KR20200135469A - 자일로스 대사 효모 - Google Patents
자일로스 대사 효모 Download PDFInfo
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- KR20200135469A KR20200135469A KR1020207030404A KR20207030404A KR20200135469A KR 20200135469 A KR20200135469 A KR 20200135469A KR 1020207030404 A KR1020207030404 A KR 1020207030404A KR 20207030404 A KR20207030404 A KR 20207030404A KR 20200135469 A KR20200135469 A KR 20200135469A
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Abstract
본 발명은 i) 자일룰로스 키나제 (XKS1), 트랜스알돌라제 (TAL1), 트랜스케톨라제 1 (TKL1) 및 트랜스케톨라제 2 (TKL2)를 코딩하는 천연 유전자의 과다발현 및 ii) 자일로스 이소머라제 (XI)를 코딩하는 기능적 이종 유전자의 발현 - 여기서 자일로스 이소머라제 (XI) 유전자는 티. 네아폴리타나, 에이. 안덴시스 및 씨. 클라리플라붐으로 이루어진 군으로부터 선택된 미생물로부터 유래됨 - 을 위한 하나 이상의 발현 구축물(들)로 형질전환된 미생물, 특히 효모에 관한 것이다. 본 발명은 또한 발현 구축물, 본 발명에 따른 미생물을 사용하여 펜토스 당을 발효시키는 방법 및 이러한 미생물을 생성하는 방법에 관한 것이다.
Description
본 발명은 i) 자일룰로스 키나제 (XKS1), 트랜스알돌라제 (TAL1), 트랜스케톨라제 1 (TKL1) 및 트랜스케톨라제 2 (TKL2)를 코딩하는 천연 유전자의 과다발현 및 ii) 자일로스 이소머라제 (XI)를 코딩하는 기능적 이종 유전자의 발현 - 여기서 자일로스 이소머라제 (XI) 유전자는 써모토가 네아폴리타나 (Thermotoga neapolitana), 안디탈레아 안덴시스 (Anditalea andensis) 및 클로스트리듐 클라리플라붐 (Clostridium clariflavum)으로 이루어진 군으로부터 선택된 미생물로부터 유래됨 - 을 위한 하나 이상의 발현 구축물(들)로 형질전환된 미생물, 특히 효모에 관한 것이다. 본 발명은 또한 자일로스 이소머라제의 트랜스제닉 발현을 위한 발현 구축물, 본 발명에 따른 미생물을 사용하여 펜토스 당을 발효시키는 방법 및 이러한 미생물을 생성하는 방법에 관한 것이다.
자일로스는 헤미셀룰로스의 가수분해 생성물이며 리그노셀룰로스 가수분해액에서 당 단량체의 상당 부분을 구성한다. 에쉐리키아 콜리 (Escherichia coli)를 포함한 많은 유기체가 자연적으로 자일로스를 탄소원으로 활용할 수 있지만, 사카로마이세스 세레비지애 (Saccharomyces cerevisiae)는 그러한 능력이 없다. 그러나, 더 높은 성장 억제제 내성 및 일반적인 견고성은 효모를 생물공학 및 바이오연료 산업을 위한 공급 원료로서의 리그노셀룰로스 바이오매스의 활용에 더 우수한 후보가 되게 한다. 지난 20년 동안 세계 최대의 에탄올 생성자인 효모가 리그노셀룰로스 가수분해물에서 당의 최대 1/3을 구성하는 5개 탄소 당을 대사할 수 있도록 상당한 노력을 기울였다. 리그노셀룰로스 물질의 5개 탄소 당 중 자일로스가 주성분이다.
자일로스의 대사 플럭스로의 통합을 가능하게 하는 2가지의 택일적 경로가 자연에 존재한다 - 자일로스 리덕타제/자일리톨 데히드로게나제 (XR/XDH) 및 자일로스 이소머라제 (XI) (도 1) (참조 8). 두 공정 모두 자일룰로스를 생성하며, 이는 자일룰로스 키나제 (XKS) 및 펜토스 포스페이트 경로 (PPP)의 산화 단계에 의해 더욱 처리된다. XI 경로는 주로 원핵생물에서 발견되고 XR/XDH 경로는 진핵생물에서 발견되며, 이 규칙에 대한 예외가 존재한다. 경험 상, 이종 유전자 발현은 유전자 발현 및 단백질 성숙 기구 뿐만 아니라 이들 유기체가 서식하는 환경의 유사성 때문에 밀접하게 관련된 유기체에서 더 효율적이다 - 즉, 초호열균으로부터의 효소는 새로운 숙주가 접근할 수 없는 온도를 필요로 할 수 있다. 이는 XR/XDH 효소의 사용이 에스. 세레비지애에서 선호될 수 있음을 제시한다. 자일로스의 자일리톨로의 환원은 XR에 의해 촉매되며 보조인자로 NADPH가 필요하다. 자일리톨의 자일룰로스로의 산화는 XDH에 의해 촉매되고 NAD+로부터 NADH를 생성한다. 탄소원으로 자일로스의 신속한 사용은 동등하게 신속한 NADP 및 NADH 불균형을 야기시켜, 성장을 억제시킨다. 따라서, XR/XDH 경로를 이용하는 작업은 종종 자일로스 대사율이 낮은 균주를 생성하고 부생성물로 상당한 양의 자일리톨을 생성하여 결과적으로 생성물 수율을 감소시킨다 (참조 9).
자일로스 이소머라제를 통해 자일로스 대사를 조작하는 다른 택일적 경로는 하나의 효소만 필요하고 XR-XDH 경로를 통한 상기에서 언급된 바와 같은 보조인자 불균형 문제가 없기 때문에 간단해 보인다. 이 경로는 박테리아에서는 흔하지만 효모와 같은 진핵생물 종에서는 드물다. 혐기성 진균인 피로마이세스 (Piromyces) 종 E2는 활성 XI 효소를 발현하는 유전자를 보유하는 공지된 극소수의 종 중 하나이다. US 7,622,284 B2는 에스. 세레비지애에서 피로마이세스 종 XI를 발현하여 낮은 비율로 자일로스를 대사할 수 있는 효모 균주를 발생시키는 방법을 기재한다.
US 8,114,974 B2에 따라, 진균 자일로스 이소머라제 및 루미노코커스 플라베파시엔스 (Ruminococcus flavefaciens) 자일로스 이소머라제의 인접한 아미노산을 포함하는 키메라 효소는 사카로마이세스 세레비지애와 같은 숙주 세포에서 발현된다. US 7,943,366 B2는 피로마이세스 종 또는 실라마이세스 아베렌시스 (Cyllamyces aberensis)와 같은 진균 또는 박테리아, 즉 박테로이데스 테타이오타오미크론 (Bacteroides thetaiotaomicron)로부터 유래될 수 있는 외인성 자일로스 이소머라제 유전자로 형질전환된 효모 세포에 관한 것이다. US 2011/0244525 A1에서 사카로마이세스 세포는 락토코쿠스 (Lactococcus) 종으로부터 유래된 자일로스 이소머라제로 형질전환되고, US 2011/0269180 A1에서 효모 세포 또는 사상 진균 세포는 클로스트리듐 피토페르멘탄스 (Clostridium phytofermentans)로부터 유래된 원핵생물 자일로스 이소머라제를 발현한다.
그러나, 박테리아 기원으로부터의 대부분의 자일로스 이소머라제 유전자의 발현은 에스. 세레비지애에서 활성 자일로스 이소머라제의 존재를 발생시키지 않으며 이에 대한 정확한 메커니즘은 완전히 이해되지 않는다 (참조 10). 효모에서 발현되는 박테리아 기원으로부터의 XI 효소 중 일부만이 기능적으로 활성인 단백질을 생성하였지만 활성이 너무 낮아 자일로스에서 혐기성 성장을 지원할 수 없다. 따라서, 생물기반 화학물질 생성을 위한 탄소원으로 자일로스를 사용할 수 있도록 충분한 활성을 갖는 효모에서 발현될 수 있는 기능적 XI 효소를 식별할 필요성이 여전히 강하다.
또한, 사카로마이세스 세레비지애 펜토스 포스페이트 경로 (PPP)는 자일룰로스를 포함하는 펜토스 당을 위한 주요 대사 경로이다. 이는 해당 경로의 초기 단계와 병행하여 작용하여 펜토스 당으로부터 글리세르알데히드-3-포스페이트 및 프룩토스-6-포스페이트를 생성한다 (도 2). 우선적으로 대사될 수 있는 헥소스 당이 존재하는 경우, PPP는 주로 리보스 당을 생성하는데 필요하다. 자일로스를 높은 비율로 효율적으로 대사할 수 있는 효모 균주를 생성하기에는 기능적 XI 유전자의 발현이 충분하지 않을 수 있는데, 이는 펜토스 포스페이트 경로로 이를 통한 플럭스가 제한 요인이 될 수 있기 때문이다. 특히, D-자일룰로스를 D-자일룰로스-5-포스페이트로 전환하여 펜토스 포스페이트 경로로의 진입을 제공하는 자일룰로스 키나제 (XKS1) 및 트랜스케톨라제 TLK1 및 TLK2 뿐만 아니라 펜토스 포스페이트 경로의 트랜스알돌라제 (TAL1)가 이러한 맥락에서 매우 중요하다.
본 발명의 목적은 자일로스와 같은 펜토스 당의 효율적인 대규모 대사를 위한 방법을 제공하는 것이다. 특히, 본 발명의 목적은 펜토스 당, 특히 자일로스를 대사할 수 있고 에탄올과 같은 대사물을 높은 수율로 생성하는 바람직하게는 사카로마이세스 세레비지애 종의 효모와 같은 억제제에 내성이 있고 일반적으로 견고한 미생물을 제공하는 것이다. 이러한 미생물은 생체내에서 높은 활성을 나타내고 원하는 생성물의 상당한 수율을 제공하는 외인성 자일로스 이소머라제를 발현해야 한다. 추가 목적은 하기의 명세서, 제공된 실시예 및 특히 첨부된 청구범위로부터 도출될 수 있다.
상기 확인된 목적은
i) 자일룰로스 키나제 (XKS1), 트랜스알돌라제 (TAL1), 트랜스케톨라제 1 (TKL1) 및 트랜스케톨라제 2 (TKL2)를 코딩하는 천연 유전자의 과다발현 및
ii) 자일로스 이소머라제 (XI)를 코딩하는 기능적 이종 유전자의 발현 - 여기서 자일로스 이소머라제 (XI) 유전자는 써모토가 네아폴리타나, 안디탈레아 안덴시스 및 클로스트리듐 클라리플라붐으로 이루어진 군으로부터 선택된 미생물로부터 유래됨 -
을 위한 하나 이상의 발현 구축물(들)로 형질전환된 미생물, 특히 바람직하게는 사카로마이세스 세레비지애 종의 효모에 의해 충족된다.
상이한 박테리아 기원으로부터의 많은 자일로스 이소머라제 (XI) 유전자가 천연 자일룰로스 키나제 및 언급된 PPP 효소를 과다발현하는 사카로마이세스 세레비지애에서 시험되었다. XI 유전자 중 단지 3개, 즉 써모토가 네아폴리타나 (티. 네아폴리타나 (T. neapolitana)), 안디탈레아 안덴시스 (에이. 안덴시스 (A. andensis)) 및 클로스트리듐 클라리플라붐 (씨. 클라리플라붐 (C. clariflavum))만이 사카로마이세스 세레비지애에서 상당한 활성을 나타내는 것으로 밝혀졌으며, 에이. 안덴시스로부터의 XI이 가장 활성적이다. 이미 상기에서 언급된 바와 같이, 박테리아 기원으로부터의 대부분의 XI 유전자가 활성 효소의 발현을 발생시키지 않는 이유는 이해되지 않는다. 따라서, 사카로마이세스 세레비지애에서 발현될 때 (활성이 조금이라도 있는 경우) 어떤 XI가 상당한 활성을 나타낼지를 예측하는 합리적인 접근법이 없는 것으로 보인다.
상술된 미생물은 바람직하게는 천연 자일로스 키나제 (XKS1), 트랜스알돌라제 (TAL1), 트랜스케톨라제 1 (TKL1) 및 트랜스케톨라제 2 (TKL2)를 함유하여 펜토스 포스페이트 경로를 통해 펜토스 당을 도입 및 대사할 수 있는 특히 사카로마이세스 세레비지애 종의 효모 세포이다. 자일룰로스 키나제 (XKS1)는 ATP를 사용하여 D-자일룰로스를 D-자일룰로스-5-포스페이트로 인산화할 수 있는 효소이다 (EC 2.7.1.17). 트랜스알돌라제 1 (TAL1)은 세도헵툴로스-7-포스페이트 및 D-글리세르알데히드-3-포스페이트의 D-프룩토스-6-포스페이트 및 D-에리트로스-4-포스페이트로의 반응을 촉매한다 (EC 2.2.1.2). 트랜스케톨라제 1 및 2 (TKL1 및 TKL2)는 세도헵툴로스-7-포스페이트 및 글리세르알데히드-3-포스페이트를 형성하기 위해 D-자일룰로스-5-포스페이트로부터 D-리보스-5-포스페이트로 2-탄소 단편의 전달을 촉매할 뿐만 아니라 D-자일룰로스-5-포스페이트로부터 에리트로스-4-포스페이트로 2-탄소 단편의 전달을 촉매하여 프룩토스-6-포스페이트 및 글리세르알데히드-3-포스페이트를 산출한다 (EC 2.2.1.1). 자일로스 이소머라제 (XI)는 D-자일로스 및 D-자일룰로스의 상호전환을 촉매하고 많은 박테리아에서 발견된다 (EC 5.3.1.5).
본 발명의 맥락에서 발현 구축물은, 각각 발현될 하나 이상의 유전자가 뒤따르는, 하나 이상의 프로모터를 포함하는 핵산 서열이다. 프로모터는 하나 이상의 유전자의 전사를 제어하는 핵산 서열이며 각각의 유전자(들)의 전사 시작 부위 근처에 위치된다. 발현될 각각의 유전자는 보통 터미네이터 서열이 뒤따른다. 과다발현이 가능한 프로모터는 바람직하게는 항상 활성인 구성적 프로모터이다. 본 발명의 맥락에서, 바람직하게는 사카로마이세스 세레비지애의 천연 유전자는 적절한 프로모터의 제어 하에 배치함으로써 과다발현되며, 이는 각각의 내인성 유전자를 발현하는 비변형된 유기체에 대한 유전자의 발현을 증가시킨다.
대조적으로, XI 유전자의 카피는 비변형된 숙주 유기체인 사카로마이세스 세레비지애에 자연적으로 존재하지 않고 공여자 유기체로부터 유래된 트랜스진으로 도입되고 발현된다. 숙주 세포에서 트랜스진의 발현을 달성하기 위해, 트랜스진의 서열은 바람직하게는 숙주 세포에 최적화된 코돈이고 숙주 세포로부터 유래된 프로모터 서열의 제어 하에 배치된다. 기능적 이종 유전자는 숙주 유기체에서 지정된 역할을 수행할 수 있는 효소의 발현을 이끈다.
XI 유전자가 유래되는 공여자 유기체는 박테리아이다. 써모토가 네아폴리타나는 온천 환경에서 발견될 수 있는 호열성 박테리아이다. 안디탈레아 안덴시스는 친알칼리성의 내염성 박테리아로 매우 알칼리성인 토양으로부터 단리되었다. 클로스트리듐 클라리플라붐은 셀룰로스를 대사할 수 있는 호열성 박테리아이다. 발현된 XI의 생체내 활성이 사카로마이세스 세레비지애에서 발현에 대해 시험된 유전자 중 가장 높기 때문에 안디탈레아 안덴시스의 XI 유전자가 본 발명의 맥락에서 특히 바람직하다.
바람직한 실시양태에 따라, 자일로스 이소머라제 (XI)는 서열식별번호 (SEQ ID No): 21, 서열식별번호: 5 또는 서열식별번호: 25와 적어도 66%, 바람직하게는 적어도 80%, 더 바람직하게는 적어도 90%, 가장 바람직하게는 적어도 95% 서열 동일성을 갖는 핵산 서열에 의해 코딩된다.
본 개시내용이 서로에 대한 핵산 또는 아미노산 서열의 동일성 백분율과 관련될 때마다, 이들 값은 핵산에 대해서는 EMBOSS 워터스 쌍별 서열 정렬 (EMBOSS Water Pairwise Sequence Alignments) (뉴클레오티드) 프로그램 (www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html) 또는 아미노산 서열에 대해서는 EMBOSS 워터스 쌍별 서열 정렬 (단백질) 프로그램 (www.ebi.ac.uk/Tools/psa/emboss_water/)을 이용하여 수득된 값을 정의한다. 본원에 사용된 정렬 또는 서열 비교는 서로 비교되는 2개의 서열의 전체 길이에 걸친 정렬을 지칭한다. 유럽 분자 생물학 실험실 (European Molecular Biology Laboratory: EMBL) 유럽 생물정보학 연구서 (European Bioinformatics Institute: EBI)에 의해 로컬 서열 정렬을 위해 제공된 도구는 변형된 스미스-워터만 (Smith-Waterman) 알고리즘을 이용한다 (www.ebi.ac.uk/Tools/psa/ and Smith, T.F. & Waterman, M.S. "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1):195-197). 정렬을 수행할 때, EMBL-EBI에 의해 정의된 디폴트 파라미터가 사용된다. 이들 파라미터는 (i) 아미노산 서열의 경우: 매트릭스 = BLOSUM62, 갭 오픈 패널티 = 10 및 갭 확장 패널티 = 0.5 또는 (ii) 핵산 서열의 경우: 매트릭스 = DNAfull, 갭 오픈 패널티 = 10 및 갭 확장 패널티 = 0.5이다.
서열식별번호: 21은 티. 네아폴리타나의 천연 XI 유전자의 핵산 서열에 상응하고, 서열식별번호: 5는 에이. 안덴시스의 천연 XI 유전자에 상응하고, 서열식별번호: 25는 씨. 클라리플라붐의 천연 XI 유전자에 상응한다. 각각 적어도 66%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99%의 서열 동일성을 갖는 서열은 기능적 자일로스 이소머라제의 발현을 허용하는 한 본 발명의 맥락에서 사용될 수 있다. 유전자는 각각의 공여자와 상이한 숙주 유기체에서 발현되기 때문에, 서열은 바람직하게는 사카로마이세스 세레비지애에서의 발현에 최적화된 코돈이다. 따라서, 상기 주어진 서열 동일성에 의해 커버되는 사카로마이세스 세레비지애에 대한 코돈-최적화로 인한 차이는 통상의 기술자에게 공지되어 있고 쉽게 식별될 수 있다.
바람직하게는, 자일로스 이소머라제 (XI)는 서열식별번호: 22, 서열식별번호: 6 또는 서열식별번호: 26에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 아미노산 서열로 나타내어진다.
서열식별번호: 22는 티. 네아폴리타나로부터의 천연 XI의 아미노산 서열에 상응하고, 서열식별번호: 6은 에이. 안덴시스의 천연 XI에 상응하고, 서열식별번호: 26은 씨. 클라리플라붐의 천연 XI에 상응한다. 각각 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99%의 서열 동일성을 갖는 서열은 기능적 자일로스 이소머라제를 구성하는 한 본 발명의 맥락에서 사용될 수 있다.
바람직한 실시양태에 따라, 자일룰로스 키나제 (XKS1)는 서열식별번호: 74에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되고, 트랜스알돌라제 (TAL1)는 서열식별번호: 77에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되고, 트랜스케톨라제 1 (TKL1)은 서열식별번호: 80에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되고, 트랜스케톨라제 2 (TKL2)는 서열식별번호: 83에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩된다.
서열식별번호: 74, 77, 80 및 83은 사카로마이세스 세레비지애의 천연 XKS1 유전자, 천연 TAL1 유전자 및 천연 TKL1 및 TKL2 유전자의 핵산 서열에 상응한다. 각각 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99%의 서열 동일성을 갖는 서열은 각각의 기능을 수행할 수 있는 효소의 발현을 허용하는 한 본 발명의 맥락에서 사용될 수 있다.
추가의 바람직한 실시 양태에서, 자일룰로스 키나제 (XKS1), 트랜스알돌라제 (TAL1), 트랜스케톨라제 1 (TKL1), 트랜스케톨라제 2 (TKL2) 및 자일로스 이소머라제 (XI)를 코딩하는 유전자 각각은 구성적 프로모터의 제어 하에 있으며, 여기서 구성적 프로모터는 서열식별번호: 73에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 TDH3, 서열식별번호: 76에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 PGK1, 서열식별번호: 79에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 CYC19, 서열식별번호: 82에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 PFK1, 서열식별번호: 90에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 말단절단된 HXT7 및 서열식별번호: 85에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 TEF로부터 선택된다.
