KR102290728B1 - Nadph 의존적 효소를 포함하는 재조합 미생물 및 이의 생산방법 - Google Patents
Nadph 의존적 효소를 포함하는 재조합 미생물 및 이의 생산방법 Download PDFInfo
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- KR102290728B1 KR102290728B1 KR1020157023535A KR20157023535A KR102290728B1 KR 102290728 B1 KR102290728 B1 KR 102290728B1 KR 1020157023535 A KR1020157023535 A KR 1020157023535A KR 20157023535 A KR20157023535 A KR 20157023535A KR 102290728 B1 KR102290728 B1 KR 102290728B1
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Abstract
본 발명은 모체 미생물에 대해 NADPH의 증가된 전체 활용도를 갖는 재조합 일산화탄소영양 클로스트리디아 미생물을 제공한다. 추가로, 본 발명은 모체 미생물에 대해 NADPH의 증가된 전체 활용도를 나타내는 재조합 일산화탄소영양 클로스트리디아 미생물의 생산방법을 제공한다. 특히 본 발명은 상기 미생물에 의한 하나 이상의 발효생성물의 생산을 증가시키기 위하여 재조합 일이산화탄소영양 클로스트리디아 미생물에서 NADPH의 전체 활용도를 증가시키는 것에 관한 것이다.
Description
본 발명은 발효에 의해 원하는 화합물의 생산을 최적화하기 위한 효소의 선택방법에 관한 것이다. 보다 구체적으로는, 그러나 배타적이지 않지만, 본 발명은 발효 경로 및 대사성 조작에 있어서 균형을 유지시키는 보조인자에 관한 것이다.
니코틴아미드 아데닌 디뉴클레오티드(NADH) 및 니코틴아미드 아데닌 디뉴클레오티드 포스페이트(NADPH)와 같은 환원 균등물은 산화환원효소 반응과 같은 효소적 산화환원 반응을 위해 중요한 조효소이고, 모든 살아있는 세포에서 발견된다. NADPH 풀(pool)이 NADH 풀에 비해 현저히 작다는 것은 일반적으로 받아들여지고 있다(G. N. Bennett & San, 2009). 글루코오스 당에서 성장한 대장균에서, NADH 풀은 NADPH 풀에 비해 20배 이상 더 크다(B. D. Bennett et al., 2009). 이러한 낮은 NADPH 이용가능성은 특히 발효 공정에서 많은 생합성 반응 및 생전환을 제한한다(R Poulsen et al., 2005). NADPH에 대한 효소의 선호는 원하는 생성물의 생산을 제한할 수 있다(G. N. Bennett & San, 2009). 이것은 미생물 내로 새로운 반응 및 경로를 조작하는 경우 문제가 되고, 생연료, 화학물질, 아미노산 또는 비타민을 포함하는 화합물의 충분한 생산 플랫폼을 생성하는데 있어서 주요 장애물 중 하나이다(Chemler, Fowler, McHugh, & Koffas, 2010).
그럼에도 불구하고, 대사성 조작은 가능하면 NADPH 의존적 반응을 제한, 회피, 또는 우회함으로써, 광범위한 연료 및 화학물질의 생산을 위해 성공적으로 입증되었다(Peralta-Yahya & Keasling, 2010). 대안적으로, 에너지-소비성 수소전달효소는 NADH와 NADPH 풀 간의 상호전달에 사용되어져 왔다. 성공적인 대사성 조작을 달성하기 위한 다른 전략은 경쟁 NADPH 의존적 반응의 제거이다. 이들 발전에도 불구하고, 이러한 신규 전략들은 종종 생산 수율 및/또는 성장률의 대가를 지불하여 추구된다(Auriol, Bestel-Corre, Claude, Soucaille, & Meynial-Salles, 2011). 또한, 이들 전략은 오로지 복합 변형을 수반하는 대규모 조작 작동에 의해서만 가능하게 된다(S. M. Ma et al., 2011). 따라서 이들 노력은 대장균(Escherichia coli) 및 사카로마이세스 세레비지애(Saccharomyces cerevisiae)와 같은 유전적으로 다루기 쉬운 유기체로만 제한되어 왔다(Peralta-Yahya & Keasling, 2010). 이들 유기체는 오로지 당을 섭취하기 때문에 제한된다. 따라서, 이들의 상업적 사용 및 실행가능성은 토지-사용, 식량-보안, 공급의 변동 및 환경 문제를 둘러싼 심각한 문제를 겪는다.
일산화탄소영양 클로스트리디아(Carboxydotrophic Clostridia)는 대장균 및 에스. 세레비지애(S. cerevisiae)에 대해 대안을 제공하고 폐기 가스 및 합성가스 상에서 성장할 수 있다. 제한된 수의 변형을 갖는 재조합 일산화탄소영양 클로스트리디아의 몇몇 예가 있다(Schiel-Bengelsdorf & Durre, 2012). 모든 알려진 예들은 NADH-의존적 반응을 사용한다.
본 발명의 목적은 선행 기술의 단점중의 하나 이상을 극복하거나 또는 개선하거나, 적어도 대중에게 유용한 선택권을 제공하는 것이다.
발명의 요약
제 1 측면에서, 본 발명은 하나 이상의 외인성 NADPH-의존적 효소를 발현하도록 개조하고 및/또는 하나 이상의 내인성 NADPH-의존적 효소를 과-발현하도록 개조된 재조합 일산화탄소영양 클로스트리디아 미생물을 제공하며, 상기 효소는, 외인성 효소가 발현되고 및/또는 내인성 효소가 과발현되는 경우, 상기 미생물에 의한 NADPH의 전체 활용도가 모체 미생물에 비해 증가되도록 선택된다.
제 2 측면에서, 본 발명은 하기 단계 a) 및 b)를 포함하는, 모체 미생물에 비해 증가된 NADPH 활용도를 나타내는 재조합 일산화탄소영양 클로스트리디아 미생물의 생산방법을 제공한다:
a. 하나 이상의 외인성 및/또는 내인성 NADPH-의존적 효소를 선택하는 단계; 및
b. 모체 미생물을 형질전환시켜 하나 이상의 NADPH-의존적 외인성 효소를 발현하도록 개조되고 및/또는 하나 이상의 NADPH-의존적 내인성 효소를 과-발현하는데 개조된 재조합 미생물을 얻는 단계.
미생물 내의 임의의 하나 이상의 NADPH-의존적 효소의 상기 발현 또는 과-발현은 모체 미생물에 비해 NADPH의 활용도에 있어서 전체적인 증가를 초래한다.
본 발명은 또한 제 2 측면의 방법에 의해 만들어진 재조합 이산화탄소영양 클로스트리디아를 제공한다.
제1 또는 제2 측면의 특정한 실시양태에서, 하나 이상의 NADPH-의존적 효소는 수소화효소(예를 들어, 서열번호 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, YP_003781016, YP_003781017, YP_003778879, YP_003779640, YP_003779893, YP_003780193 또는 이의 임의의 하나의 기능적 균등 변이체), 포르메이트 탈수소화효소 (예를 들어 AEI90721, AEI90723, AEI90725, YP_003779063, YP_003778871, YP_003780168, AEI90722, AEI90724, AEI90726 또는 이의 임의의 하나의 기능적 균등 변이체) 또는 메틸렌-THF-탈수소화효소(예를 들어 AEI90753, YP_003781891, AEI90771 또는 이의 임의의 하나의 기능적 균등 변이체)를 포함한다.
제1 또는 제2 측면의 특정한 실시양태에서, 하나 이상의 NADPH-의존적 효소는 NADH- 및 NADPH-의존적 이소형으로 존재하고 또한 재조합 미생물은 NADPH-의존적 이소형을 발현하고/하거나 과발현하도록 개조된다.
특정한 실시양태에서, 미생물은 NADPH-의존적 이소형을 발현하고/하거나 과-발현하도록 개조되고, 여기서 상응하는 NADH-의존적 이소형의 발현은 NDPH-의존적 이소형의 발현에서의 변화와 비교할 때 실질적으로 변화가 없거나, 감소되거나, 상대적으로 더 적은 증가를 나타낸다. 하나의 특정한 실시양태에서, 미생물은 개조되며, 따라서 하나 이상의 NADH-의존적 이소형의 발현이 모체 미생물에 비해 약화되거나 녹 아웃되도록 개조된다. 하나의 실시양태에서, 발현은 하나 이상의 NADH-의존적 효소를 인코딩하는 핵산을 변형시키거나 NADH-의존적 이소형을 인코딩하는 하나 이상의 핵산을 NADPH-의존적 이소형을 인코딩하는 하나 이상의 핵산과 대체시켜 약화 또는 녹 아웃된다.
제1 또는 제2 측면의 특정한 실시양태에서, NADPH의 전체적인 활용도에 있어서의 증가는 하나 이상의 NADPH-의존적 효소가 활성인 경로를 통한 NADPH 유량에서의 증가를 포함한다. 특정한 실시양태에서, 유량(flux)은 적어도 5%, 적어도 10%, 적어도 20%, 적어도 50%, 적어도 100% 증가된다. 경로를 통한 유량은 대사산물 및 생성물의 수준을 측정하고/하거나(대사체학(metabolomics)) (Patti, Yanes, & Siuzdak, 2012) C13으로서 실험을 표지화함으로써(유량학(fluxomics)) (Niittylae, Chaudhuri, Sauer, & Frommer, 2009; Tang et al., n.d.) 측정될 수 있다.
제1 또는 제2 측면의 하나의 특정한 실시양태에서, NADPH의 전체 활용도에 있어서의 증가는, 이용 시, 미생물에 의한 하나 이상의 생성물의 생성의 효율에 있어서의 증가를 초래한다.
하나의 특정한 실시양태에서, NADPH- 및 NADH-의존적 이소형으로 존재하는 하나 이상의 효소는 하이드록시메틸글루타릴-CoA (HMG-CoA) 환원효소이고, NADPH-의존적 이소형(EC 1.1.1.34; GO:0004420; 예를 들어, 사카로마이세스 세레비지애: DAA09822.1; BK006946.2:115734..118898 또는 이의 임의의 하나의 기능적 균등 변이체) 및 NADH-의존적 이소형(EC1.1.1.88; GO:0042282; 예를 들어, 슈도모나스 메발로니이(Pseudomonas mevalonii): P13702.1 또는 이의 임의의 하나의 기능적 균등 변이체)를 포함한다.
하나의 특정한 실시양태에서, NADPH- 및 NADH-의존적 이소형으로 존재하는 하나 이상의 효소는 하이드록시부티릴-CoA 탈수소화효소/ 아세토아세틸-CoA 환원효소/3-하이드록시부티릴-CoA 수화효소이고, NADPH-의존적 이소형 phaB (EC:1.1.1.36; GO:0018454; 예를 들어, 랄스토리아 유트로파(Ralstonia eutropha): YP_725942.1, 유전자번호: 4249784 유래 또는 이의 임의의 하나의 기능적 균등 변이체), NADPH 의존적 phaJ(EC 4.2.1.119; 예를 들어, 아에로모나스 펀크타타(Aeromonas punctata): BAA21816.1 유래) 및 상응하는 NADH-의존적 이소형 hbd (EC 1.1.1.157; GO:0008691; 예를 들어, 씨. 아세토부틸리쿰(C. acetobutylicum): NP_349314.1, 유전자번호:1118891 유래 또는 이의 임의의 하나의 기능적 균등 변이체)를 포함한다.
하나의 특정한 실시양태에서, NADPH- 및 NADH-의존적 이소형으로 존재하는 하나 이상의 효소는 크로토닐-CoA 환원효소/ 트랜스-2-에노일-CoA 환원효소/부티릴-CoA 탈수소화효소이고, NADPH-의존적 이소형 ccr(EC 1.3.1.86; 예를 들어, 스트렙토마이세스 콜리누스(Streptomyces collinus) 유래 또는 이의 임의의 하나의 기능적 균등 변이체) 또는 ccr Rs (EC 1.3.1.85; 예를 들어, 로도박터 스파에로이데스(Rhodobacter sphaeroides): YP_354044.1, 유전자번호: 3720751 유래) 및 상응하는 NADH-의존적 이소형 ter(EC 1.3.1.44; GO:0050343; 예를 들어, 트리포네마 덴티콜라(Treponema denticola) 유래 또는 이의 임의의 하나의 기능적 균등 변이체)를 포함한다.
추가적인 실시양태에서, 효소는 NADH 및 NADPH 의존적 이소형으로 존재하고, 또한 복수의 보조인자 의존성을 나타낸다. 제2 측면의 하나의 실시양태에서, 복수의 보조-인자 의존성을 나타내는 효소는 NADH/페레독신 이분성 효소(ferredoxin bifurcating enzyme) 또는 NADH/NADPH 상호-의존적 효소를 포함할 수 있다. 특정한 실시양태에서, 효소는 NADH/NADPH 이분성 이소형 및 NADH/페레독신 이분성 이소형으로 존재하고, 미생물은 NADH/NADPH 의존적 이소형을 발현하고/하거나 과발현하도록 개조된다. 특정한 실시양태에서, NADH/NADPH 의존적 이소형은 ter(EC 1.3.1.44; GO:0050343; 예를 들어, Euglena gracilis: AY741582.1 유래 또는 이의 임의의 하나의 기능적 균등 변이체)이다. 추가적인 실시양태에서, NADH/Fd 의존적 이소형은 NADH/페레독신 이분성 bcd- etfAB 복합체(EC 1.3.8.1; GO:0004085; 예를 들어, 씨. 아세토부틸리쿰(C. acetobutylicum): NP_349317.1; 유전자번호:1118894 유래 또는 이의 임의의 하나의 기능적 균등 변이체)이다.
제1 또는 제2 측면의 특정한 실시양태에서, 재조합 미생물은 하나 이상의 NADH-의존적 효소의 약화된 발현을 나타낸다. 이 실시양태에서, 모체 미생물 내에 효소의 NADH-의존적 이소형은 재조합 미생물 내에 효소의 NADPH-의존적 이소형에 의해 대체된 것일 수 있다.
제1 또는 제2 측면의 특정한 실시양태에서, 미생물은 모체 미생물과 비교할 때 발효 반응 동안 증가된 효율을 나타낸다.
제1 또는 제2 측면의 하나의 특정한 실시양태에서, 모체 미생물은 클로스트리듐 오토에타노게눔(Clostridium autoethanogenum), 클로스트리듐 륭달리이(Clostridium ljungdahlii), 클로스트리듐 라그스달레이(Clostridium ragsdalei), 클로스트리듐 카복시디보란스(Clostridium carboxidivorans), 클로스트리듐 드라케이(Clostridium drakei ), 클로스트리듐 스카톨로게네스 (Clostridium scatologenes), 클로스트리듐 아세티쿰(Clostridium aceticum ), 클로스트리듐 포르미코아세티쿰(Clostridium formicoaceticum ), 클로스트리듐 마그눔(Clostridium magnum)을 포함하는 이산화탄소영양 클로스트리디아의 군으로부터 선택된다.
제 1 또는 제 2 측면의 하나의 실시양태에서, 모체 미생물은 클로스트리듐 오토에타노게눔 또는 클로스트리듐 륭달리이다. 하나의 특정한 실시양태에서, 미생물은 균주 DSM10061의 유도체인 클로스트리듐 오토에타노게눔 DSM23693 이다. 다른 특정한 실시양태에서, 미생물은 클로스트리듐 륭달리이 DSM13528 (또는 ATCC55383)이다.
제1 측면의 추가적인 실시양태에서, 하나 이상의 NADPH-의존적 효소는 그의 NADH 보조인자 특이성에 비해 그의 NADPH 보조인자 특이성을 증가시키기 위해 변형된다.
제2 측면의 추가적인 실시양태에서, 상기 방법은 추가적으로 효소(들)의 NADH 보조인자 특이성이 비해 하나 이상의 NADPH-의존적 효소의 NADPH 보조인자 특이성을 증가시키는 단계를 포함한다. 하나의 실시양태에서, 이 단계는 하나 이상의 NADPH-의존적 효소를 인코딩하는 하나 이상의 핵산을 변형시키는 것을 포함한다.
특정한 실시양태에서, NADPH 보조-인자 특이성이 증가되는 하나 이상의 효소는 산화환원효소 효소(oxidoreductase enzyme), 바람직하게는, 크로토닐-CoA 환원효소/ 트랜스-2-에노일-CoA 환원효소/부티릴-CoA 탈수소화효소로 구성된 군으로부터 선택된다.
특정한 실시양태에서, 하나 이상의 외인성 또는 내인성 효소는 이분성 NADP Fe-유일(Fe-only) 수소화효소, 이분성 NADP 포르메이트 탈수소화효소, 및/또는 본 명세서에 기술된 바와 같은 포르메이트-하이드로겐 리아제 복합체(formate-hydrogen lyase complex), 또는 이의 기능적 균등 변이체를 포함한다.
추가적인 실시양태에서, 본 발명은 본 명세서에 기술된 임의의 측면에서 기술된 바와 같은 하나 이상의 변형을 갖는 제1 측면에 따른 재조합 미생물을 생성하는 방법을 제공한다
추가적인 실시양태에서, 본 발명은 본 명세서에 기술된 임의의 측면에서 기술된 바와 같은 하나 이상의 변형을 갖는 제2 측면에 따른 재조합 미생물을 생성하는 방법을 제공한다.
제3 측면에서, 본 발명은 하나 이상의 발효 생성물을 생성하는 방법을 제공하며, 상기 방법은 이산화탄소영양 미생물의 존재 하에 CO를 포함하는 기질을 혐기적으로 발효시키는 단계를 포함하고, 여기에서 이산화탄소영양 미생물은 제 1 측면에 의해 기술된 바와 같거나 제 2 측면에 의해 생성된 바와 같은 재조합 미생물이다.
특정한 실시양태에서, 하나 이상의 발효 생성물은 에탄올, 부탄올, 이소프로판올, 이소부탄올, 고급 알코올, 부탄디올, 석시네이트, 이소프레노이드, 지방산 및/또는 생중합체를 포함한다.
특정한 실시양태에서, CO를 포함하는 기질은 CO를 포함하는 가스성 기질이다. 하나의 실시양태에서, 기질은 산업 폐기 가스를 포함한다. 특정한 실시양태에서, 가스는 강철 제분 폐기물 또는 합성가스이다.
하나의 실시양태에서, 기질은 전형적으로 대부분의 CO, 예컨대 적어도 약 20 용적% 내지 약 100 용적%의 CO, 20 용적% 내지 70 용적%의 CO, 30 용적% 내지 60 용적%의 CO, 및 40 용적% 내지 55 용적%의 CO를 포함한다. 특정한 실시양태에서, 기질은 약 25 용적%, 또는 약 30 용적%, 또는 약 35 용적%, 또는 약 40 용적%, 또는 약 45 용적%, 또는 약 50 용적%의 CO, 또는 약 55 용적%의 CO, 또는 약 60 용적%의 CO를 포함한다.
제4 측면에서, 본 발명은 반응에서 복수의 보조인자를 활용하는 목적을 위해 이분성 NADP Fe-유일 수소화효소, 이분성 NADP 포르메이트 탈수소화효소, 및/또는 포르메이트-하이드로겐 리아제 복합체 또는 이의 기능적 균등 변이체의 사용을 제공한다. 바람직하게는, 복수의 보조인자는 페레독신 및 NADPH를 포함한다.
특정한 실시양태에서, 이분성 NADP 포르메이트 탈수소화효소는 AEI90721, YP_003778871, AEI90722, 및 이의 임의의 하나 이상의 기능적 균등 변이체로 구성되는 군으로부터 선택된다.
특정한 실시양태에서, 이분성 NADP Fe-유일 수소화효소는 서열번호 10, 서열번호 26 및 YP_003778879, 및 이의 임의의 하나 이상의 기능적 균등 변이체로 구성되는 군으로부터 선택된다.
특정한 실시양태에서, 이분성 포르메이트-하이드로겐 리아제 복합체는 서열번호 65 내지 67 또는 이의 기능적 균등 변이체 중 임의의 하나에 의해 인코딩된다.
제5 측면에서, 본 발명은 재조합 미생물을 제공하며, 여기에서 미생물은 외인성 이분성 NADP Fe-유일 수소화효소, 이분성 NADP 포르메이트 탈수소화효소, 및/또는 포르메이트-하이드로겐 리아제 복합체를 발현하고/하거나 내인성 이분성 NADP Fe-유일 수소화효소, 이분성 NADP 포르메이트 탈수소화효소, 및/또는 포르메이트-하이드로겐 리아제 복합체를 과발현하도록 개조되며, 따라서 상기 미생물이 반응에서 복수의 보조인자를 이용하도록 개조된다.
제 6 측면에서, 본 발명의 상기 방법은 반응에서 복수의 보조인자를 이용할 수 있는 재조합 미생물을 생산하는 방법을 제공하고, 적어도 하기 단계 a) 및 b)를 포함한다:
a) 하나 이상의 이분성 NADP Fe-유일 수소화효소, 이분성 NADP 포르메이트 탈수소화효소, 및/또는 포르메이트-하이드로겐 리아제 복합체를 선택하는 단계,
b) 모체 미생물을 형질전환시켜 반응에서 복수의 보조인자를 활용하도록 개조된 재조합 미생물을 생산하는 단계.
제5 또는 제6 측면의 하나의 실시양태에서, 복수의 보조인자는 페레독신 및 NADPH를 포함한다.
제5 또는 제6 측면의 하나의 실시양태에서, 모체 미생물은 일산화탄소영양 클로스트리디아이다. 하나의 실시양태에서, 모체 미생물은 클로스트리듐 오토에타노게눔, 클로스트리듐 륭달리이, 클로스트리듐 라그스달레이, 클로스트리듐 카복시디보란스, 클로스트리듐 드라케이, 클로스트리듐 스카톨로게네스, 클로스트리듐 아세티쿰, 클로스트리듐 포르미코아세티쿰, 클로스트리듐 마그눔을 포함하는 이산화탄소영양 클로스트리디아의 군으로부터 선택된다. 하나의 실시양태에서, 모체 미생물은 클로스트리듐 오토에타노게눔 또는 클로스트리듐 륭달리이다. 하나의 특정한 실시양태에서, 미생물은 균주 DSM10061의 유도체인 클로스트리듐 오토에타노게눔 DSM23693 이다. 다른 특정한 실시양태에서, 미생물은 클로스트리듐 륭달리이 DSM13528 (또는 ATCC55383)이다.
제 5 또는 제 6 측면의 하나의 실시양태에서, 이분성 NADP 포르메이트 탈수소화효소는 AEI90721, YP_003778871, AEI90722, 및 이의 임의의 하나 이상의 기능적 균등 변이체로 구성된 군으로부터 선택된다.
제5 또는 제6 측면의 하나의 실시양태에서, 이분성 NADP Fe-유일 수소화효소는 서열번호 10, 서열번호 26 및 YP_003778879, 및 이의 임의의 하나 이상의 기능적 균등 변이체로 구성된 군으로부터 선택된다.
제5 또는 제6 측면의 하나의 실시양태에서, 이분성 포르메이트-하이드로겐 리아제 복합체는 서열번호 65 내지 67 또는 이의 기능적 균등 변이체에 의해 인코딩된다.
제5 또는 제6 측면의 특정한 실시양태에서, 모체 미생물은 이분성 NADP Fe-유일 수소화효소, 이분성 NADP 포르메이트 탈수소화효소, 및/또는 포르메이트-하이드로겐 리아제 복합체를 인코딩하는 하나 이상의 외인성 폴리뉴클레오티드로 형질전환된다. 하나의 특정한 실시양태에서, 모체 미생물은 HQ876015, CLJU_c06990, AEI90722, 서열번호 9, 서열번호 25, CLJU_c07070, 서열번호 서열번호 65 내지 67 및 이의 임의의 하나 이상의 기능적 균등 변이체로 구성된 군으로부터 선택된 하나 이상의 외인성 폴리뉴클레오티드로 형질전환된다.
관련된 측면에서, 본 발명은 반응에서 복수의 보조인자를 활용하는 목적을 위해, 이분성 NADP Fe-유일 수소화효소, 이분성 NADP 포르메이트 탈수소화효소, 및/또는 포르메이트-하이드로겐 리아제 복합체를 포함하는 재조합 미생물의 사용을 제공한다. 바람직하게는, 복수의 보조인자는 페레독신 및 NADPH를 포함한다. 하나의 실시양태에서, 이분성 NADP Fe-유일 수소화효소, 이분성 NADP 포르메이트 탈수소화효소, 및/또는 포르메이트-하이드로겐 리아제 복합체는 제4 측면에서 기술된 바와 같다.
제7 측면에서, 본 발명은 반응의 효율을 증가시키기 위한 방법을 제공하며, 상기 방법은 이분성 NADP Fe-유일 수소화효소, 이분성 NADP 포르메이트 탈수소화효소, 및/또는 포르메이트-하이드로겐 리아제 복합체 및/또는 이를 인코딩하는 폴리뉴클레오티드, 및/또는 이를 발현하고/하거나 과발현하도록 개조된 재조합 미생물의 사용을 포함한다. 특정한 실시양태에서, 반응은 CO를 포함하는 기질의 발효이다. 효율은 NADPH 만을 활용하는 것에 비해 페레독신과 NADPH를 둘다 이용하는 이분성 효소로 인해 증가된다. 이론으로 구속받고 싶지 않지만, 본 발명자들은 NAD(P)H (E0? = -320 mV)에 대해 페레독신 (E0? = -410 mV)의 더욱 음성인 산화환원 전위를 커플링하는 것이 더 큰 에너지 전위를 제공하고 더욱 에너지방출성인 반응을 구동하므로 반응 속도 및 CO 기질 처리량을 증가시키는 것으로 여긴다.
특정한 실시양태에서, 제7 측면의 이분성 NADP Fe-유일 수소화효소, 이분성 NADP 포르메이트 탈수소화효소, 및/또는 포르메이트-하이드로겐 리아제 복합체는 제4 측면에서 기술된 것과 같다.
제8 측면에서, 본 발명은 NADH를 NADPH로 전환시키기 위해 재조합 미생물을 사용하는 것을 제공하며, 여기서 재조합 미생물은 단일 NADH-의존적인 환원된 페레독신:NADP+ 산화환원효소 (Nfn) 효소를 발현하고/하거나 과발현하도록 개조된다. 특정한 실시양태에서, Nfn 효소는 서열번호 2, 4, YP_003781852.1, CLJU_c37240 또는 적어도 76%, 80%, 85%, 90%, 95%, 또는 99% 서열 동일성을 갖는 이의 임의의 하나의 기능적 균등 변이체의 아미노산 서열을 포함한다. Nfn 효소는 NADH를 NADPH로 전환시키므로 NADH 및 NADPH-의존적 효소의 존재 하에 발현되는 경우, 효소 효율이 증가되어 더 빠른 반응 속도 및 더 빠른 NADPH의 재생 속도를 유도한다.
특정한 실시양태에서, 미생물은 일산화탄소영양 클로스트리디아 미생물을 포함한다. 추가적인 실시양태에서, 미생물은 클로스트리듐 오토에타노게눔, 클로스트리듐 륭달리이, 클로스트리듐 라그스달레이를 포함하는 이산화탄소영양 클로스트리디아의 군으로부터 선택된다.
추가적인 실시양태에서, 본 발명은 제8 측면에서 기술된 바와 같은 사용을 제공하며, 여기에서 재조합 미생물은 제5 측면에서 기술된 바와 같은 하나 이상의 변형을 포함한다.
제9 측면에서, 본 발명은 NADH를 NADPH로 전환시키는 폴리펩티드의 사용을 제공하며, 여기서 폴리펩티드는 서열번호 2, 4, YP_003781852.1, CLJU_c37240에 따른 단일 NADH-의존적 환원된 페레독신:NADP+ 산화환원효소(Nfn) 효소 또는 적어도 76%, 80%, 85%, 90%, 95%, 또는 99% 서열 동일성을 갖는 이의 기능적 균등 변이체를 포함한다.
특정한 실시양태에서, 제8 또는 제9 측면의 단일 Nfn 효소는 폴리뉴클레오티드 서열번호 1, 3, YP_003781852.1 또는 CLJU_c37240을 인코딩하는 서열, 또는 적어도 83%, 85%, 90%, 95%, 또는 99% 서열 동일성을 갖는 이의 기능적 균등 변이체에 의해 인코딩된다.
제10 측면에서, 본 발명은 서열번호 1 또는 3에 따른 폴리뉴클레오티드를 제공한다.
제11 측면에서, 본 발명은 서열번호 2 또는 4에 따른 폴리뉴클레오티드를 제공한다.
제12 측면에서, 본 발명은 제10 측면에 따른 폴리뉴클레오티드, 또는 제 11 측면에 따른 폴리펩티드를 인코딩하는 폴리뉴클레오티드를 포함하는 벡터를 제공한다.
제13 측면에서 본 발명은 제10 측면에 따른 폴리뉴클레오티드, 제11 측면에 따른 폴리펩티드를 발현하도록 변경된 재조합 미생물을 제공한다.
본 발명은 또한 본원의 명세서에 언급되거나 표시된 부분, 구성요소 및 특징으로, 개별적으로 또는 총체적으로, 두개 이상의 상기 부분, 구성요소 및 특징의 임의의 또는 모든 조합으로 하여, 이루어진다고 광범위하게 일컬어질 수 있고, 여기서 특정한 정수는 본 발명이 관련한 분야에서 공지된 등가물을 갖는 본 명세서에서 언급되고, 이러한 공지된 등가물은 개별적으로 기재된 것처럼 본 명세서에 포함된 것으로 간주된다.
이하 본 발명의 실시양태는, 단지 예로써, 첨부한 도면을 참조하여 설명될 것이다.
도 1은 그의 보조-인자 특이성을 결정하기 위한 Wood Ljungdahl 경로에서 수반된 산화환원효소 단계를 위한 효소 검정의 결과를 보여준다;
도 2는 보조인자 사용에 대한 일산화탄소영양 클로스트리듐 (예를 들어, 씨. 아우토에타노제눔(C. autoethanogenum))에서 Wood-Ljungdahl 경로를 통한 해당작용(예들 들어, 대장균에서)과 자가영양 성장 간의 차이점을 보여준다;
도 3은 포르메이트-하이드로겐 리아제 복합체를 형성할 수 있는 포르메이트 탈수소화효소 및 수소화효소 유전자의 구성을 보여준다;
도 4는 자가영양 성장 동안 높이 발현된 NADPH 의존적 반응을 강조하는, qRT-PCR 유전자 발현 결과의 분포를 보여준다;
도 5는 씨. 아우토에타노제눔의 이차 알코올 탈수화효소 및 아세톤을 기질로서 NADPH 또는 NADH를 보조-인자로서 이용하는 효소 검정의 결과를 보여준다. 활성은 NADH가 아닌 오로지 NADPH를 이용해 측정되고 이는 이 효소가 엄중하게 NADPH 의존적임을 증명한다; EH한
도 6은 NADPH 의존적 이차 알코올 탈수화효소 효소를 통한 빠른 속도의 아세톤에서 이소프로판올로의 지속적인 전환을 보여준다. 바이오리액터(bioreactor) 내로 도입된 직후 아세톤이 이소프로판올로 전환되는 것이 관찰될 수 있다. 20 g/L의 높은 농도에서조차 배양은 모든 아세톤을 이소프로판올로 전환시켰는데, 이는 NADPH 풀이 빠른 속도에서 조차 이를 유지하기에 충분함을 증명한다;
도 7은 신규한 전자-이분성 NADP Fe-유일 수소화효소 / NADP 포르메이트 탈수소화효소/ 포르메이트-하이드로겐 리아제 복합체를 통해 CO 상에서 성장하는 동안 NADPH 구동된 생성물 형성을 보여준다.
도 8은 부탄올 생합성을 위한 완전한 NADPH-의존적 경로를 보여준다. 각 단계는 이탤릭체로 주석이 달린 유전자 내에 인코딩된 효소에 의해 촉매화된다.
도 9는 씨. 아우토에타노제눔 NADPH 및 NADH 수준의 분석을 보여준다.
도 1은 그의 보조-인자 특이성을 결정하기 위한 Wood Ljungdahl 경로에서 수반된 산화환원효소 단계를 위한 효소 검정의 결과를 보여준다;
도 2는 보조인자 사용에 대한 일산화탄소영양 클로스트리듐 (예를 들어, 씨. 아우토에타노제눔(C. autoethanogenum))에서 Wood-Ljungdahl 경로를 통한 해당작용(예들 들어, 대장균에서)과 자가영양 성장 간의 차이점을 보여준다;
도 3은 포르메이트-하이드로겐 리아제 복합체를 형성할 수 있는 포르메이트 탈수소화효소 및 수소화효소 유전자의 구성을 보여준다;
도 4는 자가영양 성장 동안 높이 발현된 NADPH 의존적 반응을 강조하는, qRT-PCR 유전자 발현 결과의 분포를 보여준다;
도 5는 씨. 아우토에타노제눔의 이차 알코올 탈수화효소 및 아세톤을 기질로서 NADPH 또는 NADH를 보조-인자로서 이용하는 효소 검정의 결과를 보여준다. 활성은 NADH가 아닌 오로지 NADPH를 이용해 측정되고 이는 이 효소가 엄중하게 NADPH 의존적임을 증명한다; EH한
도 6은 NADPH 의존적 이차 알코올 탈수화효소 효소를 통한 빠른 속도의 아세톤에서 이소프로판올로의 지속적인 전환을 보여준다. 바이오리액터(bioreactor) 내로 도입된 직후 아세톤이 이소프로판올로 전환되는 것이 관찰될 수 있다. 20 g/L의 높은 농도에서조차 배양은 모든 아세톤을 이소프로판올로 전환시켰는데, 이는 NADPH 풀이 빠른 속도에서 조차 이를 유지하기에 충분함을 증명한다;
도 7은 신규한 전자-이분성 NADP Fe-유일 수소화효소 / NADP 포르메이트 탈수소화효소/ 포르메이트-하이드로겐 리아제 복합체를 통해 CO 상에서 성장하는 동안 NADPH 구동된 생성물 형성을 보여준다.
도 8은 부탄올 생합성을 위한 완전한 NADPH-의존적 경로를 보여준다. 각 단계는 이탤릭체로 주석이 달린 유전자 내에 인코딩된 효소에 의해 촉매화된다.
도 9는 씨. 아우토에타노제눔 NADPH 및 NADH 수준의 분석을 보여준다.
정의
용어 "니코틴아미드 아데닌 디뉴클레오티드"(NADH)는 NAD+ (산화된 형태) 및 NADH + H+ (환원된 형태) 둘 모두의 산화환원 결합을 나타낼 수 있다.
용어 "니코틴아미드 아데닌 디뉴클레오티드 포스페이트"(NADPH)는 NADP+ (산화된 형태) 및 NADPH + H+ (환원된 형태) 둘 모두의 산화환원 결합을 나타낼 수 있다.
본 명세서에 언급된 바와 같이, "NADPH 의존적 효소"는 대개 (필수적으로 배타적이지 않더라도) 보조-인자로서 NADPH를 이용하여 반응에 전자를 공급한다. 유사하게, NADH-의존적 효소는 대개 (필수적으로 배타적이지 않더라도) 보조-인자로서 NADH를 이용하여 반응에 전자를 공급한다. 당업자는 일부 효소가 NADPH 및 NADH를 활용할 수 있고 이중기능성 NAD(P)H-의존적 효소로서 언급될 수 있음을 또한 이해할 것이다.
본 명세서에 언급된 바와 같이, 문구 "미생물에 의해 NADPH의 전체적인 활용이 증가된", 또는 유사한 문구는 특정한 시기에 효소에 결합하는 NADPH 보조-인자의 양에 있어서의 증가를 의미한다. 특정한 실시양태에서, 증가는 적어도 5%, 적어도 10%, 적어도 20%, 적어도 50%, 또는 적어도 100%의 증가이다. 이 증가는 실시예 3에서 사용된 방법, 또는 당해 기술 분야에 알려진 다른 방법, 예를 들어 문헌 [S. Wang, Huang, Moll, & Thauer, 2010]에 따라 측정될 수 있다. 이 문구는 경로를 통한 NADPH 유량에 있어서의 증가가 있고, 상기 증가가 상기 기술된 바와 같이 동일한 양자의 증가인 것을 의미하는 것으로 또한 해석되어질 수 있다. NADPH 유량은 대사산물 및 생성물의 수준을 측정하고/하거나(metabolomics) C13으로서 실험을 표지화함으로써(fluxomics) 측정될 수 있다.
본 명세서에 사용된 바와 같이, "보조-인자 특이성"은 보조-인자가 반응 동안 효소에 결합하는 친화성의 정도를 나타낸다. 효소 및 보조-인자가 절대적인 특이성을 갖는 것을 의미하는 것으로 고려되어서는 안되며, 이것이 사실이라 하더라도, 특정효소와 다른 보조-인자에 대한 하나의 보조-인자 간의 결합을 위한 선호도를 포함한다.
본 명세서에 언급된 바와 같이, 효소의 "이소형"은 동일한 반응을 촉매화할 수 있고, 유사하지만 동일하지 않는 아미노산 서열을 갖는 임의의 두개 이상의 기능적으로 유사한 단백질이다.
본 명세서에 언급된 바와 같이, "이분성(bifurcationg) 효소"는 하나의 보조-인자가 더 낮은 반응 전위 (예를 들어, 페레독신)를 갖고 하나의 보조-인자가 더 높은 반응 전위 (예를 들어, NADH 또는 NADPH)를 갖는 복합 보조-인자를, 단지 더 높은 반응 전위를 갖는 보조-인자(예를 들어, NADH 또는 NADPH)에 의해서는 촉매화될 수 없는 반응을 촉매화하기 위한 커플링된 반응에서, 또는 반응이 더 느린 속도로 지속되는 곳에서 활용할 수 있는 효소이다. 하나의 실시양태에서 이분성 효소는 복합 보조-인자를 활용하여 반응의 속도를 증가시킬 수 있다. 이분성 효소는 본 명세서에 기술된 포르메이트 하이드로겐 리아제와 같은, 복합체일 수 있다.
용어 "개조된"은 본 발명의 재조합 미생물을 설명하기 위핸 본 명세서에 사용될 수 있으며; 예를 들어, 미생물은 특정한 효소를 발현하도록 "개조된"다. 효소의 발현과 관련하여 사용되는 경우, 이 용어는 효소가 지속적으로 발현되는 것을 의미하지 않고, 이는 효소가 발현될 수 있고 이러한 발현이 구성적일 수 있거나 유도될 수 있는 상황을 포함하는 것으로 의도된다.
본 명세서에 지칭된 바와 같이, "발효 브로쓰"는 적어도 영양 배지 및 박테리아 세포를 포함하는 배양 배지이다.
용어 "효율을 증가시키는", "증가된 효율" 등은, 발효 공정과 관련하여 사용되는 경우, 발효를 촉매화하는 미생물의 성장 속도, 증대된 생성물 농도에서의 성장 및/또는 생성물 생성 속도, 소비된 기질의 용적당 생성된 원하는 생성물의 용적, 원하는 생성물의 생성 속도 또는 생성 수준, 및 발효의 다른 부산물과 비교하여 생성된 원하는 생성물의 상대 생성 중 하나 이상의 증가를 포함하지만, 이들로 제한되지는 않는다.
구절 "일산화탄소를 포함하는 기질" 및 유사 용어는 일산화탄소가 예를 들어 성장 및/또는 발효에 대해 박테리아의 하나 이상의 균주에 이용 가능한 임의의 기질을 포함하는 것으로 이해되어야 한다.
구절 "일산화탄소를 포함하는 가스 기질" 및 유사 구절 및 용어는 일정 수준의 일산화탄소를 함유하는 임의의 가스를 포함한다. 특정한 실시양태에서, 기질은 적어도 약 20 용적% 내지 약 100 용적%의 CO, 20 용적% 내지 70 용적%의 CO, 30 용적% 내지 60 용적%의 CO, 및 40 용적% 내지 55 용적%의 CO를 포함한다. 특정한 실시양태에서, 기질은 약 25 용적%, 또는 약 30 용적%, 또는 약 35 용적%, 또는 약 40 용적%, 또는 약 45 용적%, 또는 약 50 용적%의 CO, 또는 약 55 용적%의 CO, 또는 약 60 용적%의 CO를 포함한다.
기질이 임의의 수소를 포함해야할 필요는 없지만, H2의 존재는 본 발명의 방법에 따른 생성물 형성에 해롭지 않아야 한다. 특정한 실시양태에서, 수소의 존재는 알콜 생성의 전체 효율을 개선한다. 예를 들어, 특정한 실시양태에서, 기질은 H2:CO의 대략 2:1 또는 1:1 또는 1:2 비율을 포함할 수 있다. 하나의 실시양태에서, 기질은 약 30 용적% 이하의 H2, 20 용적% 이하의 H2, 약 15 용적% 이하의 H2 또는 약 10 용적% 이하의 H2를 포함한다. 다른 실시양태에서, 기질 스트림은 낮은 농도, 예를 들어, 5% 미만 또는 4% 미만 또는 3% 미만 또는 2% 미만 또는 1% 미만의 H2를 포함하거나 실질적으로 수소가 없다. 기질은 또한 약간의 CO2, 예를 들어 약 1 용적% 내지 약 80 용적%의 CO2, 또는 1 용적% 내지 약 30 용적%의 CO2를 포함할 수 있다. 하나의 실시양태에서, 기질은 약 20 용적% 이하의 CO2를 포함한다. 특정한 실시양태에서, 기질은 약 15 용적% 이하의 CO2, 약 10 용적% 이하의 CO2, 약 5 용적% 이하의 CO2를 포함하거나 실질적으로 CO2가 없다.
하기의 설명에서, 본 발명의 실시양태는 "CO를 함유하는 가스 기질"을 전달하거나 발효시키는 것과 관련하여 기술된다. 그러나, 가스 기질이 대안적인 형태로 제공될 수 있는 것으로 이해되어야 한다. 예를 들어, CO를 함유하는 가스 기질은 액체 중에 용해되어 제공될 수 있다. 본질적으로, 액체는 일산화탄소 함유 가스로 포화되고, 이후 이 액체는 바이오리액터에 첨가된다. 이는 표준 방법론을 이용하여 달성될 수 있다. 예로써, 마이크로버블 분산액 생성기(Hensirisak et. al. Scale-up of microbubble dispersion generator for aerobic fermentation; Applied Biochemistry and Biotechnology Volume 101, Number 3 / October, 2002)가 사용될 수 있다. 추가적인 예의 방식으로, CO를 함유하는 가스 기질은 고체 지지체에 흡착될 수 있다. 이러한 대안적인 방법은 "CO를 함유하는 기질" 등의 용어의 사용에 포함된다.
본 발명의 특정한 실시양태에서, CO-함유 가스 기질은 산업 배출 가스 또는 폐기 가스이다. "산업 폐기 또는 배출 가스"는 산업 공정에 의해 생성된 CO를 포함하는 임의의 가스를 포함하는 것으로 광범위하게 여겨져야 하고, 철 금속 생성물 제조, 비-철 생성물 제조, 석유 제련 공정, 석탄의 기화, 바이오매스의 기화, 전력 생성, 카본 블랙(carbon black) 생성 및 코크스 제조의 결과로서 생성된 가스를 포함한다. 추가적인 예는 본 명세서에서 그 외 부분에서 제공될 수 있다.
문맥상 달리 필요로 하지 않는 한, 본 명세서에 사용된 바와 같은 구절 "발효하는", "발효 공정" 또는 "발효 반응" 등은 이 공정의 성장 단계 및 생성물 생합성 단계 둘 다를 포함하는 것으로 의도된다. 본 명세서에서 추가로 기술될 바와 같이, 일부 실시양태에서, 바이오리액터는 제 1 성장 반응기 및 제 2 발효 반응기를 포함할 수 있다. 그러므로, 발효 반응에 대한 금속 또는 조성물의 첨가는 이 반응기 중 하나 또는 둘 다에 대한 첨가를 포함하는 것으로 이해되어야 한다.
용어 "바이오리액터"는 연속 교반 탱크 반응기(Continuous Stirred Tank Reactor: CSTR), 부동화 세포 반응기(Immobilized Cell Reactor: ICR), 삼상층 반응기(Trickle Bed Reactor: TBR), 버블 컬럼, 가스 리프트 발효기, 정적 혼합기, 또는 다른 용기 또는 가스-액체 접촉에 적합한 다른 장치를 포함하는, 하나 이상의 용기 및/또는 탑 또는 배관 배치로 이루어진 발효 장치를 포함한다. 일부 실시양태에서, 바이오리액터는 제1 성장 반응기 및 제2 발효 반응기를 포함할 수 있다. 그러므로, 바이오리액터 또는 발효 반응에 대한 기질의 첨가를 언급할 때, 적절한 경우 이 반응기 중 하나 또는 둘 다에 대한 첨가를 포함하는 것으로 이해되어야 한다.
본 명세서에 언급된 바와 같이, "셔틀 미생물"은 메틸트랜스퍼라제 효소가 발현되고 종착점 미생물과 구분되는 미생물이다
본 명세서에 언급된 바와 같이, "종착점(destination) 미생물"은 발현 구조물/벡터 상에 포함된 유전자가 발현되고 셔틀 미생물과 구분되는 미생물이다.
