이(들)는 4-포스포-말레이트에 대한, 돌연변이 효소의 활성 및/또는 기질 친화성을 개선한다.
다른 측면에 따르면, 본 발명의 말레이트 세미알데하이드 탈수소효소는, 보고된 세미알데하이드 탈수소효소 활성을 가진, 더욱 구체적으로는 반응의 환원 측면에서 탈인산화 활성을 가진, 더욱 구체적으로는 3, 4, 또는 5개의 탄소 분자로 이루어진 유기 분자에 작용하는, 효소의 돌연변이에 의해 수득될 수 있다. 본 발명의 구체적인 측면에서, 상기 말레이트 세미알데하이드 탈수소효소는, 아스파르테이트 세미알데하이드 탈수소효소의 돌연변이에 의해 수득된다.
E. coli의 아스파르테이트 세미알데하이드 탈수소효소, Asd, 및 사카로마이세스 세레비지애의 Hom2는 천연적으로 4-포스포-말레이트 2에 대해 탈수소효소 활성을 나타낸다.
본 발명의 다른 측면에 따르면, 말레이트 세미알데하이드 탈수소효소는 아스파르테이트 세미알데하이드 탈수소효소의 돌연변이에 의해 개선될 수 있다.
도 3은, 여러 가지 생물 기원의 아스파르테이트 세미알데하이드 탈수소효소의 아미노산 서열을 정렬한 것이다. 아미노산은 모두, E. coli의 Asd 유전자에 의해 코딩되는 아스파르테이트 세미알데하이드 탈수소효소 (서열 번호 20으로 표시됨)를 근거로 참고한다. 다른 유기체 유래의, 또 다른 아스파르테이트 세미알데하이드 탈수소효소에 있는 상응하는 보존 영역의 상대적인 위치는, 후술하는 효소와 함께 도 4에서 나타낸 단순 서열 정렬을 통해, 당해 기술분야의 당업자가 쉽게 파악할 수 있다:
- EC - E. coli (서열 번호 49),
- MJ - 메타노코커스 얀나스치이 (서열 번호 94),
- TT - 써무스 써모필러스 (서열 번호 95),
- BS - 바실러스 서브틸리스 (서열 번호 96),
- CG - 코리네박테리움 글루타미쿰 (서열 번호 97),
- AT - 아라비돕시스 탈리아나 (서열 번호 98),
- SC - 사카로마이세스 세레비지애 (서열 번호 99).
상기 정렬은 ClustalW2 소프트웨어로 쉽게 수행될 수 있다.
말레이트 세미알데하이드 탈수소효소 활성이 개선된 효소는 하기와 같이 제작될 수 있다.
구체적인 측면에서, 본 발명에 따른 말레이트 세미알데하이드 탈수소효소는, 야생형 효소와 비교해, T136, Q162, I230, E241 및/또는 H274의 위치 중 하나 이상에, 하나 이상의 돌연변이를 포함하는 아스파르테이트 세미알데하이드 탈수소효소에 상응하며, 상기 위치(들)에서 천연 아미노산이, 다른 19개의 천연 단백질성 아미노산인 알라닌, 아르기닌, 아스파라긴, 아스파르트산, 시스테인, 글루타민산, 글루타민, 글리신, 히스티딘, 이소루신, 루신, 라이신, 메티오닌, 페닐알라닌, 프롤린, 세린, 트레오닌, 트립토판, 티로신, 또는 발린 중 어느 하나로 치환된다.
실시예 5에서 언급되는 바와 같이, E. coli 유래의 asd의 부위 특이적인 돌연변이유발은, 4-포스포-말레이트에 대한 돌연변이 효소의 활성 및 기질 친화성을 개선하면서, 동시에 이의 천연 기질인 4-포스포-아스파르테이트에 대한 효소의 선호도를 감소시킬 수 있다.
본 발명의 측면에 따라 4-포스포-말레이트에 대한 Asd의 활성을 개선하기 위해, E241은 부위 특이적인 돌연변이유발에 의해, 글루타민, 알라닌, 시스테인, 글리신, 히스티딘, 이소루신 또는 메티오닌 잔기로 치환되었다 (실시예 5).
본 발명의 다른 측면에서, 말레이트 세미알데하이드 탈수소효소는 서열 번호 68로 표시되며, 더욱 구체적으로는 서열 번호 54, 서열 번호 56, 서열 번호 58, 서열 번호 60, 서열 번호 62, 서열 번호 64 또는 서열 번호 66으로 표시된다.
본 발명은 또 다른 측면에서, 4-포스포-말레이트를 말레이트-4-세미알데하이드로 변환하는 말레이트 세미알데하이드 탈수소효소의 용도에 관한 것이다.
다른 측면에서, 본 발명에 따라 2,4-DHB를 제조하는 방법에서 제3 단계는, 말레이트-4-세미알데하이드를 2,4-DHB로 변환하는 것을 특징으로 하는, DHB 탈수소효소를 포함하며, 상기 효소는 경로의 생합성 측면에서, 말레이트-4-세미알데하이드 환원효소 활성을 가진다.
이미 잠재적으로는 DHB 탈수소효소 활성을 가진, 후보 (candidate) DHB 탈수소효소는, C3, C4, 또는 C5 화합물에 작용하는 베타-하이드록시산 탈수소효소 클래스에서 선택될 수 있다.
본 발명의 더욱 다른 측면에 따르면, 상기 DHB 탈수소효소 효소는 타르트로네이트 (tartronate) 세미알데하이드 환원효소, 숙시네이트 세미알데하이드 환원효소, 말로네이트 세미알데하이드 환원효소, 메틸부티르알데하이드 환원효소, 아연형 알코올 탈수소효소, L-트레오닌-3-탈수소효소, 또는 호모세린 환원효소와 같은 β-하이드록시산 탈수소효소와 구조적으로 및 메카니즘적으로 관련되어 있을 수 있다.
본 발명은 또한, 2,4-DHB에서 말레이트-4-세미알데하이드를 변환하기 위한, 메틸부티르알데하이드 환원효소 또는 숙신산 세미알데하이드 환원효소의 용도에 관한 것이다. 구체적인 실시 양태에서, 상기 메틸부티르알데하이드 환원효소는 서열 번호 74로 표시되고, 상기 숙신산 세미알데하이드 환원효소는 서열 번호 76으로 표시된다. DHB 탈수소효소 활성은 실시예 6에서 기술된 효소 테스트에 의해 측정될 수 있다 ("효소 분석" 참고).
말레이트-4-세미알데하이드에 대한, DHB 탈수소효소의 친화성은, 효소에 생긴 하나 이상의 돌연변이에 의해 증가할 수 있으며, 상기 돌연변이(들)는 말레이트-4-세미알데하이드에 대한, 돌연변이 효소의 활성 및/또는 기질 친화성을 증가시키며, 및/또는 이의 천연 기질에 대한, 활성이나 친화성을 적어도 2배 감소시킨다.
구체적인 측면에서, 본 발명에 따른 DHB 탈수소효소는, 야생형 효소와 비교해, S40, N43, H39, T49, F85, Q108, L281 및 N305의 위치 중 하나 이상에, 하나 이상의 돌연변이를 포함하는 메탈로스페라 세둘라 숙신산 세미알데하이드 환원효소 (서열 번호 76)이며, 상기 위치(들)의 천연 아미노산이, 다른 19개의 천연 단백질성 아미노산인 알라닌, 아르기닌, 아스파라긴, 아스파르트산, 시스테인, 글루타민산, 글루타민, 글리신, 히스티딘, 이소루신, 루신, 라이신, 메티오닌, 페닐알라닌, 프롤린, 세린, 트레오닌, 트립토판, 티로신, 또는 발린 중 어느 하나로 치환된다.
비-배타적인 실시예에서 입증된 바와 같이, (L)-말레이트-4-세미알데하이드에 대한, 메탈로스페라 세둘라 숙신산 세미알데하이드 환원효소의 친화성은, 부위 특이적인 돌연변이유발에 의해 이중 돌연변이 H39R N43H (서열 번호 81로 표시됨)를 도입함으로써 증가되었다. 단순 돌연변이체인 H39R (서열 번호 225) 및 N43H (서열 번호 227) 또한, 본 발명에 포함된다 (실시예 7).
DHB 탈수소효소는, 본 발명의 또 다른 측면을 구성하며, 말레이트-4-세미알데하이드를 2,4-DHB로 변환하는 데 사용될 수 있다.
유전자의 핵산 서열은 숙주 유기체의 코돈 사용에 맞게 조정하여, 이종적으로 발현되는 단백질의 생산을 증가시킬 수 있다. 이는, 본 발명의 다른 측면을 구성한다.
메탈로스페라 세둘라 숙신산 세미알데하이드 환원효소 H39R N43H를 코딩하며, 이의 뉴클레오티드 서열이 E. coli에서 상기 효소의 발현을 위해 최적화된, 합성 유전자 (서열 번호 228로 표시됨)의 합성은 본 발명의 다른 측면이다.
더욱 다른 측면에서, 본 발명은 또한, 핵산, 보다 구체적으로는 전술한 말레이트 키나아제를 코딩하는 분리된 핵산 서열에 관한 것이다.
다른 측면에서, 상기 핵산은 서열 번호 13, 서열 번호 15, 서열 번호 17, 서열 번호 19, 서열 번호 21, 서열 번호 23, 서열 번호 25, 서열 번호 27, 서열 번호 38, 서열 번호 40, 서열 번호 42 또는 서열 번호 44로 표시된다.
더욱 다른 측면에서, 본 발명은 또한, 전술한 말레이트 세미알데하이드 탈수소효소를 코딩하는 분리된 핵산 서열에 관한 것이다.
더욱 구체적으로는, 상기 핵산은 바람직하게는, 서열 번호 55, 서열 번호 57, 서열 번호 59, 서열 번호 61, 서열 번호 63, 서열 번호 65 또는 서열 번호 67로 표시된다.
더욱 다른 측면에서, 본 발명은 또한, 전술한 DHB 탈수소효소를 코딩하는 분리된 핵산 서열에 관한 것이다.
다른 측면에서, 상기 핵산은 서열 번호 73 또는 서열 번호 75, 서열 번호 224, 서열 번호 226 또는 서열 번호 82로 표시된다.
본 발명에 따르면, "핵산 서열"은 단일 또는 이중 가닥 형태의 DNA 또는 RNA 분자, 바람직하게는 DNA 분자를 지칭한다. 본원에서 사용되는 바와 같이, "분리된 DNA"는, 천연적이지 않거나, 또는 더 이상 원래 존재하였던 천연 환경에 존재하지 않는 DNA를 지칭하며, 예를 들어, 키메라 유전자 형태로 또 다른 조절 요소와 조합된 DNA 코딩 서열, 다른 숙주 세포로 이동된 DNA, 또는 임의의 자연 발생 DNA 서열과 비교해 상이한 뉴클레오티드 서열을 가진 인공적이며, 합성으로 제조된 DNA 서열을 지칭한다.
본 발명은 또한, 숙주 유기체에서 기능하는 하나 이상의 프로모터, 본 발명에 따른 말레이트 키나아제, 말레이트 세미알데하이드 탈수소효소 또는 DHB 탈수소효소를 코딩하는 폴리뉴클레오티드, 및 동일한 숙주 유기체에서 기능하는 종결자 요소를 서로 기능적으로 연결하여 포함하는, 키메라 유전자에 관한 것이다. 키메라 유전자에 포함될 수 있는 다양한 요소로는, 첫째로는, 프로모터, 신호 펩티드나 트랜지트 (transit) 펩티드를 코딩하는 서열, 또는 폴리아데닐화 신호를 구성하는 종결자 요소와 같이, 전사, 번역 및 단백질의 성숙을 조절하는 요소이며, 둘째로는, 단백질을 코딩하는 폴리뉴클레오티드이다. "서로 기능적으로 연결된"이라는 표현은, 키메라 유전자의 상기 요소들 중 어느 하나의 기능이 또 다른 요소의 기능에 의해 영향을 받도록, 이들 요소들이 서로 연결되어 있다는 것을 의미한다. 예로, 프로모터는, 코딩 서열의 발현에 영향을 미칠 수 있는 경우, 상기 코딩 서열에 기능적으로 연결되어 있다. 본 발명에 따른 키메라 유전자의 제작, 및 이의 다양한 요소들의 어셈블리는, 당해 분야의 당업자에게 잘 공지된 기술로 수행될 수 있다. 키메라 유전자를 이루는 조절 요소의 선택은, 이들이 기능해야 하는 숙주 유기체에 따라 필수적으로 다르며, 당해 분야의 당업자는 소정의 숙주 유기체에서 기능하는 조절 요소를 선택할 수 있다. "기능성"이라는 용어는, 소정의 숙주 유기체에서 기능할 수 있음을 의미하는 것이다.
본 발명에 따른 키메라 유전자에 포함될 수 있는 프로모터는 구성적 프로모터 (constitutive promoter) 또는 유도성 프로모터 (inducible promoter)이다. 예로, 박테리아의 발현에 사용되는 프로모터는 후술하는 프로모터에서 선택될 수 있다. 에스케리키아 콜라이의 발현에 대해, lac, trp, Ipp, phoA, recA, araBAD, prou, cst-l, tetA, cadA, nar, tac, trc, Ipp-lac, Psyn, cspA, PL, PL-9G-50, PR-PL, T7, [람다]PL-PT7, T3-lac, T5-lac, T4 유전자 32, nprM-lac, VHb 및 단백질 A 프로모터, 또는 심지어 Ptrp 프로모터 (WO 99/64607)를 언급할 수 있다. 코리네박테리아나 스트렙토마이세스와 같은 그람-양성 박테리아에서의 발현에 대해, PtipA 또는 PS1 및 PS2 (FR91/09870) 프로모터나, 특허 출원 제EP0629699A2 호에 기술된 것들을 언급할 수 있다. 효모와 균류에서의 발현에 대해, K. 락티스 PLAC4 프로모터 또는 K. 락티스 Ppgk 프로모터 (특허 출원 제FR 91/05294 호), 트리코데르마 (Trichoderma) tef1 또는 cbh1 프로모터 (WO 94/04673), 페니실리움 his, csl 또는 apf 프로모터 (WO 00/68401), 및 아스페르길루스 gla 프로모터를 언급할 수 있다.
본 발명에 따르면, 키메라 유전자는, 프로모터와 코딩 서열 사이에 위치하는, 전사 활성자 (인핸서)와 같은 다른 조절 서열도 포함할 수 있다.
이처럼, 본 발명의 키메라 유전자는 구체적인 실시 양태에서, 숙주 유기체에서 기능하는 하나 이상의 프로모터 조절 서열, 본 발명의 말레이트 세미알데하이드 탈수소효소의 말레이트 키나아제를 코딩하는 핵산 서열, 및 상기 숙주 유기체에서 기능하는 종결자 조절 서열을, 전사 방향으로 기능적으로 연결하여, 포함한다.
본 발명은 또한, 본 발명에 따른 키메라 유전자 또는 본 발명의 핵산 서열을 포함하는 클로닝 및/또는 발현 벡터에 관한 것이다. 본 발명에 따른 벡터는 숙주 유기체를 변환하고, 이 유기체에서 말레이트 키나아제, 말레이트 세미알데하이드 탈수소효소 및/또는 DHB 탈수소효소 중 어느 하나를 발현하는데 사용된다. 이 벡터는 플라스미드, 코스미드, 박테리오파지 또는 바이러스일 수 있다. 바람직하게는, 본 발명에 따른 형질전환 벡터는 플라스미드이다. 일반적으로, 이 벡터의 주 특징은, 숙주 유기체 세포에서 스스로 유지하고, 특히 복제 기원이 존재해서 자가복제하며, 말레이트 키나아제, 말레이트 세미알데하이드 탈수소효소 및/또는 DHB 탈수소효소 중 어느 하나를 세포 안에서 발현하는 능력이어야 한다. 숙주 유기체의 안정적인 형질전환을 위해서는, 벡터가 게놈에 삽입될 수도 있다. 이러한 벡터의 선택과, 본 발명에 따른 키메라 유전자를 상기 벡터에 삽입하는 기술은, 당해 분야의 당업자가 가지고 있는 일반적인 지식의 일부이다. 유리하게는, 본 발명에서 사용되는 벡터는, 본 발명에 따른 키메라 유전자 외에도, 선별 마커를 코딩하는 키메라 유전자도 포함한다. 상기 선별 마커는, 효과적으로 형질전환된 숙주 유기체, 즉, 벡터가 삽입된 유기체를 선별할 수 있게 한다. 본 발명의 특정 실시 양태에 따르면, 형질전환되는 숙주 유기체는 박테리아, 효모, 균류이다. 사용될 수 있는 선별 마커 중에서도, 예를 들어, 하이그로마이신 포스포트랜스퍼라아제 (hygromycin phosphotransferase) 유전자와 같은 항생제 내성 유전자를 포함하는 마커가 언급될 수 있다. 다른 마커로는, 영양요구를 보완하는 (complement) 유전자, 예컨대 pyrA, pyrB, pyrG, pyr4, arg4, argB 및 trpC 유전자, 몰리브도프테린 합성효소 유전자, 또는 아세타미다아제 (acetamidase) 유전자일 수 있다. GUS 효소와 같이 쉽게 동정될 수 있는 효소를 코딩하는 유전자나, 형질전환된 세포에서 안료를 코딩하는 유전자 또는 안료 생성을 조절하는 효소가 언급될 수도 있다. 이러한 선별 마커 유전자는 특히 특허 출원 제WO 91/02071 호, 제WO 95/06128 호, 제WO 96/38567 호, 및 제WO 97/04103 호에 기술되어 있다.
본 발명은 또한, 숙주 유기체의 게놈에 삽입되어 있거나, 염색체외 유전자 요소인 예를 들어, 플라스미드에 존재하는, 본 발명에 따른 키메라 유전자를 하나 이상 포함하는, 형질전환된 숙주 유기체에 관한 것이다. 본 발명의 더욱 구체적인 측면에서, 형질전환된 숙주 유기체는, 말레이트 키나아제를 코딩하는 본 발명의 핵산, 또는 말레이트 키나아제를 코딩하는 핵산을 포함하는 키메라 유전자, 또는 말레이트 키나아제를 코딩하는 핵산을 포함하는 발현 벡터, 및/또는 말레이트 세미알데하이드 탈수소효소를 코딩하는 핵산, 또는 말레이트 세미알데하이드 탈수소효소를 코딩하는 핵산을 포함하는 키메라 유전자, 또는 말레이트 세미알데하이드 탈수소효소를 코딩하는 핵산을 포함하는 발현 벡터, 및/또는 DHB 탈수소효소를 코딩하는 핵산, DHB 탈수소효소를 코딩하는 핵산을 포함하는 키메라 유전자, 또는 DHB 탈수소효소를 코딩하는 핵산을 포함하는 발현 벡터를 포함한다.
본 발명의 구체적인 측면에서, 말레이트 키나아제를 코딩하는 핵산은 서열 번호 13, 서열 번호 15, 서열 번호 17, 서열 번호 19, 서열 번호 21, 서열 번호 23, 서열 번호 25, 서열 번호 27, 서열 번호 38, 서열 번호 40, 서열 번호 42 또는 서열 번호 44로 표시되며, 말레이트 세미알데하이드 탈수소효소를 코딩하는 핵산은 SEQ ID 55, 서열 번호 57, 서열 번호 59, 서열 번호 61, 서열 번호 63, 서열 번호 65, 또는 서열 번호 67로 표시되고, DHB 탈수소효소를 코딩하는 핵산은 서열 번호 73, 서열 번호 75, 서열 번호 224, 서열 번호 226 또는 서열 번호 82로 표시된다.
"숙주 유기체"라는 용어는, 2,4-DHB를 제조하기 위해, 본 발명에 따른 키메라 유전자(들), 핵산(들) 또는 벡터(들)가 도입될 수 있는 임의의 저급 단세포 유기체를 의미하는 것이다. 바람직하게는, 숙주 유기체는 미생물, 특히 균류, 예를 들어, 페니실리움, 아스페르길루스, 더욱 특히 아스페르길루스 플라부스 (Aspergillus flavus), 크리소스포리움 (Chrysosporium) 또는 트리코데르마 속의 균류, 효모, 특히 사카로마이세스, 클루이베로마이세스, 또는 피치아 (Pichia) 속의 효모, 더욱 특히 자이고사카로마이세스 로욱시이, 박테리아, 예를 들어, 에스케리키아 속의 박테리아, 특히 E. coli, 또는 코리네박테리움 속의 박테리아, 더욱 특히 코리네박테리움 글루타미쿰 (Corynebacterium glutamicum), 또는 스트렙토마이세스 속의 박테리아, 또는 바쿨로바이러스이다.
숙주 유기체는, 포도당과 같은 당으로부터 말레이트나 숙시네이트를 자연적으로 과다생성하는 숙주 유기체, 또는 포도당과 같은 당으로부터 말레이트나 숙시네이트를 과다생성하도록 조작되고, 말레이트, 피루베이트, 숙시네이트, 및 푸마레이트와 같은 유기산의 유출을 용이하게 하는 모든 잠재적인 막 수송체가 결손된 숙주 유기체일 수 있다. 숙주 유기체는, DHB를 과다생성하도록 조작되고, 말레이트, 피루베이트, 숙시네이트, 및 푸마레이트와 같은 유기산의 유출을 용이하게 하는 모든 막 수송체가 결손된 유기체일 수 있다. 말레이트 및 다른 유기산의 유출을 용이하게 하는 투과효소의 예로는, 스키조사카로마이세스 폼베 (Schizosaccharomyces pombe) 유래의 Mae1 (Camarasa ef al., 2001; Grobler et al., 1995), 바실러스 서브틸리스 유래의 DctA (Groeneveld et al., 2010), E. coli 유래의 Dct 1-4, 사카로마이세스 세레비지애 유래의 Jen1 (Akita ef al., 2000)이 있다. 전문가는, 서열 상동성을 기초로, 다른 미생물에서 후보 투과효소를 동정할 수 있을 것이다. 이러한 구성은, 세포에서 말레이트와 다른 유기산이 DHB 제조에 이용될 수 있도록, 이 물질들을 유지시킬 것이다.
"형질전환된 숙주 유기체"라는 표현은, 숙주 유기체의 게놈으로, 또는 플라스미드와 같은 염색체외 유전자 요소에, 본 발명에 따른 키메라 유전자 하나 이상이 삽입되어, 결과적으로 유기체의 조직이나 배양 배지에서 말레이트 키나아제, 말레이트 세미알데하이드 탈수소효소 및/또는 DHB 탈수소효소 중 어느 하나를 생산하는 숙주 유기체를 의미하는 것이다. 본 발명에 따른 숙주 유기체를 수득하기 위해, 당해 분야의 당업자는 여러 공지된 형질전환 방법 중 하나를 이용할 수 있다.
이들 방법 중 하나는, 형질전환될 숙주 유기체의 세포를 폴리에틸렌 글리콜 (PEG)과, 그리고 본 발명에 따른 벡터와 접촉시키는 것으로 이루어진다. 형질전환될 세포와 본 발명의 벡터를 전기장에 두는 것으로 이루어진, 전기천공이 또 다른 방법이다. 다른 방법은, 미세주입에 의해 벡터를 세포나 조직에 직접 삽입하는 것으로 이루어진다. "바이오리스틱 (biolistic)" 방법이 사용될 수 있다. 이 방법은, 본 발명의 벡터가 그 위에 흡착된 입자들을 사용해, 세포나 조직을 공격함으로써 이루어진다 (미국 특허 제4,945,050 호).
박테리아를 형질전환하는 여러 방법은, 에스케리키아 콜라이와 다른 그람-음성 박테리아에 대한 문헌에 기술되어 있다. 접합이 사용될 수도 있다. 그람-양성 박테리아에 대해서는 전기천공이 사용될 수 있으며, 특히 스트렙토마이세스 속의 박테리에 대해서는 원형질 형질전환이 또한 사용될 수 있다.
균류를 형질전환하는 여러 방법이 또한 문헌에 기술되어 있다. PEG를 이용한 원형질 형질전환이 제EP 0260762 호에서 아스페르길루스를 들어 기술되어 있으며, 이 방법을 페니실리움 푸니쿨로숨 종에 맞도록 조정한 것이 제WO 00/36120 호에 기술되어 있다. 제한효소를 매개로 한 삽입, 또는 REMI에 의한 형질전환도 공지되어 있으며, 이는 아그로박테리움 속 박테리아를 사용하는 원형질 형질전환이다. 효모를 형질전환하는 기술 역시 문헌에 설명되어 있다.
다른 측면에서, 본 발명은 본 발명의 형질전환된 미생물을 배양하는 단계를 포함하는, 2,4-DHB의 제조 방법에 관한 것이다.
DHB의 제조를 위해, 포도당, 과당, 자당, 당밀, 엿당, 블랙스트랩 당밀, 전분 가수분해물 (포도당, 올리고당), 젖당, 엿당, 전분 및 전분 가수분해물, 셀룰로오스, 셀룰로오스 가수분해물, 글리세롤 및 소정의 탄화수소, 오일 및 지방 예컨대 대두유, 해바라기유, 땅콩유 및 코코넛유, 뿐만 아니라 지방산 예컨대 팔미트산, 스테아르산 및 리놀레산과 같은 다양한 탄수화물이, 각각 또는 혼합물로서 이용될 수 있다. 이들 성분들은 각각 또는 혼합물로서 사용될 수 있다.