본 발명에 따른 발현 구축물의 유전자는 바람직하게는 사카로마이세스 세레비지애의 구성적 프로모터의 제어 하에 배치된다. 각각 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99%의 서열 동일성을 갖는 서열은 본원에 기재된 유전자의 발현 또는 각각 과다발현을 촉진할 수 있는 한 본 발명의 맥락에서 사용될 수 있다.
바람직하게는, XKS1 유전자는 TDH3 프로모터의 제어 하에 배치되고/되거나 TAL1 유전자는 PGK1 프로모터의 제어 하에 배치되고/되거나 TKL1 유전자는 CYC19 프로모터의 제어 하에 배치되고 TKL2 유전자는 PFK1 프로모터의 제어 하에 배치된다. 더욱 바람직하게는, XI 유전자는 말단절단된 HXT7 또는 TEF 프로모터의 제어 하에 배치된다. 유전자 및 프로모터의 이들 조합은 다른 조합보다 높은 발현 수준을 발생시켰다.
각각의 발현 구축물(들)에서, 상술된 유전자의 서열은 바람직하게는 사카로마이세스 세레비지애로부터 유래된 터미네이터 서열이 뒤따른다. 이러한 터미네이터 서열은 tDIT1 (서열식별번호: 75), tYHI9 (서열식별번호: 78), tEFM (서열식별번호: 81), tRPL15A (서열식별번호: 84), tTEF (서열식별번호: 87), tCYC1 (서열식별번호: 91) 및 tADH1 (서열식별번호: 94)로부터 선택될 수 있다.
바람직하게는, XKS1 다음에 DIT1 터미네이터가 오고/오거나 TAL1 유전자 다음에 YHI9 터미네이터가 오고/오거나 TKL1 유전자 다음에 EFM1 터미네이터가 오고, TKL2 유전자 다음에 RPL15A 터미네이터가 온다. 더욱 바람직하게는, XI 유전자 다음에 CYC1 터미네이터 또는 ADH1 터미네이터가 온다. 상기 명시된 유전자 및 프로모터와 상기 명시된 터미네이터의 조합은 특히 높은 발현 수준을 발생시켰다.
본 발명은 또한 티. 네아폴리타나, 에이. 안덴시스 및 씨. 클라리플라붐으로 이루어진 군으로부터 선택된 미생물로부터 유래된 자일로스 이소머라제 (XI)를 코딩하는 유전자의 발현을 위한 발현 구축물에 관한 것으로서, 여기서 자일로스 이소머라제 (XI) 유전자는 사카로마이세스 세레비지애의 구성적 프로모터의 제어 하에 있다. 에이. 안덴시스로부터 유래되는 XI를 코딩하는 유전자가 특히 바람직하다.
바람직하게는, 자일로스 이소머라제 (XI)를 코딩하는 유전자는 서열식별번호: 21, 서열식별번호: 5 또는 서열식별번호: 25에 대해 적어도 66%, 바람직하게는 적어도 80%, 더 바람직하게는 적어도 90%, 가장 바람직하게는 적어도 95% 서열 동일성을 갖는 핵산 서열로 나타내어진다.
서열식별번호: 21은 티. 네아폴리타나의 천연 XI 유전자의 핵산 서열에 상응하고, 서열식별번호: 5는 에이. 안덴시스의 천연 XI 유전자에 상응하고, 서열식별번호: 25는 씨. 클라리플라붐의 천연 XI 유전자에 상응한다. 각각 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99%의 서열 동일성을 갖는 서열은 기능적 자일로스 이소머라제의 발현을 허용하는 한 본 발명의 맥락에서 사용될 수 있다. 유전자가 사카로마이세스 세레비지애에서 발현되도록 의도되기 때문에, 서열은 바람직하게는 사카로마이세스 세레비지애에 대해 최적화된 코돈이다. 상기 주어진 서열 동일성에 의해 커버되는 코돈-최적화로 인한 차이는 통상의 기술자에게 공지되어 있다.
더욱 바람직하게는, 구성적 프로모터는 서열식별번호: 90에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 말단절단된 HXT7 및 서열식별번호: 85에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 TEF로부터 선택된다.
상술된 발현 구축물의 XI 유전자는 바람직하게는 사카로마이세스 세레비지애의 구성적 프로모터의 제어 하에 배치된다. 각각 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99%의 서열 동일성을 갖는 서열은 기능적 XI의 발현을 촉진할 수 있는 한 본 발명의 맥락에서 사용될 수 있다
본 발명은 또한 상술된 바와 같은 미생물을 펜토스 당(들)을 포함하는 배양 배지에서 펜토스 당(들)이 대사될 수 있는 조건 하에서 배양하는 단계를 포함하는, 펜토스 당(들)을 발효시키는 방법에 관한 것이다.
바람직하게는, 방법은 자일로스를 발효시키는 방법이다. 이미 상기에서 언급된 바와 같이, 자일로스는 헤미셀룰로스의 가수분해 생성물이며 리그노셀룰로스 가수분해액에서 당 단량체의 상당 부분을 나타낸다. 따라서, 생물공학 및 바이오연료 산업을 위한 공급 원료로 리그노셀룰로스 바이오매스를 사용하기 위해 자일로스를 발효시킬 수 있는 것이 특히 바람직하다.
특히 바람직한 실시양태에 따라, 배양 배지는 리그노셀룰로스 바이오매스 및/또는 그의 가수분해물을 포함하거나 그로 이루어진다.
펜토스 당 발효, 특히 자일로스 발효의 유용한 생성물은 에탄올, 메탄올, 프로판올, 이소프로판올, 부탄올, 에틸렌 글리콜, 프로필렌 글리콜, 1,4-부탄디올, 글리세린, 포름산, 아세트산, 프로피온산, 부티르산, 발레르산, 카프로산, 팔미트산, 스테아르산, 옥살산, 말론산, 숙신산 또는 숙시네이트, 글루타르산, 올레산, 리놀레산, 글리콜산, 락트산 또는 락테이트, 감마-히드록시부티르산, 3-히드록시알칸산, 알라닌, 메탄, 에탄, 프로판, 펜탄, n-헥산, 피루베이트, 아스파르테이트, 말레이트, 발린, 류신 및 그의 조합이다. 바이오연료로 사용되는 것 외에도 많은 다른 용도가 있는 에탄올의 생성이 특히 바람직하다.
따라서, 상술된 방법에서 발효는 에탄올, 메탄올, 프로판올, 이소프로판올, 부탄올, 에틸렌 글리콜, 프로필렌 글리콜, 1,4-부탄디올, 글리세린, 포름산, 아세트산, 프로피온산, 부티르산, 발레르산, 카프로산, 팔미트산, 스테아르산, 옥살산, 말론산, 숙신산 또는 숙시네이트, 글루타르산, 올레산, 리놀레산, 글리콜산, 락트산 또는 락테이트, 감마-히드록시부티르산, 3-히드록시알칸산, 알라닌, 메탄, 에탄, 프로판, 펜탄, n-헥산, 피루베이트, 아스파르테이트, 말레이트, 발린 및 류신으로부터 선택되는 하나 이상의 화합물, 바람직하게는 에탄올을 생성한다.
본 발명은 또한 바람직하게는 리그노셀룰로스 바이오매스로부터 에탄올의 생성을 위한 펜토스 당(들), 특히 자일로스의 발효를 위한 상술된 미생물의 용도에 관한 것이다.
본 발명에 따른 미생물은 자일로스와 같은 펜토스 당(들)을 대규모로 효율적으로 발효시킬 수 있으므로 원하는 대사 대사물의 산업적 생성을 가능하게 한다. 유리하게는, 리그노셀룰로스 바이오매스로부터 에탄올을 생성하는데 사용될 수 있다.
또한, 본 발명은 상술된 임의의 발현 구축물(들)로 사카로마이세스 세레비지애 균주를 형질전환시키는 단계를 포함하는, 상술된 미생물을 생성하는 방법에 관한 것이다. 특히, 사카로마이세스 세레비지애 균주는
i) 자일룰로스 키나제 (XKS1), 트랜스알돌라제 (TAL1), 트랜스케톨라제 1 (TKL1) 및 트랜스케톨라제 2 (TKL2)를 코딩하는 천연 유전자의 과다발현 및
ii) 자일로스 이소머라제 (XI)를 코딩하는 기능적 이종 유전자의 발현 - 여기서 자일로스 이소머라제 (XI) 유전자는 티. 네아폴리타나, 에이. 안덴시스 및 씨. 클라리플라붐으로 이루어진 군으로부터 선택된 미생물로부터 유래됨 -
을 위한 발현 구축물(들)로 형질전환된다.
에이. 안덴시스로부터 유래되는 XI 유전자가 특히 바람직하다.
각각의 유전자 및 서열과 관련하여, 이전 설명이 그에 따라 적용된다.
발현 구축물은 플라스미드상에서 세포로 전달될 수 있고 플라스미드로부터 발현되거나 사카로마이세스 세레비지애 균주의 게놈으로 통합될 수 있다. 통상의 기술자는 적합한 형질전환 방법 및 관련 이점을 잘 알고 있다.
바람직한 실시양태에 따라, 발현 구축물은 사카로마이세스 세레비지애 균주의 염색체, 바람직하게는 16번 염색체에 통합된다. 16번 염색체에서 통합 부위를 표적화하는 PPP 경로의 바람직한 어셈블리는 도 4에 나타나 있다. 이 통합 부위가 이종 유전자에 대해 가장 높은 발현을 발생시켰다는 것이 문헌 (Flagfeldt, D.B., Siewers, V., Huang, L. and Nielsen, J. in "Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae". Yeast, 26 (10), 545-551, 2009)에 의해 입증되었다. 도 4에 나타난 어셈블리에서, 카나마이신 내성 마커는 XI 통합 모듈로 대체될 수 있다.
추가의 바람직한 실시 양태에서, 상술된 자일로스 이소머라제 (XI)를 대한 발현 구축물은 자일룰로스 키나제 (XKS1), 트랜스알돌라제 (TAL1), 트랜스케톨라제 1 (TKL1) 및 트랜스케톨라제 2 (TKL2)에 대한 천연 유전자의 과다발현을 위한 재조합 발현 구축물에 통합된다.
유리하게는, 이 실시양태는 한 단계의 형질전환을 가능하게 하는 모든 필요한 유전자를 함유하는 발현 구축물을 생성한다. 이러한 발현 구축물의 특히 바람직한 실시양태는 다음 실시예에서 설명된다. 그러나, 통상의 기술자에 의해 인식될 수 있는 바와 같이, 상기 개시내용에 따른 변형이 가능하다.
도 1은 탄소원으로 자일로스를 소비하는 2가지 경로를 나타낸다. 자일로스는 자일로스 리덕타제 및 자일리톨 데히드로게나제 (A) 또는 자일로스 이소머라제 (B)에 의해 자일룰로스로 전환된다. 두 경로 모두 ATP를 소비하지만 자일로스 리덕타제 및 자일리톨 데히드로게나제도 NADPH를 활용하고 NADH를 생성하여 보조인자 불균형을 유발한다 (참조 8).
도 2는 사카로마이세스 세레비지애에서 펜토스 포스페이트 경로의 도식적 개요를 나타낸다.
도 3은 XKS1, TAL1, TKL1 및 TKL2의 과다발현을 위해 생성된 발현 카세트 단편을 나타낸다.
도 4는 사카로마이세스 실험실 균주 SEY6210의 16번 염색체에서 통합 부위를 표적화하는 KanR 선별 마커를 갖는 펜토스 포스페이트 경로 (PPP) 어셈블리를 나타낸다.
도 5는 RT-qPCR에 의한 XKS 및 펜토스 포스페이트 경로 (PPP) 발현을 나타낸다. SEY6210은 모 사카로마이세스 실험실 균주이며; CJY21 및 CJY22는 SEY6210을 XKS-PPP 과다발현 모듈로 형질전환함으로써 단리된 2개의 동질유전자계열 클론이다.
도 6은 말단절단된 HXT7 프로모터 하에서 자일로스 이소머라제의 과다발현을 위해 생성된 유전자 발현 카세트를 나타낸다.
도 7은 KanR 내성 마커를 후보 자일로스 이소머라제 모듈의 단일 카피로 대체하기 위해 생성된 통합 카세트를 나타낸다.
도 8은 37℃ 및 42℃에서 수행된 전체 세포 추출물을 사용한 자일로스 이소머라제 활성 검정 결과를 나타낸다.
도 9는 실시예 2에서 사용된 pRS426 발현 벡터를 나타낸다.
도 10은 30℃에서 20g/L 자일로스를 갖는 YEP 배지를 사용하고 200rpm에서 진탕하는 자일로스 상에서의 (형질전환된) 균주의 호기성 성장을 나타낸다.
도 11은 30℃에서 20g/L 자일로스를 갖는 YEP 배지를 사용하고 200rpm에서 진탕하는 자일로스 상에서의 (형질전환된) 균주의 혐기성 성장을 나타낸다.
도 12는 30℃에서 20g/L 자일로스를 갖는 YEP 배지를 사용하고 200rpm에서 진탕하는 호기성 (왼쪽 컬럼) 및 혐기성 (오른쪽 컬럼) 발효에서의 자일로스 소비를 나타낸다.
도 13은 30℃에서 20g/L 자일로스를 갖는 YEP 배지를 사용하고 200rpm에서 진탕하는 호기성 (왼쪽 컬럼) 및 혐기성 (오른쪽 컬럼) 발효에서의 에탄올 생성을 나타낸다.
도 2는 사카로마이세스 세레비지애에서 펜토스 포스페이트 경로의 도식적 개요를 나타낸다.
도 3은 XKS1, TAL1, TKL1 및 TKL2의 과다발현을 위해 생성된 발현 카세트 단편을 나타낸다.
도 4는 사카로마이세스 실험실 균주 SEY6210의 16번 염색체에서 통합 부위를 표적화하는 KanR 선별 마커를 갖는 펜토스 포스페이트 경로 (PPP) 어셈블리를 나타낸다.
도 5는 RT-qPCR에 의한 XKS 및 펜토스 포스페이트 경로 (PPP) 발현을 나타낸다. SEY6210은 모 사카로마이세스 실험실 균주이며; CJY21 및 CJY22는 SEY6210을 XKS-PPP 과다발현 모듈로 형질전환함으로써 단리된 2개의 동질유전자계열 클론이다.
도 6은 말단절단된 HXT7 프로모터 하에서 자일로스 이소머라제의 과다발현을 위해 생성된 유전자 발현 카세트를 나타낸다.
도 7은 KanR 내성 마커를 후보 자일로스 이소머라제 모듈의 단일 카피로 대체하기 위해 생성된 통합 카세트를 나타낸다.
도 8은 37℃ 및 42℃에서 수행된 전체 세포 추출물을 사용한 자일로스 이소머라제 활성 검정 결과를 나타낸다.
도 9는 실시예 2에서 사용된 pRS426 발현 벡터를 나타낸다.
도 10은 30℃에서 20g/L 자일로스를 갖는 YEP 배지를 사용하고 200rpm에서 진탕하는 자일로스 상에서의 (형질전환된) 균주의 호기성 성장을 나타낸다.
도 11은 30℃에서 20g/L 자일로스를 갖는 YEP 배지를 사용하고 200rpm에서 진탕하는 자일로스 상에서의 (형질전환된) 균주의 혐기성 성장을 나타낸다.
도 12는 30℃에서 20g/L 자일로스를 갖는 YEP 배지를 사용하고 200rpm에서 진탕하는 호기성 (왼쪽 컬럼) 및 혐기성 (오른쪽 컬럼) 발효에서의 자일로스 소비를 나타낸다.
도 13은 30℃에서 20g/L 자일로스를 갖는 YEP 배지를 사용하고 200rpm에서 진탕하는 호기성 (왼쪽 컬럼) 및 혐기성 (오른쪽 컬럼) 발효에서의 에탄올 생성을 나타낸다.
실시예 1: XKS-PPP 발현 모듈의 구축
사카로마이세스 시험 균주는 효모 펜토스 포스페이트 경로를 과다발현하도록 조작되었다. 유전자 발현 카세트 단편은 TDH3 프로모터 하에서 XKS1, PGK1 프로모터 하에서 TAL1, CYC19 프로모터 하에서 TKL1 및 PFK1 프로모터 하에서 TKL2의 과다발현을 위해 생성되었다 (도 3).
XKS 발현 모듈의 구축: TDH3 프로모터는 서열식별번호: 29 및 서열식별번호: 30에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. XKS1 유전자의 코딩 영역은 서열식별번호: 31 및 서열식별번호: 32에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. DIT1 터미네이터는 서열식별번호: 33 및 서열식별번호: 34에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. 이어서, PCR 생성물은 컬럼 정제되고 깁슨 (Gibson) 등온 어셈블리 (참조 1)에 의해 SmaI-선형화된 pRS426 (참조 2) 벡터로 어셈블리되었다.
TAL1 발현 모듈의 구축: PGK1 프로모터는 서열식별번호: 35 및 서열식별번호: 36에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. TAL1 유전자의 코딩 영역은 서열식별번호: 37 및 서열식별번호: 38에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. YHI9 터미네이터는 서열식별번호: 39 및 서열식별번호: 40에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. 이어서, PCR 생성물은 컬럼 정제되고 깁슨 등온 어셈블리 (참조 1)에 의해 SmaI-선형화된 pRS426 (참조 2) 벡터로 어셈블리되었다.
TKL1 발현 모듈의 구축: CYC19 프로모터는 서열식별번호: 41 및 서열식별번호: 42에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. TKL1 유전자의 코딩 영역은 서열식별번호: 43 및 서열식별번호: 44에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. EFM1 터미네이터는 서열식별번호: 45 및 서열식별번호: 46에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. 이어서, PCR 생성물은 컬럼 정제되고 깁슨 등온 어셈블리 (참조 1)에 의해 SmaI-선형화된 pRS426 (참조 2) 벡터로 어셈블리되었다.
TKL2 발현 모듈의 구축: PFK1 프로모터는 서열식별번호: 47 및 서열식별번호: 48에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. TKL2 유전자의 코딩 영역은 서열식별번호: 49 및 서열식별번호: 50에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. RPL15A 터미네이터는 서열식별번호: 51 및 서열식별번호: 52에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 PCR 증폭되었다. 이어서, PCR 생성물은 컬럼 정제되고 깁슨 등온 어셈블리 (참조 1)에 의해 SmaI-선형화된 pRS426 (참조 2) 벡터로 어셈블리되었다.
TKL1-XKS1 발현 모듈의 구축: TKL1 카세트는 서열식별번호: 53 및 서열식별번호: 54에 의해 식별되는 프라이머를 사용하여 TKL1 발현 모듈을 함유하는 pRS426 벡터로부터 PCR 증폭되었다. XKS1 카세트는 서열식별번호: 55 및 서열식별번호: 56에 의해 식별되는 프라이머를 사용하여 XKS1 발현 모듈을 함유하는 pRS426 벡터로부터 PCR 증폭되었다. 이어서, PCR 생성물은 컬럼 정제되고 깁슨 등온 어셈블리 (참조 1)에 의해 SmaI-선형화된 pRS426 (참조 2) 벡터로 어셈블리되었다.
TKL2-TAL1 발현 모듈의 구축: TKL2 카세트는 서열식별번호: 59 및 서열식별번호: 60에 의해 식별되는 프라이머를 사용하여 TKL2 발현 모듈을 함유하는 pRS426 벡터로부터 PCR 증폭되었다. TAL1 카세트는 서열식별번호: 57 및 서열식별번호: 58에 의해 식별되는 프라이머를 사용하여 TAL1 발현 모듈을 함유하는 pRS426 벡터로부터 PCR 증폭되었다. 이어서, PCR 생성물은 컬럼 정제되고 깁슨 등온 어셈블리 (참조 1)에 의해 SmaI-선형화된 pRS426 (참조 2) 벡터로 어셈블리되었다.