"외인성 핵산"은 이 핵산이 도입되는 미생물의 외부에서 기원하는 핵산이다. 외인성 핵산은, 이들로 제한되지는 않지만, 이 핵산이 도입되는 미생물, 이 핵산이 도입되는 생물과 다른 미생물의 균주 또는 종을 포함하는 임의의 적절한 공급원로부터 유래할 수 있거나, 이는 인공으로 또는 재조합으로 생성될 수 있다. 하나의 실시양태에서, 외인성 핵산은 이 핵산이 도입되는 미생물 내에 자연적으로 존재하는 핵산 서열을 나타내고, 이는 특정한 유전자의 발현 또는 과-발현을 증가시키도록 도입된다(예를 들어, 서열(예를 들어, 유전자)의 카피수를 증가시키거나 발현을 증가시키도록 강한 또는 구성적 프로모터를 도입함으로써). 다른 실시양태에서, 외인성 핵산은 이 핵산이 도입되는 미생물 내에 자연적으로 존재하지 않고, 미생물 내에 자연적으로 존재하지 않는 생성물의 발현하는 핵산 서열을 나타내거나, (예를 들어, 조절 구성요소, 예컨대 프로모터의 도입의 경우) 미생물에 천연인 유전자의 발현 증가를 허용한다. 외인성 핵산은 이 핵산이 도입되는 미생물의 게놈을 편입하거나, 염색체외 상태로 있도록 하기에 변경될 수 있다.
"외인성"은 또한 단백질을 언급하기 사용될 수 있다. 이것은 재조합 미생물이 유래된 모체 미생물 내에 존재하지 않거나 발현될 수 없는 단백질을 나타낸다.
재조합 미생물 및 핵산과 관련하여 본 명세에 사용된 바와 같은 용어 "내인성"은 재조합 미생물이 유래되는 모체 미생물 내에 존재하는 임의의 핵산을 나타낸다. 단백질을 설명하기 위해 사용되는 경우, "내인성"은 재조합 미생물이 유래되는 모체 미생물 내에 존재하거나 발현될 수 있는 임의의 단백질을 의미하는 것으로 고려되어야 한다.
"산화환원 효소"(또한 "탈수소효소" 또는 "산화효소"로 알려짐)는 환원제, 또한 전자 공여체로서도 불리는 하나의 분자로부터 산화제, 또한 전자 수용체로도 불리는 다른 분자로의 전자의 이동을 촉매화하는 효소를 포함한다. 산화환원효소는 효소의 EC 숫자 분류 중 EC 1으로서 분류된다. 이러한 군의 효소는 보통 NADH, NADPH 또는 페레독신과 같은 보조-인자를 필요로 한다.
본 명세서에 언급된 바와 같이 효소적 "반응"은 효소에 의해 촉매화된 하나 이상의 분자 (기질)의 다른 하나 이상의 분자 (생성물)로의 전환이다.
본 명세서에 구체적으로 예시된 서열과 서열이 다른 핵산이 실질적으로 동일한 기능을 수행하는 한, 이 핵산을 사용하여 본 발명을 실행할 수 있는 것으로 이해되어야 한다. 단백질 또는 펩티드를 코딩하는 핵산 서열의 경우, 이는 코딩된 단백질 또는 펩티드가 실질적으로 동일한 기능을 갖는다는 것을 의미한다. 프로모터 서열을 나타내는 핵산 서열의 경우, 변이체 서열은 하나 이상의 유전자의 발현을 촉진하는 능력을 가질 것이다. 이러한 핵산은 본 명세서에서 "기능적 균등 변이체"라 언급될 수 있다. 예로써, 핵산의 기능적 균등 변이체는 대립형질 변이체, 유전자의 단편, 돌연변이(결실, 삽입, 뉴클레오타이드 치환 등) 및/또는 다형 등을 포함하는 유전자를 포함한다. 다른 미생물로부터의 상동성 유전자는 본 명세서에 구체적으로 예시된 서열의 기능적 균등 변이체의 예로서 또한 생각될 수 있다. 이는 클로스트리디움 아세토부틸리쿰, 클로스트리디움 바이에링키(Clostridium beijerinckii), 씨. 리융다흘리(C. ljungdahlii)와 같은 종에서 상동성 유전자를 포함하고, 이들의 상세내용은 웹사이트, 예컨대 Genbank 또는 NCBI에서 공중에게 이용 가능하다. 구절 "기능적 균등 변이체"는 또한 특정한 생물에 대한 코돈 최적화의 결과로서 서열이 변하는 핵산을 포함하는 것으로 여겨져야 한다. 문맥상 달리 필요로 하지 않는 한, 본 명세서에 핵산의 "기능적 균등 변이체"는 바람직하게는 확인된 핵산과 적어도 대략 70%, 72%, 75%, 80%, 85%, 90%, 95% 이상의 핵산 서열 동일성을 가질 것이다.
본 명세서에 구체적으로 예시화된 아미노산 서열로부터 서열이 변하는 폴리펩티드를 사용하여 본 발명을 실행할 수 있는 것으로 또한 이해되어야 한다. 이 변이체는 본 명세서에서 "기능적 균등 변이체"로서 언급될 수 있다. 문맥상 달리 필요로 하지 않는 한, 단백질 또는 펩티드의 기능적 균등 변이체는 확인된 단백질 또는 펩티드와 적어도 40%, 50%, 60%, 70%, 72%, 75%, 80%, 85%, 90%, 95% 이상의 아미노산 동일성을 공유하고 목적한 펩티드 또는 단백질과 동일한 기능을 실질적으로 갖는 단백질 또는 펩티드를 포함한다. 이러한 변이체는 이의 범위 내에 단백질 또는 펩티드의 단편을 포함하고, 단편은 폴리펩티드의 절단된 형태를 포함하고, 결실은 1개 내지 5개, 내지 10개, 내지 15개, 내지 20개, 내지 25개의 아미노산일 수 있고, 폴리펩티드의 말단 중 어느 하나에서 1번 내지 25번의 잔기로부터 연장될 수 있고, 여기에서 결실은 부위 내에 임의의 길이를 가질 수 있거나; 또는 내부 위치에 있을 수 있다. 본 명세서에서의 특정한 폴리펩티드의 기능적 균등 변이체는 또한 예를 들어 이전 문단에서 예시화된 바와 같이 박테리아의 다른 종에서 상동성 유전자에 의해 발현된 폴리펩티드를 포함하는 것으로 여겨져야 한다.
본 명세서에 사용된 바와 같은 "실질적으로 동일한 기능"은 핵산 또는 폴리펩티드가 이의 변이체인 핵산 또는 폴리펩티드의 기능을 수행할 수 있다는 것을 의미하도록 의도된다. 예를 들어, 본 발명의 효소의 변이체는 그 효소와 동일한 반응을 촉매화할 수 있다. 그러나, 변이체가 이의 변이체인 폴리펩티드 또는 핵산과 동일한 수준의 활성을 갖는 것을 의미하도록 여겨져서는 안 된다.
당업자는 기능적 균등 변이체가 이의 변이체인 핵산 또는 폴리펩티드로서 실질적으로 동일한 기능을 갖는지 여부를 당업자에게 공지된 방법을 사용하여 평가할 수 있다. 그러나, 예로써, 수소화효소, 포르메이트 탈수소화효소 또는 메틸렌-THF-탈수소화효소 활성에 대해 시험하기 위한 검정이 문헌 [Huang, Wang, Moll, & Thauer, 2012]에 기술되어 있다.
"과-발현하다", "과발현" 및 유사 용어 및 구절은, 본 발명과 관련하여 사용될 때, 동일한 조건 하에 모체 미생물의 단백질(핵산 포함)의 발현 수준과 비교하여 하나 이상의 단백질의 임의의 발현(단백질을 인코딩하는 하나 이상의 핵산의 발현 포함)의 증가를 포함하도록 광범위하게 여겨져야 한다. 단백질은 임의의 특정한 수준으로 발현되는 것으로 여겨져서는 안 된다.
본 명세서에 언급된 바와 같은 "약화된 발현"은 모체 미생물 내에서 이의 발현에 비해 저하된 핵산 또는 단백질의 발현을 의미한다. 하나의 실시양태에서, 약화된 발현은 실질적으로 전혀 발현되지 않는 것(또는 본질적으로 "제로(zero)"인 발현)을 포함할 수 있다. 이는 예를 들어, RNA 사일런싱, 발현 공정의 변형(예를 들어, 프로모터 기능의 파괴), 핵산 서열의 변화 또는 변형(하나 이상의 뉴클레오티드의 결실, 삽입 및 치환을 포함), 또는 게놈으로부터의 효소를 인코딩하는 핵산의 완전 또는 부분적 제거를 포함하는 당업계에 공지된 임의의 방법에 의해 달성될 수 있다. 유전자가 작동하지 않도록 만들어지는 경우, 이는 본 명세서에서 "녹-아웃" 또는 "녹-아웃 되었다" 등으로 언급될 수 있다.
"모체 미생물"은 본 발명의 재조합 미생물을 생성시키도록 사용되는 미생물이다. 모체 미생물은 자연에 존재하는 것 (즉, 야생형 미생물) 또는 본 발명의 대상인 이전에 변형되었지만 하나 이상의 효소를 발현하거나 과발현하지 않는 것일 수 있다. 따라서, 본 발명의 재조합 미생물은 모체 미생물에서 발현되거나 과발현되지 않은 하나 이상의 효소를 발현하거나 과발현하도록 변형된다.
하나의 실시양태에서, 미생물은 클로스트리듐 오토에타노게눔, 클로스트리듐 륭달리이, 클로스트리듐 라그스달레이, 클로스트리듐 카복시디보란스, 클로스트리듐 드라케이, 클로스트리듐 스카톨로게네스, 클로스트리듐 아세티쿰, 클로스트리듐 포르미코아세티쿰, 클로스트리듐 마그눔, 알칼리바쿨룸 바크히(Alkalibaculum bacchii), 무렐라 써모아세티카(Moorella thermoacetica), 스포로무사 오바타(Sporomusa ovata), 부티리박테리움 메틸로트로피쿰(Butyribacterium methylotrophicum), 블라우티아 프로덕타( Blautia producta), 유박테리아 리모섬(Eubacterium limosum), 써모아나에로박터 키우비(Thermoanaerobacter kiuvi )종을 포함하는 아세토제닉 이산화탄소영양 유기체의 군으로부터 선택된다.
이 이산화탄소영양 아세토젠은 아세틸-CoA, 아세테이트 및 다른 생성물을 형성하는 혐기성 조건 하에 에너지 공급원으로서 일산화탄소(CO) 및 일산화탄소(CO)를 갖는 이산화탄소(CO2) 및/또는 수소(H2)와 같은 가스성 일-탄소(C1) 공급원 상에 화학합성자가생물적(chemoautotrophically)으로 성장시키고 활용하기 위한 이의 능력에 의해 정의된다. 이는 발효, Wood-Ljungdahl 또는 환원적 아세틸-CoA 경로의 동일한 발효 방식을 공유하고, 일산화탄소 탈수소화효소(CODH), 수소화효소, 포르메이트 탈수소화효소, 포르밀-테트라히드로폴레이트 합성효소, 메틸렌-테트라히드로폴레이트 탈수소화효소, 포르밀-테트라히드로폴레이트 시클로가수분해효소, 메틸렌-테트라히드로폴레이트 환원효소, 및 일산화탄소 탈수소화효소/아세틸-CoA 합성효소 (CODH/ACS)로 구성된 효소 세트의 존재에 의해 정의되며, 이의 조합은 이 유형의 박테리아에 대해 특징적이고 고유하다(Drake, Kusel, Matthies, Wood, & Ljungdahl, 2006). 기질을 생성물이 (아세틸-CoA를 통해 또는 직접적으로) 형성되는 바이오매스, 기질, 이차 대사물질 및 피루베이트로 전환하는 당-발효 박테리아의 화학합성자가생물적 성장과 대조적으로, 아세토젠에서 기질은 아세틸-CoA 내로 직접적으로 보내지고, 이로부터, 생성물, 바이오매스, 및 이차 대사산물이 형성된다.
하나의 실시양태에서, 모체 미생물은 씨. 아우토에타노제눔, 씨. 리융다흘리 및 "씨. 라그스달레이(C. ragsdalei)"의 종 및 관련 단리물을 포함하는 이산화탄소영양 클로스트리디아의 클러스터로부터 선택된다. 이들은 균주 씨. 아우토에타노제눔 JAI-1T (DSM10061) (Abrini, Naveau, & Nyns, 1994), 씨. 아우토에타노제눔 LBS1560 (DSM19630) (WO/2009/064200), 씨. 아우토에타노제눔 LBS1561 (DSM23693), 씨. 리융다흘리 PETCT (DSM13528 = ATCC 55383) (Tanner, Miller, & Yang, 1993), 씨. 리융다흘리 ERI-2 (ATCC 55380) (미국특허 5,593,886), 씨. 리융다흘리 C-01 (ATCC 55988) (US 특허 6,368,819), 씨. 리융다흘리 O-52 (ATCC 55989) (US 특허 6,368,819), 또는 "씨. 라그스달레이 P11T" (ATCC BAA-622) (WO 2008/028055), 및 관련 단리물 예컨대 "씨. 코스카티(C. coskatii)" (미국특허 2011/0229947), "클로스트리디움 종 MT351" (Tyurin & Kiriukhin, 2012), "클로스트리디움 종 MT 653" (Berzin, Kiriukhin, & Tyurin, 2012a), "클로스트리디움 종 MT683" (Berzin, 2012), "클로스트리디움 종 MT962" (Berzin, Kiriukhin, & Tyurin, 2013) "클로스트리디움 종 MT1121" (Berzin, Kiriukhin, & Tyurin, 2012b), "클로스트리디움 종 MT1230" (Kiriukhin & Tyurin, 2013), 또는 "클로스트리디움 종 MT1962" (Berzin, Tyurin, & Kiriukhin, 2013), 및 이들의 돌연변이 균주 예컨대, 씨. 리흉다홀리 OTA-1 (Tirado-Acevedo O. Production of Bioethanol from Synthesis Gas Using Clostridium ljungdahlii. PhD thesis, North Carolina State University, 2010) 또는 "클로스트리움 종 MT896" (Berzin, Kiriukhin, & Tyurin, 2012c)을 포함하지만 이들로 제한되지는 않는다.
DNA-DNA 연계 및 DNA 핑거프린팅 실험에 의해 결저된 바와 같은 별개의 종이라고 하더라도, 이 균주는 16S rRNA 유전자 수준 상에 적어도 99% 동일성을 갖는, 클로스트리디아 rRNA 클러스터 I (Collins et al., 1994) 내의 하부클러스터를 형성한다(WO 2008/028055, US patent 2011/0229947).
이 클러스터의 균주는 유사한 유전자형 및 표현형 둘 모두를 갖는, 공통 특징에 의해 정의되며, 이들은 모두 동일한 방식의 에너지 보존 및 발효 물질대사를 공유한다. 이 클러스터의 균주는 사이토크롬이 없고 Rnf 복합체를 통해 에너지를 보존한다.
이 클러스터의 모든 균주는 대략 4.2 MBp(Kopke et al., 2010)의 게놈 크기및 대략 32 %몰의 GC 조성물을 가지고(Abrini et al., 1994; Kopke et al., 2010; Tanner et al., 1993)(WO 2008/028055; 미국특허 2011/0229947), Wood-Ljungdahl 경로 (일산화탄소 탈수소화효소, 포르밀-테트라히드로폴레이트 합성효소, 메틸렌-테트라히드로폴레이트 탈수소화효소, 포르밀-테트라히드로폴레이트 시클로가수분해효소, 메틸렌-테트라히드로폴레이트 환원효소, 및 일산화탄소 탈수소화효소/아세틸-CoA 합성효소)의 효소, 수소화효소, 포르메이트 탈수소화효소, Rnf 복합체 (rnfCDGEAB), 피루베이트:페레독신 산화환원효소, 알데히드:페레독신 산화환원효소 를 인코딩하는 필수적인 주요 유전자 오페론을 보존하였다(Kopke et al., 2010, 2011). 가스 흡수에 책임이 있는, Wood-Ljungdahl 경로 유전자의 조성 및 수는 핵산 및 아미노산 서열에 있어서의 차이에도 불구하고, 모든 종에서 동일한 것으로 밝혀졌다(Kopke et al., 2011).
이 클러스터의 모든 종은 유사한 형태 및 크기(대수로 성장하는 세포는 0.5 내지 0.7 x 3-5 ㎛임)를 갖고, 중온성(30 내지 37℃의 최적 성장 온도)이고, 엄격히 혐기성 생물이다(Abrini et al., 1994; Tanner et al., 1993)(WO 2008/028055). 또한, 이들은 모두 동일한 주요한 계통발생 특징, 예컨대 동일한 pH 범위(pH 4-7.5, 5.5-6의 최적 초기 pH), 특정한 조건 하에 형성된 소량의 2,3-부탄디올 및 락트산 및 주요 발효 최종 생성물로서의 에탄올 및 아세트산과 유사한 성장 속도 및 유사한 대사 프로필을 갖는 CO 함유 가스에서 강한 독립영양 성장을 공유한다(Abrini et al., 1994; Kopke et al., 2011; Tanner et al., 1993)(WO는 다양한 당(예를 들어, 람노스, 아라비노스), 산(예를 들어, 글루코네이트, 시트레이트), 아미노산(예를 들어, 아르기닌, 히스티딘) 또는 다른 기질(예를 들어, 베타인, 부탄올)의 기질 활용이 상이하다. 일부 종은 특정한 비타민(예를 들어, 티아민, 비오틴)에 대해 영양요구성인 것으로 밝혀진 반면, 다른 것은 그렇지 않다. 카르복실산의 이의 상응하는 알코올로의 환원은 이들 유기체의 범위 내에서 나타났다(Perez, Richter, Loftus, & Angenent, 2012).
그러므로 기술된 특성은 씨. 아우토에타노제눔 또는 씨. 리융다흘리와 같은 하나의 유기체에 특이적인 것이 아니라, 오히려 일산화탄소영양, 에탄올-합성 클로스트리디아에 대해 일반적인 특성이다. 따라서, 본 발명은 수행면에서 차이가 있다고 하더라도, 이들 균주에 걸쳐 작동하는 것으로 기대되어질 수 있다.
특정한 실시양태에서, 모체 미생물은 클로스트리듐 오토에타노게눔 클로스트리디움 리융다흘리, 및 클로스트리듐 라그스달레이를 포함하는 군으로부터 선택된다. 하나의 실시양태에서, 군은 또한 클로스트리듐 코스카티(Clostridium coskatii)를 포함한다. 하나의 특정한 실시양태에서, 모체 미생물은 클로스트리듐 오토에타노게눔 DSM23693이다.
용어 핵산 "작제물" 또는 "벡터" 등 용어는 유전 재료를 세포에 전달하기 위한 비히클로서 사용하기에 적합한 임의의 핵산(DNA 및 RNA를 포함)을 포함하는 것으로 광범위하게 여겨져야 한다. 상기 용어는 플라스미드, 바이러스(박테리오파지를 포함), 코스미드(cosmid) 및 인공 염색체를 포함하는 것으로 여겨져야 한다. 작제물 또는 벡터는 하나 이상의 조절 구성요소, 복제 기원, 멀티클로닝 부위 및/또는 선택 가능한 마커를 포함할 수 있다. 특정한 하나의 실시양태에서, 작제물 또는 벡터는 작제물 또는 벡터에 의해 인코딩된 하나 이상의 유전자의 발현을 허용하도록 변경된다. 핵산 작제물 또는 벡터는 네이키드 핵산 및 세포(예를 들어, 리포솜 접합 핵산, 핵산이 함유된 생물)로의 전달을 용이하게 하는 하나 이상의 제제로 제형화된 핵산을 포함한다.
폐기 가스 및 합성가스 상에 성장하는 이산화탄소영양 클로스트리디아의 모든 알려진 예는 NADH-의존적 반응을 사용한다. 산화환원 쌍 NADPH + H+/NADP+은 NADH+ H+/NAD+ 산화환원 쌍 보다 더욱 음성인 산화환원 전위를 가진다(Auriol et al., 2011). 생체내 조건 하에서 NAD+/NADH 커플의 산화환원 전위 E'는 약 -280 mV (Eo' = -320 mV)인 반면 NADP+/NADPH 커플의 E'는 약 -360 mV (Eo' = -320 mV)이다. 본 발명자들은 CO, CO2, 및 H2 가스의 흡수 및 활용을 위한 자가영양 성장에 수반되는 많은 효소(예를 들어, 수소화효소 효소 및 Wood-Ljungdahl 경로 효소)가 NADH 보다 NADPH를 활용하는 쪽으로 명백하게 편향성을 보임을 놀랍게도 밝혀냈다.이것은 예를 들어, 대부분의 박테리아성 공정을 위한 방식으로서 작용하고 완전히 NADH 편향된 대장균과 같은 박테리아를 활용하는 당의 해당작용에 완벽하게 반대된다. 이 대장균 기반 반응은 NADPH 의존적 반응 단계를 포함하지 않지만 몇몇 NADH 의존적 단계는 포함한다(글루코오스 + 2 NAD+ + 2 ADP + 2 Pi → 2 피루베이트 + 2 NADH + 2 H+ + 2 ATP + 2 H2O; 도 2).
대장균 내의 NADPH-의존적 반응은 NADPH 풀을 빠르게 고갈시키고 세포 성장 억제 및 사멸을 유도하는 것으로 밝혀졌다. 대장균 내의 NADPH 용량의 이러한 부재는 NADPH 의존성을 감소시키기 위한 시도에 대한 이전 연구를 이끌어 냈으며, 따라서 상기 연구는 NADPH 활용을 증가시키는 것이 발효 반응에서 바람직하지 않을 수 있음을 제안한다. 그러므로 본 명세서에 언급된 이산화탄소영양 클로스트리디아가 NADPH-의존적 반응을 지속하기 위해 상대적으로 큰 용량을 가진다는 것을 본 발명자들이 밝혀낸 것은 놀라운 일이었다.
본 발명자들은 NADPH-의존적 효소에 의해 바이오리액터 내에서 아세톤 전환을 모니터링하는 실험에 의해 일산화탄소영양 클로스트리듐 미생물 내에 NADPH 풀의 상대적으로 큰 용량을 증명하였다(실시예 3 참조). 따라서, 본 발명자들은 NADH 보다 NADPH를 사용하는 것이 발효 공정에서 효소 반응을 구동시키기에 더 유리할 수 있음을 보여주었다.
따라서 NADH 의존적 반응을 사용하고 NADPH 의존적 반응 (생산 수율의 감소를 초래하고 광범위한 변형이 필요함)을 우회하는, 대장균에 대한 기존 전략은 일산화탄소영양 클로스트리디디아에서는 생산적이지 않다. 본 명세서에 기술된 바와 같이 본 발명은 물질대사성 조작을 위해 최대 생산 수율을 달성하기 위한 일산화탄소영양 클로스트리디아에서 NADPH 의존적 반응에 대해 우선적으로 선택함으로써 이를 극복하는 전략을 제공한다. NADPH 의존적 반응의 용량 및 전위는 대장균을 활용하는 당에 차이 뿐만 아니라 실시예 3에서 나타나 있다. 유사하게, 이 전략은 이종성 경로에 적용되어 최대 생산 수율 및 유량을 달성할 수 있다.
더 나아가, 본 발명자들은 NADPH 의존적 반응이 일산화탄소영양 미생물에서 확산되는 것을 확인하였다. 이는 NADPH 풀을 사용하는 효소 및 유전를 확인하고 특성화하기 위한 선택 기술의 개발을 가능하게 한다. 이 기술에 따라 선택된 효소를 발현하거나 과-발현할 수 있는 재조합 미생물은 일산화탄소영양 미생물의 효율을 개선하고 이의 원하는 생성물의 생성을 증가시키는데 있어서 활용성을 갖는다.
대장균과 같은 당 활용 유기체와 관련하여 선행 기술에 의해 교시된 것과 대조적으로, 본 발명자들은 일산화탄소영양 미생물을 고려하는 경우 NADPH 의존적 반응이 원치않는 보틀넥(bottleneck)이 아닌 것으로 생각한다. 본 발명자들은 사실상 NADPH를 활용하는 효소가 NADH의 존해 하에 이의 활성과 비교하는 경우 이의 존재에서 증가된 활성을 가지기 때문에 양성적으로 바람직하다는 것을 믿는다.
NADPH-의존적 효소가 원하는 생성물의 생성을 구동하는데 사용될 수 있다는 발견은 본 발명자들에게 이들 효소를 발현하거나 과-발현할 수 있는 신규한 재조합 미생물을 조작하도록 유도하였다. 이들 재조합 미생물은 신규 경로가 탐색되고 원하는 생성물이 생성되도록 한다. 특정한 실시양태에서, 재조합 미생물은 일산화탄소영양 미생물이다. NADPH 의존적 효소가 생략되거나 우회되어야 한다는 이전에 생각과는 반대로, 본 발명자들은 놀랍게도 일산화탄소영양 미생물에서 이들 효소의 활용이 미생물 성장 및/또는 생성에 있어서의 감소를 초래하지 않고 이러한 효소를 생략하기 위한 광범위한 조작이 필요하지 않다는 것으로 밝혀냈다.
본 발명의 제1 측면에 따르면, 하나 이상의 외인성 NADPH-의존적 효소를 발현하도록 개조하고 및/또는 하나 이상의 내인성 효소를 과-발현하도록 개조된 재조합 일산화탄소영양 클로스트리디아 미생물을 제공하며, 상기 효소는 외인성 효소가 발현되고/되거나 내인성 효소가 과발현되도록 선택되고, 미생물에 의한 NADPH의 전체 활용이 모체 미생물에 비해 증가된다.
추가적인 측면에서, 본 발명은 또한 모체 미생물에 비해 증가된 NADPH 활용을 나타내는 재조합 이산화탄소영양 클로스트리디아 미생물을 생산하는 방법을 제공하며, 상기 방법은 다음 단계를 포함한다:
a. 하나 이상의 외인성 및/또는 내인성 NADPH-의존적 효소를 선택하는 단계;
b. 모체 미생물을 형질전환시켜 하나 이상의 외인성 효소를 발현하고/하거나 하나 이상의 내인성 효소를 과-발현하도록 개조된 재조합 미생물을 생산하는 단계.
미생물 내의 임의의 하나 이상의 NADPH-의존적 효소의 발현 또는 과-발현은 하나 이상의 효소가 발현되지 않거나 과-발현되지 않는 미생물에 비해 NADPH의 활용에 있어서 전체적인 증가를 초래한다.
하나 이상의 효소는 NADH 및 NADPH 의존적 이소형으로 존재할 수 있다. 특정한 실시양태에서 재조합 미생물은 NADPH-의존적 이소형을 발현하고 및/또는 과발현하도록 개조된다. 본 발명의 방법은 NADPH 및 NADH의 활용도가 유사한 범위 내인 경우, 즉 이것이 NADH 보조인자에 결합하는 경우 보조-인자를 활용하는 이소형의 활성이 이것이 NADHP 보조인자에 결합하는 경우 이소형의 활성과 유사한, 특별한 활성의 것이다.
특정한 실시양태에서, 하나 이상의 NADPH-의존적 효소는 수소화효소 (예를 들어, 서열번호 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, YP_003781016, YP_003781017, YP_003778879, YP_003779640, YP_003779893, YP_003780193, 또는 이의 임의의 하나의 기능적 균등 변이체에 따른 아미노산 서열을 가짐), 포르메이트 탈수소화효소 (예를 들어, AEI90721, AEI90723, AEI90725, YP_003779063, YP_003778871, YP_003780168, AEI90722, AEI90724, AEI90726, 또는 이의 임의의 하나의 기능적 균등 변이체의 아미노산 서열을 가짐) 또는 메틸렌-THF-탈수소화효소 (예를 들어, AEI90753, YP_003781891, AEI90771 또는 이의 기능적 균등 변이체의 아미노산 서열을 가짐)을 포함한다.
본 발명의 특정한 실시양태에서, NADPH 및 NADH 의존적 반응의 선택이 존재한다. 본 발명은 모체 미생물에서 NADPH 활용에 비해 NADPH의 전체 활용도를 증가시키는 방법에 의해서 NADH 의존적 이소형과 비교하여 NADPH-의존적 이소형의 사용을 우선적으로 만드는 방법 및 재조합 미생물을 제공한다. 이러한 경로 및 산화환원효소 반응의 예는 다음을 포함한다:
■ 이소프레노이드 생성을 위한 메발로네이트(Mevalonate) 경로
○ 하이드록시메틸글루타릴-CoA (HMG-CoA) 환원효소 (S. M. Ma et al., 2011):
■ NADPH-의존적 효소 (EC 1.1.1.34; GO:0004420; 예를 들어, 사카로마이세스 세레비지애: DAA09822.1; BK006946.2:115734..118898) 및
■ NADH-의존적 효소 (EC1.1.1.88; GO:0042282; 예를 들어, 슈도모나스 메발로니이: P13702.1)
■ 부탄올/PHB 경로 (Bond-Watts, Bellerose, & Chang, 2011):
○ 3-하이드록시부티릴-CoA 탈수소화효소/ 아세토아세틸-CoA 환원효소 / 3-하이드록시부티릴-CoA 수화효소:
■ NADPH 의존적 phaB (EC:1.1.1.36; GO:0018454; 예를 들어, 랄스토니아 유트로파: YP_725942.1, 유전자번호:4249784 유래) 및
■ NADPH 의존적 phaJ (EC 4.2.1.119; 예를 들어, 아에로모나스 펀크타타: BAA21816.1 유래)
■ NADH 의존적 hbd (EC 1.1.1.157; GO:0008691; 예를 들어, 씨. 아세토부틸리쿰: NP_349314.1, 유전자번호:1118891 유래)
○ 크로토닐-CoA 환원효소/ 크로토닐-CoA 카르복실라제-환원효소 / 트랜스-2-에노일-CoA 환원효소/부티릴-CoA 탈수소화효소 (Hu et al., 2012):
■ NADPH 의존적 ccr (EC 1.3.1.86; 예를 들어, 스트렙토마이세스 콜리누스 유래) 또는 ccr Rs (EC 1.3.1.85; 예를 들어, 로도박터 스파에로이데스: YP_354044.1, 유전자번호: 3720751 유래)
■ NADH 의존적 ter (EC 1.3.1.44; GO:0050343; 예를 들어, 트레포네마 덴티콜라 유래)
■ NADH/페레독신 이분성 bcd- etfAB 복합체 (EC 1.3.8.1; GO:0004085; 예를 들어, 씨. 아세토부틸리쿰: NP_349317.1; 유전자번호:1118894 유래) (Li et al., 2008) 또는
■ NADH / NADPH 이중기능성 의존적 ter (EC 1.3.1.44; GO:0050343; 예를 들어, 유글레나 그라실리스: AY741582.1 유래) (Hoffmeister, Piotrowski, Nowitzki, & Martin, 2005)
탈수소화효소(예를 들어, 에탄올 또는 부탄올에 대한 알코올 탈수소화효소, 또는 부탄디올에 대한 디올 탈수소화효소) 및 산화효소를 수반하는 대부분의 산화환원효소 반응으로부터, NADH 또는 NADPH 의존적 효소 중 하나의 선택이 이용가능하며 개별 효소는 Braunschweig 효소 데이터베이스 BRENDA (http://www.brenda-enzymes.info/)와 같은 데이터 베이스를 사용해 확인될 수 있다(Scheer et al., 2011).
특정한 실시양태에서 미생물은 NADPH-의존적 이소형을 발현하고/하거나 과-발현하도록 개조되는 반면 상응하는 NADH-의존적 이소형의 발현은 NADPH-의존적 이소형의 발현에 있어서의 변화와 비교할 때 변화가 없거나, 감소하거나, 상대적으로 적은 증가를 나타낸다. 이런 방식으로, NADPH의 전체 활용은 모체 미생물에 비해 증가된다.
특정한 실시양태에서, 본 발명은 하나 이상의 NADH-의존적 효소의 발현이 약화되거나 제로화된 재조합 미생물을 제공한다. 하나의 특정한 실시양태에서, 하나 이상의 NADH-의존적 이소형의 발현은 모체 미생물에 비해 약화되거나 녹아웃되었다. 약화/녹아웃은 하나 이상의 NADH-의존적 효소를 인코딩하는 핵산을 변형시키거나 NADH-의존적 효소를 인코딩하는 하나 이상의 핵산을 NADPH-의존적 이소형을 인코딩하는 하나 이상의 핵산으로 대체함으로써 달성될 수 있다. 효소의 약화 또는 녹-아웃은 임의의 수의 알려진 형질전환 및 재조합 핵산 기술을 사용하여 본 발명의 미생물에 도달하기 위해 모체 미생물을 형질전환시켜 달성될 수 있다. 이산화탄소영양 아세토젠에서 약화 또는 녹-아웃을 달성할 수 있는 특정한 방법은 문헌 [Leang, Ueki, & Lovley, 2011]에 기술되어 있고 추가적인 기술은 예를 들어, 문헌 [Sambrook et al, (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)]에 기술되어 있다. 일반적인 예의 방법으로서, 돌연변이를 유전자 내로 도입하는 경우, 또는 다르게는 유전자를 파괴하거나 녹 아웃시키는 경우, 적절한 핵산 작제물 또는 벡터가 모체 미생물의 게놈 내로 편입되도록 고안되어 유전자를 파괴할 수 있다. 이러한 작제물은 전형적으로 파괴되는 유전자 내부 또는 측면 부위에 상동인 핵산 서열 (암; arm)을 포함할 것이며, 이는 상동성 재조합이 일어나도록 허용하고, 돌연변이의 도입, 유전자로부터 핵산 부위의 절제, 또는 반대편에 핵산을 갖는 유전자 부위의 치환이 일어나도록 한다. 작제물 상의 암이, 필수적인 것은 아니지만, 표적되는 게놈 내의 부위에 100% 상보적인 것이 바람직한 반면, 단 서열은 관심의 유전 부위를 갖는 표적된 재조합을 허용하도록 충분히 상보적이다. 전형적으로, 암은 문헌 [Sambrook et al 1989]에 정의된 바와 같이, 엄중한 조건 하에 표적 부위에 하이브리드화를 허용하는 수준의 상동성을 가질 것이다. 숙련가는 본 명세서에 기술된 바와 같은 본 발명에서 수반된 효소에 대한 이용 가능한 서열 정보를 갖는 모체 미생물의 게놈 내로 외의성 핵산의 표적된 상동성 재조합 및 편입을 허용하기에 충분하도록 이해할 것이다.
하나의 실시양태에서, 효소는 복합 보조-인자 의존성을 나타낼 수 있다. 이러한 효소는 NADH/페레독신 이분성 효소(ferredoxin bifurcating enzyme) 또는 NADH/NADPH 상호-의존적 효소를 포함할 수 있다. 특정한 실시양태에서, 효소는 NADH/NADPH 이분성 이소형 및 NADH/페레독신 이분성 이소형으로 존재하고 미생물은 NADH / NADPH 의존적 이소형을 발현하고 및/또는 과발현하도록 개조된다. 특정한 실시양태에서, NADH/NADPH 의존적 이소형은 ter 이다(EC 1.3.1.44; GO:0050343; 유글레나 그라실리스: AY741582.1 유래 또는 이의 기능적 균등 변이체). 추가적인 실시양태에서, NADH/Fd 의존적 이소형은 NADH/페레독신 이분성 bcd- etfAB 복합체이다(EC 1.3.8.1; GO:0004085; 예를 들어, 씨. 아세토부틸리쿰: NP_349317.1; 유전자번호:1118894 유래 또는 이의 기능적 균등 변이체).
본 발명의 특정한 실시양태에서, 미생물은 모체 미생물과 비교할 때 발효 반응 동안 증가된 효율을 나타낸다. 발효 생성물의 생성에 관련된 미생물은 발효 생성물의 성장 및 생성과 관련된 반응을 구동하기 위한 보조-인자로서 NADPH를 사용한다. 이러한 미생물이 NADH 보조인자와 비교할 때 NADPH 보조인자에 대해 높은 친화성을 갖는 효소를 발현하는 경우, NADPH의 활용이 증가된다면 이의 효율(상기 정의 참고)이 증가될 잠재력이 존재한다.
본 발명의 효소는 많은 생성물을 생성하기 위한 생합성 경로에 수반된다. 특정한 실시양태에서, 경로는 메발로네이트 경로 또는 부탄올 합성 경로이다.
본 발명의 하나의 실시양태에서, 하나 이상의 NADPH-의존적 효소의 보조-인자 특이성은 이의 NADH 보조인자 특이성과 비교하여 이의 NADPH 보조인자 특이성을 증가시키도록 개조될 수 있다.
특정한 실시양태에서, 본 발명은 효소의 NADH 보조-인자 특이성과 비교하여 산화환원효소 효소의 NADPH 보조인자 특이성을 증가시켜 이산화탄소영양 미생물의 효율을 증가시키기 위한 방법을 제공한다.
산화환원효소 효소의 보조인자 특이성은 아미노산 서열을 변형시킴으로써, 특히 각각의 NADH 및 NADPH 결합 포켓의 일부분을 형성하거나 이에 기여하는 효소의 부위에서 NADH에서 NADPH(또는 그 반대)로 변형될 수 있다. NADH/NADPH 결합 포켓은 당해 기술분야에 알려진 다른 방법 중 변형될 수 있다.
아미노산 서열의 변형은 하나 이상의 아미노산 잔기의 삽입, 결실 및/또는 치환, 또는 당해 기술분야에 손쉬울 수 있는 하나 이상의 다른 변형을 포함할 수 있다. 변형은 효소의 임의의 부위에서 발생할 수 있다. 그러나, 하나의 실시양태에서 이는 NADH 결합 포켓 내이다.
특정한 실시양태에서, 아미노산 서열의 변형은 NADH 결합 포켓 내의 특정 아미노산 잔기(들), 예를 들어 부티릴-CoA 탈수소화효소 효소에 대한 글루탐산 잔기의 변형을 포함한다. 특정한 실시양태에서, 글루탐산 잔기는 씨. 아세토부틸리쿰의 bcd 내의 Glu75 및/또는 T. denticola 내의 Glu80이다. 하나의 실시양태에서, 당해 기술분야에 공지된 원하는 보조-인자 특이성을 달성하기 위한 변형, 예를 들어 문헌 [Hu et al (2012)]에 기술된 변형이 사용될 수 있다.
특정한 실시양태에서, 본 발명자들은 보조-인자 특이성의 변형이 다양한 종 유래의 상기-언급된 크로토닐-CoA 환원효소/ 트랜스-2-에노일-CoA 환원효소/부티릴-CoA 탈수소화효소의 구조적 분석 및 NADPH에 대해 NADH를 구분하는데 있어서 중요한 역할을 하는 Glu(씨. 아세토부틸리쿰의 bcd 내의 Glu75 및 티. 덴티콜라 내의 Glu80)의 전환을 통해 달성될 수 있다고 가정한다. 이론에 구속받는 것을 원하지 않으면서, 이는 NADH의 아데닌 리보스의 2'-OH를 인식하는 효소에 의해 일어나는 것으로 생각된다. NADPH 의존적 효소에서, 이 잔기는 변형된다(Hu et al., 2012).
보조-인자 특이성의 변형을 달성하기 위한 방법은 당업자에게 공지될 것이다. 그러나, 예의 방식으로, 다양한 산화환원효소 효소에 대핸 보조-인자 특이성의 변화와 관련된 하기 실시예에서 사용되는 방법이 사용될 것이다:
● 1,3-프로판디올 산화환원효소 (C. Ma, Zhang, Dai, & Xiu, 2010)
● p-하이드록시벤조에이트 수산화효소 (Eppink, Overkamp, Schreuder, & Van Berkel, 1999)
● 17β-하이드록시스테로이드 탈수소화효소 (McKeever et al., 2002)
● 케톨산 레덕토아이소머라제(Ketol Acid Reductoisomerase)(Rane & Calvo, 1997)
신규
이분성
효소
전자-이분성(Electron-bifurcation)은 혐기성 박테리아 및 고박테리아의 세포질에서 에너지방출성 산화환원 반응에 대해 에너지방출성 산화환원 반응을 커플링하는 최근에 발견된 기전이다. 현재까지, 단지 몇몇 전자-이분성 효소 복합체가 확인되었고, 그 중 4개의 특징이 밝혀졌다(Herrmann, Jayamani, Mai, & Buckel, 2008; Huang et al., 2012; Li et al., 2008; Schuchmann & Mueller, 2012; Schut & Adams, 2009; G. Wang & Wang, 1984). 2008년에, 클로스트리디아를 형성하는 부틸산에서 NADH (Eo' = -320 MV)로의 크로토닐-CoA (Eo' = -10 mV)의 에너지방출성 환원이 NADH로의 페레독신 (Eo' = -400 mV)의 에너지흡수성 환원과 커플링되고, 이 커플링 반응이 세포질 부티릴-CoA 탈수소화효소-전자 이동 플라보단백질 복합체 Bcd-EtfAB에 의해 촉매화된다는 것이 밝혀졌다(Herrmann, Jayamani, Mai, & Buckel, 2008; Li et al., 2008). 이는 이 공정이 플라빈-기반이고: 단백질 결합된 플라빈은 후속적으로 페레독신 (Fd)를 환원시키기에 충분히 음성인 산화환원 전위를 갖는 세미퀴논 라디칼로 크로토닐-CoA에 의해 재-산화되는 하이드로퀴논이 되도록 NADH에 의해 환원되는 됨을 제안한다. 현재까지, 많지 않은 전자-이분성 효소 복합체가 확인되었고 그중 4개의 특징이 밝혀졌다(Herrmann et al., 2008; Huang et al., 2012; Li et al., 2008; Schuchmann & Mueller, 2012; Schut & Adams, 2009; G. Wang & Wang, 1984). 반응 1의 Bcd-EtfAB 복합체 외에도, 반응 2를 촉매화하는 메탄생성 고박테리아으로부터의 MvhADG-HdrABC 복합체, 반응 3을 촉매화는 박테리아 및 고박테리아으로부터의 NfnAB 복합체 및 반응 4를 촉매화하는 박테리아로부터의 HydABC 복합체가 있다.
(1) 2NADH + Fdox + 크로토닐-CoA → 2 NAD+ + Fdred 2- + 부티릴-CoA
△G°′= -44 kJ/몰*
(2) 2H2 + CoM-S-S-CoB + Fdox → CoM-SH + CoB-SH + Fdred 2- + 2H+
△G°′= -50 kJ/몰*
(3) NADH + Fdred 2- + 2NADP+ + H+ → NAD+ + Fdox + 2NADPH
△G°′= - 16 kJ/몰*
(4) NADH + Fdred 2- + 3H+ → NAD+ + Fdox + 2H2
△G°′= +21 kJ/몰*
* -400 mV의 Eo'를 사용하는 표준 조건하에서(1M 농도의 기질 및 생성물; 가스의 부분압력 = 1 바; pH = 7)
이들 복합체 모두는 NADH 의존적으로, 그 중 2개의 헤테로메릭(heteromeric) Fe-유일 수소화효소는 H2로의 페레독신의 에너지흡수성 환원과 H2로의 NAD의 에너지방출성 환원을 가역적을 커플링한다(Schuchmann & Mueller, 2012; Schut & Adams, 2009). 본 발명자들은 NADPH 의존적 이분성 효소(반응 5), NAD 보다 NADP 특이적인 신규한 전자-이분성 [FeFe]-수소화효소를 최초로 확인하였다.
(5) NADPH + Fdred 2- + 3H+ 
NADP+ + Fdox + 2H2
△G°′= +21 kJ/몰*
* -400 mV의 Eo'를 사용하는 표준 조건 하에서(1M 농도의 기질 및 생성물; 가스의 부분 압력 = 1 바; pH = 7)
이론에 구속받는 것을 원하지 않으면서, 수소화효소와 더불어 이 복합체의 역할은 포르메이트:하이드로겐 리아제로서 작용하는 것이다. 이 기능에서 이는 Fdox/Fdred 2- 커플 및 NADP+/NADPH 커플의 세포내 산화환원 전위가 CO 과환원으로 인해 너무 낮아지는 경우, 각각 양성자 및 CO2의 환원에 의해 H2 및 포르메이트를 형성함으로써 NADPH 구동된 생성물 형성을 위한 전자 과유량 밸브를 나타낸다.
생체내 조건 하에서 NAD+/NADH 커플의 산화환원 전위 E'는 약 -280 mV (Eo' = -320 mV)이고 NADP+/NADPH 커플의 E'는 약 -360 mV (Eo' = -320 mV)이다. Fdox/Fdred2- 커플의 산화환원 전위 E'는 더 낮은 것으로 고려되는 -400 mV (씨. 파스튜리아눔(C. pasteurianum) 으로부터의 Eo')이다. 이처럼, 본 발명의 이 이분성 공정 측면은 더 빠른 반응 속도와 같은 장점을 제공한다.