기체성 또는 수성 암모니아와 같은 무기 화합물, 황산암모늄, 질산암모늄, 인산암모늄, 염화암모늄, 아세트산암모늄 및 탄산암모늄과 같은 무기 또는 유기산의 암모늄염을 비롯하여 다양한 질소원이, 상용화와 중간 시험 규모의 제조에, 각각 또는 혼합물로 이용될 수 있다. 대안적으로는, 대두-가수분해물, 콩 단백질 HCl-가수분해물 (총 질소량은 약 7%임), 콩가루, 콩깻묵 가수분해물, 옥수수 침지액, 카제인 가수분해물, 효모 추출물, 육류 추출물, 맥아 추출물, 요소, 펩톤 및 아미노산과 같은, 천연 질소 포함 유기 물질이 이용될 수도 있다.
제조 공정은 호기성, 혐기성 및 산소 제한 조건 하에 수행될 수 있다. 공정은 유가식 공정 또는 회분식 공정으로 수행될 수 있다.
증식이 잘 되게 하는 말레이트 (또는 피루베이트, 숙시네이트나 푸마레이트와 같은 다른 유기산)를 단독으로, 또는 다른 탄소원과 함께 첨가한 매질에서 숙주 유기체를 배양함으로써, 상기 2,4-DHB를 제조할 수 있다. 말레이트 (및 다른 유기산)는 직접 첨가될 수 있거나, 또는, 제1 공정 단계에서, 말레이트-과다생성 미생물에 의해 말레이트 (또는 다른 유기산)가 생성되고, 후속한 단계에서, 본 발명에 따른 숙주 유기체에 의해 2,4-DHB가 생성되는 2-단계 발효 공정을 설계하여, 첨가될 수 있다.
생성물 분리 및 정제는 총 공정 효율과 생성물 가격에 크게 영향을 미치는 매우 중요한 요소이다. 생성물 회수 방법은 보편적으로, 세포 분리, 뿐만 아니라 생성물 정제, 농축 및 건조 단계를 각각 포함한다.
세포 분리
발효 매질에서 세포를 분리하기 위해, 한외여과 및 원심분리가 사용될 수 있다. 발효 매질에서 세포를 분리하는 것은, 높은 매질 점성으로 인해 종종 복잡하다. 따라서, 본 발명자들은, 세포 분리를 최적화하기 위해, 무기산이나 알칼리염과 같은 첨가제를 첨가하거나, 배양 배지 (culture broth)를 열처리할 수 있다.
생성물 회수
바이오매스 제거 전이나 후에, 다양한 이온-교환 크로마토그래피 방법이 DHB 분리에 이용될 수 있다. 이 방법들은, 생성물을 이들의 등전점에 따라 분리하는 것을 용이하게 하는, 1차 양이온 교환 수지의 사용을 포함한다. 전형적으로, 상기 수지에는 용액이 채워져 있으며, 보유 생성물은 용리액에서 (예를 들어, 수산화암모늄의 첨가에 의한) pH 증가에 따라 개별적으로 용리된다. 다른 가능성으로는, 고정층 또는 모사 이동층 수지를 사용하는 이온-교환 크로마토그래피의 이용이다. 적합한 생성물 순도를 수득하기 위해서는, 여러 가지 크로마토그래피 단계가 조합되어야 한다. 이들 정제 방법은 고비용의 결정화 단계에 비해 더 경제적이며, 최종 산물의 형태와 관련해 부가적인 이점이라든지 유연성을 제공하기도 한다.
생성물 농축 및 건조
정제 공정은, 분무 과립 건조기, 분무 건조기, 드럼 건조기, 회전 건조기, 및 터널 건조기와 같은 임의의 적절한 건조 수단을 수반할 수 있는 건조 단계를 포함할 수도 있다. 농축된 DHB 용액은, 다목적 농축기나 박막 증발기를 사용하는 경우, 130℃에서 증기로, 감압 하에 발효조를 가열함으로써 수득될 수 있다.
효율적인 DHB 제조는, 숙주 유기체의 대사 네트워크에서 탄소 흐름의 재분배를 최적화하고, DHB 경로의 3 가지 효소에 NADPH와 ATP를 충분히 공급해줌으로써, 보장될 수 있다. 원하는 대사 경로로 탄소 흐름을 채널링하고, NAD(P)H 보조인자를 공급하는 것은 보편적으로, 경쟁적 천연 발효 경로를 생략하거나 감소시킴으로써 용이해 진다. 비-배타적인 예로는,
- (피루베이트 탈카르복실화효소의 결손에 의해) 에탄올 형성을 방해함으로써, 사카로마이세스 세레비지애에서 말레이트 생성을 최적화하는 것 (Zelle et al., 2008; Zelle et al., 2010),
- 락테이트 형성 (예를 들어, IdhA의 결손), 아세테이트 형성 (예를 들어, pta, ackA의 결손), 에탄올 형성 (예를 들어, adhE의 결손), 포르메이트 형성 (예를 들어, pflB, focA의 결손), 피루베이트 산화 (예를 들어, poxB의 결손), 말레이트 분해 (maeB 및 scfA의 결손), 숙시네이트 형성 (예를 들어, frdBC의 결손), 메틸글리옥살 형성 (mgsA의 결손) (Jantama et al, 2008a; Jantama et al., 2008b; Lin et al., 2005; Sanchez et al., 2005a; Zhang et al., 2011)을 방해함으로써, E. coli에서 숙시네이트나 말레이트 생성을 최적화하는 것이 있다.
유기산 제조를 위해 탄소 흐름과 ATP 공급을 증가시킬 수 있는 다른 방법은, 포스포에놀피루베이트 (PEP)/피루베이트/옥살로아세테이트 브랜치 노드 (branch node)를 조작하는 것이다 (Sauer & Eikmanns, 2005에서 리뷰됨). 포스포에놀피루베이트에서 옥살로아세테이트로의 탄소 흐름을 확실히 증가시키는 대사 공학 계획에 대한 비-배타적인 예로는,
- 피루베이트 키나아제의 작용을 방해하고, PEP 카르복시키나아제의 활성을 증가시킴으로써, 사카로마이세스 세레비지애에서 말레이트 생성을 최적화하는 것 (Zelle et al., 2010).
- 천연적으로 또는 이종적으로 발현되는 PEP 카르복실라아제, PEP 카르복시키나아제, 또는 피루베이트 카르복실라아제의 활성을 증가시킴으로써, E. coli에서 숙시네이트 생성을 최적화하는 것 (Millard et al., 1996; Sanchez et al., 2005b; Zhang et al., 2009)이다.
포도당을 처음으로 인산화시키는 단계에 PEP-소모 포스포트랜스퍼라아제 시스템 (PTS)을 이용하는 E. coli와 또 다른 박테리아에서, 유기산의 제조를 위해 탄소 흐름과 ATP 공급을 증가시키는 다른 방법은, PTS 시스템의 필수 성분들 (예를 들어, pts1 또는 ptsG)을 결손시키는 것이다 (Lin et al., 2005; Zhang et al., 2009). PTS 시스템에 결손 돌연변이를 가진 돌연변이체에서 포도당 흡수를 더욱 확실히 하기 위해서는, 대체 포도당 흡수 시스템 (예를 들어 GalP)의 활성이 보장되어야 한다.
유기산을 제조하는 바람직한 반응경로로 향하는 탄소 흐름을 증가시킬 다른 방법은, 시트르산 및 글리옥실레이트 회로를 조작하는 것이다. 비-배타적인 예로는,
- 이소시트레이트 분해효소의 활성을 증가시킴으로써 (전사 억제자 iclR의 결손), E. coli에서 숙신산 생성을 최적화하는 것 (Lin et al., 2005; Sanchez ef al., 2005a).
- 이소시트레이트 탈수소효소, 및/또는 숙시네이트 탈수소효소의 결손에 의해, 숙신산 생성을 최적화하는 것 (Lin et al., 2005)이다.
DHB를 제조하는 바람직한 반응경로로 향하는 탄소 흐름을 증가시킬 다른 방법은, 생성 유기체에서 적절한 피루베이트 탈수소효소 및 시트레이트 합성효소가 발현되는 것이다. E. coli의 천연 피루베이트 탈수소효소 및 시트레이트 합성효소는, 혐기성 조건 하에 이들 효소의 활성을 감소시키는, 고농도의 세포내 NADH에 의해 저해된다. E. coli에서, NADH에 둔감한 피루베이트 탈수소효소 돌연변이체가 발현되면, 혐기성 조건 하에 아세틸-CoA가 과다생성되고, 발효 최종 산물 (아세테이트, 락테이트, 에탄올, 포르메이트, 및 피루베이트) 사이에 탄소 흐름 재분배가 변경된다 (Wang et al., 2010). NADH에 둔감한 바실러스 서브틸리스 시트레이트 합성효소의 이종성 발현은, 조작된 E. coli 균주에서 숙신산 생성을 증가시킨다 (Sanchez et al., 2005a). 전술한 돌연변이와 더불어, 적절한 피루베이트 탈수소효소 및 시트레이트 합성효소 (NADH 민감성 또는 둔감성)를 사용하면, 호기성 및 혐기성 조건 시, 글리옥실레이트 및 시트르산 회로 반응과 발효 경로 사이에 탄소 흐름 재분배를 조정할 수 있다.
DHB 경로를 통한 탄소 흐름을 증가시킬 다른 방법은, 반응경로 중간체인 4-포스포말레이트, 4-말레이트 세미알데하이드를 분해할 수 있는 효소 반응을 생략하는 것이다. 말레이트 세미알데하이드를 분해할 수 있는 후보 효소는 숙신산 세미알데하이드 탈수소효소 (sad, gabD)와, 말단 알데하이드기로 C4 분자를 산화시킬 수 있는 다른 탈수소효소들이다.
숙주 유기체의 DHB 생산성을 증가시킬 다른 방법은, DHB를 분해하는 대사 반응을 생략하는 것이다. DHB는 말산 효소의 경쟁적 저해자로서, 이 효소의 활성 부위에 대한 친화성이 상당히 높다 (Rognstad & Katz, 1979). 따라서, DHB는 다른 효소에 의해 인지되어 잠재적으로 분해될 수 있다. 이들 효소는 숙주 유기체에서 동정되고 결손될 수 있다.
2,4-DHB 생성이 말레이트 또는 다른 유기산의 첨가를 기본으로 할 경우, 2,4-DHB-생성 미생물은, 말레이트 (또는 피루베이트, 숙시네이트 등과 같은 다른 유기산)의 흡수를 용이하게 하는, 막 수송 단백질을 기능적으로 발현해야 한다.
하기 실시예는 본 발명을 예시한다. 이들 실시예는 예시를 목적으로 할 뿐, 어떤 방식으로 본 발명의 범위를 한정하려는 것이 아니다.
도 1 : (i) (L)-말레이트가 (L)-2,4-디하이드록시부티레이트 (DHB)로 전환되고, (ii) 유사하게, (L)-아스파르테이트가 (L)-호모세린으로 전환되는 것을 설명하는 반응 도식.
도 2: 여러 가지 유기체 유래의 아스파르테이트 키나아제의 아미노산 서열 정렬. (Ec_AKIII - E. coli 유래의 아스파르테이트 키나아제 III (서열 번호 4), LysC, Ec_AKI (서열 번호 87) - E. coli 유래의 아스파르테이트 키나아제 I, ThrA, Ec_AKII (서열 번호 88) - E. coli 유래의 아스파르테이트 키나아제 II, MetL, Mj - 메타노코커스 얀나스치이 (Methanococcus jannaschii) (서열 번호 89), Tt - 써무스 써모필러스 (Thermus thermophilus) (서열 번호 90), Cg -코리네박테리움 글루타미쿰 (Corynebacterium glutamicum) (서열 번호 91), At - 아라비돕시스 탈리아나 (Arabidopsis thaliana) (서열 번호 92), Sc - 사카로마이세스 세레비지애 (서열 번호 93)). 상기 도는 ClustalW2로 제작되었다 (Larkin et al, 2007).
도 3: 여러 가지 유기체 유래의 아스파르테이트 세미알데하이드 탈수소효소의 아미노산 서열 정렬 (Ec - E. coli (서열 번호 49), Mj -메타노코커스 얀나스치이 (서열 번호 94), Tt - 써무스 써모필러스 (서열 번호 95), Bs - 바실러스 서브틸리스 (Bacillus subtilis) (서열 번호 96), Cg - 코리네박테리움 글루타미쿰 (서열 번호 97), At - 아라비돕시스 탈리아나 (서열 번호 98), Sc -사카로마이세스 세레비지애 (서열 번호 99)). 상기 도는 ClustalW2로 제작되었다 (Larkin et al, 2007).
도 4: 하기를 도시하는, DHB의 체류 시간에 해당되는 영역을 확대한 GC 크로마토그램: (A) DHB 표준물 (농도 = 1 mM); (B) 말레이트 키나아제 (MK), 말레이트 세미알데하이드 탈수소효소 (MSA-Dh), 및 말레이트 세미알데하이드 환원효소 (MSA-Red)를 포함하는 반응 A의 조성물 ; (C) 대조군 반응 B의 조성물 (A와 동일하지만 MSA-Red가 결핍되어 있음); (D) 대조군 반응 C의 조성물 (A와 동일하지만 MSA-Dh가 결핍되어 있음).
도 5: 정제된 LysC E119G, LysC E119G E250K, LysC E119G T344M, LysC E119G S345L, LysC E119G T344M, 및 LysC E119G T352I 돌연변이체의, 반응 완충액 중 라이신 농도에 대한, 상대적인 활성.
도 2: 여러 가지 유기체 유래의 아스파르테이트 키나아제의 아미노산 서열 정렬. (Ec_AKIII - E. coli 유래의 아스파르테이트 키나아제 III (서열 번호 4), LysC, Ec_AKI (서열 번호 87) - E. coli 유래의 아스파르테이트 키나아제 I, ThrA, Ec_AKII (서열 번호 88) - E. coli 유래의 아스파르테이트 키나아제 II, MetL, Mj - 메타노코커스 얀나스치이 (Methanococcus jannaschii) (서열 번호 89), Tt - 써무스 써모필러스 (Thermus thermophilus) (서열 번호 90), Cg -코리네박테리움 글루타미쿰 (Corynebacterium glutamicum) (서열 번호 91), At - 아라비돕시스 탈리아나 (Arabidopsis thaliana) (서열 번호 92), Sc - 사카로마이세스 세레비지애 (서열 번호 93)). 상기 도는 ClustalW2로 제작되었다 (Larkin et al, 2007).
도 3: 여러 가지 유기체 유래의 아스파르테이트 세미알데하이드 탈수소효소의 아미노산 서열 정렬 (Ec - E. coli (서열 번호 49), Mj -메타노코커스 얀나스치이 (서열 번호 94), Tt - 써무스 써모필러스 (서열 번호 95), Bs - 바실러스 서브틸리스 (Bacillus subtilis) (서열 번호 96), Cg - 코리네박테리움 글루타미쿰 (서열 번호 97), At - 아라비돕시스 탈리아나 (서열 번호 98), Sc -사카로마이세스 세레비지애 (서열 번호 99)). 상기 도는 ClustalW2로 제작되었다 (Larkin et al, 2007).
도 4: 하기를 도시하는, DHB의 체류 시간에 해당되는 영역을 확대한 GC 크로마토그램: (A) DHB 표준물 (농도 = 1 mM); (B) 말레이트 키나아제 (MK), 말레이트 세미알데하이드 탈수소효소 (MSA-Dh), 및 말레이트 세미알데하이드 환원효소 (MSA-Red)를 포함하는 반응 A의 조성물 ; (C) 대조군 반응 B의 조성물 (A와 동일하지만 MSA-Red가 결핍되어 있음); (D) 대조군 반응 C의 조성물 (A와 동일하지만 MSA-Dh가 결핍되어 있음).
도 5: 정제된 LysC E119G, LysC E119G E250K, LysC E119G T344M, LysC E119G S345L, LysC E119G T344M, 및 LysC E119G T352I 돌연변이체의, 반응 완충액 중 라이신 농도에 대한, 상대적인 활성.
실시예
1 :
아스파르테이트
및
말레이트
키나아제
활성에 대한, 각각 에스케리키아
콜라이
및
사카로마이세스
세레비지애
유래의
아스파르테이트
키나아제
LysC
및 Hom3의 테스트
아스파르테이트 키나아제의 야생형 유전자를 포함하는 플라스미드의 제작: 개시 코돈의 상류과 정지 코돈의 하류 각각에 NdeI 및 BamHI 제한효소 부위를 도입하는, 정방향 및 역방향 프라이머 5'CACGAGGTACATATGTCTGAAATTGTTGTCTCC3' (서열 번호 1) 및 5'CTTCCAGGGGATCCAGT-ATTTACTCAAAC3' (서열 번호 2)와, 하이 피델리티 중합효소 Phusion™ (high fidelity polymerase Phusion™) (핀자임스 (Finnzymes) 사 제품)을 사용한 PCR에 의해, lysC 유전자를 증폭시켜, 플라스미드 pLYSCwt를 제작하였다. E. coli DH5α의 게놈 DNA를 주형으로 사용하였다. PCR 산물을 NdeI 및 BamHI으로 잘라, T4 DNA 리가아제 (바이오랩스 (Biolabs) 사 제품)를 사용해 pET28a (노바겐 (Novagen) 사 제품) 발현 벡터의 해당 부위에 연결하고, 이를 E. coli DH5α 세포에 형질전환하였다. 제조되는 pAKIIIwt 플라스미드를 분리하고, 올바른 서열 (서열 번호 3)을 가진 전장 lysC 유전자를 포함하는지를, DNA 시퀀싱으로 확인하였다. 해당 단백질은 서열 번호 4로 표시된다.
개시 코돈의 상류과 정지 코돈의 하류 각각에 NheI 및 EcoRI 제한효소 부위를 도입하는, 정방향 및 역방향 프라이머 5'TATAATGCTAGCATGCCAATGGATTTCCAACC3' (서열 번호 5) 및 5'TATAATGAATTCT- TAAATTCCAAGTCTTTTCAATTGTTC3' (서열 번호 6)과, 하이 피델리티 중합효소 Phusion™ (핀자임스 사 제품)을 사용한 PCR에 의해, HOM3 유전자를 증폭시켜, 플라스미드 pHOM3wt를 제작하였다. 사카로마이세스 세레비지애 BY4741wt 유래의 게놈 DNA를 주형으로 사용하였다. PCR 산물을 NheI 및 EcoRI으로 절단하고, T4 DNA 리가아제 (바이오랩스 사 제품)를 사용해 pET28a (노바겐 사 제품) 발현 벡터의 해당 부위에 연결하고, 이를 E. coli DH5α 세포에 형질전환하였다. 제조되는 pHOM3wt 플라스미드를 분리하고, 올바른 서열 (서열 번호 7)을 가진 전장 HOM3 유전자를 포함하는지를, DNA 시퀀싱으로 확인하였다. 해당 단백질은 서열 번호 8로 표시된다.
효소 발현: E. coli BL21 D3 스타 세포 (star cell)를 적절한 플라스미드로 형질전환하였다. OD600 0.1에서 하룻밤 배양시킨 배양물을 접종하여 OD600 0.6으로 증식시킨 후, 1 mM 이소프로필 β-D-1-티오갈락토파리노시드 (IPTG)를 배양 배지에 첨가하여 단백질 발현을 유도하여, 250 mL LB 배양액에서, N-말단 헥사-His 태그가 붙은 효소를 발현시켰다. 단백질 발현 3 시간 후, 13000 g에서 10 분 동안 원심분리해서 세포를 회수하고, 다음 분석까지 -20℃에 보관하였다. 증식 및 단백질 발현은 37℃에서 수행하였다. 배양 배지에는 50 ㎍/L 카나마이신이 첨가되었다.
효소 정제: 발현 배양물 중 동결시킨 세포 펠렛을 0.5 mL의 분해 완충액 (50 mM Hepes, 300mM NaCl, pH 7.5)에 재현탁시키고, 전력 출력을 30%로 세팅한 초음파 파쇄법을 4 회 연속으로 진행시켜, 분해하였다 (바이오블락 사이언티픽 (Bioblock Scientific) 사 제품, 바이브라셀™ 72437 (Vibracell™ 72437)). 조 (crude) 추출물을 4℃, 13000 g에서 15 분 동안 원심분리하여 세포 잔해를 제거하고, 맑은 상층액을 수득하였다. 15 mg/mL 스트렙토마이신 (시그마 (Sigma) 사 제품)을 첨가하고, 샘플을 4℃, 13000 g에서 10 분 동안 원심분리하여, 상층액을 수득함으로써, RNA 및 DNA를 추출물에서 제거하였다. 맑은 단백질 추출물을, 베드 (bed) 부피가 0.75 mL인 탤론™ 코발트 친화성 수지 (Talon™ Cobalt affinity resin) (클론테크 (Clontech) 사 제품)로 4℃에서 1 시간 동안 인큐베이션하였다. 현탁액을 테이블 탑 원심분리기, 700 g에서 원심분리하고, 상층액을 제거하였다. 0.5 mL의 용리 완충액 (50 mM Hepes, 300 mM NaCl, 500 mM 이미다졸, pH 7.5)으로 아스파르테이트 키나아제를 용리하기 전에, 10 베드 부피의 세정 완충액 (50 mM Hepes, 300 mM NaCl, 15 mM 이미다졸, pH 7.5)으로 수지를 세정하였다. 용리된 효소의 순도는 SDS-PAGE 분석으로 확인하였다.
효소 분석: 포스포에놀피루베이트, 피루베이트 키나아제, 및 락테이트 탈수소효소의 존재 하에, 키나아제 반응의 ADP 생성을 NADH 산화와 커플링함으로써, 아스파르테이트 또는 말레이트 키나아제 활성을 분석하였다.
반응 도식:
아스파르테이트 (또는 말레이트) 키나아제
아스파르테이트 (또는 말레이트) + ATP → 4-포스포-(L)-아스파르테이트 (또는 4-포스포-(L)-말레이트) + ADP
피루베이트 키나아제
ADP + 포스포에놀피루베이트 → ATP + 피루베이트
락테이트 탈수소효소
피루베이트 + NADH → NAD+ + 락테이트
분석 혼합물은 50 mM Hepes (pH 7.5), 50 mM KCl, 5 mM MgCl2, 0.24 mM NADH, 0.96 mM ATP, 0.96 mM PEP, 9 ㎍/mL의 락테이트 탈수소효소 (시그마 사 제품, L2500), 12.4 ㎍/mL 피루베이트 키나아제 (시그마 사 제품, P1506), 및 적당량의 정제된 아스파르테이트 (말레이트) 키나아제를 포함한다. 50 mM (L)-아스파르테이트 또는 (L)-말레이트를 첨가하여 반응을 개시하였다. 효소 분석은, 30℃의, 96-웰 평판 마이크로타이터 플레이트에, 최종 부피 250 ㎕에서 수행하였다. 다음으로, 340 nm에서의 NADH의 특징적인 흡광 반응을 마이크로플레이트 판독기 (바이오라드 680XR (BioRad 680XR))에서 수행하였다.
하이드록사메이트 분석: 야생형 또는 돌연변이형 아스파르테이트 키나아제에 의한 기질의 인산화, 즉, 아실포스페이트 무수물의 형성을 확인하기 위해, 키나아제 반응 생성물을 하이드록실아민과 인큐베이션하여, 상응하는 아스파르테이트 또는 말레이트 하이드록사메이트 유도체를 제조하였다. 분석 혼합물에는, 120 mM Hepes (pH 8), 200 mM KCl, 10 mM ATP, 200 mM 하이드록실아민, 10 mM 아스파르테이트 또는 말레이트와, 적당량의 정제 단백질이 들어 있었다. 30 분 후에, 1 M 염산 중 1.7% (w/v) FeC를 동일한 부피로 첨가하여, 반응을 중지시켰다. 마이크로타이터 플레이트 판독기에서, 540 nm에서의 하이드록사메이트-철 착체의 특징적인 흡광도를 측정하여, 상기 착체의 형성을 확인하였다. ATP를 제외한 모든 성분이 들어 있는 분석 혼합물을 블랭크로 사용하였다.
결과: 정제된 LysC (His-태그 부착 안됨, 서열 번호 4) 및 Hom3 (His-태그 부착 안됨, 서열 번호 7) 효소는 아스파르테이트 키나아제 활성을 나타내었으나, 하이드록사메이트 분석으로 확인한 바와 같이 말레이트를 인산화하지는 못하였다 (Keng & Viola, 1996). 아스파르테이트에 대한 LysC 및 Hom3의 최대 활성은 각각 4.5 μmol/(min*mgprot) 및 1.6 μmol/(min*mgprot)이었다. 아스파르테이트에 대한 Km 값은, 에디 호프스티 (Eadie and Hofstee) 식으로, 여러 가지 기질 농도 (c)에서 초기 반응 속도 (v)를 측정하고, v/c 플롯에 대한 v의 기울기를 구해서, 산출하였다. 정제된 His-태깅된 LysC의 Km은 대략 0.6 mM로서, His-태깅된 단백질은, 0.6 mM로 보고된 비-태깅된 정제 효소의 기질 친화성과 동일한 것으로 나타났다 (Marco-Marin et al., 2003).