XKS-PPP 발현 모듈의 구축: The TKL1-XKS1 카세트는 서열식별번호: 61 및 서열식별번호: 62에 의해 식별되는 프라이머를 사용하여 TKL1-XKS1 발현 모듈을 함유하는 pRS426 벡터로부터 PCR 증폭되었다. KanR 선별 마커 (서열식별번호: 86)는 서열식별번호: 63 및 서열식별번호: 64에 의해 식별되는 프라이머를 사용하여 pRS42K (참조 1)로부터 PCR 증폭되었다. TAL1-TKL2 카세트는 서열식별번호: 65 및 서열식별번호: 66에 의해 식별되는 프라이머를 사용하여 TAL1-TKL2 발현 모듈을 함유하는 pRS426 벡터로부터 PCR 증폭되었다. 이어서, PCR 생성물은 컬럼 정제되고 깁슨 등온 어셈블리 (참조 1)에 의해 SmaI-선형화된 pRS426 (참조 2) 벡터로 어셈블리되었다.
XKS-PPP 16번 염색체 통합 모듈의 구축: 사카로마이세스 실험실 균주 SEY6210의 16번 염색체를 표적화하는 5' 상동성 아암, CHR16-업 (서열식별번호: 88)은 서열식별번호: 69 및 서열식별번호: 70에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 증폭되었다. PPP-KanR 모듈은 서열식별번호: 67 및 서열식별번호: 68에 의해 식별되는 프라이머를 사용하여 PPP-KanR을 함유하는 pRS426 벡터로부터 증폭되었다. 사카로마이세스 실험실 균주 SEY6210의 16번 염색체를 표적화하는 3' 상동성 아암, CHR16-다운 (서열식별번호: 89)은 서열식별번호: 71 및 서열식별번호: 72에 의해 식별되는 프라이머를 사용하여 SEY6210 게놈 DNA로부터 증폭되었다. PCR 생성물을 컬럼 정제하고 깁슨 등온 어셈블리 (참조 1)에 의해 SmaI-선형화된 pRS426 (참조 2) 벡터로 어셈블리하였다. 상동성 아암, PPP 및 KanR 마커를 함유하는 통합 카세트를 BamHI 및 SalI으로 플라스미드 백본에서 분리하여 선형 재조합 카세트를 생성한 후 (도 4) SEY6210으로 형질전환하였다. 형질전환체를 스크리닝하여 동질유전자계열 클론 CJY21 및 CJY22가 생성되었다. XKS 및 PPP 유전자의 과다발현은 RT-qPCR에 의해 확인되었다 (도 5).
실시예 2: XI 후보 스크리닝
총 14개의 자일로스 이소머라제 효소 후보는 뉴클레오티드 서열식별번호: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 및 27로부터 단백질 서열 서열식별번호: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 및 28로 판독되며, 사카로마이세스에 코돈-최적화되고, 합성된다 (IDT). 이어서, 이들 합성된 후보 유전자는 말단절단된 구성적 HXT77-391 (참조 3) 프로모터 (서열식별번호: 90) 및 에스. 세레비지애로부터의 CYC1 터미네이터 (서열식별번호: 91)의 제어 하에 pRS4272로 클로닝된다 (도 6).
HXT7 프로모터-XI 유전자-CYC1 터미네이터 카세트는 PPP 카세트의 KANR 마커의 5' 말단에 있는 TEF 프로모터와 상동성인 TEF 프로모터 (서열식별번호: 87), NATR 내성 마커 (참조 4) (서열식별번호: 93), ADH1 터미네이터 (서열식별번호: 94) 및 PPP 카세트의 KANR 마커의 3' 말단과 상동성인 표적-다운 서열 (서열식별번호: 92)을 갖는 통합 벡터에 추가로 서브클로닝되었다 (도 7). 이어서, 통합 모듈은 CJY21로 형질전환되어 KANR 내성 마커를 후보 자일로스 이소머라제 모듈의 단일 카피로 대체하였다.
자일로스 이소머라제 후보는 시험관내에서 효소 활성에 대해 검정되었다. 균주를 5 ml의 YPD에서 밤새 성장시키고, 수거하고, 세척하고, XI 검정 용해 완충제 (50 mM 트리스 pH 7.5, 150 mM NaCl, 0.01% 트리톤 X-100, 10 mM MgCl2, 50 μM CoCl2, 50 μM MnCl2, 피어스 (Pierce) 프로테아제 억제제 [피어스 88666] 포함)에서 기계적 비드 비팅 (MP 바이오메디컬 패스트프렙(MP Biomedical Fastprep))에 의해 용해시켰다. 단백질 농도는 브래드퍼드 (Bradford) 검정 (참조 5)에 의해 결정되었다. 50 μl의 정화된 전체-세포 추출물 (WCE)을 50 μl의 100 mM D-자일로스와 함께 각각 37℃ 및 42℃에서 16시간 동안 인큐베이션한 다음, 95℃로 5분 동안 가열하여 중단시키고 원심분리로 제거하였다.
자일룰로스의 정량화는 소르비톨 데히드로게나제 (SDH)-기반의 NADH-연결된 검정 (참조 6)에 의해 수행되었다. 96-웰 플레이트 (코닝 (Corning) # 3635)에서 SDH 완충제 (메가짐스 (Megazymes)) 및 150 μM NADH를 총 부피 200 μl로 10 μl의 검정된 당 용액과 조합하고, 혼합한 다음, A340에 대해 스캐닝하였다. 3.5 μl의 SDH 용액 (메가짐스)을 첨가하고, 혼합하고, 실온에서 15분 동안 인큐베이션하였다. 이어서, 플레이트를 A340에 대해 다시 스캐닝하였다. 검정 용액의 NADH 농도를 A340 (6220 M-1 cm-1) 및 경로 길이 0.58 cm에서 흡광 계수를 사용하여 결정한 다음, 자일룰로스 농도를 계산하는데 사용하였다. 이어서, D-자일로스의 자일룰로스로의 효소적 전환에 대한 자일로스 이소머라제 반응 속도 [μmole/min/mg WCE]를 계산하였다. 3개의 후보를 사카로마이세스에서 발현 시 시험관내 자일로스 이소머라제 활성으로 식별하였다 (도 8).
이어서, 티. 네아폴리타나 (서열식별번호: 22), 에이. 안덴시스 (서열식별번호: 6) 및 씨. 클라리플라붐 (서열식별번호: 26)으로부터의 초기 스크린에서 강력한 활성을 나타내는 XI 후보는 강력한 구성적 pTEF 프로모터 (서열식별번호: 85)의 제어 하에 pRS426 (도 9)으로 서브클로닝되었다.
이어서, 이들 플라스미드는 CJY21로 형질전환되고, 성장은 YEP + 20g/l D-자일로스에서 호기성 진탕 플라스크 (도 10) 및 혐기성 가압 병 (도 11)에서 600 nm에서의 흡광도를 측정함으로써 결정되었다. 자일로스 소비 및 에탄올 형성은 또한 48시간의 발효 시간에서 HPLC로 모니터링되었다 (에이. 안덴시스의 경우, 120시간 샘플도 에탄올 생성에 대해 측정됨) (도 12 및 13).
티. 네아폴리타나 (서열식별번호: 22), 에이. 안덴시스 (서열식별번호: 6) 및 씨. 클라리플라붐 (서열식별번호: 26)으로부터의 XI를 발현하는 pRS426 발현 벡터를 함유하는 균주 CJY21에 대한 효소 동역학을 30℃에서 30분 반응에서 50 mM, 25 mM, 5 mM 및 1 mM의 D-자일로스 농도를 사용하여 상기와 같이 반복하였다. Vmax는 미하엘리스-멘텐 (Michaelis-Menten) 운동 방정식을 사용하여 계산되었으며, 문헌으로부터 취한 피로마이세스 종 및 클로스트리듐 피토페르멘타스 (Clostridium phytofermentas) 자일로스 이소머라제에 대한 참조 XI 활성과 함께 표 1에 나타낸다.
<표 1>
참조문헌
SEQUENCE LISTING
<110> BASF SE
<120> Xylose metabolizing yeast
<130> PF170268
<160> 94
<170> PatentIn version 3.5
<210> 1
<211> 1320
<212> DNA
<213> Thermonanaerobacterium xylanolyticum
<400> 1
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ccttattctt ttaaatttta caatcctgag gaagtaatcg atggtaagac gatggaggag 120
catcttcgct tttctatagc ttattggcac acttttactg ctgatggaac agatcaattt 180
ggcaaagcta ccatgcaaag gccatggaat cactatacag atcctatgga catagctaaa 240
gcaagggtag aggcagcatt tgagtttttt gataagataa atgcaccgta tttctgcttc 300
catgatagag atattgcccc tgaaggagac actcttagag agacgaacaa aaatttagat 360
acaatagttg ctatgataaa ggattacttg aagaccagca agacgaaagt tttgtggggt 420
actgcgaatc ttttctccaa tccaagattt gtgcatggtg catcaacgtc ttgcaatgcc 480
gatgttttcg catattctgc atcacaagtc aaaaaagcac ttgagattac taaggagctt 540
ggtggcgaaa actacgtatt ctggggtgga agagaaggat atgagacact tctcaataca 600
gatatggagt ttgagcttga taattttgca agatttttgc acatggctgt tgattatgca 660
aaggaaatcg gctttgaagg ccagctcttg attgagccga agccaaagga gcctacaaag 720
catcaatacg actttgacgt ggcaaatgta ttggcattct tgagaaaata cgatcttgac 780
aaatatttca aagttaatat cgaagcaaat catgcaacat tagcattcca tgatttccag 840
catgagctaa gatacgccag aataaacggt gtattaggat cgattgacgc aaatacaggt 900
gatatgctat taggctggga tacagatcag ttccctacag atatacgcat gacaacactt 960
gctatgtatg aagtcataaa gatgggcgga tttgacaaag gtggactcaa tttcgatgcg 1020
aaagtaagac gtgcttcatt tgagccagaa gatcttttct tgggtcacat agcaggaatg 1080
gatgcttttg caaaaggctt caaagtggct tacaagcttg taaaagatgg cgtttttgac 1140
aagttcatcg aggaaagata tgcaagctac aaagatggca taggtgcaga tattgtaagt 1200
ggaaaagctg attttagaag ccttgagaag tacgcattag agcacagcca gattgtcaac 1260
aaatcaggaa gacaagagct attagaatca atcctaaatc agtatttgtt tgcagaataa 1320
<210> 2
<211> 439
<212> PRT
<213> Thermonanaerobacterium xylanolyticum
<400> 2
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Tyr Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ser Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Phe Glu Leu Asp Asn
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Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
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Phe Glu Gly Gln Leu Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
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His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
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Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
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Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
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Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
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Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
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Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
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Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
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Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
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Glu Arg Tyr Ala Ser Tyr Lys Asp Gly Ile Gly Ala Asp Ile Val Ser
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Gly Lys Ala Asp Phe Arg Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
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Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
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Asn Gln Tyr Leu Phe Ala Glu
435
<210> 3
<211> 1320
<212> DNA
<213> Pseudobacteroides cellulosolvens
<400> 3
atgtcagaat tttttagtaa tgtttcaaag attcaatatg aaggtaagaa ctctgataat 60
ccattggctt ttaagtatta taacccagat gaggttatag gcggaaagac aatgaaggat 120
catttgagat tcgcagttgc ttactggcat acattccagg gaacaggcgg agatccattc 180
ggacctggta cagcagtaag accatgggac aatataacag atccaatgga acttgctaaa 240
gcaaaagtag ctgcaaactt cgagttctgt gaaaaattag gtgtaccgtt ctactgtttc 300
catgacaggg atatagcacc tgaagcttca actcttagag aaacaaataa gagacttgat 360
gaaatagttg ctcttatgaa ggaatatatg aaaaccagca gtgttaagct cctctgggga 420
actacaaatg cattcggtaa cccaagattt gtacacggtg cttcaacatc accaaatgcc 480
gatgtttttg catttgcagc agctcaggtt aaaaaagcaa tggaaataac tttagagctt 540
ggcggacaga actatgtatt ctggggtgga agagaaggtt atgagaccct attaaacaca 600
gacatgaagc ttgagcttga caacatggga agattcttaa gaatggctgt tgattacgca 660
aaagaaatag gctttaaagg acaattcctc attgaaccaa agccgaagga acctacaaaa 720
caccagtatg atttcgatac agctacagtt gttggtttct taagagctca tggtcttgaa 780
aacgatttca agatgaacat agaagcaaac catgctaccc ttgctgctca taccttccag 840
catgaagtat atactgcaag agtaaacaat gtattcggaa gtattgatgc aaatcaggga 900
gacttgctct taggatggga tacagaccaa ttcccaacta atatttatga tacaacactt 960
tgcatgtatg aagttcttaa agcaggcggt ttcacaaccg gcggattaaa cttcgactct 1020
aaagtaagaa gaggttcatt tgagccaatc gatcttttct atgcacatat tgcaggaatg 1080
gatgcttttg ctaagggtct taagattgct tacaagatgg tttcagaagg caagttcgat 1140
aaagttattg aagaccgtta tgcaagctac aaaagcggta ttggtagcga tatagttaat 1200
ggaaaagttg gatttaaaga attggaaaaa tatgcattgg agcatgatca ggttaagaac 1260
gtatcaggaa gacaggaagt tcttgaaagc atgctgaaca agtatatttt agaagattaa 1320
<210> 4
<211> 439
<212> PRT
<213> Pseudobacteroides cellulosolvens
<400> 4
Met Ser Glu Phe Phe Ser Asn Val Ser Lys Ile Gln Tyr Glu Gly Lys
1 5 10 15
Asn Ser Asp Asn Pro Leu Ala Phe Lys Tyr Tyr Asn Pro Asp Glu Val
20 25 30
Ile Gly Gly Lys Thr Met Lys Asp His Leu Arg Phe Ala Val Ala Tyr
35 40 45
Trp His Thr Phe Gln Gly Thr Gly Gly Asp Pro Phe Gly Pro Gly Thr
50 55 60
Ala Val Arg Pro Trp Asp Asn Ile Thr Asp Pro Met Glu Leu Ala Lys
65 70 75 80
Ala Lys Val Ala Ala Asn Phe Glu Phe Cys Glu Lys Leu Gly Val Pro
85 90 95
Phe Tyr Cys Phe His Asp Arg Asp Ile Ala Pro Glu Ala Ser Thr Leu
100 105 110
Arg Glu Thr Asn Lys Arg Leu Asp Glu Ile Val Ala Leu Met Lys Glu
115 120 125
Tyr Met Lys Thr Ser Ser Val Lys Leu Leu Trp Gly Thr Thr Asn Ala
130 135 140
Phe Gly Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Pro Asn Ala
145 150 155 160
Asp Val Phe Ala Phe Ala Ala Ala Gln Val Lys Lys Ala Met Glu Ile
165 170 175
Thr Leu Glu Leu Gly Gly Gln Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Lys Leu Glu Leu Asp Asn
195 200 205
Met Gly Arg Phe Leu Arg Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Lys Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Thr Ala Thr Val Val Gly Phe Leu Arg Ala
245 250 255
His Gly Leu Glu Asn Asp Phe Lys Met Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Ala His Thr Phe Gln His Glu Val Tyr Thr Ala Arg Val
275 280 285
Asn Asn Val Phe Gly Ser Ile Asp Ala Asn Gln Gly Asp Leu Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp Thr Thr Leu
305 310 315 320
Cys Met Tyr Glu Val Leu Lys Ala Gly Gly Phe Thr Thr Gly Gly Leu
325 330 335
Asn Phe Asp Ser Lys Val Arg Arg Gly Ser Phe Glu Pro Ile Asp Leu
340 345 350
Phe Tyr Ala His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Leu Lys
355 360 365
Ile Ala Tyr Lys Met Val Ser Glu Gly Lys Phe Asp Lys Val Ile Glu
370 375 380
Asp Arg Tyr Ala Ser Tyr Lys Ser Gly Ile Gly Ser Asp Ile Val Asn
385 390 395 400
Gly Lys Val Gly Phe Lys Glu Leu Glu Lys Tyr Ala Leu Glu His Asp
405 410 415
Gln Val Lys Asn Val Ser Gly Arg Gln Glu Val Leu Glu Ser Met Leu
420 425 430
Asn Lys Tyr Ile Leu Glu Asp
435
<210> 5
<211> 770
<212> DNA
<213> Anditalea andensis
<400> 5
atgtctaaaa cctattttcc atcaattgaa aaaattaaat tcgaaggaag ggattccaaa 60
aatccttttg ctttcaaatt ttacgatgaa aaccgtgtag tagggggtaa aagcatgaag 120
gagcacttca agtttgccat cgcatactgg cattcattca atgccaaagg ggatgatcct 180
tttggtccag gaaccaaaac ttttgaatgg gatgagtcat ccgatgctgt tcagagagcc 240
aaagataaaa tggatgctgc atttgaattt attcaaaaga taggagcacc atattactgc 300
ttccatgatg tggatctggt agatgaaggt gattctatag aggaatatga aagaaggatg 360
aaggccatag tcgagtatgc taagcaaaag cagcaagata ctggtatcaa gcttctttgg 420
ggcacagcca atgttttcag taacccacgt tatatgaatg gtgcttcgac taaccccgat 480
tttaatgtag tttcatgggc agctactcaa gttaagaatt ctattgatgc tactatagcc 540
ctaggtgggg aaaactatgt attctggggt ggaagagaag gatatatgtc tttactcaat 600
accgatatga aacgagaaac agaacattta gctcagtttc ttaccatggc gcgggattat 660
gcgcgtcagc agggttttaa aggtaatttc cttatagagc caaaaccaat ggagcctacc 720
aaacaccagt atgatttcga ttctgctacg gtagccggtt ttctaagact 770
<210> 6
<211> 437
<212> PRT
<213> Anditalea andensis
<400> 6
Met Ser Lys Thr Tyr Phe Pro Ser Ile Glu Lys Ile Lys Phe Glu Gly
1 5 10 15
Arg Asp Ser Lys Asn Pro Phe Ala Phe Lys Phe Tyr Asp Glu Asn Arg
20 25 30
Val Val Gly Gly Lys Ser Met Lys Glu His Phe Lys Phe Ala Ile Ala
35 40 45
Tyr Trp His Ser Phe Asn Ala Lys Gly Asp Asp Pro Phe Gly Pro Gly
50 55 60
Thr Lys Thr Phe Glu Trp Asp Glu Ser Ser Asp Ala Val Gln Arg Ala
65 70 75 80
Lys Asp Lys Met Asp Ala Ala Phe Glu Phe Ile Gln Lys Ile Gly Ala
85 90 95
Pro Tyr Tyr Cys Phe His Asp Val Asp Leu Val Asp Glu Gly Asp Ser
100 105 110
Ile Glu Glu Tyr Glu Arg Arg Met Lys Ala Ile Val Glu Tyr Ala Lys