생체내 조건 하에서, 페레독신의 산화환원 전위는 -500 mV 근처, NADP 산화환원 전위에서 -370 mV 근처이고 NAD 산화환원 전위에서 -280 mV 근처일 것으로 예상된다. 약 200 mV의 Fdox/Fdred 2 - 커플과 NAD+/NADH 커플의 간의 산화환원 전위 차이는 Rnf 복합체 및 FoF1 ATP 합성효소와 연관된 막에 의해 매개되는 전자 이동 인산화로 커플링 되기에 충분히 크다. 페레독신과의 NAD+의 환원은 CO 상에 성장하는 C.아우토에타노제눔의 에너지 물질대사에서 주요 커플링 부위이다. NAD+는 이후에 생성물 형성을 구동할 수 있는 NADPH를 산출하는 반응 촉매화된 Nfn을 통해 지속적으로 재생된다(도 7).
본 발명자들은 NAD 보다 NADP에 특이적인 신규 전자-이분성 [FeFe]-수소화효소를 확인하였다. 본 발명자들은 또한 씨. 아우토에타노제눔에서 발현되는 포르메이트 탈수화효소가 NADPH만을 활용하기 보다는 페레독신 및 NADPH 둘 모두를 활용할 수 있다는 것을 확인하였다(이분성 포르메이트 탈수소화효소로서 본 명세서에 지칭됨).
이 효소의 신규한 기능은 이전에 알려지지 않았고 이들 효소는 확인된 최초의 NADPH-의존적 이분성 NADP Fe-유일 수소화효소 및 이분성 NADP 포르메이트 탈수소화효소 효소이다. 본 발명자들에 의한 추가적인 연구는 NADP Fe-유일 수소화효소 및 NADP 포르메이트 탈수화효소가 포르메이트-하이드로겐 리아제 복합체로서 본 명세서에 대해 언급된, 효소 복합체를 형성하는 것을 나타냈다. 특정한 실시양태에서, 이 복합체는 또한 복수의 보조인자 의존성을 달성하기 위한 재조합 미생물의 생성에 있어서 활용된다.
따라서, 본 발명은 재조합 미생물, 폴리펩티드, 또는 반응에서 복합 보조-인자(예를 들어, 페레독신 및 NADPH) 활용 목적을 위해 상기 효소를 인코딩하거나 발현하는 효소의 사용을 제공한다. 특정한 실시양태에서, 폴리펩티드는 AEI90721, YP_003778871, AEI90722, 또는 이의 임의의 하나의 기능적 균등 변이체에 따른 이분성 NADP 포르메이트 탈수소화효소를 포함한다.
특정한 실시양태에서, 이분성 NADP Fe-유일 수소화효소는 서열번호 10, 서열번호 26 및 YP_003778879, 및 이의 하나 이상의 기능적 균등 변이체로 구성된 군으로부터 선택된다.
특정한 실시양태에서, 이분성 포르메이트-하이드로겐 리아제 복합체는 서열번호 65 내지 67 또는 이의 기능적 균등 변이체에 의해서 인코딩된다.
특정한 실시양태에서, 이분성 NADP Fe-유일 수소화효소, NADP 포르메이트 탈수소화효소 또는 포르메이트-하이드로겐 리아제 복합체를 인코딩하는 폴리뉴클레오티드는 HQ876015, CLJU_c06990, AEI90722, 서열번호 9, 서열번호 25, CLJU_c07070, 서열번호 67 내지 69 및 이의 하나 이상의 기능적 균등 변이체로 구성된 군으로부터 선택된 하나 이상의 폴리뉴클레오티드를 포함한다.
이분성 NADP 포르메이트 탈수소화효소 및 이분성 NADP Fe-유일 수소화효소에 대한 유전자를 인코딩하는 단백질은 NADP 결합 부위를 갖는 철-황 플라보단백질, 철-황 (FeS) 단백질 및 셀레노시스테인- 및 몰리도프레틴-함유 포르메이트 탈수소화효소에 대한 유전자와 함께, 단일 유전자 클러스터에서 본 발명자들에 의해 밝혀졌다(도 3). 이들 유전자가 포르메이트 하이드로겐 리아제로서 본 명세서에 언급될 기능적 복합체를 인코딩한다는 것이 본 발명자들에 의해 제안된다(실시예 1 참고). 유전자 클러스터를 구성하는 철-황 플라보단백질, 철-황 (FeS) 단백질 및 포르메이트 및 포르메이트 및 몰리브데넘 악세사리 단백질은 하기 표 1에 나타낸 바와 같은 폴리펩티드에 의해 인코딩된다:
본 발명은 또한 반응에서 다중 보조인자를 이용할 목적으로 사용되는 경우, 신규한 이분성 (bifurcating) NADP Fe-유일 수소화효소, 이분성 NADP 포름산염 탈수소화효소 및/또는 포름산염-하이드로겐 리아제를 발현하는 재조합 일산화탄소영양 미생물을 제공한다. 바람직하게는, 다중 보조인자에는 페레독신 및 NADPH가 포함된다.
본 발명은 또한 전술된 바와 같은 이분성 수소화효소를 발현하는 재조합 일산화탄소영양 미생물을 이용함으로써, CO-함유 기질의 발효 효율을 증가시키는 방법을 제공한다. NADPH만을 이용하기 보다 페레독신 및 NADPH 둘 모두를 이용하는 이분성 효소로 인해, 그 효율이 증가된다. NADPH와 비교하여, 더 큰 음성 산화 환원 전위의 페레독신은 반응에 더 강한 전위를 제공하며, 이에 따라 반응 속도 및 CO 기질 처리량이 증가된다.
또한, 본 발명자들은 씨. 아우토에타노제눔, 씨. 리융다흘리 및 씨. 라그스달레이를 포함하는 일산화탄소영양 클로스트리듐 종에서 신규한 Nfn 효소를 발견하였다. 이 Nfn 효소는 NADH+ + H+의 소모시, 풀을 보충하기 위해, NADP+를 NADPH + H+로 환원시킬 수 있다 (또는 그 역의 경우도 가능)(반응 2):
(2) Fdred 2- + NADH + 2 NADP+ + H+ 
Fdox + NAD+ + 2 NADPH
이 효소는, 지금까지 복합체를 형성하는 두 개의 하위 단위 NfnA 및 NfnB로 이루어진 1 종의 유기물에 대해서만 씨. 클루이베리(C. kluyveri)(S. Wang et al., 2010)에서 기술되었다. 본 발명자들은 씨. 아우토에타노제눔(C.autoethanogenum) 세포에서의 활성을 동정하였고, 해당 유전자를 발견하였다 (실시예 4). 이에 따라, 일산화탄소영양 유기체에서, 단일 Nfn 유전자가 처음으로 발견되었고, Nfn 효소가 최초로 발견되었다. 2 종의 하위 단위의 복합체가 아닌 1 종의 하위 단위만을 가짐으로써, 효소의 생성 및 개질에 있어서 이점을 갖는다.
이러한 이론에 결부시키고자 하는 것은 아니나, 본 발명자들은, 두 가지 신규한 복합체 전자-분기 NADP Fe-단독 수소화효소 / NADP 포름산염 탈수소화효소/포름산염-하이드로겐 리아제 및 Nfn 복합체가, 반응 7-9의 개시된 페레독신 의존성 일산화탄소 탈수소화효소 (CODH), FoF1 Rnf 복합체 (Kopke et al., 2010; Tremblay, Zhang, Dar, Leang, & Lovley, 2012)에 따라, NADPH에 의해 유도되는, CO로부터의 환원 생성물의 생성 및 에너지 보존에서 주요 역할을 한다고 생각한다 (도 7).
(7) CO + H2O + Fdox ↔ CO2 + Fdred2- + 2 H+
(8) Fred 2- + NAD+ + H+ 
.Fdox + NADH + △μH+
(9) △μH+ + 0.5 ADP + 0.5 Pi 
0.5 ATP + 0.5 H2O
생체 내에서 페레독신은 -400 mV 이하의 음성 산화 환원 전위에서 작용하고, NADP는 -360 mV 근처의 산화 환원 전위에서 작용하며, NAD는 -280 mV 근처의 산화 환원 전위에서 작용한다. 120 mV 이상의 NAD+/NADH 쌍과 Fdox/Fdred 2 - 쌍 사이의 산화 환원 전위차는 막 결합 Rnf 복합체 (반응 8) 및 an FOF1 ATP 생성효소 (반응 9)에 의해 매개되는 전자 전달 인산화 반응에 의해 커플링되기에 충분히 크다. NAD+는 NADPH를 생성시키는 Nfn 복합체 촉매 반응 6 및 기타 NADH 의존성 반응을 통해 지속적으로 재생된다. NADPH는 추후, 확인된 다른 NADPH 의존성 반응에 따른 생성물 생성을 유도하는데 이용될 수 있다 (도 7).
CO의 강한 음성 산화 환원 전위 (- 520 mV)로 인해, 전자 운반체가 충분히 신속하게 재산화될 수 없는 경우, 페레독신 및 NADP가 과환원되는 경향이 있다. 페레독신- 및 NADPH 재산화율을 증가시키기 위한 한 가지 방법은 NADPH 의존성 반응을 선택하여, 환원 생성물의 생성율을 증가시키는 것이다.
표 2의 결과는, Nfn 효소를 발현하는 일산화탄소영양 미생물이 NADPH-의존성 효소에 의해 NADH를 NADPH로 전환시키는 능력을 가졌다는 것을 나타낸다.
| 반응 기질 | 효소 활성 | |
| Nfn |
NADPH + NAD+ + Fdox | 0.7 |
| NADPH + Fdox-RS + NAD+ | 0.3 | |
| NADPH + NAD+ | 0.09 |
본 발명자들은 Nfn 효소가 씨. 클루이베리에서와 같이 두 개가 아니라, 단일 유전자/단백질 (씨. 아우토에타노제눔에서 각각 서열번호 1 및 2)로 전사된다는 것을 발견하였다. 이 효소를 인코딩하는 유사 유전자가 또한 씨. 리융다흘리 (YP_003781852.1; CLJU_c37240)(글루타메이트 생성효소로서 주해됨) 및 씨. 라그스달레이 (서열번호: 3 및 4)에 존재한다.
본 발명자들은, 재조합 미생물에서, Nfn 유전자 또는 그의 기능적 변이체의 발현을 증진시킴으로써, NADPH-의존성 효소의 효율이 증가될 수 있고, 발효 반응의 생성물의 고수율로 유도할 수 있을 것이라고 생각한다.
따라서, 특정 측면에서, 본 발명은 Nfn 효소 복합체의 발현 또는 과발현에 의해, 미생물의 생성 효율을 증가시키는 방법을 제공한다.
특정 실시형태에서, 본 발명은, NADH를 NADPH로 전환시켜 NADPH 풀 크기를 증가시키기 위해, 단일 Nfn 효소의 발현 및/또는 과발현에 적응된 재조합 미생물을 사용하는 방법을 제공한다. 특정 실시형태에서, Nfn 효소는 서열번호 2, 4, YP_003781852.1, CLJU_c37240 또는 적어도 76%, 80%, 85%, 90%, 95%, 또는 99% 서열 동일성을 갖는 기능적 등가 변이체 중 임의의 하나의 아미노산 서열을 포함한다. Nfn 효소는 NADH를 NADPH로 전환시킴으로써, NADH 및 NADPH-의존성 효소의 존재 하에 발현되는 경우, 효소 효율이 증가하여, 반응 속도 및 NADPH의 재생율이 증가된다.
특정 실시형태에서, 상기 미생물에는 일산화탄소영양 클로스트리듐 미생물이 포함된다. 추가의 실시형태에서, 상기 미생물은 클로스트리듐 오토에타노게눔, 클로스트리듐 륭달리이, 클로스트리듐 라그스달레이를 포함하는 일산화탄소영양 클로스트리듐의 그룹으로부터 선택된다.
본 발명은 또한 NADH를 NADPH로 전환시키기 위해, 서열번호: 2, 4, YP_003781852.1, CLJU_c37240, 또는 기능적 등가 변이체 중 임의의 하나에 따른 단일 Nfn 효소를 포함하는 폴리펩티드를 사용하는 방법을 제공한다. 또한, 본 발명은 NADH를 NADPH로 전환시키기 위해 폴리뉴클레오티드를 사용하는 방법을 제공하며, 여기서 폴리뉴클레오티드는 단일 Nfn 효소를 인코딩하고, CLJU_c37240 또는 YP_003781852.1을 인코딩하는 서열인 서열번호: 1, 3 또는 적어도 83%, 85%, 90%, 95%, 또는 99% 서열 동일성을 갖는 그의 기능적 등가 변이체를 포함한다.
추가의 측면에서, 본 발명은 서열번호 1 또는 3에 따른 폴리뉴클레오티드를 제공한다.
추가의 측면에서, 본 발명은 서열번호 2 또는 4에 따른 폴리뉴클레오티드를 제공한다. 추가의 측면에서, 본 발명은 본 발명의 신규한 Nfn 폴리펩티드를 인코딩하는 폴리뉴클레오티드 및/또는 신규한 Nfn 폴리뉴클레오티드를 포함하는 벡터 및/또는 재조합 미생물을 제공한다.
특정 실시형태에서, 본 발명은 NADH-의존성 반응의 최적화를 위한 방법을 제공한다. 이 경우, 각각의 NADPH 의존성 수소화효소, 포름산염 탈수소화효소, 포름산염-하이드로겐 리아제, 및/또는 메틸렌-THF-탈수소화효소는, 예를 들어 무렐라 써모아세티카 또는 에이. 우디이(A. woodii)의 해당 NADH-의존성 효소를 대체할 수 있다. 이것은 NADH를 위해 설계 및 최적화된 경로를 통해, 흐름을 최적화할 수 있다. 이 실시형태는, NADPH-의존성 효소가 존재하지 않거나 또는, NADPH-의존성 효소를 포함하는 재조합 유기체가 효율적으로 작제될 수 없는 경우, 특히 유용할 수 있다.
특정 효소의 발현을 증가시키기 위해, 상기 효소를 인코딩하는 핵산의 발현을 증가시킨다. 목적 효소를 인코딩하는 핵산의 발현을 증가시키는 방법이 하기에서 개괄된다. 당업자는 기타 이용될 수 있는 기법을 용이하게 인식할 것이다.
본 발명은 본 명세서에서 언급되는 단백질 및 펩티드를 인코딩하는 핵산을 포함할 수 있고, 본 발명에서 이용되는 단백질 및 펩티드를 인코딩하는 핵산을 이용할 수 있다. 일 실시형태에서, 핵산은 핵산 작제물 또는 벡터이다. 특정 일 실시형태에서, 상기 핵산 작제물 또는 벡터는 발현 작제물 또는 벡터이나, 본 발명에는 클로닝에 이용될 수 있는 것과 같은 기타 작제물 및 벡터가 포함된다. 특정 일 실시형태에서, 상기 발현 작제물 또는 벡터는 플라스미드이다.
본 발명의 발현 작제물/벡터는, 바람직한 경우, 또한 단백질의 발현에 적합한 추가적 유전자뿐만 아니라, 또한 프로모터에 임의의 수의 조절 인자를 포함할 수 있다는 것이 인식될 것이다. 일 실시형태에서, 상기 발현 작제물/벡터는 하나의 프로모터를 포함한다. 다른 실시형태에서, 상기 발현 작제물/벡터는 두 개 이상의 프로모터를 포함한다. 특정의 일 실시형태에서, 상기 발현 작제물/벡터는 각각의 발현 대상 유전자 당 하나의 프로모터를 포함한다. 일 실시형태에서, 상기 발현 작제물/벡터는 하나 이상의 리보솜 결합 자리, 바람직하게는 각각의 발현 대상 유전자의 리보솜 결합 자리를 포함한다.
당업자는, 본 명세서에서 개시되는 핵산 서열 및 작제물/벡터가 리보솜 결합 자리 및/또는 제한 부위에 필요한 것과 같은 기준 링커 뉴클레오티드를 포함할 수 있다. 이러한 링커 서열은 필수적인 것으로 인식되어서는 안되며, 정의 서열을 제한하지 않는다.
본 발명의 발현 작제물/벡터를 포함하는 핵산 및 핵산 작제물은 임의의 수의 당업계 표준 기법을 이용하여 작제될 수 있다. 예를 들어, 화학 합성법 또는 재조합 기법이 이용될 수 있다. 이러한 기법은, 예를 들어 문헌 [Sambrook et al (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)]에 개시되어 있다. 또한, 예시 기법이 후술되는 실시예 부분에서 개시된다. 실질적으로, 개별 유전자 및 조절 인자는, 상기 유전자가 발현되어, 목적 단백질이 생성될 수 있도록, 서로 작동적으로 연결된다. 당업자는 본 발명에 이용되기에 적합한 벡터를 인식할 것이다. 그러나, 예를 들어, 하기 벡터가 적합할 수 있다: pMTL80000 벡터, pIMP1, pJIR750, 및 후술되는 실시예 부분에서 예시되는 플라스미드.
본 발명의 핵산이 RNA, DNA, 또는 cDNA를 포함하는 임의의 적절한 형태일 수 있다는 것이 인식되어야 한다.
본 발명은 또한 본 명세서에서 개시되는 핵산 중 하나 이상의 임의의 것을 포함하는, 바이러스, 박테리아 및 효모를 비롯한 숙주 유기체, 특히 미생물을 제공한다.
재조합 미생물의 제조 방법
하나 이상의 외인성 핵산이 기본 핵산으로서 모체 미생물 (parental microorganism)에 전달될 수 있거나 또는, 형질전환 과정을 촉진시키기 위한 하나 이상의 제제 (예를 들어, 리포좀-컨쥬게이션 핵산, 핵산이 포함된 유기체)에 의해 제형화될 수 있다. 상기 하나 이상의 핵산은 DNA, RNA, 또는 적절한 경우, 그의 조합일 수 있다. 제한 저해물질이 특정 실시형태에서 이용될 수 있다; 예를 들어, 문헌 [Murray, N.E. et al. (2000) Microbial. Molec. Biol. Rev. 64, 412.)]을 참조한다.
본 발명의 미생물은 임의의 수의, 재조합 미생물을 제조하기 위한 당업계의 공지 기법을 이용하여, 모 미생물 및 하나 이상의 외인성 핵산으로부터 제조될 수 있다. 단지 예로서, 형질전환 (형질도입 또는 형질주입 포함)은 전기천공법, 초음파처리, 폴리에틸렌 글리콜-매개 형질전환, 화학적 또는 자연적 기능 (chemical or natural competence), 프로토플라스트 형질전환, 프로파지 유도 또는 컨쥬게이션에 의해 성취될 수 있다. 적합한 형질전환 기법이, 예를 들어 문헌 [Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, Cold Spring Harbour, 1989]에 개시되어 있다.
전기천공법은 씨. 리융다흘리 (Kopke et al. 2010, Poc. Nat. Acad. Sci. U.S.A. 107: 13087-92; (Leang et al., 2011) PCT/NZ2011/000203; WO2012/053905), 씨. 아우토에타노제눔 (PCT/NZ2011/000203; WO2012/053905), Acetobacterium woodii (Straetz et al., 1994, Appl. Environ. Microbiol. 60:1033-37) 또는 무렐라 써모아세티카(Kita et al., 2012)로서 다수의 일산화탄소영양 아세토젠에서 기술되었으며, 씨. 아세토부틸리쿰(Mermelstein et al., 1992, Biotechnology, 10, 190-195), 씨. 셀룰로리티쿰(C. cellulolyticum) (Jennert et al., 2000, Microbiology, 146: 3071-3080) 또는 씨. 써모셀륨(C. thermocellum) (Tyurin et al., 2004, Appl. Environ. Microbiol. 70: 883-890)과 같은 많은 클로스트리디아에서 이용되는 표준 방법이다. 프로파지 유도는 씨. 스카토로제네스(C. scatologenes) (Prasanna Tamarapu Parthasarathy, 2010, Development of a genetic Modification System in Clostridium scatologenes ATCC 25775 for generation of Mutants, Masters Project Western Kentucky University)의 경우의 일산화탄소영양 아세토젠에서 입증되었으며, 컨쥬게이션은 클로스트리듐 디피실(Clostridium difficile) (Herbert et al., 2003, FEMS Microbiol. Lett. 229: 103-110) 또는 또는 씨. 아세토부일리쿰(C. Acetobuylicum)(Williams et al., 1990, J. Gen. Microbiol. 136: 819-826)을 포함하는 많은 클로스트리디아에서 선택 방법으로서 기술되었고, 일산화탄소영양 아세토젠과 유사한 방식으로 이용될 수 있다.
특정 실시형태에서, 형질전환되는 미생물에서 활성인 제한 시스템으로 인해, 미생물에 도입되는 핵산의 메틸화가 필요하다. 이것은 하기에서 기술되고, 후술되는 실시예 부분에서 예시되는 것들을 포함한 다양한 기법을 이용하여 이루어질 수 있다.
예로서, 일 실시형태에서, 본 발명의 재조합 미생물은 하기 단계를 포함하는 방법에 의해 제조된다:
a. (i) 본 명세서에서 기술된 바와 같은 발현 작제물/벡터 및 (ii) 메틸전달효소 유전자를 포함하는 메틸화 작제물/벡터의 셔틀 미생물 (shuttle microorganism)로의 도입 단계;
b. 메틸전달효소 유전자의 발현 단계;
c. 상기 셔틀 미생물로부터 하나 이상의 작제물/벡터의 분리 단계; 및
d. 목표 미생물로의 하나 이상의 작제물/벡터의 도입 단계.
일 실시형태에서, 단계 B의 메틸전달효소 유전자는 실질적으로 발현된다. 다른 실시형태에서, 단계 B의 메틸전달효소 유전자의 발현이 유도된다.
셔틀 미생물은 상기 발현 작제물/벡터를 구성하는 핵산 서열의 메틸화를 촉진하는 미생물, 바람직하게는 제한 음성 미생물이다. 특정의 실시형태에서, 셔틀 미생물은 제한 음성 대장균, 바실러스 서브틸리스(Bacillus subtillis), 또는 락토코쿠스 락티스(Lactococcus lactis) 이다.
메틸화 작제물/벡터는 메틸전달효소를 인코딩하는 핵산을 포함한다.
상기 발현 작제물/벡터 및 메틸화 작제물/벡터가 셔틀 미생물에 도입될 때, 메틸화 작제물/벡터에 존재하는 메틸전달효소 유전자가 도입된다. 본 발명의 특정 일 실시형태에서, 메틸화 작제물/벡터가 유도성 lac 프로모터를 포함하고, 락토오스 또는 그의 유사체, 더욱 바람직하게는 이소프로필-β-D-티오-갈락토시드 (IPTG)의 첨가에 의해 유도된다 할지라도, 임의의 적절한 프로모터 시스템에 의해 유도될 수 있다. 기타 적절한 프로모터에는 ara, tet, 또는 T7 시스템이 포함된다. 본 발명의 추가의 실시형태에서, 메틸화 작제물/벡터 프로모터는 구성요소 프로모터이다.
특정 실시형태에서, 메틸화 작제물/벡터는 그에 존재하는 임의의 유전자가 셔틀 미생물에서 발현될 수 있도록 하는, 셔틀 미생물의 정체에 특이적인 복제 원점을 갖는다. 바람직하게는, 상기 발현 작제물/벡터는 그에 존재하는 임의의 유전자가 목표 미생물에서 발현될 수 있도록 하는, 목표 미생물의 정체에 특이적인 복제 원점을 갖는다.
메틸전달효소 효소의 발현에 의해, 발현 작제물/벡터에 존재하는 유전자가 메틸화된다. 그 후, 발현 작제물/벡터는 공지 방법 중 임의의 하나에 따라 셔틀 미생물로부터 분리할 수 있다. 단지 예시로서, 후술되는 실시예 부분에서 개시되는 방법론이 발현 작제물/벡터를 분리하기 위해 이용될 수 있다.
특정 일 실시형태에서, 작제물/벡터 둘 모두가 동시에 분리된다.
상기 발현 작제물/벡터는 임의의 수의 공지 방법을 이용하여, 목표 미생물로 도입시킬 수 있다. 그러나, 예로서, 후술되는 실시예 부분에서 개시되는 방법론이 이용될 수 있다. 발현 작제물/벡터의 메틸화로 인해, 발현 작제물/벡터에 존재하는 핵산 서열이 목표 미생물내로 혼입될 수 있고, 성공적으로 발현될 수 있다.
메틸전달효소 유전자가 셔틀 미생물내로 도입되어, 과발현될 수 있을 것으로 예상된다. 따라서, 일 실시형태에서, 생성된 메틸전달효소 효소는 공지 방법에 의해 수집될 수 있고, 시험관 내에서 발현 플라스미드의 메틸화에 이용될 수 있다. 그후, 발현 작제물/벡터는 발현시키기 위해, 목표 미생물로 도입시킬 수 있다. 다른 실시형태에서, 메틸전달효소 유전자를 셔틀 미생물의 게놈으로 도입시켜, 셔틀 미생물로 발현 작제물/벡터를 도입시키고, 셔틀 미생물로부터 하나 이상의 작제물/벡터를 분리한 다음, 목표 미생물로 발현 작제물/벡터을 도입시킨다.
전술된 바와 같은 발현 작제물/벡터 및 메틸화 작제물/벡터를 조합시켜, 일정 조성물을 제공할 수 있을 것이다. 이러한 조성물은 특히, 본 발명의 재조합 미생물을 제조하는데 있어서, 제한 장벽 기전 (restriction barrier mechanism)을 피하기 위해 이용될 수 있다.
특정 일 실시형태에서, 상기 발현 작제물/벡터 및/또는 메틸화 작제물/벡터는 플라스미드이다.
당업자는 본 발명의 미생물의 제조에 이용될 수 있는 다양한 적절한 메틸전달효소를 인식할 것이다. 그러나, 예로서, B바실러스 서브틸리스 파지 ΦT1 메틸전달효소 및 후술되는 실시예에서 제시되는 메틸전달효소가 이용될 수 있다. 일 실시형태에서, 메틸전달효소는 WO/2012/053905에 개시되었다.
메틸전달효소 유전자의 발현에 적용된 임의의 수의 작제물/벡터가 메틸화 작제물/벡터를 생성시키기 위해 이용될 수 있다. 그러나, 예로서, 후술되는 실시예 부분에서 개시되는 플라스미드가 이용될 수 있다.
제조 방법
본 발명의 일 실시형태에서, 미생물에 의해 발효되는 가스 기질은 CO 함유 가스 기질이다. 가스 기질은 자동차 배기가스와 같은 기타 일부 공급원으로부터 또는 산업적 과정의 부산물로서 얻어지는 CO-함유 폐기가스일 수 있다. 특정 실시형태에서, 상기 산업적 과정은 제강 과정, 비철 제품 제조과정, 석유 정제 과정, 석탄 가스화 과정, 전력 생성 과정, 카본 블랙 생성 과정, 암모니아 생성 과정, 메탄올 생성 과정 및 코크스 제조 과정과 같은 철 금속 제품 제조 과정으로 이루어진 그룹으로부터 선택된다. 이들 실시형태에서, CO-함유 가스는 임의의 통상의 방법을 이용하여, 대기로 방출되기 전에 산업적 과정으로부터 포집될 수 있다. CO는 합성가스 (일산화탄소 및 수소 포함 가스)의 성분일 수 있다. 산업적 과정으로부터 생성되는 CO는 정상적으로 타올라서, CO2가 생성되므로, 본 발명은 CO2 온실가스 배출의 감축 및 바이오연료 생성을 위해 특히 유용할 수 있다. 가스 CO - 함유 기질의 조성에 따라, 발효 과정으로의 도입 전에, 더스트 입자와 같은 바람직하지 않은 임의의 불순물을 제거하기 위해 처리하는 것이 바람직할 수 있다. 예를 들어, 가스 기질은 공지 방법에 의해 여과 또는 스크러브될 수 있다.
박테리아 성장 및 생성물 생성을 위해, 또한 CO-함유 기질 가스에, 적합한 액체 영양 배지가 바이오리액터에 공급될 필요가 있다는 것이 이해될 것이다.
방법 측면의 특정 실시형태에서, 발효는 수성 배양 배지에서 이루어진다. 방법 측면의 특정 실시형태에서, 기질의 발효는 바이오리액터에서 이루어진다.
기질 및 배지가 지속적으로, 배치 (batch) 또는 배치 공급 방식으로 바이오리액터에 공급될 수 있다. 영양 배지에는 이용되는 미생물의 성장에 충분한 비타민 및 미네랄이 포함될 것이다. CO에 의한 발효에 적절한 혐기성 배지는 당업계에 공지되어 있다. 예를 들어, Biebel (2001)에 적절한 배지가 개시되어 있다. 본 발명의 일 실시형태에서, 상기 배지는 후술되는 실시예 부분에서 개시되는 바와 같다.
발효는 바이오연료의 생성을 위해 적절한 발효 조건 하에서 바람직하게 이루어져야 한다. 고려되어야 하는 반응 조건에는 압력, 온도, 가스 유량률, 액체 유량률, 배지 pH, 배지 산화 환원 전위, 교반 속도 (연속 교반 탱크 반응기를 사용하는 경우), 접종물 농도, 액체상의 CO가 제한되지 않을 수 있는 최대 가스 기질 농도 및 생성물 억제를 피할 수 있는 최대 생성물 농도가 포함된다.
또한, 종종, 기질 흐름의 CO 농도 (또는 가스 기질의 CO 분압)를 증가시킴으로써, CO가 기질인 발효 반응의 효율을 증가시키는 것이 바람직할 수 있다. 증가된 압력에서 반응시킴으로써, 발효 과정을 위한 탄소원으로서 미생물에 의해 섭취될 수 있는 것으로서, 가스상으로부터 액체상으로의 CO 전달률이 현저하게 증가될 수 있다. 결국, 이것은, 바이오리액터가 대기압 이상의 압력에서 유지되는 경우, 보유 시간 (바이오리액터의 액체 용적을 유입 기체 유량률로 나눈 값으로 정의됨)이 단축될 수 있다는 것을 의미한다. 최적의 반응 조건은 이용되는 본 발명의 특정 미생물에 부분적으로 의존적이다. 그러나, 일반적으로, 주위 압력 이상의 압력에서 발효가 이루어지는 것이 바람직하다. 또한, CO 전환율이 부분적으로 기질 보유 시간의 함수이므로, 바람직한 보유 시간의 달성에 의해, 필요한 바이오리액터의 용적이 획득되고, 가압계를 이용함으로써, 필요한 바이오리액터의 용적이 현저하게 감소될 수 있으며, 결과적으로, 발효 장비의 자본비가 감축될 수 있게 된다. 미국 특허 제 5,593,886호의 예에 따르면, 반응기에서, 반응기 용적은 반응 압력의 증가에 선비례하여 감소될 수 있다. 즉, 10 대기압에서 작동되는 바이오리액터는 1 대기압에서 작동되는 바이오리액터 용적의 단지 1/10일 수 있다.
예로서, 증가된 압력에서, 가스-에탄올 발효의 이점이 개시되어 있다. 예를 들어, WO 02/08438은 30 psig 및 75 psig의 압력 하에서 이루어져, 각각 150 g/L/일 및 369 g/L/일의 에탄올 생성률을 제공하는 가스-에탄올 발효에 대해 기술하고 있다. 그러나, 유사한 배지 및 대기압에서의 유입 가스 조성을 이용한 예시적 발효에 의해, 10 배 내지 20 배의 에탄올/리터/일이 생성된다는 것이 발견되었다.
또한, 액체상의 CO 농도가 제한되지 않을 수 있는 CO-함유 가스 기질의 도입률이 바람직할 수 있다. 왜냐하면, CO-제한 조건의 결과, 하나 이상의 생성물이 배양 과정에서 소모될 수 있기 때문이다.
발효 반응에 공급하기 위해 이용되는 가스 흐름의 조성물은 반응의 효율 및/또는 비용에 상당한 영향을 미친다. 예를 들어, O2는 혐기성 발효 과정의 효율을 저하시킬 수 있다. 발효 과정 전 또는 후에 발효 과정의 단계에서 원치않거나 또는 불필요한 가스의 처리는 이러한 단계의 부담을 증가시킬 수 있다 (예를 들어, 가스 흐름이 바이오리액터 도입 전에 압축되는 경우, 발효 과정에 필요하지 않은 가스의 압축에 불필요한 에너지가 사용될 수 있음). 따라서, 원치않는 성분을 제거하고, 바람직한 성분의 농도를 증가시키기 위해, 기질 흐름, 특히 산업적 공급원으로부터 유래하는 기질 흐름을 처리하는 것이 바람직할 수 있다.
특정 실시형태에서, 본 발명의 박테리아 배양은 수성 배양 배지에서 유지된다. 바람직하게는, 수성 배양 배지는 혐기성 미생물 최소 성장 배지이다. 적절한 배지가 당업계에 공지되어 있으며, 예를 들어 미국 특허 제 5,173,429호 및 제 5,593,886호 및 WO 02/08438에 개시되어 있으며, 후술되는 실시예 부분에서 기술되는 바와 같다.
또한, 활성탄으로의 아세트산의 흡수를 증진시키기 위해, 브로쓰의 pH를 전술된 바와 같이 조정하는 경우, 바이오리액터로의 재유입 전에, 발효 바이오리액터의 브로쓰의 pH와 유사하게 pH를 재조정해야 한다.
실시예
실시예 1
Wood-Ljungdahl 경로의 산화환원효소 5 단계 전부를 다른 기질의 존재 하에서 그 활성을 측정하기 위해, 분석하였다. 이들 효소에서, 반응 유도를 위해 공동 인자가 이용될 수 있다. 상기 효소는 CO, CO2, 및 H2 가스의 흡수 및 이용을 포함하는 자가 영양 성장에 포함된다.
분석 효소 및 그 활성은 도 1에 상세하게 개시된다. 시행된 모든 분석은 대조군으로서, 합성 산화 환원 염료, 메틸 비올로겐 (MV) 또는 벤질 비올로겐 (BV)을 이용하여 시험하였다. 공동 인자 페레독신 (Fd), NADH 및 NADPH 또는 그의 조합이 시험되었다. 효소 분석은 CO 및 수소에서 자가 영양 성장되는 통상의 반응기로부터의 미정제 추출물을 이용하여 시행하였다.
발효
씨. 아우토에타노제눔 DSM23693에 의한 발효를, 후술되는 바와 같은 유일한 에너지 및 탄소원으로서, CO-함유 제강 기체 및 37℃의 1.5L 바이오리액터에서 반응시켰다. 배지 성장을 위해, 리터 당 MgCl, CaCl2 (0.5mM), KCl (2mM), H3PO4 (5mM), Fe (100μM), Ni, Zn (5μM), Mn, B, W, Mo, Se(2μM)를 함유하는 정의된 배지를 사용하였다. 배지를 바이오리액터로 옮기고, 121℃에서 45 분 동안 오토클레이브하였다. 오토클레이브 후, 배지에, 티아민, 판토테네이트 (0.05mg), 비오틴 (0.02mg)을 보충하고, 3mM 시스테인-HCl으로 환원시켰다. 무산소화를 달성하기 위해, 반응 용기를 0.2 μm 필터를 통해 질소로 스파징 (sparging)하였다. 접종 전에, 가스를 CO-함유 제강 가스로 교체하여, 반응기로 연속 공급하였다. 공급 가스의 조성은 2% H2 42% CO 20% CO2 36% N2이었다. 배양액 pH는 5 및 5.2로 유지하였다.
세포 수집
세포 수집시, 가스 소모율은 5 몰 CO L-1 일-1 및 10 밀리몰 H2 L-1 일-1이고, 그후 대사물 14 g L-1 일-1 아세테이트 및 19.5 g L-1 일-1 에탄올이 생성되었다. 배양액 pH를 K2CO3에 의해 pH 6으로 조정하고, 반응기를 얼음 욕조에서 냉각시켰다. ~1.2 L의 배양액을 얼음에서 수집하였다. 배양액을 2x 1-L 원심분리용 병으로 나누고 (효소의 불활성화를 방지하기 위해, 무산소 조건이 되도록 하기 위해, 이 단계 및 그후 모든 단계를 무산소 챔버에서 수행하였음), 세포를 10 분 동안 5000 rpm에서 펠렛팅하였다. 상청액을 붓고, 잔여액을 제거하였다. 각 펠렛을 10 mM DTT가 포함된, pH 7.0의 50 mM K PO4 ~30 mL 중에 재현탁시켰다. 재현탁액을 일정 중량의 50-mL-팔콘-튜브로 옮기고, 세포를 15 분 동안 최대 속도 (5000g)에서 재펠렛팅하였다. 튜브를 무산소 챔버로부터 제거하고, 분석 전에 액체 N2에서 즉시 동결시켰다.
미정제 세포 추출물의 제조 및 효소 분석
세포를 무산소 조건에서 연속 반응기로부터 수집하였다. 수집된 세포를 문헌 [Huang et al., 2012]에 기술된 바와 같이, 프렌치 프레스 (French press)를 통해 세 개의 경로로 분리시켰다.
지정된 경우를 제외하고, 모든 분석은 문헌 [Huang et al., 2012]에 기술된 바와 같이, 1.2 x 105 Pa에서, 0.8 ml 반응 혼합물 및 0.7 ml N2 또는 H2 또는 CO가 충진된, 고무 마개로 밀봉된 1.5-ml-무산소 큐벳, 37℃에서 수행하였다.
CO 탈수소화효소, 포름산염 탈수소화효소, 메틸렌-THF 탈수소화효소, 및 메틸렌-THF 환원 효소를 모두 [Huang et al., 2012]에 기술된 바와 같이 분석하였다.
CO 탈수소화효소를 100 mM Tris/HCl (pH 7.5), 2 mM DTT 및 약 30 μM 페레독신 및/또는 1 mM NAD+ 또는 1 mM NADP+가 포함된 분석 혼합물을 이용하여 측정하였다. 가스 상은 100% CO이었다.
수소화효소 활성을 개시된 바와 같이 측정하고, H2에 의한 NADP+ 의존성 페레독신 환원율을 추가로 측정하였다. 반응 혼합물에, 페레독신 (30 μM) 및 1 mM NADP를 보충하였다. 가스상은 100% H2이었다. 반응 개시 후에 효소 페레독신 환원은 430 nm (εΔox -red 
13.1 mM- 1 cm- 1)에서 수행하였다.
포름산염-하이드로겐 리아제 활성을 1.2 x 105 Pa에서 0.8 ml 반응 혼합물 및 4.2 ml N2가 충진된, 고무 마개에 의해 밀봉된 5-ml 무산소 세럼 바틀에서 측정하였다. 반응 혼합물에는 100 mM Tris-HCl pH 7.5 및 20 mM 포름산염이 함유되었다. 효소 첨가에 의한 반응 개시 후, H2 생성을 가스 크로마토그래피에 의해 모니터링하였다. 포름산염으로 H2에 의한 CO2의 환원을 위한 포름산염-하이드로겐 리아제 활성을 100 mM 인산 칼륨, 2 mM DTT, 및 30 mM [14C]K2CO3 (24,000 dpm/μmol)이 함유된 분석 혼합물에 의해 측정하였다. 가스상은 100% H2이었다. 액체상과 가스상의 평형을 확실히하기 위해, 세럼 바틀을 200 rpm에서 지속적으로 교반시켰다. 효소에 의한 반응 개시 후, 100 μl 액체 샘플을 1.5 분 마다 회수하여, 산성화에 의해 반응을 정지시키기 위해, 100 μl의 150 mM 아세트산이 함유된 1.5-ml 안전 밀봉 마이크로 튜브에 첨가하였다. 이어서, 200 μl 혼합물을 Thermomixer에서 1,400 rpm에서 교반하면서, 40℃에서 10 분 동안 인큐베이션시켜서, 14C-포름산염 생성 후 방출된 모든 14CO2를 제거하였다. 그후, 100 μl의 혼합물을 5 ml의 Quicksave A 신틸레이션 유체 (Zinsser Analytic, Frankfurt, Germany)에 첨가하고, Beckman LS6500 액체 신틸레이션 카운터 (Fullerton, CA)에서 14C 방사성에 대해 분석하였다.
포름산염 탈수소화효소 측정을 100 mM Tris/HCl (pH 7.5) 또는 100 mM 인산 칼륨, 2 mM DTT, 20 mM 포름산염 및 지정된 경우, 25 μM 페레독신, 1 mM NADP+, 1 mM NAD+ 및/또는 10 mM 메틸 비올로겐이 함유된 분석 혼합물로 수행하였다. 가스상은 100% N2이었다.
메틸렌-H4F 탈수소화효소를 100 mM MOPS/KOH (pH 6.5), 50 mM 2-메르캅토에탄올, 0.4 mM 테트라하이드로폴레이트, 10 mM 포름알데하이드 및 0.5 mM NADP+ 또는 0.5 mM NAD+가 함유된 분석 혼합물을 이용하여 측정하였다. 가스상은 100% N2이었다.
메틸렌-H4F 환원 효소를 하기 조건에서 분석하였다. 분석 혼합물에는 100 mM Tris/HCl (pH 7.5), 20 mM 아스코르브산염, 10 μM FAD. 20 mM 벤질 비올로겐 및 1 mM 메틸-H4F가 함유되었다. 효소에 의한 반응 개시 후, 벤질 비올로겐을 디티온산 나트륨으로 0.3의 △A555로 환원시켰다.
알데하이드:페레독신 산화환원효소를 100 mM Tris/HCl (pH 7.5), 2 mM DTT, 1.1 mM 아세트알데하이드, 및 약 25 μM 페레독신이 함유된 혼합물을 이용하여 분석하였다. 가스상은 100% N2이었다.
CoA 아세틸화 아세트알데하이드 탈수소화효소를 100 mM Tris/HCl (pH 7.5), 2 mM DTT, 1.1 mM 아세트알데하이드, 1 mM 조효소 A, 및 1 mM NADP+ 또는 1 mM NAD+가 함유된 혼합물을 이용하여 측정하였다. 가스상은 100% N2이었다.
알코올 및 부탄디올 탈수소화효소를 각각 100 mM 인산 칼륨 (pH 6), 2 mM DTT, 1.1 mM 아세트알데하이드 또는 아세토인 및 1 mM NADPH 또는 1 mM NADH에 의한 분석에서 측정하였다. 가스상은 100% N2이었다.
페레독신을 문헌 [Schonheit, Wascher, & Thauer, 1978]에 개시된 바와 같이, C. pasteurianum로부터 정제하였다.
결과
수소화효소: 이 효소는 에너지원으로서 수소 흡수를 위해 중요하며, CO2에서 일산화탄소영양 미생물의 성장을 위해 필수적이다. 이 효소는 또한 수소를 생성시킬 수 있으며, 포름산염 하이드로겐 리아제로서, 포름산염 탈수소화효소에 의한 컨쥬게이션에서 작용할 수 있다.
씨. 아우토에타노제눔의 게놈에, 7 수소화효소 유전자 (6 Fe-단독 수소화효소 및 하나의 NiFe 수소화효소; 서열번호 5-20)가 존재한다. 이들 5 개의 유전자의 상동체가 씨. 리융다흘리의 게놈에 존재하고 (Kopke et al., 2010) (YP_003781016/CLJU_c26060; YP_003781017/CLJU_c26070; CLJU_c07070/YP_003778879; CLJU_c14700/YP_003779640; CLJU_c17280/YP_003779893; CLJU_c20290/YP_003780193), 또한 씨. 라그스달레이의 게놈에서 동정할 수 있다 (서열번호 21-32) (표 3).
| 씨. 아우토에타노제눔 | 씨. 리융다흘리 | 씨. 라그스달레이 | |
| [NiFe] 수소화효소 | 서열번호 5-8 | YP_003781016-17; CLJU_c26060-70 |
서열번호 21-24 |
| [FeFe] 수소화효소 | 서열번호 9-10 | CLJU_c07070; YP_003778879 |
서열번호 25-26 |
| [FeFe] 수소화효소 | 서열번호 11-12 | CLJU_c14700; YP_003779640 |
서열번호 27-28 |
| [FeFe] 수소화효소 | 서열번호 13-14 | ||
| [FeFe] 수소화효소 | 서열번호 15-16 | CLJU_c20290; YP_003780193 |
서열번호 29-30 |
| [FeFe] 수소화효소 | 서열번호 17-18 | CLJU_c17280; YP_003779893 |
서열번호 31-32 |
| [FeFe] 수소화효소 | 서열번호 19-20 |
단일 보조인자를 이용하여, NADPH (0.2 U/mg)에 의해 활성을 관찰하였으며, NADH (0.05 U/mg) 또는 페레독신 (<0.01 U/mg)에 의해, 0 또는 매우 낮은 활성이 관찰되었다. 이것은, 수소화효소가 NADPH 특이적이라는 것을 입증하는 것이다.
보조인자의 조합을 이용하는 경우, 최고 활성이 확인되었다. 페레독신의 존재 하에, NADPH에 의해, 0.68 U/mg이 측정되었다. 반대로, NADH (<0.01 U/mg)의 경우, 측정가능한 활성이 관찰되지 않았으며, 이는, NADPH에 대한 이 효소의 고 특이성을 재확인시키는 것이다. 이 데이타는, 이분성 수소화효소가 써모토가 마리티마(Thermotoga maritima)(Schut & Adams, 2009) 또는 아세토박테리움 우디 (Schuchmann & Mueller, 2012) 또는 무렐라 써모아세티카 (Huang et al., 2012)에 존재한다는 것을 시사한다. 그러나, 이들 다른 유기체에서, 상기 효소는 NADH 의존성이다. 이와 같이, 이것은 발견된 최초의 NADPH 의존성 이분성 수소화효소이다.