실시예
2: 에스케리키아
콜라이
유래의 아스파르테이트
키나아제
LysC
의 부위 특이적인 돌연변이유발, 및 돌연변이 효소의,
말레이트
키나아제
활성에 대한 테스트
표 1에서 열거된 올리고뉴클레오티드 쌍과, 주형으로 pLYSCwt (서열 번호 3) 플라스미드를 사용하여, 부위 특이적인 돌연변이유발을 수행하였다. 아미노산 서열을 바꾸기 위해, 표 1에 열거된 올리고뉴클레오티드 쌍을 사용한 PCR (Phusion 1 U, HF 완충액 20% (v/v), dNTPs 2.5 mM, 정방향 및 역방향 프라이머 각각 1 μM, 주형 플라스미드 200 ng, 물)로 점 돌연변이를 도입하였다. PCR로 제작된 플라스미드는 새로운 Nco1 제한효소 부위 (침묵 돌연변이로 도입됨)외에도, 돌연변이된 클론의 동정을 용이하게 하는 기능성 돌연변이를 포함하였다. 주형 DNA를 제거하기 위해, PCR 산물을 37℃에서 1 시간 동안 Dpnl로 분해시키고, NEB 5-알파 컴피턴트 E. coli 세포 (NEB)에 형질전환시켰다. 돌연변이된 플라스미드를 제한효소 부위 분석으로 동정하고, 원하는 돌연변이가 생겼는지를 DNA 시퀀싱으로 확인하였다.
위치 119에 돌연변이를 나타내는 서열은 서열 번호 9로 표시될 수 있으며, 위치 119의 잔기는 X이며, 이 X는 19개의 천연 아미노산 (글루타민 제외) 중 어느 하나이다.
실시예 1에서 기술된 바와 같이, 돌연변이 효소를 발현시키고, 정제하고, 아스파르테이트 및 말레이트 키나아제 활성을 테스트하였다. 결과를 하기 표 2에 요약하였다.
표 2에 열거된 돌연변이들 어느 것도 아스파르테이트에 대한 활성을 나타내지 않았다.
그 결과, 위치 119의 보존된 (conserved) 글루타메이트를 시스테인, 글리신, 아스파라긴, 프롤린, 글루타민, 세린, 트레오닌, 또는 발린으로 치환함으로써, 아스파르테이트 키나아제를 말레이트 키나아제로 변환시킬 수 있다.
표 2에 열거된 효소에 상응하는 핵산 서열은 서열 번호 13, 서열 번호 15, 서열 번호 17, 서열 번호 19, 서열 번호 21, 서열 번호 23, 서열 번호 25 및 서열 번호 27이다.
실시예
3: 라이신 저해에 대한 민감도가
크게 감소된
말레이트
키나아제의
구축
표 3에 열거된 올리고뉴클레오티드 쌍과, 주형으로서 pLYSC_E119G 플라스미드를 사용하여, 부위 특이적인 돌연변이유발을 수행하였다. pLYSC_E119G 플라스미드는 실시예 2에서 기술된 바와 같이, lysC 유전자의 DNA 서열 (서열 번호 15)에 하기 변화를 도입하여 수득하였다. 아미노산 서열을 바꾸기 위해, 표 1에 열거된 올리고뉴클레오티드 쌍을 사용한 PCR (Phusion 1 U, HF 완충액 20% (v/v), dNTPs 2.5 mM, 정방향 및 역방향 프라이머 각각 1 μM, 주형 플라스미드 200 ng, 물)로 점 돌연변이를 도입하였다. 가능한 경우, PCR로 제작된 플라스미드는 새로운 제한효소 부위 (침묵 돌연변이로 도입됨)외에도, 돌연변이된 클론의 동정을 용이하게 하는 기능성 돌연변이를 포함하였다. 주형 DNA를 제거하기 위해, PCR 산물을 37℃에서 1 시간 동안 Dpnl로 분해시키고, NEB 5-알파 컴피턴트 E. coli 세포 (NEB)에 형질전환하였다.
돌연변이된 플라스미드를 제한효소 부위 분석으로 동정하고, 원하는 돌연변이가 생겼는지를 DNA 시퀀싱으로 확인하였다.
(i) 위치 250의 글루타민산이 라이신으로 치환된 부가적인 돌연변이를 포함하는 단백질 LysC E119G의 핵산 서열은 서열 번호 38로 표시되고; 이의 상응하는 아미노산 서열은 서열 번호 39로 표시되며; (ii) 위치 344의 트레오닌이 메티오닌으로 치환된 것은 서열 번호 40으로 표시되고; 이의 상응하는 아미노산 서열은 서열 번호 41로 표시되며; (iii) 위치 352에서 트레오닌이 이소루신으로 치환된 것은 서열 번호 42로 표시되고; 이의 상응하는 아미노산 서열은 서열 번호 43으로 표시되며; (iv) 위치 345의 세린이 루신으로 치환된 것은 서열 번호 44로 표시되며; 이의 상응하는 아미노산 서열은 서열 번호 45로 표시된다.
효소의 발현 및 정제: His-태깅된 효소 LysC E119G, LysC E119G E250K, LysC E119G T344M, LysC E119G S345L, LysC E119G T352I에 대한 단백질 발현을 실시예 1에서 기술된 바와 같이 수행하였다.
효소 분석: 실시예 1에서 기술된 바와 같이, 말레이트 키나아제 활성을 분석하였다. 반응 완충액 내 라이신 농도는 다양하게 하였다.
결과: 돌연변이 E250K, T344M 또는 S345L을 LysC E119G에 도입하면, 라이신 농도가 증가했음에도, 라이신에 대한 말레이트 키나아제 활성이 크게 둔감해졌다 (도 4 참고).
실시예
4: 에스케리키아
콜라이
유래의 아스파르테이트
세미알데하이드
탈수소효소
Asd
의, 아스파르테이트 및
말레이트
세미알데하이드
탈수소효소 활성에 대한 테스트
아스파르테이트 세미알데하이드 탈수소효소의 야생형 유전자를 포함하는 플라스미드의 제작: 개시 코돈의 상류과 정지 코돈의 하류 각각에 NheI 및 BamHI 제한효소 부위를 도입하는, 정방향 및 역방향 프라이머 5'TATAATGCTAGCATGAAAAATGTTGGTTTTATCGG3' (서열 번호 46) 및 5'TATAATGGATCCTTACGCCAGTTGACGAAGC3' (서열 번호 47)과, 하이 피델리티 중합효소 Phusion™ (핀자임스 사 제품)을 사용한 PCR에 의해, E. coli의 asd 유전자를 증폭시켜, 플라스미드 pASDwt를 제작하였다. E. coli DH5α 유래의 게놈 DNA를 주형으로 사용하였다. PCR 산물을 NheI 및 BamHI으로 분해하고, T4 DNA 리가아제 (바이오랩스 사 제품)로 pET28a (노바겐 사 제품) 발현 벡터의 상응하는 자리에 연결하고, E. coli DH5α 세포에 형질전환하였다. 제조되는 pASDwt 플라스미드를 분리하고, 올바른 서열 (서열 번호 48)을 가진 전장 asd 유전자를 포함하는지를, DNA 시퀀싱으로 확인하였다. 상기 효소에 상응하는 아미노산 서열은 서열 번호 49로 표시된다.
효소의 발현 및 정제: 실시예 1에서 기술된 바와 같이 His-태깅된 효소 Asd에 대한 단백질 발현을 수행하였다.
효소 분석: 아스파르테이트 또는 말레이트 세미알데하이드가 4-포스포-(L)-아스파르테이트 또는 4-포스포-(L)-말레이트로 각각 산화되는 중에 이루어지는 NADP의 환원을 추적함으로써, 아스파르테이트 또는 말레이트 세미알데하이드 탈수소효소 활성을 생합성의 역방향으로 분석하였다 (Roberts et al., 2003).
(L)-아스파르테이트 세미알데하이드 (또는 (L)-말레이트 세미알데하이드) + NADP + Pi → 4-포스포-(L)-아스파르테이트 (또는 4-포스포-(L)-말레이트) + NADPH
분석 혼합물에는 200 mM Hepes (pH 9), 50 mM K2HPO4, 0,25 mM NADP가 포함되었다. (L)-아스파르테이트 세미알데하이드 또는 (L)-말레이트 세미알데하이드를 첨가하여, 반응을 개시하였다. (L)-아스파르테이트 세미알데하이드는, 호모세린 탈수소효소 및 아스파르테이트 세미알데하이드 탈수소효소의 효소 테스트에 적합한 기질인 L-아스파르트산 β-세미알데하이드 하이드레이트 트리플루오로아세테이트 (분해를 방지하기 위해 pH3에서 유지됨) 형태로 첨가하였다 (Roberts et al., 2003). 효소 테스트 전에, 안정한 말레이트 세미알데하이드 유도체인 2-[(4S)-2,2-디메틸-5-옥소-1,3-디옥솔란-4-일]아세트알데하이드 (DMODA)의 탈보호에 의해, 불안정한 말레이트 세미알데하이드를 새로 제조하였다. 25℃에서 15 분 동안 DMODA를 2 M 염산 중에 인큐베이션하고, 방출된 아세톤을 증발시켜 (35℃, 50 mbar), 말레이트 세미알데하이드를 수득하였다. 말레이트 세미알데하이드 용액의 pH는 중탄산나트륨을 이용해 3으로 고정시켰다.
효소 분석은, 30℃, 96-웰 평판 마이크로타이터 플레이트에서 250 ㎕의 최종 부피로 수행하였다. 반응 후, 340 nm에서의 NADPH의 특징적인 흡광도를 마이크로플레이트 판독기 (바이오라드 680XR)에서 측정하였다.
결과: His-태깅된 야생형 아스파르테이트 세미알데하이드 탈수소효소, Asd는 (L)-아스파르테이트 세미알데하이드를 4-포스포-(L)-아스파르테이트로 산화하였으며, 최대 비활성은 160 μmol/(min*mgprot)이었다. (L)-말레이트 세미알데하이드에 대한 효소의 활성은 0.01 μmol/(min*mgprot)이었다.
실시예 5: 에스케리키아 콜라이 유래의 아스파르테이트 세미알데하이드 탈수소효소 Asd 의 부위 특이적인 돌연변이유발, 및 상기 돌연변이 효소의, 말레이트 세미알데 하이드 탈수소효소 활성에 대한 테스트.
pASDwt 플라스미드를 주형으로 사용하고 실시예 2의 프로토콜에 따라, Asd의 아미노산 서열에 점 돌연변이를 도입하였다. 표 4에 열거된 올리고뉴클레오티드 쌍을 사용해, 위치 241의 글루타메이트 잔기와, 위치 136의 트레오닌 잔기를 돌연변이시켰다. 돌연변이된 플라스미드는 제한효소 부위 분석으로 동정하였고, 원하는 돌연변이가 생겼는지를 DNA 시퀀싱으로 확인하였다.
위치 241에서 돌연변이된 Asd 단백질은 서열 번호 68로 표시될 수 있으며, 위치 241의 잔기는 X이고, 이 X는 다른 19개의 생물학적으로 형성되는 아미노산 (글루타민 제외) 중 어느 하나이다.
결과: 위치 E241에서 돌연변이된 Asd의 활성 및 Km 값을 하기 표 5에 요약하였다. 위치 241의 글루타메이트를 알라닌, 시스테인, 글리신, 히스티딘, 이소루신, 메티오닌, 또는 글루타민으로 치환한 Asd 돌연변이체는 (L)-아스파르테이트-4-세미알데하이드를 4-포스포-(L)-아스파르테이트로 산화하였으며, 최대 비활성이 야생형 효소보다 훨씬 더 높았다. 이중 돌연변이체 Asd E241Q T136N (서열 번호 231)의 최대 비활성은 0.25 μmol/(min*mgprot)이었고, Km은 0.25 mM이었다.
상응하는 핵산은 서열 번호 55, 서열 번호 57, 서열 번호 48, 서열 번호 59, 서열 번호 61, 서열 번호 63, 서열 번호 65 및 서열 번호 67로 표시된다.
이중 돌연변이체인 Asd E241Q T136N은 서열 번호 230으로 표시된 핵산 서열을 가진다.
실시예
6: 2,4
DHB
탈수소효소의 동정
적합한 2,4 DHB 탈수소효소를 동정하기 위해, 말레이트 세미알데하이드를 환원하는, 여러 가지 생물 기원의 베타-하이드록시산 탈수소효소의 능력을 테스트하였다. 테스트한 효소 중에는, 사카로마이세스 세레비지애 유래의 메틸부티르알데하이드 환원효소, Ypr1 (Ford & Ellis, 2002) (서열 번호 73 및 서열 번호 74); 및 메탈로스패라 세둘라 (Metallosphaera sedula) 유래의 숙신산 세미알데하이드 환원효소, Ms-Ssr (Kockelkorn & Fuchs, 2009) (서열 번호 75 및 서열 번호 76)이 있었다. 표 6에 열거된 프라이머를 사용해 유전자 YPR1 및 Ms-SSR을 증폭시키고, 벡터 pET28 (제한 효소는 상기 표 3을 참고함)에 형질전환시켜, 각각 플라스미드 pYPR1 및 pMs-SSR을 수득하였다. 실시예 1에서 기술된 바와 같이, 단백질을 발현시키고 정제하였다.
말레이트 세미알데하이드 환원효소 활성에 대한 테스트:
반응:
(L)-
말레이트
세미알데하이드
+
NAD
(P)H → (L)-2,4-
디하이드록시부티르산
+
NAD
(P)
분석 혼합물에는, 200 mM Hepes (pH 7.5), 50 mM KCl, 5 mM MgCl2, 0.24 mM NADH 또는 NADPH, 및 적당량의 정제 효소가 포함되었다. 10 mM (L)-말레이트 세미알데하이드 (실시예 4를 참고로, 각 테스트를 위해, 말레이트 세미알데하이드를 새로 제조하였음)를 첨가하여, 반응을 개시하였다. 효소 분석은, 30℃, 96-웰 평판 마이크로타이터 플레이트에서, 250 ㎕의 최종 부피로 수행하였다. 반응 후, 340 nm에서의 NAD(P)H의 특징적인 흡광도를 마이크로플레이트 판독기 (바이오라드 680XR)에서 측정하였다. 결과를 하기 표 7에 요약하였다.
메탈로스페라 세둘라 유래의 숙신산 세미알데하이드 탈수소효소 및 사카로마이세스 세레비지애 유래의 메틸부티르알데하이드 환원효소는 말레이트 세미알데하이드 환원효소 활성을 가졌다. 말레이트 세미알데하이드에 대한 Ms-SSR의 Km은 1.1 mM이었다.
실시예 7: 메탈로스페라 세둘라 유래의 숙신산 세미알데하이드 환원효소의 부위 특이적인 돌연변이유발
표 8에서 열거된 올리고뉴클레오티드 쌍과, 주형으로 pMs-SSR 플라스미드를 사용하여 부위 특이적인 돌연변이유발을 수행하였다. 아미노산 서열을 바꾸기 위해, PCR (Phusion 1 U, HF 완충액 20% (v/v), dNTPs 2.5 mM, 정방향 및 역방향 프라이머 각각 1 μM, 주형 플라스미드 200 ng, 물)로 점 돌연변이를 도입하였다. 가능한 경우, PCR로 제작된 플라스미드는 새로운 제한효소 부위 (침묵 돌연변이로 도입됨)외에도, 돌연변이된 클론의 동정을 용이하게 하는 기능성 돌연변이를 포함하였다. 주형 DNA를 제거하기 위해, PCR 산물을 37℃에서 1 시간 동안 Dpnl로 분해시키고, NEB 5-알파 컴피턴트 E. coli 세포 (NEB)에 형질전환하였다. 돌연변이된 플라스미드를 제한효소 부위 분석으로 동정하고, 원하는 돌연변이가 생겼는지를 DNA 시퀀싱으로 확인하였다. 표 9에 돌연변이체의 카이네틱 파라미터를 요약하였다. 결과에 따르면, 이중 돌연변이체 Ms-SSR H39R N43H (서열 번호 81, 서열 번호 82)의, 말레이트 세미알데하이드에 대한 친화성이, 야생형 효소와 비교해 개선되었다.
상응하는 핵산 서열은 서열 번호 224, 서열 번호 226 및 서열 번호 82로 표시된다.
실시예
8:
DHB
의
시험관내
제조
실시예 1에서 기술된 바와 같이, 효소 말레이트 키나아제 (LysC E119G, 서열 번호 15), 말레이트 세미알데하이드 탈수소효소 (Asd E241Q; 서열 번호 67), 및 말레이트 세미알데하이드 환원효소 (Ms SSrR, 서열 번호 76)를 발현시켜 정제하였다. 50 mM Hepes (pH 7.5), 50 mM KCl, 5 mM MgCl2, 1 mM NADPH, 180 ㎍/mL의 말레이트 키나아제 (Lys E119G), 325 ㎍/mL의 말레이트 세미알데하이드 탈수소효소 (Asd E241Q), 및 130 ㎍/mL의 말레이트 세미알데하이드 환원효소 (Ms_Ssr)가 포함된 반응 혼합물에 50 mM 말레이트를 첨가함으로써, DHB의 시험관내 생산을 입증하였다 (반응 A). 대조군 반응물에는, 말레이트 세미알데하이드 환원효소를 제외한 모든 성분이 포함되어 있거나 (반응 B), 또는 말레이트 세미알데하이드 탈수소효소를 제외한 모든 성분이 포함되어 있었다 (반응 C). 30℃에서 30 분 동안 인큐베이션시킨 후, 반응 혼합물을 기체 크로마토그래피 [CPG 배리안 시리즈 430 (CPG Varian Series 430); FID 검출기; 오토샘플러 CP8400; 스플릿리스 주입기 (splitless injector) 1177 (230℃); 칼럼: CP-WAX58/FFAP, 30 m x 0.53 mm, df 0.50 ㎛; 및 라이너 (liner): 투입구 슬리브 (Inlet Sleeve), 구스넥 (gooseneck) 6.5 mm x 78.5 mm x 4 mm GWOL이 장착되어 있음 (배리안 (Varian) 사 제품)]로 분석하였다. 캐리어 기체는 질소로, 유속은 25 mL/분이었다. 공기-수소 혼합물을 이용해 불꽃 이온화 반응 (유속은 각각 300 mL/분 및 30 mL/분이었음)을 수행하였다. 검출기 온도는 240℃였다. 주입된 샘플 부피는 1 ㎕였다. 온도 프로그램은 하기 표 10에 나타내었다.
DHB 제조는 반응물 A (모든 효소가 존재함)에서 검출되었으나, 대조군 반응물 B와 C에서는 이루어지지 않았다 (도 5).
실시예 9: E. coli 에서 메탈로스페라 세둘라 숙신산 세미알데하이드 환원효소를 발현시키기 위한, 상기 효소의 코딩 서열의 최적화.
돌연변이 H39R 및 N43H를 포함한 메탈로스페라 세둘라 숙신산 세미알데하이드 환원효소의 코딩 서열이, 진옵티마이저® (GeneOptimizer®) 소프트웨어를 이용해, E. coli에서 최대로 발현되도록 최적화하였다. 진아트® 진 신세시스 (GeneArt® Gene Synthesis) (인비트로겐 라이프 테크놀로지 (Invitrogen Life Technologie) 사 제품)로, 합성 유전자를 제조하였다. NheI 및 EcoRI 제한효소 부위를 개시 코돈의 상류과, 정지 코돈의 하류 각각에 도입하여, pET28a+ (노바겐 사 제품)에 직접 클로닝하였다.
제조되는 pSSR-H39RN43H-opt 플라스미드를 분리하고, 올바른 서열 (서열 번호 228)을 가진 전장 메탈로스페라 세둘라 SSR H39R N43H 유전자를 포함하는지를 DNA 시퀀싱으로 확인하였다.
실시예 10: E. coli 를 숙주 유기체로 사용한, 말레이트 키나아제 (E. coli 유래의 lysC 유전자의 돌연변이체), 말레이트 세미알데하이드 탈수소효소, (E. coli 유래의 asd 유전자의 돌연변이체), 및 DHB 탈수소효소 (메탈로스페라 세둘라 숙신산 세미알데하이드 환원효소 유전자의 돌연변이체)의 동시 발현이 용이한 플라스미드의 제작.
오페론 제작용 백본으로 플라스미드 pLYSC-E119G E250K (서열 번호 38)를 사용하였다. 주형으로 pASD-E241 Q (서열 번호 55), 및 rbs의 상류과 정지 코돈의 하류 각각에 BamHI 및 EcoRI 제한효소 부위를 도입하는 정방향 및 역방향 프라이머 5'TATAAGGATCCGTTTAACTTTAAGAAGGAGATATACCATGGG3' (서열 번호 83) 및 5'TATAAGAATTCTTACGCCAGTTGACGAAG3' (서열 번호 84)를 사용한 PCR (하이 피델리티 중합효소 Phusion™ (핀자임스 사 제품))로, pET28 (노바겐 사 제품) 리보좀 결합 부위 (rbs), 및 ASD-E241Q의 코딩 영역을 포함하는 DNA 절편을 수득하였다. PCR 산물을 BamHI 및 EcoRI으로 분해하고, T4 DNA 리가아제 (바이오랩스 사 제품)를 이용해 pLYSC-E119G E250K의 상응하는 자리에 연결하고, 이를 E. coli DH5α 세포에 형질전환하였다. 제조되는 pLYSC-E119G-E250K_ASD-E241 Q 플라스미드를 분리하고, 올바른 서열을 가지는지를 DNA 시퀀싱으로 확인하였다.
주형으로 pSSR-H39RN43H-opt와, rbs의 상류과 정지 코돈의 하류 각각에 NotI 및 PspXI 제한효소 부위를 도입하는 정방향 및 역방향 프라이머 5'TATAAGCGGCCGCGTTTAACTTTAAGAAGGAGATAT3' (서열 번호 85) and 5'TATAAACTCGAGCTTACGGAATAATCAGG3' (서열 번호 86)를 사용한 PCR에 의해, pET28 리보좀 결합 부위 (rbs), 및 코돈-최적화된 Ms-SSR-H39RN43H-opt의 코딩 서열을 포함하는 DNA 절편을 수득하였다. PCR 산물을 NotI 및 PspXI으로 분해하고, T4 DNA 리가아제 (바이오랩스 사 제품)를 이용해 pLYSC-E119G-E250K_ASD-E241Q의 상응하는 자리에 연결하고, 이를 E. coli DH5α 세포에 형질전환하였다. 제조되는 pET28-DHB 플라스미드 (서열 번호 229)를 분리하고, 올바른 서열을 가지는지를 DNA 시퀀싱으로 확인하였다.
pET28-DHB를 SphI 및 XbaI으로 분해하고, 적절한 제한효소 부위에 다른 프로모터 영역을 클로닝하여, 3개 유전자의 발현을 동시에 조절하는 5' 상류 프로모터 영역 (즉, pET28-DHB의 T7 프로모터)을, 유도성 또는 구성적인 임의의 다른 프로모터로 치환할 수 있다. 유도성 프로모터를 사용하는 예로, pET28-DHB 백본의 T7 프로모터를, 포도당의 존재 시, 단백질을 발현할 수 있게 하는 특징을 가진 tac 프로모터로 치환하였다 (de Boer et al., 1983). 플라스미드를 SphI 및 XbaI로 분해하여, 플라스미드 pEXT20 (Dykxhoorn et al., 1996)에서 tac 프로모터를 수득하였다. 상기 프로모터를 포함하는 DNA 절편을 정제하고, 이를, SphI 및 XbaI으로 분해한 pET28-DHB 플라스미드에 클로닝하였다. 제조되는 pTAC-DHB 플라스미드를 분리하고, 올바른 서열을 가지는지 DNA 시퀀싱으로 확인하였다.
실시예 11: 발효에 의해 DHB 를 제조하기 위해, 탄소 흐름의 재분배 및 NADPH -보조인자 공급을 최적화하기 위한, E. coli 균주의 제작.
DHB 제조를 위한 탄소 흐름 재분배 및 보조인자 공급을 최적화하기 위해, E. coli 균주 MG1655에서 유전자 수종을 파괴하였다. Datsenko et al. (Datsenko & Wanner, 2000)에 따라, 람다 레드 재조합효소 방법으로 유전자를 결손시켰다.
하이 피델리티 중합효소 Phusion™ (핀자임스 사 제품), 및 주형으로 플라스미드 pKD4의 FRT-플랭크된 카나마이신 내성 유전자 (kan)를 사용한 PCR에 의해, 결손 카세트를 제조하였다 (Datsenko & Wanner, 2000). 센스 프라이머는, 각 표적 유전자 (밑줄이 그어져 있음)의 5' 말단에 상응하는 서열과, 뒤이어, pKD4의 FRT-kan-FRT 카세트에 상응하는 20 bp를 포함하였다. 안티-센스 프라이머는, 각 표적 유전자 (밑줄이 그어져 있음)의 3' 말단 영역에 상응하는 서열과, 뒤이어 카세트에 상응하는 20 bp를 포함하였다. 하기 표 12에 프라이머를 기술하였다. 형질전환하기 전에, PCR 산물을 DpnI으로 분해하고, 정제하였다.
E. coli MG1655 균주는, 세포를 37℃의 LB 액상 매질에서 OD600 0.6으로 증식시키고, 상기 세포를 100-배 농축시킨 다음, 아이스콜드 10% 글리세롤로 2 회 세정함으로써, 일렉트로-컴피턴트 (electro-competent)로 만들었다. 전기천공 (2.5 kV, 200 Ω, 25 μF, 2 mm 갭 큐벳 (gap cuvettes)에서)에 의해, 세포를 플라스미드 pKD46 (Datsenko & Wanner, 2000)으로 형질전환하였다. 형질전환체를, 30℃에서, 앰피실린 (100 ㎍/mL)이 든 LB 고형 매질에서 선별하였다.