115 120 125
Gln Lys Gln Gln Asp Thr Gly Ile Lys Leu Leu Trp Gly Thr Ala Asn
130 135 140
Val Phe Ser Asn Pro Arg Tyr Met Asn Gly Ala Ser Thr Asn Pro Asp
145 150 155 160
Phe Asn Val Val Ser Trp Ala Ala Thr Gln Val Lys Asn Ser Ile Asp
165 170 175
Ala Thr Ile Ala Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly Gly Arg
180 185 190
Glu Gly Tyr Met Ser Leu Leu Asn Thr Asp Met Lys Arg Glu Thr Glu
195 200 205
His Leu Ala Gln Phe Leu Thr Met Ala Arg Asp Tyr Ala Arg Gln Gln
210 215 220
Gly Phe Lys Gly Asn Phe Leu Ile Glu Pro Lys Pro Met Glu Pro Thr
225 230 235 240
Lys His Gln Tyr Asp Phe Asp Ser Ala Thr Val Ala Gly Phe Leu Arg
245 250 255
Leu Tyr Gly Leu Asp Lys Asp Phe Lys Leu Asn Ile Glu Val Asn His
260 265 270
Ala Thr Leu Ala Gly His Thr Phe Gln His Glu Leu Gln Val Ala Ala
275 280 285
Asp Ala Gly Met Leu Gly Ser Ile Asp Ala Asn Arg Gly Asp Tyr Gln
290 295 300
Asn Gly Trp Asp Thr Asp Gln Phe Ala Leu Asn Leu Gln Glu Leu Thr
305 310 315 320
Glu Ala Met Leu Val Ile Leu Glu Ala Gly Gly Ile Gln Gly Gly Gly
325 330 335
Val Asn Phe Asp Ala Lys Leu Arg Arg Asn Ser Thr Asp Leu Glu Asp
340 345 350
Leu Phe His Ala His Ile Gly Ser Met Asp Ala Phe Ala Arg Ala Leu
355 360 365
Leu Ile Ala Gln Asp Ile Leu Asp Asn Ser Asp Tyr Arg Ala Met Arg
370 375 380
Lys Ala Arg Tyr Ala Ser Phe Asp Glu Gly Lys Gly Lys Glu Phe Glu
385 390 395 400
Ser Gly Lys Leu Thr Leu Glu Asp Leu Arg Glu His Ala Leu Ala Thr
405 410 415
Gly Glu Pro Lys Ser Ile Ser Gly Arg Gln Glu Met Tyr Glu Asn Leu
420 425 430
Leu Asn Gln Phe Ile
435
<210> 7
<211> 1317
<212> DNA
<213> Algibacter lectus
<400> 7
atggctacta aagaatattt taaaggcatt agcaacatta aatttgaagg taaggaatct 60
gataatccat tagcattcaa atattacaat ccggaccagg ttgtagcagg aaaaaccatg 120
aaagaatggt ttaaattttc aattgcttac tggcatacat tctgtgggca aggtggagat 180
ccatttggtc caggaacaca aagttttgag tgggataaat catcagatcc aattcaagcg 240
gcaaaagata aagccgatgc agctttcgaa tttattggaa aaatgggatt cgattatttc 300
tgtttccacg atttcgattt aattcaagaa ggtgcaacat ttgcagagtc agaaagtaga 360
ttagagacta tcacagatta cataaaaggt aaacaagccg aaagtggtgt aaaattactt 420
tggggaacag caaactgttt ttctaaccca cgttacatga atggtgcttc tacaaatcca 480
gatttcgatg tggtagctag agcaggcgga caagtaaaat tagctttaga tgcgactatt 540
aaattaggtg gtgaaaacta cgtattctgg ggaggtcgtg aaggttacat gtctttatta 600
aatactgata tggggcgtga attagatcac atggggcaat ttttaaccat ggctagagat 660
tacgcaagag ctcaaggttt taaaggaaac ttttttatcg agcctaagcc aatggagcca 720
tctaaacacc aatacgattt cgattcggct acagctatcg gtttcttaag agaatatggt 780
ttagataaag atttcaaaat aaacatagaa gtaaaccatg ctacattagc acaacatacg 840
ttccaacacg aaattgaaac ggctgcaaaa gctggtatgt taggtagctt agatgctaac 900
cgtggcgatt accaaaatgg ttgggatacc gatcaattcc caaacaatat tcaagaaaca 960
acagaagcta tgttagtttt catgaaagct ggtggtttac aaggtggtgg tgttaatttc 1020
gatgctaaaa ttagaagaaa ctcaaccgat ttagacgatg ttttccatgc acatattggt 1080
ggagcagata cttttgctag agcattatta acagccgata aaattattac agattcagct 1140
tacgataaat tacgtaaaga gcgttacagt tctttcgatg ctggaaaagg taaagatttt 1200
gaagctggta aattaaactt acaagatttg tataaaattg ctcaagataa tggtgaactg 1260
caattacaaa gcggtaagca agaattgttt gagaatatta tcaatcagta tatctag 1317
<210> 8
<211> 438
<212> PRT
<213> Algibacter lectus
<400> 8
Met Ala Thr Lys Glu Tyr Phe Lys Gly Ile Ser Asn Ile Lys Phe Glu
1 5 10 15
Gly Lys Glu Ser Asp Asn Pro Leu Ala Phe Lys Tyr Tyr Asn Pro Asp
20 25 30
Gln Val Val Ala Gly Lys Thr Met Lys Glu Trp Phe Lys Phe Ser Ile
35 40 45
Ala Tyr Trp His Thr Phe Cys Gly Gln Gly Gly Asp Pro Phe Gly Pro
50 55 60
Gly Thr Gln Ser Phe Glu Trp Asp Lys Ser Ser Asp Pro Ile Gln Ala
65 70 75 80
Ala Lys Asp Lys Ala Asp Ala Ala Phe Glu Phe Ile Gly Lys Met Gly
85 90 95
Phe Asp Tyr Phe Cys Phe His Asp Phe Asp Leu Ile Gln Glu Gly Ala
100 105 110
Thr Phe Ala Glu Ser Glu Ser Arg Leu Glu Thr Ile Thr Asp Tyr Ile
115 120 125
Lys Gly Lys Gln Ala Glu Ser Gly Val Lys Leu Leu Trp Gly Thr Ala
130 135 140
Asn Cys Phe Ser Asn Pro Arg Tyr Met Asn Gly Ala Ser Thr Asn Pro
145 150 155 160
Asp Phe Asp Val Val Ala Arg Ala Gly Gly Gln Val Lys Leu Ala Leu
165 170 175
Asp Ala Thr Ile Lys Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly Gly
180 185 190
Arg Glu Gly Tyr Met Ser Leu Leu Asn Thr Asp Met Gly Arg Glu Leu
195 200 205
Asp His Met Gly Gln Phe Leu Thr Met Ala Arg Asp Tyr Ala Arg Ala
210 215 220
Gln Gly Phe Lys Gly Asn Phe Phe Ile Glu Pro Lys Pro Met Glu Pro
225 230 235 240
Ser Lys His Gln Tyr Asp Phe Asp Ser Ala Thr Ala Ile Gly Phe Leu
245 250 255
Arg Glu Tyr Gly Leu Asp Lys Asp Phe Lys Ile Asn Ile Glu Val Asn
260 265 270
His Ala Thr Leu Ala Gln His Thr Phe Gln His Glu Ile Glu Thr Ala
275 280 285
Ala Lys Ala Gly Met Leu Gly Ser Leu Asp Ala Asn Arg Gly Asp Tyr
290 295 300
Gln Asn Gly Trp Asp Thr Asp Gln Phe Pro Asn Asn Ile Gln Glu Thr
305 310 315 320
Thr Glu Ala Met Leu Val Phe Met Lys Ala Gly Gly Leu Gln Gly Gly
325 330 335
Gly Val Asn Phe Asp Ala Lys Ile Arg Arg Asn Ser Thr Asp Leu Asp
340 345 350
Asp Val Phe His Ala His Ile Gly Gly Ala Asp Thr Phe Ala Arg Ala
355 360 365
Leu Leu Thr Ala Asp Lys Ile Ile Thr Asp Ser Ala Tyr Asp Lys Leu
370 375 380
Arg Lys Glu Arg Tyr Ser Ser Phe Asp Ala Gly Lys Gly Lys Asp Phe
385 390 395 400
Glu Ala Gly Lys Leu Asn Leu Gln Asp Leu Tyr Lys Ile Ala Gln Asp
405 410 415
Asn Gly Glu Leu Gln Leu Gln Ser Gly Lys Gln Glu Leu Phe Glu Asn
420 425 430
Ile Ile Asn Gln Tyr Ile
435
<210> 9
<211> 1329
<212> DNA
<213> Epilithonimonas lactis
<400> 9
atggcaatta cgacaggaaa caaagagtac tttaaaggaa tagaaaaaat caagtttgag 60
ggaagagaat cagataatcc gttggcattc aaattttacg atgagaattt ggtcgttcgt 120
ggaaaaacga tgaaagaata tttcaagttt gcatctgctt actggcatac attctgcgca 180
actggtggag acccgtttgg tgcaggaact cagcaatttg actggttaac ggcttctgat 240
gcaaaacaga gagcaacaga aaaaatggat gccgctttcg aatttttcac caaattaggt 300
gttccttact actgtttcca cgattacgat ttgattgacg aggcagataa ctttacagaa 360
tctaccaaaa gactggaatt tattactgat tacgctaaag gaaagcaggc tgcttctggt 420
gtgaaactgc tttggggaac ttccaactgt ttctcaaacc caagatttat gaacggtgcg 480
gcaaccaatc cttcatttga cgttttggcg tacgcaggtg gacaggtgaa aaatgcttta 540
gatgctacga taaaattagg tggcgaaaac tatgtattct ggggcggccg tgaaggttat 600
atgtctttat taaacacgaa tatgaagcgt gagcaagagc acatggcgaa gtttttacat 660
ttggctaaag attacgcaag agctcaggga ttcaaaggaa ctttcttcat cgagccaaaa 720
ccgatggagc ctacaaaaca ccagtacgac ttcgatgctg cgacttgttt aaatttcctt 780
cgtcagtacg atttattgaa tgattttaaa ttaaatcttg aagttaatca cgctactttg 840
gctcaacata ctttcgagca cgaacttcag gtagctgcag ataacaatgt tttgggaagc 900
attgatgcga acagaggaga ttatcaaaac ggttgggata cagatcagtt cccggttgat 960
ttgtacgaaa tgactcaggc gatgttggtg attatccagg ctggaggttt ccagggcgga 1020
ggtgttaact ttgatgcaaa aatcagaaga aactcaaccg acctggaaga tattttcatc 1080
gctcacatca gcggaatgga caattttgca agatcgttcc tggctgctga taaaatttta 1140
gaaaaatcaa aatattctga gatcagaacc aacagatatt cttcgtttga ttctggaaaa 1200
ggtaaagatt tcgaaaacgg aagcttatct ttaacagatc ttgccactta tgctcaagga 1260
ttaggtgaag ttggaagaga gagcggaaag caggaatatc ttgagagtat tattaatcag 1320
tatttataa 1329
<210> 10
<211> 442
<212> PRT
<213> Epilithonimonas lactis
<400> 10
Met Ala Ile Thr Thr Gly Asn Lys Glu Tyr Phe Lys Gly Ile Glu Lys
1 5 10 15
Ile Lys Phe Glu Gly Arg Glu Ser Asp Asn Pro Leu Ala Phe Lys Phe
20 25 30
Tyr Asp Glu Asn Leu Val Val Arg Gly Lys Thr Met Lys Glu Tyr Phe
35 40 45
Lys Phe Ala Ser Ala Tyr Trp His Thr Phe Cys Ala Thr Gly Gly Asp
50 55 60
Pro Phe Gly Ala Gly Thr Gln Gln Phe Asp Trp Leu Thr Ala Ser Asp
65 70 75 80
Ala Lys Gln Arg Ala Thr Glu Lys Met Asp Ala Ala Phe Glu Phe Phe
85 90 95
Thr Lys Leu Gly Val Pro Tyr Tyr Cys Phe His Asp Tyr Asp Leu Ile
100 105 110
Asp Glu Ala Asp Asn Phe Thr Glu Ser Thr Lys Arg Leu Glu Phe Ile
115 120 125
Thr Asp Tyr Ala Lys Gly Lys Gln Ala Ala Ser Gly Val Lys Leu Leu
130 135 140
Trp Gly Thr Ser Asn Cys Phe Ser Asn Pro Arg Phe Met Asn Gly Ala
145 150 155 160
Ala Thr Asn Pro Ser Phe Asp Val Leu Ala Tyr Ala Gly Gly Gln Val
165 170 175
Lys Asn Ala Leu Asp Ala Thr Ile Lys Leu Gly Gly Glu Asn Tyr Val
180 185 190
Phe Trp Gly Gly Arg Glu Gly Tyr Met Ser Leu Leu Asn Thr Asn Met
195 200 205
Lys Arg Glu Gln Glu His Met Ala Lys Phe Leu His Leu Ala Lys Asp
210 215 220
Tyr Ala Arg Ala Gln Gly Phe Lys Gly Thr Phe Phe Ile Glu Pro Lys
225 230 235 240
Pro Met Glu Pro Thr Lys His Gln Tyr Asp Phe Asp Ala Ala Thr Cys
245 250 255
Leu Asn Phe Leu Arg Gln Tyr Asp Leu Leu Asn Asp Phe Lys Leu Asn
260 265 270
Leu Glu Val Asn His Ala Thr Leu Ala Gln His Thr Phe Glu His Glu
275 280 285
Leu Gln Val Ala Ala Asp Asn Asn Val Leu Gly Ser Ile Asp Ala Asn
290 295 300
Arg Gly Asp Tyr Gln Asn Gly Trp Asp Thr Asp Gln Phe Pro Val Asp
305 310 315 320
Leu Tyr Glu Met Thr Gln Ala Met Leu Val Ile Ile Gln Ala Gly Gly
325 330 335
Phe Gln Gly Gly Gly Val Asn Phe Asp Ala Lys Ile Arg Arg Asn Ser
340 345 350
Thr Asp Leu Glu Asp Ile Phe Ile Ala His Ile Ser Gly Met Asp Asn
355 360 365
Phe Ala Arg Ser Phe Leu Ala Ala Asp Lys Ile Leu Glu Lys Ser Lys
370 375 380
Tyr Ser Glu Ile Arg Thr Asn Arg Tyr Ser Ser Phe Asp Ser Gly Lys
385 390 395 400
Gly Lys Asp Phe Glu Asn Gly Ser Leu Ser Leu Thr Asp Leu Ala Thr
405 410 415
Tyr Ala Gln Gly Leu Gly Glu Val Gly Arg Glu Ser Gly Lys Gln Glu
420 425 430
Tyr Leu Glu Ser Ile Ile Asn Gln Tyr Leu
435 440
<210> 11
<211> 1338
<212> DNA
<213> Xanthomonas campestris
<400> 11
atgagcaaca ccgtctacat cggcgccaaa gagtatttcc ccggcatcgg caagatcggc 60
ttcgaaggcc gcgactccga caacccgctc gcgttcaagg tttacgacgc caacaagaag 120
atcggcgcca agaccatggc cgagcacctg cgctttgccg tggcctactg gcacagcttc 180
tgcggcaacg gcgccgatcc gttcggcccg ggcacgcgtg cgtatccgtg ggatatcggc 240
aacagcgcgc tcgatcgtgc cgaggccaag gccgatgccg cgttcgaatt cttcaccaag 300
ctcggcgtgc cgtattactg ctttcacgat atcgacctgt cgccggatgc cgacgacatc 360
ggcgagtacg aaagcaacct caagcacatg gtgggcatcg ccaagcagcg ccaggccgac 420
accggcatca agctgctctg gggcaccgcc aacctgttct cgcacccgcg ctacatgaat 480
ggtgcatcga ccaacccgga cttcaatgtg gtggcgcgtg ccgcggtgca ggtcaaggcg 540
gcgatcgatg ccacggtgga actgggcggt gaaaactacg tgttctgggg cggccgcgaa 600
ggctatgcct gcctgcacaa cacgcagatg aagcgcgagc aggacaacat ggcgcgcttc 660
ctcaccctgg cacgcgacta cggccgcgcg atcggcttca aaggcaactt cctgatcgag 720
cccaagccca tggagccgat gaagcaccaa tacgacttcg acagcgccac ggtgatcggc 780
ttcctgcgtc agcatggcct ggaccaggat ttcaagctca atatcgaggc caaccacgcc 840
accctgtccg gtcacagctt cgagcacgat ctgcaggttg ccagtgatgc cggcctgctc 900
ggcagcatcg atgccaaccg cggcaacccg cagaatggct gggataccga ccagttcccg 960
accgacctgt acgacaccgt cggcgcgatg ctggtggtgc tgcgccaggg cgggctggca 1020
ccgggtggcc tgaatttcga cgccaaggtg cgccgcgagt cgtccgaccc gcaggacctg 1080
ttcctggcgc acatcggtgg catggacgcg ttcgcacgcg ggctggaagt ggccaatgcg 1140
ctgctgacgt cttcgccgct ggagacctgg cgcgccgagc gttacgccag cttcgacagc 1200
ggcgccggtg ccgactttgc caacggcacc agcacgctgg cggatctggc caagtacgcc 1260
gccggtaatg cgcccaagca actcagcggc cgtcaggaag cctacgaaaa cctgatcaat 1320
cagtatctga tccggtga 1338
<210> 12
<211> 445
<212> PRT
<213> Xanthomonas campestris
<400> 12
Met Ser Asn Thr Val Tyr Ile Gly Ala Lys Glu Tyr Phe Pro Gly Ile
1 5 10 15
Gly Lys Ile Gly Phe Glu Gly Arg Asp Ser Asp Asn Pro Leu Ala Phe
20 25 30
Lys Val Tyr Asp Ala Asn Lys Lys Ile Gly Ala Lys Thr Met Ala Glu
35 40 45
His Leu Arg Phe Ala Val Ala Tyr Trp His Ser Phe Cys Gly Asn Gly
50 55 60
Ala Asp Pro Phe Gly Pro Gly Thr Arg Ala Tyr Pro Trp Asp Ile Gly
65 70 75 80
Asn Ser Ala Leu Asp Arg Ala Glu Ala Lys Ala Asp Ala Ala Phe Glu
85 90 95
Phe Phe Thr Lys Leu Gly Val Pro Tyr Tyr Cys Phe His Asp Ile Asp
100 105 110
Leu Ser Pro Asp Ala Asp Asp Ile Gly Glu Tyr Glu Ser Asn Leu Lys
115 120 125
His Met Val Gly Ile Ala Lys Gln Arg Gln Ala Asp Thr Gly Ile Lys
130 135 140
Leu Leu Trp Gly Thr Ala Asn Leu Phe Ser His Pro Arg Tyr Met Asn
145 150 155 160
Gly Ala Ser Thr Asn Pro Asp Phe Asn Val Val Ala Arg Ala Ala Val
165 170 175
Gln Val Lys Ala Ala Ile Asp Ala Thr Val Glu Leu Gly Gly Glu Asn
180 185 190
Tyr Val Phe Trp Gly Gly Arg Glu Gly Tyr Ala Cys Leu His Asn Thr
195 200 205
Gln Met Lys Arg Glu Gln Asp Asn Met Ala Arg Phe Leu Thr Leu Ala
210 215 220
Arg Asp Tyr Gly Arg Ala Ile Gly Phe Lys Gly Asn Phe Leu Ile Glu
225 230 235 240
Pro Lys Pro Met Glu Pro Met Lys His Gln Tyr Asp Phe Asp Ser Ala
245 250 255
Thr Val Ile Gly Phe Leu Arg Gln His Gly Leu Asp Gln Asp Phe Lys
260 265 270
Leu Asn Ile Glu Ala Asn His Ala Thr Leu Ser Gly His Ser Phe Glu
275 280 285
His Asp Leu Gln Val Ala Ser Asp Ala Gly Leu Leu Gly Ser Ile Asp
290 295 300
Ala Asn Arg Gly Asn Pro Gln Asn Gly Trp Asp Thr Asp Gln Phe Pro