포름산염 탈수소화효소: 이 효소는 Wood-Ljungdahl 경로의 메틸 가지에서 포름산염으로 CO2의 환원을 촉매하고, CO 또는 CO2 및 H2에서 아세토젠에 의한 자가 영양 성장에 필수적이다.
3 개의 유전자는 셀레노 및 비-셀레노 포름산염 탈수소화효소를 인코딩하고, 씨. 아우토에타노제눔 (AEI90721, AEI90723, AEI90725; HQ876015, HQ876017, HQ876019), 씨. 리융다흘리 (YP_003779063, YP_003778871, YP_003780168; CLJU_c08930, CLJU_c06990, CLJU_c20040) 및 씨. 라그스달레이 (AEI90722, AEI90724, AEI90726; HQ876016, HQ876018, HQ876020) (Kopke et al., 2010, 2011)의 게놈에 존재한다.
단지 하나의 공동 인자를 이용하는 경우, NAD가 아닌 NADPH에 대한 특이성이 검출되었다: 0.2 U/mg 이상, 매우 작은 경우 0.03 U/mg.
그러나, 두 개의 공동 인자의 조합의 이용시, 상당한 고활성이 검출되었고: NADPH 및 페레독신의 경우, 1.10 U/mg이 검출되었으며, NADPH 대신 NADH에 의한 경우, 단지 0.07 U/mg이 검출되었다. 이것은, 개시된 적이 없는 효소인, 이분성 NADP 포름산염 탈수소화효소의 존재를 시사하는 것이다.
포름산염-하이드로겐 리아제: H2에 의한 경우, 2.4 U/mg의 고활성이 검출되었으며, 이는 이분성 NADP 포름산염 탈수소화효소가 NADPH 이분성 수소화효소와 포름산염-수소 복합체를 형성할 수 있다는 것을 시사한다.
이분성 NADP 포름산염 탈수소화효소 (AEI90721, HQ876015; YP_003778871, CLJU_c08930; AEI90722, HQ876016) 및 이분성 NADP Fe-단독 수소화효소 (서열번호 9-10; CLJU_c07070, YP_003778879 ; SeqID 25-26)의 단백질 인코딩 유전자가 NADP 결합 자리의 철-황 플라빈단백질, 인-황 (FeS) 단백질 및 셀레노시스테인- 및 몰리브도프테린-함유 포름산염 탈수소화효소의 유전자를 따라, 하나의 유전자 클러스터에서 발견되었다 (도 3). 두 개의 효소의 유전자가 게놈에서 차례로 존재하며, 전사 단위를 형성할 수 있다는 발견은 기능성 복합체 형성을 반영한다.
CO2 및 H2로부터 포름산염 방향으로 작용하는 포름산염-하이드로겐 리아제는 이전에 개시된 적이 없으며, 일산화탄소영양 클로스트리듐에 있어서 신규한 것이다 (도 1). 또한 이 반응의 가역성이 입증되었고, 포름산염으로부터 수소 및 CO2가 방출되었다. 이 효소를 이용함으로써, 그후 재방출될 수 있는 수소를 이용한 포름산염의 생성에서 CO2가 포집될 수 있다. 정제 효소에 의해, CO2+H2로부터 포름산염의 생성의 경우, 41 U/mg 및 포름산염으로부터 수소 생성의 경우, 40 U/mg의 포름산염 하이드로겐 리아제 활성이 측정되었다 (표 4).
| 기질 | 특이적 활성 (U/mg) |
| H2 + NADP+ + Fdox | pH 6.5에서 32 (pH 7.5에서 29.2) |
| H2 + NAD+ + Fdox | <0.2 |
| H2 + NADP+ | 1.6 |
| H2 + Fdox | <0.2 |
| H2 + NAD+ | <0.1 |
| NADPH + Fdred2- (H2 생성) | pH 6에서 26.5 (pH 7.5에서 8.7) |
| NADPH (H2 생성) | <0.1 |
| Fdred2- (H2 생성) | 0.9 |
| 포름산염 + NADP+ + Fdox | pH 7.5에서 15.2 (pH 6.5에서 13) |
| 포름산염 + NAD+ + Fdox | 0.2 |
| 포름산염 + NADP+ | 2 |
| 포름산염 + Fdox | 0.2 |
| 포름산염 + NAD+ | <0.1 |
| CO2 + Fdred2- + NADPH (포름산염 생성) | pH 7.5에서 7 (본문 참조) |
| CO2 + H2 (포름산염 생성) | pH 7.0에서 41 (pH 7.5에서 35) |
| CO2 + H2 + Fdox + NADP+ (포름산염 생성) | 40 |
| 포름산염 (H2 생성) | pH 6에서 40 (pH 7.5에서 23) |
| H2 + MV | pH 7.5에서 18,000 |
| 포름산염 + MV | 170 |
| NADPH + MV | 27 |
| NADH + MV | <0.1 |
표 4와 관련하여, 씨. 아우토에타노제눔의 포름산염 하이드로겐 리아제 복합체의 정제를 실온에서 엄격한 무산소 조건 하에서 수행하였다. 2 mM DTT, 5 μM FAD, 및 5 μM FMN이 함유된 무산소 50 mM Tris-HCl (pH 7.6)(완충액 A)을 전 과정을 통해 사용하였다. 대략 47 mg 단백질 ml-1이 포함된 세포질 분획을 함유하는 150,000 x g 상청을 황산 암모늄으로 분별하였다. 40%와 55% 황산 암모늄 포화 사이의 분획을 4℃에서 30 분 동안 30,000 × g에서 원심분리에 의해 수집하였다. 침전물을 0.8 M 황산 암모늄이 함유된 7 ml 완충액 A 중에 용해시켰다. 원심분리에 의해 용해되지 않은 단백질을 제거 한 후, 상청액을, 0.8 M 황산 암모늄이 함유된 완충액 A로 평형화된 페닐 세파로오스(Phenyl Sepharose) 고성능 칼럼 (2.6 cm, 12 cm)에 부하하였다. 단백질을 5 ml 분-1의 유속에서 단계적 황산 암모늄 구배 (완충액 A 중에 각각 0.80, 0.64, 0.48, 0.32, 0.16, 및 0 M; 100 ml)로 용리시켰다. 수소화효소 활성을 0.48 M 황산 암모늄의 피크에서 용리하였다. 풀 (pool) 분획을 농축하고, 50-kDa-컷오프 막 (cutoff membrane)의 아미콘(Amicon) 세포로 탈염시켰다. 이어서, 농축물을 완충액 A에 의해 평형화된 Q 세파로오스 고성능 칼럼 (1.6 cm, 13 cm)에 적용하였다. 이어서, 칼럼을 90 ml 완충액 A로 세척하였다. 단백질을 5 ml 분-1의 유속에서 0 내지 1 M NaCl 선형 구배로 용리하였다. 수소화효소 활성을 약 0.4 M NaCl 용리 단일 피크에서 회수하였다. 분획을 농축하고, 50-kDa-컷오프 아미콘 필터로 탈염한 다음, 사용시까지, 95% N2/5% H2의 대기 하에서, -20℃의 완충액 A에 저장하였다.
활성을 37℃에서 지정 pH의 100 mM 인산 칼륨에서 측정하였다. 포름산염 (포름산염 하이드로겐 리아제 활성)으로부터 H2가 생성된 경우, 분석 혼합물은 100 mM Tris-HCl (pH 7.5) (표 1) 또는 100 mM 인산 칼륨 (지정 pH) (표 3), 2 mM DTT 및 20 mM 포름산 나트륨을 함유한다. 가스상은 100% N2이었다. 세럼 바틀을 200 rpm에서 연속 교반하여, 액체상으로부터 가스상으로 H2가 확실히 전달되도록 하였다. 가스 샘플 (0.2 ml)을 1 분 마다 회수하고, H2를 가스 크로마토그래피에 의해 정량화하였다. 포름산염으로 H2에 의한 CO2의 환원이 측정된 경우, 분석 혼합물은 100 mM 인산 칼륨 (최종 지정 pH), 2 mM DTT, 및 30 mM [14C]K2CO3 (24,000 dpm/μmol)를 함유한다. 가스상은 100% H2이었다. 세럼 바틀을 200 rpm에서 연속 교반하여, 액체상과 가스상의 평형을 확실히 하였다. 효소에 의한 반응 개시 후, 100 μl 액체 샘플을 1.5 분 마다 회수하고, 100 μl의 150 mM 아세트산이 함유된 1.5-ml 안전 밀봉 마이크로 튜브에 첨가하여, 산성화에 의해 반응을 정지시켰다. 이어서, 200 μl 혼합물을 Thermomixer (유형 5436, Eppendorf, Germany)에서 1,400 rpm에서 교반하면서 10 분 동안 40℃에서 인큐베이션시켜서, 14C-포름산염 생성 후 방출된 모든 14CO2를 제거하였다. 그후, 100 μl의 혼합물을 5 ml의 Quicksave A 신틸레이션 유체 (Zinsser Analytic, Frankfurt, Germany)에 첨가하고, Beckman LS6500 액체 신틸레이션 카운터 (Fullerton, CA, USA)에서 14C 방사성에 대해 분석하였다. 포름산염으로 환원된 페레독신 및 NADPH에 의해 CO2가 환원된 경우, 분석 혼합물은 100 mM 인산 칼륨 (최종 지정된 바와 같음), 2 mM DTT, 30 mM [14C]K2CO3 (24,000 dpm/μmol), 1 mM NADPH, 및 환원된 페레독신-생성계 (10 mM 피루베이트, 0.1 mM 티아민 피로포스페이트, 1 mM 조효소 A, 25 μM 씨. 파스투리아눔(C. pasteurianum ) 페레독신, 1 U 피루베이트:페레독신 산화환원효소, 및 5 U 인산아세틸기전달효소)를 함유한다. 가스상은 100% N2이었다. 세럼 바틀을 200 rpm에서 연속 교반하여, 액체상과 가스상의 평형을 확실히 하였다. 효소에 의한 반응 개시 후, 100 μl 액체 분취량을 1.5 분 마다 회수하고, 포름산염에 대해 분석하였다. 포름산염으로 환원된 페레독신 및 NADPH에 의해 CO2가 환원된 경우, 분석 혼합물은 100 mM 인산 칼륨 (최종 지정된 바와 같음), 2 mM DTT, 30 mM [14C]K2CO3 (24,000 dpm/μmol), 1 mM NADPH, 및 환원된 페레독신-생성계 (10 mM 피루베이트, 0.1 mM 티아민 피로포스페이트, 1 mM 조효소 A, 25 μM 씨. 파스투리아눔 페레독신, 1 U 피루베이트:페레독신 산화환원효소, 및 5 U 인산아세틸기전달효소)를 함유한다. 가스상은 100% N2이었다. 세럼 바틀을 200 rpm에서 연속 교반하여, 액체상과 가스상의 평형을 확실히 하였다. 효소에 의한 반응 개시 후, 100 μl 액체 분취량을 1.5 분 마다 회수하고, 전술된 바와 같이, 포름산염에 대해 분석하였다. 씨. 파스투리아눔 DSM 525로부터 정제된 페레독신 (Fd)을 문헌 [Schonheit et al (Rapid procedure for purification of ferredoxin from clostridia using polyethyleneimine. FEBS Lett. 1978, 89:219-222)]에 따라 제조하여 사용하였다. 일 단위 (U)는 분 당 전달된 2 μmol 전자이다.
메틸렌-THF-탈수소화효소: 이 효소는 5,10-메틸렌테트라하이드로폴레이트로부터 5,10-메텐일테트라하이드로폴레이트로의 반응을 촉매하고, 자가 영양 성장에 필수적이다. 이것은 Wood-Ljungdahl 경로의 일부이며, 명백하게 NADPH 특이적인 것으로 확인되었다 (NADPH의 경우, 1.12 U/mg, NADH 또는 페레독신의 경우, 활성 검출불가능).
이 효소 및 각각의 유전자는 2기능 메틸렌-테트라하이드로폴레이트 탈수소화효소/포르밀-테트라하이드로폴레이트 시클로가수분해효소로서 씨. 아우토에타노제눔 (AEI90753; HQ876031, GI:338225353), 씨. 리융다흘리 (YP_003781891; CLJU_c37630) 및 씨. 라그스달레이 (AEI90771; HQ876032, GI:338225372)에서 발견되었다.
이 효소는 종래에 무렐라 써모아세티카에서 NADPH-의존성인 것으로 나타났으며, 다른 반응은 이 유기체에서 NADH 또는 페레독신 의존성이다 (Huang et al., 2012).
보조인자의 메틸렌-THF 환원 효소에 대한 시험관 내 분석에서 측정가능한 활성은 검출되지 않는다 (합성 염료 단독). 그러나, 본 발명자들은, 이 결과가, 씨. 리융다흘리 또는 에이. 우디이 (Kopke et al., 2010; Poehlein et al., 2012)와 같은 다른 효소에서 제안된 바와 같은 추가의 효소로서, 미지의 커플링 위치가 필요한 효소에 의해 설명될 수 있다고 생각한다. 이 커플링 메카니즘은 NADPH 의존성일 수 있다. CO 탈수소화효소 반응은 이 종류의 효소에서 종래에 보고된 바와 같이, 페레독신 의존성인 것으로 나타났다.
일산화탄소영양 클로스트리듐 클로스트리듐 오토에타노게눔에서 Wood-Ljungdahl 경로의 5 가지의 시험된 산화환원효소 반응 모두로부터, 놀랍게도, NADH 의존성이 아니었으며, 대부분은 오히려 NADPH 의존성인 것으로 나타났다. 이러한 사실은, 예를 들어 대장균와 같은 박테리아을 이용한 당의 해당과정과 완전히 반대되는 것이다 (도 2). 따라서, 대장균에 대한 전략으로서, NADH 의존성 반응 및 우회 NADPH 의존성 반응 (생성물 수율이 저하되고, 대규모 변형이 요구됨)을 이용하는 경우, 일산화탄소영양 클로스트리듐에서 생산적이지 않다. 본 명세서에서 개시된 바와 같이, 본 발명은 대사 조작에서 최대 생성물 수율을 달성하기 위해, 일산화탄소영양 클로스트리듐에서 NADPH 의존성 반응에 대해 우선 선별함에 의한 극복 전략을 제공한다. 대장균에 의한 경우, 당에 있어서의 차이점뿐만 아니라, NADPH 의존성 반응의 능력 및 가능성이 실시예 3에서 제시된다. 유사하게, 이 전략은 최대 생성물 수율 및 흐름을 달성하기 위해, 이종 경로에 적용될 수 있다.
실시예 2
200 개 이상의 유전자 씨. 아우토에타노제눔 유전자의 상대 발현을 최대 발현의 유전자를 측정하기 위해, 실시간 정량 PCR을 이용하여 분석하였다.
발효
씨. 아우토에타노제눔 DSM23693에 의한 발효를, 후술되는 바와 같은 유일한 에너지 및 탄소원으로서, CO-함유 제강 기체 및 37℃의 1.5L 바이오리액터에서 반응시켰다. 배지 성장을 위해, 리터 당 MgCl, CaCl2 (0.5mM), KCl (2mM), H3PO4 (5mM), Fe (100μM), Ni, Zn (5μM), Mn, B, W, Mo, Se(2μM)를 함유하는 정의된 배지를 사용하였다. 배지를 바이오리액터로 옮기고, 121℃에서 45 분 동안 오토클레이브하였다. 오토클레이브 후, 배지에, 티아민, 판토테네이트 (0.05mg), 비오틴 (0.02mg)을 보충하고, 3mM 시스테인-HCl으로 환원시켰다. 무산소화를 달성하기 위해, 반응 용기를 0.2 μm 필터를 통해 질소로 스파징하였다. 접종 전에, 가스를 CO-함유 제강 가스로 교체하여, 반응기로 연속 공급하였다. 가스 흐름을 처음에 80 ml/분으로 설정하고, 중간 지수기 (mid-exponential phase) 과정 동안, 200 rpm에서 350 rpm으로 증가시켜, 교반하면서, 200 ml/분으로 증가시켰다. Na2S를 0.25 ml/hr에서 바이오리액터에 제공하였다. OD600이 0.5가 되면, 바이오리액터를 1.0 ml/분의 속도에서, 연속 모드로 교체하였다 (희석률 0.96 d-1). 바이오매스 및 대사물의 측정을 위해, 배지 샘플을 취하고, 유입 및 유출 가스의 헤드스페이스 분석 (headspace analysis)을 정규 기준에서 수행하였다.
qRT-PCR
200 개 이상의 유전자에 의한 qRT-PCR 연구를 적절한 프라이머를 이용하여 수행하였다. 샘플을, 전 성장 기간 (4 일) 동안 전술된 바와 같은 통상의 1.5L 공급-배치 발효 과정으로부터 취하였다. 샘플을 원심분리 (6,000 x g, 5 분, 4 ℃)에 의해 수집하고, 세포 펠렛을 액체 질소에서 스냅 동결시키고, 사용시까지 -80 ℃에 저장하였다. RNA를 얼음 상에서 세포 펠렛을 해동시킴으로써 분리하고, 100 μL의 라이소자임 용액 (50,000 U 라이소자임, 0.5 μL 10% SDS, 10 mM Tris-HCl, 0.1 mM EDTA; pH 8) 중에 현탁시켰다. 5 분 후, 350 μL의 용해 완충액 (함유 10 μL의 2-메르캅토에탄올)을 첨가하였다. 세포 현탁액을 18-21 게이지 주사 바늘을 통해 5 회 통과시킴으로써, 기계적으로 파쇄하였다. 이어서, RNA를 PureLink™ RNA 미니 키트 (Invitrogen, Carlsbad, CA 92008, USA)를 이용하여 분리하고, 100 μL의 RNase-무함유 물에서 용리시켰다. RNA를 PCR 및 겔 전기영동을 통해 검사하고, 분광광도법에 의해 정량화한 다음, 필요한 경우, DNase I (Roche)으로 처리하였다. 역전사 단계를 SuperScript III 역전사 효소 키트 (Invitrogen, Carlsbad, CA 92008, USA)를 이용하여 수행하였다. RT-PCR 반응을 25 ng의 cDNA 주형, 67 nM 의 각각의 프라이머, 및 1x iQ SYBR Green Supermix (Bio-Rad Labratories, Hercules, CA 94547, USA)가 포함된 15 μL의 반응 용적에서, MyiQ 단일 색 실시간 PCR 검출 시스템 (Bio-Rad Labratories, Hercules, CA 94547, USA)에서 수행하였다. 구아닐레이트 키나아제 (GnK) 및 포름산염 테트라하이드로폴레이트 리가아제 (FoT4L)를 항존유전자 (housekeeping gene)로서 사용하였고, 비주형 대조군을 포함시켰다. 반응 조건은 95 ℃에서 3 분 동안 후, 95℃에서 15 초, 55℃에서 15 초 및 72℃에서 30 초 동안 40 회이었다. 용융 곡선 분석을, 프라이머 이량체화 및 기타 증폭 인공물의 검출에 대해, qRT PCR (1 ℃/s에서 58 ℃ 내지 95 ℃ 38 회) 직후 수행하였다. 발현 수준에 대한 데이타를 Biorad iQ5 2.0 소프트웨어에 의해 계산된 바와 같이, PCR 기준선 차감 곡선 피트 방법에 기초한 한계 순환 (threshold cycle, Ct)의 형태로 컴퓨터 분석하였다. 원 Ct 값을 또한 상대 발현 소프트웨어 툴 (RESTⓒ) 2008 V2.0.7을 이용하여 분석하였다.
결과
자가 영양 성장시, 일산화탄소영양 미생물은 탄소 및 에너지원으로서 기능하는 가스를 흡수한다. 도 4는 씨. 아우토에타노제눔에서 발현된 유전자의 상대 발현을 나타낸다. 확인된 3 개의 효소는 자가 영양 성장 및 가스 흡수에 포함되며, 본 발명자들은 이들이 미생물에서 가장 많이 발현되는 유전자 중 하나라는 것을 발견하였다. 실시예 1에서 제시된 바와 같이, 이와 동일한 효소가 NADH에 비하여, NADPH를 많이 이용하거나 또는 배타적으로 이용한다는 것이 발견되었다. NADH-의존성 효소의 발현은 매우 낮은 수준에서 이루어진다. 이들 유전자가 인코딩하는 효소가 NADPH 의존성인 경우, 이는, NADPH 풀이 매우 중요하고 (당을 이용하는 대장균와 같은 유기체의 경우와 다름), NADPH 의존성 반응이 장애가 되지 않는다는 것을 시사한다. 일산화탄소영양 클로스트리듐 세포에서의 경로의 조작에 있어서, NADPH 의존성 반응을 선택하는 것이 가능하고, 이들 반응을 피해야 하거나 또는 우회하지 않아도 된다는 큰 이점이 있다. 또한, NADPH 풀은 고갈되지 않을만큼 충분히 크고, 광범위한 조작이 필요하지 않는다.
실시예
3
일차-이차 알코올 탈수소화효소 (ADH)는 엄격한 NADPH-의존성 효소로서, 아세톤을 이소프로판올로 전환시킨다. 0.2mM의 NADH 또는 NADPH뿐만아니라 아세톤이 함유된 발효 브로스로부터 제조된 미정제 추출물에 의한 효소 분석을 이용하여 그 활성이 입증되었다 (Ismaiel, Zhu, Colby, & Chen, 1993)
고속의 효과적인, 아세톤의 이소프로판올로의 NADPH 의존성 전환을 입증하기 위해, 씨. 아우토에타노제눔에 의한 반응기 연구를 수행하였다. 안정한 바이오매스 및 대사 생성의 연속 방식으로, 아세톤을 바이오리액터 및 공급 배지 둘 모두에 첨가하였다. 아세톤을 일정 속도로 반응기에 첨가한 다음, 연속 공급하였다. 처음에, 1 g/L의 아세톤을 첨가한 후, 대사물 농도가 안정화되면, 농도를 5 g/L, 15 g/L로 증가시키고, 제2 실험의 경우에는 20 g/L로 증가시켰다.
재료 및 방법
대사물 분석
아세톤, 이소프로판올 및 기타 대사물의 HPLC 분석을 35 ℃에서 작동하는 RID가 장착된 Agilent 1100 Series HPLC 시스템 (Refractive Index Detector) 및 60 ℃에서 유지되는 Alltech IOA-2000 유기 산 칼럼 (150 x 6.5 mm, 입자 크기 5 μm)을 이용하여 수행하였다. 약산성화된 물을 0.7 ml/분의 유속으로 이동상으로 이용하였다 (0.005M H2SO4). 단백질 및 기타 세포 잔여물을 제거하기 위해, 400 μl 샘플을 100 μl의 2 % (w/v) 5-술포살리실산과 혼합하고, 3분 동안 14,000 x g에서 원심분리하여, 침전 잔여물을 분리시켰다. 이어서, 10 μl의 상청액을 분석용 HPLC에 주입하였다.
아세톤, 이소프로판올 및 기타 대사물의 GC 분석을 불꽃 이온화 검출기 (FID), Alltech EC-1000 (30m x 0.25mm x 0.25 μm) 칼럼 및 Supelco PDMS 100 1cm 피버 (fiber)가 장착된 Agilent 6890N 헤드스페이스 GC를 이용하여 수행하였다. 5 ml 샘플을 Hungate 튜브로 옮기고, 물 욕조에서 40 ℃로 가열하고, 정확히 5 분 동안 피버에 노출시켰다. 주입기를 250 ℃에서 유지시키고, 1 ml/분의 일정한 유량의 헬륨을 담체 가스로서 사용하였다. 오븐 프로그램은 40 ℃에서 5 분 후, 200 ℃ 이하로 10 ℃/분으로 증가시켰다. 이어서, 온도를 50 ℃/분의 속도로 220 ℃로 증가시킨 후, 5 분 동안 이 온도를 유지한 다음, 온도를 50 ℃/분의 속도로 40 ℃로 감소시키고, 최종 1 분 동안 이를 유지시켰다. FID를 구성 가스로서 40 ml/분 수소, 450 ml/분 공기 및 15 ml/분 질소에 의해 250℃에서 유지하였다.
헤드스페이스 분석
2 개의 채널이 구비된 Varian CP-4900 마이크로 GC에서 측정을 수행하였다. 채널 1은 70 ℃, 200kPa 아르곤 및 4.2 초의 블랙플러쉬 시간 (backflush time)에서 구동되는 10m Mol-체 칼럼이고, 채널 2는 90 ℃, 150kPa 헬륨 및 블랙플러쉬 부재에서 구동되는 10m PPQ 칼럼이었다. 두 채널 모두의 주입기 온도는 70 ℃이었다. 작동 시간은 120 초로 설정하였고, 모든 관심 피크는 일반적으로 100 초 전에 용리한다.
세포 수집
에탄올, 아세테이트 및 2,3-부탄디올을 생성하는 것으로서, 광학 밀도 (OD)가 4인 CO 및 H2를 이용하여, 1.5L 바이오리액터로부터 세포를 고무마개로 밀봉된 2 L 바틀로 튜브를 통해 옮기고, 우선 N2로 충진하였다. 마개를 통해 주사기 방식으로 과압을 방출하였다. 바틀을 0 ℃ 이하의 온도에서 유지하고, 배양액을 천천히 옮긴 후, 배양액을 최대한 신속하게 0 ℃로 냉각시켰다. 모두 옮긴 다음, 바틀을 무산소 텐트에 배치하였다. 튜브를 원심분리한 다음, 상청액을 따라버리고, 잔여액을 여과지에 의해 제거하였다. 펠렛을 50 mM 무산소 인산 칼륨 pH 7 함유 10 mM 디티오트레이톨 중에 현탁시켰다. 다수의 바틀 현탁액을 결합하고, 원심분리한 다음, 건조하여, 중량을 잰 후, 드라이 아이스에 저장하였다.
효소 분석
효소 분석을 문헌 [Huang (Huang et al., 2012) 및 Ismaiel (Ismaiel et al., 1993)]에 개괄된 방법에 따라 수행하였다.
결과
아세톤의 이소프로판올로의 환원은 도 5에 제시된 바와 같이, 엄격한 NADPH 의존성 이차 알코올 탈수소화효소 효소의 기능으로 나타났다. 활성을 NADH를 제외한 NADPH 단독에 의해 측정하였고, 이에 의해, 이 효소가 엄격하게 NADPH 의존성이라는 것이 입증되었다.
CO에서 자가 영양 성장 과정 동안 NADPH 풀의 성능을 입증하기 위해, 아세톤에서 성장되는 반응기로 아세톤을 지속적으로 공급하였다. 아세톤이, 고속에서 이 NADPH 의존성 이차 알코올 탈수소화효소 효소를 통해, 이소프로판올로 효과적으로 전환된다는 것이 확인되었다. 도 6은, 아세톤이 바이오리액터로의 도입 직후, 이소프로판올로 전환되는 것을 나타낸다. 20 g/L의 고농도에서도, 모든 아세톤이 이소프로판올로 전환된 배양액에 의해, NADPH 풀이 이러한 고속을 유지하기에 충분하다는 것이 입증되었다.
이 실험에 의해, 일산화탄소영양 클로스트리듐 미생물의 NADPH-의존성 반응을 유지시키는 능력이 입증되었다. 대장균에서, NADPH 능력은 푸르푸랄 연구에서 제시된 바와 같이 (E N Miller et al., 2009; Elliot N Miller et al., 2009), 상당히 낮은 수준이다.
실시예 4
NADH 및 NADPH 의존성 효소 사이에 선택을 제공하는 다수의 경로, 예를 들어 부탄올 경로가 존재한다. 대부분의 조작 노력은 NADH 의존성 반응에 집중되었고, NADPH 의존성 반응은 배제되었다. 이로 인해, 경로 선택이 제한되었고, NADPH에 의해 제공되는 추가적 구동력이 무시되었다.
티올라아제 (EC 2.3.1.9; btkB, 예를 들어 랄스토니아 유트로파(Ralstonia eutropha): YP_725948.1, 유전자ID:4248815; phaA, 예를 들어 랄스토니아 유트로파: YP_725941.1, 유전자 ID: 4249783), an NADPH 의존성 R-3-히드록시부티릴-CoA 탈수소화효소 (EC:1.1.1.36; phaB GO:0018454; 예를 들어 랄스토니아 유트로파: YP_725942.1, 유전자ID:4249784) 및 3-히드록시부티릴-CoA 디하이드로타아제 (EC 4.2.1.119; phaJ, 예를 들어 아에로모나스 펀크타타 : BAA21816.1), NADPH 의존성 크로토닐-CoA 카르복실라아제/환원 효소 (EC 1.3.1.86; ccr, 예를 들어 스트렙토마이세스(Streptomyces collinus); EC 1.3.1.85; ccrRs, 예를 들어 로도박테르 스파에로이드스(Rhodobacter sphaeroides): YP_354044.1, 유전자 ID: 3720751) 및 NADPH 의존성 에틸말로닐-CoA 디카르복실라아제 (EC 4.1.1.41; 예를 들어 무스 무스쿨루스(Mus musculus): NP_001103665.1, 유전자ID:52665)로 이루어진, 부티릴-CoA로의 부탄올 생합성을 위한 신규의 완전한 NADPH 의존성 경로로서, 부티릴-CoA가 그후, 직접 알데하이드/알코올 탈수소화효소를 통하거나 또는 포스포트란스카에틸라아제 및 부티레이트 키나아제, 알데하이드 페레독신 옥시도레두아가타아제 및 알코올 탈수소화효소를 통해 부티레이트에 의해 부탄올로 전환될 수 있는 경로가 설계되었다. NADPH 의존성 부티릴-CoA 환원 효소 (EC 1.1.2.10; bldh, 예를 들어 클로스트리듐 사카로퍼부틸아세토니쿰(Clostridium saccharoperbutylacetonicm) N1-4: AGF59413.1, 유전자ID: Cspa_c56880) 및 알데하이드 환원 효소 (EC 1.1.1.1; adhA, 예를 들어 시네코시스티스 종(Synechocystis sp.) PCC 6803: NP_443028.1, 유전자ID:951896) (도 7)가 이용될 수 있다.
2 분자의 아세틸-CoA가, 랄스토니아 유트로파로부터의 phaABJ에 의해 인코딩되는 3 개의 효소에 의해, 크로토닐-CoA로 전환된다. 두 개의 아세틸-CoA가 티올라아제에 의해 아세토아세틸-CoA로 응축된 후, NADPH 특이적 R-3-히드록시부티릴-CoA 탈수소화효소에 의해 R-3 히드록시부티릴-CoA로 환원된다. 이어서, R-3-히드록시부티릴-CoA가 R-3-히드록시부티릴-CoA 디하이드라타아제에 의해 크로토닐-CoA로 전환된다.
로도박테르 스파에로이드스 (Erb et al., 2007)의 크로토닐-CoA 카르복실라아제/환원 효소 및 무스 무스쿨루스 (mouse) (Linster et al., 2011)의 에틸말로닐-CoA 디카르복실라아제 조합은 처음에 이산화 탄소와 크로토닐-CoA이 응축되고, NADPH가 소모되면서 에틸말로닐-CoA가 생성된 다음, 에틸말로닐-CoA가 부티릴-CoA로 탈카르복실화되는 과정을 촉매한다.
클로스트리듐 사카로퍼부틸아세토니쿰 NI-4의 부티릴-CoA 환원 효소는 부티릴-CoA로부터 CoA 모이어티(moiety)를 분리시켜서, 부티르알데하이드를 생성시킨다. 이 효소는, 클로스티븀 베이어린키이(Clostridium beijerinkii) NRRL B592의 동족체가 NADPH에 의해 가장 활성인 것으로 보아 (Yan 및 Chen, 1990), NADPH 의존성인 것으로 추정된다.
시아노박테리움 시네코시스티스 종(Synechocystis sp .) PCC 6803의 알데하이드 환원 효소는 중간 사슬 길이의 방향족 알데하이드의 알코올로의 NADPH 환원에 대해 강한 선호도를 갖는다 (Vidal et al., 2009). 부티르알데하이드의 부탄올로의 환원성 (preference for reduction): 부탄올의 산화성은 환원도 (favour of reduction)에서 251:1이다.
실시예 5
글루코오스 당에서 성장된 대장균 세포에서, NADH 풀이 NADPH 풀 보다 20 배 이상 크고 (B. D. Bennett et al., 2009), 특히 발효 과정에서 많은 생합성 반응 및 생물전환을 제한한다는 것이 입증되었다 (R Poulsen et al., 2005). NADPH 및 NADH 풀을 카복시도트로픽 아세토제닉 Clostridium에서 측정하였다.
실시예 2에서 개시된 바와 같은 클로스트리듐 오토에타노게눔에 의한 연속 발효로부터 샘플을 취하여 분석하였다. 5 mL 배양 샘플을 원심분리 (13000 rpm, -10℃, 5 분)에 의해 신속하게 펠렛팅하고, 상청액을 제거한 다음, 세포 펠렛을 액체 질소 중에 스냅 동결시킨 후, 분석시까지 -80℃에서 저장하였다. 대사물 분석을 문헌 [B. D. Bennett et al., 2009; Yang et al, Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress. BMC Genomics 2012, 13:336; Marcellin E, Quantitative analysis of intracellular sugar phosphates and sugar nucleotides in encapsulated streptococci using HPAEC-PAD, Biotechnol J 2009, 4, 58-63]에 개시된 바와 같이, 미생물 펠렛에서 수행하였다.
대장균와 대조적으로, 씨. 아우토에타노제눔에서, NADPH 풀은 NADH 풀 보다 더 크고, 그 비율은 2.2:1 NADPH+H+ 및 NADP 대 NADH+H+ 및 NADH, 각각 36.8:1 NADPH+H+ 대 NADH+H+인 것으로 확인되었고 (도 7), 이에 의해, 기질로서 CO를 이용하는 아세토젠 일산화탄소영양 클로스트리디아의 NADPH의 구동력이 입증되었다.
법적 기준에 대한 명시
본 발명은 본 명세서에서, 과도한 실험을 하지 않고도 본 발명을 실행할 수 있도록 하기 위해, 바람직한 특정 실시형태를 참조로 기술되었다. 그러나, 당업자는 본 발명의 범위를 벗어나지 않고, 다양한 성분 및 파라미터가 일정 범위에서 다양화되거나 또는 변형될 수 있고, 공지된 균등물에 의해 치환될 수 있다는 것을 용이하게 인식할 것이다. 각각 설명된 바와 같이, 이러한 변형 및 균등물이 본 명세서에 혼입된다는 것이 이해되어야 한다. 제목, 표목 등은 본 명세서의 이해를 증진시키기 위해 제공되는 것으로, 본 발명의 범위를 제한하는 것으로 해석되어서는 안된다.
상기 및 하기에서 인용되는 모든 출원, 특허 및 공보의 전문은 본 명세서에 참조로서 포함된다. 그러나, 본 명세서의 임의의 출원, 특허 및 공보에 대한 참조는, 이들이 유효한 종래 기술을 구성하거나 또는 임의의 국가에서 통상의 일반 지식의 일부를 형성한다는 임의의 형태의 의견 또는 자인으로 인식되지 않고, 그렇게 인식되지 않아야 한다.
명세서 및 하기 특허청구범위 전체에서, 문맥상 별도로 요구되지 않는 한, 단어 "~를 포함하다", "~를 포함하는" 등은, 이른바 "포함하나, 제한하는 것은 아님"의 의미로, 배타적 의미에 반대되는 것으로서, 포괄적 의미로 이해되어야 한다.