람다 레드 재조합효소-발현 플라스미드 pKD46을 가진 일렉트로-컴피턴트 E. coli 균주를 파괴 카세트로 형질전환하였다. 상기 세포를 앰피실린 (100 ㎍/mL)이 첨가된, 30℃, 액상 SOB 매질에서 증식시켰다. 배양물의 OD600이 0.1에 도달했을 때, 10 mM의 아라비노스를 첨가하여, 람다 레드 재조합효소 시스템을 유도하였다. 원심분리로 세포를 회수하기 전에, 상기 세포를 0.6의 OD600으로 더 증식시키고, 아이스콜드 10% 글리세롤로 2회 세정하고, 전기천공에 의해 파괴 카세트로 형질전환하였다. 30℃, LB 액상 매질에서 밤새 표현형이 발현되도록 한 다음, 상기 세포를 25 ㎍/mL 카나마이신이 첨가된 고형 LB 매질에 평판 배양하였다. 30℃에서 배양한 후, 형질전환체를 선별하였다.
크림슨 택 중합효소 (Crimson Taq polymerase) (NEB 사 제품)를 사용한 콜로니 PCR에 의해, 유전자 치환을 확인하였다. 동시적인, 모 절편의 소실과 신규 돌연변이체 특이 절편의 생성을 확인하기 위해, 플랭킹 유전자 좌-특이 (flanking locus-specific) 프라이머 (표 13 참고)로 제1 반응을 수행하였다. FRT-카나마이신 내성 카세트에 근접한 유전자 좌-특이 프라이머와, 각각 보편적인 테스트 프라이머인 k1rev, 또는 k2for (표 13 참고) (센스 유전자 좌 프라이머/k1rev 및 k2for/역방향 유전자 좌 프라이머)을 사용해, 2회의 추가적인 반응을 수행하였다.
이어서, FLP 재조합효소를 가지는 플라스미드 pCP20 (Cherepanov & Wackernagel, 1995)을 사용해 내성 유전자 (FRT-kan-FRT)를 염색체에서 절단하였고, 이로써, FRT 부위가 하나 포함된 스카 영역 (scar region)이 형성되었다. pCP20은 앰피실린 및 CmR 플라스미드로서, 열 유도성 FLP 재조합효소 합성과 온도-민감성 복제를 나타낸다. 카나마이신 내성 돌연변이체를 pCP20으로 형질전환하고, 30℃에서 앰피실린-내성 형질전환체를 선별하였다. 다음, 형질전환체를 37℃, 고형 LB 매질에서 증식시키고, 항생제 내성이 모두 소실되었는지 테스트하였다. 크림슨 택 중합효소와 플랭킹 유전자 좌-특이 프라이머를 사용한 콜로니 PCR에 의해, FRT-카나마이신 카세트의 절단을 분석하였다 (표 13). 전술한 단계의 반복으로, 다중 결손이 이루어졌다.
결손이 하나 또는 여러 개 있는 균주는, 전술한 바와 같이 일렉트로-컴피턴트한 상태로 만든 후, 상기 균주를, IPTG에 의해 유도되어 DHB 경로의 효소를 발현 하는 pTAC-DHB 플라스미드로 형질전환한 다음 (실시예 10 참조), 50 ㎍/mL 카나마이신이 첨가된 고형 LB 매질에서 선별하였다.
주형으로 E. coli MG1655 유래의 게놈 DNA와, 각각 정방향 및 역방향 프라이머인 5'TATAATCCCGGGATGCGCGTTAACAATGGTTTGACC3' (서열 번호 100) 및 5'TATAATTCTAGATTACAGTTTCGGACCAGCCG3' (서열 번호 101)를 사용하여, pck 코딩 서열을 증폭시켜, E. coli의 PEP 카르복시키나아제 코딩 pck 유전자를 가지는 플라스미드 pACT3-pck를 제작하였다. DNA 절편을 XmaI 및 XbaI으로 절단하고, T4 DNA 리가아제 (바이오랩스 사 제품)를 사용해 pACT3 발현 벡터 (Dykxhoorn et al., 1996)의 해당 자리에 연결하고, 이를 E. coli DH5α 세포에 형질전환하였다. 클로람페니콜 (25 ㎍/mL)이 첨가된 고형 LB 매질에서 형질전환체를 선별하였다. 제조되는 플라스미드를 분리하고, pck 유전자가 올바르게 삽입되었는지 시퀀싱으로 확인하였다. 각각 aceA, ppc, galP, 또는 pykA (모두 E. coli 유래) 또는 락토코커스 락티스 (Lactococcus lactis) 유래의 pycA를 가지는 플라스미드 pACT3-aceA, pACT3-ppc, pACT3-galP, pACT3-pykA 및 pACT3-pyc를, 하기 표 14에서 열거한 프라이머를 사용해 유사하게 제작하였다.
하기 표 12에 열거한 결손의 조합을 포함하는 E. coli MG1655 돌연변이체를, 전술한 pACT3-유래의 플라스미드와 pTAC-DHB 플라스미드로 형질전환하였다. 클로람페니콜 (25 ㎍/mL) 및 카나마이신 (50 ㎍/mL)이 첨가된 고형 LB 매질에서, 플라스미드 둘 모두를 포함하는 형질전환체를 선별하였다. 제작된 균주의 예를 하기 표 15에 열거하였다.
실시예
12: 포도당 발효에 의한 2,4-
디하이드록시부티르산의
제조
균주 및 배양 조건: 말레이트 키나아제, 말레이트 세미알데하이드 탈수소효소, 및 플라스미드 pTAC-DHB 유래의 DHB 탈수소효소를 공동-발현하는 E. coli ECE1 균주 (실시예 11 참고)와, 빈 플라스미드 (즉, 전술한 효소의 코딩 서열이 존재하지 않는 pTAC 백본)만을 포함하는 동종 (isogenic) 대조군 균주로 실험을 수행하였다. 1 리터 배양 배지에는, 20 g 포도당, 18 g Na2HP04 * 12 H20, 3 g KH2P04, 0.5 g NaCl, 2 g NH4Cl, 0.5 g MgS04 * 7 H20, 0.015 CaCl2 * 2 H20, 농축 HCl에서 100 배 희석되어 제조된, 1 mL의, 0.06 mol/L FeCl3 스탁 용액, 2 mL의 10 mM 티아민 HCl 스탁 용액, 20 g MOPS, 50 ㎍ 카나마이신 설페이트, 및 1 mL의 미량 원소 용액 (리터 당, 0.04 g Na2EDTA * 2H20, 0.18 g CoCl2 * 6 H20, ZnSO4 * 7 H20, 0.04 g Na2MoO4 * 2 H20, 0.01 g H3BO3, 0.12 g MnSO4 * H20, 0.12 g CuCl2 * H20이 포함되었음)이 들어 있었다. pH를 7로 맞추고, 매질을 여과 멸균시켰다. 37℃, 170 rpm으로 운행되는 인포스 (Infors) 회전 쉐이커에서 모든 배양을 수행하였다. 글리세롤 스탁 유래의 하룻밤 배양물 (테스트 튜브 내 3 mL 매질)을 접종하였으며, 이를 이용하여 500 mL 쉐이크 플라스크 내 100 mL 증식 배양물의 OD600을 0.05로 적정하였다. 배양 배지의 OD600이 0.2에 도달했을 때, IPTG를 1 mmol/L의 농도로 첨가하였다.
LC - MS / MS 분석에 의한 DHB 농도 측정: 원심분리 (베크만-쿨터 알레그라 21R (Beckmann-Coulter Allegra 21R), 로터 베크만 S4180 (Rotor Beckmann S4180), 10 분, 4800 rpm)로 배양 배지를 세포와 분리하였다. 다음 분석 때까지, 맑은 상층액을 -20℃에 보관하였다. 질량 민감성 검출기 (TQ, 와터스 (Waters) 사 제품, ESI 모드, 모세관 전압 (capillary voltage): 2.5 kV, 콘 전압 (cone voltage): 25 V, 추출기 전압 (extractor voltage): 3V, 소스 온도 (source temperature): 150℃, 디졸베이션 온도 (desolvation temperature): 450℃, 콘 가스 유속 (cone gas flow): 50 L/h, 디졸베이션 가스 유속 (desolvation gas flow): 750 L/h)에 연결된, 액퀴티 (ACQUITY) UPLC BEH 칼럼 (C18, 1.7 ㎛, 100 mm x 2.1 mm, 와터스 사 제품)이 장착된 HPLC (와터스 사 제품)로, DHB 함량을 정량화하였다. 칼럼 온도를 30℃에서 유지하였다. 이동상은 0.08% 테트라-n-부틸암모늄 수산화물 용액 88%와, 아세토니트릴 12%의 혼합물이었다. 유속은 0.4 mL/min에서 유지하였다. 샘플 주입 부피는 5 ㎕이었다.
결과:
IPTG에 의해, 말레이트 키나아제, 아스파르테이트 세미알데하이드 탈수소효소, 및 DHB 탈수소효소의 발현을 유도한 지 8 시간 및 24 시간 후, E. coli ECE1 균주와 대조군 균주의 배양 배지 내 DHB 함량을 측정하였다. 하기 표 16에서 알 수 있듯이, DHB 경로의 효소를 발현하는 ECE1 균주는 대조군 균주보다 훨씬 더 다량의 DHB를 생산하였으며, 이는 도 1 (i)에서 도시된 대사 경로를 통해 DHB를 발효에 의해 제조할 수 있음을 의미한다.
SEQUENCE LISTING
<110> ADISSEO France SAS
<120> A method of production of 2,4-dihydroxybutyric acid
<130> BR073152
<150> PCT/IB2010/003153
<151> 2010-10-28
<150> PCT/IB2011/001559
<151> 2011-05-23
<160> 231
<170> PatentIn version 3.5
<210> 1
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplication
<400> 1
cacgaggtac atatgtctga aattgttgtc tcc 33
<210> 2
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 2
cttccagggg atccagtatt tactcaaac 29
<210> 3
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 3
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca cggcgagctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 4
<211> 449
<212> PRT
<213> Escherichia coli
<400> 4
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 5
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 5
tataatgcta gcatgccaat ggatttccaa cc 32
<210> 6
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 6
tataatgaat tcttaaattc caagtctttt caattgttc 39
<210> 7
<211> 1584
<212> DNA
<213> Saccharomyces cerevisiae
<400> 7
atgccaatgg atttccaacc tacatcaagt cattcgaact gggtcgtgca aaagttcggt 60
ggtacatctg tcggtaaatt tcccgtccaa atagtggatg acattgtgaa gcactattct 120
aaacctgacg gcccaaacaa taatgtcgct gtcgtttgtt ccgcccgttc ttcatacacc 180
aaggctgaag gtaccacttc tcgtcttttg aaatgttgtg atttggcttc gcaagaatct 240
gaatttcaag acattatcga agttatcaga caagaccata tcgataatgc cgaccgcttc 300
attctcaatc ctgccttgca agccaagtta gtggatgata ccaataaaga acttgaactg 360
gtcaagaaat atttaaatgc ttcaaaagtt ttgggtgaag tgagttcacg tacagtagat 420
ctggtgatgt catgtggtga gaagttgagt tgtttgttca tgactgcttt atgtaatgac 480
cgtggctgta aggccaaata tgtggatttg agccacattg ttccctctga tttcagtgcc 540
agcgctttgg ataacagttt ctacactttc ctggttcaag cattgaaaga aaaattggcc 600
ccctttgtaa gtgctaaaga gcgtatcgtt ccagtcttta cagggttttt tggtttagtt 660
ccaactggtc ttctgaatgg tgttggtcgt ggctataccg atttatgtgc cgctttgata 720
gcagttgctg taaatgctga tgaactacaa gtttggaagg aagttgatgg tatatttact 780
gctgatcctc gtaaggttcc tgaagcacgt ttgctagaca gtgttactcc agaagaagct 840
tctgaattaa catattatgg ttccgaagtt atacatcctt ttacgatgga acaagttatt 900
agggctaaga ttcctattag aatcaagaat gttcaaaatc cattaggtaa cggtaccatt 960
atctacccag ataatgtagc aaagaagggt gaatctactc caccacatcc tcctgagaac 1020
ttatcctcat ctttctatga aaagagaaag agaggtgcca ctgctatcac caccaaaaat 1080
gacattttcg tcatcaacat tcattccaat aagaaaaccc tatcccatgg tttcctagct 1140
caaatattta ccatcctgga taagtacaag ttagtcgtag atttaatatc tacttctgaa 1200
gttcatgttt cgatggcttt gcccattcca gatgcagact cattaaaatc tctgagacaa 1260
gctgaggaaa aattgagaat tttaggttct gttgatatca caaagaagtt gtctattgtt 1320
tcattagttg gtaaacatat gaaacaatac atcggcattg ctggtaccat gtttactact 1380
cttgctgaag aaggcatcaa cattgaaatg atttctcaag gggcaaatga aataaacata 1440
tcctgcgtta tcaatgaatc tgactccata aaagcgctac aatgtattca tgccaagtta 1500
ctaagtgagc ggacaaatac ttcaaaccaa tttgaacatg ccattgatga acgtttagaa 1560
caattgaaaa gacttggaat ttaa 1584
<210> 8
<211> 527
<212> PRT
<213> Saccharomyces cerevisiae
<400> 8
Met Pro Met Asp Phe Gln Pro Thr Ser Ser His Ser Asn Trp Val Val
1 5 10 15
Gln Lys Phe Gly Gly Thr Ser Val Gly Lys Phe Pro Val Gln Ile Val
20 25 30
Asp Asp Ile Val Lys His Tyr Ser Lys Pro Asp Gly Pro Asn Asn Asn
35 40 45
Val Ala Val Val Cys Ser Ala Arg Ser Ser Tyr Thr Lys Ala Glu Gly
50 55 60
Thr Thr Ser Arg Leu Leu Lys Cys Cys Asp Leu Ala Ser Gln Glu Ser
65 70 75 80
Glu Phe Gln Asp Ile Ile Glu Val Ile Arg Gln Asp His Ile Asp Asn
85 90 95
Ala Asp Arg Phe Ile Leu Asn Pro Ala Leu Gln Ala Lys Leu Val Asp
100 105 110
Asp Thr Asn Lys Glu Leu Glu Leu Val Lys Lys Tyr Leu Asn Ala Ser
115 120 125
Lys Val Leu Gly Glu Val Ser Ser Arg Thr Val Asp Leu Val Met Ser
130 135 140
Cys Gly Glu Lys Leu Ser Cys Leu Phe Met Thr Ala Leu Cys Asn Asp
145 150 155 160
Arg Gly Cys Lys Ala Lys Tyr Val Asp Leu Ser His Ile Val Pro Ser
165 170 175
Asp Phe Ser Ala Ser Ala Leu Asp Asn Ser Phe Tyr Thr Phe Leu Val
180 185 190
Gln Ala Leu Lys Glu Lys Leu Ala Pro Phe Val Ser Ala Lys Glu Arg
195 200 205
Ile Val Pro Val Phe Thr Gly Phe Phe Gly Leu Val Pro Thr Gly Leu
210 215 220
Leu Asn Gly Val Gly Arg Gly Tyr Thr Asp Leu Cys Ala Ala Leu Ile
225 230 235 240
Ala Val Ala Val Asn Ala Asp Glu Leu Gln Val Trp Lys Glu Val Asp
245 250 255
Gly Ile Phe Thr Ala Asp Pro Arg Lys Val Pro Glu Ala Arg Leu Leu
260 265 270
Asp Ser Val Thr Pro Glu Glu Ala Ser Glu Leu Thr Tyr Tyr Gly Ser
275 280 285
Glu Val Ile His Pro Phe Thr Met Glu Gln Val Ile Arg Ala Lys Ile
290 295 300
Pro Ile Arg Ile Lys Asn Val Gln Asn Pro Leu Gly Asn Gly Thr Ile
305 310 315 320
Ile Tyr Pro Asp Asn Val Ala Lys Lys Gly Glu Ser Thr Pro Pro His
325 330 335
Pro Pro Glu Asn Leu Ser Ser Ser Phe Tyr Glu Lys Arg Lys Arg Gly
340 345 350
Ala Thr Ala Ile Thr Thr Lys Asn Asp Ile Phe Val Ile Asn Ile His
355 360 365
Ser Asn Lys Lys Thr Leu Ser His Gly Phe Leu Ala Gln Ile Phe Thr
370 375 380
Ile Leu Asp Lys Tyr Lys Leu Val Val Asp Leu Ile Ser Thr Ser Glu
385 390 395 400
Val His Val Ser Met Ala Leu Pro Ile Pro Asp Ala Asp Ser Leu Lys
405 410 415
Ser Leu Arg Gln Ala Glu Glu Lys Leu Arg Ile Leu Gly Ser Val Asp
420 425 430
Ile Thr Lys Lys Leu Ser Ile Val Ser Leu Val Gly Lys His Met Lys
435 440 445
Gln Tyr Ile Gly Ile Ala Gly Thr Met Phe Thr Thr Leu Ala Glu Glu
450 455 460
Gly Ile Asn Ile Glu Met Ile Ser Gln Gly Ala Asn Glu Ile Asn Ile
465 470 475 480
Ser Cys Val Ile Asn Glu Ser Asp Ser Ile Lys Ala Leu Gln Cys Ile
485 490 495
His Ala Lys Leu Leu Ser Glu Arg Thr Asn Thr Ser Asn Gln Phe Glu
500 505 510
His Ala Ile Asp Glu Arg Leu Glu Gln Leu Lys Arg Leu Gly Ile
515 520 525
<210> 9
<211> 449
<212> PRT
<213> Escherichia coli
<220>
<221> MISC_FEATURE
<222> (119)..(119)
<223> X being any of amino acid except E
<400> 9
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Xaa Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 10
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<220>
<221> misc_feature
<222> (17)..(19)
<223> nnn encoding anyone of the other 19 naturally existing
proteinogenic amino acids, except glutamine
<400> 10
gctggtcagc catggcnnnc tgatgtcgac cctgc 35
<210> 11
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<220>
<221> misc_feature
<222> (17)..(19)
<223> nnn encoding anyone of the other 19 naturally existing
proteinogenic amino acids, except glutamine
<400> 11
gcagggtcga catcagnnng ccatggctga ccagc 35
<210> 12
<211> 449
<212> PRT
<213> Escherichia coli
<400> 12
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Cys Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 13
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 13
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggctgtctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 14
<211> 449
<212> PRT
<213> Escherichia coli
<400> 14
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Gly Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 15
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 15
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggcggcctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 16
<211> 449
<212> PRT
<213> Escherichia coli
<400> 16
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Asn Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 17
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 17
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggcaatctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 18
<211> 449
<212> PRT
<213> Escherichia coli
<400> 18
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Pro Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 19
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 19
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggcccgctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 20
<211> 449
<212> PRT
<213> Escherichia coli
<400> 20
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Gln Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 21
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 21
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggccagctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 22
<211> 449
<212> PRT
<213> Escherichia coli
<400> 22
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Ser Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 23
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 23
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggctcgctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 24
<211> 449
<212> PRT
<213> Escherichia coli
<400> 24
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Thr Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 25
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 25
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggcactctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 26
<211> 449
<212> PRT
<213> Escherichia coli
<400> 26
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Val Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 27
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 27
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggcgtgctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 28
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 28
gcgtttgccg aagcggcaaa gatggccact tttg 34
<210> 29
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 29
caaaagtggc catctttgcc gcttcggcaa acgc 34
<210> 30
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 30
ggtagatcta atcaccatgt cagaagtgag cgtgg 35
<210> 31
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 31
ccacgctcac ttctgacatg gtgattagat ctacc 35
<210> 32
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 32
ggtagatcta atcaccacgt tagaagtgag cgtggc 36
<210> 33
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 33
gccacgctca cttctaacgt ggtgattaga tctacc 36
<210> 34
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 34
ggtagatcta atcaccatgt cagaagtgag cgtgg 35
<210> 35
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 35
ccacgctcac ttctgacatg gtgattagat ctacc 35
<210> 36
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 36
gtcagaagtg agcgtggcat taattctaga taccac 36
<210> 37
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 37
gtggtatcta gaattaatgc cacgctcact tctgac 36
<210> 38
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 38
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggcggcctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcaaag atggccactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 39
<211> 449
<212> PRT
<213> Escherichia coli
<400> 39
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Gly Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Lys Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 40
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 40
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggcggcctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagat 1020
ctaatcacca tgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 41
<211> 449
<212> PRT
<213> Escherichia coli
<400> 41
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Gly Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Met Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 42
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 42
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggcggcctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaattctag ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 43
<211> 449
<212> PRT
<213> Escherichia coli
<400> 43
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Gly Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Ile
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 44
<211> 1350
<212> DNA
<213> Escherichia coli
<400> 44
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca tggcggcctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagat 1020
ctaatcacca cgttagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 45
<211> 449
<212> PRT
<213> Escherichia coli
<400> 45
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Gly Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Leu Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 46
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 46
tataatgcta gcatgaaaaa tgttggtttt atcgg 35
<210> 47
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 47
tataatggat ccttacgcca gttgacgaag c 31
<210> 48
<211> 1104
<212> DNA
<213> Escherichia coli
<400> 48
atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60
atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120
ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180
gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240
atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300
ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360
gacggattaa ataatggcat caggactttt gttggcggta actgtaccgt aagcctgatg 420
ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480
taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540
catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600
gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660
ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagccgc 720
gaagagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780
gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840
aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900
tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960
gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020
ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080
cggatgcttc gtcaactggc gtaa 1104
<210> 49
<211> 367
<212> PRT
<213> Escherichia coli
<400> 49
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Glu Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu
340 345 350
Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gln Leu Ala
355 360 365
<210> 50
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<220>
<221> misc_feature
<222> (24)..(26)
<223> nnn encoding anyone of the other 19 naturally existing
proteinogenic amino acids, except glutamine
<400> 50
agctcgataa cggtcagagt cgannngagt ggaaagggca ggcgg 45
<210> 51
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<220>
<221> misc_feature
<222> (20)..(22)
<223> nnn encoding anyone of the other 19 naturally existing
proteinogenic amino acids, except glutamine
<400> 51
ccgcctgccc tttccactcn nntcgactct gaccgttatc gagct 45
<210> 52
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 52
ttttgttggc ggtaactgta acgtgtccct gatgttg 37
<210> 53
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 53
caacatcagg gacacgttac agttaccgcc aacaaaa 37
<210> 54
<211> 367
<212> PRT
<213> Escherichia coli
<400> 54
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Ala Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu
340 345 350
Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gln Leu Ala
355 360 365
<210> 55
<211> 1104
<212> DNA
<213> Escherichia coli
<400> 55
atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60
atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120
ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180
gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240
atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300
ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360
gacggattaa ataatggcat caggactttt gttggcggta actgtaccgt aagcctgatg 420
ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480
taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540
catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600
gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660
ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagtcga 720
gctgagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780
gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840
aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900
tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960
gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020
ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080
cggatgcttc gtcaactggc gtaa 1104
<210> 56
<211> 367
<212> PRT
<213> Escherichia coli
<400> 56
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Cys Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu
340 345 350
Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gln Leu Ala
355 360 365
<210> 57
<211> 1104
<212> DNA
<213> Escherichia coli
<400> 57
atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60
atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120
ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180
gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240
atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300
ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360
gacggattaa ataatggcat caggactttt gttggcggta actgtaccgt aagcctgatg 420
ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480
taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540
catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600
gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660
ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagtcga 720
tgtgagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780
gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840
aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900
tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960
gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020
ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080
cggatgcttc gtcaactggc gtaa 1104
<210> 58
<211> 367
<212> PRT
<213> Escherichia coli
<400> 58
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Gly Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu
340 345 350
Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gln Leu Ala
355 360 365
<210> 59
<211> 1104
<212> DNA
<213> Escherichia coli
<400> 59
atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60
atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120
ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180
gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240
atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300
ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360
gacggattaa ataatggcat caggactttt gttggcggta actgtaccgt aagcctgatg 420
ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480
taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540
catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600
gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660
ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagtcga 720
ggggagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780
gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840
aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900
tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960
gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020
ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080
cggatgcttc gtcaactggc gtaa 1104
<210> 60
<211> 367
<212> PRT
<213> Escherichia coli
<400> 60
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
His Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu
340 345 350
Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gln Leu Ala
355 360 365
<210> 61
<211> 1104
<212> DNA
<213> Escherichia coli
<400> 61
atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60
atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120
ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180
gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240
atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300
ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360
gacggattaa ataatggcat caggactttt gttggcggta actgtaccgt aagcctgatg 420
ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480
taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540
catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600
gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660
ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagtcga 720
catgagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780
gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840
aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900
tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960
gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020
ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080
cggatgcttc gtcaactggc gtaa 1104
<210> 62
<211> 367
<212> PRT
<213> Escherichia coli
<400> 62
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Ile Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu
340 345 350
Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gln Leu Ala
355 360 365
<210> 63
<211> 1104
<212> DNA
<213> Escherichia coli
<400> 63
atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60
atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120
ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180
gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240
atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300
ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360
gacggattaa ataatggcat caggactttt gttggcggta actgtaccgt aagcctgatg 420
ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480
taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540
catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600
gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660
ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagtcga 720
attgagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780
gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840
aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900
tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960
gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020
ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080
cggatgcttc gtcaactggc gtaa 1104
<210> 64
<211> 367
<212> PRT
<213> Escherichia coli
<400> 64
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Met Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu
340 345 350
Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gln Leu Ala
355 360 365
<210> 65
<211> 1104
<212> DNA
<213> Escherichia coli
<400> 65
atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60
atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120
ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180
gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240
atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300
ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360
gacggattaa ataatggcat caggactttt gttggcggta actgtaccgt aagcctgatg 420
ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480
taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540
catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600
gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660
ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagtcga 720
atggagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780
gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840
aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900
tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960
gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020
ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080
cggatgcttc gtcaactggc gtaa 1104
<210> 66
<211> 367
<212> PRT
<213> Escherichia coli
<400> 66
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Gln Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu
340 345 350
Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gln Leu Ala
355 360 365
<210> 67
<211> 1104
<212> DNA
<213> Escherichia coli
<400> 67
atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60
atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120
ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180
gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240
atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300
ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360
gacggattaa ataatggcat caggactttt gttggcggta actgtaccgt aagcctgatg 420
ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480
taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540
catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600
gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660
ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagtcga 720
caggagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780
gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840
aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900
tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960
gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020
ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080
cggatgcttc gtcaactggc gtaa 1104
<210> 68
<211> 367
<212> PRT
<213> Escherichia coli
<220>
<221> MISC_FEATURE
<222> (241)..(241)
<223> X being any other aminoacid the glutamine
<400> 68
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Xaa Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu
340 345 350
Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gln Leu Ala
355 360 365
<210> 69
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 69
tataatgcta gcatgaaagc tgcagtactt ca 32
<210> 70
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 70
tataatgaat tcttacggga ttatgagact tc 32
<210> 71
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 71
tataatgcta gcatgcctgc tacgttaaag aa 32
<210> 72
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 72
tataatgagc tctcattgga aaattgggaa gg 32
<210> 73
<211> 939
<212> DNA
<213> Saccharomyces cerevisiae
<400> 73
atgcctgcta cgttaaagaa ttcttctgct acattaaaac taaatactgg tgcctccatt 60
ccagtgttgg gtttcggcac ttggcgttcc gttgacaata acggttacca ttctgtaatt 120
gcagctttga aagctggata cagacacatt gatgctgcgg ctatctattt gaatgaagaa 180
gaagttggca gggctattaa agattccgga gtccctcgtg aggaaatttt tattactact 240
aagctttggg gtacggaaca acgtgatccg gaagctgctc taaacaagtc tttgaaaaga 300
ctaggcttgg attatgttga cctatatctg atgcattggc cagtgccttt gaaaaccgac 360
agagttactg atggtaacgt tctgtgcatt ccaacattag aagatggcac tgttgacatc 420
gatactaagg aatggaattt tatcaagacg tgggagttga tgcaagagtt gccaaagacg 480
ggcaaaacta aagccgttgg tgtctctaat ttttctatta acaacattaa agaattatta 540
gaatctccaa ataacaaggt ggtaccagct actaatcaaa ttgaaattca tccattgcta 600
ccacaagacg aattgattgc cttttgtaag gaaaagggta ttgttgttga agcctactca 660
ccatttggga gtgctaatgc tcctttacta aaagagcaag caattattga tatggctaaa 720
aagcacggcg ttgagccagc acagcttatt atcagttgga gtattcaaag aggctacgtt 780
gttctggcca aatcggttaa tcctgaaaga attgtatcca attttaagat tttcactctg 840
cctgaggatg atttcaagac tattagtaac ctatccaaag tgcatggtac aaagagagtc 900
gttgatatga agtggggatc cttcccaatt ttccaatga 939
<210> 74
<211> 312
<212> PRT
<213> Saccharomyces cerevisiae
<400> 74
Met Pro Ala Thr Leu Lys Asn Ser Ser Ala Thr Leu Lys Leu Asn Thr
1 5 10 15
Gly Ala Ser Ile Pro Val Leu Gly Phe Gly Thr Trp Arg Ser Val Asp
20 25 30
Asn Asn Gly Tyr His Ser Val Ile Ala Ala Leu Lys Ala Gly Tyr Arg
35 40 45
His Ile Asp Ala Ala Ala Ile Tyr Leu Asn Glu Glu Glu Val Gly Arg
50 55 60
Ala Ile Lys Asp Ser Gly Val Pro Arg Glu Glu Ile Phe Ile Thr Thr
65 70 75 80
Lys Leu Trp Gly Thr Glu Gln Arg Asp Pro Glu Ala Ala Leu Asn Lys
85 90 95
Ser Leu Lys Arg Leu Gly Leu Asp Tyr Val Asp Leu Tyr Leu Met His
100 105 110
Trp Pro Val Pro Leu Lys Thr Asp Arg Val Thr Asp Gly Asn Val Leu
115 120 125
Cys Ile Pro Thr Leu Glu Asp Gly Thr Val Asp Ile Asp Thr Lys Glu
130 135 140
Trp Asn Phe Ile Lys Thr Trp Glu Leu Met Gln Glu Leu Pro Lys Thr
145 150 155 160
Gly Lys Thr Lys Ala Val Gly Val Ser Asn Phe Ser Ile Asn Asn Ile
165 170 175
Lys Glu Leu Leu Glu Ser Pro Asn Asn Lys Val Val Pro Ala Thr Asn
180 185 190
Gln Ile Glu Ile His Pro Leu Leu Pro Gln Asp Glu Leu Ile Ala Phe
195 200 205
Cys Lys Glu Lys Gly Ile Val Val Glu Ala Tyr Ser Pro Phe Gly Ser
210 215 220
Ala Asn Ala Pro Leu Leu Lys Glu Gln Ala Ile Ile Asp Met Ala Lys
225 230 235 240
Lys His Gly Val Glu Pro Ala Gln Leu Ile Ile Ser Trp Ser Ile Gln
245 250 255
Arg Gly Tyr Val Val Leu Ala Lys Ser Val Asn Pro Glu Arg Ile Val
260 265 270
Ser Asn Phe Lys Ile Phe Thr Leu Pro Glu Asp Asp Phe Lys Thr Ile
275 280 285
Ser Asn Leu Ser Lys Val His Gly Thr Lys Arg Val Val Asp Met Lys
290 295 300
Trp Gly Ser Phe Pro Ile Phe Gln
305 310
<210> 75
<211> 1083
<212> DNA
<213> Metallosphaera sedula
<400> 75
atgaaagctg cagtacttca tacgtataag gaaccgctgt ccattgagga cgtgaatatc 60
tcccaaccta aggctgggga agtcaagatc aaggtcaagg caaccgggct ctgtcactcc 120
gacgtcaatg tctttgaggg gaaaacccca gttcctcccc cagtggttgc tggacacgaa 180
atatcaggga ttgtggagga agtgggacct ggggtgacca gggttaaacc aggtgatagg 240
gtgatttcag cgtttattca cccctgtggt aaatgcggta actgcgttgc aggaaaggag 300
aatctgtgtg agaccttctc ccaggtcaga ctcaagggag taatgccaga tggaacgtca 360
aggctgtcaa aggacggaaa ggagataagg actttccttg gaggcggttt cgcggagtac 420
gccattgtgg gagagaacgc gctaaccagg gttccagagg acatggacct agagaaggta 480
gctgtcctag gttgtgctgg gttaacaggg tacggtgcca tatcatcatc caagattgag 540
cctggagaca ctgtggccgt gataggcgta ggaggagtgg gtttgtccac aatacaactc 600
ctaagggcct cgggtgccgg gaggataatc gccgtgggaa cgaaaaagtg gaaacttgac 660
agggccatgg agctaggtgc aactgacgtg gtaaactcga aggagataga tcccgtcaaa 720
gcaataaagg agatcacggg tggagggcca caggtggtga tagaggctgg aggaaatgag 780
gatacgattc atatggcgct ggattcagtt agaattggag gaaaggtggt tctggtaggg 840
ttacctccag caacggccat gatacccatc agggtagcgt caatagttag gggaggcata 900
gaggttgtgg ggaattacgg aggaagacct agggttgata tgcccaagct tctcgagcta 960
gtgaggcagg gaagatacga tccgtctagg cttgtgacgg gtagattcag gttggaggaa 1020
ataaatgagg cagtcaaaat gcttgaggaa ggagaggcca taagaagtct cataatcccg 1080
taa 1083
<210> 76
<211> 360
<212> PRT
<213> Metallosphaera sedula
<400> 76
Met Lys Ala Ala Val Leu His Thr Tyr Lys Glu Pro Leu Ser Ile Glu
1 5 10 15
Asp Val Asn Ile Ser Gln Pro Lys Ala Gly Glu Val Lys Ile Lys Val
20 25 30
Lys Ala Thr Gly Leu Cys His Ser Asp Val Asn Val Phe Glu Gly Lys
35 40 45
Thr Pro Val Pro Pro Pro Val Val Ala Gly His Glu Ile Ser Gly Ile
50 55 60
Val Glu Glu Val Gly Pro Gly Val Thr Arg Val Lys Pro Gly Asp Arg
65 70 75 80
Val Ile Ser Ala Phe Ile His Pro Cys Gly Lys Cys Gly Asn Cys Val
85 90 95
Ala Gly Lys Glu Asn Leu Cys Glu Thr Phe Ser Gln Val Arg Leu Lys
100 105 110
Gly Val Met Pro Asp Gly Thr Ser Arg Leu Ser Lys Asp Gly Lys Glu
115 120 125
Ile Arg Thr Phe Leu Gly Gly Gly Phe Ala Glu Tyr Ala Ile Val Gly
130 135 140
Glu Asn Ala Leu Thr Arg Val Pro Glu Asp Met Asp Leu Glu Lys Val
145 150 155 160
Ala Val Leu Gly Cys Ala Gly Leu Thr Gly Tyr Gly Ala Ile Ser Ser
165 170 175
Ser Lys Ile Glu Pro Gly Asp Thr Val Ala Val Ile Gly Val Gly Gly
180 185 190
Val Gly Leu Ser Thr Ile Gln Leu Leu Arg Ala Ser Gly Ala Gly Arg
195 200 205
Ile Ile Ala Val Gly Thr Lys Lys Trp Lys Leu Asp Arg Ala Met Glu
210 215 220
Leu Gly Ala Thr Asp Val Val Asn Ser Lys Glu Ile Asp Pro Val Lys
225 230 235 240
Ala Ile Lys Glu Ile Thr Gly Gly Gly Pro Gln Val Val Ile Glu Ala
245 250 255
Gly Gly Asn Glu Asp Thr Ile His Met Ala Leu Asp Ser Val Arg Ile
260 265 270
Gly Gly Lys Val Val Leu Val Gly Leu Pro Pro Ala Thr Ala Met Ile
275 280 285
Pro Ile Arg Val Ala Ser Ile Val Arg Gly Gly Ile Glu Val Val Gly
290 295 300
Asn Tyr Gly Gly Arg Pro Arg Val Asp Met Pro Lys Leu Leu Glu Leu
305 310 315 320
Val Arg Gln Gly Arg Tyr Asp Pro Ser Arg Leu Val Thr Gly Arg Phe
325 330 335
Arg Leu Glu Glu Ile Asn Glu Ala Val Lys Met Leu Glu Glu Gly Glu
340 345 350
Ala Ile Arg Ser Leu Ile Ile Pro
355 360
<210> 77
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 77
gtcaaggcaa ccggtctctg tcgctccgac gtcaatg 37
<210> 78
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 78
cattgacgtc ggagcgacag agaccggttg ccttgac 37
<210> 79
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 79
ggctctgtca ctccgacgta catgtctttg aggggaaaac 40
<210> 80
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 80
gttttcccct caaagacatg tacgtcggag tgacagagcc 40
<210> 81
<211> 360
<212> PRT
<213> Metallosphaera sedula
<400> 81
Met Lys Ala Ala Val Leu His Thr Tyr Lys Glu Pro Leu Ser Ile Glu
1 5 10 15
Asp Val Asn Ile Ser Gln Pro Lys Ala Gly Glu Val Lys Ile Lys Val
20 25 30
Lys Ala Thr Gly Leu Cys Arg Ser Asp Val His Val Phe Glu Gly Lys
35 40 45
Thr Pro Val Pro Pro Pro Val Val Ala Gly His Glu Ile Ser Gly Ile
50 55 60
Val Glu Glu Val Gly Pro Gly Val Thr Arg Val Lys Pro Gly Asp Arg
65 70 75 80
Val Ile Ser Ala Phe Ile His Pro Cys Gly Lys Cys Gly Asn Cys Val
85 90 95
Ala Gly Lys Glu Asn Leu Cys Glu Thr Phe Ser Gln Val Arg Leu Lys
100 105 110
Gly Val Met Pro Asp Gly Thr Ser Arg Leu Ser Lys Asp Gly Lys Glu
115 120 125
Ile Arg Thr Phe Leu Gly Gly Gly Phe Ala Glu Tyr Ala Ile Val Gly
130 135 140
Glu Asn Ala Leu Thr Arg Val Pro Glu Asp Met Asp Leu Glu Lys Val
145 150 155 160
Ala Val Leu Gly Cys Ala Gly Leu Thr Gly Tyr Gly Ala Ile Ser Ser
165 170 175
Ser Lys Ile Glu Pro Gly Asp Thr Val Ala Val Ile Gly Val Gly Gly
180 185 190
Val Gly Leu Ser Thr Ile Gln Leu Leu Arg Ala Ser Gly Ala Gly Arg
195 200 205
Ile Ile Ala Val Gly Thr Lys Lys Trp Lys Leu Asp Arg Ala Met Glu
210 215 220
Leu Gly Ala Thr Asp Val Val Asn Ser Lys Glu Ile Asp Pro Val Lys
225 230 235 240
Ala Ile Lys Glu Ile Thr Gly Gly Gly Pro Gln Val Val Ile Glu Ala
245 250 255
Gly Gly Asn Glu Asp Thr Ile His Met Ala Leu Asp Ser Val Arg Ile
260 265 270
Gly Gly Lys Val Val Leu Val Gly Leu Pro Pro Ala Thr Ala Met Ile
275 280 285
Pro Ile Arg Val Ala Ser Ile Val Arg Gly Gly Ile Glu Val Val Gly
290 295 300
Asn Tyr Gly Gly Arg Pro Arg Val Asp Met Pro Lys Leu Leu Glu Leu
305 310 315 320
Val Arg Gln Gly Arg Tyr Asp Pro Ser Arg Leu Val Thr Gly Arg Phe
325 330 335
Arg Leu Glu Glu Ile Asn Glu Ala Val Lys Met Leu Glu Glu Gly Glu
340 345 350
Ala Ile Arg Ser Leu Ile Ile Pro
355 360
<210> 82
<211> 1083
<212> DNA
<213> Metallosphaera sedula
<400> 82
atgaaagcag cagttctgca tacctataaa gaaccgctga gcattgaaga tgtgaatatt 60
tcacagccga aagccggtga agtgaaaatc aaagttaaag caaccggtct gtgtcgtagt 120
gatgttcatg tttttgaagg taaaacaccg gttccgcctc cggttgttgc aggtcatgaa 180
attagcggta ttgttgaaga ggttggtccg ggtgttaccc gtgttaaacc gggtgatcgt 240
gttattagcg catttattca tccgtgtggt aaatgcggta attgtgttgc cggtaaagaa 300
aatctgtgtg aaacctttag ccaggttcgt ctgaaaggtg ttatgccgga tggcaccagc 360
cgtctgagca aagatggcaa agaaattcgt acctttctgg gtggtggttt tgcagaatat 420
gcaattgttg gtgaaaatgc actgacccgt gttccggaag atatggatct ggaaaaagtt 480
gcagttctgg gttgtgccgg tctgaccggt tatggtgcaa ttagcagcag caaaattgaa 540
cctggtgata ccgttgcagt tattggtgtt ggtggtgtgg gtctgagcac cattcagctg 600
ctgcgtgcaa gcggtgcagg tcgtattatt gcagttggca ccaaaaaatg gaaactggat 660
cgtgcaatgg aactgggtgc aaccgatgtt gttaacagta aagaaattga tccggtgaaa 720
gccatcaaag aaatcaccgg tggtggtccg caggttgtta ttgaagccgg tggtaatgaa 780
gataccattc acatggcact ggatagcgtt cgtattggtg gtaaagttgt tctggttggt 840
ctgcctccgg caaccgcaat gattccgatt cgtgttgcaa gcattgttcg tggtggtatt 900
gaagttgttg gtaattatgg tggtcgtccg cgtgttgata tgccgaaact gctggaactg 960
gttcgtcagg gtcgttatga tccgagccgt ctggttaccg gtcgttttcg tctggaagaa 1020
attaatgaag ccgtcaaaat gctggaagaa ggtgaagcaa ttcgtagcct gattattccg 1080
taa 1083
<210> 83
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 83
tataaggatc cgtttaactt taagaaggag atataccatg gg 42
<210> 84
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 84
tataagaatt cttacgccag ttgacgaag 29
<210> 85
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 85
tataagcggc cgcgtttaac tttaagaagg agatat 36
<210> 86
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 86
tataaactcg agcttacgga ataatcagg 29
<210> 87
<211> 841
<212> PRT
<213> Escherichia coli
<400> 87
Met Lys Asn Leu Arg Leu Cys Arg Arg Ile Phe Ile Ser Thr Lys Gly
1 5 10 15
Asn Glu Val Thr Thr Met Arg Val Leu Lys Phe Gly Gly Thr Ser Val
20 25 30
Ala Asn Ala Glu Arg Phe Leu Arg Val Ala Asp Ile Leu Glu Ser Asn
35 40 45
Ala Arg Gln Gly Gln Val Ala Thr Val Leu Ser Ala Pro Ala Lys Ile
50 55 60
Thr Asn His Leu Val Ala Met Ile Glu Lys Thr Ile Ser Gly Gln Asp
65 70 75 80
Ala Leu Pro Asn Ile Ser Asp Ala Glu Arg Ile Phe Ala Glu Leu Leu
85 90 95
Thr Gly Leu Ala Ala Ala Gln Pro Gly Phe Pro Leu Ala Gln Leu Lys
100 105 110
Thr Phe Val Asp Gln Glu Phe Ala Gln Ile Lys His Val Leu His Gly
115 120 125
Ile Ser Leu Leu Gly Gln Cys Pro Asp Ser Ile Asn Ala Ala Leu Ile
130 135 140
Cys Arg Gly Glu Lys Met Ser Ile Ala Ile Met Ala Gly Val Leu Glu
145 150 155 160
Ala Arg Gly His Asn Val Thr Val Ile Asp Pro Val Glu Lys Leu Leu
165 170 175
Ala Val Gly His Tyr Leu Glu Ser Thr Val Asp Ile Ala Glu Ser Thr
180 185 190
Arg Arg Ile Ala Ala Ser Arg Ile Pro Ala Asp His Met Val Leu Met
195 200 205