305 310 315 320
Thr Asp Leu Tyr Asp Thr Val Gly Ala Met Leu Val Val Leu Arg Gln
325 330 335
Gly Gly Leu Ala Pro Gly Gly Leu Asn Phe Asp Ala Lys Val Arg Arg
340 345 350
Glu Ser Ser Asp Pro Gln Asp Leu Phe Leu Ala His Ile Gly Gly Met
355 360 365
Asp Ala Phe Ala Arg Gly Leu Glu Val Ala Asn Ala Leu Leu Thr Ser
370 375 380
Ser Pro Leu Glu Thr Trp Arg Ala Glu Arg Tyr Ala Ser Phe Asp Ser
385 390 395 400
Gly Ala Gly Ala Asp Phe Ala Asn Gly Thr Ser Thr Leu Ala Asp Leu
405 410 415
Ala Lys Tyr Ala Ala Gly Asn Ala Pro Lys Gln Leu Ser Gly Arg Gln
420 425 430
Glu Ala Tyr Glu Asn Leu Ile Asn Gln Tyr Leu Ile Arg
435 440 445
<210> 13
<211> 1341
<212> DNA
<213> Treponema primitia
<400> 13
atggcaaatt attttaccgg cggcaaggaa tattttcccg gcataggaaa aatcccttat 60
gaaggaagcg gatcaaaaaa tcccctggcc tttaagtatt atgacgccga aaaaacggtg 120
cgtggaaaaa aaacaaagga ttggcttcgt tttgcaattg cgtattggca tagtttctgc 180
ggtgatggtg cggacccctt tggctccgct acccatatct tcccatggaa cagtaccaat 240
gaacccctgc agaacgctaa aaataaagcg gacgcggctt ttgaatttat caccaagatc 300
ggtgctccct actattgctg gcatgaccgg gatatagccc ccgaaggcaa ggaccccgat 360
gaaaccgcca agaacctcgg tattattgtt gatgagttga agaagcggca ggatgctacg 420
ggggtaaaac tcctctgggc aacggccaat gtgtttacca atccccggtt catgaacggg 480
gcggcgacca accctgattt taacattgtg gttcaggccg ccaatcaggt gaaacatgct 540
attgacggcg ccataaagct cggcgccgaa gggtacacct tctggggcgg ccgcgagggt 600
tatatgtctt tgcttaacac ggacatgaaa cgggaaaagg aacacctcgc catattcctg 660
accattgcac gggattatgc acgcaaacaa ggttttaaag gttctttcta tatcgaaccg 720
aaaccgatgg aaccgaccaa acatcagtat gattttgatt ccgaaacggt tatcggtttt 780
ttaaaagccc acggccttga gaaggacttt aagttgaata ttgaggctaa ccacgcggaa 840
cttgcgggcc atgatttcta tcatgaactg tcggtctgtg ttgataacga tatgctcgga 900
tcggttgacg caaaccgcgg cgaaccccgt aacggctggg atacggatca attcccctcc 960
agcgtttatg agaccaccct ggcgatgctt actatcctcc gcatgggcgg tttcaaaacc 1020
ggggggctta atttcgatgc aaaaatccgc cgcaactcaa ttgatcctga ggatcttttt 1080
atcgcccaca tcggcggtat ggacaccttt gcctacggac ttgaaaaggc ctctgcggtc 1140
cttgatgacg ggcgtattcc ggatctgatt aaaaaacgtt actcctcctt tgattcaggg 1200
gatggcgcga aatttgagaa gagcggattt accctggacg cgttggccgc tcttgccaag 1260
gattacggta aagccggctg gaccagcggc aagcaggaac tgtttgaaaa tctcttttct 1320
gatattatat tgttaaaata a 1341
<210> 14
<211> 446
<212> PRT
<213> Treponema primitia
<400> 14
Met Ala Asn Tyr Phe Thr Gly Gly Lys Glu Tyr Phe Pro Gly Ile Gly
1 5 10 15
Lys Ile Pro Tyr Glu Gly Ser Gly Ser Lys Asn Pro Leu Ala Phe Lys
20 25 30
Tyr Tyr Asp Ala Glu Lys Thr Val Arg Gly Lys Lys Thr Lys Asp Trp
35 40 45
Leu Arg Phe Ala Ile Ala Tyr Trp His Ser Phe Cys Gly Asp Gly Ala
50 55 60
Asp Pro Phe Gly Ser Ala Thr His Ile Phe Pro Trp Asn Ser Thr Asn
65 70 75 80
Glu Pro Leu Gln Asn Ala Lys Asn Lys Ala Asp Ala Ala Phe Glu Phe
85 90 95
Ile Thr Lys Ile Gly Ala Pro Tyr Tyr Cys Trp His Asp Arg Asp Ile
100 105 110
Ala Pro Glu Gly Lys Asp Pro Asp Glu Thr Ala Lys Asn Leu Gly Ile
115 120 125
Ile Val Asp Glu Leu Lys Lys Arg Gln Asp Ala Thr Gly Val Lys Leu
130 135 140
Leu Trp Ala Thr Ala Asn Val Phe Thr Asn Pro Arg Phe Met Asn Gly
145 150 155 160
Ala Ala Thr Asn Pro Asp Phe Asn Ile Val Val Gln Ala Ala Asn Gln
165 170 175
Val Lys His Ala Ile Asp Gly Ala Ile Lys Leu Gly Ala Glu Gly Tyr
180 185 190
Thr Phe Trp Gly Gly Arg Glu Gly Tyr Met Ser Leu Leu Asn Thr Asp
195 200 205
Met Lys Arg Glu Lys Glu His Leu Ala Ile Phe Leu Thr Ile Ala Arg
210 215 220
Asp Tyr Ala Arg Lys Gln Gly Phe Lys Gly Ser Phe Tyr Ile Glu Pro
225 230 235 240
Lys Pro Met Glu Pro Thr Lys His Gln Tyr Asp Phe Asp Ser Glu Thr
245 250 255
Val Ile Gly Phe Leu Lys Ala His Gly Leu Glu Lys Asp Phe Lys Leu
260 265 270
Asn Ile Glu Ala Asn His Ala Glu Leu Ala Gly His Asp Phe Tyr His
275 280 285
Glu Leu Ser Val Cys Val Asp Asn Asp Met Leu Gly Ser Val Asp Ala
290 295 300
Asn Arg Gly Glu Pro Arg Asn Gly Trp Asp Thr Asp Gln Phe Pro Ser
305 310 315 320
Ser Val Tyr Glu Thr Thr Leu Ala Met Leu Thr Ile Leu Arg Met Gly
325 330 335
Gly Phe Lys Thr Gly Gly Leu Asn Phe Asp Ala Lys Ile Arg Arg Asn
340 345 350
Ser Ile Asp Pro Glu Asp Leu Phe Ile Ala His Ile Gly Gly Met Asp
355 360 365
Thr Phe Ala Tyr Gly Leu Glu Lys Ala Ser Ala Val Leu Asp Asp Gly
370 375 380
Arg Ile Pro Asp Leu Ile Lys Lys Arg Tyr Ser Ser Phe Asp Ser Gly
385 390 395 400
Asp Gly Ala Lys Phe Glu Lys Ser Gly Phe Thr Leu Asp Ala Leu Ala
405 410 415
Ala Leu Ala Lys Asp Tyr Gly Lys Ala Gly Trp Thr Ser Gly Lys Gln
420 425 430
Glu Leu Phe Glu Asn Leu Phe Ser Asp Ile Ile Leu Leu Lys
435 440 445
<210> 15
<211> 1329
<212> DNA
<213> Pedobacter heparinus
<400> 15
atgacaaaac ttactgcaga caacgaatat ttcaaaggga tcggacagat cagctttgaa 60
ggacaggaaa cagacaaccc gctggctttc agatggtaca atcctgaaca ggtggttgcc 120
ggcaaaaaga tgaaagagca cctgcgtttt gccggtgctt actggcattc tttctgcgga 180
aatggtacag atccctttgg cggtccgaca catatttttc cctgggacgc gaaagcggat 240
gtactggatc gtgcaaagga caaaatggat gcagcctttg aatttctgac caaaatgaac 300
ctgccctatt actgctttca tgatgtggat gtggtagatt atggcaacga catcaaagaa 360
aatgaaagac ggatgcagat catgaccgat tatgcaaaag ccaaacaggc agaaacaggt 420
gtaaaattgc tttggggtac ggctaatctt ttctctcacc gcaggtatat gaacggagcg 480
gctaccaatc ccgactttca tgtgctgagc catggcgcag cacaggtaaa agcagccctt 540
gatgccacca tagcccttaa tggggaaaat tatgtattct ggggtggccg cgaaggttac 600
atgagcctcc tgaacaccaa tatgaaacgc gaacaggaac atctggcaaa atttctgcat 660
acagccaaag attatgcccg taaaaatggt ttcaaaggca ccttctttat tgagcccaaa 720
ccttgtgaac ccaccaagca ccagtacgat tacgatgcag caaccgtact tggctttctc 780
cgtcagtacg acctgctggg tgattttaaa ctgaacctgg aagttaacca tgctacgctg 840
gccggacata ccttccagca tgagctgcag gtggctgctg atgccggaat gctgggctct 900
attgatgcca accgcggcga cgaacaaaat ggctgggata cagaccagtt tccaaacaac 960
atcaatgagg ttacagaatc catgctgatc atcctggaag cagggggcct gcaaggtggg 1020
ggtataaatt tcgatgccaa gatccgcagg aattcaacgg atccggccga ccttttccat 1080
gcacatattg gtggaatgga tattttcgcc cgggccctga ttaccgccga ccgcatcctt 1140
cagcattctg aatacaaaaa aataagggca gaaagatatg cgtcttacga cagtggaaaa 1200
ggcaaagcct ttgaagaagg gagcttaagc ctggaagacc tgcgcgatta tgcagtggca 1260
cagggcgaac cgcaaaccat cagcggcaaa caggaattcc tggaaaacct gatcaacagg 1320
tatatttaa 1329
<210> 16
<211> 442
<212> PRT
<213> Pedobacter heparinus
<400> 16
Met Thr Lys Leu Thr Ala Asp Asn Glu Tyr Phe Lys Gly Ile Gly Gln
1 5 10 15
Ile Ser Phe Glu Gly Gln Glu Thr Asp Asn Pro Leu Ala Phe Arg Trp
20 25 30
Tyr Asn Pro Glu Gln Val Val Ala Gly Lys Lys Met Lys Glu His Leu
35 40 45
Arg Phe Ala Gly Ala Tyr Trp His Ser Phe Cys Gly Asn Gly Thr Asp
50 55 60
Pro Phe Gly Gly Pro Thr His Ile Phe Pro Trp Asp Ala Lys Ala Asp
65 70 75 80
Val Leu Asp Arg Ala Lys Asp Lys Met Asp Ala Ala Phe Glu Phe Leu
85 90 95
Thr Lys Met Asn Leu Pro Tyr Tyr Cys Phe His Asp Val Asp Val Val
100 105 110
Asp Tyr Gly Asn Asp Ile Lys Glu Asn Glu Arg Arg Met Gln Ile Met
115 120 125
Thr Asp Tyr Ala Lys Ala Lys Gln Ala Glu Thr Gly Val Lys Leu Leu
130 135 140
Trp Gly Thr Ala Asn Leu Phe Ser His Arg Arg Tyr Met Asn Gly Ala
145 150 155 160
Ala Thr Asn Pro Asp Phe His Val Leu Ser His Gly Ala Ala Gln Val
165 170 175
Lys Ala Ala Leu Asp Ala Thr Ile Ala Leu Asn Gly Glu Asn Tyr Val
180 185 190
Phe Trp Gly Gly Arg Glu Gly Tyr Met Ser Leu Leu Asn Thr Asn Met
195 200 205
Lys Arg Glu Gln Glu His Leu Ala Lys Phe Leu His Thr Ala Lys Asp
210 215 220
Tyr Ala Arg Lys Asn Gly Phe Lys Gly Thr Phe Phe Ile Glu Pro Lys
225 230 235 240
Pro Cys Glu Pro Thr Lys His Gln Tyr Asp Tyr Asp Ala Ala Thr Val
245 250 255
Leu Gly Phe Leu Arg Gln Tyr Asp Leu Leu Gly Asp Phe Lys Leu Asn
260 265 270
Leu Glu Val Asn His Ala Thr Leu Ala Gly His Thr Phe Gln His Glu
275 280 285
Leu Gln Val Ala Ala Asp Ala Gly Met Leu Gly Ser Ile Asp Ala Asn
290 295 300
Arg Gly Asp Glu Gln Asn Gly Trp Asp Thr Asp Gln Phe Pro Asn Asn
305 310 315 320
Ile Asn Glu Val Thr Glu Ser Met Leu Ile Ile Leu Glu Ala Gly Gly
325 330 335
Leu Gln Gly Gly Gly Ile Asn Phe Asp Ala Lys Ile Arg Arg Asn Ser
340 345 350
Thr Asp Pro Ala Asp Leu Phe His Ala His Ile Gly Gly Met Asp Ile
355 360 365
Phe Ala Arg Ala Leu Ile Thr Ala Asp Arg Ile Leu Gln His Ser Glu
370 375 380
Tyr Lys Lys Ile Arg Ala Glu Arg Tyr Ala Ser Tyr Asp Ser Gly Lys
385 390 395 400
Gly Lys Ala Phe Glu Glu Gly Ser Leu Ser Leu Glu Asp Leu Arg Asp
405 410 415
Tyr Ala Val Ala Gln Gly Glu Pro Gln Thr Ile Ser Gly Lys Gln Glu
420 425 430
Phe Leu Glu Asn Leu Ile Asn Arg Tyr Ile
435 440
<210> 17
<211> 876
<212> DNA
<213> Pyrolobus fumarii
<400> 17
gtggggggcc cactcgtgcc ggagaggata cgcttcggac cagcgggtaa gccggtcggc 60
atgaagagtg gcgactatgt caaggctatc gagtatgtgg cgaacgaggg tctcgacgca 120
ctcgagtatg aggcggtgcg cggcgtgcgt atcagcgaga agaaggctgt tgagataaag 180
agggctgcct tggagcacgg tatccttctc tcgatgcacg cgccctactt catcaaccta 240
gcgtcgccca acgaggacac cgttaagaag agccaacaga ggcttctcga cgcgctcaag 300
gcggctaact ggatgggcgc ctatgtggtc gtcttccacc cgggctacta caaggacaac 360
ccgagtaaag aagccgccct caagagggtg atcgagaacc tgaagcccgt tgtagagcag 420
gctaagcagc tcgggatcaa gggtgtcgag ctgggccccg agactaccgg gaagagagcc 480
caggtcggcg atatagacga ggtgatcaca atctgcaggg aggttgagat gtgccgcccg 540
gtggtagact gggcgcacat ctacgccagg taccggggcc aacacgtgac cagcatcgac 600
caggtgctca aggtgataga gaagattgag aaggagcttg ggagtcgcgc tgtcaacccg 660
ctacacactc acttctcgcg catcgagtac ggggagggag gagagaggga gcaccatacg 720
ctcgacgagg cggagtatgg accggagttt aggatagtgt gtgaggctta caaacaagcc 780
gggatacgcg cagtgataat ctcggagagc ccgatactag accaggacgc actcaagatg 840
aagaagattt gttgcgagga gctaggctac tgctag 876
<210> 18
<211> 291
<212> PRT
<213> Pyrolobus fumarii
<400> 18
Val Gly Gly Pro Leu Val Pro Glu Arg Ile Arg Phe Gly Pro Ala Gly
1 5 10 15
Lys Pro Val Gly Met Lys Ser Gly Asp Tyr Val Lys Ala Ile Glu Tyr
20 25 30
Val Ala Asn Glu Gly Leu Asp Ala Leu Glu Tyr Glu Ala Val Arg Gly
35 40 45
Val Arg Ile Ser Glu Lys Lys Ala Val Glu Ile Lys Arg Ala Ala Leu
50 55 60
Glu His Gly Ile Leu Leu Ser Met His Ala Pro Tyr Phe Ile Asn Leu
65 70 75 80
Ala Ser Pro Asn Glu Asp Thr Val Lys Lys Ser Gln Gln Arg Leu Leu
85 90 95
Asp Ala Leu Lys Ala Ala Asn Trp Met Gly Ala Tyr Val Val Val Phe
100 105 110
His Pro Gly Tyr Tyr Lys Asp Asn Pro Ser Lys Glu Ala Ala Leu Lys
115 120 125
Arg Val Ile Glu Asn Leu Lys Pro Val Val Glu Gln Ala Lys Gln Leu
130 135 140
Gly Ile Lys Gly Val Glu Leu Gly Pro Glu Thr Thr Gly Lys Arg Ala
145 150 155 160
Gln Val Gly Asp Ile Asp Glu Val Ile Thr Ile Cys Arg Glu Val Glu
165 170 175
Met Cys Arg Pro Val Val Asp Trp Ala His Ile Tyr Ala Arg Tyr Arg
180 185 190
Gly Gln His Val Thr Ser Ile Asp Gln Val Leu Lys Val Ile Glu Lys
195 200 205
Ile Glu Lys Glu Leu Gly Ser Arg Ala Val Asn Pro Leu His Thr His
210 215 220
Phe Ser Arg Ile Glu Tyr Gly Glu Gly Gly Glu Arg Glu His His Thr
225 230 235 240
Leu Asp Glu Ala Glu Tyr Gly Pro Glu Phe Arg Ile Val Cys Glu Ala
245 250 255
Tyr Lys Gln Ala Gly Ile Arg Ala Val Ile Ile Ser Glu Ser Pro Ile
260 265 270
Leu Asp Gln Asp Ala Leu Lys Met Lys Lys Ile Cys Cys Glu Glu Leu
275 280 285
Gly Tyr Cys
290
<210> 19
<211> 972
<212> DNA
<213> Geobacillus species
<400> 19
atgaaagtag gcgtatttac cgtcttgtat caacagctgc cgttggaaga catgctcgac 60
aaagtcgccg ccatgggcat tgaggccgtt gagcttggca ccggcaatta cccgggcagc 120
gcccattgcg atcccgacgc gctgttggac cagccggaaa acatcaaagc gttgaaaaaa 180
gccgtcgccg accgcggcct tgtcatcagc gccttaagct gccatggcaa tccgcttcat 240
ccggacaaaa cgttcgcgaa acagtcgcat gacacgtggc ggaaaactgt caggctcgcc 300
gagcagcttg aagtcccggt catcaacgcc ttctccggct gcccgggcga ccatcccggc 360
gccaaatacc cgaactgggt cacatgctcc tggccgccgg attacttgga aattttaaaa 420
tggcaatggg aagaagtcgt catcccgtac tggcgcgaag aagcagcgtt cgccaaggag 480
cacggcatca cgcaaatcgc ctttgaaatg catccgggct tcgtcgtcta caacccggaa 540
acgctcctca aactgcgcga acacgtcggt gaagcgatcg gcgccaactt tgacccgagc 600
cacttgcttt ggcaaggcat cgacccggtt gaggcgatca aactgctcgg ccgcgaaaaa 660
gcgattttcc acgtccatgc gaaagacacg tacttagacg aagcgaacat ccgcaaaaac 720
ggcgtgctcg atacgaaaca ttacagccaa attctcgatc gctcatgggt gttccgcacc 780
gtcggctacg ggcaaagcga aaaaatgtgg cgcgacatcg tcagcgccct gcgcgccgtc 840
ggctacgact acgtgctgtc aatcgaacac gaagatatgc tcgcttcgat cgatgaaggg 900
ctgtcaaagg ccgtcgccct cttgaaaaag gtgttgttca aagaagaact gccggagatg 960
tggtgggcat aa 972
<210> 20
<211> 323
<212> PRT
<213> Geobacillus species
<400> 20
Met Lys Val Gly Val Phe Thr Val Leu Tyr Gln Gln Leu Pro Leu Glu
1 5 10 15
Asp Met Leu Asp Lys Val Ala Ala Met Gly Ile Glu Ala Val Glu Leu
20 25 30
Gly Thr Gly Asn Tyr Pro Gly Ser Ala His Cys Asp Pro Asp Ala Leu
35 40 45
Leu Asp Gln Pro Glu Asn Ile Lys Ala Leu Lys Lys Ala Val Ala Asp
50 55 60