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SEQUENCE LISTING
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Thr Leu Gly Ala Ser Thr Lys Lys Met Ala Lys Tyr Glu Val Gly Glu
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Tyr Phe Glu Asp Phe Val Gly Pro Leu Gly His Cys Ser Glu Leu Val
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Glu Met Asp Leu Lys Glu Leu Lys Lys Lys Asn Ile Met Phe Val Ala
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Gly Gly Val Gly Thr Ala Pro Val Tyr Pro Gln Val Lys Trp Leu His
100 105 110
Gln His Gly Val Glu Ala Asp Val Ile Val Gly Cys Lys Ser Lys Asp
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Tyr Leu Leu Phe Glu Asp Glu Leu Lys Pro Ile Cys Gly Asn Leu Tyr
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Ile Ala Thr Asp Asp Gly Ser Tyr Gly Tyr Lys Gly Phe Val Thr Asp
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Leu Leu Lys Glu Leu Ile Asp Lys Lys Asp Lys Glu Tyr Asp Cys Val
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Val Ala Ile Gly Pro Met Ile Met Met Lys Phe Ile Thr Gln Val Thr
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Lys Gln Tyr Gly Ile Lys Thr Ile Val Ser Leu Asn Thr Ile Met Val
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Asp Gly Thr Gly Met Cys Gly Ala Cys Arg Val Thr Val Gly Gly Glu
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Leu Lys Phe Ala Cys Val Asp Gly Pro Glu Phe Asp Gly His Leu Val
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Asn Phe Asp Glu Ala Met Arg Arg Gln Ala Met Tyr Lys Thr Glu Glu
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Gly Lys Lys Leu Leu Lys Glu Glu Glu Gly Asp Thr Phe Asp Arg Lys
260 265 270
Gly Cys Glu Cys His Asn Glu Asp Lys Ala Ala Arg Met Lys Arg Val
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Pro Ile Lys Glu Gln Asp Pro Lys Val Arg Ala Thr Asn Phe Asp Glu
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Val Cys Leu Gly Tyr Thr Glu Glu Glu Ala Val Lys Glu Ala Ser Arg
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Cys Leu Asn Cys Lys Lys Pro Met Cys Val Thr Gln Cys Pro Val Thr
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Ile Thr Ile Pro Lys Phe Val Glu Gln Val Lys Asn Arg Asn Phe Glu
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Glu Ala Ala Lys Ile Ile Ala Glu Ser Ser Ala Leu Pro Ala Val Cys
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Gly Arg Val Cys Pro Gln Glu Thr Gln Cys Glu Gly Lys Cys Val Leu
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Gly Lys Lys Gly Asp Ala Val Ala Ile Gly Lys Leu Glu Arg Phe Val
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Ala Asp Trp Ser Arg Lys Asn Asn Ile Asp Leu Ser Lys Thr Leu Pro
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Lys Asn Gly Lys Lys Val Ala Val Ile Gly Ser Gly Pro Ser Gly Leu
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Thr Cys Ala Gly Asp Leu Ala Lys Leu Gly Tyr Asp Val Thr Ile Phe
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Glu Ala Leu His Glu Ala Gly Gly Val Leu Val Tyr Gly Ile Pro Glu
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Phe Arg Leu Pro Lys Asp Thr Val Val Lys His Glu Val Glu Asn Val
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Lys Lys Leu Gly Val Lys Ile Glu Thr Asp Val Ile Ile Gly Arg Thr
485 490 495
Val Thr Ile Asp Glu Leu Val Glu Lys Glu Lys Phe Asp Ala Val Phe
500 505 510
Ile Gly Ser Gly Ala Gly Leu Pro Arg Phe Met Gly Ile Pro Gly Glu
515 520 525
Asn Leu Asn Gly Val Phe Ser Ala Asn Glu Phe Leu Thr Arg Ser Asn
530 535 540
Leu Met Lys Ala Tyr Arg Asp Asp Tyr Ala Thr Pro Ile Lys Ala Gly
545 550 555 560
Lys Lys Val Ala Val Val Gly Gly Gly Asn Val Ala Met Asp Ser Ala
565 570 575
Arg Thr Ala Leu Arg Leu Gly Ala Glu Val Tyr Ile Val Tyr Arg Arg
580 585 590
Ser Glu Ala Glu Leu Pro Ala Arg Ala Glu Glu Val His His Ala Lys
595 600 605
Glu Glu Gly Ile Lys Phe Asn Leu Leu Thr Asn Pro Val Glu Ile Leu
610 615 620
Gly Asp Glu Lys Gly Trp Val Asn Gly Ile Arg Cys Ile Lys Met Glu
625 630 635 640
Leu Gly Glu Pro Asp Ala Ser Gly Arg Arg Lys Pro Val Ala Ile Lys
645 650 655
Gly Ser Glu Phe Asp Leu Asp Val Asp Thr Val Ile Met Ala Leu Gly
660 665 670
Thr Ser Pro Asn Pro Leu Ile Ser Thr Thr Thr Lys Gly Leu Glu Met
675 680 685
Asn Lys Arg Lys Cys Leu Ile Ala Glu Glu Glu Thr Gly Leu Thr Thr
690 695 700
Arg Glu Gly Ile Tyr Ala Gly Gly Asp Ala Val Thr Gly Ala Ala Thr
705 710 715 720
Val Ile Leu Ala Met Gly Ala Gly Lys Lys Ala Ala Lys Ala Ile Asp
725 730 735
Glu Tyr Leu Lys Lys
740
<210> 3
<211> 2163
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 3
atggatgata aaggggaaag aatacctctt actatctgtg attatgatgc agaaaaagga 60
actgttacta ttgtatttca gacattagga gcttcaacta aaaaaatggc taaatatgaa 120
gtgggagagt attttgaaga ttttgttgga ccacttggac attgctcaga acttgtagag 180
atggatctta aagaacttaa gaagaaaaat ataatgtttg tagcaggggg tgtaggtact 240
gcaccagttt atccacaggt aaaatggctt catgaacatg gagttgctgc agatgttata 300
gttggatgta aatcaaaaga tcttttatta tttgaagatg aattgaaacc aatttgtgga 360
aatttatata tagcaacgga tgatgggagt tatggatata agggctttgt tacgaatctt 420
ttgaaagaac ttattgacaa gaaagataaa gaatatgatt gtgtaattgc cataggacct 480
atgataatga tgaagtttat aactcaagtt acgaaaccat atggaattaa aacaatagta 540
agtttaaata ctataatggt agatggaact ggaatgtgcg gagcttgtag ggttactgta 600
ggtggagaat taaaatttgc ctgtgtagat ggtcctgaat ttgatggtca tcttgtaaat 660
tttgatgaag ctatgagaag acaagctatg tacaagactg aagaaggaaa gaaacttctg 720
caagaagaag aaggggacac tggaagtaga gaaggtaaaa agtgtagagc agaagaaaaa 780
cttgaaagaa tgaaaagagt acctataaag gaacaggatc ctaaagttag agctactaat 840
tttgatgaag tttgcttggg atacactgaa gaagaagctg taaaagaagc ttcaagatgc 900
ttgaattgta aaaagcctat gtgtgttact cagtgccctg ttacaataac tatacctaag 960
tttgttgaac aggtaaaaaa tagaaacttt gaagaagctg ctaaaataat agcagaatca 1020
agtgcacttc ctgctgtatg tggaagagta tgtcctcagg aaactcagtg tgaaggaaaa 1080
tgtgtacttg gcaaaaaagg tgatgctgtt gctataggta agctggaaag atttgtagca 1140
gattggtcaa gaaagaataa tatcgattta tctaagactt tacctaaaaa cggcaaaaaa 1200
gtagctgtta taggaagtgg tccttcagga cttacttgtg caggagattt agcaaagctt 1260
ggatatgacg ttactatatt tgaagcactt catgaagcag gaggagtact tgtatatggt 1320
attccagagt tcagacttcc aaaggatact gtagtaaaac atgaagttga aaatgtaaag 1380
aaattaggag taaaaataga gacagatgta ataataggaa gaactgttac tatagatgaa 1440
ctagtagaaa aagaaaaatt tgatgctgta tttataggtt caggagcagg actaccaagg 1500
tttatgggaa tacctggaga aaacttaaat ggagtattct ctgcaaatga attcttaaca 1560
agaagtaatt taatgaaagc atatagggat gattatgcaa ctcctataaa agctggtaag 1620
aaagtagctg tagtaggagg cggaaacgta gctatggact ctgcaaggac agctctaaga 1680
cttggagcag aagtatacat agtatacaga agatccgaag cagaacttcc agcaagagca 1740
gaggaagtac accatgcaaa agaggaagga ataaagttca atcttttaac taatcctgta 1800
gaaatattag gagatgaaaa aggttgggtt aatggaatac gctgtattaa gatggaactt 1860
ggagaaccag atgcatctgg aagaagaaaa ccagttgcaa taaagggatc agaatttgat 1920
ttagatgtag atactgtaat tatggccctt ggtacttcac caaatccact tatatcaact 1980
acaacaaaag gacttgaaat gaataagcgt aaatgcttaa tagcagaaga agagactgga 2040
ctcactacaa gagaaggaat atatgcaggt ggagatgcag taacaggtgc tgctactgta 2100
atacttgcaa tgggtgcagg taaaaaagct gctaaggcta tagatgaata tctgaaaaaa 2160
taa 2163
<210> 4
<211> 720
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 4
Met Asp Asp Lys Gly Glu Arg Ile Pro Leu Thr Ile Cys Asp Tyr Asp
1 5 10 15
Ala Glu Lys Gly Thr Val Thr Ile Val Phe Gln Thr Leu Gly Ala Ser
20 25 30
Thr Lys Lys Met Ala Lys Tyr Glu Val Gly Glu Tyr Phe Glu Asp Phe
35 40 45
Val Gly Pro Leu Gly His Cys Ser Glu Leu Val Glu Met Asp Leu Lys
50 55 60
Glu Leu Lys Lys Lys Asn Ile Met Phe Val Ala Gly Gly Val Gly Thr
65 70 75 80
Ala Pro Val Tyr Pro Gln Val Lys Trp Leu His Glu His Gly Val Ala
85 90 95
Ala Asp Val Ile Val Gly Cys Lys Ser Lys Asp Leu Leu Leu Phe Glu
100 105 110
Asp Glu Leu Lys Pro Ile Cys Gly Asn Leu Tyr Ile Ala Thr Asp Asp
115 120 125
Gly Ser Tyr Gly Tyr Lys Gly Phe Val Thr Asn Leu Leu Lys Glu Leu
130 135 140
Ile Asp Lys Lys Asp Lys Glu Tyr Asp Cys Val Ile Ala Ile Gly Pro
145 150 155 160
Met Ile Met Met Lys Phe Ile Thr Gln Val Thr Lys Pro Tyr Gly Ile
165 170 175
Lys Thr Ile Val Ser Leu Asn Thr Ile Met Val Asp Gly Thr Gly Met
180 185 190
Cys Gly Ala Cys Arg Val Thr Val Gly Gly Glu Leu Lys Phe Ala Cys
195 200 205
Val Asp Gly Pro Glu Phe Asp Gly His Leu Val Asn Phe Asp Glu Ala
210 215 220
Met Arg Arg Gln Ala Met Tyr Lys Thr Glu Glu Gly Lys Lys Leu Leu
225 230 235 240
Gln Glu Glu Glu Gly Asp Thr Gly Ser Arg Glu Gly Lys Lys Cys Arg
245 250 255
Ala Glu Glu Lys Leu Glu Arg Met Lys Arg Val Pro Ile Lys Glu Gln
260 265 270
Asp Pro Lys Val Arg Ala Thr Asn Phe Asp Glu Val Cys Leu Gly Tyr
275 280 285
Thr Glu Glu Glu Ala Val Lys Glu Ala Ser Arg Cys Leu Asn Cys Lys
290 295 300
Lys Pro Met Cys Val Thr Gln Cys Pro Val Thr Ile Thr Ile Pro Lys
305 310 315 320
Phe Val Glu Gln Val Lys Asn Arg Asn Phe Glu Glu Ala Ala Lys Ile
325 330 335
Ile Ala Glu Ser Ser Ala Leu Pro Ala Val Cys Gly Arg Val Cys Pro
340 345 350
Gln Glu Thr Gln Cys Glu Gly Lys Cys Val Leu Gly Lys Lys Gly Asp
355 360 365
Ala Val Ala Ile Gly Lys Leu Glu Arg Phe Val Ala Asp Trp Ser Arg
370 375 380
Lys Asn Asn Ile Asp Leu Ser Lys Thr Leu Pro Lys Asn Gly Lys Lys
385 390 395 400
Val Ala Val Ile Gly Ser Gly Pro Ser Gly Leu Thr Cys Ala Gly Asp
405 410 415
Leu Ala Lys Leu Gly Tyr Asp Val Thr Ile Phe Glu Ala Leu His Glu
420 425 430
Ala Gly Gly Val Leu Val Tyr Gly Ile Pro Glu Phe Arg Leu Pro Lys
435 440 445
Asp Thr Val Val Lys His Glu Val Glu Asn Val Lys Lys Leu Gly Val
450 455 460
Lys Ile Glu Thr Asp Val Ile Ile Gly Arg Thr Val Thr Ile Asp Glu
465 470 475 480
Leu Val Glu Lys Glu Lys Phe Asp Ala Val Phe Ile Gly Ser Gly Ala
485 490 495
Gly Leu Pro Arg Phe Met Gly Ile Pro Gly Glu Asn Leu Asn Gly Val
500 505 510
Phe Ser Ala Asn Glu Phe Leu Thr Arg Ser Asn Leu Met Lys Ala Tyr
515 520 525
Arg Asp Asp Tyr Ala Thr Pro Ile Lys Ala Gly Lys Lys Val Ala Val
530 535 540
Val Gly Gly Gly Asn Val Ala Met Asp Ser Ala Arg Thr Ala Leu Arg
545 550 555 560
Leu Gly Ala Glu Val Tyr Ile Val Tyr Arg Arg Ser Glu Ala Glu Leu
565 570 575
Pro Ala Arg Ala Glu Glu Val His His Ala Lys Glu Glu Gly Ile Lys
580 585 590
Phe Asn Leu Leu Thr Asn Pro Val Glu Ile Leu Gly Asp Glu Lys Gly
595 600 605
Trp Val Asn Gly Ile Arg Cys Ile Lys Met Glu Leu Gly Glu Pro Asp
610 615 620
Ala Ser Gly Arg Arg Lys Pro Val Ala Ile Lys Gly Ser Glu Phe Asp
625 630 635 640
Leu Asp Val Asp Thr Val Ile Met Ala Leu Gly Thr Ser Pro Asn Pro
645 650 655
Leu Ile Ser Thr Thr Thr Lys Gly Leu Glu Met Asn Lys Arg Lys Cys
660 665 670
Leu Ile Ala Glu Glu Glu Thr Gly Leu Thr Thr Arg Glu Gly Ile Tyr
675 680 685
Ala Gly Gly Asp Ala Val Thr Gly Ala Ala Thr Val Ile Leu Ala Met
690 695 700
Gly Ala Gly Lys Lys Ala Ala Lys Ala Ile Asp Glu Tyr Leu Lys Lys
705 710 715 720
<210> 5
<211> 882
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 5
atgaatgctc gaagcaaggt tatatgtcct ttaatcgtag ataaggaacg cagttcaaag 60
gcttttacta gtgaagctat agatttaatt gaaaggagaa agacgaaaaa attaaatgct 120
atatggcttg aagtaacagg atgttcagga aatatcattt cttttttaaa tagtgaaaat 180
cctggactcg attatatttt ggaaaaactc attaatttaa aatacaacaa tactctaatg 240
acttcagaag gggagtatgc ctttaaacaa ttcttagata cattgaatac tgaatttata 300
ctattagtag atggagcagt atctactgcc cagaacggtt tttataatat tgttgccaat 360
tatgaaggaa accctgttac tgcacttgaa gctgtaaaaa tggcaggaga aaaagcaaag 420
catgttctct gtgtaggaac ttgtgcatcc tatggtggaa tttctgccgc caggccaaac 480
ccttcagaaa gcaaaagtgt taaagaaata ctaaatcgtg aagtcataag acttccaggc 540
tgtccatgcc acccggattg ggtagttgga actttagcac acttggttgc ttttggaaaa 600
ccacaattgg ataaagaagg aagacctctt cttttttatg gaattaccat tcatgatagt 660
tgtacaagaa gaggattttt tgataacaga atttttgcaa aaaaatttgg agaaaatgga 720
tgcatgttta aacttggatg cagggggcct gtaactaaaa cagattgtcc taggagaaag 780
tggaatgggt acgtgaactg gcctgttgaa gacaatacca actgtatagg atgtgcaaat 840
tctagatttc cagatggtat ggaaccattt gtaaggtatt ag 882
<210> 6
<211> 293
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 6
Met Asn Ala Arg Ser Lys Val Ile Cys Pro Leu Ile Val Asp Lys Glu
1 5 10 15
Arg Ser Ser Lys Ala Phe Thr Ser Glu Ala Ile Asp Leu Ile Glu Arg
20 25 30
Arg Lys Thr Lys Lys Leu Asn Ala Ile Trp Leu Glu Val Thr Gly Cys
35 40 45
Ser Gly Asn Ile Ile Ser Phe Leu Asn Ser Glu Asn Pro Gly Leu Asp
50 55 60
Tyr Ile Leu Glu Lys Leu Ile Asn Leu Lys Tyr Asn Asn Thr Leu Met
65 70 75 80
Thr Ser Glu Gly Glu Tyr Ala Phe Lys Gln Phe Leu Asp Thr Leu Asn
85 90 95
Thr Glu Phe Ile Leu Leu Val Asp Gly Ala Val Ser Thr Ala Gln Asn
100 105 110
Gly Phe Tyr Asn Ile Val Ala Asn Tyr Glu Gly Asn Pro Val Thr Ala
115 120 125
Leu Glu Ala Val Lys Met Ala Gly Glu Lys Ala Lys His Val Leu Cys
130 135 140
Val Gly Thr Cys Ala Ser Tyr Gly Gly Ile Ser Ala Ala Arg Pro Asn
145 150 155 160
Pro Ser Glu Ser Lys Ser Val Lys Glu Ile Leu Asn Arg Glu Val Ile
165 170 175
Arg Leu Pro Gly Cys Pro Cys His Pro Asp Trp Val Val Gly Thr Leu
180 185 190
Ala His Leu Val Ala Phe Gly Lys Pro Gln Leu Asp Lys Glu Gly Arg
195 200 205
Pro Leu Leu Phe Tyr Gly Ile Thr Ile His Asp Ser Cys Thr Arg Arg
210 215 220
Gly Phe Phe Asp Asn Arg Ile Phe Ala Lys Lys Phe Gly Glu Asn Gly
225 230 235 240
Cys Met Phe Lys Leu Gly Cys Arg Gly Pro Val Thr Lys Thr Asp Cys
245 250 255
Pro Arg Arg Lys Trp Asn Gly Tyr Val Asn Trp Pro Val Glu Asp Asn
260 265 270
Thr Asn Cys Ile Gly Cys Ala Asn Ser Arg Phe Pro Asp Gly Met Glu
275 280 285
Pro Phe Val Arg Tyr
290
<210> 7
<211> 1386
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 7
atgaaaaaga aaattacaat tgatccaatt acgagaataa gcggtttttt ggaaactaaa 60
gtgcaagtag aaaaaaatat tatagtagat gctgaaacta gtggattgct ttttagagga 120
tttgaaaaaa tgttaaagaa cagagagccg ctggatgcag tatattttac agaaagaatt 180
tgtggaatat gttcaacagc tcatgccgtg gcggctgcta cagctcttga agatgctttg 240
aagataaaaa ttagtgtaaa tgattcgtat atgcgtaatt taatacatgg ttttgaattt 300
atacaaaatc atataagaca tttttataat ttgaccatac caagttatgt gaagatgccc 360
gatataaatc ctctttcttc aaatcaatat gaagattata gattgcctta taacctaaat 420
aaaaagataa gtgaagatta tattgaaagt attaaataca gcaggttagc ccatgaaggg 480
ttggctatcc ttggaggaaa agccccccat aatcatggaa tttttgttgg aggagttacc 540
ataaatatag atccatataa actcacaaaa gttaaatcta ttatttctca aattaataaa 600
tttgtaagta gtgttatgtt agaggacatg aacataattt caaaatacta tgctgattat 660
tttaaaatgg gaggagctta tggaaacttt atgacttatg gaatttttga caagtatgct 720
gatcctgaga taagttatgt aggaccttct gtattaataa atggacgaaa gtataatttc 780
aatagtaata aaattacgga aaacatactc cacacctggt atacaagcga tgatgaaacg 840
ataaatttat ctaaagaaac aggttacagc tttataaaat cgccaaccta taatggatat 900
tctatggaag taggacctct agcaagattg atactttcag gtgagtatac tggtggaagt 960
tcatgtatgg acagaaatgt tgccagagta cttgaaacaa aaaagatttt agaaattatg 1020
caaggacttg cagatagaat taagcttatt ccagcagaac aaagaatata tcaaatacca 1080
gataaagcat ttggtgcagg attaattgac acaactagag gatccttggg acactggata 1140
agtatagaag ataaatttat aaagcattac aatattataa ctcctacagt gtggaatatg 1200
gggccaagaa atcaatcagg tgcgcttgga attggagaaa aatctttagt tggaacgaaa 1260
ataaaagata taaagcagcc tatagaagtt gggagaatta tgagatcttt tgatccttgt 1320
gtttcctgtg caacgcatct tgtaagtgat aaatatgaac cagtggatgt acaggttata 1380
gtatga 1386
<210> 8
<211> 461
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 8
Met Lys Lys Lys Ile Thr Ile Asp Pro Ile Thr Arg Ile Ser Gly Phe
1 5 10 15
Leu Glu Thr Lys Val Gln Val Glu Lys Asn Ile Ile Val Asp Ala Glu
20 25 30
Thr Ser Gly Leu Leu Phe Arg Gly Phe Glu Lys Met Leu Lys Asn Arg
35 40 45
Glu Pro Leu Asp Ala Val Tyr Phe Thr Glu Arg Ile Cys Gly Ile Cys
50 55 60
Ser Thr Ala His Ala Val Ala Ala Ala Thr Ala Leu Glu Asp Ala Leu
65 70 75 80
Lys Ile Lys Ile Ser Val Asn Asp Ser Tyr Met Arg Asn Leu Ile His
85 90 95
Gly Phe Glu Phe Ile Gln Asn His Ile Arg His Phe Tyr Asn Leu Thr
100 105 110
Ile Pro Ser Tyr Val Lys Met Pro Asp Ile Asn Pro Leu Ser Ser Asn
115 120 125
Gln Tyr Glu Asp Tyr Arg Leu Pro Tyr Asn Leu Asn Lys Lys Ile Ser
130 135 140
Glu Asp Tyr Ile Glu Ser Ile Lys Tyr Ser Arg Leu Ala His Glu Gly
145 150 155 160
Leu Ala Ile Leu Gly Gly Lys Ala Pro His Asn His Gly Ile Phe Val
165 170 175
Gly Gly Val Thr Ile Asn Ile Asp Pro Tyr Lys Leu Thr Lys Val Lys
180 185 190
Ser Ile Ile Ser Gln Ile Asn Lys Phe Val Ser Ser Val Met Leu Glu
195 200 205
Asp Met Asn Ile Ile Ser Lys Tyr Tyr Ala Asp Tyr Phe Lys Met Gly
210 215 220
Gly Ala Tyr Gly Asn Phe Met Thr Tyr Gly Ile Phe Asp Lys Tyr Ala
225 230 235 240
Asp Pro Glu Ile Ser Tyr Val Gly Pro Ser Val Leu Ile Asn Gly Arg
245 250 255
Lys Tyr Asn Phe Asn Ser Asn Lys Ile Thr Glu Asn Ile Leu His Thr
260 265 270
Trp Tyr Thr Ser Asp Asp Glu Thr Ile Asn Leu Ser Lys Glu Thr Gly
275 280 285
Tyr Ser Phe Ile Lys Ser Pro Thr Tyr Asn Gly Tyr Ser Met Glu Val
290 295 300
Gly Pro Leu Ala Arg Leu Ile Leu Ser Gly Glu Tyr Thr Gly Gly Ser
305 310 315 320
Ser Cys Met Asp Arg Asn Val Ala Arg Val Leu Glu Thr Lys Lys Ile
325 330 335
Leu Glu Ile Met Gln Gly Leu Ala Asp Arg Ile Lys Leu Ile Pro Ala
340 345 350
Glu Gln Arg Ile Tyr Gln Ile Pro Asp Lys Ala Phe Gly Ala Gly Leu
355 360 365
Ile Asp Thr Thr Arg Gly Ser Leu Gly His Trp Ile Ser Ile Glu Asp
370 375 380
Lys Phe Ile Lys His Tyr Asn Ile Ile Thr Pro Thr Val Trp Asn Met
385 390 395 400
Gly Pro Arg Asn Gln Ser Gly Ala Leu Gly Ile Gly Glu Lys Ser Leu
405 410 415
Val Gly Thr Lys Ile Lys Asp Ile Lys Gln Pro Ile Glu Val Gly Arg
420 425 430
Ile Met Arg Ser Phe Asp Pro Cys Val Ser Cys Ala Thr His Leu Val
435 440 445
Ser Asp Lys Tyr Glu Pro Val Asp Val Gln Val Ile Val
450 455 460
<210> 9
<211> 1398
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 9
atgttaaata tgccaactag tacttctatg ataaatatag atgaagaatt atgtacaggc 60
tgcagacgat gtgcggatgt ctgccctgta gatgctatag aaggtgaaca gggtaaacct 120
cagaagataa atactgaaaa gtgtgttatg tgcggacaat gcattcaagt ttgtaaaggc 180
tatcaatctg tatacgacga tgttcctact ccagttagca aaaggttatt tgatagagga 240
ttgttaaagg aagtagatga accattattt gcagcatata ataaaggtca ggtaaagagt 300
gttaaagaaa ttttacaaaa caaagatgta tttaaaattg tgcaatgtgc acctgctgta 360
agagttgcta taggagagga ttttggaatg cctcttggaa ctttaagtga aggaaaaatg 420
gcagctgcac tcagaaaatt aggatttgac aaagtatatg atacaaactt tggtgcagat 480
cttactataa tggaagaagg tagtgagtta ctaaaaagag tagctgaagg cggagttttg 540
ccaatgttta cttcttgttg tccagcatgg gtaaaatatg cagaacaaac atatccagaa 600
cttttacctc atctttcaag ttgtaagtct ccaaatcaga tggctggagc tatatttaaa 660
acttatggag cagagataaa taaggttaat ccggctaaaa tttataatgt atctgttatg 720
ccatgtacat gcaaggaatt tgaaagtgaa agagaagaaa tgcatgacag tggacacagg 780
gatgtagatg cagttataac tacaagggaa ttagcacaac tgttcaaaga tgctgatata 840
gattttaata ctattgaaga agaacagttt gatactcctc ttggtatgta taccggtgca 900
ggaactatat ttggtgctac aggtggagtt atggaagcag cacttagaac tggatatgaa 960
ctttatacta aaaaaactat tccaagtata gatcttacta tggtaagagg tggagaaggt 1020
tttagaactg ctgaagtaga tttaggggat attagactaa aagtaggagt agtttccggc 1080
ttaaaaaatg taaaagacgt tatggaatca gtaaaggcag gcaaatgtga tttgcacttt 1140
atagaggtta tgacctgtcc tcaaggatgt ataagtggtg gaggacaacc taaagttata 1200
cttgattcag ataaagagga agcttataat aataggaaaa agggactata taatcatgac 1260
gctaatctta cttatagaaa atcacatgaa aatccagaaa taaagaaaat atatgatgag 1320
ttcttagaca aaccattagg agctaagtct catgaattat tgcacactaa atatatctca 1380
agaaaaaagg agagttaa 1398
<210> 10
<211> 465
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 10
Met Leu Asn Met Pro Thr Ser Thr Ser Met Ile Asn Ile Asp Glu Glu
1 5 10 15
Leu Cys Thr Gly Cys Arg Arg Cys Ala Asp Val Cys Pro Val Asp Ala
20 25 30
Ile Glu Gly Glu Gln Gly Lys Pro Gln Lys Ile Asn Thr Glu Lys Cys
35 40 45
Val Met Cys Gly Gln Cys Ile Gln Val Cys Lys Gly Tyr Gln Ser Val
50 55 60
Tyr Asp Asp Val Pro Thr Pro Val Ser Lys Arg Leu Phe Asp Arg Gly
65 70 75 80
Leu Leu Lys Glu Val Asp Glu Pro Leu Phe Ala Ala Tyr Asn Lys Gly
85 90 95
Gln Val Lys Ser Val Lys Glu Ile Leu Gln Asn Lys Asp Val Phe Lys
100 105 110
Ile Val Gln Cys Ala Pro Ala Val Arg Val Ala Ile Gly Glu Asp Phe
115 120 125
Gly Met Pro Leu Gly Thr Leu Ser Glu Gly Lys Met Ala Ala Ala Leu
130 135 140
Arg Lys Leu Gly Phe Asp Lys Val Tyr Asp Thr Asn Phe Gly Ala Asp
145 150 155 160
Leu Thr Ile Met Glu Glu Gly Ser Glu Leu Leu Lys Arg Val Ala Glu
165 170 175
Gly Gly Val Leu Pro Met Phe Thr Ser Cys Cys Pro Ala Trp Val Lys
180 185 190
Tyr Ala Glu Gln Thr Tyr Pro Glu Leu Leu Pro His Leu Ser Ser Cys
195 200 205
Lys Ser Pro Asn Gln Met Ala Gly Ala Ile Phe Lys Thr Tyr Gly Ala
210 215 220
Glu Ile Asn Lys Val Asn Pro Ala Lys Ile Tyr Asn Val Ser Val Met
225 230 235 240
Pro Cys Thr Cys Lys Glu Phe Glu Ser Glu Arg Glu Glu Met His Asp
245 250 255
Ser Gly His Arg Asp Val Asp Ala Val Ile Thr Thr Arg Glu Leu Ala
260 265 270
Gln Leu Phe Lys Asp Ala Asp Ile Asp Phe Asn Thr Ile Glu Glu Glu
275 280 285
Gln Phe Asp Thr Pro Leu Gly Met Tyr Thr Gly Ala Gly Thr Ile Phe
290 295 300
Gly Ala Thr Gly Gly Val Met Glu Ala Ala Leu Arg Thr Gly Tyr Glu
305 310 315 320
Leu Tyr Thr Lys Lys Thr Ile Pro Ser Ile Asp Leu Thr Met Val Arg
325 330 335
Gly Gly Glu Gly Phe Arg Thr Ala Glu Val Asp Leu Gly Asp Ile Arg
340 345 350
Leu Lys Val Gly Val Val Ser Gly Leu Lys Asn Val Lys Asp Val Met
355 360 365
Glu Ser Val Lys Ala Gly Lys Cys Asp Leu His Phe Ile Glu Val Met
370 375 380
Thr Cys Pro Gln Gly Cys Ile Ser Gly Gly Gly Gln Pro Lys Val Ile
385 390 395 400
Leu Asp Ser Asp Lys Glu Glu Ala Tyr Asn Asn Arg Lys Lys Gly Leu
405 410 415
Tyr Asn His Asp Ala Asn Leu Thr Tyr Arg Lys Ser His Glu Asn Pro
420 425 430
Glu Ile Lys Lys Ile Tyr Asp Glu Phe Leu Asp Lys Pro Leu Gly Ala
435 440 445
Lys Ser His Glu Leu Leu His Thr Lys Tyr Ile Ser Arg Lys Lys Glu
450 455 460
Ser
465
<210> 11
<211> 1680
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 11
atgagtggac aatttatgat tatagataat attcctgtag agataaatgg tgaaaaaaat 60
attcttgaac taattagaaa agctggcatt gatttaccta cattttgcta tcattcggag 120
ctttcagttt atggtgcatg ccgtatgtgc atggttgaag ataaacgcgg ccgtatgcaa 180
gctgcatgtt ctactcctcc tcaagctggt atagaaatat atacaaatac tccaagactt 240
agaaaataca gaaaaaacat tcttgagttg ttactcgcaa atcattgcag agattgtaca 300
acttgtgaaa aaaatgagca ttgcaaacta caagatttag caaagcgttt taaaataaag 360
aaagtaagat ttaaaaatac ttctataaat aaaaaaattg ataattcatc agtatgcatt 420
gtaagaaata ggagtaaatg tatcttatgc ggtgactgtg taagagtgtg tgaagaagta 480
caaaatgttg gagctattga ttttgttaaa agaggttcta atatgactgt aactactgca 540
tttgatgaac ctatagcaaa ttcgaattgt gttggatgtg gtcaatgtgc ggcagtatgc 600
cctactggtg ctattgtagt aaaggatgat acagctgaat tatgggaagc acttagtgat 660
aagaatacaa aggttgtagc tcaaattgcg cccgctgtaa gagttggtct taatgaggaa 720
ttaggtgagg aaaacggcga aaacgaaatg ggtaaaatag tagctgcact tagaagaatg 780
ggatttgatg aagtttttga tacttcaacg gcagcagatc ttacagtttt ggaagaaaca 840
gcagaattta cttcaagact tgaaaaaaat gaaagtttac cattgtttac atcctgttgt 900
tctgcatggg taaattatgt agagaataca catccagagt taatgaaata tgtttctact 960
tgcaaatcac ctatggaaat gtttgcttct gtacttaagg agtactataa aaatagtgat 1020
aaaaaaattg tagttgtagc agttatgcct tgtacagcta aaaaatatga agcaaaacga 1080
gaagaatttt caaaaaatgg tgtacctgat gtagattatg taataactac acaggagctt 1140
ataagtatga taagacaagc aggaattgta tttcctgaat tagagcctga agcagttgat 1200
atgccatttg atcttagcag tggagctgga gttatatttg gagtaacagg tggtgttaca 1260
gaggctgtta tacgtaaagt tttagctgat aaatcaaatg ctgcattacg tgcaattgtg 1320
tttaatggtg ttaggggcat ggaaggtact aaagaagcta gcattactgt tggtgatcgt 1380
gaaataaaaa tagcaatagt aagcggtctt agaaatgcag aaaatcttat acagaaaata 1440
caatctggtg aatcaaaata tgatttcgtt gaagttatgg catgtccagg tggatgcatt 1500
tctggtggtg gacaaccatt tgaaaaactt gaaggaaagc taaaacgtag tgctggaata 1560
tatcaatcag ataaaatgag cactataaaa cgtacagctg acaatccgct tatgaaatca 1620
ctgtattcag gattgttaaa aggtaaaaac cacgaactat tacatgtaaa ccgcaaatag 1680
<210> 12
<211> 559
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 12
Met Ser Gly Gln Phe Met Ile Ile Asp Asn Ile Pro Val Glu Ile Asn
1 5 10 15
Gly Glu Lys Asn Ile Leu Glu Leu Ile Arg Lys Ala Gly Ile Asp Leu
20 25 30
Pro Thr Phe Cys Tyr His Ser Glu Leu Ser Val Tyr Gly Ala Cys Arg
35 40 45
Met Cys Met Val Glu Asp Lys Arg Gly Arg Met Gln Ala Ala Cys Ser
50 55 60
Thr Pro Pro Gln Ala Gly Ile Glu Ile Tyr Thr Asn Thr Pro Arg Leu
65 70 75 80
Arg Lys Tyr Arg Lys Asn Ile Leu Glu Leu Leu Leu Ala Asn His Cys
85 90 95
Arg Asp Cys Thr Thr Cys Glu Lys Asn Glu His Cys Lys Leu Gln Asp
100 105 110
Leu Ala Lys Arg Phe Lys Ile Lys Lys Val Arg Phe Lys Asn Thr Ser
115 120 125
Ile Asn Lys Lys Ile Asp Asn Ser Ser Val Cys Ile Val Arg Asn Arg
130 135 140
Ser Lys Cys Ile Leu Cys Gly Asp Cys Val Arg Val Cys Glu Glu Val
145 150 155 160
Gln Asn Val Gly Ala Ile Asp Phe Val Lys Arg Gly Ser Asn Met Thr
165 170 175
Val Thr Thr Ala Phe Asp Glu Pro Ile Ala Asn Ser Asn Cys Val Gly
180 185 190
Cys Gly Gln Cys Ala Ala Val Cys Pro Thr Gly Ala Ile Val Val Lys
195 200 205
Asp Asp Thr Ala Glu Leu Trp Glu Ala Leu Ser Asp Lys Asn Thr Lys
210 215 220
Val Val Ala Gln Ile Ala Pro Ala Val Arg Val Gly Leu Asn Glu Glu
225 230 235 240
Leu Gly Glu Glu Asn Gly Glu Asn Glu Met Gly Lys Ile Val Ala Ala
245 250 255
Leu Arg Arg Met Gly Phe Asp Glu Val Phe Asp Thr Ser Thr Ala Ala
260 265 270
Asp Leu Thr Val Leu Glu Glu Thr Ala Glu Phe Thr Ser Arg Leu Glu
275 280 285
Lys Asn Glu Ser Leu Pro Leu Phe Thr Ser Cys Cys Ser Ala Trp Val
290 295 300
Asn Tyr Val Glu Asn Thr His Pro Glu Leu Met Lys Tyr Val Ser Thr
305 310 315 320
Cys Lys Ser Pro Met Glu Met Phe Ala Ser Val Leu Lys Glu Tyr Tyr
325 330 335
Lys Asn Ser Asp Lys Lys Ile Val Val Val Ala Val Met Pro Cys Thr
340 345 350
Ala Lys Lys Tyr Glu Ala Lys Arg Glu Glu Phe Ser Lys Asn Gly Val
355 360 365
Pro Asp Val Asp Tyr Val Ile Thr Thr Gln Glu Leu Ile Ser Met Ile
370 375 380
Arg Gln Ala Gly Ile Val Phe Pro Glu Leu Glu Pro Glu Ala Val Asp
385 390 395 400
Met Pro Phe Asp Leu Ser Ser Gly Ala Gly Val Ile Phe Gly Val Thr
405 410 415
Gly Gly Val Thr Glu Ala Val Ile Arg Lys Val Leu Ala Asp Lys Ser
420 425 430
Asn Ala Ala Leu Arg Ala Ile Val Phe Asn Gly Val Arg Gly Met Glu
435 440 445
Gly Thr Lys Glu Ala Ser Ile Thr Val Gly Asp Arg Glu Ile Lys Ile
450 455 460
Ala Ile Val Ser Gly Leu Arg Asn Ala Glu Asn Leu Ile Gln Lys Ile
465 470 475 480
Gln Ser Gly Glu Ser Lys Tyr Asp Phe Val Glu Val Met Ala Cys Pro
485 490 495
Gly Gly Cys Ile Ser Gly Gly Gly Gln Pro Phe Glu Lys Leu Glu Gly
500 505 510
Lys Leu Lys Arg Ser Ala Gly Ile Tyr Gln Ser Asp Lys Met Ser Thr
515 520 525
Ile Lys Arg Thr Ala Asp Asn Pro Leu Met Lys Ser Leu Tyr Ser Gly
530 535 540
Leu Leu Lys Gly Lys Asn His Glu Leu Leu His Val Asn Arg Lys
545 550 555
<210> 13
<211> 1917
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 13
gtgaataaaa gtcctgtaac tgttttaaaa gaaaaatgta caggttgtaa taagtgtata 60
agaacttgtc ctattcttgg tgcaaatgtt acagcaactg aaaatggtgt aagtaaggtt 120
tacatagatg aggaaagatg tattggatgc ggtgaatgtg taaaggtttg cgaacatggg 180
gctagagact ttaacgatag tacacaagac ttttttaagg atttgaaaaa aggcaaaaaa 240
ataacagtaa tagctgcacc atctattata gttaatatta aaaattacaa gaaatttttt 300
ggatatctta aatctttggg agtatctata atttatgatg tttctttcgg tgcagatatt 360
actacctggg cttatctaaa agcaatgaag gagaaaaata tttcttcttt aatatcacag 420
ccctgtccta tcgtggttaa ttatattgaa aaatataagc ctgaattaat agaatattta 480
gcgcctatac acagtcctat gatgtgtact gctgtttatc tgaaaaaata taagcatata 540
tgtgaagata tagcttttct gtcaccttgt attggcaagc taattgaaat caatgataag 600
aatacagatg ggtatgtgaa atataatgta acgtacaaaa agattttaga ttatttgaga 660
gataataatg tgaatttgaa caattacgat gaagttgaat ttgataatgt tcctgcttct 720
ttgggggttg tttatagttt accaggtgga ttaaaagcaa atgtaaaagc tagaactgaa 780
gaactacatg ttcttcagat agaaggacac aaagaggcaa ttgagtactt gaataagtat 840
tctgatagag ttaaagctaa taaacttata cctagtttac ttgatatttt aaattgcaaa 900
aatggatgta atataggtac agcttcctta gacaatttaa cggaatatga tattcaatat 960
aggtttcatg atataaaggt ggaaaagtta agagaaaaaa ctggcttgtt taagaaaaaa 1020
atcaaatcaa tagacgagta ctttgataaa aatcttaatt taaacgattt tgtaagaaag 1080
tatactgcac agaaggttaa aaaaataatt gaacctaccc aaaaagatta tgacaacata 1140
tttgatgaga tgatgaaaac tacaacattg ggaaaagaat tcaattgttc cgcttgtgga 1200
tacagcactt gcaaagaaat ggtaaagatg atttttaacg gcataaattc taaggaaaat 1260
tgtatttatt atgtgaagaa aaaaatcaat atggaatata gcgaactgga agaaaaaaat 1320
gaagaggtca aagagtctat aaacaaaata actgtactag ctgaggaaag acaacgaaaa 1380
tcagatgaaa taattaaatt tgcaaatact ctattggcag ctataaatga agtaagtaaa 1440
gggaatgagg aaagcgcatc tgcaattcaa gatatagtag aagaattaaa gtcaataatg 1500
gatatatcaa gtaagttaaa ggaaaacatt catcagataa atgaaaagtt agacaaattc 1560
acggattcat cagatagtat tgtggctatt tcagagcaga ctaatttact ttcgctaaat 1620
gccgcaatag aagcagcacg ggcaaatgaa catggaaaag gttttgcagt agtagcagat 1680
gaagtaaaga agctagcaga gcaatctaaa actaccgcac aatctactaa aaacgaagaa 1740
aatgaaatga tgcaatctat tttaaaggtt attgaagtat ctgatttact tcagaacaaa 1800
atggataata taaataacga tatattaact atatcggaaa ctatccaaga gataagtgct 1860
aagagtcagg agatagtttc tagctctgaa aagttaataa acagtgagtc acattaa 1917
<210> 14
<211> 638
<212> PRT
<213> C. autoethanogenum
<400> 14
Met Asn Lys Ser Pro Val Thr Val Leu Lys Glu Lys Cys Thr Gly Cys
1 5 10 15
Asn Lys Cys Ile Arg Thr Cys Pro Ile Leu Gly Ala Asn Val Thr Ala
20 25 30
Thr Glu Asn Gly Val Ser Lys Val Tyr Ile Asp Glu Glu Arg Cys Ile
35 40 45
Gly Cys Gly Glu Cys Val Lys Val Cys Glu His Gly Ala Arg Asp Phe
50 55 60
Asn Asp Ser Thr Gln Asp Phe Phe Lys Asp Leu Lys Lys Gly Lys Lys
65 70 75 80
Ile Thr Val Ile Ala Ala Pro Ser Ile Ile Val Asn Ile Lys Asn Tyr
85 90 95
Lys Lys Phe Phe Gly Tyr Leu Lys Ser Leu Gly Val Ser Ile Ile Tyr
100 105 110
Asp Val Ser Phe Gly Ala Asp Ile Thr Thr Trp Ala Tyr Leu Lys Ala
115 120 125
Met Lys Glu Lys Asn Ile Ser Ser Leu Ile Ser Gln Pro Cys Pro Ile
130 135 140
Val Val Asn Tyr Ile Glu Lys Tyr Lys Pro Glu Leu Ile Glu Tyr Leu
145 150 155 160
Ala Pro Ile His Ser Pro Met Met Cys Thr Ala Val Tyr Leu Lys Lys
165 170 175
Tyr Lys His Ile Cys Glu Asp Ile Ala Phe Leu Ser Pro Cys Ile Gly
180 185 190
Lys Leu Ile Glu Ile Asn Asp Lys Asn Thr Asp Gly Tyr Val Lys Tyr
195 200 205
Asn Val Thr Tyr Lys Lys Ile Leu Asp Tyr Leu Arg Asp Asn Asn Val
210 215 220
Asn Leu Asn Asn Tyr Asp Glu Val Glu Phe Asp Asn Val Pro Ala Ser
225 230 235 240
Leu Gly Val Val Tyr Ser Leu Pro Gly Gly Leu Lys Ala Asn Val Lys
245 250 255
Ala Arg Thr Glu Glu Leu His Val Leu Gln Ile Glu Gly His Lys Glu
260 265 270
Ala Ile Glu Tyr Leu Asn Lys Tyr Ser Asp Arg Val Lys Ala Asn Lys
275 280 285
Leu Ile Pro Ser Leu Leu Asp Ile Leu Asn Cys Lys Asn Gly Cys Asn
290 295 300
Ile Gly Thr Ala Ser Leu Asp Asn Leu Thr Glu Tyr Asp Ile Gln Tyr
305 310 315 320
Arg Phe His Asp Ile Lys Val Glu Lys Leu Arg Glu Lys Thr Gly Leu
325 330 335
Phe Lys Lys Lys Ile Lys Ser Ile Asp Glu Tyr Phe Asp Lys Asn Leu
340 345 350
Asn Leu Asn Asp Phe Val Arg Lys Tyr Thr Ala Gln Lys Val Lys Lys
355 360 365
Ile Ile Glu Pro Thr Gln Lys Asp Tyr Asp Asn Ile Phe Asp Glu Met
370 375 380
Met Lys Thr Thr Thr Leu Gly Lys Glu Phe Asn Cys Ser Ala Cys Gly
385 390 395 400
Tyr Ser Thr Cys Lys Glu Met Val Lys Met Ile Phe Asn Gly Ile Asn
405 410 415
Ser Lys Glu Asn Cys Ile Tyr Tyr Val Lys Lys Lys Ile Asn Met Glu
420 425 430
Tyr Ser Glu Leu Glu Glu Lys Asn Glu Glu Val Lys Glu Ser Ile Asn
435 440 445
Lys Ile Thr Val Leu Ala Glu Glu Arg Gln Arg Lys Ser Asp Glu Ile
450 455 460
Ile Lys Phe Ala Asn Thr Leu Leu Ala Ala Ile Asn Glu Val Ser Lys
465 470 475 480