Ala Gly Phe Thr Ala Gly Asn Glu Lys Gly Glu Leu Val Val Leu Gly
210 215 220
Arg Asn Gly Ser Asp Tyr Ser Ala Ala Val Leu Ala Ala Cys Leu Arg
225 230 235 240
Ala Asp Cys Cys Glu Ile Trp Thr Asp Val Asp Gly Val Tyr Thr Cys
245 250 255
Asp Pro Arg Gln Val Pro Asp Ala Arg Leu Leu Lys Ser Met Ser Tyr
260 265 270
Gln Glu Ala Met Glu Leu Ser Tyr Phe Gly Ala Lys Val Leu His Pro
275 280 285
Arg Thr Ile Thr Pro Ile Ala Gln Phe Gln Ile Pro Cys Leu Ile Lys
290 295 300
Asn Thr Gly Asn Pro Gln Ala Pro Gly Thr Leu Ile Gly Ala Ser Arg
305 310 315 320
Asp Glu Asp Glu Leu Pro Val Lys Gly Ile Ser Asn Leu Asn Asn Met
325 330 335
Ala Met Phe Ser Val Ser Gly Pro Gly Met Lys Gly Met Val Gly Met
340 345 350
Ala Ala Arg Val Phe Ala Ala Met Ser Arg Ala Arg Ile Ser Val Val
355 360 365
Leu Ile Thr Gln Ser Ser Ser Glu Tyr Ser Ile Ser Phe Cys Val Pro
370 375 380
Gln Ser Asp Cys Val Arg Ala Glu Arg Ala Met Gln Glu Glu Phe Tyr
385 390 395 400
Leu Glu Leu Lys Glu Gly Leu Leu Glu Pro Leu Ala Val Thr Glu Arg
405 410 415
Leu Ala Ile Ile Ser Val Val Gly Asp Gly Met Arg Thr Leu Arg Gly
420 425 430
Ile Ser Ala Lys Phe Phe Ala Ala Leu Ala Arg Ala Asn Ile Asn Ile
435 440 445
Val Ala Ile Ala Gln Gly Ser Ser Glu Arg Ser Ile Ser Val Val Val
450 455 460
Asn Asn Asp Asp Ala Thr Thr Gly Val Arg Val Thr His Gln Met Leu
465 470 475 480
Phe Asn Thr Asp Gln Val Ile Glu Val Phe Val Ile Gly Val Gly Gly
485 490 495
Val Gly Gly Ala Leu Leu Glu Gln Leu Lys Arg Gln Gln Ser Trp Leu
500 505 510
Lys Asn Lys His Ile Asp Leu Arg Val Cys Gly Val Ala Asn Ser Lys
515 520 525
Ala Leu Leu Thr Asn Val His Gly Leu Asn Leu Glu Asn Trp Gln Glu
530 535 540
Glu Leu Ala Gln Ala Lys Glu Pro Phe Asn Leu Gly Arg Leu Ile Arg
545 550 555 560
Leu Val Lys Glu Tyr His Leu Leu Asn Pro Val Ile Val Asp Cys Thr
565 570 575
Ser Ser Gln Ala Val Ala Asp Gln Tyr Ala Asp Phe Leu Arg Glu Gly
580 585 590
Phe His Val Val Thr Pro Asn Lys Lys Ala Asn Thr Ser Ser Met Asp
595 600 605
Tyr Tyr His Gln Leu Arg Tyr Ala Ala Glu Lys Ser Arg Arg Lys Phe
610 615 620
Leu Tyr Asp Thr Asn Val Gly Ala Gly Leu Pro Val Ile Glu Asn Leu
625 630 635 640
Gln Asn Leu Leu Asn Ala Gly Asp Glu Leu Met Lys Phe Ser Gly Ile
645 650 655
Leu Ser Gly Ser Leu Ser Tyr Ile Phe Gly Lys Leu Asp Glu Gly Met
660 665 670
Ser Phe Ser Glu Ala Thr Thr Leu Ala Arg Glu Met Gly Tyr Thr Glu
675 680 685
Pro Asp Pro Arg Asp Asp Leu Ser Gly Met Asp Val Ala Arg Lys Leu
690 695 700
Leu Ile Leu Ala Arg Glu Thr Gly Arg Glu Leu Glu Leu Ala Asp Ile
705 710 715 720
Glu Ile Glu Pro Val Leu Pro Ala Glu Phe Asn Ala Glu Gly Asp Val
725 730 735
Ala Ala Phe Met Ala Asn Leu Ser Gln Leu Asp Asn Leu Phe Ala Ala
740 745 750
Arg Val Ala Lys Ala Arg Asp Glu Gly Lys Val Leu Arg Tyr Val Gly
755 760 765
Asn Ile Asp Glu Asp Gly Val Cys Arg Val Lys Ile Ala Glu Val Asp
770 775 780
Ser Asn Asp Pro Leu Phe Lys Val Lys Asn Gly Glu Asn Ala Leu Ala
785 790 795 800
Phe Tyr Ser His Tyr Tyr Gln Pro Leu Pro Leu Val Leu Arg Gly Tyr
805 810 815
Gly Ala Gly Asn Asp Val Thr Ala Ala Gly Val Phe Ala Asp Leu Leu
820 825 830
Arg Thr Leu Ser Trp Lys Leu Gly Val
835 840
<210> 88
<211> 810
<212> PRT
<213> Escherichia coli
<400> 88
Met Ser Val Ile Ala Gln Ala Gly Ala Lys Gly Arg Gln Leu His Lys
1 5 10 15
Phe Gly Gly Ser Ser Leu Ala Asp Val Lys Cys Tyr Leu Arg Val Ala
20 25 30
Gly Ile Met Ala Glu Tyr Ser Gln Pro Asp Asp Met Met Val Val Ser
35 40 45
Ala Ala Gly Ser Thr Thr Asn Gln Leu Ile Asn Trp Leu Lys Leu Ser
50 55 60
Gln Thr Asp Arg Leu Ser Ala His Gln Val Gln Gln Thr Leu Arg Arg
65 70 75 80
Tyr Gln Cys Asp Leu Ile Ser Gly Leu Leu Pro Ala Glu Glu Ala Asp
85 90 95
Ser Leu Ile Ser Ala Phe Val Ser Asp Leu Glu Arg Leu Ala Ala Leu
100 105 110
Leu Asp Ser Gly Ile Asn Asp Ala Val Tyr Ala Glu Val Val Gly His
115 120 125
Gly Glu Val Trp Ser Ala Arg Leu Met Ser Ala Val Leu Asn Gln Gln
130 135 140
Gly Leu Pro Ala Ala Trp Leu Asp Ala Arg Glu Phe Leu Arg Ala Glu
145 150 155 160
Arg Ala Ala Gln Pro Gln Val Asp Glu Gly Leu Ser Tyr Pro Leu Leu
165 170 175
Gln Gln Leu Leu Val Gln His Pro Gly Lys Arg Leu Val Val Thr Gly
180 185 190
Phe Ile Ser Arg Asn Asn Ala Gly Glu Thr Val Leu Leu Gly Arg Asn
195 200 205
Gly Ser Asp Tyr Ser Ala Thr Gln Ile Gly Ala Leu Ala Gly Val Ser
210 215 220
Arg Val Thr Ile Trp Ser Asp Val Ala Gly Val Tyr Ser Ala Asp Pro
225 230 235 240
Arg Lys Val Lys Asp Ala Cys Leu Leu Pro Leu Leu Arg Leu Asp Glu
245 250 255
Ala Ser Glu Leu Ala Arg Leu Ala Ala Pro Val Leu His Ala Arg Thr
260 265 270
Leu Gln Pro Val Ser Gly Ser Glu Ile Asp Leu Gln Leu Arg Cys Ser
275 280 285
Tyr Thr Pro Asp Gln Gly Ser Thr Arg Ile Glu Arg Val Leu Ala Ser
290 295 300
Gly Thr Gly Ala Arg Ile Val Thr Ser His Asp Asp Val Cys Leu Ile
305 310 315 320
Glu Phe Gln Val Pro Ala Ser Gln Asp Phe Lys Leu Ala His Lys Glu
325 330 335
Ile Asp Gln Ile Leu Lys Arg Ala Gln Val Arg Pro Leu Ala Val Gly
340 345 350
Val His Asn Asp Arg Gln Leu Leu Gln Phe Cys Tyr Thr Ser Glu Val
355 360 365
Ala Asp Ser Ala Leu Lys Ile Leu Asp Glu Ala Gly Leu Pro Gly Glu
370 375 380
Leu Arg Leu Arg Gln Gly Leu Ala Leu Val Ala Met Val Gly Ala Gly
385 390 395 400
Val Thr Arg Asn Pro Leu His Cys His Arg Phe Trp Gln Gln Leu Lys
405 410 415
Gly Gln Pro Val Glu Phe Thr Trp Gln Ser Asp Asp Gly Ile Ser Leu
420 425 430
Val Ala Val Leu Arg Thr Gly Pro Thr Glu Ser Leu Ile Gln Gly Leu
435 440 445
His Gln Ser Val Phe Arg Ala Glu Lys Arg Ile Gly Leu Val Leu Phe
450 455 460
Gly Lys Gly Asn Ile Gly Ser Arg Trp Leu Glu Leu Phe Ala Arg Glu
465 470 475 480
Gln Ser Thr Leu Ser Ala Arg Thr Gly Phe Glu Phe Val Leu Ala Gly
485 490 495
Val Val Asp Ser Arg Arg Ser Leu Leu Ser Tyr Asp Gly Leu Asp Ala
500 505 510
Ser Arg Ala Leu Ala Phe Phe Asn Asp Glu Ala Val Glu Gln Asp Glu
515 520 525
Glu Ser Leu Phe Leu Trp Met Arg Ala His Pro Tyr Asp Asp Leu Val
530 535 540
Val Leu Asp Val Thr Ala Ser Gln Gln Leu Ala Asp Gln Tyr Leu Asp
545 550 555 560
Phe Ala Ser His Gly Phe His Val Ile Ser Ala Asn Lys Leu Ala Gly
565 570 575
Ala Ser Asp Ser Asn Lys Tyr Arg Gln Ile His Asp Ala Phe Glu Lys
580 585 590
Thr Gly Arg His Trp Leu Tyr Asn Ala Thr Val Gly Ala Gly Leu Pro
595 600 605
Ile Asn His Thr Val Arg Asp Leu Ile Asp Ser Gly Asp Thr Ile Leu
610 615 620
Ser Ile Ser Gly Ile Phe Ser Gly Thr Leu Ser Trp Leu Phe Leu Gln
625 630 635 640
Phe Asp Gly Ser Val Pro Phe Thr Glu Leu Val Asp Gln Ala Trp Gln
645 650 655
Gln Gly Leu Thr Glu Pro Asp Pro Arg Asp Asp Leu Ser Gly Lys Asp
660 665 670
Val Met Arg Lys Leu Val Ile Leu Ala Arg Glu Ala Gly Tyr Asn Ile
675 680 685
Glu Pro Asp Gln Val Arg Val Glu Ser Leu Val Pro Ala His Cys Glu
690 695 700
Gly Gly Ser Ile Asp His Phe Phe Glu Asn Gly Asp Glu Leu Asn Glu
705 710 715 720
Gln Met Val Gln Arg Leu Glu Ala Ala Arg Glu Met Gly Leu Val Leu
725 730 735
Arg Tyr Val Ala Arg Phe Asp Ala Asn Gly Lys Ala Arg Val Gly Val
740 745 750
Glu Ala Val Arg Glu Asp His Pro Leu Ala Ser Leu Leu Pro Cys Asp
755 760 765
Asn Val Phe Ala Ile Glu Ser Arg Trp Tyr Arg Asp Asn Pro Leu Val
770 775 780
Ile Arg Gly Pro Gly Ala Gly Arg Asp Val Thr Ala Gly Ala Ile Gln
785 790 795 800
Ser Asp Ile Asn Arg Leu Ala Gln Leu Leu
805 810
<210> 89
<211> 473
<212> PRT
<213> Methanococcus jannaschii
<400> 89
Met Thr Thr Val Met Lys Phe Gly Gly Thr Ser Val Gly Ser Gly Glu
1 5 10 15
Arg Ile Arg His Val Ala Lys Ile Val Thr Lys Arg Lys Lys Glu Asp
20 25 30
Asp Asp Val Val Val Val Val Ser Ala Met Ser Glu Val Thr Asn Ala
35 40 45
Leu Val Glu Ile Ser Gln Gln Ala Leu Asp Val Arg Asp Ile Ala Lys
50 55 60
Val Gly Asp Phe Ile Lys Phe Ile Arg Glu Lys His Tyr Lys Ala Ile
65 70 75 80
Glu Glu Ala Ile Lys Ser Glu Glu Ile Lys Glu Glu Val Lys Lys Ile
85 90 95
Ile Asp Ser Arg Ile Glu Glu Leu Glu Lys Val Leu Ile Gly Val Ala
100 105 110
Tyr Leu Gly Glu Leu Thr Pro Lys Ser Arg Asp Tyr Ile Leu Ser Phe
115 120 125
Gly Glu Arg Leu Ser Ser Pro Ile Leu Ser Gly Ala Ile Arg Asp Leu
130 135 140
Gly Glu Lys Ser Ile Ala Leu Glu Gly Gly Glu Ala Gly Ile Ile Thr
145 150 155 160
Asp Asn Asn Phe Gly Ser Ala Arg Val Lys Arg Leu Glu Val Lys Glu
165 170 175
Arg Leu Leu Pro Leu Leu Lys Glu Gly Ile Ile Pro Val Val Thr Gly
180 185 190
Phe Ile Gly Thr Thr Glu Glu Gly Tyr Ile Thr Thr Leu Gly Arg Gly
195 200 205
Gly Ser Asp Tyr Ser Ala Ala Leu Ile Gly Tyr Gly Leu Asp Ala Asp
210 215 220
Ile Ile Glu Ile Trp Thr Asp Val Ser Gly Val Tyr Thr Thr Asp Pro
225 230 235 240
Arg Leu Val Pro Thr Ala Arg Arg Ile Pro Lys Leu Ser Tyr Ile Glu
245 250 255
Ala Met Glu Leu Ala Tyr Phe Gly Ala Lys Val Leu His Pro Arg Thr
260 265 270
Ile Glu Pro Ala Met Glu Lys Gly Ile Pro Ile Leu Val Lys Asn Thr
275 280 285
Phe Glu Pro Glu Ser Glu Gly Thr Leu Ile Thr Asn Asp Met Glu Met
290 295 300
Ser Asp Ser Ile Val Lys Ala Ile Ser Thr Ile Lys Asn Val Ala Leu
305 310 315 320
Ile Asn Ile Phe Gly Ala Gly Met Val Gly Val Ser Gly Thr Ala Ala
325 330 335
Arg Ile Phe Lys Ala Leu Gly Glu Glu Glu Val Asn Val Ile Leu Ile
340 345 350
Ser Gln Gly Ser Ser Glu Thr Asn Ile Ser Leu Val Val Ser Glu Glu
355 360 365
Asp Val Asp Lys Ala Leu Lys Ala Leu Lys Arg Glu Phe Gly Asp Phe
370 375 380
Gly Lys Lys Ser Phe Leu Asn Asn Asn Leu Ile Arg Asp Val Ser Val
385 390 395 400
Asp Lys Asp Val Cys Val Ile Ser Val Val Gly Ala Gly Met Arg Gly
405 410 415
Ala Lys Gly Ile Ala Gly Lys Ile Phe Thr Ala Val Ser Glu Ser Gly
420 425 430
Ala Asn Ile Lys Met Ile Ala Gln Gly Ser Ser Glu Val Asn Ile Ser
435 440 445
Phe Val Ile Asp Glu Lys Asp Leu Leu Asn Cys Val Arg Lys Leu His
450 455 460
Glu Lys Phe Ile Glu Lys Thr Asn Ser
465 470
<210> 90
<211> 405
<212> PRT
<213> Thermus thermophilus
<400> 90
Met Ala Leu Val Val Gln Lys Tyr Gly Gly Thr Ser Val Gly Asp Leu
1 5 10 15
Glu Arg Ile His Lys Val Ala Gln Arg Ile Ala His Tyr Arg Glu Lys
20 25 30
Gly His Arg Leu Ala Val Val Val Ser Ala Met Gly His Thr Thr Asp
35 40 45
Glu Leu Ile Ala Leu Ala Lys Arg Val Asn Pro Arg Pro Pro Phe Arg
50 55 60
Glu Leu Asp Leu Leu Thr Thr Thr Gly Glu Gln Val Ser Val Ala Leu
65 70 75 80
Leu Ser Met Gln Leu Trp Ala Met Gly Ile Pro Ala Lys Gly Phe Val
85 90 95
Gln His Gln Ile Gly Ile Thr Thr Asp Gly Arg Tyr Gly Asp Ala Arg
100 105 110
Ile Leu Glu Val Asn Pro Ala Arg Ile Arg Glu Ala Leu Asp Gln Gly
115 120 125
Phe Val Ala Val Ile Ala Gly Phe Met Gly Thr Thr Pro Glu Gly Glu
130 135 140
Ile Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala Ile
145 150 155 160
Ala Ala Ala Leu Gly Ala Lys Glu Cys Glu Ile Tyr Thr Asp Thr Glu
165 170 175
Gly Val Tyr Thr Thr Asp Pro His Leu Ile Pro Glu Ala Arg Lys Leu
180 185 190
Ser Val Ile Gly Tyr Asp Gln Met Leu Glu Met Ala Ala Leu Gly Ala
195 200 205
Arg Val Leu His Pro Arg Ala Val Tyr Tyr Ala Lys Arg Tyr Gly Val
210 215 220
Val Leu His Val Arg Ser Ser Phe Ser Tyr Asn Pro Gly Thr Leu Val
225 230 235 240
Lys Glu Val Ala Met Glu Met Asp Lys Ala Val Thr Gly Val Ala Leu
245 250 255
Asp Leu Asp His Ala Gln Ile Gly Leu Ile Gly Ile Pro Asp Gln Pro
260 265 270
Gly Ile Ala Ala Lys Val Phe Gln Ala Leu Ala Glu Arg Gly Ile Ala
275 280 285
Val Asp Met Ile Ile Gln Gly Val Pro Gly His Asp Pro Ser Arg Gln
290 295 300
Gln Met Ala Phe Thr Val Lys Lys Asp Phe Ala Gln Glu Ala Leu Glu
305 310 315 320
Ala Leu Glu Pro Val Leu Ala Glu Ile Gly Gly Glu Ala Ile Leu Arg
325 330 335
Pro Asp Ile Ala Lys Val Ser Ile Val Gly Val Gly Leu Ala Ser Thr
340 345 350
Pro Glu Val Pro Ala Lys Met Phe Gln Ala Val Ala Ser Thr Gly Ala
355 360 365
Asn Ile Glu Met Ile Ala Thr Ser Glu Val Arg Ile Ser Val Ile Ile
370 375 380
Pro Ala Glu Tyr Ala Glu Ala Ala Leu Arg Ala Val His Gln Ala Phe
385 390 395 400
Glu Leu Asp Lys Ala
405
<210> 91
<211> 420
<212> PRT
<213> Corynebacterium glutamicum
<400> 91
Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala
1 5 10 15
Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala
20 25 30
Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp
35 40 45
Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg
50 55 60
Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu
65 70 75 80
Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr
85 90 95
Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg
100 105 110
Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly
115 120 125
Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg
130 135 140
Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160
Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val
165 170 175
Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys
180 185 190
Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly
195 200 205
Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn
210 215 220
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu
225 230 235 240
Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr
245 250 255
Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile
260 265 270
Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp
275 280 285
Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu
290 295 300
Asp Gly Thr Thr Asp Ile Thr Phe Thr Cys Pro Arg Ser Asp Gly Arg
305 310 315 320
Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr
325 330 335
Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala
340 345 350
Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu
355 360 365
Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg
370 375 380
Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala
385 390 395 400
Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr
405 410 415
Ala Gly Thr Gly
420
<210> 92
<211> 569
<212> PRT
<213> Arabidopsis thaliana
<400> 92
Met Ala Ala Thr Arg Val Arg Cys Cys His Ser Asn Ala Ala Phe Thr
1 5 10 15
Arg Leu Pro Leu Thr Arg His Arg Asn Ser Pro Thr Leu Pro Ile Ser
20 25 30
Leu Asn Arg Val Asp Phe Pro Thr Leu Lys Lys Leu Ser Leu Pro Ile
35 40 45
Gly Asp Gly Ser Ser Ile Arg Lys Val Ser Gly Ser Gly Ser Arg Asn
50 55 60
Ile Val Arg Ala Val Leu Glu Glu Lys Lys Thr Glu Ala Ile Thr Glu
65 70 75 80
Val Asp Glu Lys Gly Ile Thr Cys Val Met Lys Phe Gly Gly Ser Ser
85 90 95
Val Ala Ser Ala Glu Arg Met Lys Glu Val Ala Asp Leu Ile Leu Thr
100 105 110
Phe Pro Glu Glu Ser Pro Val Ile Val Leu Ser Ala Met Gly Lys Thr
115 120 125
Thr Asn Asn Leu Leu Leu Ala Gly Glu Lys Ala Val Ser Cys Gly Val
130 135 140
Ser Asn Ala Ser Glu Ile Glu Glu Leu Ser Ile Ile Lys Glu Leu His
145 150 155 160
Ile Arg Thr Val Lys Glu Leu Asn Ile Asp Pro Ser Val Ile Leu Thr
165 170 175
Tyr Leu Glu Glu Leu Glu Gln Leu Leu Lys Gly Ile Ala Met Met Lys
180 185 190
Glu Leu Thr Leu Arg Thr Arg Asp Tyr Leu Val Ser Phe Gly Glu Cys
195 200 205
Leu Ser Thr Arg Ile Phe Ala Ala Tyr Leu Asn Thr Ile Gly Val Lys
210 215 220
Ala Arg Gln Tyr Asp Ala Phe Glu Ile Gly Phe Ile Thr Thr Asp Asp
225 230 235 240
Phe Thr Asn Gly Asp Ile Leu Glu Ala Thr Tyr Pro Ala Val Ala Lys
245 250 255
Arg Leu Tyr Asp Asp Trp Met His Asp Pro Ala Val Pro Ile Val Thr
260 265 270
Gly Phe Leu Gly Lys Gly Trp Lys Thr Gly Ala Val Thr Thr Leu Gly
275 280 285
Arg Gly Gly Ser Asp Leu Thr Ala Thr Thr Ile Gly Lys Ala Leu Gly
290 295 300
Leu Lys Glu Ile Gln Val Trp Lys Asp Val Asp Gly Val Leu Thr Cys
305 310 315 320
Asp Pro Thr Ile Tyr Lys Arg Ala Thr Pro Val Pro Tyr Leu Thr Phe
325 330 335
Asp Glu Ala Ala Glu Leu Ala Tyr Phe Gly Ala Gln Val Leu His Pro
340 345 350
Gln Ser Met Arg Pro Ala Arg Glu Gly Glu Ile Pro Val Arg Val Lys
355 360 365
Asn Ser Tyr Asn Pro Lys Ala Pro Gly Thr Ile Ile Thr Lys Thr Arg
370 375 380
Asp Met Thr Lys Ser Ile Leu Thr Ser Ile Val Leu Lys Arg Asn Val
385 390 395 400
Thr Met Leu Asp Ile Ala Ser Thr Arg Met Leu Gly Gln Val Gly Phe
405 410 415
Leu Ala Lys Val Phe Ser Ile Phe Glu Glu Leu Gly Ile Ser Val Asp
420 425 430
Val Val Ala Thr Ser Glu Val Ser Ile Ser Leu Thr Leu Asp Pro Ser
435 440 445
Lys Leu Trp Ser Arg Glu Leu Ile Gln Gln Glu Leu Asp His Val Val
450 455 460
Glu Glu Leu Glu Lys Ile Ala Val Val Asn Leu Leu Lys Gly Arg Ala
465 470 475 480
Ile Ile Ser Leu Ile Gly Asn Val Gln His Ser Ser Leu Ile Leu Glu
485 490 495
Arg Ala Phe His Val Leu Tyr Thr Lys Gly Val Asn Val Gln Met Ile
500 505 510
Ser Gln Gly Ala Ser Lys Val Asn Ile Ser Phe Ile Val Asn Glu Ala
515 520 525
Glu Ala Glu Gly Cys Val Gln Ala Leu His Lys Ser Phe Phe Glu Ser
530 535 540
Gly Asp Leu Ser Glu Leu Leu Ile Gln Pro Arg Leu Gly Asn Gly Ser
545 550 555 560
Pro Val Arg Thr Leu Gln Val Glu Asn
565
<210> 93
<211> 527
<212> PRT
<213> Saccharomyces cerevisiae
<400> 93
Met Pro Met Asp Phe Gln Pro Thr Ser Ser His Ser Asn Trp Val Val
1 5 10 15
Gln Lys Phe Gly Gly Thr Ser Val Gly Lys Phe Pro Val Gln Ile Val
20 25 30
Asp Asp Ile Val Lys His Tyr Ser Lys Pro Asp Gly Pro Asn Asn Asn
35 40 45
Val Ala Val Val Cys Ser Ala Arg Ser Ser Tyr Thr Lys Ala Glu Gly
50 55 60
Thr Thr Ser Arg Leu Leu Lys Cys Cys Asp Leu Ala Ser Gln Glu Ser
65 70 75 80
Glu Phe Gln Asp Ile Ile Glu Val Ile Arg Gln Asp His Ile Asp Asn
85 90 95
Ala Asp Arg Phe Ile Leu Asn Pro Ala Leu Gln Ala Lys Leu Val Asp
100 105 110
Asp Thr Asn Lys Glu Leu Glu Leu Val Lys Lys Tyr Leu Asn Ala Ser
115 120 125
Lys Val Leu Gly Glu Val Ser Ser Arg Thr Val Asp Leu Val Met Ser
130 135 140
Cys Gly Glu Lys Leu Ser Cys Leu Phe Met Thr Ala Leu Cys Asn Asp
145 150 155 160
Arg Gly Cys Lys Ala Lys Tyr Val Asp Leu Ser His Ile Val Pro Ser
165 170 175
Asp Phe Ser Ala Ser Ala Leu Asp Asn Ser Phe Tyr Thr Phe Leu Val
180 185 190
Gln Ala Leu Lys Glu Lys Leu Ala Pro Phe Val Ser Ala Lys Glu Arg
195 200 205
Ile Val Pro Val Phe Thr Gly Phe Phe Gly Leu Val Pro Thr Gly Leu
210 215 220
Leu Asn Gly Val Gly Arg Gly Tyr Thr Asp Leu Cys Ala Ala Leu Ile
225 230 235 240
Ala Val Ala Val Asn Ala Asp Glu Leu Gln Val Trp Lys Glu Val Asp
245 250 255
Gly Ile Phe Thr Ala Asp Pro Arg Lys Val Pro Glu Ala Arg Leu Leu
260 265 270
Asp Ser Val Thr Pro Glu Glu Ala Ser Glu Leu Thr Tyr Tyr Gly Ser
275 280 285
Glu Val Ile His Pro Phe Thr Met Glu Gln Val Ile Arg Ala Lys Ile
290 295 300
Pro Ile Arg Ile Lys Asn Val Gln Asn Pro Leu Gly Asn Gly Thr Ile
305 310 315 320
Ile Tyr Pro Asp Asn Val Ala Lys Lys Gly Glu Ser Thr Pro Pro His
325 330 335
Pro Pro Glu Asn Leu Ser Ser Ser Phe Tyr Glu Lys Arg Lys Arg Gly
340 345 350
Ala Thr Ala Ile Thr Thr Lys Asn Asp Ile Phe Val Ile Asn Ile His
355 360 365
Ser Asn Lys Lys Thr Leu Ser His Gly Phe Leu Ala Gln Ile Phe Thr
370 375 380
Ile Leu Asp Lys Tyr Lys Leu Val Val Asp Leu Ile Ser Thr Ser Glu
385 390 395 400
Val His Val Ser Met Ala Leu Pro Ile Pro Asp Ala Asp Ser Leu Lys
405 410 415
Ser Leu Arg Gln Ala Glu Glu Lys Leu Arg Ile Leu Gly Ser Val Asp
420 425 430
Ile Thr Lys Lys Leu Ser Ile Val Ser Leu Val Gly Lys His Met Lys
435 440 445
Gln Tyr Ile Gly Ile Ala Gly Thr Met Phe Thr Thr Leu Ala Glu Glu
450 455 460
Gly Ile Asn Ile Glu Met Ile Ser Gln Gly Ala Asn Glu Ile Asn Ile
465 470 475 480
Ser Cys Val Ile Asn Glu Ser Asp Ser Ile Lys Ala Leu Gln Cys Ile
485 490 495
His Ala Lys Leu Leu Ser Glu Arg Thr Asn Thr Ser Asn Gln Phe Glu
500 505 510
His Ala Ile Asp Glu Arg Leu Glu Gln Leu Lys Arg Leu Gly Ile
515 520 525
<210> 94
<211> 354
<212> PRT
<213> Methanococcus jannaschii
<400> 94
Met Ser Lys Gly Glu Lys Met Lys Ile Lys Val Gly Val Leu Gly Ala
1 5 10 15
Thr Gly Ser Val Gly Gln Arg Phe Val Gln Leu Leu Ala Asp His Pro
20 25 30
Met Phe Glu Leu Thr Ala Leu Ala Ala Ser Glu Arg Ser Ala Gly Lys
35 40 45
Lys Tyr Lys Asp Ala Cys Tyr Trp Phe Gln Asp Arg Asp Ile Pro Glu
50 55 60
Asn Ile Lys Asp Met Val Val Ile Pro Thr Asp Pro Lys His Glu Glu
65 70 75 80
Phe Glu Asp Val Asp Ile Val Phe Ser Ala Leu Pro Ser Asp Leu Ala
85 90 95
Lys Lys Phe Glu Pro Glu Phe Ala Lys Glu Gly Lys Leu Ile Phe Ser
100 105 110
Asn Ala Ser Ala Tyr Arg Met Glu Glu Asp Val Pro Leu Val Ile Pro
115 120 125
Glu Val Asn Ala Asp His Leu Glu Leu Ile Glu Ile Gln Arg Glu Lys
130 135 140
Arg Gly Trp Asp Gly Ala Ile Ile Thr Asn Pro Asn Cys Ser Thr Ile
145 150 155 160
Cys Ala Val Ile Thr Leu Lys Pro Ile Met Asp Lys Phe Gly Leu Glu
165 170 175
Ala Val Phe Ile Ala Thr Met Gln Ala Val Ser Gly Ala Gly Tyr Asn
180 185 190
Gly Val Pro Ser Met Ala Ile Leu Asp Asn Leu Ile Pro Phe Ile Lys
195 200 205
Asn Glu Glu Glu Lys Met Gln Thr Glu Ser Leu Lys Leu Leu Gly Thr
210 215 220
Leu Lys Asp Gly Lys Val Glu Leu Ala Asn Phe Lys Ile Ser Ala Ser
225 230 235 240
Cys Asn Arg Val Ala Val Ile Asp Gly His Thr Glu Ser Ile Phe Val
245 250 255
Lys Thr Lys Glu Gly Ala Glu Pro Glu Glu Ile Lys Glu Val Met Asp
260 265 270
Lys Phe Asp Pro Leu Lys Asp Leu Asn Leu Pro Thr Tyr Ala Lys Pro
275 280 285
Ile Val Ile Arg Glu Glu Ile Asp Arg Pro Gln Pro Arg Leu Asp Arg
290 295 300
Asn Glu Gly Asn Gly Met Ser Ile Val Val Gly Arg Ile Arg Lys Asp