Arg Gly Leu Val Ile Ser Ala Leu Ser Cys His Gly Asn Pro Leu His
65 70 75 80
Pro Asp Lys Thr Phe Ala Lys Gln Ser His Asp Thr Trp Arg Lys Thr
85 90 95
Val Arg Leu Ala Glu Gln Leu Glu Val Pro Val Ile Asn Ala Phe Ser
100 105 110
Gly Cys Pro Gly Asp His Pro Gly Ala Lys Tyr Pro Asn Trp Val Thr
115 120 125
Cys Ser Trp Pro Pro Asp Tyr Leu Glu Ile Leu Lys Trp Gln Trp Glu
130 135 140
Glu Val Val Ile Pro Tyr Trp Arg Glu Glu Ala Ala Phe Ala Lys Glu
145 150 155 160
His Gly Ile Thr Gln Ile Ala Phe Glu Met His Pro Gly Phe Val Val
165 170 175
Tyr Asn Pro Glu Thr Leu Leu Lys Leu Arg Glu His Val Gly Glu Ala
180 185 190
Ile Gly Ala Asn Phe Asp Pro Ser His Leu Leu Trp Gln Gly Ile Asp
195 200 205
Pro Val Glu Ala Ile Lys Leu Leu Gly Arg Glu Lys Ala Ile Phe His
210 215 220
Val His Ala Lys Asp Thr Tyr Leu Asp Glu Ala Asn Ile Arg Lys Asn
225 230 235 240
Gly Val Leu Asp Thr Lys His Tyr Ser Gln Ile Leu Asp Arg Ser Trp
245 250 255
Val Phe Arg Thr Val Gly Tyr Gly Gln Ser Glu Lys Met Trp Arg Asp
260 265 270
Ile Val Ser Ala Leu Arg Ala Val Gly Tyr Asp Tyr Val Leu Ser Ile
275 280 285
Glu His Glu Asp Met Leu Ala Ser Ile Asp Glu Gly Leu Ser Lys Ala
290 295 300
Val Ala Leu Leu Lys Lys Val Leu Phe Lys Glu Glu Leu Pro Glu Met
305 310 315 320
Trp Trp Ala
<210> 21
<211> 1335
<212> DNA
<213> Thermotoga neapolitana
<400> 21
atggctgaat tctttccaga aatcccgaaa gtgcagttcg aaggcaaaga aagcacaaat 60
ccacttgcgt tcaagttcta cgatccagaa gagatcatcg acggcaaacc cctcaaggac 120
catctgaagt tctccgttgc cttctggcac accttcgtga acgagggaag ggatcccttc 180
ggagacccaa cggccgatcg tccctggaac aggtacaccg atcccatgga caaggctttt 240
gcaagggtgg acgccctttt tgaattctgc gaaaaactca acatcgagta cttctgcttc 300
cacgacagag acatcgctcc cgagggaaaa acgctgaggg agacaaacaa aattttggac 360
aaagtagtgg agagaatcaa agagagaatg aaagacagca acgtgaagct cctctggggt 420
actgcaaacc tcttttccca cccaaggtac atgcatggtg cagcgacaac ctgcagtgct 480
gatgtttttg cgtacgcggc cgcccaggtg aaaaaagccc ttgagatcac caaagaactt 540
ggaggagaag ggtacgtctt ctggggtgga agagaaggat acgaaacact cctcaacacg 600
gaccttggat tcgaacttga aaacctcgcc cgcttcctca gaatggctgt ggattatgca 660
aaaaggatcg gtttcaccgg acagttcctc atcgaaccaa aaccgaaaga acccaccaaa 720
caccagtacg acttcgacgt tgcaaccgcc tatgccttcc tgaagagcca cggtctcgat 780
gaatacttca aattcaacat cgaggcaaac cacgccacac tcgccggtca caccttccag 840
cacgaactga gaatggcaag gatccttgga aaactcggaa gcatcgatgc aaaccaggga 900
gaccttcttc ttggatggga caccgatcag ttcccaacaa acgtctacga tacaaccctt 960
gcaatgtacg aagtgataaa agcgggaggc ttcacaaaag gtgggctcaa cttcgatgcg 1020
aaggtgagga gggcttctta caaagtggag gacctcttca tagggcacat agcgggaatg 1080
gacacctttg cactcggttt caaggtggca tacaaactcg tgaaggatgg tgttctggac 1140
aaattcatcg aagaaaagta cagaagtttc agggagggca ttggaaggga catcgtcgaa 1200
ggtaaagtgg attttgaaaa acttgaagag tatataatag acaaagaaac gatagaactt 1260
ccatctggaa agcaagaata cctggaaagc ctcatcaaca gttacatagt gaagaccatt 1320
ctggaactga ggtga 1335
<210> 22
<211> 444
<212> PRT
<213> Thermotoga neapolitana
<400> 22
Met Ala Glu Phe Phe Pro Glu Ile Pro Lys Val Gln Phe Glu Gly Lys
1 5 10 15
Glu Ser Thr Asn Pro Leu Ala Phe Lys Phe Tyr Asp Pro Glu Glu Ile
20 25 30
Ile Asp Gly Lys Pro Leu Lys Asp His Leu Lys Phe Ser Val Ala Phe
35 40 45
Trp His Thr Phe Val Asn Glu Gly Arg Asp Pro Phe Gly Asp Pro Thr
50 55 60
Ala Asp Arg Pro Trp Asn Arg Tyr Thr Asp Pro Met Asp Lys Ala Phe
65 70 75 80
Ala Arg Val Asp Ala Leu Phe Glu Phe Cys Glu Lys Leu Asn Ile Glu
85 90 95
Tyr Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Lys Thr Leu
100 105 110
Arg Glu Thr Asn Lys Ile Leu Asp Lys Val Val Glu Arg Ile Lys Glu
115 120 125
Arg Met Lys Asp Ser Asn Val Lys Leu Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser His Pro Arg Tyr Met His Gly Ala Ala Thr Thr Cys Ser Ala
145 150 155 160
Asp Val Phe Ala Tyr Ala Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Gly Glu Gly Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Leu Gly Phe Glu Leu Glu Asn
195 200 205
Leu Ala Arg Phe Leu Arg Met Ala Val Asp Tyr Ala Lys Arg Ile Gly
210 215 220
Phe Thr Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Thr Ala Tyr Ala Phe Leu Lys Ser
245 250 255
His Gly Leu Asp Glu Tyr Phe Lys Phe Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Gly His Thr Phe Gln His Glu Leu Arg Met Ala Arg Ile
275 280 285
Leu Gly Lys Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Leu Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asn Val Tyr Asp Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Ala Gly Gly Phe Thr Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Tyr Lys Val Glu Asp Leu
340 345 350
Phe Ile Gly His Ile Ala Gly Met Asp Thr Phe Ala Leu Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Leu Asp Lys Phe Ile Glu
370 375 380
Glu Lys Tyr Arg Ser Phe Arg Glu Gly Ile Gly Arg Asp Ile Val Glu
385 390 395 400
Gly Lys Val Asp Phe Glu Lys Leu Glu Glu Tyr Ile Ile Asp Lys Glu
405 410 415
Thr Ile Glu Leu Pro Ser Gly Lys Gln Glu Tyr Leu Glu Ser Leu Ile
420 425 430
Asn Ser Tyr Ile Val Lys Thr Ile Leu Glu Leu Arg
435 440
<210> 23
<211> 663
<212> DNA
<213> Thermotoga maritima
<400> 23
gtgtggacca ttctgtgcga taaagatagc ggaggagttc tcttgagaaa aggtgtatcc 60
acgagcatca taagaagcaa tcccgatctg cttgaagcac tcccgaaagc agaactttac 120
gaactgggtt ttttcaaggc ggaagacttc gagaaggtcc tccgcttttt tcacgacaaa 180
aattttggaa tccacgctcc tttcatctac aggtacagat accaccatcc gaatccgacc 240
tctctgaacg aggaagaaag agaggacacc ttttctgtga acaaaaaatg cgctgagctt 300
gccaggaaga tcggcgcaga atacatgata attcacttcc caaatgccct tcagaaagaa 360
aactggcttt ctgtttacag agaggtggag agagaattct ccgagcttgc gggtgtcatc 420
agcgttcgag tggagaacgt ttatggaaac gatcatttcc actccgctga agattacagg 480
acctttcttg aaaacacagg ttgtaagatg tgcgttgaca tcggccatct tcttctagac 540
gctgaggttt acggtttttc tcccatcgaa ttcatagaaa aactctctga ttttgtagaa 600
gaatttcaca tttacacgcg gatttcgaaa cctacaaaaa tgccatcacg ctccctgggg 660
tga 663
<210> 24
<211> 220
<212> PRT
<213> Thermotoga maritima
<400> 24
Val Trp Thr Ile Leu Cys Asp Lys Asp Ser Gly Gly Val Leu Leu Arg
1 5 10 15
Lys Gly Val Ser Thr Ser Ile Ile Arg Ser Asn Pro Asp Leu Leu Glu
20 25 30
Ala Leu Pro Lys Ala Glu Leu Tyr Glu Leu Gly Phe Phe Lys Ala Glu
35 40 45
Asp Phe Glu Lys Val Leu Arg Phe Phe His Asp Lys Asn Phe Gly Ile
50 55 60
His Ala Pro Phe Ile Tyr Arg Tyr Arg Tyr His His Pro Asn Pro Thr
65 70 75 80
Ser Leu Asn Glu Glu Glu Arg Glu Asp Thr Phe Ser Val Asn Lys Lys
85 90 95
Cys Ala Glu Leu Ala Arg Lys Ile Gly Ala Glu Tyr Met Ile Ile His
100 105 110
Phe Pro Asn Ala Leu Gln Lys Glu Asn Trp Leu Ser Val Tyr Arg Glu
115 120 125
Val Glu Arg Glu Phe Ser Glu Leu Ala Gly Val Ile Ser Val Arg Val
130 135 140
Glu Asn Val Tyr Gly Asn Asp His Phe His Ser Ala Glu Asp Tyr Arg
145 150 155 160
Thr Phe Leu Glu Asn Thr Gly Cys Lys Met Cys Val Asp Ile Gly His
165 170 175
Leu Leu Leu Asp Ala Glu Val Tyr Gly Phe Ser Pro Ile Glu Phe Ile
180 185 190
Glu Lys Leu Ser Asp Phe Val Glu Glu Phe His Ile Tyr Thr Arg Ile
195 200 205
Ser Lys Pro Thr Lys Met Pro Ser Arg Ser Leu Gly
210 215 220
<210> 25
<211> 1320
<212> DNA
<213> Clostridium clariflavum
<400> 25
atgtcagagt attttaaagg aatatcaaaa atacagtatg aaggaaagga ttcagacaat 60
cctttagcct ttaagtacta taatcctgat gaggttgtcg gagacaagac aatgaaagaa 120
cacctcaggt ttgctgttgc ttattggcat acattccagg gcacaggagc agacccattc 180
ggtgtaggca cagctcaaag accgtgggaa aatattactg atccaatgga tttggcaaaa 240
gcaaaggtag aagctaactt tgaattttgt gaaaagttag gggttccttt cttctgcttc 300
catgacagag atatagctcc tgaagctgac aatctcagag agacaaataa aagacttgat 360
gagattgtag cagtaataaa ggatcgcatg aagaacagcc ctgtaaaact tctctgggga 420
acaaccaatg cgtttggcaa tccaagattt gttcatgggg cttcaacttc tccaaatgca 480
gatgtatttg catatgcagc tgcccaagta aagaaagcta tggagataac taaggaactt 540
ggcggtcaga actatgtatt ctggggcgga agagaaggtt atgagacact gctcaatacc 600
gatatgaagc ttgagttgga caatatggca agattcttaa gaatggctgt ggaatataaa 660
aaggaaatag gatttgacgg ccagctctta attgagccta agccaaagga acctacaaaa 720
catcagtatg attttgatac tgctacagtg atcggattct tgagaaccta cggacttgaa 780
aaagaattta aaatgaacat tgaggctaac catgctaccc tcgctgctca cacattccag 840
catgaactta gggtggcagc tataaacaat gcattaggaa gcattgacgc aaatcagggt 900
gacttgttgt taggatggga tactgaccaa ttcccgacaa acttatatga tacaaccctc 960
gcaatgtatg aagtattgaa ggccggcgga tttacaaaag gcggtttgaa ctttgactcg 1020
aaagtgagaa gaggttcctt tgaaccggtt gatctcttct atgctcatat tgcaggtatg 1080
gatgcttttg caagaggctt gaaagttgct tacaagatgc ttcaggacgg taaatttgaa 1140
aagttcattg aagaaagata ccagagctat aagaccggaa tcggaaaaga tattgttgaa 1200
ggaaaagttg gatttaaaga actcgaaaag tatgttttag agcttgaaac ggtaaaaaat 1260
acatccggta ggcaggaagt tcttgaagca atgttgaata aatatattat tgaaagctaa 1320
<210> 26
<211> 439
<212> PRT
<213> Clostridium clariflavum
<400> 26
Met Ser Glu Tyr Phe Lys Gly Ile Ser Lys Ile Gln Tyr Glu Gly Lys
1 5 10 15
Asp Ser Asp Asn Pro Leu Ala Phe Lys Tyr Tyr Asn Pro Asp Glu Val
20 25 30
Val Gly Asp Lys Thr Met Lys Glu His Leu Arg Phe Ala Val Ala Tyr
35 40 45
Trp His Thr Phe Gln Gly Thr Gly Ala Asp Pro Phe Gly Val Gly Thr
50 55 60
Ala Gln Arg Pro Trp Glu Asn Ile Thr Asp Pro Met Asp Leu Ala Lys
65 70 75 80
Ala Lys Val Glu Ala Asn Phe Glu Phe Cys Glu Lys Leu Gly Val Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Ala Asp Asn Leu
100 105 110
Arg Glu Thr Asn Lys Arg Leu Asp Glu Ile Val Ala Val Ile Lys Asp
115 120 125
Arg Met Lys Asn Ser Pro Val Lys Leu Leu Trp Gly Thr Thr Asn Ala
130 135 140
Phe Gly Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Pro Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ala Ala Ala Gln Val Lys Lys Ala Met Glu Ile
165 170 175
Thr Lys Glu Leu Gly Gly Gln Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Lys Leu Glu Leu Asp Asn
195 200 205
Met Ala Arg Phe Leu Arg Met Ala Val Glu Tyr Lys Lys Glu Ile Gly
210 215 220
Phe Asp Gly Gln Leu Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Thr Ala Thr Val Ile Gly Phe Leu Arg Thr
245 250 255
Tyr Gly Leu Glu Lys Glu Phe Lys Met Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Ala His Thr Phe Gln His Glu Leu Arg Val Ala Ala Ile
275 280 285
Asn Asn Ala Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Leu Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asn Leu Tyr Asp Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Leu Lys Ala Gly Gly Phe Thr Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ser Lys Val Arg Arg Gly Ser Phe Glu Pro Val Asp Leu
340 345 350
Phe Tyr Ala His Ile Ala Gly Met Asp Ala Phe Ala Arg Gly Leu Lys
355 360 365
Val Ala Tyr Lys Met Leu Gln Asp Gly Lys Phe Glu Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Gln Ser Tyr Lys Thr Gly Ile Gly Lys Asp Ile Val Glu
385 390 395 400
Gly Lys Val Gly Phe Lys Glu Leu Glu Lys Tyr Val Leu Glu Leu Glu
405 410 415
Thr Val Lys Asn Thr Ser Gly Arg Gln Glu Val Leu Glu Ala Met Leu
420 425 430
Asn Lys Tyr Ile Ile Glu Ser
435
<210> 27
<211> 1329
<212> DNA
<213> Chryseobacterium sp.
<400> 27
atgaacactt taacaggtac aaaagagttt tttacaggta ttgaaaaaat taagtttgag 60
gggaaggaaa gcaggaatcc gttggcattc cgttattatg atgctgaaaa gatcgtaatg 120
ggaaaaccaa tgaaagactg gaccagattt gcaatggcat ggtggcatac cttatgtgca 180
aacggaagcg atccattcgg aggacctact atccaccacc catgggatat cggaaatgat 240
cctgtgacca gagcaatgca taagatggat gcaggctttg aattcatgtc taaaatgggc 300
ttcaattatt actgtttcca tgatatcgat ttggtagacc ccgccaataa ttggaaagac 360
tatgagaaga atatgcagac tattgtggag tatgcaaaac aaaagcagaa ggaaacagga 420
attaaacttt tatggggaac agcaaatgtt ttcacgcatg aaagatacat gaatggagct 480
tctaccaatc ccaattttga tgttgtagcc tgcgcaggaa cccaggtgaa aaattcaata 540
gatgccacca ttgcacttgg aggtgaaaac tatgttttct ggggtggaag agaaggatat 600
atgagtcttt taaataccga tatgaagcgt gaaaaagatc atctggcccg tttcctttcc 660
atgtcgagag attatgcccg tcagcaagga tttaaaggaa ctttccttat tgaacctaaa 720
ccaatggagc ctaccaaaca tcagtatgat tatgactctg aaaccgtaat cggattcctg 780
agacactatg gactagacaa agactttaaa ctgaatatcg aagtgaatca tgctacattg 840
gcaggtcata catttgaaca tgaacttcag gttgctgttg atgcagggct tttaggaagt 900
attgatgcga acagaggaga ttatcaaaac ggctgggata cggatcagtt tccgatcgat 960
tattatgata tggttcaggc atggttggta ctgcttccgg caggaggtct gggaaccgga 1020
ggcgtaaact ttgatgccaa aatcagaaga aattctattg atgctgaaga tttattcatt 1080
tctcatattt caggaatgga tgtattcgct aaaggtcttc ttgcggcagc ggatattttt 1140
gaaaattcgg attacaaaaa actgaaaaca aaccgttatg cttcttttga taacggaagt 1200
ggaaaagcat tcgaggaagg tacgcttacc ttggaagatc ttcagagaat tgctcacgaa 1260
ataggcgaac cacagccaaa aagcggaaaa caggaactgt ttgaggccat cgtgaatatg 1320
tatatataa 1329
<210> 28
<211> 442
<212> PRT
<213> Chryseobacterium sp.