Gly Asn Glu Glu Ser Ala Ser Ala Ile Gln Asp Ile Val Glu Glu Leu
485 490 495
Lys Ser Ile Met Asp Ile Ser Ser Lys Leu Lys Glu Asn Ile His Gln
500 505 510
Ile Asn Glu Lys Leu Asp Lys Phe Thr Asp Ser Ser Asp Ser Ile Val
515 520 525
Ala Ile Ser Glu Gln Thr Asn Leu Leu Ser Leu Asn Ala Ala Ile Glu
530 535 540
Ala Ala Arg Ala Asn Glu His Gly Lys Gly Phe Ala Val Val Ala Asp
545 550 555 560
Glu Val Lys Lys Leu Ala Glu Gln Ser Lys Thr Thr Ala Gln Ser Thr
565 570 575
Lys Asn Glu Glu Asn Glu Met Met Gln Ser Ile Leu Lys Val Ile Glu
580 585 590
Val Ser Asp Leu Leu Gln Asn Lys Met Asp Asn Ile Asn Asn Asp Ile
595 600 605
Leu Thr Ile Ser Glu Thr Ile Gln Glu Ile Ser Ala Lys Ser Gln Glu
610 615 620
Ile Val Ser Ser Ser Glu Lys Leu Ile Asn Ser Glu Ser His
625 630 635
<210> 15
<211> 1347
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 15
ttgagagatg attataggaa tctatttaaa ttcataataa aggcatatta tagtggaaat 60
tttgaagaag aagtgatgtc atttttatta gagtctaaaa tggataaaca ggaattgtgt 120
aagattatat ctacgttgtg cggcactaat atagattata gttccaattt tatagaaaat 180
ttgaaaaaag caataaagtc ttataaacaa gaaggtaaag tagtcaataa agttagagac 240
tgttccatgg aatgtgtgga tgaaaaaggt gagatacttt gtcaaaaaac atgtcctttt 300
gatgcaattt ttatagacaa taagaaaaat tgtgcttaca tagataaaga aaaatgtacc 360
gattgtggtt tttgtgtaga tgtttgtcct actgggggaa taatggataa agttcagttc 420
attcctcttg ctgatatttt aaaaagcaaa tctccagttg tggctatagt ggctcctgcc 480
ataataggac agtttggcga agatgttact atggatcaac ttaggactgc ttttaaaaaa 540
ctgggattta ctgatatgat tgaagtggca ttttttgcag atatgcttac tttaaaggag 600
tctattgaat ttgacaatca tgtaaaagat gaaaaagatt ttatgataac ctcctgctgc 660
tgccctatgt gggtagctat ggtaaaaaag gtatacagta atttggttaa acacgtatcc 720
ccctctgtat ctccaatggt tgcaggagga agagtactta aaaagttaaa cccttactgc 780
aaggtagtgt ttataggacc atgtattgct aaaaaatctg aggcaaagga agaagacata 840
aaaggagcaa tagatttcgt acttactttt gaagaattaa gagatatatt tgatgctttt 900
catatagttc catctaaact tgaaggagat ttttcctcta aatatgcgtc taggggtgga 960
agattatatg ctcgtacagg gggagtttct attgcagtaa gcgaagctgt ggaaagaatt 1020
ttccctgaaa agcataaact atttagtgca attcaggcaa atggcattag agaatgtaga 1080
gaaatgctta ccaaggtgca aaatggagaa ataaaagcta attttattga aggaatgggc 1140
tgcattggcg gatgtgtagg tggtcccaaa gcaattgtac ttaaggatga aggtagggat 1200
cgagtaaata aatttgcaca agattctgaa ataaaagttg ctgtagatag tgaatgcatg 1260
catggagtat tacatgcttt ggatatacat tctatagatg attttaagga tgagaaaaaa 1320
atagaactgt tagaacgaga attttaa 1347
<210> 16
<211> 448
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 16
Met Arg Asp Asp Tyr Arg Asn Leu Phe Lys Phe Ile Ile Lys Ala Tyr
1 5 10 15
Tyr Ser Gly Asn Phe Glu Glu Glu Val Met Ser Phe Leu Leu Glu Ser
20 25 30
Lys Met Asp Lys Gln Glu Leu Cys Lys Ile Ile Ser Thr Leu Cys Gly
35 40 45
Thr Asn Ile Asp Tyr Ser Ser Asn Phe Ile Glu Asn Leu Lys Lys Ala
50 55 60
Ile Lys Ser Tyr Lys Gln Glu Gly Lys Val Val Asn Lys Val Arg Asp
65 70 75 80
Cys Ser Met Glu Cys Val Asp Glu Lys Gly Glu Ile Leu Cys Gln Lys
85 90 95
Thr Cys Pro Phe Asp Ala Ile Phe Ile Asp Asn Lys Lys Asn Cys Ala
100 105 110
Tyr Ile Asp Lys Glu Lys Cys Thr Asp Cys Gly Phe Cys Val Asp Val
115 120 125
Cys Pro Thr Gly Gly Ile Met Asp Lys Val Gln Phe Ile Pro Leu Ala
130 135 140
Asp Ile Leu Lys Ser Lys Ser Pro Val Val Ala Ile Val Ala Pro Ala
145 150 155 160
Ile Ile Gly Gln Phe Gly Glu Asp Val Thr Met Asp Gln Leu Arg Thr
165 170 175
Ala Phe Lys Lys Leu Gly Phe Thr Asp Met Ile Glu Val Ala Phe Phe
180 185 190
Ala Asp Met Leu Thr Leu Lys Glu Ser Ile Glu Phe Asp Asn His Val
195 200 205
Lys Asp Glu Lys Asp Phe Met Ile Thr Ser Cys Cys Cys Pro Met Trp
210 215 220
Val Ala Met Val Lys Lys Val Tyr Ser Asn Leu Val Lys His Val Ser
225 230 235 240
Pro Ser Val Ser Pro Met Val Ala Gly Gly Arg Val Leu Lys Lys Leu
245 250 255
Asn Pro Tyr Cys Lys Val Val Phe Ile Gly Pro Cys Ile Ala Lys Lys
260 265 270
Ser Glu Ala Lys Glu Glu Asp Ile Lys Gly Ala Ile Asp Phe Val Leu
275 280 285
Thr Phe Glu Glu Leu Arg Asp Ile Phe Asp Ala Phe His Ile Val Pro
290 295 300
Ser Lys Leu Glu Gly Asp Phe Ser Ser Lys Tyr Ala Ser Arg Gly Gly
305 310 315 320
Arg Leu Tyr Ala Arg Thr Gly Gly Val Ser Ile Ala Val Ser Glu Ala
325 330 335
Val Glu Arg Ile Phe Pro Glu Lys His Lys Leu Phe Ser Ala Ile Gln
340 345 350
Ala Asn Gly Ile Arg Glu Cys Arg Glu Met Leu Thr Lys Val Gln Asn
355 360 365
Gly Glu Ile Lys Ala Asn Phe Ile Glu Gly Met Gly Cys Ile Gly Gly
370 375 380
Cys Val Gly Gly Pro Lys Ala Ile Val Leu Lys Asp Glu Gly Arg Asp
385 390 395 400
Arg Val Asn Lys Phe Ala Gln Asp Ser Glu Ile Lys Val Ala Val Asp
405 410 415
Ser Glu Cys Met His Gly Val Leu His Ala Leu Asp Ile His Ser Ile
420 425 430
Asp Asp Phe Lys Asp Glu Lys Lys Ile Glu Leu Leu Glu Arg Glu Phe
435 440 445
<210> 17
<211> 1374
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 17
ttgacagtta aaagtgaagg tattgtcaaa attgataaag agttgtgtac aggatgtaga 60
cgatgtgcag atatttgtcc tgtagatgct atagaaggtg agaagggaca acctcaaaca 120
attaatactg aacgctgtgt tttgtgtggt cagtgtgtac aaatttgcag tgcttatgca 180
tctgcatttg atgaagatat tactcctcat aaggaaaaga taaaagagcg taatatgctt 240
ccatctgtta aagagccctt atttgcatcc tactatagag gagatgctcc agcagtaaaa 300
gaggccttag caaattctaa actttttact atggttcaat gcgcaccagc agtacgtgtg 360
gctattgccg aagaatttgg tatgccactt ggaagtttaa caccagggaa aatggcagct 420
gcgctaagag agttaggttt tgatcgaatt tatgatacta attttgctgc tgatctaact 480
attatggagg aaggcagtga acttattaaa agggttactg aaggtggagt actgccaatg 540
ttcacttcat gttgtcctgc ttgggtaaaa tttattgagc aggattatcc ggaacttatt 600
ccacatctgt cttcttgtaa atctccgcag caaatggaag gtgctctgct taaaacatat 660
ggtgcacagg ttgatggtgt agatgctggc aagatttata gtgtttcagt tatgccttgt 720
atttgcaaaa aatttgaatg tgaacgtcct gaaatgaaag acagtggata tcaggatgta 780
gatgctgtaa ttaccacacg ggaacttgca caattaatca aggatgatgg cattgatttt 840
aatggtttac ctgaaaaaga atttgacaag ccacttggaa cttattctgg tgcaggcact 900
attttctgtg ctactggtgg tgttatggaa gctgccctgc gtacggcata taaattgatt 960
actaaagaag agattccaga tgtcgatctc aaattcataa gaggaggcga aggcgcaaga 1020
agttcagaaa ttaaagtagg agatttaaca ctaaaagtag cagtagttgc tggtctgaaa 1080
aatgttgtac cagttttgga agcaattaaa actgggaaag cagatttcca tttcattgaa 1140
gtgatgacct gtccagttgg atgtgttagc gggggcggac aacctaaggt attgatacct 1200
gatgaaaaag ctgattcgta tactaatcgt acatgcagta cgtatgtaca tgatgaaaat 1260
atggaatata gaaaatcaca tgataatcct gaaatacaga aaatttataa agaattcttg 1320
gtagaagata atattcatca tttgcttcat actacgtata cgccaaggag gtaa 1374
<210> 18
<211> 457
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 18
Met Thr Val Lys Ser Glu Gly Ile Val Lys Ile Asp Lys Glu Leu Cys
1 5 10 15
Thr Gly Cys Arg Arg Cys Ala Asp Ile Cys Pro Val Asp Ala Ile Glu
20 25 30
Gly Glu Lys Gly Gln Pro Gln Thr Ile Asn Thr Glu Arg Cys Val Leu
35 40 45
Cys Gly Gln Cys Val Gln Ile Cys Ser Ala Tyr Ala Ser Ala Phe Asp
50 55 60
Glu Asp Ile Thr Pro His Lys Glu Lys Ile Lys Glu Arg Asn Met Leu
65 70 75 80
Pro Ser Val Lys Glu Pro Leu Phe Ala Ser Tyr Tyr Arg Gly Asp Ala
85 90 95
Pro Ala Val Lys Glu Ala Leu Ala Asn Ser Lys Leu Phe Thr Met Val
100 105 110
Gln Cys Ala Pro Ala Val Arg Val Ala Ile Ala Glu Glu Phe Gly Met
115 120 125
Pro Leu Gly Ser Leu Thr Pro Gly Lys Met Ala Ala Ala Leu Arg Glu
130 135 140
Leu Gly Phe Asp Arg Ile Tyr Asp Thr Asn Phe Ala Ala Asp Leu Thr
145 150 155 160
Ile Met Glu Glu Gly Ser Glu Leu Ile Lys Arg Val Thr Glu Gly Gly
165 170 175
Val Leu Pro Met Phe Thr Ser Cys Cys Pro Ala Trp Val Lys Phe Ile
180 185 190
Glu Gln Asp Tyr Pro Glu Leu Ile Pro His Leu Ser Ser Cys Lys Ser
195 200 205
Pro Gln Gln Met Glu Gly Ala Leu Leu Lys Thr Tyr Gly Ala Gln Val
210 215 220
Asp Gly Val Asp Ala Gly Lys Ile Tyr Ser Val Ser Val Met Pro Cys
225 230 235 240
Ile Cys Lys Lys Phe Glu Cys Glu Arg Pro Glu Met Lys Asp Ser Gly
245 250 255
Tyr Gln Asp Val Asp Ala Val Ile Thr Thr Arg Glu Leu Ala Gln Leu
260 265 270
Ile Lys Asp Asp Gly Ile Asp Phe Asn Gly Leu Pro Glu Lys Glu Phe
275 280 285
Asp Lys Pro Leu Gly Thr Tyr Ser Gly Ala Gly Thr Ile Phe Cys Ala
290 295 300
Thr Gly Gly Val Met Glu Ala Ala Leu Arg Thr Ala Tyr Lys Leu Ile
305 310 315 320
Thr Lys Glu Glu Ile Pro Asp Val Asp Leu Lys Phe Ile Arg Gly Gly
325 330 335
Glu Gly Ala Arg Ser Ser Glu Ile Lys Val Gly Asp Leu Thr Leu Lys
340 345 350
Val Ala Val Val Ala Gly Leu Lys Asn Val Val Pro Val Leu Glu Ala
355 360 365
Ile Lys Thr Gly Lys Ala Asp Phe His Phe Ile Glu Val Met Thr Cys
370 375 380
Pro Val Gly Cys Val Ser Gly Gly Gly Gln Pro Lys Val Leu Ile Pro
385 390 395 400
Asp Glu Lys Ala Asp Ser Tyr Thr Asn Arg Thr Cys Ser Thr Tyr Val
405 410 415
His Asp Glu Asn Met Glu Tyr Arg Lys Ser His Asp Asn Pro Glu Ile
420 425 430
Gln Lys Ile Tyr Lys Glu Phe Leu Val Glu Asp Asn Ile His His Leu
435 440 445
Leu His Thr Thr Tyr Thr Pro Arg Arg
450 455
<210> 19
<211> 1737
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 19
atggtaaatt taactataaa cgatataaag gtttctgtcc cagaaggcac tacaatttta 60
aacgctgcaa aaaaagtaaa cataaatata cctactctct gctatcttga tcttcacgat 120
ataaaaatgg taaatagaac ttcctcctgc agagtttgtc ttgttgaaat tgaaggaaga 180
cgaaatcttg caccttcatg ttctacagaa gctttcgagg gtatgatagt tagaacaaac 240
agtgccagag ctataaaagc aaggcgtacc atggtagaac ttttattatc agatcatcct 300
accgactgcc ttgtatgtga aaagaatact caatgccaac ttcaattaat tgctgctgaa 360
ttaggtataa ggaaaataag atataaaggt gctatgtcta attataagaa ggattcctcc 420
agcggtgctc tatatagaaa tctggataag tgcataatgt gcagacgatg cgaaaccatg 480
tgcaatgaag ttcaaacctg tcaggtttac tctgcagtag atagaggctt cgaaactgta 540
gtatcccctg catttggtcg tcctatggtt gacacgcaat gcacattttg cggtcaatgt 600
gtatcagtat gtccaaccgc tgcattaact caagttagta atgtagctaa ggtatgggaa 660
gtactaactg atcctgataa atatgtagta gttcaaactg cccctgctat aagagttact 720
ttaggtgaaa aattcggtat ggaacctgga actattgtaa ctggcaaaat ggttgcagcc 780
ttaagaagat tgggttttga taaggtatgt gatacagact ttgcagcaga tgtaactatt 840
ttagaagaag ctcatgaatt tatagataga cttcaaaatg gtggaagact tccaatactc 900
acaagctgct gtcccagctg ggttaaattt atagaacatc aatttcctga tcttttagat 960
ataccttcaa cttgtaaatc tccacacata atgtttggta ctttagctaa aacatatatg 1020
gcagaaaaat taaatattga tccatctaaa attgtagtag tttcagttat gccctgtatt 1080
gcaaaaaaat atgaaataag cagaaaagag cttcaatatg aaggtcataa aaatgttgat 1140
cttgtggtta ccacaagaga gcttgcagat atgataatgg aagcaggaat agattttaac 1200
aaacttcctg atgaagattt tgataatcca cttggagaat ccacaggtgc ctctgtaata 1260
tttggaacta ccggcggcgt aattgaagca gctcttagaa ctgcttatga atggattact 1320
ggagagactt taaaagaagt agaatttcat agtgtaagag gtcttgacgg acttaaagaa 1380
gccagtataa atattggtgg taaaaaaata aacatcggtg tagcacacgg tcttggcaac 1440
gcaagaaaac ttcttgagga aatagaatct ggtgaatcaa aatatcatgc tatagaaata 1500
atggcctgtc ctggaggatg tattgacgga ggaggtcagc catatcactt tggagattta 1560
gatatcgtaa agaaaagaat ggaagcttta tatagagaag atagaaacaa acctctcaga 1620
aaatctcatg aaaatcctga agttcaagct ctatataaag aatttattgg tgatgtaggt 1680
ggaaaaaaag ctcatgatct ccttcacacc cattatataa aaaggcaaaa attataa 1737
<210> 20
<211> 578
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 20
Met Val Asn Leu Thr Ile Asn Asp Ile Lys Val Ser Val Pro Glu Gly
1 5 10 15
Thr Thr Ile Leu Asn Ala Ala Lys Lys Val Asn Ile Asn Ile Pro Thr
20 25 30
Leu Cys Tyr Leu Asp Leu His Asp Ile Lys Met Val Asn Arg Thr Ser
35 40 45
Ser Cys Arg Val Cys Leu Val Glu Ile Glu Gly Arg Arg Asn Leu Ala
50 55 60
Pro Ser Cys Ser Thr Glu Ala Phe Glu Gly Met Ile Val Arg Thr Asn
65 70 75 80
Ser Ala Arg Ala Ile Lys Ala Arg Arg Thr Met Val Glu Leu Leu Leu
85 90 95
Ser Asp His Pro Thr Asp Cys Leu Val Cys Glu Lys Asn Thr Gln Cys
100 105 110
Gln Leu Gln Leu Ile Ala Ala Glu Leu Gly Ile Arg Lys Ile Arg Tyr
115 120 125
Lys Gly Ala Met Ser Asn Tyr Lys Lys Asp Ser Ser Ser Gly Ala Leu
130 135 140
Tyr Arg Asn Leu Asp Lys Cys Ile Met Cys Arg Arg Cys Glu Thr Met
145 150 155 160
Cys Asn Glu Val Gln Thr Cys Gln Val Tyr Ser Ala Val Asp Arg Gly
165 170 175
Phe Glu Thr Val Val Ser Pro Ala Phe Gly Arg Pro Met Val Asp Thr
180 185 190
Gln Cys Thr Phe Cys Gly Gln Cys Val Ser Val Cys Pro Thr Ala Ala
195 200 205
Leu Thr Gln Val Ser Asn Val Ala Lys Val Trp Glu Val Leu Thr Asp
210 215 220
Pro Asp Lys Tyr Val Val Val Gln Thr Ala Pro Ala Ile Arg Val Thr
225 230 235 240
Leu Gly Glu Lys Phe Gly Met Glu Pro Gly Thr Ile Val Thr Gly Lys
245 250 255
Met Val Ala Ala Leu Arg Arg Leu Gly Phe Asp Lys Val Cys Asp Thr
260 265 270
Asp Phe Ala Ala Asp Val Thr Ile Leu Glu Glu Ala His Glu Phe Ile
275 280 285
Asp Arg Leu Gln Asn Gly Gly Arg Leu Pro Ile Leu Thr Ser Cys Cys
290 295 300
Pro Ser Trp Val Lys Phe Ile Glu His Gln Phe Pro Asp Leu Leu Asp
305 310 315 320
Ile Pro Ser Thr Cys Lys Ser Pro His Ile Met Phe Gly Thr Leu Ala
325 330 335
Lys Thr Tyr Met Ala Glu Lys Leu Asn Ile Asp Pro Ser Lys Ile Val
340 345 350
Val Val Ser Val Met Pro Cys Ile Ala Lys Lys Tyr Glu Ile Ser Arg
355 360 365
Lys Glu Leu Gln Tyr Glu Gly His Lys Asn Val Asp Leu Val Val Thr
370 375 380
Thr Arg Glu Leu Ala Asp Met Ile Met Glu Ala Gly Ile Asp Phe Asn
385 390 395 400
Lys Leu Pro Asp Glu Asp Phe Asp Asn Pro Leu Gly Glu Ser Thr Gly
405 410 415
Ala Ser Val Ile Phe Gly Thr Thr Gly Gly Val Ile Glu Ala Ala Leu
420 425 430
Arg Thr Ala Tyr Glu Trp Ile Thr Gly Glu Thr Leu Lys Glu Val Glu
435 440 445
Phe His Ser Val Arg Gly Leu Asp Gly Leu Lys Glu Ala Ser Ile Asn
450 455 460
Ile Gly Gly Lys Lys Ile Asn Ile Gly Val Ala His Gly Leu Gly Asn
465 470 475 480
Ala Arg Lys Leu Leu Glu Glu Ile Glu Ser Gly Glu Ser Lys Tyr His
485 490 495
Ala Ile Glu Ile Met Ala Cys Pro Gly Gly Cys Ile Asp Gly Gly Gly
500 505 510
Gln Pro Tyr His Phe Gly Asp Leu Asp Ile Val Lys Lys Arg Met Glu
515 520 525
Ala Leu Tyr Arg Glu Asp Arg Asn Lys Pro Leu Arg Lys Ser His Glu
530 535 540
Asn Pro Glu Val Gln Ala Leu Tyr Lys Glu Phe Ile Gly Asp Val Gly
545 550 555 560
Gly Lys Lys Ala His Asp Leu Leu His Thr His Tyr Ile Lys Arg Gln
565 570 575
Lys Leu
<210> 21
<211> 882
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 21
atgaatgttc gaaacaaggg tatatgtcct ttaatcgtag ataaggaacg cagttcaaag 60
gcttttacta gtgaagctat agatttaatt aaaaggggaa agacgaaaaa attaaatgct 120
atatggcttg aagtaacagg atgttcagga aatattattt cttttttaaa tagtgaaaat 180
cctggactcg attatatttt agaaaaactc attaatttaa aatacaacaa tactctaatg 240
acttcagaag gtgagtatgc ttttaaacaa ttcttagata cattggatac tgaatttata 300
ctactagtag atggagcggt atctactgct caaaatggtt tttataatat tgttgccaat 360
tatgaaggaa aacctgttac tgcacttgaa gctgtaaaaa tggcaggaga aaaagcaaag 420
tatgttctct gtgtaggaac ttgtgcatcc tatggtggaa tttctgccgc caggccaaac 480
ccatcagaaa gcaaaagtgt taaagaaata ctaaatcgtg aagtcataag acttccaggc 540
tgtccatgcc atccggattg ggtagttgga actttagcac atttagttgc ttttggcaaa 600
ccgcaattgg atgaagatgg aagacctctt cttttttatg gaattaccat tcatgatagg 660
tgtacaagaa ggggattttt tgataacaga atttttgcaa aaaaatttgg agaagatgga 720
tgtatgttta aacttggatg cagggggcct gtaactaaaa cagattgtcc taggagaaag 780
tggaatggat atgtgaactg gcctgttgaa gacaatacca actgtatagg atgtgcaaat 840
tctagatttc cagatggtat ggaaccattt gtaaggtatt ag 882
<210> 22
<211> 293
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 22
Met Asn Val Arg Asn Lys Gly Ile Cys Pro Leu Ile Val Asp Lys Glu
1 5 10 15
Arg Ser Ser Lys Ala Phe Thr Ser Glu Ala Ile Asp Leu Ile Lys Arg
20 25 30
Gly Lys Thr Lys Lys Leu Asn Ala Ile Trp Leu Glu Val Thr Gly Cys
35 40 45
Ser Gly Asn Ile Ile Ser Phe Leu Asn Ser Glu Asn Pro Gly Leu Asp
50 55 60
Tyr Ile Leu Glu Lys Leu Ile Asn Leu Lys Tyr Asn Asn Thr Leu Met
65 70 75 80
Thr Ser Glu Gly Glu Tyr Ala Phe Lys Gln Phe Leu Asp Thr Leu Asp
85 90 95
Thr Glu Phe Ile Leu Leu Val Asp Gly Ala Val Ser Thr Ala Gln Asn
100 105 110
Gly Phe Tyr Asn Ile Val Ala Asn Tyr Glu Gly Lys Pro Val Thr Ala
115 120 125
Leu Glu Ala Val Lys Met Ala Gly Glu Lys Ala Lys Tyr Val Leu Cys
130 135 140
Val Gly Thr Cys Ala Ser Tyr Gly Gly Ile Ser Ala Ala Arg Pro Asn
145 150 155 160
Pro Ser Glu Ser Lys Ser Val Lys Glu Ile Leu Asn Arg Glu Val Ile
165 170 175
Arg Leu Pro Gly Cys Pro Cys His Pro Asp Trp Val Val Gly Thr Leu
180 185 190
Ala His Leu Val Ala Phe Gly Lys Pro Gln Leu Asp Glu Asp Gly Arg
195 200 205
Pro Leu Leu Phe Tyr Gly Ile Thr Ile His Asp Arg Cys Thr Arg Arg
210 215 220
Gly Phe Phe Asp Asn Arg Ile Phe Ala Lys Lys Phe Gly Glu Asp Gly
225 230 235 240
Cys Met Phe Lys Leu Gly Cys Arg Gly Pro Val Thr Lys Thr Asp Cys
245 250 255
Pro Arg Arg Lys Trp Asn Gly Tyr Val Asn Trp Pro Val Glu Asp Asn
260 265 270
Thr Asn Cys Ile Gly Cys Ala Asn Ser Arg Phe Pro Asp Gly Met Glu
275 280 285
Pro Phe Val Arg Tyr
290
<210> 23
<211> 1386
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 23
atgaaaaaga aaattaccat tgatccaatt acgagaataa gtggtttttt ggaaactaaa 60
gtgcaagtag aaaaaaatat tatagtagat gctgaaacta gtggattgct ttttagagga 120
tttgaaaaaa tgttaaaaaa cagacagccg ctggatgcag tatattttac agaaagaatt 180
tgtgggatat gttcaacagc tcatgctgtg gcagctgcta cagctcttga agatgctttg 240
aagataaaaa ttagtgtaaa tgattcgtat atgcgtaatt taatacatgg ttttgaattt 300
atacaaaatc atataagaca tttttataat ttgactatac caagttatgt gaagatgccc 360
aatataaatc ctcttttttc agatcaatat gaagattata gattacctta taacttaaat 420
aaaaagataa gtgaagatta tattgaaagt attaaataca gtaggttagc ccatgaagga 480
ttggctactc ttggaggaaa ggctccccat aatcacggaa tttttgttgg aggagttacc 540
ataaatatag atccatataa acttacaaaa gttaaatcta ttatttctca aattaatgaa 600
ttcgtaagta gtgttatgtt agaggacatg aacataattt caaaatacta tgctgattat 660
tttaaaatgg gaaaagccta tggaaacttt atgacttatg gaatttttga taagtatgct 720
gatcctgaga taagttatgt aggaccttct gtcttaataa atggacaaaa gcataacttt 780
aatagtaata aaattacaga gaatatactt tacacctggt acatgaatga tgatgaaaca 840
ataaatttat ctaaagaaac aggttacagc tttataaaat cgccaactta tgatggctat 900
tctatggaag taggacctct agcaagattg atactttcag gtgagtatac tggtggaagt 960
tcatgtatgg acagaaatgt tgccagagta cttgaaacaa aaaagatttt agaaattatg 1020
caaggacttg cagatagaat taagcttatt ccagcagaac aaagaatata tgaaatccca 1080
gataaagcat ttggtgcagg attaattgac acaactagag gatccttggg acactggata 1140
agtatagaag ataaatttat aaagcattac aatattataa ctcctacagt gtggaacatg 1200
gggccaagaa atcaatcagg tgcgcttgga attggagaaa aatctttact tggaacgaaa 1260
ataaaagata taaagcagcc tatagaagtt gggagaatta tgaggtcctt tgatccttgt 1320
gtttcctgtg caactcattt gataagtgat gcatatgaac cagtggacgt acaggttata 1380
gtatga 1386
<210> 24
<211> 461
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 24
Met Lys Lys Lys Ile Thr Ile Asp Pro Ile Thr Arg Ile Ser Gly Phe
1 5 10 15
Leu Glu Thr Lys Val Gln Val Glu Lys Asn Ile Ile Val Asp Ala Glu
20 25 30
Thr Ser Gly Leu Leu Phe Arg Gly Phe Glu Lys Met Leu Lys Asn Arg
35 40 45
Gln Pro Leu Asp Ala Val Tyr Phe Thr Glu Arg Ile Cys Gly Ile Cys
50 55 60
Ser Thr Ala His Ala Val Ala Ala Ala Thr Ala Leu Glu Asp Ala Leu
65 70 75 80
Lys Ile Lys Ile Ser Val Asn Asp Ser Tyr Met Arg Asn Leu Ile His
85 90 95
Gly Phe Glu Phe Ile Gln Asn His Ile Arg His Phe Tyr Asn Leu Thr
100 105 110
Ile Pro Ser Tyr Val Lys Met Pro Asn Ile Asn Pro Leu Phe Ser Asp
115 120 125
Gln Tyr Glu Asp Tyr Arg Leu Pro Tyr Asn Leu Asn Lys Lys Ile Ser
130 135 140
Glu Asp Tyr Ile Glu Ser Ile Lys Tyr Ser Arg Leu Ala His Glu Gly
145 150 155 160
Leu Ala Thr Leu Gly Gly Lys Ala Pro His Asn His Gly Ile Phe Val
165 170 175
Gly Gly Val Thr Ile Asn Ile Asp Pro Tyr Lys Leu Thr Lys Val Lys
180 185 190
Ser Ile Ile Ser Gln Ile Asn Glu Phe Val Ser Ser Val Met Leu Glu
195 200 205
Asp Met Asn Ile Ile Ser Lys Tyr Tyr Ala Asp Tyr Phe Lys Met Gly
210 215 220
Lys Ala Tyr Gly Asn Phe Met Thr Tyr Gly Ile Phe Asp Lys Tyr Ala
225 230 235 240
Asp Pro Glu Ile Ser Tyr Val Gly Pro Ser Val Leu Ile Asn Gly Gln
245 250 255
Lys His Asn Phe Asn Ser Asn Lys Ile Thr Glu Asn Ile Leu Tyr Thr
260 265 270
Trp Tyr Met Asn Asp Asp Glu Thr Ile Asn Leu Ser Lys Glu Thr Gly
275 280 285
Tyr Ser Phe Ile Lys Ser Pro Thr Tyr Asp Gly Tyr Ser Met Glu Val
290 295 300
Gly Pro Leu Ala Arg Leu Ile Leu Ser Gly Glu Tyr Thr Gly Gly Ser
305 310 315 320
Ser Cys Met Asp Arg Asn Val Ala Arg Val Leu Glu Thr Lys Lys Ile
325 330 335
Leu Glu Ile Met Gln Gly Leu Ala Asp Arg Ile Lys Leu Ile Pro Ala
340 345 350
Glu Gln Arg Ile Tyr Glu Ile Pro Asp Lys Ala Phe Gly Ala Gly Leu
355 360 365
Ile Asp Thr Thr Arg Gly Ser Leu Gly His Trp Ile Ser Ile Glu Asp
370 375 380
Lys Phe Ile Lys His Tyr Asn Ile Ile Thr Pro Thr Val Trp Asn Met
385 390 395 400
Gly Pro Arg Asn Gln Ser Gly Ala Leu Gly Ile Gly Glu Lys Ser Leu
405 410 415
Leu Gly Thr Lys Ile Lys Asp Ile Lys Gln Pro Ile Glu Val Gly Arg
420 425 430
Ile Met Arg Ser Phe Asp Pro Cys Val Ser Cys Ala Thr His Leu Ile
435 440 445
Ser Asp Ala Tyr Glu Pro Val Asp Val Gln Val Ile Val
450 455 460
<210> 25
<211> 1398
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 25
atgttaaata tgccaactag tacttctatg ataaatatag atgaagaatt atgtacaggc 60
tgcagacgat gtgcggatgt ctgccctgta gatgctatag aaggtgaaca gggtaaacct 120
cagaagataa atactgaaaa gtgtgttatg tgcggacaat gcattcaagt ttgtaaaggc 180
tatcaatctg tatacgatga tattcctact ccagttagca aaaggttatt tgatagagga 240
ttgttaaagg aagtagatga accattattt gcagcatata ataaaggtca ggcaaagaga 300
gttaaagaaa ttttacaaaa caaagatgta tttaaaattg tgcaatgtgc acctgctgta 360
agagttgcta taggagagga ttttggaatg cctcttggaa ctttaagtga aggaaaaatg 420
gcagctgcac tcagaaaatt aggatttgac aaagtatatg atacaaactt tggtgcagat 480
cttactataa tggaagaagg tagtgagtta ctaaaaagag tagctgaagg tggagttttg 540
ccaatgttta cttcttgttg tccagcatgg gtaaaatatg cagaacaaac atatccagaa 600
cttttacctc atctttcaag ttgtaagtct ccaaatcaga tggctggagc tatatttaaa 660
acttatggag cagagataaa taaggttaat ccggctaaaa tttataatgt atctgttatg 720
ccatgtacat gcaaggaatt tgaaagtgaa agagaagaaa tgcatgacag tggacacaga 780
gatgtagatg cagttataac tacaagggaa ttagcacaac tgttcaaaga tgctgatata 840
gattttaata ctattgaaga agaacagttt gatactcctc ttggtatgta tactggtgca 900
ggaactatat ttggtgctac aggtggagtt atggaagcag cacttagaac tggatatgaa 960
ctttatacta aaaaaactat tccaagtata gatcttacta tggtaagagg tggagaaggt 1020
tttagaactg ctgaagtaga tttaggagat attagactaa aagtaggagt agtttccggc 1080
ttaaaaaatg taaaagatgt tatggaatca gtaaaggcag gtaaatgtga tttacacttt 1140
atagaggtta tgacttgtcc tcaaggatgt ataagtggtg gaggacaacc taaggttata 1200
cttgattcag ataaagaaga agcttataat aataggaaaa agggactata taatcatgac 1260
gctaatctta cttatagaaa atcacatgaa aatccagaaa taaagaaaat atatgatgag 1320
ttcttagaca aaccattagg agctaagtct catgaattat tgcacactaa atatatctca 1380
agaaaaaagg agagttaa 1398
<210> 26
<211> 465
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 26
Met Leu Asn Met Pro Thr Ser Thr Ser Met Ile Asn Ile Asp Glu Glu
1 5 10 15
Leu Cys Thr Gly Cys Arg Arg Cys Ala Asp Val Cys Pro Val Asp Ala
20 25 30
Ile Glu Gly Glu Gln Gly Lys Pro Gln Lys Ile Asn Thr Glu Lys Cys
35 40 45
Val Met Cys Gly Gln Cys Ile Gln Val Cys Lys Gly Tyr Gln Ser Val
50 55 60
Tyr Asp Asp Ile Pro Thr Pro Val Ser Lys Arg Leu Phe Asp Arg Gly
65 70 75 80
Leu Leu Lys Glu Val Asp Glu Pro Leu Phe Ala Ala Tyr Asn Lys Gly
85 90 95
Gln Ala Lys Arg Val Lys Glu Ile Leu Gln Asn Lys Asp Val Phe Lys
100 105 110
Ile Val Gln Cys Ala Pro Ala Val Arg Val Ala Ile Gly Glu Asp Phe
115 120 125
Gly Met Pro Leu Gly Thr Leu Ser Glu Gly Lys Met Ala Ala Ala Leu
130 135 140
Arg Lys Leu Gly Phe Asp Lys Val Tyr Asp Thr Asn Phe Gly Ala Asp
145 150 155 160
Leu Thr Ile Met Glu Glu Gly Ser Glu Leu Leu Lys Arg Val Ala Glu
165 170 175
Gly Gly Val Leu Pro Met Phe Thr Ser Cys Cys Pro Ala Trp Val Lys
180 185 190
Tyr Ala Glu Gln Thr Tyr Pro Glu Leu Leu Pro His Leu Ser Ser Cys
195 200 205
Lys Ser Pro Asn Gln Met Ala Gly Ala Ile Phe Lys Thr Tyr Gly Ala
210 215 220
Glu Ile Asn Lys Val Asn Pro Ala Lys Ile Tyr Asn Val Ser Val Met
225 230 235 240
Pro Cys Thr Cys Lys Glu Phe Glu Ser Glu Arg Glu Glu Met His Asp
245 250 255
Ser Gly His Arg Asp Val Asp Ala Val Ile Thr Thr Arg Glu Leu Ala
260 265 270
Gln Leu Phe Lys Asp Ala Asp Ile Asp Phe Asn Thr Ile Glu Glu Glu
275 280 285
Gln Phe Asp Thr Pro Leu Gly Met Tyr Thr Gly Ala Gly Thr Ile Phe
290 295 300
Gly Ala Thr Gly Gly Val Met Glu Ala Ala Leu Arg Thr Gly Tyr Glu
305 310 315 320
Leu Tyr Thr Lys Lys Thr Ile Pro Ser Ile Asp Leu Thr Met Val Arg
325 330 335
Gly Gly Glu Gly Phe Arg Thr Ala Glu Val Asp Leu Gly Asp Ile Arg
340 345 350
Leu Lys Val Gly Val Val Ser Gly Leu Lys Asn Val Lys Asp Val Met
355 360 365
Glu Ser Val Lys Ala Gly Lys Cys Asp Leu His Phe Ile Glu Val Met
370 375 380
Thr Cys Pro Gln Gly Cys Ile Ser Gly Gly Gly Gln Pro Lys Val Ile
385 390 395 400
Leu Asp Ser Asp Lys Glu Glu Ala Tyr Asn Asn Arg Lys Lys Gly Leu
405 410 415
Tyr Asn His Asp Ala Asn Leu Thr Tyr Arg Lys Ser His Glu Asn Pro
420 425 430
Glu Ile Lys Lys Ile Tyr Asp Glu Phe Leu Asp Lys Pro Leu Gly Ala
435 440 445
Lys Ser His Glu Leu Leu His Thr Lys Tyr Ile Ser Arg Lys Lys Glu
450 455 460
Ser
465
<210> 27
<211> 1737
<212> DNA
<213> C. ragsdalei
<400> 27
atggtaaatt taactataaa cgatataaag gtttctgtcc cagaaggcac tacaatttta 60
aacgctgcaa aaaaagtaaa cataaatata cctactctct gctatcttga tcttcacgat 120
ataaaaatgg taaataggac ttcctcctgc agagtctgcc ttgttgaaat tgaaggcagg 180
cgaaatcttg caccttcatg ttctacagaa gctttcgaag gtatgatagt tagaacaaat 240
agtgccagag ctataaaagc aaggcgtact atggtagaac ttttattatc agatcatcct 300
accgactgcc ttgtatgtga aaagaatact caatgtcaac ttcaattaat cgctgctgaa 360
ttaggtataa gaaaaataag atataaaggt gctatgtcta attacaaaaa ggattcatca 420
agtggtgcta tatatagaaa tcttgataaa tgtataatgt gcagacgatg tgaaaccatg 480
tgcaatgaag ttcaaacctg tcaggtttac tctgcagtag atagaggctt cgaaactgta 540
gtatcccctg catttggtcg tcccatggtt gacacgcaat gcacattttg cggtcaatgt 600
gtatccgtat gcccaactgc tgcattaact caagttagta atgtagctaa ggtatgggaa 660
gtactaactg atcctgataa atatgtagta gttcaaactg cccctgctat aagagttact 720
ttaggtgaaa aattcggtat ggaacctgga actattgtaa ctggcaaaat ggtatctgct 780
cttagaagat tgggctttga taaggtatgt gataccgatt ttgcagcaga tgtaactatt 840
ttagaagaag ctcatgaatt tatagataga cttcaaaacg gcggaagact tccaatactc 900
acaagctgct gtcccagctg ggttaaattt atagaacatc aatttcctga tcttttagat 960
ataccttcaa cttgtaagtc tccacacata atgtttggta ctttagctaa aacatatatg 1020
gcagaaaaat taaatattga tccatctaaa attgtaatag tttcagttat gccatgtatt 1080
gcaaaaaaat atgaagtaag cagaaaagaa cttcaatatg aaggtcataa aaatgttgat 1140
cttgtagtta ccacaagaga gcttgcagat atgataatgg aagcaggaat agattttaat 1200
aaacttcctg atgaagactt tgataaacct tttggagaat ccacaggtgc ttctgtaata 1260
tttggaacta ccggcggtgt aattgaagca gctcttagaa ctgcttatga atggattact 1320
ggagagactt taaaagaagt agaatttcat ggtgtaagag gacttgatgg acttaaagaa 1380
gccagtataa atattggtgg taaagaaata aacattggcg tagctcacgg tcttggcaac 1440
gcaagaaaac ttcttgagga aatagaatct ggtgaatcaa aatatcacgc tatagaaata 1500
atggcatgtc ctggaggatg tattgacgga ggaggtcagc cgtatcattt tggagattta 1560
gatattgtaa agaaaagaat ggacgcttta tatagagaag atagaaacaa acctctcaga 1620
aaatctcatg agaatcctga agttcaagct ctatataaag aatttattgg agatgtaggc 1680
ggaaaaaaag ctcatgatct ccttcacact cattatataa aaaggcaaaa gttataa 1737
<210> 28
<211> 578
<212> PRT
<213> C. ragsdalei
<400> 28
Met Val Asn Leu Thr Ile Asn Asp Ile Lys Val Ser Val Pro Glu Gly
1 5 10 15
Thr Thr Ile Leu Asn Ala Ala Lys Lys Val Asn Ile Asn Ile Pro Thr
20 25 30
Leu Cys Tyr Leu Asp Leu His Asp Ile Lys Met Val Asn Arg Thr Ser
35 40 45
Ser Cys Arg Val Cys Leu Val Glu Ile Glu Gly Arg Arg Asn Leu Ala
50 55 60
Pro Ser Cys Ser Thr Glu Ala Phe Glu Gly Met Ile Val Arg Thr Asn
65 70 75 80
Ser Ala Arg Ala Ile Lys Ala Arg Arg Thr Met Val Glu Leu Leu Leu
85 90 95
Ser Asp His Pro Thr Asp Cys Leu Val Cys Glu Lys Asn Thr Gln Cys
100 105 110
Gln Leu Gln Leu Ile Ala Ala Glu Leu Gly Ile Arg Lys Ile Arg Tyr
115 120 125
Lys Gly Ala Met Ser Asn Tyr Lys Lys Asp Ser Ser Ser Gly Ala Ile
130 135 140
Tyr Arg Asn Leu Asp Lys Cys Ile Met Cys Arg Arg Cys Glu Thr Met
145 150 155 160
Cys Asn Glu Val Gln Thr Cys Gln Val Tyr Ser Ala Val Asp Arg Gly
165 170 175
Phe Glu Thr Val Val Ser Pro Ala Phe Gly Arg Pro Met Val Asp Thr
180 185 190
Gln Cys Thr Phe Cys Gly Gln Cys Val Ser Val Cys Pro Thr Ala Ala
195 200 205
Leu Thr Gln Val Ser Asn Val Ala Lys Val Trp Glu Val Leu Thr Asp
210 215 220
Pro Asp Lys Tyr Val Val Val Gln Thr Ala Pro Ala Ile Arg Val Thr
225 230 235 240
Leu Gly Glu Lys Phe Gly Met Glu Pro Gly Thr Ile Val Thr Gly Lys
245 250 255
Met Val Ser Ala Leu Arg Arg Leu Gly Phe Asp Lys Val Cys Asp Thr
260 265 270
Asp Phe Ala Ala Asp Val Thr Ile Leu Glu Glu Ala His Glu Phe Ile
275 280 285
Asp Arg Leu Gln Asn Gly Gly Arg Leu Pro Ile Leu Thr Ser Cys Cys
290 295 300
Pro Ser Trp Val Lys Phe Ile Glu His Gln Phe Pro Asp Leu Leu Asp
305 310 315 320
Ile Pro Ser Thr Cys Lys Ser Pro His Ile Met Phe Gly Thr Leu Ala
325 330 335
Lys Thr Tyr Met Ala Glu Lys Leu Asn Ile Asp Pro Ser Lys Ile Val
340 345 350
Ile Val Ser Val Met Pro Cys Ile Ala Lys Lys Tyr Glu Val Ser Arg
355 360 365
Lys Glu Leu Gln Tyr Glu Gly His Lys Asn Val Asp Leu Val Val Thr
370 375 380
Thr Arg Glu Leu Ala Asp Met Ile Met Glu Ala Gly Ile Asp Phe Asn
385 390 395 400
Lys Leu Pro Asp Glu Asp Phe Asp Lys Pro Phe Gly Glu Ser Thr Gly
405 410 415
Ala Ser Val Ile Phe Gly Thr Thr Gly Gly Val Ile Glu Ala Ala Leu
420 425 430
Arg Thr Ala Tyr Glu Trp Ile Thr Gly Glu Thr Leu Lys Glu Val Glu
435 440 445
Phe His Gly Val Arg Gly Leu Asp Gly Leu Lys Glu Ala Ser Ile Asn
450 455 460
Ile Gly Gly Lys Glu Ile Asn Ile Gly Val Ala His Gly Leu Gly Asn
465 470 475 480
Ala Arg Lys Leu Leu Glu Glu Ile Glu Ser Gly Glu Ser Lys Tyr His
485 490 495
Ala Ile Glu Ile Met Ala Cys Pro Gly Gly Cys Ile Asp Gly Gly Gly
500 505 510
Gln Pro Tyr His Phe Gly Asp Leu Asp Ile Val Lys Lys Arg Met Asp
515 520 525
Ala Leu Tyr Arg Glu Asp Arg Asn Lys Pro Leu Arg Lys Ser His Glu
530 535 540
Asn Pro Glu Val Gln Ala Leu Tyr Lys Glu Phe Ile Gly Asp Val Gly
545 550 555 560
Gly Lys Lys Ala His Asp Leu Leu His Thr His Tyr Ile Lys Arg Gln
565 570 575
Lys Leu
<210> 29
<211> 1101
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 29
atggaatgtg tggatgaaaa aggtgagata ctatgtcaaa aaacatgtcc ttttgatgca 60
atttttatag acaataagaa aaattgtgct tacatagata aagaaaagtg taccgattgt 120
ggtttgtgtg tagatgtttg ccctactggg ggaataatgg ataaagttca gttcattcct 180
attttggata ttttaaaaag taaatctcca gttgtggcta tagtggctcc tgccataata 240
ggacagtttg gggaagatgt tactatggat caacttagga ccgcttttaa aaaactggga 300
tttactgata tgattgaagt ggcatttttt gcagatatgc ttactttaaa ggaatctatt 360
gaatttgaca atcatgtaaa agatgaaaaa gattttatga taacttcctg ctgttgtcct 420
atgtgggtgg ctatggtaaa aaaggtatac agtaacttgg ttaaacatgt atctccctct 480
gtatctccga tggttgcagg aggaagagta cttaaaaagt taagtcctta ctgcaaggta 540
gtgtttatag gcccatgtat tgctaaaaaa tctgaggcaa aggaagaaga tataaaagga 600
gcaatagatt ttgtacttac ttttgaagaa ttaagagata tatttgatgc ttttcatata 660
gttccatcta aacttgaagg agatttttca tctaaatatg cttctagagg tggaagatta 720
tatgcccgta caggtggagt ttctattgca gtaagcgaag ctgtggaaaa gatttttcct 780
gaaaagcata aactatttag tgcaattcag gcaaatggca ttagagaatg taaagaaatg 840
cttaccaagg tgcaaaatgg agaaataaaa gctaatttta ttgaaggaat gggctgtatt 900
ggtggatgtg taggtggtcc caaagcaatt gcatctaagg atgaaggtag ggatcgagta 960
aataaatttg cacaagattc tgaaataaaa gttgctgtag atagtgaatg tatgcatgga 1020
gtattacatg ctttggatat acattctata gatgatttta aggatgagaa aaaaatagaa 1080
ctgttagaac gagaatttta a 1101
<210> 30
<211> 366
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 30
Met Glu Cys Val Asp Glu Lys Gly Glu Ile Leu Cys Gln Lys Thr Cys
1 5 10 15
Pro Phe Asp Ala Ile Phe Ile Asp Asn Lys Lys Asn Cys Ala Tyr Ile
20 25 30
Asp Lys Glu Lys Cys Thr Asp Cys Gly Leu Cys Val Asp Val Cys Pro
35 40 45