305 310 315 320
Pro Ile Phe Asp Val Lys Tyr Thr Ala Leu Glu His Asn Thr Ile Arg
325 330 335
Gly Ala Ala Gly Ala Ser Val Leu Asn Ala Glu Tyr Phe Val Lys Lys
340 345 350
Tyr Ile
<210> 95
<211> 331
<212> PRT
<213> Thermus thermophilus
<400> 95
Met Arg Val Ala Val Val Gly Ala Thr Gly Ala Val Gly Arg Glu Ile
1 5 10 15
Leu Lys Val Leu Glu Ala Arg Asp Phe Pro Leu Ser Asp Leu Arg Leu
20 25 30
Tyr Ala Ser Pro Arg Ser Ala Gly Val Arg Leu Ala Phe Arg Gly Glu
35 40 45
Glu Ile Pro Val Glu Pro Leu Pro Glu Gly Pro Leu Pro Val Asp Leu
50 55 60
Val Leu Ala Ser Ala Gly Gly Gly Ile Ser Lys Ala Lys Ala Leu Val
65 70 75 80
Trp Ala Glu Gly Gly Ala Leu Val Val Asp Asn Ser Ser Ala Trp Arg
85 90 95
Tyr Glu Pro Trp Val Pro Leu Val Val Pro Glu Val Asn Arg Glu Lys
100 105 110
Ile Phe Gln His Arg Gly Ile Ile Ala Asn Pro Asn Cys Thr Thr Ala
115 120 125
Ile Leu Ala Met Ala Leu Trp Pro Leu His Arg Ala Phe Gln Ala Lys
130 135 140
Arg Val Ile Val Ala Thr Tyr Gln Ala Ala Ser Gly Ala Gly Ala Lys
145 150 155 160
Ala Met Glu Glu Leu Leu Thr Glu Thr His Arg Phe Leu His Gly Glu
165 170 175
Ala Pro Lys Ala Glu Ala Phe Ala His Pro Leu Pro Phe Asn Val Ile
180 185 190
Pro His Ile Asp Ala Phe Gln Glu Asn Gly Tyr Thr Arg Glu Glu Met
195 200 205
Lys Val Val Trp Glu Thr His Lys Ile Phe Gly Asp Asp Thr Ile Arg
210 215 220
Ile Ser Ala Thr Ala Val Arg Val Pro Thr Leu Arg Ala His Ala Glu
225 230 235 240
Ala Val Ser Val Glu Phe Ala Arg Pro Val Thr Pro Glu Ala Ala Arg
245 250 255
Glu Val Leu Lys Glu Ala Pro Gly Val Glu Val Val Asp Glu Pro Glu
260 265 270
Ala Lys Arg Tyr Pro Met Pro Leu Thr Ala Ser Gly Lys Trp Asp Val
275 280 285
Glu Val Gly Arg Ile Arg Lys Ser Leu Ala Phe Glu Asn Gly Leu Asp
290 295 300
Phe Phe Val Val Gly Asp Gln Leu Leu Lys Gly Ala Ala Leu Asn Ala
305 310 315 320
Val Gln Ile Ala Glu Glu Trp Leu Lys Gly Ala
325 330
<210> 96
<211> 346
<212> PRT
<213> Bacillus subtilis
<400> 96
Met Gly Arg Gly Leu His Val Ala Val Val Gly Ala Thr Gly Ala Val
1 5 10 15
Gly Gln Gln Met Leu Lys Thr Leu Glu Asp Arg Asn Phe Glu Met Asp
20 25 30
Thr Leu Thr Leu Leu Ser Ser Lys Arg Ser Ala Gly Thr Lys Val Thr
35 40 45
Phe Lys Gly Gln Glu Leu Thr Val Gln Glu Ala Ser Pro Glu Ser Phe
50 55 60
Glu Gly Val Asn Ile Ala Leu Phe Ser Ala Gly Gly Ser Val Ser Gln
65 70 75 80
Ala Leu Ala Pro Glu Ala Val Lys Arg Gly Ala Ile Val Ile Asp Asn
85 90 95
Thr Ser Ala Phe Arg Met Asp Glu Asn Thr Pro Leu Val Val Pro Glu
100 105 110
Val Asn Glu Ala Asp Leu His Glu His Asn Gly Ile Ile Ala Asn Pro
115 120 125
Asn Cys Ser Thr Ile Gln Met Val Ala Ala Leu Glu Pro Ile Arg Lys
130 135 140
Ala Tyr Gly Leu Asn Lys Val Ile Val Ser Thr Tyr Gln Ala Val Ser
145 150 155 160
Gly Ala Gly Asn Glu Ala Val Lys Glu Leu Tyr Ser Gln Thr Gln Ala
165 170 175
Ile Leu Asn Lys Glu Glu Ile Glu Pro Glu Ile Met Pro Val Lys Gly
180 185 190
Asp Lys Lys His Tyr Gln Ile Ala Phe Asn Ala Ile Pro Gln Ile Asp
195 200 205
Lys Phe Gln Asp Asn Gly Tyr Thr Phe Glu Glu Met Lys Met Ile Asn
210 215 220
Glu Thr Lys Lys Ile Met His Met Pro Asp Leu Gln Val Ala Ala Thr
225 230 235 240
Cys Val Arg Leu Pro Ile Gln Thr Gly His Ser Glu Ser Val Tyr Ile
245 250 255
Glu Ile Asp Arg Asp Asp Ala Thr Val Glu Asp Ile Lys Asn Leu Leu
260 265 270
Lys Glu Ala Pro Gly Val Thr Leu Gln Asp Asp Pro Ser Gln Gln Leu
275 280 285
Tyr Pro Met Pro Ala Asp Ala Val Gly Lys Asn Asp Val Phe Val Gly
290 295 300
Arg Ile Arg Lys Asp Leu Asp Arg Ala Asn Gly Phe His Leu Trp Val
305 310 315 320
Val Ser Asp Asn Leu Leu Lys Gly Ala Ala Trp Asn Ser Val Gln Ile
325 330 335
Ala Glu Ser Leu Lys Lys Leu Asn Leu Val
340 345
<210> 97
<211> 344
<212> PRT
<213> Corynebacterium glutamicum
<400> 97
Met Thr Thr Ile Ala Val Val Gly Ala Thr Gly Gln Val Gly Gln Val
1 5 10 15
Met Arg Thr Leu Leu Glu Glu Arg Asn Phe Pro Ala Asp Thr Val Arg
20 25 30
Phe Phe Ala Ser Pro Arg Ser Ala Gly Arg Lys Ile Glu Phe Arg Gly
35 40 45
Thr Glu Ile Glu Val Glu Asp Ile Thr Gln Ala Thr Glu Glu Ser Leu
50 55 60
Lys Asp Ile Asp Val Ala Leu Phe Ser Ala Gly Gly Thr Ala Ser Lys
65 70 75 80
Gln Tyr Ala Pro Leu Phe Ala Ala Ala Gly Ala Thr Val Val Asp Asn
85 90 95
Ser Ser Ala Trp Arg Lys Asp Asp Glu Val Pro Leu Ile Val Ser Glu
100 105 110
Val Asn Pro Ser Asp Lys Asp Ser Leu Val Lys Gly Ile Ile Ala Asn
115 120 125
Pro Asn Cys Thr Thr Met Ala Ala Met Pro Val Leu Lys Pro Leu His
130 135 140
Asp Ala Ala Gly Leu Val Lys Leu His Val Ser Ser Tyr Gln Ala Val
145 150 155 160
Ser Gly Ser Gly Leu Ala Gly Val Glu Thr Leu Ala Lys Gln Val Ala
165 170 175
Ala Val Gly Asp His Asn Val Glu Phe Val His Asp Gly Gln Ala Ala
180 185 190
Asp Ala Gly Asp Val Gly Pro Tyr Val Ser Pro Ile Ala Tyr Asn Val
195 200 205
Leu Pro Phe Ala Gly Asn Leu Val Asp Asp Gly Thr Phe Glu Thr Asp
210 215 220
Glu Glu Gln Lys Leu Arg Asn Glu Ser Arg Lys Ile Leu Gly Leu Pro
225 230 235 240
Asp Leu Lys Val Ser Gly Thr Cys Val Arg Val Pro Val Phe Thr Gly
245 250 255
His Thr Leu Thr Ile His Ala Glu Phe Asp Lys Ala Ile Thr Val Asp
260 265 270
Gln Ala Gln Glu Ile Leu Gly Ala Ala Ser Gly Val Lys Leu Val Asp
275 280 285
Val Pro Thr Pro Leu Ala Ala Ala Gly Ile Asp Glu Ser Leu Val Gly
290 295 300
Arg Ile Arg Gln Asp Ser Thr Val Asp Asp Asn Arg Gly Leu Val Leu
305 310 315 320
Val Val Ser Gly Asp Asn Leu Arg Lys Gly Ala Ala Leu Asn Thr Ile
325 330 335
Gln Ile Ala Glu Leu Leu Val Lys
340
<210> 98
<211> 340
<212> PRT
<213> Arabidopsis thaliana
<400> 98
Glu Ser Ala Pro Ser Leu Ala Val Val Gly Val Thr Gly Ala Val Gly
1 5 10 15
Gln Glu Phe Leu Ser Val Leu Ser Asp Arg Asp Phe Pro Tyr Ser Ser
20 25 30
Ile Lys Met Leu Ala Ser Lys Arg Ser Ala Gly Lys Arg Val Ala Phe
35 40 45
Asp Gly His Glu Tyr Thr Val Glu Glu Leu Thr Ala Asp Ser Phe Asn
50 55 60
Gly Val Asp Ile Ala Leu Phe Ser Ala Gly Gly Ser Ile Ser Lys Glu
65 70 75 80
Phe Gly Pro Leu Ala Ala Glu Lys Gly Thr Ile Val Val Asp Asn Ser
85 90 95
Ser Ala Phe Arg Met Val Asp Gly Val Pro Leu Val Ile Pro Glu Val
100 105 110
Asn Pro Glu Ala Met Lys Gly Ile Lys Val Gly Met Gly Lys Gly Ala
115 120 125
Leu Ile Ala Asn Pro Asn Cys Ser Thr Ile Ile Cys Leu Met Ala Val
130 135 140
Thr Pro Leu His His His Ala Lys Val Lys Arg Met Val Val Ser Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Ala Gly Ala Ala Ala Met Glu Glu Leu Val
165 170 175
Gln Gln Thr Arg Glu Val Leu Glu Gly Lys Pro Pro Thr Cys Asn Ile
180 185 190
Phe Gly Gln Gln Tyr Ala Phe Asn Leu Phe Ser His Asn Ala Pro Ile
195 200 205
Leu Asp Asn Gly Tyr Asn Glu Glu Glu Met Lys Leu Val Lys Glu Thr
210 215 220
Arg Lys Ile Trp Asn Asp Thr Glu Val Lys Val Thr Ala Thr Cys Ile
225 230 235 240
Arg Val Pro Val Met Arg Ala His Ala Glu Ser Val Asn Leu Gln Phe
245 250 255
Glu Asn Pro Leu Asp Glu Asn Thr Ala Arg Glu Ile Leu Lys Lys Ala
260 265 270
Pro Gly Val Tyr Ile Ile Asp Asp Arg Ala Ser Asn Thr Phe Pro Thr
275 280 285
Pro Leu Asp Val Ser Asn Lys Asp Asp Val Ala Val Gly Arg Ile Arg
290 295 300
Arg Asp Val Ser Gln Asp Gly Asn Phe Gly Leu Asp Ile Phe Val Cys
305 310 315 320
Gly Asp Gln Ile Arg Lys Gly Ala Ala Leu Asn Ala Val Gln Ile Ala
325 330 335
Glu Met Leu Leu
340
<210> 99
<211> 365
<212> PRT
<213> Saccharomyces cerevisiae
<400> 99
Met Ala Gly Lys Lys Ile Ala Gly Val Leu Gly Ala Thr Gly Ser Val
1 5 10 15
Gly Gln Arg Phe Ile Leu Leu Leu Ala Asn His Pro His Phe Glu Leu
20 25 30
Lys Val Leu Gly Ala Ser Ser Arg Ser Ala Gly Lys Lys Tyr Val Asp
35 40 45
Ala Val Asn Trp Lys Gln Thr Asp Leu Leu Pro Glu Ser Ala Thr Asp
50 55 60
Ile Ile Val Ser Glu Cys Lys Ser Glu Phe Phe Lys Glu Cys Asp Ile
65 70 75 80
Val Phe Ser Gly Leu Asp Ala Asp Tyr Ala Gly Ala Ile Glu Lys Glu
85 90 95
Phe Met Glu Ala Gly Ile Ala Ile Val Ser Asn Ala Lys Asn Tyr Arg
100 105 110
Arg Glu Gln Asp Val Pro Leu Ile Val Pro Val Val Asn Pro Glu His
115 120 125
Leu Asp Ile Val Ala Gln Lys Leu Asp Thr Ala Lys Ala Gln Gly Lys
130 135 140
Pro Arg Pro Gly Phe Ile Ile Cys Ile Ser Asn Cys Ser Thr Ala Gly
145 150 155 160
Leu Val Ala Pro Leu Lys Pro Leu Ile Glu Lys Phe Gly Pro Ile Asp
165 170 175
Ala Leu Thr Thr Thr Thr Leu Gln Ala Ile Ser Gly Ala Gly Phe Ser
180 185 190
Pro Gly Val Pro Gly Ile Asp Ile Leu Asp Asn Ile Ile Pro Tyr Ile
195 200 205
Gly Gly Glu Glu Asp Lys Met Glu Trp Glu Thr Lys Lys Ile Leu Ala
210 215 220
Pro Leu Ala Glu Asp Lys Thr His Val Lys Leu Leu Thr Pro Glu Glu
225 230 235 240
Ile Lys Val Ser Ala Gln Cys Asn Arg Val Ala Val Ser Asp Gly His
245 250 255
Thr Glu Cys Ile Ser Leu Arg Phe Lys Asn Arg Pro Ala Pro Ser Val
260 265 270
Glu Gln Val Lys Thr Cys Leu Lys Glu Tyr Val Cys Asp Ala Tyr Lys
275 280 285
Leu Gly Cys His Ser Ala Pro Lys Gln Thr Ile His Val Leu Glu Gln
290 295 300
Pro Asp Arg Pro Gln Pro Arg Leu Asp Arg Asn Arg Asp Ser Gly Tyr
305 310 315 320
Gly Val Ser Val Gly Arg Ile Arg Glu Asp Pro Leu Leu Asp Phe Lys
325 330 335
Met Val Val Leu Ser His Asn Thr Ile Ile Gly Ala Ala Gly Ser Gly
340 345 350
Val Leu Ile Ala Glu Ile Leu Leu Ala Arg Asn Leu Ile
355 360 365
<210> 100
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 100
tataatcccg ggatgcgcgt taacaatggt ttgacc 36
<210> 101
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 101
tataattcta gattacagtt tcggaccagc cg 32
<210> 102
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 102
gaaggttgcg cctacactaa gcatagttgt tgatgagtgt aggctggagc tgcttc 56
<210> 103
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 103
ttaaaccagt tcgttcgggc aggtttcgcc tttttcatgg gaattagcca tggtcc 56
<210> 104
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 104
atggctgtta ctaatgtcgc tgaacttaac gcactcgtag agcgtgtgta ggctggagct 60
gcttc 65
<210> 105
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 105
ttaagcggat tttttcgctt ttttctcagc tttagccgga gcagccatat gaatatcctc 60
cttag 65
<210> 106
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 106
atgtcgagta agttagtact ggttctgaac tgcggtagtt cttcagtgta ggctggagct 60
gcttc 65
<210> 107
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 107
tcaggcagtc aggcggctcg cgtcttgcgc gataaccagt tcttccatat gaatatcctc 60
cttag 65
<210> 108
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 108
ttactccgta tttgcataaa aaccatgcga gttacgggcc tataagtgta ggctggagct 60
gcttc 65
<210> 109
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 109
atagattgag tgaaggtacg agtaataacg tcctgctgct gttctcatat gaatatcctc 60
cttag 65
<210> 110
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 110
gtgtcccgta ttattatgct gatccctacc ggaaccagcg tcggtgtgta ggctggagct 60
gcttc 65
<210> 111
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 111
ttactgctgc tgtgcagact gaatcgcagt cagcgcgatg gtgtacatat gaatatcctc 60
cttag 65
<210> 112
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 112
atgaaacaaa cggttgcagc ttatatcgcc aaaacactcg aatcggtgta ggctggagct 60
gcttc 65
<210> 113
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 113
ttaccttagc cagtttgttt tcgccagttc gatcacttca tcacccatat gaatatcctc 60
cttag 65
<210> 114
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 114
atgaccatta ctccggcaac tcatgcaatt tcgataaatc ctgccgtgta ggctggagct 60
gcttc 65
<210> 115
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 115
tcagatccgg tctttccaca ccgtctggat attacagaat tcgtgcatat gaatatcctc 60
cttag 65
<210> 116
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 116
atgaaactta acgacagtaa cttattccgc cagcaggcgt tgattgtgta ggctggagct 60
gcttc 65
<210> 117
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 117
ttaaagaccg atgcacatat atttgatttc taagtaatct tcgatcatat gaatatcctc 60
cttag 65
<210> 118
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 118
atggaccaga agctgttaac ggatttccgc tcagaactac tcgatgtgta ggctggagct 60
gcttc 65
<210> 119
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 119
tcaggtgtgt ttaaagctgt tctgctgggc aataccctgc agtttcatat gaatatcctc 60
cttag 65
<210> 120
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 120
atggataaga agcaagtaac ggatttaagg tcggaactac tcgatgtgta ggctggagct 60
gcttc 65
<210> 121
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 121
tcaggtatgt ttaaagctgt tctgttgggc aataccctgc agtttcatat gaatatcctc 60
cttag 65
<210> 122
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 122
atggctacat cagtacagac aggtaaagct aagcagctca cattagtgta ggctggagct 60
gcttc 65
<210> 123
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 123
ttagtgtttc ttgtcattca tcacaatata gtgtggtgaa cgtgccatat gaatatcctc 60
cttag 65
<210> 124
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 124
atggaaccaa aaacaaaaaa acagcgttcg ctttatatcc cttacgtgta ggctggagct 60
gcttc 65
<210> 125
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 125
ttagatggag gtacggcggt agtcgcggta ttcggcttgc cagaacatat gaatatcctc 60
cttag 65
<210> 126
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 126
atggatgacc agttaaaaca aagtgcactt gatttccatg aatttgtgta ggctggagct 60
gcttc 65
<210> 127
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 127
ttacagcggt tgggtttgcg cttctaccac ggccagcgcc accatcatat gaatatcctc 60
cttag 65
<210> 128
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 128
atgaacgaac aatattccgc attgcgtagt aatgtcagta tgctcgtgta ggctggagct 60
gcttc 65
<210> 129
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 129
ttagccggta ttacgcatac ctgccgcaat cccggcaata gtgaccatat gaatatcctc 60
cttag 65
<210> 130
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 130
atgtccagaa ggcttcgcag aacaaaaatc gttaccacgt taggcgtgta ggctggagct 60
gcttc 65
<210> 131
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 131
ttactctacc gttaaaatac gcgtggtatt agtagaaccc acggtcatat gaatatcctc 60
cttag 65
<210> 132
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 132
atgaaaaaga ccaaaattgt ttgcaccatc ggaccgaaaa ccgaagtgta ggctggagct 60
gcttc 65
<210> 133
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 133
ttacaggacg tgaacagatg cggtgttagt agtgccgctc ggtaccatat gaatatcctc 60
cttag 65
<210> 134
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 134
atggaactga cgactcgcac tttacctgcg cggaaacata ttgcggtgta ggctggagct 60
gcttc 65
<210> 135
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 135
ttacttcaga cggtccgcga gataacgctg ataatcgggg atcagcatat gaatatcctc 60
cttag 65
<210> 136
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 136
atggtcgcac ccattcccgc gaaacgcggc agaaaacccg ccgttgtgta ggctggagct 60
gcttc 65
<210> 137
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 137
tcagcgcatt ccaccgtacg ccagcgtcac ttccttcgcc gctttcatat gaatatcctc 60
cttag 65
<210> 138
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 138
atggaaagta aagtagttgt tccggcacaa ggcaagaaga tcaccgtgta ggctggagct 60
gcttc 65
<210> 139
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 139
ttacatgttt tcgatgatcg cgtcaccaaa ctctgaacat ttcagcatat gaatatcctc 60
cttag 65
<210> 140
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 140
atgcagaaca gcgctttgaa agcctggttg gactcttctt acctcgtgta ggctggagct 60
gcttc 65
<210> 141
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 141
ttattcgacg ttcagcgcgt cattaaccag atcttgttgc tgtttcatat gaatatcctc 60
cttag 65
<210> 142
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 142
atgagtagcg tagatattct ggtccctgac ctgcctgaat ccgtagtgta ggctggagct 60
gcttc 65
<210> 143
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 143
ctacacgtcc agcagcagac gcgtcggatc ttccagcaac tctttcatat gaatatcctc 60
cttag 65
<210> 144
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 144
gtgcaaacct ttcaagccga tcttgccatt gtaggcgccg gtggcgtgta ggctggagct 60
gcttc 65
<210> 145
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 145
tcagccattc gccttctcct tcttattggc tgcttccgcc ttatccatat gaatatcctc 60
cttag 65
<210> 146
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 146
atggctgaga tgaaaaacct gaaaattgag gtggtgcgct ataacgtgta ggctggagct 60
gcttc 65
<210> 147
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 147
ttagcgtggt ttcagggtcg cgataagaaa gtctttcgaa ctttccatat gaatatcctc 60
cttag 65
<210> 148
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 148
atgacgacta aacgtaaacc gtatgtacgg ccaatgacgt ccaccgtgta ggctggagct 60
gcttc 65
<210> 149
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 149
ttaccagtac agggcaacaa acaggattac gatggtggca accaccatat gaatatcctc 60
cttag 65
<210> 150
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 150
atgattaatc caaatccaaa gcgttctgac gaaccggtat tctgggtgta ggctggagct 60
gcttc 65
<210> 151
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 151
ttagattgta acgacaccaa tcagcgtgac aactgtcagg atagccatat gaatatcctc 60
cttag 65
<210> 152
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 152
atgtttaaga atgcatttgc taacctgcaa aaggtcggta aatcggtgta ggctggagct 60
gcttc 65
<210> 153
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 153
ttagtggtta cggatgtact catccatctc ggttttcagg ttatccatat gaatatcctc 60
cttag 65
<210> 154
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 154
atgatttcag gcattttagc atccccgggt atcgctttcg gtaaagtgta ggctggagct 60
gcttc 65
<210> 155
<211> 65
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 155
ttagcagatt gttttttctt caatgaactt gttaaccagc gtcatcatat gaatatcctc 60
cttag 65
<210> 156
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 156
cggtgccctg aatgaactgc 20
<210> 157
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 157
cagtcatagc cgaatagcct 20
<210> 158
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 158
atacgtgtcc cgagcggtag 20
<210> 159
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 159
tacacatccc gccatcagca 20
<210> 160
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 160
gaagtaaacg ggaaaatcaa 20
<210> 161
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 161
agaagtggca taagaaaacg 20
<210> 162
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 162
ccattggctg aaaattacgc 20
<210> 163
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 163
gttccattgc acggatcacg 20
<210> 164
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 164
atgccgtaga agccgccagt 20
<210> 165
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 165
tgttggtgcg cagctcgaag 20
<210> 166
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 166
gcaaatctgg tttcatcaac 20
<210> 167
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 167
tcccttgcac aaaacaaagt 20
<210> 168
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 168
ggatttggtt ctcgcataat 20
<210> 169
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 169
agcattaacg gtagggtcgt 20
<210> 170
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 170
gctgattctc gcgaataaac 20
<210> 171
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 171
aaaaacgttc ttgcgcgtct 20
<210> 172
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 172
tctgtttgtc accaccccgc 20
<210> 173
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 173
aagccagcac ctggaagcag 20
<210> 174
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 174
aagagctgcc gcaggaggat 20
<210> 175
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 175
gccgccctct taagtcaaat 20
<210> 176
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 176
ggattttagc aatattcgct 20
<210> 177
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 177
cctaatagca ggaagaagac 20
<210> 178
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 178
gctgaactgt tgctggaaga 20
<210> 179
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 179
ggcgtgcttt tacaactaca 20
<210> 180
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 180
tagtaaataa cccaaccggc 20
<210> 181
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 181
tcagtgagcg cagtgtttta 20
<210> 182
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 182
attaatggtg agagtttgga 20
<210> 183
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 183
tgcttttttt tattattcgc 20
<210> 184
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 184
gctttataaa agacgacgaa 20
<210> 185
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 185
gtaacgacaa ttccttaagg 20
<210> 186
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 186
tttatatgcc catggtttct 20
<210> 187
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 187
atctgttaga ggcggatgat 20
<210> 188
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 188
ctggaacgtt aaatctttga 20
<210> 189
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 189
ccagtttagt agctttcatt 20
<210> 190
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 190