<400> 28
Met Asn Thr Leu Thr Gly Thr Lys Glu Phe Phe Thr Gly Ile Glu Lys
1 5 10 15
Ile Lys Phe Glu Gly Lys Glu Ser Arg Asn Pro Leu Ala Phe Arg Tyr
20 25 30
Tyr Asp Ala Glu Lys Ile Val Met Gly Lys Pro Met Lys Asp Trp Thr
35 40 45
Arg Phe Ala Met Ala Trp Trp His Thr Leu Cys Ala Asn Gly Ser Asp
50 55 60
Pro Phe Gly Gly Pro Thr Ile His His Pro Trp Asp Ile Gly Asn Asp
65 70 75 80
Pro Val Thr Arg Ala Met His Lys Met Asp Ala Gly Phe Glu Phe Met
85 90 95
Ser Lys Met Gly Phe Asn Tyr Tyr Cys Phe His Asp Ile Asp Leu Val
100 105 110
Asp Pro Ala Asn Asn Trp Lys Asp Tyr Glu Lys Asn Met Gln Thr Ile
115 120 125
Val Glu Tyr Ala Lys Gln Lys Gln Lys Glu Thr Gly Ile Lys Leu Leu
130 135 140
Trp Gly Thr Ala Asn Val Phe Thr His Glu Arg Tyr Met Asn Gly Ala
145 150 155 160
Ser Thr Asn Pro Asn Phe Asp Val Val Ala Cys Ala Gly Thr Gln Val
165 170 175
Lys Asn Ser Ile Asp Ala Thr Ile Ala Leu Gly Gly Glu Asn Tyr Val
180 185 190
Phe Trp Gly Gly Arg Glu Gly Tyr Met Ser Leu Leu Asn Thr Asp Met
195 200 205
Lys Arg Glu Lys Asp His Leu Ala Arg Phe Leu Ser Met Ser Arg Asp
210 215 220
Tyr Ala Arg Gln Gln Gly Phe Lys Gly Thr Phe Leu Ile Glu Pro Lys
225 230 235 240
Pro Met Glu Pro Thr Lys His Gln Tyr Asp Tyr Asp Ser Glu Thr Val
245 250 255
Ile Gly Phe Leu Arg His Tyr Gly Leu Asp Lys Asp Phe Lys Leu Asn
260 265 270
Ile Glu Val Asn His Ala Thr Leu Ala Gly His Thr Phe Glu His Glu
275 280 285
Leu Gln Val Ala Val Asp Ala Gly Leu Leu Gly Ser Ile Asp Ala Asn
290 295 300
Arg Gly Asp Tyr Gln Asn Gly Trp Asp Thr Asp Gln Phe Pro Ile Asp
305 310 315 320
Tyr Tyr Asp Met Val Gln Ala Trp Leu Val Leu Leu Pro Ala Gly Gly
325 330 335
Leu Gly Thr Gly Gly Val Asn Phe Asp Ala Lys Ile Arg Arg Asn Ser
340 345 350
Ile Asp Ala Glu Asp Leu Phe Ile Ser His Ile Ser Gly Met Asp Val
355 360 365
Phe Ala Lys Gly Leu Leu Ala Ala Ala Asp Ile Phe Glu Asn Ser Asp
370 375 380
Tyr Lys Lys Leu Lys Thr Asn Arg Tyr Ala Ser Phe Asp Asn Gly Ser
385 390 395 400
Gly Lys Ala Phe Glu Glu Gly Thr Leu Thr Leu Glu Asp Leu Gln Arg
405 410 415
Ile Ala His Glu Ile Gly Glu Pro Gln Pro Lys Ser Gly Lys Gln Glu
420 425 430
Leu Phe Glu Ala Ile Val Asn Met Tyr Ile
435 440
<210> 29
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ5_pTDH3_fwd
<400> 29
atatcgaatt cctgcagccc acagtttatt cctggcatc 39
<210> 30
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ6_pTDH3_rev
<400> 30
aacacaacat tttgtttgtt tatgtgtgtt tattc 35
<210> 31
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ7_XKS1_fwd
<400> 31
aacaaacaaa atgttgtgtt cagtaattca g 31
<210> 32
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ8_XKS1_rev
<400> 32
tcttacttta ttagatgaga gtcttttcca g 31
<210> 33
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ9_tDIT1_fwd
<400> 33
tctcatctaa taaagtaaga gcgctacatt g 31
<210> 34
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ10_tDIT1_rev
<400> 34
ctagaactag tggatccccc gaaattcaaa atatcatctt tgacag 46
<210> 35
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ11_pPGK1_fwd
<400> 35
atatcgaatt cctgcagccc tgtttgcaaa aagaacaaaa c 41
<210> 36
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ12_pPGK1_rev
<400> 36
gttcagacat tgttttatat ttgttgtaaa aagtagataa ttac 44
<210> 37
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ13_TAL1_fwd
<400> 37
aatataaaac aatgtctgaa ccagctcaaa ag 32
<210> 38
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ14_TAL1_rev
<400> 38
gtttagaatc ttaagcggta actttctttt c 31
<210> 39
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ15_tYHI9_fwd
<400> 39
taccgcttaa gattctaaac gcatagttgt aaggttgatg 40
<210> 40
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ16_tYHI9_rev
<400> 40
ctagaactag tggatccccc tcaataccgc ctccggcg 38
<210> 41
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ17_pCYC19_fwd
<400> 41
atatcgaatt cctgcagccc acagattggg agattttcat ag 42
<210> 42
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ18_pCYC19_rev
<400> 42
attgagtcat tgtgatgatg ttttatttgt tttg 34
<210> 43
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ19_TKL1_fwd
<400> 43
catcatcaca atgactcaat tcactgacat tg 32
<210> 44
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ20_TKL1_rev
<400> 44
cagatcaaag ttagaaagct tttttcaaag gag 33
<210> 45
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ21_tEFM1_fwd
<400> 45
agctttctaa ctttgatctg tagcctaagt ataaaattc 39
<210> 46
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ22_tEFM1_rev
<400> 46
ctagaactag tggatccccc tagataatat cattggccta ttatcaaatg 50
<210> 47
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ23_pPFK1_fwd
<400> 47
atatcgaatt cctgcagccc gaaaaatata aggatgagaa agtgaaatc 49
<210> 48
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ24_pPFK1_rev
<400> 48
actgtgccat ctttgatatg attttgtttc agatttttta tataaaag 48
<210> 49
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ25_TKL2_fwd
<400> 49
catatcaaag atggcacagt tctccgac 28
<210> 50
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ26_TKL2_rev
<400> 50
atcaaccagc ttagaaagct cttcccatag g 31
<210> 51
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ27_tRPL15A_fwd
<400> 51
agctttctaa gctggttgat ggaaaatata attttattg 39
<210> 52
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ28_tRPL15A_rev
<400> 52
ctagaactag tggatccccc gcttgatagc agaataaaag tac 43
<210> 53
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ29_module_TKL1_fwd
<400> 53
gcttgatatc gaattcctgc agccctagat aatatcattg gcctattatc aaatg 55
<210> 54
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ30_module_TKL1_rev
<400> 54
aggaataaac tgtacagatt gggagatttt catag 35
<210> 55
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ31_module_XKS1_fwd
<400> 55
tctcccaatc tgtacagttt attcctggca tc 32
<210> 56
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ32_module_XKS1_rev
<400> 56
ccgctctaga actagtggat cccccgaaat tcaaaatatc atctttgaca g 51
<210> 57
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ33_module_TAL1_fwd
<400> 57
gcttgatatc gaattcctgc agccctcaat accgcctccg gcg 43
<210> 58
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ34_module_TAL1_rev
<400> 58
ccttatattt ttctgtttgc aaaaagaaca aaactgaaaa aacc 44
<210> 59
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ35_module_TKL2_fwd
<400> 59
ctttttgcaa acagaaaaat ataaggatga gaaagtgaaa tc 42
<210> 60
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ36_module_TKL2_rev
<400> 60
ccgctctaga actagtggat cccccgcttg atagcagaat aaaagtac 48
<210> 61
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ39_mTKL1-mXKS1_fwd
<400> 61
gcttgatatc gaattcctgc agccctagat aatatcattg gcctattatc aaatg 55
<210> 62
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ40_mTKL1-mXKS1_rev
<400> 62
ctccatgtcg ctggaaattc aaaatatcat ctttgacag 39
<210> 63
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ41_KANMX6_fwd
<400> 63
tattttgaat ttccagcgac atggaggccc a 31
<210> 64
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ42_KANMX6_rev
<400> 64
gaggcggtat tgatcgacac tggatggcgg c 31
<210> 65
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ43_mTKL2-mTAL1_fwd
<400> 65
catccagtgt cgatcaatac cgcctccggc g 31
<210> 66
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ44_mTKL2-mTAL1_rev
<400> 66
ccgctctaga actagtggat cccccgcttg atagcagaat aaaagtacag ctc 53
<210> 67
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ49_PPP-KAN_fwd
<400> 67
cctgcaaatc gtgtagataa tatcattggc ctattatcaa atg 43
<210> 68
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ50_PPP-KAN_rev
<400> 68
ttgataaatt actgcttgat agcagaataa aagtac 36
<210> 69
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ53_CHR16-UP-1000_fwd
<400> 69
gcttgatatc gaattcctgc agcccgagaa tagaatacgt gtctataggt g 51
<210> 70
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ54_CHR16-UP-1000_rev
<400> 70
atgatattat ctacacgatt tgcaggacag tttac 35
<210> 71
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ55_CHR16_DOWN-1000_fwd
<400> 71
tctgctatca agcagtaatt tatcaagctt taataagttt g 41
<210> 72
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> CJ56_CHR16_DOWN-1000_rev
<400> 72
ccgctctaga actagtggat ccccctggtt tagaatcctg aacc 44
<210> 73
<211> 500
<212> DNA
<213> Saccharomyces cerevisae
<400> 73
acagtttatt cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag 60
aaaaaaaaag aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt 120
ccattctctt agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac 180
ctcaatggag tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc 240
atgtatctat ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa 300
agctgaaaaa aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt 360
atataaagac ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat 420
tctactttta tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata 480
aacacacata aacaaacaaa 500
<210> 74
<211> 1803
<212> DNA
<213> Saccharomyces cerevisae
<400> 74
atgttgtgtt cagtaattca gagacagaca agagaggttt ccaacacaat gtctttagac 60
tcatactatc ttgggtttga tctttcgacc caacaactga aatgtctcgc cattaaccag 120
gacctaaaaa ttgtccattc agaaacagtg gaatttgaaa aggatcttcc gcattatcac 180
acaaagaagg gtgtctatat acacggcgac actatcgaat gtcccgtagc catgtggtta 240
gaggctctag atctggttct ctcgaaatat cgcgaggcta aatttccatt gaacaaagtt 300
atggccgtct cagggtcctg ccagcagcac gggtctgtct actggtcctc ccaagccgaa 360
tctctgttag agcaattgaa taagaaaccg gaaaaagatt tattgcacta cgtgagctct 420
gtagcatttg caaggcaaac cgcccccaat tggcaagacc acagtactgc aaagcaatgt 480
caagagtttg aagagtgcat aggtgggcct gaaaaaatgg ctcaattaac agggtccaga 540
gcccatttta gatttactgg tcctcaaatt ctgaaaattg cacaattaga accagaagct 600
tacgaaaaaa caaagaccat ttctttagtg tctaattttt tgacttctat cttagtgggc 660
catcttgttg aattagagga ggcagatgcc tgtggtatga acctttatga tatacgtgaa 720
agaaaattca gtgatgagct actacatcta attgatagtt cttctaagga taaaactatc 780
agacaaaaat taatgagagc acccatgaaa aatttgatag cgggtaccat ctgtaaatat 840
tttattgaga agtacggttt caatacaaac tgcaaggtct ctcccatgac tggggataat 900
ttagccacta tatgttcttt acccctgcgg aagaatgacg ttctcgtttc cctaggaaca 960
agtactacag ttcttctggt caccgataag tatcacccct ctccgaacta tcatcttttc 1020
attcatccaa ctctgccaaa ccattatatg ggtatgattt gttattgtaa tggttctttg 1080
gcaagggaga ggataagaga cgagttaaac aaagaacggg aaaataatta tgagaagact 1140
aacgattgga ctctttttaa tcaagctgtg ctagatgact cagaaagtag tgaaaatgaa 1200
ttaggtgtat attttcctct gggggagatc gttcctagcg taaaagccat aaacaaaagg 1260
gttatcttca atccaaaaac gggtatgatt gaaagagagg tggccaagtt caaagacaag 1320
aggcacgatg ccaaaaatat tgtagaatca caggctttaa gttgcagggt aagaatatct 1380
cccctgcttt cggattcaaa cgcaagctca caacagagac tgaacgaaga tacaatcgtg 1440
aagtttgatt acgatgaatc tccgctgcgg gactacctaa ataaaaggcc agaaaggact 1500
ttttttgtag gtggggcttc taaaaacgat gctattgtga agaagtttgc tcaagtcatt 1560
ggtgctacaa agggtaattt taggctagaa acaccaaact catgtgccct tggtggttgt 1620
tataaggcca tgtggtcatt gttatatgac tctaataaaa ttgcagttcc ttttgataaa 1680
tttctgaatg acaattttcc atggcatgta atggaaagca tatccgatgt ggataatgaa 1740
aattgggatc gctataattc caagattgtc cccttaagcg aactggaaaa gactctcatc 1800
taa 1803
<210> 75
<211> 300
<212> DNA
<213> Saccharomyces cerevisae
<400> 75
taaagtaaga gcgctacatt ggtctacctt tttgttcttt tacttaaaca ttagttagtt 60
cgttttcttt ttctcatttt tttatgtttc ccccccaaag ttctgatttt ataatatttt 120
atttcacaca attccattta acagaggggg aatagattct ttagcttaga aaattagtga 180
tcaatatata tttgcctttc ttttcatctt ttcagtgata ttaatggttt cgagacactg 240
caatggccct agttgtctaa gaggatagat gttactgtca aagatgatat tttgaatttc 300
<210> 76
<211> 500
<212> DNA
<213> Saccharomyces cerevisae
<400> 76
tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct gtcttcctat 60
tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca caggttttgt 120
aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat gctatgatgc 180
ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc tctttcaaac 240
agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt tcttctaacc 300
aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat atataaactt 360
gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt agtttttcaa 420
gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa ttatctactt 480
tttacaacaa atataaaaca 500
<210> 77
<211> 1008
<212> DNA
<213> Saccharomyces cerevisae
<400> 77
atgtctgaac cagctcaaaa gaaacaaaag gttgctaaca actctctaga acaattgaaa 60
gcctccggca ctgtcgttgt tgccgacact ggtgatttcg gctctattgc caagtttcaa 120
cctcaagact ccacaactaa cccatcattg atcttggctg ctgccaagca accaacttac 180
gccaagttga tcgatgttgc cgtggaatac ggtaagaagc atggtaagac caccgaagaa 240
caagtcgaaa atgctgtgga cagattgtta gtcgaattcg gtaaggagat cttaaagatt 300
gttccaggca gagtctccac cgaagttgat gctagattgt cttttgacac tcaagctacc 360
attgaaaagg ctagacatat cattaaattg tttgaacaag aaggtgtctc caaggaaaga 420
gtccttatta aaattgcttc cacttgggaa ggtattcaag ctgccaaaga attggaagaa 480
aaggacggta tccactgtaa tttgactcta ttattctcct tcgttcaagc agttgcctgt 540
gccgaggccc aagttacttt gatttcccca tttgttggta gaattctaga ctggtacaaa 600
tccagcactg gtaaagatta caagggtgaa gccgacccag gtgttatttc cgtcaagaaa 660
atctacaact actacaagaa gtacggttac aagactattg ttatgggtgc ttctttcaga 720
agcactgacg aaatcaaaaa cttggctggt gttgactatc taacaatttc tccagcttta 780
ttggacaagt tgatgaacag tactgaacct ttcccaagag ttttggaccc tgtctccgct 840
aagaaggaag ccggcgacaa gatttcttac atcagcgacg aatctaaatt cagattcgac 900
ttgaatgaag acgctatggc cactgaaaaa ttgtccgaag gtatcagaaa attctctgcc 960
gatattgtta ctctattcga cttgattgaa aagaaagtta ccgcttaa 1008
<210> 78
<211> 120
<212> DNA
<213> Saccharomyces cerevisae
<400> 78
gattctaaac gcatagttgt aaggttgatg tatatatata tatatatatg tatatattaa 60
ttacaataat atgctcccgc ccaaattttt ctccttcaat accgccggag gcggtattga 120
<210> 79
<211> 500
<212> DNA
<213> Saccharomyces cerevisae
<400> 79
acagattggg agattttcat agtagaattc agcatgatag ctacgtaaat gtgttccgca 60
ccgtcacaaa gtgttttcta ctgttctttc ttctttcgtt cattcagttg agttgagtga 120
gtgctttgtt caatggatct tagctaaaat gcatattttt tctcttggta aatgaatgct 180
tgtgatgtct tccaagtgat ttcctttcct tcccatatga tgctaggtac ctttagtgtc 240
ttcctaaaaa aaaaaaaagg ctcgccatca aaacgatatt cgttggcttt tttttctgaa 300
ttataaatac tctttggtaa cttttcattt ccaagaacct cttttttcca gttatatcat 360
ggtccccttt caaagttatt ctctactctt tttcatattc attctttttc atcctttggt 420
tttttattct taacttgttt attattctct cttgtttcta tttacaagac accaatcaaa 480
acaaataaaa catcatcaca 500
<210> 80
<211> 2043
<212> DNA
<213> Saccharomyces cerevisae
<400> 80
atgactcaat tcactgacat tgataagcta gccgtctcca ccataagaat tttggctgtg 60
gacaccgtat ccaaggccaa ctcaggtcac ccaggtgctc cattgggtat ggcaccagct 120
gcacacgttc tatggagtca aatgcgcatg aacccaacca acccagactg gatcaacaga 180
gatagatttg tcttgtctaa cggtcacgcg gtcgctttgt tgtattctat gctacatttg 240
actggttacg atctgtctat tgaagacttg aaacagttca gacagttggg ttccagaaca 300
ccaggtcatc ctgaatttga gttgccaggt gttgaagtta ctaccggtcc attaggtcaa 360
ggtatctcca acgctgttgg tatggccatg gctcaagcta acctggctgc cacttacaac 420
aagccgggct ttaccttgtc tgacaactac acctatgttt tcttgggtga cggttgtttg 480
caagaaggta tttcttcaga agcttcctcc ttggctggtc atttgaaatt gggtaacttg 540
attgccatct acgatgacaa caagatcact atcgatggtg ctaccagtat ctcattcgat 600
gaagatgttg ctaagagata cgaagcctac ggttgggaag ttttgtacgt agaaaatggt 660
aacgaagatc tagccggtat tgccaaggct attgctcaag ctaagttatc caaggacaaa 720
ccaactttga tcaaaatgac cacaaccatt ggttacggtt ccttgcatgc cggctctcac 780
tctgtgcacg gtgccccatt gaaagcagat gatgttaaac aactaaagag caaattcggt 840
ttcaacccag acaagtcctt tgttgttcca caagaagttt acgaccacta ccaaaagaca 900
attttaaagc caggtgtcga agccaacaac aagtggaaca agttgttcag cgaataccaa 960
aagaaattcc cagaattagg tgctgaattg gctagaagat tgagcggcca actacccgca 1020
aattgggaat ctaagttgcc aacttacacc gccaaggact ctgccgtggc cactagaaaa 1080
ttatcagaaa ctgttcttga ggatgtttac aatcaattgc cagagttgat tggtggttct 1140
gccgatttaa caccttctaa cttgaccaga tggaaggaag cccttgactt ccaacctcct 1200
tcttccggtt caggtaacta ctctggtaga tacattaggt acggtattag agaacacgct 1260
atgggtgcca taatgaacgg tatttcagct ttcggtgcca actacaaacc atacggtggt 1320
actttcttga acttcgtttc ttatgctgct ggtgccgtta gattgtccgc tttgtctggc 1380
cacccagtta tttgggttgc tacacatgac tctatcggtg tcggtgaaga tggtccaaca 1440
catcaaccta ttgaaacttt agcacacttc agatccctac caaacattca agtttggaga 1500
ccagctgatg gtaacgaagt ttctgccgcc tacaagaact ctttagaatc caagcatact 1560
ccaagtatca ttgctttgtc cagacaaaac ttgccacaat tggaaggtag ctctattgaa 1620
agcgcttcta agggtggtta cgtactacaa gatgttgcta acccagatat tattttagtg 1680
gctactggtt ccgaagtgtc tttgagtgtt gaagctgcta agactttggc cgcaaagaac 1740
atcaaggctc gtgttgtttc tctaccagat ttcttcactt ttgacaaaca acccctagaa 1800
tacagactat cagtcttacc agacaacgtt ccaatcatgt ctgttgaagt tttggctacc 1860
acatgttggg gcaaatacgc tcatcaatcc ttcggtattg acagatttgg tgcctccggt 1920
aaggcaccag aagtcttcaa gttcttcggt ttcaccccag aaggtgttgc tgaaagagct 1980
caaaagacca ttgcattcta taagggtgac aagctaattt ctcctttgaa aaaagctttc 2040
taa 2043
<210> 81
<211> 120
<212> DNA
<213> Saccharomyces cerevisae
<400> 81
ctttgatctg tagcctaagt ataaaattct acgtatgtat atatttacat gcaatttttt 60
ctttttccaa ttcatgttaa tgttcttcat catttgataa taggccaatg atattatcta 120
<210> 82
<211> 500
<212> DNA
<213> Saccharomyces cerevisae
<400> 82
gaaaaatata aggatgagaa agtgaaatcg gttttttttt tccattgtcg tcatcaacat 60
gattttttaa ataaataaat acgatttttt attttttttc ccttctttgt ttttgttttg 120
cttattccca tcttcattat taaattcttc cgctcttaat aaaggagttt ttttattatc 180
ttcttgtgta atcatccttt ttctttaatt ttcttccttt tctttttctc tttactggtt 240