Thr Gly Gly Ile Met Asp Lys Val Gln Phe Ile Pro Ile Leu Asp Ile
50 55 60
Leu Lys Ser Lys Ser Pro Val Val Ala Ile Val Ala Pro Ala Ile Ile
65 70 75 80
Gly Gln Phe Gly Glu Asp Val Thr Met Asp Gln Leu Arg Thr Ala Phe
85 90 95
Lys Lys Leu Gly Phe Thr Asp Met Ile Glu Val Ala Phe Phe Ala Asp
100 105 110
Met Leu Thr Leu Lys Glu Ser Ile Glu Phe Asp Asn His Val Lys Asp
115 120 125
Glu Lys Asp Phe Met Ile Thr Ser Cys Cys Cys Pro Met Trp Val Ala
130 135 140
Met Val Lys Lys Val Tyr Ser Asn Leu Val Lys His Val Ser Pro Ser
145 150 155 160
Val Ser Pro Met Val Ala Gly Gly Arg Val Leu Lys Lys Leu Ser Pro
165 170 175
Tyr Cys Lys Val Val Phe Ile Gly Pro Cys Ile Ala Lys Lys Ser Glu
180 185 190
Ala Lys Glu Glu Asp Ile Lys Gly Ala Ile Asp Phe Val Leu Thr Phe
195 200 205
Glu Glu Leu Arg Asp Ile Phe Asp Ala Phe His Ile Val Pro Ser Lys
210 215 220
Leu Glu Gly Asp Phe Ser Ser Lys Tyr Ala Ser Arg Gly Gly Arg Leu
225 230 235 240
Tyr Ala Arg Thr Gly Gly Val Ser Ile Ala Val Ser Glu Ala Val Glu
245 250 255
Lys Ile Phe Pro Glu Lys His Lys Leu Phe Ser Ala Ile Gln Ala Asn
260 265 270
Gly Ile Arg Glu Cys Lys Glu Met Leu Thr Lys Val Gln Asn Gly Glu
275 280 285
Ile Lys Ala Asn Phe Ile Glu Gly Met Gly Cys Ile Gly Gly Cys Val
290 295 300
Gly Gly Pro Lys Ala Ile Ala Ser Lys Asp Glu Gly Arg Asp Arg Val
305 310 315 320
Asn Lys Phe Ala Gln Asp Ser Glu Ile Lys Val Ala Val Asp Ser Glu
325 330 335
Cys Met His Gly Val Leu His Ala Leu Asp Ile His Ser Ile Asp Asp
340 345 350
Phe Lys Asp Glu Lys Lys Ile Glu Leu Leu Glu Arg Glu Phe
355 360 365
<210> 31
<211> 1374
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 31
ttgacagtta agactgaagg tattgtcaaa attgataaag agttgtgtac aggatgtagg 60
cgatgtgcag atgtttgtcc tgtagatgct atagaaggtg aaaaaggaca acctcaaaaa 120
attaatactg aacgttgtgt tttgtgtggt cagtgtgtac aaatttgcag tgcttatgca 180
tctgcatttg atgaagatat tactcctcgt gagcaaaaga taaaagagcg taatatgctc 240
ccctctgtta aagagccctt atttgcatcc tactatacag gccatgccat agaagtaaaa 300
gaggccttag caaattctaa actttttact atggttcaat gtgcaccagc agtacgtgta 360
gctattgctg aagaatttgg tatgccactt ggaagtttaa caccagggaa aatggcagct 420
gcactaagag agctaggttt tgatcgaatt tatgatacta attttgctgc tgatctcact 480
attatggagg aaggtaatga acttattaaa agggttactg aaggtggagt gctgcccatg 540
ttcacttcat gttgtcctgc ttgggtaaaa tttattgagc aggattatcc ggagcttatt 600
cctcatctgt cttcttgtaa atctccacaa caaatggaag gtgctctgct taaaacatat 660
ggtgcacagg ttgatggtgt agatgctggc aagatttata gtgtttcagt tatgccttgt 720
atttgcaaaa aatttgaatg tgaacgtcct gaaatgaaag acagtggata tcaggatgta 780
gattctgtaa ttaccacacg ggaacttgga caattaatca aagatgctgg cattgatttt 840
aatgctttac ctgaaaatga atttgacaag ccacttggga cttattctgg ggcaggtact 900
attttttgcg ctactggagg tgttatggaa gctgccctgc gtacggcata taaattgatt 960
actaaagaag agattccaga tgtcaatctc aaatttataa gaggcggcga aggagtaaga 1020
agttcagaag ttaaagtagg agatctgaca ttaaaagtag cagtagttgc tggtctgaaa 1080
aatgttgtac cagttttgga agcaattaaa gctggaaaag cagatttcca tttcattgaa 1140
gtgatgacct gtccagttgg atgtgttagt ggagggggac aacctaagat attgatacct 1200
gatgaaaaaa ctgatgctta tactaatcgt acgtgcagta cgtatgtaca tgatgaaaat 1260
atggaatata gaaaatcaca tgataatcct gagatacaga aaatttataa agaattcttg 1320
gtagaagata atattcatca tttgcttcat actacgtata caccaaggag gtaa 1374
<210> 32
<211> 457
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 32
Met Thr Val Lys Thr Glu Gly Ile Val Lys Ile Asp Lys Glu Leu Cys
1 5 10 15
Thr Gly Cys Arg Arg Cys Ala Asp Val Cys Pro Val Asp Ala Ile Glu
20 25 30
Gly Glu Lys Gly Gln Pro Gln Lys Ile Asn Thr Glu Arg Cys Val Leu
35 40 45
Cys Gly Gln Cys Val Gln Ile Cys Ser Ala Tyr Ala Ser Ala Phe Asp
50 55 60
Glu Asp Ile Thr Pro Arg Glu Gln Lys Ile Lys Glu Arg Asn Met Leu
65 70 75 80
Pro Ser Val Lys Glu Pro Leu Phe Ala Ser Tyr Tyr Thr Gly His Ala
85 90 95
Ile Glu Val Lys Glu Ala Leu Ala Asn Ser Lys Leu Phe Thr Met Val
100 105 110
Gln Cys Ala Pro Ala Val Arg Val Ala Ile Ala Glu Glu Phe Gly Met
115 120 125
Pro Leu Gly Ser Leu Thr Pro Gly Lys Met Ala Ala Ala Leu Arg Glu
130 135 140
Leu Gly Phe Asp Arg Ile Tyr Asp Thr Asn Phe Ala Ala Asp Leu Thr
145 150 155 160
Ile Met Glu Glu Gly Asn Glu Leu Ile Lys Arg Val Thr Glu Gly Gly
165 170 175
Val Leu Pro Met Phe Thr Ser Cys Cys Pro Ala Trp Val Lys Phe Ile
180 185 190
Glu Gln Asp Tyr Pro Glu Leu Ile Pro His Leu Ser Ser Cys Lys Ser
195 200 205
Pro Gln Gln Met Glu Gly Ala Leu Leu Lys Thr Tyr Gly Ala Gln Val
210 215 220
Asp Gly Val Asp Ala Gly Lys Ile Tyr Ser Val Ser Val Met Pro Cys
225 230 235 240
Ile Cys Lys Lys Phe Glu Cys Glu Arg Pro Glu Met Lys Asp Ser Gly
245 250 255
Tyr Gln Asp Val Asp Ser Val Ile Thr Thr Arg Glu Leu Gly Gln Leu
260 265 270
Ile Lys Asp Ala Gly Ile Asp Phe Asn Ala Leu Pro Glu Asn Glu Phe
275 280 285
Asp Lys Pro Leu Gly Thr Tyr Ser Gly Ala Gly Thr Ile Phe Cys Ala
290 295 300
Thr Gly Gly Val Met Glu Ala Ala Leu Arg Thr Ala Tyr Lys Leu Ile
305 310 315 320
Thr Lys Glu Glu Ile Pro Asp Val Asn Leu Lys Phe Ile Arg Gly Gly
325 330 335
Glu Gly Val Arg Ser Ser Glu Val Lys Val Gly Asp Leu Thr Leu Lys
340 345 350
Val Ala Val Val Ala Gly Leu Lys Asn Val Val Pro Val Leu Glu Ala
355 360 365
Ile Lys Ala Gly Lys Ala Asp Phe His Phe Ile Glu Val Met Thr Cys
370 375 380
Pro Val Gly Cys Val Ser Gly Gly Gly Gln Pro Lys Ile Leu Ile Pro
385 390 395 400
Asp Glu Lys Thr Asp Ala Tyr Thr Asn Arg Thr Cys Ser Thr Tyr Val
405 410 415
His Asp Glu Asn Met Glu Tyr Arg Lys Ser His Asp Asn Pro Glu Ile
420 425 430
Gln Lys Ile Tyr Lys Glu Phe Leu Val Glu Asp Asn Ile His His Leu
435 440 445
Leu His Thr Thr Tyr Thr Pro Arg Arg
450 455
<210> 33
<211> 1200
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 33
gtgatagacg tacatgaagc attcaatata gtaatgaata acacaaaact gcttaaaggt 60
gaagatatat cgttgataaa ttctcttaac agggtattgg cagaggatat aagctcaaaa 120
gataatcttc ccccatttga caaatcctgt atggatgggt atgctttaaa aagtgaagat 180
actaaggaaa aaatgtcaaa atttcgaatt aagggaagca taaaggcggg agatttttct 240
gatatagtat tgaaaaatgg tgaagccata aaaataatga caggagctcc agtaccaaaa 300
ggtgcagatg cagttattca aatagaaaaa gtaaaagtag aaggaaaaga acttcatgta 360
ttagaaaata tatctcctgg aaccaacata tttaaaactg gtgaggagat aaaaattggt 420
gatgttgctt taagaaaagg aaagattttg agacctgcag aaatagggtt attggcatca 480
ctaggttata ctaaaataaa atgttataaa gcccctaaaa ttataataat aaatactggg 540
gatgaactta taaatataga tcaaaactta atgcaaggta aaataaggaa ttgtaatgaa 600
tatacattaa ttgcccttat taaaaattta aatgcagaag ttaaatcata tgggataata 660
agagatgata aggataagat ttttaatgct ataaaaactg catttgaaga gggagatata 720
atcataacta ctggaggagc atctgtaggt gattacgatt ttatagaaga tgttcttcag 780
aaaataggaa cagatataaa gtttacttcg gtagctatta aaccaggaaa accagttgtt 840
tttgcaactt ttaaagataa attgttcttt ggacttccag gaaatccgct ttcggtaata 900
aattcatttg aaagttttgt agcaccatct attaaaaaaa tgattggaag agatgatgcg 960
tttcctgaag aatttcctgt aactttaaaa gatgatttta aatcaggaaa agaaagagac 1020
tgctatatgt atgtaaacat aaaaaaggaa gataaccgtt attatgccta tgatgtagga 1080
agacaagatt ccaatgggct ttttacgctt actaagtcaa atggagtcgt catcatggaa 1140
aagggaacta gtatagcaaa agctggagat attttaaatg gaaaatttat attcaaataa 1200
<210> 34
<211> 399
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 34
Val Ile Asp Val His Glu Ala Phe Asn Ile Val Met Asn Asn Thr Lys
1 5 10 15
Leu Leu Lys Gly Glu Asp Ile Ser Leu Ile Asn Ser Leu Asn Arg Val
20 25 30
Leu Ala Glu Asp Ile Ser Ser Lys Asp Asn Leu Pro Pro Phe Asp Lys
35 40 45
Ser Cys Met Asp Gly Tyr Ala Leu Lys Ser Glu Asp Thr Lys Glu Lys
50 55 60
Met Ser Lys Phe Arg Ile Lys Gly Ser Ile Lys Ala Gly Asp Phe Ser
65 70 75 80
Asp Ile Val Leu Lys Asn Gly Glu Ala Ile Lys Ile Met Thr Gly Ala
85 90 95
Pro Val Pro Lys Gly Ala Asp Ala Val Ile Gln Ile Glu Lys Val Lys
100 105 110
Val Glu Gly Lys Glu Leu His Val Leu Glu Asn Ile Ser Pro Gly Thr
115 120 125
Asn Ile Phe Lys Thr Gly Glu Glu Ile Lys Ile Gly Asp Val Ala Leu
130 135 140
Arg Lys Gly Lys Ile Leu Arg Pro Ala Glu Ile Gly Leu Leu Ala Ser
145 150 155 160
Leu Gly Tyr Thr Lys Ile Lys Cys Tyr Lys Ala Pro Lys Ile Ile Ile
165 170 175
Ile Asn Thr Gly Asp Glu Leu Ile Asn Ile Asp Gln Asn Leu Met Gln
180 185 190
Gly Lys Ile Arg Asn Cys Asn Glu Tyr Thr Leu Ile Ala Leu Ile Lys
195 200 205
Asn Leu Asn Ala Glu Val Lys Ser Tyr Gly Ile Ile Arg Asp Asp Lys
210 215 220
Asp Lys Ile Phe Asn Ala Ile Lys Thr Ala Phe Glu Glu Gly Asp Ile
225 230 235 240
Ile Ile Thr Thr Gly Gly Ala Ser Val Gly Asp Tyr Asp Phe Ile Glu
245 250 255
Asp Val Leu Gln Lys Ile Gly Thr Asp Ile Lys Phe Thr Ser Val Ala
260 265 270
Ile Lys Pro Gly Lys Pro Val Val Phe Ala Thr Phe Lys Asp Lys Leu
275 280 285
Phe Phe Gly Leu Pro Gly Asn Pro Leu Ser Val Ile Asn Ser Phe Glu
290 295 300
Ser Phe Val Ala Pro Ser Ile Lys Lys Met Ile Gly Arg Asp Asp Ala
305 310 315 320
Phe Pro Glu Glu Phe Pro Val Thr Leu Lys Asp Asp Phe Lys Ser Gly
325 330 335
Lys Glu Arg Asp Cys Tyr Met Tyr Val Asn Ile Lys Lys Glu Asp Asn
340 345 350
Arg Tyr Tyr Ala Tyr Asp Val Gly Arg Gln Asp Ser Asn Gly Leu Phe
355 360 365
Thr Leu Thr Lys Ser Asn Gly Val Val Ile Met Glu Lys Gly Thr Ser
370 375 380
Ile Ala Lys Ala Gly Asp Ile Leu Asn Gly Lys Phe Ile Phe Lys
385 390 395
<210> 35
<211> 570
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 35
atggaaaatt tatattcaaa taagaggtca gtaatttcta ttatttcatc tagttcaaat 60
tcgggtaaga ctaccttgat agaaggaata ataagaattc taaaaagcag aggatataag 120
gttggtgcaa taaagaatga tgctcataag ttacagatag attacccagg aaaagacagc 180
tttagattta cagaggcagg tgcggacaat gttgttattg catcggataa tacagtggct 240
atgataaaaa aagtaagtgg acccaaaagt atagaagaac tactgttgct ttttcaagat 300
gtagatattg taatagtgga aggcttcaag ggtaacgaat ttcctaaaat agaagtatac 360
aggaaaaatg caagcaaatg tttactttac aaaaattcta aatataattt tcaaaatttt 420
gtagctattg taaccaatga aaacttaata actgatattc ctgtatttga tataaatgat 480
acaaaaaaaa tagctgattt tattgagaac gactttatag gaggtaacaa aaatggagaa 540
aaccatggaa cttcatgtag tcaaatatga 570
<210> 36
<211> 189
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 36
Met Glu Asn Leu Tyr Ser Asn Lys Arg Ser Val Ile Ser Ile Ile Ser
1 5 10 15
Ser Ser Ser Asn Ser Gly Lys Thr Thr Leu Ile Glu Gly Ile Ile Arg
20 25 30
Ile Leu Lys Ser Arg Gly Tyr Lys Val Gly Ala Ile Lys Asn Asp Ala
35 40 45
His Lys Leu Gln Ile Asp Tyr Pro Gly Lys Asp Ser Phe Arg Phe Thr
50 55 60
Glu Ala Gly Ala Asp Asn Val Val Ile Ala Ser Asp Asn Thr Val Ala
65 70 75 80
Met Ile Lys Lys Val Ser Gly Pro Lys Ser Ile Glu Glu Leu Leu Leu
85 90 95
Leu Phe Gln Asp Val Asp Ile Val Ile Val Glu Gly Phe Lys Gly Asn
100 105 110
Glu Phe Pro Lys Ile Glu Val Tyr Arg Lys Asn Ala Ser Lys Cys Leu
115 120 125
Leu Tyr Lys Asn Ser Lys Tyr Asn Phe Gln Asn Phe Val Ala Ile Val
130 135 140
Thr Asn Glu Asn Leu Ile Thr Asp Ile Pro Val Phe Asp Ile Asn Asp
145 150 155 160
Thr Lys Lys Ile Ala Asp Phe Ile Glu Asn Asp Phe Ile Gly Gly Asn
165 170 175
Lys Asn Gly Glu Asn His Gly Thr Ser Cys Ser Gln Ile
180 185
<210> 37
<211> 777
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 37
atggaacttc atgtagtcaa atatgacaaa gaatgtaaaa agacagtaga ggaatcaact 60
atttgtgaat atcctttaaa tgtatttgta aatggtgaac atttgacagt actcttatgt 120
acgcctgaaa agcttaagga attaacaata ggtttcttga cctttagagg tgttataaaa 180
tctctagatg aaataaaatc tctagagata gatgaaaaaa gtggagcgtc cagggtaact 240
ttgaaaaata gccaatttaa taaagagttg tattcaaagc aagtgcttcc tataacattt 300
agtgaaaatg agaaaagtaa gttcttttcg tatattattg attccatgga aattagtata 360
atcaacaatg ataatgttta cattcatgtc gataaaatct atgatctaat gatggacaat 420
cttggatatt ccaagacgtt taaactcact ggaggaacac attgtgcagc tctttgtgat 480
gaagataaag taatatctat ttgtgaggat gtggctagac acaatgctgt agacaagctt 540
ataggtgagg catttataaa aaatatttat ttaaaggata aaataatatt tgtgagcagc 600
agagtatctt ttgaaatggt atataaaatt gctaggctag gggtacctat aataatatct 660
aaatctgcac ctacaagtct ttctatagaa tttgcaaaag ctttaaatgt tacattaatt 720
ggatttgtaa ggggagaaag aatgaatgta tatacaaatc cacagagaat aatatag 777
<210> 38
<211> 258
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 38
Met Glu Leu His Val Val Lys Tyr Asp Lys Glu Cys Lys Lys Thr Val
1 5 10 15
Glu Glu Ser Thr Ile Cys Glu Tyr Pro Leu Asn Val Phe Val Asn Gly
20 25 30
Glu His Leu Thr Val Leu Leu Cys Thr Pro Glu Lys Leu Lys Glu Leu
35 40 45
Thr Ile Gly Phe Leu Thr Phe Arg Gly Val Ile Lys Ser Leu Asp Glu
50 55 60
Ile Lys Ser Leu Glu Ile Asp Glu Lys Ser Gly Ala Ser Arg Val Thr
65 70 75 80
Leu Lys Asn Ser Gln Phe Asn Lys Glu Leu Tyr Ser Lys Gln Val Leu
85 90 95
Pro Ile Thr Phe Ser Glu Asn Glu Lys Ser Lys Phe Phe Ser Tyr Ile
100 105 110
Ile Asp Ser Met Glu Ile Ser Ile Ile Asn Asn Asp Asn Val Tyr Ile
115 120 125
His Val Asp Lys Ile Tyr Asp Leu Met Met Asp Asn Leu Gly Tyr Ser
130 135 140
Lys Thr Phe Lys Leu Thr Gly Gly Thr His Cys Ala Ala Leu Cys Asp
145 150 155 160
Glu Asp Lys Val Ile Ser Ile Cys Glu Asp Val Ala Arg His Asn Ala
165 170 175
Val Asp Lys Leu Ile Gly Glu Ala Phe Ile Lys Asn Ile Tyr Leu Lys
180 185 190
Asp Lys Ile Ile Phe Val Ser Ser Arg Val Ser Phe Glu Met Val Tyr
195 200 205
Lys Ile Ala Arg Leu Gly Val Pro Ile Ile Ile Ser Lys Ser Ala Pro
210 215 220
Thr Ser Leu Ser Ile Glu Phe Ala Lys Ala Leu Asn Val Thr Leu Ile
225 230 235 240
Gly Phe Val Arg Gly Glu Arg Met Asn Val Tyr Thr Asn Pro Gln Arg
245 250 255
Ile Ile
<210> 39
<211> 486
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 39
atgttaacta aacagcaaaa tgaagacctg tctggacaag atgtaattga aaaatatcct 60
aaagagcaga gatttactct tgctatacta caggatatac agagaaagta caaatatata 120
ccaagagaag cactggagaa tttagctaag tatttggaca cgcctgtaag tagactgtat 180
ggtatggcta ctttttataa ggcattgagc cttactccaa aaggggaaaa cataataact 240
gtatgtgatg gaaccgcttg ccatgttgct ggttctatgg ttgtaatgga tgaacttgaa 300
aaggcaatag gaattaaacc aggtgaaact acagaggatc tcaaattttc aataaataca 360
gttaactgta taggatgctg tgcaatagct cctgtcatga tgataaatga caaatattat 420
ggaaatttaa cacctaaact ggttgaagaa attcttagtg agtataggag tgagagtgat 480
gagtga 486
<210> 40
<211> 161
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 40
Met Leu Thr Lys Gln Gln Asn Glu Asp Leu Ser Gly Gln Asp Val Ile
1 5 10 15
Glu Lys Tyr Pro Lys Glu Gln Arg Phe Thr Leu Ala Ile Leu Gln Asp
20 25 30
Ile Gln Arg Lys Tyr Lys Tyr Ile Pro Arg Glu Ala Leu Glu Asn Leu
35 40 45
Ala Lys Tyr Leu Asp Thr Pro Val Ser Arg Leu Tyr Gly Met Ala Thr
50 55 60
Phe Tyr Lys Ala Leu Ser Leu Thr Pro Lys Gly Glu Asn Ile Ile Thr
65 70 75 80
Val Cys Asp Gly Thr Ala Cys His Val Ala Gly Ser Met Val Val Met
85 90 95
Asp Glu Leu Glu Lys Ala Ile Gly Ile Lys Pro Gly Glu Thr Thr Glu
100 105 110
Asp Leu Lys Phe Ser Ile Asn Thr Val Asn Cys Ile Gly Cys Cys Ala
115 120 125
Ile Ala Pro Val Met Met Ile Asn Asp Lys Tyr Tyr Gly Asn Leu Thr
130 135 140
Pro Lys Leu Val Glu Glu Ile Leu Ser Glu Tyr Arg Ser Glu Ser Asp
145 150 155 160
Glu
<210> 41
<211> 1800
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 41
atgagtgata aaaaaactgt caatatatgt tgtgggacag gttgtttagc taaaggcagt 60
atggaagtat atgaagaaat gaaggcacaa atagctaaat taggggcaaa tgcagaagta 120
aatgttaaat taaaagcaac aggttgcgat ggattgtgtg agaaaggtcc tgtactgaaa 180
atatatccag atgacattgc atattttaaa gttaaagtag aagatgtaga agacgtagta 240
aaaaagacat tgatgaatgg ggaaataatt gaaaaattat tatattttga gactgctaca 300
aaacagagat taagaaatca taaagaaagt gaattttgta aaagacaata caaaattgct 360
ctcagaaatg ttggtgaaat agatccaata agtttggaag attatgttga aagaggcgga 420
tacaaagctc ttaaaaaagc aataagcagc atgaaacctg aagatgtgct tgaagaaata 480
acaaaatcag gtcttagagg aagaggtgga gcaggattcc caacaggacg taaatggaaa 540
actgctgcag atattgatac atcacctata tatgtagtat gtaatggtga tgaaggagat 600
cctggagcat ttatggatag aagtataatg gagggagatc ctaacagtgt tatagaaggt 660
atgacattat gtgcttatgc agtaggaggt acaaatggat ttgcttatat aagagatgaa 720
tatggacttg ctgtagaaaa tatgcagaaa gctattaata aagcaaaaga tgaaaattta 780
ttaggtaata atatattagg aactgatttt tccttcgata tacagatagt aagaggtgga 840
ggagcttttg tatgtggtga atctactgca cttatgtcgt ctatagaagg tatggtaggt 900
gaacctagag ctaaatatat acacactaca gaaaaaggat tgtggggaca acctacagtt 960
ttaaataatg tagaaacttg ggccaatgta cctataataa ttgaaaaagg cggagattgg 1020
tatcatgcta taggaactat ggagaagagt aagggaacaa aggtattctc attagttgga 1080
aaagttaaga atactggact tgtagaagta cctatgggaa ctactcttag agaaataata 1140
tatgatattg gcggtggagt attaaatgat agaaagttta aggcagttca aataggtgga 1200
ccttcaggtg gatgtttacc agctgaatat ttagatttgc cagtagatta tgatactttg 1260
gttaaagcag attccatgat gggttcaggc ggaatgatcg taatggatga tagaacctgt 1320
atggtagatg taactagata ttacctgagc ttcttggctg aagaatcttg tggaaagtgt 1380
gtaccttgta gagaaggcgt aaagaggatg cttgaaatac tcactgacat atgcaatggt 1440
gatggaaaag aaggagacat agaagagctt ctcgaaatat gttccatgac aagcaaggca 1500
tctctgtgca gtcttggtaa gagtgctcca aatccagtaa ttgcttctat aagatatttt 1560
agagatgaat ttgaagagca tataaagaat aagagatgta gagcaggagt ttgtaagaaa 1620
cttactacat ttggtataga cgaggataaa tgtaagggat gcgatatgtg taaaaagaat 1680
tgtccagctg attgtataac aggggaaatt aagaaaccac atacaataga tgctgataag 1740
tgcttgagat gcggtaactg catgaacatc tgtaagtttg atgctgttaa ggttctatag 1800
<210> 42
<211> 599
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 42
Met Ser Asp Lys Lys Thr Val Asn Ile Cys Cys Gly Thr Gly Cys Leu
1 5 10 15
Ala Lys Gly Ser Met Glu Val Tyr Glu Glu Met Lys Ala Gln Ile Ala
20 25 30
Lys Leu Gly Ala Asn Ala Glu Val Asn Val Lys Leu Lys Ala Thr Gly
35 40 45
Cys Asp Gly Leu Cys Glu Lys Gly Pro Val Leu Lys Ile Tyr Pro Asp
50 55 60
Asp Ile Ala Tyr Phe Lys Val Lys Val Glu Asp Val Glu Asp Val Val
65 70 75 80
Lys Lys Thr Leu Met Asn Gly Glu Ile Ile Glu Lys Leu Leu Tyr Phe
85 90 95
Glu Thr Ala Thr Lys Gln Arg Leu Arg Asn His Lys Glu Ser Glu Phe
100 105 110
Cys Lys Arg Gln Tyr Lys Ile Ala Leu Arg Asn Val Gly Glu Ile Asp
115 120 125
Pro Ile Ser Leu Glu Asp Tyr Val Glu Arg Gly Gly Tyr Lys Ala Leu
130 135 140
Lys Lys Ala Ile Ser Ser Met Lys Pro Glu Asp Val Leu Glu Glu Ile
145 150 155 160
Thr Lys Ser Gly Leu Arg Gly Arg Gly Gly Ala Gly Phe Pro Thr Gly
165 170 175
Arg Lys Trp Lys Thr Ala Ala Asp Ile Asp Thr Ser Pro Ile Tyr Val
180 185 190
Val Cys Asn Gly Asp Glu Gly Asp Pro Gly Ala Phe Met Asp Arg Ser
195 200 205
Ile Met Glu Gly Asp Pro Asn Ser Val Ile Glu Gly Met Thr Leu Cys
210 215 220
Ala Tyr Ala Val Gly Gly Thr Asn Gly Phe Ala Tyr Ile Arg Asp Glu
225 230 235 240
Tyr Gly Leu Ala Val Glu Asn Met Gln Lys Ala Ile Asn Lys Ala Lys
245 250 255
Asp Glu Asn Leu Leu Gly Asn Asn Ile Leu Gly Thr Asp Phe Ser Phe
260 265 270
Asp Ile Gln Ile Val Arg Gly Gly Gly Ala Phe Val Cys Gly Glu Ser
275 280 285
Thr Ala Leu Met Ser Ser Ile Glu Gly Met Val Gly Glu Pro Arg Ala
290 295 300
Lys Tyr Ile His Thr Thr Glu Lys Gly Leu Trp Gly Gln Pro Thr Val
305 310 315 320
Leu Asn Asn Val Glu Thr Trp Ala Asn Val Pro Ile Ile Ile Glu Lys
325 330 335
Gly Gly Asp Trp Tyr His Ala Ile Gly Thr Met Glu Lys Ser Lys Gly
340 345 350
Thr Lys Val Phe Ser Leu Val Gly Lys Val Lys Asn Thr Gly Leu Val
355 360 365
Glu Val Pro Met Gly Thr Thr Leu Arg Glu Ile Ile Tyr Asp Ile Gly
370 375 380
Gly Gly Val Leu Asn Asp Arg Lys Phe Lys Ala Val Gln Ile Gly Gly
385 390 395 400
Pro Ser Gly Gly Cys Leu Pro Ala Glu Tyr Leu Asp Leu Pro Val Asp
405 410 415
Tyr Asp Thr Leu Val Lys Ala Asp Ser Met Met Gly Ser Gly Gly Met
420 425 430
Ile Val Met Asp Asp Arg Thr Cys Met Val Asp Val Thr Arg Tyr Tyr
435 440 445
Leu Ser Phe Leu Ala Glu Glu Ser Cys Gly Lys Cys Val Pro Cys Arg
450 455 460
Glu Gly Val Lys Arg Met Leu Glu Ile Leu Thr Asp Ile Cys Asn Gly
465 470 475 480
Asp Gly Lys Glu Gly Asp Ile Glu Glu Leu Leu Glu Ile Cys Ser Met
485 490 495
Thr Ser Lys Ala Ser Leu Cys Ser Leu Gly Lys Ser Ala Pro Asn Pro
500 505 510
Val Ile Ala Ser Ile Arg Tyr Phe Arg Asp Glu Phe Glu Glu His Ile
515 520 525
Lys Asn Lys Arg Cys Arg Ala Gly Val Cys Lys Lys Leu Thr Thr Phe
530 535 540
Gly Ile Asp Glu Asp Lys Cys Lys Gly Cys Asp Met Cys Lys Lys Asn
545 550 555 560
Cys Pro Ala Asp Cys Ile Thr Gly Glu Ile Lys Lys Pro His Thr Ile
565 570 575
Asp Ala Asp Lys Cys Leu Arg Cys Gly Asn Cys Met Asn Ile Cys Lys
580 585 590
Phe Asp Ala Val Lys Val Leu
595
<210> 43
<211> 768
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 43
gtgaatgtag atatgaaaat tacaatagat ggaaaagctt gtgaagctga aaaaggagaa 60
ttcatattac aaatagcaag aagaaataac atatatatac ctacattatg tcacagcgat 120
gcattgcctg ggcttgctag ctgtagacta tgtatagtta aagtagtaga taggggacgt 180
gcaaagatag taacttcctg tatattccct gtaagtaagg aagtagaagt tataactaat 240
gacgatgaaa taaagagaat gagaaaaaac atagttatgc ttttaaaagt aagatgccct 300
gaaaataaag aggtaaatga attagctaaa gcctttggag tagaggaaaa gagagtaaag 360
aggttcaaat tggatccaga acaaaattgt gttttgtgcg gactttgtgc aaaagcttgc 420
aaggaattag gtactggagc aatttcaaca gttaataggg gtatgtataa agaagtagca 480
actccatatc acgaatcttc accggaatgt ataggatgtg cttcctgtgc aaatgtttgt 540
ccaactaatg caataaaagt tgtggataaa gatggagaaa gagaaatatg gggcaaaaaa 600
ttcaagatgg ttaagtgtga tttgtgcgga gaatattttg ctacagaaga acatgtaaaa 660
tatgcttaca ataggcttgg aaaagagcag ccagaaaaac ttatgtgtag cagctgcaag 720
aagaaagtta cagccaaaga tgtcaaaaat atttttgaga acgtgtga 768
<210> 44
<211> 255
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 44
Val Asn Val Asp Met Lys Ile Thr Ile Asp Gly Lys Ala Cys Glu Ala
1 5 10 15
Glu Lys Gly Glu Phe Ile Leu Gln Ile Ala Arg Arg Asn Asn Ile Tyr
20 25 30
Ile Pro Thr Leu Cys His Ser Asp Ala Leu Pro Gly Leu Ala Ser Cys
35 40 45
Arg Leu Cys Ile Val Lys Val Val Asp Arg Gly Arg Ala Lys Ile Val
50 55 60
Thr Ser Cys Ile Phe Pro Val Ser Lys Glu Val Glu Val Ile Thr Asn
65 70 75 80
Asp Asp Glu Ile Lys Arg Met Arg Lys Asn Ile Val Met Leu Leu Lys
85 90 95
Val Arg Cys Pro Glu Asn Lys Glu Val Asn Glu Leu Ala Lys Ala Phe
100 105 110
Gly Val Glu Glu Lys Arg Val Lys Arg Phe Lys Leu Asp Pro Glu Gln
115 120 125
Asn Cys Val Leu Cys Gly Leu Cys Ala Lys Ala Cys Lys Glu Leu Gly
130 135 140
Thr Gly Ala Ile Ser Thr Val Asn Arg Gly Met Tyr Lys Glu Val Ala
145 150 155 160
Thr Pro Tyr His Glu Ser Ser Pro Glu Cys Ile Gly Cys Ala Ser Cys
165 170 175
Ala Asn Val Cys Pro Thr Asn Ala Ile Lys Val Val Asp Lys Asp Gly
180 185 190
Glu Arg Glu Ile Trp Gly Lys Lys Phe Lys Met Val Lys Cys Asp Leu
195 200 205
Cys Gly Glu Tyr Phe Ala Thr Glu Glu His Val Lys Tyr Ala Tyr Asn
210 215 220
Arg Leu Gly Lys Glu Gln Pro Glu Lys Leu Met Cys Ser Ser Cys Lys
225 230 235 240
Lys Lys Val Thr Ala Lys Asp Val Lys Asn Ile Phe Glu Asn Val
245 250 255
<210> 45
<211> 555
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 45
atgaaaccag agtttaattc ttttgtaata gccgatcctg acaagtgcat aggctgtaga 60
tcttgtgaga ttgcctgtgc tgcaaaacat agagaagata ctcaaggaaa aaccattgga 120
actatgaata ataaagttac tccaaggtta ttctttgtta aaaataaagg aaatgtaatg 180
ccagtacaat gcagacattg tgaggatgca ccatgtctaa atgcctgccc agttaatgct 240
atagttgaaa aagatggaag tatcattata aatgaaagtg catgtatagg atgtcagacc 300
tgtacaatag tatgtccggt aggtgctgta agtttactgc ctagaactca aggtaaagta 360
gttacaggag gaattcaggt taaagtaaga gcagcagctt ataaatgtga tttatgtaag 420
gaagagggag gagaacctgc ttgcgtcaaa gaatgtccaa aagaggcctt gaggttagta 480
gatcctagag aagataaaaa agatcgtagt gtgaaagctg ctatggaact gttaaatata 540
aacgcaaatc tctaa 555
<210> 46
<211> 184
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 46
Met Lys Pro Glu Phe Asn Ser Phe Val Ile Ala Asp Pro Asp Lys Cys
1 5 10 15
Ile Gly Cys Arg Ser Cys Glu Ile Ala Cys Ala Ala Lys His Arg Glu
20 25 30
Asp Thr Gln Gly Lys Thr Ile Gly Thr Met Asn Asn Lys Val Thr Pro
35 40 45
Arg Leu Phe Phe Val Lys Asn Lys Gly Asn Val Met Pro Val Gln Cys
50 55 60
Arg His Cys Glu Asp Ala Pro Cys Leu Asn Ala Cys Pro Val Asn Ala
65 70 75 80
Ile Val Glu Lys Asp Gly Ser Ile Ile Ile Asn Glu Ser Ala Cys Ile
85 90 95
Gly Cys Gln Thr Cys Thr Ile Val Cys Pro Val Gly Ala Val Ser Leu
100 105 110
Leu Pro Arg Thr Gln Gly Lys Val Val Thr Gly Gly Ile Gln Val Lys
115 120 125
Val Arg Ala Ala Ala Tyr Lys Cys Asp Leu Cys Lys Glu Glu Gly Gly
130 135 140
Glu Pro Ala Cys Val Lys Glu Cys Pro Lys Glu Ala Leu Arg Leu Val
145 150 155 160
Asp Pro Arg Glu Asp Lys Lys Asp Arg Ser Val Lys Ala Ala Met Glu
165 170 175
Leu Leu Asn Ile Asn Ala Asn Leu
180
<210> 47
<211> 567
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 47
atgaagaatt gcctcgtagt agcagatcct aataaatgca taggatgtag gacttgtgaa 60
gcagcttgtg gtattgcaca ttcaggaggg gactttttta acacaaatgt atccaagatt 120
aattttaatc ctcgcttaaa tgtgataaaa actgctaaag taagtgctcc tgttcaatgc 180
agacaatgcg aagatgcacc ttgtggtaaa gcttgtccag ttaacgctat ttcaaatgaa 240
aatggttatg ttagtgtaaa taaagatgta tgtgttggat gtaaaatctg catgttagct 300
tgtccttttg gagctattga attagcttct caatataggg atggagaagt tgtagaccaa 360
aagggactta agatgagtga ggaaggtaat cctactgtga atggaaaagg aagagtggta 420
gcaaataagt gtgatctctg ccaggatagg gatggagggc ctgcttgtat agaagtttgt 480
cctacaaaat ctctcaaact agttacttat gatgacaata ataatatagt tgaaaaaaaa 540
gatgacgacg aacgtgaagt aagctaa 567
<210> 48
<211> 188
<212> PRT
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 48
Met Lys Asn Cys Leu Val Val Ala Asp Pro Asn Lys Cys Ile Gly Cys
1 5 10 15
Arg Thr Cys Glu Ala Ala Cys Gly Ile Ala His Ser Gly Gly Asp Phe
20 25 30
Phe Asn Thr Asn Val Ser Lys Ile Asn Phe Asn Pro Arg Leu Asn Val
35 40 45
Ile Lys Thr Ala Lys Val Ser Ala Pro Val Gln Cys Arg Gln Cys Glu
50 55 60
Asp Ala Pro Cys Gly Lys Ala Cys Pro Val Asn Ala Ile Ser Asn Glu
65 70 75 80
Asn Gly Tyr Val Ser Val Asn Lys Asp Val Cys Val Gly Cys Lys Ile
85 90 95
Cys Met Leu Ala Cys Pro Phe Gly Ala Ile Glu Leu Ala Ser Gln Tyr
100 105 110
Arg Asp Gly Glu Val Val Asp Gln Lys Gly Leu Lys Met Ser Glu Glu
115 120 125
Gly Asn Pro Thr Val Asn Gly Lys Gly Arg Val Val Ala Asn Lys Cys
130 135 140
Asp Leu Cys Gln Asp Arg Asp Gly Gly Pro Ala Cys Ile Glu Val Cys
145 150 155 160
Pro Thr Lys Ser Leu Lys Leu Val Thr Tyr Asp Asp Asn Asn Asn Ile
165 170 175
Val Glu Lys Lys Asp Asp Asp Glu Arg Glu Val Ser
180 185
<210> 49
<211> 957
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 49
gtgatagacg tacatgaagc attcaatata gtaatgaata atacaaaact gcttaaaagt 60
gaagatatgt ccctgataaa ttctcttaac agggtattgg cagaggatat aagctcaaaa 120
gataatcttc ccccatttga caaatcctgt atggatgggt atgctttaaa aagtgaagat 180
actaaggaaa aaatgtcaaa atttcaaatt aagggaagca taaaggcggg agatttttct 240
gatatagtat taaaaaatgg tgaagccata aaaataatga caggagctcc agtaccaaaa 300
ggtgcagatg caattattca aatagaaaaa gtaaaagtag aaggaaaaga acttcatgta 360
ttggaaaagg tatctcctgg aaccaacata tttaaaactg gtgaggagat aaaaattggt 420
gatgttgctt taaaaaaggg aaaaatcgta agacctgcag aaatagggtt attggcatca 480
ctaggttata ctaaaataaa atgctacaaa gtccctaaaa ttataataat aaatacaggg 540
gatgaactta taaatataga tcaaaactta atgcaaggta aaataagaaa ttgtaatgaa 600
tacacattaa ttgcccttat taaaaattta aatgcagaag ttaaatcgta tgggataata 660
agagatgata agaataaaat ttttaatgct ataaaaactg catttgaaga gggagatata 720
atcataacta ctggaggagc atctgtgggt gattacgatt ttatagaaga tgttcttaag 780
gaaataggaa cagatataaa atttacttcg gtagctatta aaccaggaaa accagttgtt 840
tttgcaactt ttaaagataa attattcttt ggacttccag gaaatccact ttcagtaata 900
aattcatttg aaagttttgt agcaccatct attaaaaaaa atgattggaa gagatga 957
<210> 50
<211> 318
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 50
Met Ile Asp Val His Glu Ala Phe Asn Ile Val Met Asn Asn Thr Lys
1 5 10 15
Leu Leu Lys Ser Glu Asp Met Ser Leu Ile Asn Ser Leu Asn Arg Val
20 25 30
Leu Ala Glu Asp Ile Ser Ser Lys Asp Asn Leu Pro Pro Phe Asp Lys
35 40 45
Ser Cys Met Asp Gly Tyr Ala Leu Lys Ser Glu Asp Thr Lys Glu Lys
50 55 60
Met Ser Lys Phe Gln Ile Lys Gly Ser Ile Lys Ala Gly Asp Phe Ser
65 70 75 80
Asp Ile Val Leu Lys Asn Gly Glu Ala Ile Lys Ile Met Thr Gly Ala
85 90 95
Pro Val Pro Lys Gly Ala Asp Ala Ile Ile Gln Ile Glu Lys Val Lys
100 105 110
Val Glu Gly Lys Glu Leu His Val Leu Glu Lys Val Ser Pro Gly Thr
115 120 125
Asn Ile Phe Lys Thr Gly Glu Glu Ile Lys Ile Gly Asp Val Ala Leu
130 135 140
Lys Lys Gly Lys Ile Val Arg Pro Ala Glu Ile Gly Leu Leu Ala Ser
145 150 155 160
Leu Gly Tyr Thr Lys Ile Lys Cys Tyr Lys Val Pro Lys Ile Ile Ile
165 170 175
Ile Asn Thr Gly Asp Glu Leu Ile Asn Ile Asp Gln Asn Leu Met Gln
180 185 190
Gly Lys Ile Arg Asn Cys Asn Glu Tyr Thr Leu Ile Ala Leu Ile Lys
195 200 205
Asn Leu Asn Ala Glu Val Lys Ser Tyr Gly Ile Ile Arg Asp Asp Lys
210 215 220
Asn Lys Ile Phe Asn Ala Ile Lys Thr Ala Phe Glu Glu Gly Asp Ile
225 230 235 240
Ile Ile Thr Thr Gly Gly Ala Ser Val Gly Asp Tyr Asp Phe Ile Glu
245 250 255
Asp Val Leu Lys Glu Ile Gly Thr Asp Ile Lys Phe Thr Ser Val Ala
260 265 270
Ile Lys Pro Gly Lys Pro Val Val Phe Ala Thr Phe Lys Asp Lys Leu
275 280 285
Phe Phe Gly Leu Pro Gly Asn Pro Leu Ser Val Ile Asn Ser Phe Glu
290 295 300
Ser Phe Val Ala Pro Ser Ile Lys Lys Asn Asp Trp Lys Arg
305 310 315
<210> 51
<211> 570
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 51
atggaaaatt tatattcaaa taagaggtca gtaatttcta ttatttcatc tagttcaaat 60
tcaggtaaga ctaccttaat agaaggaata ataagaattt taaaaagcag aggatataag 120
gttggtgcaa taaagaatga tgctcataag ttacagatag attatccagg aaaagacagc 180
tttagattta cagaggcagg tgcagacaat gttgttattg catcggataa tacagtggct 240
atgataaaaa aagtaagtgg accaaaaagt atagaagaat tgctgttgct ttttcaggat 300
gtagatattg taatagtgga aggttttaag ggcaacgaat ttcctaaaat agaagtatac 360
aggaaaaatg caagtaaatg tttactctac aaaaattctg aatataattt tcaaaatttt 420
gtagctattg taaccaatga aaatttaata actgatattc ctgtatttga tataaatgat 480
acaaaaaaag tagctgattt tattgaaagc aactttatag gaggtaacaa aaatggagaa 540
aaccatggaa cttcatgtag tcaaatatga 570
<210> 52
<211> 189
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 52
Met Glu Asn Leu Tyr Ser Asn Lys Arg Ser Val Ile Ser Ile Ile Ser
1 5 10 15
Ser Ser Ser Asn Ser Gly Lys Thr Thr Leu Ile Glu Gly Ile Ile Arg
20 25 30
Ile Leu Lys Ser Arg Gly Tyr Lys Val Gly Ala Ile Lys Asn Asp Ala
35 40 45
His Lys Leu Gln Ile Asp Tyr Pro Gly Lys Asp Ser Phe Arg Phe Thr
50 55 60
Glu Ala Gly Ala Asp Asn Val Val Ile Ala Ser Asp Asn Thr Val Ala
65 70 75 80
Met Ile Lys Lys Val Ser Gly Pro Lys Ser Ile Glu Glu Leu Leu Leu
85 90 95
Leu Phe Gln Asp Val Asp Ile Val Ile Val Glu Gly Phe Lys Gly Asn
100 105 110
Glu Phe Pro Lys Ile Glu Val Tyr Arg Lys Asn Ala Ser Lys Cys Leu
115 120 125
Leu Tyr Lys Asn Ser Glu Tyr Asn Phe Gln Asn Phe Val Ala Ile Val
130 135 140
Thr Asn Glu Asn Leu Ile Thr Asp Ile Pro Val Phe Asp Ile Asn Asp
145 150 155 160
Thr Lys Lys Val Ala Asp Phe Ile Glu Ser Asn Phe Ile Gly Gly Asn
165 170 175
Lys Asn Gly Glu Asn His Gly Thr Ser Cys Ser Gln Ile
180 185
<210> 53
<211> 789
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 53
atggagaaaa ccatggaact tcatgtagtc aaatatgaca aagaatgtaa aaagacagtg 60
gaggaatcaa ctatttgcga atatccttta aatgtatttg taaatggtga acatttgaca 120
gtgctcttat gtactcctga aaagcttaag gaattaacaa taggtttctt ggtctttaaa 180
ggtattataa aatctctaga tgaaataaaa tctttagaga tagatgaaaa aagtggagcg 240
tccagggtaa tcttgaaaaa tagacaattt aataaagagt tgtattcaaa gcaagtactt 300
cctacaacat ttaatgaaaa tgaaaaaagt aaattctttt catatattat tgattccatg 360
aaaattagtc taatcaataa tgataatgtt tacatacatg ttgataaaat ctatggctta 420
atgatggaca atcttggata ttccaagact tttaaactca ctggaggagc acattgtgca 480
gctctttgtg atgaagataa agtaatatct atttgtgagg atgtggccag acacaatgct 540
gtagacaagc ttataggtga ggcatttata aaaaatattt gtttaaaaga taaaataata 600
tttgtgagta gcagggtatc ttttgaaatg gtatataaaa ttgctagact tggggtacct 660
ataataatat ctaaatctgc acctacaaat ctttctatag aatttgcaaa agctttaaat 720
gttacattaa ttggatttgt aaggggagaa agaatgaatg tatatacaaa tccacagaga 780
ataatatag 789
<210> 54
<211> 262