gatttgttca acattaactc atcgg 25
<210> 191
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 191
tgcgattaac agacaccctt 20
<210> 192
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 192
tctcaggtgc tcacagaaca 20
<210> 193
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 193
tatggaagag gcgctactgc 20
<210> 194
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 194
cgacctgctg cataaacacc 20
<210> 195
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 195
tgaacgctaa ggtgattgca 20
<210> 196
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 196
acgtagacaa gagctcgcaa 20
<210> 197
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 197
catcacgtac gactgcgtcg 20
<210> 198
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 198
tgcaactttg tgctgagca 19
<210> 199
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 199
tatcgcttcc gggcattgtc 20
<210> 200
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 200
aaatcgatct cgtcaaattt cagac 25
<210> 201
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 201
aggaaccaca aatcgccata 20
<210> 202
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 202
gacgtgaaga ttactacgct 20
<210> 203
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 203
agttcaatgc tgaaccacac 20
<210> 204
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 204
tagccgcgac cacggtaaga aggag 25
<210> 205
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 205
cagcgcatca cccggaaaca 20
<210> 206
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 206
atcgtgatca ttaacctgat 20
<210> 207
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 207
ttaccctgat aaattaccgc 20
<210> 208
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 208
ccatccgttg aatgagtttt 20
<210> 209
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 209
tggtgttaac tggcaaaatc 20
<210> 210
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 210
gtgacttcca acggcaaaag 20
<210> 211
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 211
ccgttggttt gatagcaata 20
<210> 212
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 212
tataatcccg ggatgcgcgt taacaatggt ttgacc 36
<210> 213
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 213
tataattcta gattacagtt tcggaccagc cg 32
<210> 214
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 214
tataatcccg ggatgaacga acaatattcc 30
<210> 215
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 215
tataattcta gattagccgg tattacgcat 30
<210> 216
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 216
tataatcccg ggatgtccag aaggcttcgc agaaca 36
<210> 217
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 217
tataattcta gattactcta ccgttaaaat ac 32
<210> 218
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 218
tataatcccg ggatgaaaac ccgtacacaa caaatt 36
<210> 219
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 219
tataattcta gattagaact gcgattcttc ag 32
<210> 220
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 220
tataatcccg ggatgaaaaa actactcgtc gccaat 36
<210> 221
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 221
tataattcta gattaattaa tttcgattaa ca 32
<210> 222
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 222
tataatcccg ggatgcctga cgctaaaaaa caggggcggt 40
<210> 223
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for amplification
<400> 223
tataattcta gattaatcgt gagcgcctat ttc 33
<210> 224
<211> 1083
<212> DNA
<213> Metallosphaera sedula
<400> 224
atgaaagctg cagtacttca tacgtataag gaaccgctgt ccattgagga cgtgaatatc 60
tcccaaccta aggctgggga agtcaagatc aaggtcaagg caaccggtct ctgtcgctcc 120
gacgtcaatg tctttgaggg gaaaacccca gttcctcccc cagtggttgc tggacacgaa 180
atatcaggga ttgtggagga agtgggacct ggggtgacca gggttaaacc aggtgatagg 240
gtgatttcag cgtttattca cccctgtggt aaatgcggta actgcgttgc aggaaaggag 300
aatctgtgtg agaccttctc ccaggtcaga ctcaagggag taatgccaga tggaacgtca 360
aggctgtcaa aggacggaaa ggagataagg actttccttg gaggcggttt cgcggagtac 420
gccattgtgg gagagaacgc gctaaccagg gttccagagg acatggacct agagaaggta 480
gctgtcctag gttgtgctgg gttaacaggg tacggtgcca tatcatcatc caagattgag 540
cctggagaca ctgtggccgt gataggcgta ggaggagtgg gtttgtccac aatacaactc 600
ctaagggcct cgggtgccgg gaggataatc gccgtgggaa cgaaaaagtg gaaacttgac 660
agggccatgg agctaggtgc aactgacgtg gtaaactcga aggagataga tcccgtcaaa 720
gcaataaagg agatcacggg tggagggcca caggtggtga tagaggctgg aggaaatgag 780
gatacgattc atatggcgct ggattcagtt agaattggag gaaaggtggt tctggtaggg 840
ttacctccag caacggccat gatacccatc agggtagcgt caatagttag gggaggcata 900
gaggttgtgg ggaattacgg aggaagacct agggttgata tgcccaagct tctcgagcta 960
gtgaggcagg gaagatacga tccgtctagg cttgtgacgg gtagattcag gttggaggaa 1020
ataaatgagg cagtcaaaat gcttgaggaa ggagaggcca taagaagtct cataatcccg 1080
taa 1083
<210> 225
<211> 360
<212> PRT
<213> Metallosphaera sedula
<400> 225
Met Lys Ala Ala Val Leu His Thr Tyr Lys Glu Pro Leu Ser Ile Glu
1 5 10 15
Asp Val Asn Ile Ser Gln Pro Lys Ala Gly Glu Val Lys Ile Lys Val
20 25 30
Lys Ala Thr Gly Leu Cys Arg Ser Asp Val Asn Val Phe Glu Gly Lys
35 40 45
Thr Pro Val Pro Pro Pro Val Val Ala Gly His Glu Ile Ser Gly Ile
50 55 60
Val Glu Glu Val Gly Pro Gly Val Thr Arg Val Lys Pro Gly Asp Arg
65 70 75 80
Val Ile Ser Ala Phe Ile His Pro Cys Gly Lys Cys Gly Asn Cys Val
85 90 95
Ala Gly Lys Glu Asn Leu Cys Glu Thr Phe Ser Gln Val Arg Leu Lys
100 105 110
Gly Val Met Pro Asp Gly Thr Ser Arg Leu Ser Lys Asp Gly Lys Glu
115 120 125
Ile Arg Thr Phe Leu Gly Gly Gly Phe Ala Glu Tyr Ala Ile Val Gly
130 135 140
Glu Asn Ala Leu Thr Arg Val Pro Glu Asp Met Asp Leu Glu Lys Val
145 150 155 160
Ala Val Leu Gly Cys Ala Gly Leu Thr Gly Tyr Gly Ala Ile Ser Ser
165 170 175
Ser Lys Ile Glu Pro Gly Asp Thr Val Ala Val Ile Gly Val Gly Gly
180 185 190
Val Gly Leu Ser Thr Ile Gln Leu Leu Arg Ala Ser Gly Ala Gly Arg
195 200 205
Ile Ile Ala Val Gly Thr Lys Lys Trp Lys Leu Asp Arg Ala Met Glu
210 215 220
Leu Gly Ala Thr Asp Val Val Asn Ser Lys Glu Ile Asp Pro Val Lys
225 230 235 240
Ala Ile Lys Glu Ile Thr Gly Gly Gly Pro Gln Val Val Ile Glu Ala
245 250 255
Gly Gly Asn Glu Asp Thr Ile His Met Ala Leu Asp Ser Val Arg Ile
260 265 270
Gly Gly Lys Val Val Leu Val Gly Leu Pro Pro Ala Thr Ala Met Ile
275 280 285
Pro Ile Arg Val Ala Ser Ile Val Arg Gly Gly Ile Glu Val Val Gly
290 295 300
Asn Tyr Gly Gly Arg Pro Arg Val Asp Met Pro Lys Leu Leu Glu Leu
305 310 315 320
Val Arg Gln Gly Arg Tyr Asp Pro Ser Arg Leu Val Thr Gly Arg Phe
325 330 335
Arg Leu Glu Glu Ile Asn Glu Ala Val Lys Met Leu Glu Glu Gly Glu
340 345 350
Ala Ile Arg Ser Leu Ile Ile Pro
355 360
<210> 226
<211> 1083
<212> DNA
<213> Metallosphaera sedula
<400> 226
atgaaagctg cagtacttca tacgtataag gaaccgctgt ccattgagga cgtgaatatc 60
tcccaaccta aggctgggga agtcaagatc aaggtcaagg caaccgggct ctgtcactcc 120
gacgtacatg tctttgaggg gaaaacccca gttcctcccc cagtggttgc tggacacgaa 180
atatcaggga ttgtggagga agtgggacct ggggtgacca gggttaaacc aggtgatagg 240
gtgatttcag cgtttattca cccctgtggt aaatgcggta actgcgttgc aggaaaggag 300
aatctgtgtg agaccttctc ccaggtcaga ctcaagggag taatgccaga tggaacgtca 360
aggctgtcaa aggacggaaa ggagataagg actttccttg gaggcggttt cgcggagtac 420
gccattgtgg gagagaacgc gctaaccagg gttccagagg acatggacct agagaaggta 480
gctgtcctag gttgtgctgg gttaacaggg tacggtgcca tatcatcatc caagattgag 540
cctggagaca ctgtggccgt gataggcgta ggaggagtgg gtttgtccac aatacaactc 600
ctaagggcct cgggtgccgg gaggataatc gccgtgggaa cgaaaaagtg gaaacttgac 660
agggccatgg agctaggtgc aactgacgtg gtaaactcga aggagataga tcccgtcaaa 720
gcaataaagg agatcacggg tggagggcca caggtggtga tagaggctgg aggaaatgag 780
gatacgattc atatggcgct ggattcagtt agaattggag gaaaggtggt tctggtaggg 840
ttacctccag caacggccat gatacccatc agggtagcgt caatagttag gggaggcata 900
gaggttgtgg ggaattacgg aggaagacct agggttgata tgcccaagct tctcgagcta 960
gtgaggcagg gaagatacga tccgtctagg cttgtgacgg gtagattcag gttggaggaa 1020
ataaatgagg cagtcaaaat gcttgaggaa ggagaggcca taagaagtct cataatcccg 1080
taa 1083
<210> 227
<211> 360
<212> PRT
<213> Metallosphaera sedula
<400> 227
Met Lys Ala Ala Val Leu His Thr Tyr Lys Glu Pro Leu Ser Ile Glu
1 5 10 15
Asp Val Asn Ile Ser Gln Pro Lys Ala Gly Glu Val Lys Ile Lys Val
20 25 30
Lys Ala Thr Gly Leu Cys His Ser Asp Val His Val Phe Glu Gly Lys
35 40 45
Thr Pro Val Pro Pro Pro Val Val Ala Gly His Glu Ile Ser Gly Ile
50 55 60
Val Glu Glu Val Gly Pro Gly Val Thr Arg Val Lys Pro Gly Asp Arg
65 70 75 80
Val Ile Ser Ala Phe Ile His Pro Cys Gly Lys Cys Gly Asn Cys Val
85 90 95
Ala Gly Lys Glu Asn Leu Cys Glu Thr Phe Ser Gln Val Arg Leu Lys
100 105 110
Gly Val Met Pro Asp Gly Thr Ser Arg Leu Ser Lys Asp Gly Lys Glu
115 120 125
Ile Arg Thr Phe Leu Gly Gly Gly Phe Ala Glu Tyr Ala Ile Val Gly
130 135 140
Glu Asn Ala Leu Thr Arg Val Pro Glu Asp Met Asp Leu Glu Lys Val
145 150 155 160
Ala Val Leu Gly Cys Ala Gly Leu Thr Gly Tyr Gly Ala Ile Ser Ser
165 170 175
Ser Lys Ile Glu Pro Gly Asp Thr Val Ala Val Ile Gly Val Gly Gly
180 185 190
Val Gly Leu Ser Thr Ile Gln Leu Leu Arg Ala Ser Gly Ala Gly Arg
195 200 205
Ile Ile Ala Val Gly Thr Lys Lys Trp Lys Leu Asp Arg Ala Met Glu
210 215 220
Leu Gly Ala Thr Asp Val Val Asn Ser Lys Glu Ile Asp Pro Val Lys
225 230 235 240
Ala Ile Lys Glu Ile Thr Gly Gly Gly Pro Gln Val Val Ile Glu Ala
245 250 255
Gly Gly Asn Glu Asp Thr Ile His Met Ala Leu Asp Ser Val Arg Ile
260 265 270
Gly Gly Lys Val Val Leu Val Gly Leu Pro Pro Ala Thr Ala Met Ile
275 280 285
Pro Ile Arg Val Ala Ser Ile Val Arg Gly Gly Ile Glu Val Val Gly
290 295 300
Asn Tyr Gly Gly Arg Pro Arg Val Asp Met Pro Lys Leu Leu Glu Leu
305 310 315 320
Val Arg Gln Gly Arg Tyr Asp Pro Ser Arg Leu Val Thr Gly Arg Phe
325 330 335
Arg Leu Glu Glu Ile Asn Glu Ala Val Lys Met Leu Glu Glu Gly Glu
340 345 350
Ala Ile Arg Ser Leu Ile Ile Pro
355 360
<210> 228
<211> 1083
<212> DNA
<213> Artificial Sequence
<220>
<223> M. sedula codon optimized sequence
<400> 228
atgaaagcag cagttctgca tacctataaa gaaccgctga gcattgaaga tgtgaatatt 60
tcacagccga aagccggtga agtgaaaatc aaagttaaag caaccggtct gtgtcgtagt 120
gatgttcatg tttttgaagg taaaacaccg gttccgcctc cggttgttgc aggtcatgaa 180
attagcggta ttgttgaaga ggttggtccg ggtgttaccc gtgttaaacc gggtgatcgt 240
gttattagcg catttattca tccgtgtggt aaatgcggta attgtgttgc cggtaaagaa 300
aatctgtgtg aaacctttag ccaggttcgt ctgaaaggtg ttatgccgga tggcaccagc 360
cgtctgagca aagatggcaa agaaattcgt acctttctgg gtggtggttt tgcagaatat 420
gcaattgttg gtgaaaatgc actgacccgt gttccggaag atatggatct ggaaaaagtt 480
gcagttctgg gttgtgccgg tctgaccggt tatggtgcaa ttagcagcag caaaattgaa 540
cctggtgata ccgttgcagt tattggtgtt ggtggtgtgg gtctgagcac cattcagctg 600
ctgcgtgcaa gcggtgcagg tcgtattatt gcagttggca ccaaaaaatg gaaactggat 660
cgtgcaatgg aactgggtgc aaccgatgtt gttaacagta aagaaattga tccggtgaaa 720
gccatcaaag aaatcaccgg tggtggtccg caggttgtta ttgaagccgg tggtaatgaa 780
gataccattc acatggcact ggatagcgtt cgtattggtg gtaaagttgt tctggttggt 840
ctgcctccgg caaccgcaat gattccgatt cgtgttgcaa gcattgttcg tggtggtatt 900
gaagttgttg gtaattatgg tggtcgtccg cgtgttgata tgccgaaact gctggaactg 960
gttcgtcagg gtcgttatga tccgagccgt ctggttaccg gtcgttttcg tctggaagaa 1020
attaatgaag ccgtcaaaat gctggaagaa ggtgaagcaa ttcgtagcct gattattccg 1080
taa 1083
<210> 229
<211> 4107
<212> DNA
<213> Artificial Sequence
<220>
<223> chimeric gene for the expression of malate kinase, malate semi
aldehyde dehydrognase and DHB dehydrogenase
<400> 229
ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca 60
caggaaacag aattcgagct cggtacccgg ggatcctcta gaaataattt tgtttaactt 120
taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc agcggcctgg 180
tgccgcgcgg cagccatatg tctgaaattg ttgtctccaa atttggcggt accagcgtag 240
ctgattttga cgccatgaac cgcagcgctg atattgtgct ttctgatgcc aacgtgcgtt 300
tagttgtcct ctcggcttct gctggtatca ctaatctgct ggtcgcttta gctgaaggac 360
tggaacctgg cgagcgattc gaaaaactcg acgctatccg caacatccag tttgccattc 420
tggaacgtct gcgttacccg aacgttatcc gtgaagagat tgaacgtctg ctggagaaca 480
ttactgttct ggcagaagcg gcggcgctgg caacgtctcc ggcgctgaca gatgagctgg 540
tcagccatgg cggcctgatg tcgaccctgc tgtttgttga gatcctgcgc gaacgcgatg 600
ttcaggcaca gtggtttgat gtacgtaaag tgatgcgtac caacgaccga tttggtcgtg 660
cagagccaga tatagccgcg ctggcggaac tggccgcgct gcagctgctc ccacgtctca 720
atgaaggctt agtgatcacc cagggattta tcggtagcga aaataaaggt cgtacaacga 780
cgcttggccg tggaggcagc gattatacgg cagccttgct ggcggaggct ttacacgcat 840
ctcgtgttga tatctggacc gacgtcccgg gcatctacac caccgatcca cgcgtagttt 900
ccgcagcaaa acgcattgat gaaatcgcgt ttgccgaagc ggcaaagatg gccacttttg 960
gtgcaaaagt actgcatccg gcaacgttgc tacccgcagt acgcagcgat atcccggtct 1020
ttgtcggctc cagcaaagac ccacgcgcag gtggtacgct ggtgtgcaat aaaactgaaa 1080
atccgccgct gttccgcgct ctggcgcttc gtcgcaatca gactctgctc actttgcaca 1140
gcctgaatat gctgcattct cgcggtttcc tcgcggaagt tttcggcatc ctcgcgcggc 1200
ataatatttc ggtagactta atcaccacgt cagaagtgag cgtggcatta acccttgata 1260
ccaccggttc aacctccact ggcgatacgt tgctgacgca atctctgctg atggagcttt 1320
ccgcactgtg tcgggtggag gtggaagaag gtctggcgct ggtcgcgttg attggcaatg 1380
acctgtcaaa agcctgcggc gttggcaaag aggtattcgg cgtactggaa ccgttcaaca 1440
ttcgcatgat ttgttatggc gcatccagcc ataacctgtg cttcctggtg cccggcgaag 1500
atgccgagca ggtggtgcaa aaactgcata gtaatttgtt tgagtaaata ctggatccgt 1560
ttaactttaa gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc 1620
ggcctggtgc cgcgcggcag ccatatggct agcatgaaaa atgttggttt tatcggctgg 1680
cgcggtatgg tcggctccgt tctcatgcaa cgcatggttg aagagcgcga cttcgacgcc 1740
attcgccctg tcttcttttc tacttctcag cttggccagg ctgcgccgtc ttttggcgga 1800
accactggca cacttcagga tgcctttgat ctggaggcgc taaaggccct cgatatcatt 1860
gtgacctgtc agggcggcga ttataccaac gaaatctatc caaagcttcg tgaaagcgga 1920
tggcaaggtt actggattga cgcagcatcg tctctgcgca tgaaagatga cgccatcatc 1980
attcttgacc ccgtcaatca ggacgtcatt accgacggat taaataatgg catcaggact 2040
tttgttggcg gtaactgtac cgtaagcctg atgttgatgt cgttgggtgg tttattcgcc 2100
aatgatcttg ttgattgggt gtccgttgca acctaccagg ccgcttccgg cggtggtgcg 2160
cgacatatgc gtgagttatt aacccagatg ggccatctgt atggccatgt ggcagatgaa 2220
ctcgcgaccc cgtcctctgc tattctcgat atcgaacgca aagtcacaac cttaacccgt 2280
agcggtgagc tgccggtgga taactttggc gtgccgctgg cgggtagcct gattccgtgg 2340
atcgacaaac agctcgataa cggtcagagt cgacaggagt ggaaagggca ggcggaaacc 2400
aacaagatcc tcaacacatc ttccgtaatt ccggtagatg gtttatgtgt gcgtgtcggg 2460
gcattgcgct gccacagcca ggcattcact attaaattga aaaaagatgt gtctattccg 2520
accgtggaag aactgctggc tgcgcacaat ccgtgggcga aagtcgttcc gaacgatcgg 2580
gaaatcacta tgcgtgagct aaccccagct gccgttaccg gcacgctgac cacgccggta 2640
ggccgcctgc gtaagctgaa tatgggacca gagttcctgt cagcctttac cgtgggcgac 2700
cagctgctgt ggggggccgc ggagccgctg cgtcggatgc ttcgtcaact ggcgtaagaa 2760
ttcgagctcc gtcgacaagc ttgcggccgc gtttaacttt aagaaggaga tataccatgg 2820
gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatgg 2880
ctagcatgaa agcagcagtt ctgcatacct ataaagaacc gctgagcatt gaagatgtga 2940
atatttcaca gccgaaagcc ggtgaagtga aaatcaaagt taaagcaacc ggtctgtgtc 3000
gtagtgatgt tcatgttttt gaaggtaaaa caccggttcc gcctccggtt gttgcaggtc 3060
atgaaattag cggtattgtt gaagaggttg gtccgggtgt tacccgtgtt aaaccgggtg 3120
atcgtgttat tagcgcattt attcatccgt gtggtaaatg cggtaattgt gttgccggta 3180
aagaaaatct gtgtgaaacc tttagccagg ttcgtctgaa aggtgttatg ccggatggca 3240
ccagccgtct gagcaaagat ggcaaagaaa ttcgtacctt tctgggtggt ggttttgcag 3300
aatatgcaat tgttggtgaa aatgcactga cccgtgttcc ggaagatatg gatctggaaa 3360
aagttgcagt tctgggttgt gccggtctga ccggttatgg tgcaattagc agcagcaaaa 3420
ttgaacctgg tgataccgtt gcagttattg gtgttggtgg tgtgggtctg agcaccattc 3480
agctgctgcg tgcaagcggt gcaggtcgta ttattgcagt tggcaccaaa aaatggaaac 3540
tggatcgtgc aatggaactg ggtgcaaccg atgttgttaa cagtaaagaa attgatccgg 3600
tgaaagccat caaagaaatc accggtggtg gtccgcaggt tgttattgaa gccggtggta 3660
atgaagatac cattcacatg gcactggata gcgttcgtat tggtggtaaa gttgttctgg 3720
ttggtctgcc tccggcaacc gcaatgattc cgattcgtgt tgcaagcatt gttcgtggtg 3780
gtattgaagt tgttggtaat tatggtggtc gtccgcgtgt tgatatgccg aaactgctgg 3840
aactggttcg tcagggtcgt tatgatccga gccgtctggt taccggtcgt tttcgtctgg 3900
aagaaattaa tgaagccgtc aaaatgctgg aagaaggtga agcaattcgt agcctgatta 3960
ttccgtaagc tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga 4020
aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc 4080
tctaaacggg tcttgagggg ttttttg 4107
<210> 230
<211> 1104
<212> DNA
<213> Escherichia coli
<400> 230
atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60
atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120
ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180
gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240
atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300
ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360
gacggattaa ataatggcat caggactttt gttggcggta actgtaacgt gtccctgatg 420
ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480
taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540
catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600
gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660
ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagtcga 720
caggagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780
gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840
aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900
tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960
gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020
ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080
cggatgcttc gtcaactggc gtaa 1104
<210> 231
<211> 367
<212> PRT
<213> Escherichia coli
<400> 231
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Asn Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Gln Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln Leu Leu
340 345 350
Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gln Leu Ala
355 360 365
Claims (60)
- 하기 단계를 포함하는, 2,4-디하이드록시부티르산 (2,4-DHB)의 제조 방법:
- 말레이트 키나아제에 의해 말레이트를 4-포스포-말레이트로 변환하는 제1 단계로서, 상기 말레이트 키나아제가 서열번호 9, 서열번호 12, 서열번호 14, 서열번호 16, 서열번호 18, 서열번호 20, 서열번호 22, 서열번호 24, 서열번호 26, 서열번호 39, 서열번호 41, 서열번호 43 또는 서열번호 45로 표시되는, 단계,
- 말레이트 세미알데하이드 탈수소효소에 의해 4-포스포-말레이트를 말레이트-4-세미알데하이드로 변환하는 제2 단계로서, 상기 말레이트 세미알데하이드 탈수소효소가 서열번호 68, 서열번호 54, 서열번호 56, 서열번호 58, 서열번호 60, 서열번호 62, 서열번호 64, 서열번호 66 또는 서열번호 231로 표시되는, 단계, 및
- DHB 탈수소효소에 의해 말레이트-4-세미알데하이드를 2,4-DHB로 변환하는 제3 단계로서, 상기 DHB 탈수소효소가 서열번호 74, 서열번호 76, 서열번호 81, 서열번호 225 또는 서열번호 227로 표시되는, 단계. - 전사 방향으로, 기능적으로 연결된,
- 숙주 유기체에서 기능하는 프로모터 조절 서열,
- 말레이트를 4-포스포-말레이트로 변환하는 것을 특징으로 하는 말레이트 키나아제를 코딩하는 핵산 서열,
- 4-포스포-말레이트를 말레이트-4-세미알데하이드로 변환하는 것을 특징으로 하는 말레이트 세미알데하이드 탈수소효소를 코딩하는 핵산 서열,
- 말레이트-4-세미알데하이드를 2,4-DHB로 변환하는 것을 특징으로 하는 DHB 탈수소효소를 코딩하는 핵산 서열, 또는 서열 번호 73 또는 서열 번호 75 또는 서열 번호 82로 표시되는 핵산 서열, 및
- 상기 숙주 유기체에서 기능하는 종결자 조절 서열을 포함하는 것을 특징으로 하는, 키메라 유전자로서,
상기 말레이트 키나아제가 서열번호 9, 서열번호 12, 서열번호 14, 서열번호 16, 서열번호 18, 서열번호 20, 서열번호 22, 서열번호 24, 서열번호 26, 서열번호 39, 서열번호 41, 서열번호 43 또는 서열번호 45로 표시되고,
상기 말레이트 세미알데하이드 탈수소효소가 서열번호 68, 서열번호 54, 서열번호 56, 서열번호 58, 서열번호 60, 서열번호 62, 서열번호 64, 서열번호 66 또는 서열번호 231로 표시되고,
상기 DHB 탈수소효소가 서열번호 74, 서열번호 76, 서열번호 81, 서열번호 225 또는 서열번호 227로 표시되는 것을 특징으로 하는, 키메라 유전자. - 제2 항에 있어서, 서열 번호 229로 표시되는 것을 특징으로 하는, 키메라 유전자.
- 제2항 또는 제3항에 따른 키메라 유전자를 포함하는, 발현 벡터.
- 제2항 또는 제3항에 따른 키메라 유전자 또는 이를 포함하는 발현 벡터로 형질전환된, 숙주 미생물.
- 말레이트 키나아제, 말레이트 세미알데하이드 탈수소효소, 및 DHB 탈수소효소를 발현하는 제5항에 따른 숙주 미생물을 배양하는 단계를 포함하는, 2,4-DHB의 제조 방법.
- 제6항에 있어서, 숙주 유기체가, 말레이트, 또는 피루베이트, 숙시네이트 또는 푸마레이트와 같은 다른 유기산이 첨가된 매질에서 배양되는 것을 특징으로 하는, 2,4-DHB의 제조 방법.
- 제7항에 있어서, 배양 배지가 다른 탄소원을 추가로 포함하는 것을 특징으로 하는, 방법.
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CN105505892B (zh) | 2019-03-26 |
CN105505892A (zh) | 2016-04-20 |
CN103270155B (zh) | 2016-02-10 |
RU2013123481A (ru) | 2014-12-10 |
JP6345747B2 (ja) | 2018-06-20 |
US10358663B2 (en) | 2019-07-23 |
KR20140091463A (ko) | 2014-07-21 |
JP2017070285A (ja) | 2017-04-13 |
CN103270155A (zh) | 2013-08-28 |
US20130273623A1 (en) | 2013-10-17 |
RU2626531C2 (ru) | 2017-07-28 |
US9238829B2 (en) | 2016-01-19 |
JP2013543727A (ja) | 2013-12-09 |
BR112013010268A2 (pt) | 2016-07-05 |
US20160153013A1 (en) | 2016-06-02 |
WO2012056318A1 (en) | 2012-05-03 |
TWI626311B (zh) | 2018-06-11 |
JP6071887B2 (ja) | 2017-02-01 |
AR083589A1 (es) | 2013-03-06 |
ES2631554T3 (es) | 2017-09-01 |
BR112013010268B1 (pt) | 2020-09-08 |
TW201221648A (en) | 2012-06-01 |
EP2633038A1 (en) | 2013-09-04 |
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