tttttacttc tttattctca accatctaaa gaatattatt gctttctacc aataaaatct 300
gttaattcta tttggattgt cgtctactca agtctcgcct agtaaataaa cgataaacaa 360
atttgaagta agaataacaa tatagggaga gaaatttttc tatttttaat ttcgaaacag 420
gtaccaaaaa atctaagttc actttagcac tatttgggaa agcttttata taaaaaatct 480
gaaacaaaat catatcaaag 500
<210> 83
<211> 2046
<212> DNA
<213> Saccharomyces cerevisae
<400> 83
atggcacagt tctccgacat tgataaactt gcggtttcca ctttaagatt actttccgtt 60
gaccaggtgg aaagcgcaca atctggccac ccaggtgcac cactaggatt ggcaccagtt 120
gcccatgtaa ttttcaagca actgcgctgt aaccctaaca atgaacattg gatcaataga 180
gacaggtttg ttctgtcgaa cggtcactca tgcgctcttc tgtactcaat gctccatcta 240
ttaggatacg attactctat cgaggacttg agacaattta gacaagtaaa ctcaaggaca 300
ccgggtcatc cagaattcca ctcagcggga gtggaaatca cttccggtcc gctaggccag 360
ggtatctcaa atgctgttgg tatggcaata gcgcaggcca actttgccgc cacttataac 420
gaggatggct ttcccatttc cgactcatat acgtttgcta ttgtagggga tggttgctta 480
caagagggtg tttcttcgga gacctcttcc ttagcgggac atctgcaatt gggtaacttg 540
attacgtttt atgacagtaa tagcatttcc attgacggta aaacctcgta ctcgttcgac 600
gaagatgttt tgaagcgata cgaggcatat ggttgggaag tcatggaagt cgataaagga 660
gacgacgata tggaatccat ttctagcgct ttggaaaagg caaaactatc gaaggacaag 720
ccaaccataa tcaaggtaac tactacaatt ggatttgggt ccctacaaca gggtactgct 780
ggtgttcatg ggtccgcttt gaaggcagat gatgttaaac agttgaagaa gaggtggggg 840
tttgacccaa ataaatcatt tgtagtacct caagaggtgt acgattatta taagaagact 900
gttgtggaac ccggtcaaaa acttaatgag gaatgggata ggatgtttga agaatacaaa 960
accaaatttc ccgagaaggg taaagaattg caaagaagat tgaatggtga gttaccggaa 1020
ggttgggaaa agcatttacc gaagtttact ccggacgacg atgctctggc aacaagaaag 1080
acatcccagc aggtgctgac gaacatggtc caagttttgc ctgaattgat cggtggttct 1140
gccgatttga caccttcgaa tctgacaagg tgggaaggcg cggtagattt ccaacctccc 1200
attacccaac taggtaacta tgcaggaagg tacattagat acggtgtgag ggaacacgga 1260
atgggtgcca ttatgaacgg tatctctgcc tttggtgcaa actacaagcc ttacggtggt 1320
acctttttga acttcgtctc ttatgctgca ggagccgtta ggttagccgc cttgtctggt 1380
aatccagtca tttgggttgc aacacatgac tctatcgggc ttggtgagga tggtccaacg 1440
caccaaccta ttgaaactct ggctcacttg agggctattc caaacatgca tgtatggaga 1500
cctgctgatg gtaacgaaac ttctgctgcg tattattctg ctatcaaatc tggtcgaaca 1560
ccatctgttg tggctttatc acgacagaat cttcctcaat tggagcattc ctcttttgaa 1620
aaagccttga agggtggcta tgtgatccat gacgtggaga atcctgatat tatcctggtg 1680
tcaacaggat cagaagtctc catttctata gatgcagcca aaaaattgta cgatactaaa 1740
aaaatcaaag caagagttgt ttccctgcca gacttttata cttttgacag gcaaagtgaa 1800
gaatacagat tctctgttct accagacggt gttccgatca tgtcctttga agtattggct 1860
acttcaagct ggggtaagta tgctcatcaa tcgttcggac tcgacgaatt tggtcgttca 1920
ggcaaggggc ctgaaattta caaattgttc gatttcacag cggacggtgt tgcgtcaagg 1980
gctgaaaaga caatcaatta ctacaaagga aagcagttgc tttctcctat gggaagagct 2040
ttctaa 2046
<210> 84
<211> 168
<212> DNA
<213> Saccharomyces cerevisae
<400> 84
gctggttgat ggaaaatata attttattgg gcaaactttt gtttatctga tgtgttttat 60
actattatct ttttaattaa tgattctata tacaaacctg tatatttttt ctttaaccaa 120
tttttttttt tatagaccta gagctgtact tttattctgc tatcaagc 168
<210> 85
<211> 348
<212> DNA
<213> Saccharomyces cerevisae
<400> 85
cagcgacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc 60
atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc 120
catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg 180
cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta 240
gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa 300
aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaacc 348
<210> 86
<211> 810
<212> DNA
<213> Saccharomyces cerevisae
<400> 86
atgggtaagg aaaagactca cgtttcgagg ccgcgattaa attccaacat ggatgctgat 60
ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga 120
ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 180
aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 240
accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 300
ggcaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 360
gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 420
agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat 480
gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 540
cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 600
aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 660
gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 720
ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 780
tttcatttga tgctcgatga gtttttctaa 810
<210> 87
<211> 240
<212> DNA
<213> Saccharomyces cerevisae
<400> 87
tcagtactga caataaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt 60
atattgtagt tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg 120
acatcatctg cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta 180
tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta acgccgccat ccagtgtcga 240
<210> 88
<211> 1000
<212> DNA
<213> Saccharomyces cerevisae
<400> 88
gagaatagaa tacgtgtcta taggtgctac tatgtgatta agaggttgca aagtgaaagg 60
cagtatttga ttcctgcaag gatcttccgt gcaggacaat agggtttaat gccacattgt 120
actctgcgtg ctatgcgaat aaaggaagcg cacgccgaat ctgaaatagg caatttggta 180
caaaaatcac gttattccat taagcagagg cagttcatat tactttggcc tgtcttacga 240
atgttcttct gaatacccaa ttctcctgag aacatctatc atataatttt tgagtttagg 300
cagacttgag gaaaaagtgg gttttgaggt ggttgtttgg agtctatctc tgataagaat 360
ggctttattg catatattct aacaggccct ctcgtaggta aaggaatccc caaaaaagag 420
tgggcagctt tacatggtaa aattacaatt cgttctttcg tttcacacgt cggcacttac 480
tatcctatta cattattaat ccttacattt cagcttccac taaattcgat ggccgtttct 540
cgtcatttat gtgatatcat aacaccatat atggcagtac atcaggcata agcactaatc 600
cgtagaaatt agttgatccc aagtttaacg gactcgaagt cctgttaatt atgtgagccg 660
aagcgtagga atattaatgt aatagaatca ataaatgact gtatattaaa acgaagaacg 720
aaagaatttt accactttgt aaaatattag attgcgttga ggggcttgtg gtcacctgtc 780
ataggatgcc tatgttcccc ccaaaaattt aattctgaag taagtttttg ttgagtactt 840
caactttatt tccttcaatt gtgaaatgtt gataactagc atctattact atccgataac 900
gccaggcgcc tttatatcat ataattaaga cacaaaagga taaaacaaag gtgttaacta 960
ttctgcatac tcactatcgt aaactgtcct gcaaatcgtg 1000
<210> 89
<211> 1000
<212> DNA
<213> Saccharomyces cerevisae
<400> 89
agtaatttat caagctttaa taagtttggg tagtttaact gtgcaaaaag gtatttacct 60
tacatactga atcttgtctg tttggtagcg gctgctttat gggtgtttca tagatgtcca 120
aaatatattg agatattgag atataacatt ctaggataat caaaattacc ataattcaaa 180
agctcgtatg gcgcagtggt agcgcagcag attgcaaatc tgttggtcct tagttcgatc 240
ctgagtgcga gctttctttt ttagactact aattttattt tgctagtcat ttttttttta 300
tactcaaaaa gtaaaaagac tacgagtata ttcaaagtaa aaaacgaacg tcaaactatc 360
tcgattaaaa cttgtcatac tgtgggtatc atattctgtg ccctcagtga aaagaaccag 420
caaaagaacg cgcatctcga gtgaagacgc gcccttgatg gtacaaaatt taacgggaag 480
gcgcgtcgtg atgttcacgc gctttgccca cattgggata gcgcccacag catatctgtg 540
ctaaactcac ttttcctagt gactgccgat agctactgcc atctaccgcg aagggaactt 600
catttgcgtt catcggttta ttagaagcta cttggaacta attcttaagc ttctcaagaa 660
aagttttttt tctgtctatc tattgaagtc tttttgtctt tgtacttcaa gagactcaat 720
cacctaaagc ttttcacggc caattagttg tctcacacaa agcaaaataa gcttaataat 780
tagcagtaac gcgcttttcc ctgtatttaa agccgctgaa cacctttact gaacaatggg 840
agagaaccac gaccatgagc agagtattaa aagaaattct atgatttata atgaaaatga 900
gaggcagttg tgcaattcaa acctaaagat tcttcaaaat aaaagggccc tttcaaaaaa 960
tgacagctct agtaagcagc aggttcagga ttctaaacca 1000
<210> 90
<211> 391
<212> DNA
<213> Saccharomyces cerevisae
<400> 90
ctcgtaggaa caatttcggg cccctgcgtg ttcttctgag gttcatcttt tacatttgct 60
tctgctggat aattttcaga ggcaacaagg aaaaattaga tggcaaaaag tcgtctttca 120
aggaaaaatc cccaccatct ttcgagatcc cctgtaactt attggcaact gaaagaatga 180
aaaggaggaa aatacaaaat atactagaac tgaaaaaaaa aaagtataaa tagagacgat 240
atatgccaat acttcacaat gttcgaatct attcttcatt tgcagctatt gtaaaataat 300
aaaacatcaa gaacaaacaa gctcaacttg tcttttctaa gaacaaagaa taaacacaaa 360
aacaaaaagt ttttttaatt ttaatcaaaa a 391
<210> 91
<211> 248
<212> DNA
<213> Saccharomyces cerevisae
<400> 91
tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg 60
aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 120
tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 180
acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 240
taatttgc 248
<210> 92
<211> 278
<212> DNA
<213> Saccharomyces cerevisae
<400> 92
tcaataccgc ctccggcggt attgaaggag aaaaatttgg gcgggagcat attattgtaa 60
ttaatatata catatatata tatatatata catcaacctt acaactatgc gtttagaatc 120
ttaagcggta actttctttt caatcaagtc gaatagagta acaatatcgg cagagaattt 180
tctgatacct tcggacaatt tttcagtggc catagcgtct tcattcaagt cgaatctgaa 240
tttagattcg tcgctgatgt aagaaatctt gtcgccgg 278
<210> 93
<211> 576
<212> DNA
<213> Artificial Sequence
<220>
<223> NATR
<400> 93
atgggtacca ctcttgacga cacggcttac cggtaccgca ccagtgtccc gggggacgcc 60
gaggccatcg aggcactgga tgggtccttc accaccgaca ccgtcttccg cgtcaccgcc 120
accggggacg gcttcaccct gcgggaggtg ccggtggacc cgcccctgac caaggtgttc 180
cccgacgacg aatcggacga cgaatcggac gacggggagg acggcgaccc ggactcccgg 240
acgttcgtcg cgtacgggga cgacggcgac ctggcgggct tcgtggtcat ctcgtactcg 300
gcgtggaacc gccggctgac cgtcgaggac atcgaggtcg ccccggagca ccgggggcac 360
ggggtcgggc gcgcgttgat ggggctcgcg acggagttcg ccggcgagcg gggcgccggg 420
cacctctggc tggaggtcac caacgtcaac gcaccggcga tccacgcgta ccggcggatg 480
gggttcaccc tctgcggcct ggacaccgcc ctgtacgacg gcaccgcctc ggacggcgag 540
cggcaggcgc tctacatgag catgccctgc ccctaa 576
<210> 94
<211> 306
<212> DNA
<213> Saccharomyces cerevisae
<400> 94
gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 60
tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttgttctt gagtaactct 120
ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc 180
tctaccggca tgccgagcaa atgcctgcaa atcgctcccc atttcaccca attgtagata 240
tgctaactcc agcaatgagt tgatgaatct cggtgtgtat tttatgtcct cagaggacaa 300
cacctg 306
Claims (15)
- i) 자일룰로스 키나제 (XKS1), 트랜스알돌라제 (TAL1), 트랜스케톨라제 1 (TKL1) 및 트랜스케톨라제 2 (TKL2)를 코딩하는 천연 유전자의 과다발현 및
ii) 자일로스 이소머라제 (XI)를 코딩하는 기능적 이종 유전자의 발현 - 여기서 자일로스 이소머라제 (XI) 유전자는 써모토가 네아폴리타나 (Thermotoga neapolitana), 안디탈레아 안덴시스 (Anditalea andensis) 및 클로스트리듐 클라리플라붐 (Clostridium clariflavum)으로 이루어진 군으로부터 선택된 미생물로부터 유래됨 -
을 위한 하나 이상의 발현 구축물(들)로 형질전환된 미생물, 특히 바람직하게는 사카로마이세스 세레비지애 (Saccharomyces cerevisiae) 종의 효모. - 제1항에 있어서, 자일로스 이소머라제 (XI)가 서열식별번호: 21, 서열식별번호: 5 또는 서열식별번호: 25에 대해 적어도 66%, 바람직하게는 적어도 80%, 더 바람직하게는 적어도 90%, 가장 바람직하게는 적어도 95% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 것인 미생물.
- 제1항 또는 제2항에 있어서, 자일로스 이소머라제 (XI)가 서열식별번호: 22, 서열식별번호: 6 또는 서열식별번호: 26에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 아미노산 서열로 나타내어지는 것인 미생물.
- 제1항 내지 제3항 중 어느 한 항에 있어서, 자일룰로스 키나제 (XKS1)가 서열식별번호: 74에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되고, 트랜스알돌라제 (TAL1)가 서열식별번호: 77에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되고, 트랜스케톨라제 1 (TKL1)이 서열식별번호: 80에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되고, 트랜스케톨라제 2 (TKL2)가 서열식별번호: 83에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 것인 미생물.
- 제1항 내지 제4항 중 어느 한 항에 있어서, 자일룰로스 키나제 (XKS1), 트랜스알돌라제 (TAL1), 트랜스케톨라제 1 (TKL1), 트랜스케톨라제 2 (TKL2) 및 자일로스 이소머라제 (XI)를 코딩하는 유전자 각각이 구성적 프로모터의 제어 하에 있고, 여기서 구성적 프로모터는 서열식별번호: 73에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 TDH3, 서열식별번호: 76에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 PGK1, 서열식별번호: 79에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 CYC19, 서열식별번호: 82에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 PFK1, 서열식별번호: 90에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 말단절단된 HXT7 및 서열식별번호: 85에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 TEF로부터 선택되는 것인 미생물.
- 티. 네아폴리타나 (T. neapolitana), 에이. 안덴시스 (A. andensis) 및 씨. 클라리플라붐 (C. clariflavum)으로 이루어진 군으로부터 선택된 미생물로부터 유래된 자일로스 이소머라제 (XI)를 코딩하는 유전자의 발현을 위한 발현 구축물이며, 여기서 자일로스 이소머라제 (XI) 유전자는 사카로마이세스 세레비지애의 구성적 프로모터의 제어 하에 있는 것인 발현 구축물.
- 제6항에 있어서, 자일로스 이소머라제 (XI)를 코딩하는 유전자가 서열식별번호: 21, 서열식별번호: 5 또는 서열식별번호: 25에 대해 적어도 66%, 바람직하게는 적어도 80%, 더 바람직하게는 적어도 90%, 가장 바람직하게는 적어도 95% 서열 동일성을 갖는 핵산 서열로 나타내어지는 것인 발현 구축물.
- 제6항 또는 제7항에 있어서, 구성적 프로모터가 서열식별번호: 90에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 말단절단된 HXT7 및 서열식별번호: 85에 대해 적어도 80%, 바람직하게는 적어도 90%, 더 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 서열 동일성을 갖는 핵산 서열에 의해 코딩되는 TEF로부터 선택되는 것인 발현 구축물.
- 제1항 내지 제5항 중 어느 한 항에 따른 미생물을 펜토스 당(들)을 포함하는 배양 배지에서 펜토스 당(들)이 대사될 수 있는 조건 하에서 배양하는 단계를 포함하는, 펜토스 당을 발효시키는 방법.
- 제9항에 있어서, 배양 배지가 리그노셀룰로스 바이오매스 및/또는 그의 가수분해물을 포함하거나 그로 이루어지는 것인 방법.
- 제9항 또는 제10항에 있어서, 발효가 에탄올, 메탄올, 프로판올, 이소프로판올, 부탄올, 에틸렌 글리콜, 프로필렌 글리콜, 1,4-부탄디올, 글리세린, 포름산, 아세트산, 프로피온산, 부티르산, 발레르산, 카프로산, 팔미트산, 스테아르산, 옥살산, 말론산, 숙신산 또는 숙시네이트, 글루타르산, 올레산, 리놀레산, 글리콜산, 락트산 또는 락테이트, 감마-히드록시부티르산, 3-히드록시알칸산, 알라닌, 메탄, 에탄, 프로판, 펜탄, n-헥산, 피루베이트, 아스파르테이트, 말레이트, 발린 및 류신으로부터 선택되는 하나 이상의 화합물, 바람직하게는 에탄올을 생성하는 것인 방법.
- 펜토스 당(들), 특히 자일로스의 발효, 바람직하게는 리그노셀룰로스 바이오매스로부터 에탄올의 생성을 위한 제1항 내지 제5항 중 어느 한 항에 따른 미생물의 용도.
- 사카로마이세스 세레비지애 균주를 제1항에서 정의된 바와 같은 또는 제6항 내지 제8항 중 어느 한 항에 따른 발현 구축물(들)로 형질전환시키는 단계를 포함하는, 미생물, 바람직하게는 제1항 내지 제5항 중 어느 한 항에 따른 미생물을 생성하는 방법.
- 제13항에 있어서, 제6항 내지 제8항 중 어느 한 항에 따른 발현 구축물이 사카로마이세스 세레비지애 균주의 염색체로 통합되는 것인 방법.
- 제13항 또는 제14항에 있어서, 제6항 내지 제8항 중 어느 한 항에 따른 발현 구축물이 자일룰로스 키나제 (XKS1), 트랜스알돌라제 (TAL1), 트랜스케톨라제 1 (TKL1) 및 트랜스케톨라제 2 (TKL2)에 대한 천연 유전자의 과다발현을 위한 재조합 발현 구축물로 통합되는 것인 방법.
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US201862648619P | 2018-03-27 | 2018-03-27 | |
US62/648,619 | 2018-03-27 | ||
PCT/EP2019/057753 WO2019185737A1 (en) | 2018-03-27 | 2019-03-27 | Xylose metabolizing yeast |
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KR20200135469A true KR20200135469A (ko) | 2020-12-02 |
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US (1) | US20210017526A1 (ko) |
EP (1) | EP3775219A1 (ko) |
KR (1) | KR20200135469A (ko) |
CN (1) | CN112004930A (ko) |
WO (1) | WO2019185737A1 (ko) |
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BR0306740A (pt) | 2002-01-23 | 2004-12-28 | Royal Nedalco B V | Célula hospedeira transformada com um construto de ácido nucléico, molécula de ácido nucleico isolada, e, processos para a produção de etanol, e de um produto de fermentação |
AU2003219671A1 (en) * | 2002-01-23 | 2003-09-02 | Michigan State University | Thermotoga neapolitana xylose isomerase polypeptides and nucleic acids encoding same |
CN1922198B (zh) | 2003-05-02 | 2012-07-11 | 卡吉尔公司 | 基因修饰酵母物种和使用基因修饰酵母的发酵方法 |
FR2898131B1 (fr) * | 2006-03-01 | 2012-08-31 | Mane Fils V | Systeme de production de molecules aromatiques par bioconversion |
US7998722B2 (en) * | 2008-03-27 | 2011-08-16 | E. I. Du Pont De Nemours And Company | Zymomonas with improved xylose utilization |
DE102008031350B4 (de) | 2008-07-02 | 2011-02-10 | Johann Wolfgang Goethe-Universität Frankfurt am Main | Prokaryotische Xylose-Isomerase zur Konstruktion Xylose-vergärender Hefen |
GB0812318D0 (en) * | 2008-07-04 | 2008-08-13 | Terranol As | Microorganism |
GB0822937D0 (en) | 2008-12-16 | 2009-01-21 | Terranol As | Microorganism |
EP2451960A2 (en) | 2009-07-09 | 2012-05-16 | Verdezyne, Inc. | Engineered microorganisms with enhanced fermentation activity |
WO2011078262A1 (ja) * | 2009-12-22 | 2011-06-30 | 株式会社豊田中央研究所 | キシロースイソメラーゼ及びその利用 |
BR112012028290B1 (pt) * | 2010-05-05 | 2021-02-02 | Lallemand Hungary Liquidity Management Llc. | levedura recombinante, processo para converter biomassa em etanol e meio de fermentação compreendendo dita levedura |
AR087423A1 (es) * | 2011-08-04 | 2014-03-19 | Dsm Ip Assets Bv | Celula capaz de fermentar azucares pentosas |
EP2877576B1 (en) * | 2012-07-24 | 2019-06-05 | BP Corporation North America Inc. | Xylose isomerases and their uses |
US9200291B2 (en) * | 2012-12-19 | 2015-12-01 | Helge Zieler | Compositions and methods for creating altered and improved cells and organisms |
WO2014133092A1 (ja) * | 2013-02-27 | 2014-09-04 | トヨタ自動車株式会社 | 組換え酵母を用いたエタノールの製造方法 |
AR097480A1 (es) * | 2013-08-29 | 2016-03-16 | Dsm Ip Assets Bv | Células de levadura convertidoras de glicerol y ácido acético con una conversión de ácido acético mejorada |
US10619174B2 (en) * | 2014-07-25 | 2020-04-14 | Alderys | Microorganism strains for the production of 2.3-butanediol |
DK3209677T3 (da) * | 2014-10-22 | 2020-08-03 | Lallemand Hungary Liquidity Man Llc | Varianter af gal2-transporter og anvendelser deraf |
PL3026116T3 (pl) * | 2014-11-26 | 2017-09-29 | Clariant International Ltd | Sekwencja oligonukleotydowa do stosowania w modyfikacji szlaku |
US10612032B2 (en) * | 2016-03-24 | 2020-04-07 | The Board Of Trustees Of The Leland Stanford Junior University | Inducible production-phase promoters for coordinated heterologous expression in yeast |
WO2017164388A1 (ja) * | 2016-03-25 | 2017-09-28 | 国立研究開発法人産業技術総合研究所 | 変異型キシロース代謝酵素とその利用 |
US10689670B2 (en) * | 2016-06-14 | 2020-06-23 | Dsm Ip Assets B.V. | Recombinant yeast cell |
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- 2019-03-27 US US17/041,320 patent/US20210017526A1/en active Pending
- 2019-03-27 EP EP19714403.3A patent/EP3775219A1/en active Pending
- 2019-03-27 WO PCT/EP2019/057753 patent/WO2019185737A1/en unknown
- 2019-03-27 CN CN201980022435.7A patent/CN112004930A/zh active Pending
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CN112004930A (zh) | 2020-11-27 |
US20210017526A1 (en) | 2021-01-21 |
EP3775219A1 (en) | 2021-02-17 |
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