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 54
Met Glu Lys Thr Met Glu Leu His Val Val Lys Tyr Asp Lys Glu Cys
1 5 10 15
Lys Lys Thr Val Glu Glu Ser Thr Ile Cys Glu Tyr Pro Leu Asn Val
20 25 30
Phe Val Asn Gly Glu His Leu Thr Val Leu Leu Cys Thr Pro Glu Lys
35 40 45
Leu Lys Glu Leu Thr Ile Gly Phe Leu Val Phe Lys Gly Ile Ile Lys
50 55 60
Ser Leu Asp Glu Ile Lys Ser Leu Glu Ile Asp Glu Lys Ser Gly Ala
65 70 75 80
Ser Arg Val Ile Leu Lys Asn Arg Gln Phe Asn Lys Glu Leu Tyr Ser
85 90 95
Lys Gln Val Leu Pro Thr Thr Phe Asn Glu Asn Glu Lys Ser Lys Phe
100 105 110
Phe Ser Tyr Ile Ile Asp Ser Met Lys Ile Ser Leu Ile Asn Asn Asp
115 120 125
Asn Val Tyr Ile His Val Asp Lys Ile Tyr Gly Leu Met Met Asp Asn
130 135 140
Leu Gly Tyr Ser Lys Thr Phe Lys Leu Thr Gly Gly Ala His Cys Ala
145 150 155 160
Ala Leu Cys Asp Glu Asp Lys Val Ile Ser Ile Cys Glu Asp Val Ala
165 170 175
Arg His Asn Ala Val Asp Lys Leu Ile Gly Glu Ala Phe Ile Lys Asn
180 185 190
Ile Cys Leu Lys Asp Lys Ile Ile Phe Val Ser Ser Arg Val Ser Phe
195 200 205
Glu Met Val Tyr Lys Ile Ala Arg Leu Gly Val Pro Ile Ile Ile Ser
210 215 220
Lys Ser Ala Pro Thr Asn Leu Ser Ile Glu Phe Ala Lys Ala Leu Asn
225 230 235 240
Val Thr Leu Ile Gly Phe Val Arg Gly Glu Arg Met Asn Val Tyr Thr
245 250 255
Asn Pro Gln Arg Ile Ile
260
<210> 55
<211> 486
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 55
atgttaacta aacagcaaaa tgaagacctg tctggacaag atgtaattga aaaatatcct 60
aaagagcaga gatttactct tgctatacta caggatatac agagaaagta caaatatata 120
cccagagaag cactggagaa tttagctaag tatttggaca cgcctgtaag tagactgtat 180
ggtatggcta ctttttataa ggcattgagc cttactccaa aaggggaaaa cataataact 240
gtatgtgatg gaaccgcttg ccatgttgct ggttctatgg ttgtaatgga tgaacttgaa 300
aaggcaatag gaattaaacc aggtgaaact acagaagatc ttaaattttc aataaataca 360
gttaactgta taggatgctg tgcaatagct cctgtcatga tgataaatga caaatatttt 420
ggaaatttaa cacctaaact ggttgaagaa attcttagtg agtataggag tgaaagccat 480
gagtga 486
<210> 56
<211> 161
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 56
Met Leu Thr Lys Gln Gln Asn Glu Asp Leu Ser Gly Gln Asp Val Ile
1 5 10 15
Glu Lys Tyr Pro Lys Glu Gln Arg Phe Thr Leu Ala Ile Leu Gln Asp
20 25 30
Ile Gln Arg Lys Tyr Lys Tyr Ile Pro Arg Glu Ala Leu Glu Asn Leu
35 40 45
Ala Lys Tyr Leu Asp Thr Pro Val Ser Arg Leu Tyr Gly Met Ala Thr
50 55 60
Phe Tyr Lys Ala Leu Ser Leu Thr Pro Lys Gly Glu Asn Ile Ile Thr
65 70 75 80
Val Cys Asp Gly Thr Ala Cys His Val Ala Gly Ser Met Val Val Met
85 90 95
Asp Glu Leu Glu Lys Ala Ile Gly Ile Lys Pro Gly Glu Thr Thr Glu
100 105 110
Asp Leu Lys Phe Ser Ile Asn Thr Val Asn Cys Ile Gly Cys Cys Ala
115 120 125
Ile Ala Pro Val Met Met Ile Asn Asp Lys Tyr Phe Gly Asn Leu Thr
130 135 140
Pro Lys Leu Val Glu Glu Ile Leu Ser Glu Tyr Arg Ser Glu Ser His
145 150 155 160
Glu
<210> 57
<211> 1821
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 57
gtgagtatag gagtgaaagc catgagtgat aaaaaaattg tcaatatatg ttgtggaaca 60
ggttgcttag ctaaaggcag caaggaagta tatgaagaaa tgaaggcaca aatagctaaa 120
ttaggggcaa atgcagaagt aaatgttaaa ttaaaagcaa caggttgcga tggattgtgt 180
gagaaaggtc ctgtactgaa aatatatcca gatgacattg catattttaa agttaaagta 240
gaagatgtag aagacgtagt aaaaaagaca ttgatgaatg gggaaataat tgaaaaatta 300
ttatattttg aaactgctac aaaacagaga ttaagaaatc ataaagaaag tgaattttgt 360
aaaagacaat acaaaattgc tctcagaaat gttggtgaaa tagatccaat aagtttggaa 420
gattatgttg aaagaggcgg atataaagct cttaaaaaag caataagcag catgaaacct 480
gaagatgtgc ttgaagaaat aacaaaatca ggtcttagag gaagaggtgg agcaggattc 540
ccaacaggac gtaaatggaa aactgctgca gatattgata catcacctat atatgtagta 600
tgcaatggtg atgagggaga tcctggagca tttatggata gaagtataat ggagggagat 660
cctaacagtg ttatagaagg tatgacattg tgtgcctatg cagtaggagg tacaaacggc 720
tttgcttata taagagatga atatggactt gctgtagaaa atatgcagaa agctattaat 780
aaagcaaaag atgaaaattt attaggtaat aatatattag gaactgactt ttccttcgat 840
atacagatag taagaggtgg aggagctttt gtatgtggtg agtctactgc acttatgtca 900
tctatagaag gtatggtagg agaacctaga gctaaatata tacacactac agaaaaagga 960
ttgtggggac aacctactgt tttaaataat gtagaaactt gggccaatgt acctataata 1020
attgaaaaag gtggagattg gtatcatgct ataggaacta tggagaagag taagggaaca 1080
aaagtattct cattagttgg aaaagttaag aatactggac ttgtagaagt acctatggga 1140
actactctta gagaaataat atatgatatt ggcggtggag tattaaacga cagaaagttt 1200
aaggcagttc aaataggtgg accttcagga ggatgtttac catctgaata tttagacttg 1260
ccagtagatt atgatacttt ggttaaagcg gattctatga tgggttccgg cggaatgatc 1320
gtaatggatg atagaacctg tatggtagat gtaactagat attacttgag tttcttagct 1380
gaagaatctt gtggaaagtg tgtaccttgt agagaaggcg taaagagaat gcttgaaata 1440
ctcactgata tatgcaatgg tgatggaaaa gaaggagaca tagaagagct tcttgaaata 1500
tgttccatga caagcaaggc atctctgtgc agtcttggta agagtgctcc aaatccagta 1560
aaagcagcta taagatattt tagagatgaa tttgaagaac atataaagaa taagagatgt 1620
agagcaggag tttgtaagaa acttactaca tttggtatag atcaagataa atgtaaggga 1680
tgcgatatgt gtaaaaagaa ttgtccagct gattgtataa caggggaaat taagaaacca 1740
catacaatag atgctgataa gtgcttgaga tgcggtaact gcatgaacat ctgtaagttt 1800
gatgctgtta aggttttata g 1821
<210> 58
<211> 606
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 58
Met Ser Ile Gly Val Lys Ala Met Ser Asp Lys Lys Ile Val Asn Ile
1 5 10 15
Cys Cys Gly Thr Gly Cys Leu Ala Lys Gly Ser Lys Glu Val Tyr Glu
20 25 30
Glu Met Lys Ala Gln Ile Ala Lys Leu Gly Ala Asn Ala Glu Val Asn
35 40 45
Val Lys Leu Lys Ala Thr Gly Cys Asp Gly Leu Cys Glu Lys Gly Pro
50 55 60
Val Leu Lys Ile Tyr Pro Asp Asp Ile Ala Tyr Phe Lys Val Lys Val
65 70 75 80
Glu Asp Val Glu Asp Val Val Lys Lys Thr Leu Met Asn Gly Glu Ile
85 90 95
Ile Glu Lys Leu Leu Tyr Phe Glu Thr Ala Thr Lys Gln Arg Leu Arg
100 105 110
Asn His Lys Glu Ser Glu Phe Cys Lys Arg Gln Tyr Lys Ile Ala Leu
115 120 125
Arg Asn Val Gly Glu Ile Asp Pro Ile Ser Leu Glu Asp Tyr Val Glu
130 135 140
Arg Gly Gly Tyr Lys Ala Leu Lys Lys Ala Ile Ser Ser Met Lys Pro
145 150 155 160
Glu Asp Val Leu Glu Glu Ile Thr Lys Ser Gly Leu Arg Gly Arg Gly
165 170 175
Gly Ala Gly Phe Pro Thr Gly Arg Lys Trp Lys Thr Ala Ala Asp Ile
180 185 190
Asp Thr Ser Pro Ile Tyr Val Val Cys Asn Gly Asp Glu Gly Asp Pro
195 200 205
Gly Ala Phe Met Asp Arg Ser Ile Met Glu Gly Asp Pro Asn Ser Val
210 215 220
Ile Glu Gly Met Thr Leu Cys Ala Tyr Ala Val Gly Gly Thr Asn Gly
225 230 235 240
Phe Ala Tyr Ile Arg Asp Glu Tyr Gly Leu Ala Val Glu Asn Met Gln
245 250 255
Lys Ala Ile Asn Lys Ala Lys Asp Glu Asn Leu Leu Gly Asn Asn Ile
260 265 270
Leu Gly Thr Asp Phe Ser Phe Asp Ile Gln Ile Val Arg Gly Gly Gly
275 280 285
Ala Phe Val Cys Gly Glu Ser Thr Ala Leu Met Ser Ser Ile Glu Gly
290 295 300
Met Val Gly Glu Pro Arg Ala Lys Tyr Ile His Thr Thr Glu Lys Gly
305 310 315 320
Leu Trp Gly Gln Pro Thr Val Leu Asn Asn Val Glu Thr Trp Ala Asn
325 330 335
Val Pro Ile Ile Ile Glu Lys Gly Gly Asp Trp Tyr His Ala Ile Gly
340 345 350
Thr Met Glu Lys Ser Lys Gly Thr Lys Val Phe Ser Leu Val Gly Lys
355 360 365
Val Lys Asn Thr Gly Leu Val Glu Val Pro Met Gly Thr Thr Leu Arg
370 375 380
Glu Ile Ile Tyr Asp Ile Gly Gly Gly Val Leu Asn Asp Arg Lys Phe
385 390 395 400
Lys Ala Val Gln Ile Gly Gly Pro Ser Gly Gly Cys Leu Pro Ser Glu
405 410 415
Tyr Leu Asp Leu Pro Val Asp Tyr Asp Thr Leu Val Lys Ala Asp Ser
420 425 430
Met Met Gly Ser Gly Gly Met Ile Val Met Asp Asp Arg Thr Cys Met
435 440 445
Val Asp Val Thr Arg Tyr Tyr Leu Ser Phe Leu Ala Glu Glu Ser Cys
450 455 460
Gly Lys Cys Val Pro Cys Arg Glu Gly Val Lys Arg Met Leu Glu Ile
465 470 475 480
Leu Thr Asp Ile Cys Asn Gly Asp Gly Lys Glu Gly Asp Ile Glu Glu
485 490 495
Leu Leu Glu Ile Cys Ser Met Thr Ser Lys Ala Ser Leu Cys Ser Leu
500 505 510
Gly Lys Ser Ala Pro Asn Pro Val Lys Ala Ala Ile Arg Tyr Phe Arg
515 520 525
Asp Glu Phe Glu Glu His Ile Lys Asn Lys Arg Cys Arg Ala Gly Val
530 535 540
Cys Lys Lys Leu Thr Thr Phe Gly Ile Asp Gln Asp Lys Cys Lys Gly
545 550 555 560
Cys Asp Met Cys Lys Lys Asn Cys Pro Ala Asp Cys Ile Thr Gly Glu
565 570 575
Ile Lys Lys Pro His Thr Ile Asp Ala Asp Lys Cys Leu Arg Cys Gly
580 585 590
Asn Cys Met Asn Ile Cys Lys Phe Asp Ala Val Lys Val Leu
595 600 605
<210> 59
<211> 768
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 59
gtgaatgtag atatgaaaat tacaatagat ggaaaagctt gtgaagctga aaaaggagaa 60
ttcatattac aaatagcaag aagaaataat atatatatac ctacactgtg ccacagtgat 120
gcattgcctg ggcttgctag ctgtagactg tgtatagtta aagtagtaga taggggacgt 180
gcaaagatag taacttcctg tatattccct gtaagtaagg aagtagaagt tataactaat 240
gacgatgaaa taaagagaat gagaaaaaac atagttatgc ttttaaaagt aagatgccct 300
gaaaataaag aagtaaatga attagctaaa gcctttggag tagaggaaaa gagagtaaag 360
aggttcaaat tggatccaga acaaaattgt gttttgtgcg gactttgtgc aaaagcttgc 420
aaggaattag gtactggagc aatctcaaca gttaataggg gtatgtataa agaagtagca 480
actccatatc acgaatcttc accagaatgt ataggatgtg cttcctgtgc aaatgtttgt 540
ccaactaatg caataaaagt tgtggataaa gatggagaaa gagaaatatg gggcaaaaaa 600
ttcaagatgg ttaaatgtga tttgtgcgga gaatattttg ctacagaaga acacgtaaaa 660
tatgcttaca ataggcttgg aaaagagcag ccagaaaagc ttatgtgcag cagctgcaag 720
aagaaagtta cagccaaaga tgtcaaaaat atttttgaga acgtgtga 768
<210> 60
<211> 255
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 60
Met Asn Val Asp Met Lys Ile Thr Ile Asp Gly Lys Ala Cys Glu Ala
1 5 10 15
Glu Lys Gly Glu Phe Ile Leu Gln Ile Ala Arg Arg Asn Asn Ile Tyr
20 25 30
Ile Pro Thr Leu Cys His Ser Asp Ala Leu Pro Gly Leu Ala Ser Cys
35 40 45
Arg Leu Cys Ile Val Lys Val Val Asp Arg Gly Arg Ala Lys Ile Val
50 55 60
Thr Ser Cys Ile Phe Pro Val Ser Lys Glu Val Glu Val Ile Thr Asn
65 70 75 80
Asp Asp Glu Ile Lys Arg Met Arg Lys Asn Ile Val Met Leu Leu Lys
85 90 95
Val Arg Cys Pro Glu Asn Lys Glu Val Asn Glu Leu Ala Lys Ala Phe
100 105 110
Gly Val Glu Glu Lys Arg Val Lys Arg Phe Lys Leu Asp Pro Glu Gln
115 120 125
Asn Cys Val Leu Cys Gly Leu Cys Ala Lys Ala Cys Lys Glu Leu Gly
130 135 140
Thr Gly Ala Ile Ser Thr Val Asn Arg Gly Met Tyr Lys Glu Val Ala
145 150 155 160
Thr Pro Tyr His Glu Ser Ser Pro Glu Cys Ile Gly Cys Ala Ser Cys
165 170 175
Ala Asn Val Cys Pro Thr Asn Ala Ile Lys Val Val Asp Lys Asp Gly
180 185 190
Glu Arg Glu Ile Trp Gly Lys Lys Phe Lys Met Val Lys Cys Asp Leu
195 200 205
Cys Gly Glu Tyr Phe Ala Thr Glu Glu His Val Lys Tyr Ala Tyr Asn
210 215 220
Arg Leu Gly Lys Glu Gln Pro Glu Lys Leu Met Cys Ser Ser Cys Lys
225 230 235 240
Lys Lys Val Thr Ala Lys Asp Val Lys Asn Ile Phe Glu Asn Val
245 250 255
<210> 61
<211> 555
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 61
atgaaaccag agtttaattc ttttgtaata gccgatcctg acaagtgcat aggctgtaga 60
tcttgtgaga ttgcctgtgc tgcaaaacat agagaagata ctcaaggaaa aactattgga 120
actatgaata ataaagttac tccaaggtta ttctttgtta aaaataaagg aaatgtaatg 180
ccggtacaat gcagacattg tgaggatgca ccatgtctaa atgcctgccc agttaatgct 240
atagttgaaa aagatggaag tatcattata aatgaaagtg catgtatagg gtgtcagacc 300
tgtacaatag tatgtccggt aggtgctgta agtttacttc ctagaactca aggtaaagta 360
gttacaggag gaattcaggt taaagtaaga gcagcagctt ataaatgtga tttatgtaag 420
gaagagggag gagaacctgc ctgcgtcaaa gaatgtccaa aagaggcctt aaggttagta 480
gatcctagag aagataaaaa agatcgtagt gtgaaagctg ctatggaact gttaaatata 540
aacgcaaatc tctaa 555
<210> 62
<211> 184
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 62
Met Lys Pro Glu Phe Asn Ser Phe Val Ile Ala Asp Pro Asp Lys Cys
1 5 10 15
Ile Gly Cys Arg Ser Cys Glu Ile Ala Cys Ala Ala Lys His Arg Glu
20 25 30
Asp Thr Gln Gly Lys Thr Ile Gly Thr Met Asn Asn Lys Val Thr Pro
35 40 45
Arg Leu Phe Phe Val Lys Asn Lys Gly Asn Val Met Pro Val Gln Cys
50 55 60
Arg His Cys Glu Asp Ala Pro Cys Leu Asn Ala Cys Pro Val Asn Ala
65 70 75 80
Ile Val Glu Lys Asp Gly Ser Ile Ile Ile Asn Glu Ser Ala Cys Ile
85 90 95
Gly Cys Gln Thr Cys Thr Ile Val Cys Pro Val Gly Ala Val Ser Leu
100 105 110
Leu Pro Arg Thr Gln Gly Lys Val Val Thr Gly Gly Ile Gln Val Lys
115 120 125
Val Arg Ala Ala Ala Tyr Lys Cys Asp Leu Cys Lys Glu Glu Gly Gly
130 135 140
Glu Pro Ala Cys Val Lys Glu Cys Pro Lys Glu Ala Leu Arg Leu Val
145 150 155 160
Asp Pro Arg Glu Asp Lys Lys Asp Arg Ser Val Lys Ala Ala Met Glu
165 170 175
Leu Leu Asn Ile Asn Ala Asn Leu
180
<210> 63
<211> 618
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 63
atgaattatt gcacactaaa tatatctcaa gaaaaaagga gagttaataa aatgaagaat 60
tgcctcgtag tagcagatcc taataaatgc ataggatgta ggacttgtga agcagcttgt 120
ggtattgcac attcaggagg ggactttttt aatacaaatg tatccaaaat taattttaat 180
cctcgcttaa atgtgataaa aactgctaaa gtaagtgctc ctgttcaatg cagacaatgc 240
gaagatgcac cttgtggtaa agcttgtcca gttaacgcta tttcaaatga aaatggttat 300
gttagtgtag ataaagatgt atgtgttgga tgtaaaatct gcatgttagc ttgtcctttt 360
ggagctattg aattagcttc tcaatataag gatggagaag ttgtagacca aaagggactt 420
aagatgagtg aggaaggtaa tcctactgta aatggaaaag gaagagtggt agcaaataag 480
tgtgatcttt gccaggatag ggatggagga cctgcttgca tagaagtttg tcctacaaaa 540
tctctcaaat tagttactta tgatgacaat aataatatag ttgaaaaaaa agatgacgac 600
gaacgtgaag taggctaa 618
<210> 64
<211> 205
<212> PRT
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 64
Met Asn Tyr Cys Thr Leu Asn Ile Ser Gln Glu Lys Arg Arg Val Asn
1 5 10 15
Lys Met Lys Asn Cys Leu Val Val Ala Asp Pro Asn Lys Cys Ile Gly
20 25 30
Cys Arg Thr Cys Glu Ala Ala Cys Gly Ile Ala His Ser Gly Gly Asp
35 40 45
Phe Phe Asn Thr Asn Val Ser Lys Ile Asn Phe Asn Pro Arg Leu Asn
50 55 60
Val Ile Lys Thr Ala Lys Val Ser Ala Pro Val Gln Cys Arg Gln Cys
65 70 75 80
Glu Asp Ala Pro Cys Gly Lys Ala Cys Pro Val Asn Ala Ile Ser Asn
85 90 95
Glu Asn Gly Tyr Val Ser Val Asp Lys Asp Val Cys Val Gly Cys Lys
100 105 110
Ile Cys Met Leu Ala Cys Pro Phe Gly Ala Ile Glu Leu Ala Ser Gln
115 120 125
Tyr Lys Asp Gly Glu Val Val Asp Gln Lys Gly Leu Lys Met Ser Glu
130 135 140
Glu Gly Asn Pro Thr Val Asn Gly Lys Gly Arg Val Val Ala Asn Lys
145 150 155 160
Cys Asp Leu Cys Gln Asp Arg Asp Gly Gly Pro Ala Cys Ile Glu Val
165 170 175
Cys Pro Thr Lys Ser Leu Lys Leu Val Thr Tyr Asp Asp Asn Asn Asn
180 185 190
Ile Val Glu Lys Lys Asp Asp Asp Glu Arg Glu Val Gly
195 200 205
<210> 65
<211> 10640
<212> DNA
<213> Unknown
<220>
<223> Clostridium autoethanogenum
<400> 65
atggataaaa aagttttaac tgtttgtcct tactgtggcg ctggttgtaa tttatacttg 60
catgtaaaga atggcaaaat aattaaagca gagcctgcta atggtaggac aaatgaagga 120
tcactgtgtt taaaaggaca ctttggttgg gattttttaa acgatcctaa aatattgaca 180
tctagaatta aacatccgat gataagaaaa aacggagagc tagaagaggt aagctgggat 240
gaagctatta gttttacggc ttcaagattg tcacaaataa aagagaaata tggacctgat 300
tccataatgg gaacaggatg tgctaggggt tctggaaacg aagcaaacta cataatgcaa 360
aagtttatga gggcggttat tggaaccaat aacgtagatc actgtgccag agtttgacat 420
gctccttctg tagccggtct ggcttacgtt ttaggaaatg gtgctatgtc aaatggtata 480
catgaaatag atgatacaga tttactactt atttttggat ataatggagc agcttcgcat 540
ccaatagttg ctaagagaat agttagggca aaacaaaagg gtgcaaaggt aatagttgta 600
gatccacgta taacagagtc tggtaggata gcagatttat ggctccctat aaaaaatgga 660
acaaatatgg ttcttgtaaa tacttttgcc aacatactta taaataaaca gttttataac 720
aaacaatatg tagaagatca tactgttggt tttgaagaat atagatctat agttgaaaat 780
tatactcctg aatatgcaga aaaagttact ggcatacctt cagaggatat agtagaagct 840
atgaaaatgt actcaggtgc taaaaatgcc atgatattat atggtatggg agtatgtcaa 900
tttgctcaag ctgtagatgt agttaaggga ctagcttcta tagcattatt aactggtaat 960
tttggaagac ctaatgtagg tataggacct gtaagaggcc agaacaatgt tcaaggtgct 1020
tgtgatatgg gagcacttcc taatgtatac ccaggttatc aaagtgtaac tgacgatgca 1080
attaggcaaa aatttgaaaa agcttggggt gttaaacttc caaacaaagt tggttatcac 1140
ctgacacaag ttcctgaatt aacgcttaaa gaggataaaa taaaggcata ttatataatg 1200
ggtgaagatc cagttcaaag tgatcctgat tctaatgaaa tgagagagac actggataaa 1260
atggaacttg taatagttca ggatatattt atgaataaaa ctgcactcca tgcagatgta 1320
attttacctt ccacgtcttg gggagaacat gaaggagtct ttagttctgc agatagagga 1380
ttccagagat ttagaaaagc tgtagaacct aagggagatg ttaaaccaga ttgggaaata 1440
atttcaaaaa ttgcctgtgc tatgggttat aatatgcatt ataacaatac tgaggaaata 1500
tggaatgaac ttataaattt atgtccaaat ttcaaaggag caacttataa gagactcgaa 1560
gaattaggag gaatccaatg gccttgtcca tctgaaaatc atcctggaac ttcttatctc 1620
tacaaaggta ataaatttaa tacacctact ggaaaagcaa acttatttgc agcagaatgg 1680
agacctcctg tagagcagac agataaagat tatccactcg ttctttctac agttagagaa 1740
gtaggacatt attctgtaag aacaatgaca ggaaactgta gggcacttca gcagttagcc 1800
gatgaaccag gatatgtaca agttaatcca atggatgcaa aggctaaggg aataatagat 1860
ggtgagctta tgagaataag ttcacgaaga ggttctgtgg ttgcccgtgc acttattact 1920
gaaagggcaa ataaaggagc agtctatatg acctatcaat ggtgggtagg cgcatgtaat 1980
gaacttacat ctaataatct agatccagta tcaaaaactc ctgaattaaa gtattgtgca 2040
gtaaaaatag aagctataaa agatcagaaa gaagctgaaa agtttataaa agatcaatat 2100
gatcttttaa agaaaaagat gaatgtttaa tattttaata taaagatggc taaaaagacc 2160
ttgattaaga ggtcttttag ccaaaagctt taaatcaata ggagttgata gactgtgata 2220
gacgtacatg aagcattcaa tatagtaatg aataacacaa aactgcttaa aggtgaagat 2280
atatcgttga taaattctct taacagggta ttggcagagg atataagctc aaaagataat 2340
cttcccccat ttgacaaatc ctgtatggat gggtatgctt taaaaagtga agatactaag 2400
gaaaaaatgt caaaatttcg aattaaggga agcataaagg cgggagattt ttctgatata 2460
gtattgaaaa atggtgaagc cataaaaata atgacaggag ctccagtacc aaaaggtgca 2520
gatgcagtta ttcaaataga aaaagtaaaa gtagaaggaa aagaacttca tgtattagaa 2580
aatatatctc ctggaaccaa catatttaaa actggtgagg agataaaaat tggtgatgtt 2640
gctttaagaa aaggaaagat tttgagacct gcagaaatag ggttattggc atcactaggt 2700
tatactaaaa taaaatgtta taaagcccct aaaattataa taataaatac tggggatgaa 2760
cttataaata tagatcaaaa cttaatgcaa ggtaaaataa ggaattgtaa tgaatataca 2820
ttaattgccc ttattaaaaa tttaaatgca gaagttaaat catatgggat aataagagat 2880
gataaggata agatttttaa tgctataaaa actgcatttg aagagggaga tataatcata 2940
actactggag gagcatctgt aggtgattac gattttatag aagatgttct tcagaaaata 3000
ggaacagata taaagtttac ttcggtagct attaaaccag gaaaaccagt tgtttttgca 3060
acttttaaag ataaattgtt ctttggactt ccaggaaatc cgctttcggt aataaattca 3120
tttgaaagtt ttgtagcacc atctattaaa aaaatgattg gaagagatga tgcgtttcct 3180
gaagaatttc ctgtaacttt aaaagatgat tttaaatcag gaaaagaaag agactgctat 3240
atgtatgtaa acataaaaaa ggaagataac cgttattatg cctatgatgt aggaagacaa 3300
gattccaatg ggctttttac gcttactaag tcaaatggag tcgtcatcat ggaaaaggga 3360
actagtatag caaaagctgg agatatttta aatggaaaat ttatattcaa ataagaggtc 3420
agtaatttct attatttcat ctagttcaaa ttcgggtaag actaccttga tagaaggaat 3480
aataagaatt ctaaaaagca gaggatataa ggttggtgca ataaagaatg atgctcataa 3540
gttacagata gattacccag gaaaagacag ctttagattt acagaggcag gtgcggacaa 3600
tgttgttatt gcatcggata atacagtggc tatgataaaa aaagtaagtg gacccaaaag 3660
tatagaagaa ctactgttgc tttttcaaga tgtagatatt gtaatagtgg aaggcttcaa 3720
gggtaacgaa tttcctaaaa tagaagtata caggaaaaat gcaagcaaat gtttacttta 3780
caaaaattct aaatataatt ttcaaaattt tgtagctatt gtaaccaatg aaaacttaat 3840
aactgatatt cctgtatttg atataaatga tacaaaaaaa atagctgatt ttattgagaa 3900
cgactttata ggaggtaaca aaaatggaga aaaccatgga acttcatgta gtcaaatatg 3960
acaaagaatg taaaaagaca gtagaggaat caactatttg tgaatatcct ttaaatgtat 4020
ttgtaaatgg tgaacatttg acagtactct tatgtacgcc tgaaaagctt aaggaattaa 4080
caataggttt cttgaccttt agaggtgtta taaaatctct agatgaaata aaatctctag 4140
agatagatga aaaaagtgga gcgtccaggg taactttgaa aaatagccaa tttaataaag 4200
agttgtattc aaagcaagtg cttcctataa catttagtga aaatgagaaa agtaagttct 4260
tttcgtatat tattgattcc atggaaatta gtataatcaa caatgataat gtttacattc 4320
atgtcgataa aatctatgat ctaatgatgg acaatcttgg atattccaag acgtttaaac 4380
tcactggagg aacacattgt gcagctcttt gtgatgaaga taaagtaata tctatttgtg 4440
aggatgtggc tagacacaat gctgtagaca agcttatagg tgaggcattt ataaaaaata 4500
tttatttaaa ggataaaata atatttgtga gcagcagagt atcttttgaa atggtatata 4560
aaattgctag gctaggggta cctataataa tatctaaatc tgcacctaca agtctttcta 4620
tagaatttgc aaaagcttta aatgttacat taattggatt tgtaagggga gaaagaatga 4680
atgtatatac aaatccacag agaataatat agtaatttct gttaacatat ttttgaatat 4740
aatcttaaaa aattaatcat atagttatat aaaaataata taatatttaa tgttaataat 4800
tagtccctaa ttaaatagga ctaagtttat attttaattt ttaaataagg taattaaaat 4860
aattttaaat taaatgtgtt atatgtttta aaattatttc ttaagtatag aggctcaaat 4920
ctttggttta gagctaatat cttattcctt ctaatatttt aagggggaaa tcaattataa 4980
tattcaaatg ggagggtgaa gtatttaatg ttaactaaac agcaaaatga agacctgtct 5040
ggacaagatg taattgaaaa atatcctaaa gagcagagat ttactcttgc tatactacag 5100
gatatacaga gaaagtacaa atatatacca agagaagcac tggagaattt agctaagtat 5160
ttggacacgc ctgtaagtag actgtatggt atggctactt tttataaggc attgagcctt 5220
actccaaaag gggaaaacat aataactgta tgtgatggaa ccgcttgcca tgttgctggt 5280
tctatggttg taatggatga acttgaaaag gcaataggaa ttaaaccagg tgaaactaca 5340
gaggatctca aattttcaat aaatacagtt aactgtatag gatgctgtgc aatagctcct 5400
gtcatgatga taaatgacaa atattatgga aatttaacac ctaaactggt tgaagaaatt 5460
cttagtgagt ataggagtga gagtgatgag tgataaaaaa actgtcaata tatgttgtgg 5520
gacaggttgt ttagctaaag gcagtatgga agtatatgaa gaaatgaagg cacaaatagc 5580
taaattaggg gcaaatgcag aagtaaatgt taaattaaaa gcaacaggtt gcgatggatt 5640
gtgtgagaaa ggtcctgtac tgaaaatata tccagatgac attgcatatt ttaaagttaa 5700
agtagaagat gtagaagacg tagtaaaaaa gacattgatg aatggggaaa taattgaaaa 5760
attattatat tttgagactg ctacaaaaca gagattaaga aatcataaag aaagtgaatt 5820
ttgtaaaaga caatacaaaa ttgctctcag aaatgttggt gaaatagatc caataagttt 5880
ggaagattat gttgaaagag gcggatacaa agctcttaaa aaagcaataa gcagcatgaa 5940
acctgaagat gtgcttgaag aaataacaaa atcaggtctt agaggaagag gtggagcagg 6000
attcccaaca ggacgtaaat ggaaaactgc tgcagatatt gatacatcac ctatatatgt 6060
agtatgtaat ggtgatgaag gagatcctgg agcatttatg gatagaagta taatggaggg 6120
agatcctaac agtgttatag aaggtatgac attatgtgct tatgcagtag gaggtacaaa 6180
tggatttgct tatataagag atgaatatgg acttgctgta gaaaatatgc agaaagctat 6240
taataaagca aaagatgaaa atttattagg taataatata ttaggaactg atttttcctt 6300
cgatatacag atagtaagag gtggaggagc ttttgtatgt ggtgaatcta ctgcacttat 6360
gtcgtctata gaaggtatgg taggtgaacc tagagctaaa tatatacaca ctacagaaaa 6420
aggattgtgg ggacaaccta cagttttaaa taatgtagaa acttgggcca atgtacctat 6480
aataattgaa aaaggcggag attggtatca tgctatagga actatggaga agagtaaggg 6540
aacaaaggta ttctcattag ttggaaaagt taagaatact ggacttgtag aagtacctat 6600
gggaactact cttagagaaa taatatatga tattggcggt ggagtattaa atgatagaaa 6660
gtttaaggca gttcaaatag gtggaccttc aggtggatgt ttaccagctg aatatttaga 6720
tttgccagta gattatgata ctttggttaa agcagattcc atgatgggtt caggcggaat 6780
gatcgtaatg gatgatagaa cctgtatggt agatgtaact agatattacc tgagcttctt 6840
ggctgaagaa tcttgtggaa agtgtgtacc ttgtagagaa ggcgtaaaga ggatgcttga 6900
aatactcact gacatatgca atggtgatgg aaaagaagga gacatagaag agcttctcga 6960
aatatgttcc atgacaagca aggcatctct gtgcagtctt ggtaagagtg ctccaaatcc 7020
agtaattgct tctataagat attttagaga tgaatttgaa gagcatataa agaataagag 7080
atgtagagca ggagtttgta agaaacttac tacatttggt atagacgagg ataaatgtaa 7140
gggatgcgat atgtgtaaaa agaattgtcc agctgattgt ataacagggg aaattaagaa 7200
accacataca atagatgctg ataagtgctt gagatgcggt aactgcatga acatctgtaa 7260
gtttgatgct gttaaggttc tatagggagg tgaatgtaga tatgaaaatt acaatagatg 7320
gaaaagcttg tgaagctgaa aaaggagaat tcatattaca aatagcaaga agaaataaca 7380
tatatatacc tacattatgt cacagcgatg cattgcctgg gcttgctagc tgtagactat 7440
gtatagttaa agtagtagat aggggacgtg caaagatagt aacttcctgt atattccctg 7500
taagtaagga agtagaagtt ataactaatg acgatgaaat aaagagaatg agaaaaaaca 7560
tagttatgct tttaaaagta agatgccctg aaaataaaga ggtaaatgaa ttagctaaag 7620
cctttggagt agaggaaaag agagtaaaga ggttcaaatt ggatccagaa caaaattgtg 7680
ttttgtgcgg actttgtgca aaagcttgca aggaattagg tactggagca atttcaacag 7740
ttaatagggg tatgtataaa gaagtagcaa ctccatatca cgaatcttca ccggaatgta 7800
taggatgtgc ttcctgtgca aatgtttgtc caactaatgc aataaaagtt gtggataaag 7860
atggagaaag agaaatatgg ggcaaaaaat tcaagatggt taagtgtgat ttgtgcggag 7920
aatattttgc tacagaagaa catgtaaaat atgcttacaa taggcttgga aaagagcagc 7980
cagaaaaact tatgtgtagc agctgcaaga agaaagttac agccaaagat gtcaaaaata 8040
tttttgagaa cgtgtgaaaa ttaaaagagg ggtggtaatt taaatgaaac cagagtttaa 8100
ttcttttgta atagccgatc ctgacaagtg cataggctgt agatcttgtg agattgcctg 8160
tgctgcaaaa catagagaag atactcaagg aaaaaccatt ggaactatga ataataaagt 8220
tactccaagg ttattctttg ttaaaaataa aggaaatgta atgccagtac aatgcagaca 8280
ttgtgaggat gcaccatgtc taaatgcctg cccagttaat gctatagttg aaaaagatgg 8340
aagtatcatt ataaatgaaa gtgcatgtat aggatgtcag acctgtacaa tagtatgtcc 8400
ggtaggtgct gtaagtttac tgcctagaac tcaaggtaaa gtagttacag gaggaattca 8460
ggttaaagta agagcagcag cttataaatg tgatttatgt aaggaagagg gaggagaacc 8520
tgcttgcgtc aaagaatgtc caaaagaggc cttgaggtta gtagatccta gagaagataa 8580
aaaagatcgt agtgtgaaag ctgctatgga actgttaaat ataaacgcaa atctctaatt 8640
tataagttat aaaaatccgg aaaagaaagg aatgttaaat atgccaacta gtacttctat 8700
gataaatata gatgaagaat tatgtacagg ctgcagacga tgtgcggatg tctgccctgt 8760
agatgctata gaaggtgaac agggtaaacc tcagaagata aatactgaaa agtgtgttat 8820
gtgcggacaa tgcattcaag tttgtaaagg ctatcaatct gtatacgacg atgttcctac 8880
tccagttagc aaaaggttat ttgatagagg attgttaaag gaagtagatg aaccattatt 8940
tgcagcatat aataaaggtc aggtaaagag tgttaaagaa attttacaaa acaaagatgt 9000
atttaaaatt gtgcaatgtg cacctgctgt aagagttgct ataggagagg attttggaat 9060
gcctcttgga actttaagtg aaggaaaaat ggcagctgca ctcagaaaat taggatttga 9120
caaagtatat gatacaaact ttggtgcaga tcttactata atggaagaag gtagtgagtt 9180
actaaaaaga gtagctgaag gcggagtttt gccaatgttt acttcttgtt gtccagcatg 9240
ggtaaaatat gcagaacaaa catatccaga acttttacct catctttcaa gttgtaagtc 9300
tccaaatcag atggctggag ctatatttaa aacttatgga gcagagataa ataaggttaa 9360
tccggctaaa atttataatg tatctgttat gccatgtaca tgcaaggaat ttgaaagtga 9420
aagagaagaa atgcatgaca gtggacacag ggatgtagat gcagttataa ctacaaggga 9480
attagcacaa ctgttcaaag atgctgatat agattttaat actattgaag aagaacagtt 9540
tgatactcct cttggtatgt ataccggtgc aggaactata tttggtgcta caggtggagt 9600
tatggaagca gcacttagaa ctggatatga actttatact aaaaaaacta ttccaagtat 9660
agatcttact atggtaagag gtggagaagg ttttagaact gctgaagtag atttagggga 9720
tattagacta aaagtaggag tagtttccgg cttaaaaaat gtaaaagacg ttatggaatc 9780
agtaaaggca ggcaaatgtg atttgcactt tatagaggtt atgacctgtc ctcaaggatg 9840
tataagtggt ggaggacaac ctaaagttat acttgattca gataaagagg aagcttataa 9900
taataggaaa aagggactat ataatcatga cgctaatctt acttatagaa aatcacatga 9960
aaatccagaa ataaagaaaa tatatgatga gttcttagac aaaccattag gagctaagtc 10020
tcatgaatta ttgcacacta aatatatctc aagaaaaaag gagagttaat aaaatgaaga 10080
attgcctcgt agtagcagat cctaataaat gcataggatg taggacttgt gaagcagctt 10140
gtggtattgc acattcagga ggggactttt ttaacacaaa tgtatccaag attaatttta 10200
atcctcgctt aaatgtgata aaaactgcta aagtaagtgc tcctgttcaa tgcagacaat 10260
gcgaagatgc accttgtggt aaagcttgtc cagttaacgc tatttcaaat gaaaatggtt 10320
atgttagtgt aaataaagat gtatgtgttg gatgtaaaat ctgcatgtta gcttgtcctt 10380
ttggagctat tgaattagct tctcaatata gggatggaga agttgtagac caaaagggac 10440
ttaagatgag tgaggaaggt aatcctactg tgaatggaaa aggaagagtg gtagcaaata 10500
agtgtgatct ctgccaggat agggatggag ggcctgcttg tatagaagtt tgtcctacaa 10560
aatctctcaa actagttact tatgatgaca ataataatat agttgaaaaa aaagatgacg 10620
acgaacgtga agtaagctaa 10640
<210> 66
<211> 10640
<212> DNA
<213> Unknown
<220>
<223> Clostridium ljungdahlii
<400> 66
atggataaaa aagttttaac tgtttgtcct tactgtggcg ctggttgtaa tttatacttg 60
catgtaaaga atggcaaaat aattaaagca gagcctgcta atggtaggac aaatgaagga 120
tcactgtgtt taaaaggaca ctttggttgg gattttttaa acgatcctaa aatattgaca 180
tctagaatta aacatccgat gataagaaaa aacggagagc tagaagaggt aagctgggat 240
gaagctatta gttttacggc ttcaagattg tcacaaataa aagagaaata tggacctgat 300
tccataatgg gaacaggatg tgctaggggt tctggaaacg aagcaaacta cataatgcaa 360
aagtttatga gggcggttat tggaaccaat aacgtagatc actgtgccag agtttgacat 420
gctccttctg tagccggtct ggcttacgtt ttaggaaatg gtgctatgtc aaatggtata 480
catgaaatag atgatacaga tttactactt atttttggat ataatggagc agcttcgcat 540
ccaatagttg ctaagagaat agttagggca aaacaaaagg gtgcaaaggt aatagttgta 600
gatccacgta taacagagtc tggtaggata gcagatttat ggctccctat aaaaaatgga 660
acaaatatgg ttcttgtaaa tacttttgcc aacatactta taaataaaca gttttataac 720
aaacaatatg tagaagatca tactgttggt tttgaagaat atagatctat agttgaaaat 780
tatactcctg aatatgcaga aaaagttact ggcatacctt cagaggatat agtagaagct 840
atgaaaatgt actcaggtgc taaaaatgcc atgatattat atggtatggg agtatgtcaa 900
tttgctcaag ctgtagatgt agttaaggga ctagcttcta tagcattatt aactggtaat 960
tttggaagac ctaatgtagg tataggacct gtaagaggcc agaacaatgt tcaaggtgct 1020
tgtgatatgg gagcacttcc taatgtatac ccaggttatc aaagtgtaac tgacgatgca 1080
attagggaaa aatttgaaaa agcttggggt gttaaacttc caaacaaagt tggttatcac 1140
ctgacacaag ttcctgaatt aacgcttaaa gaggataaaa taaaggcata ttatataatg 1200
ggtgaagatc cagttcaaag tgatcctgat tctaatgaaa tgagagagac actggataaa 1260
atggaacttg taatagttca ggatatattt atgaataaaa ctgcactcca tgcagatgta 1320
attttacctt ccacgtcttg gggagaacat gaaggagtct ttagttctgc agatagagga 1380
ttccagagat ttagaaaagc tgtagaacct aagggagatg ttaaaccaga ttgggaaata 1440
atttcaaaaa ttgcctgtgc tatgggttat aatatgcatt ataacaatac tgaggaaata 1500
tggaatgaac ttataaattt atgtccaaat ttcaaaggag caacttataa gagactcgaa 1560
gaattaggag gaatccaatg gccttgtcca tctgaaaatc atcctggaac ttcttatctc 1620
tacaaaggta ataaatttaa tacacctact ggaaaagcaa acttatttgc agcagaatgg 1680
agacctcctg tagagcagac agataaagat tatccacttg ttctttctac agttagagaa 1740
gtaggacatt attctgtaag aacaatgaca ggaaactgta gggcacttca gcagttagcc 1800
gatgaaccag gatatgtaca agttaatcca atggatgcaa aggctaaggg aataatagat 1860
ggtgagctta tgagaataag ttcacgaaga ggttctgtgg ttgcccgtgc acttattact 1920
gaaagggcaa ataaaggagc agtctatatg acctatcaat ggtgggtagg cgcatgtaat 1980
gaacttacat ctaataatct agatccagta tcaaaaactc ctgaattaaa gtattgtgca 2040
gtaaaaatag aagctataaa agatcagaaa gaagctgaaa agtttataaa agatcaatat 2100
gatcttttaa agaaaaagat gaatgtttaa tattttaata taaagatggc taaaaagacc 2160
ttgattaaga ggtcttttag ccaaaagctt taaatcaata ggagttgata gactgtgata 2220
gacgtacatg aagcattcaa tatagtaatg aataacacaa aactgcttaa aggtgaagat 2280
atatcgttga taaattctct taacagggta ttggcagagg atataagctc aaaagataat 2340
cttcccccat ttgacaaatc ctgtatggat gggtatgctt taaaaagtga agatactaag 2400
gaaaaaatgt caaaatttcg aattaaggga agcataaagg cgggagattt ttctgatata 2460
gtattgaaaa atggtgaagc cataaaaata atgacaggag ctccagtacc aaaaggtgca 2520
gatgcagtta ttcaaataga aaaagtaaaa gtagaaggaa aagaacttca tgtattagaa 2580
aatgtatctc ctggaaccaa catatttaaa actggtgagg agataaaaat tggtgatgtt 2640
gctttaagaa aaggaaagat tttgagacct gcagaaatag ggttattggc atcactaggt 2700
tatactaaaa taaaatgtta taaagcccct aaaattataa taataaatac tggggatgaa 2760
cttataaata tagatcaaaa cttaatgcaa ggtaaaataa ggaattgtaa tgaatataca 2820
ttaattgccc ttattaaaaa tttaaatgca gaagttaaat catatgggat aataagagat 2880
gataaggata agatttttaa tgctataaaa actgcatttg aagagggaga tataatcata 2940
actactggag gagcatctgt aggtgattac gattttatag aagatgttct tcagaaaata 3000
ggaacagata taaagtttac ttcggtagct attaaaccag gaaaaccagt tgtttttgca 3060
acttttaaag ataaattgtt ctttggactt ccaggaaatc cgctttcggt aataaattca 3120
tttgaaagtt ttgtagcacc atctattaaa aaaatgattg gaagagatga tgcgtttcct 3180
gaagaatttc ctgtaacttt aaaagatgat tttaaatcag gaaaagaaag agactgctat 3240
atgtatgtaa acataaaaaa ggaagataac cgttattatg cctatgatgt aggaagacaa 3300
gattccaatg ggctttttac gcttactaag tcaaatggag tcgtcatcat ggaaaaggga 3360
actagtatag caaaagctgg agatatttta aatggaaaat ttatattcaa ataagaggtc 3420
agtaatttct attatttcat ctagttcaaa ttcgggtaag actaccttga tagaaggaat 3480
aataagaatt ctaaaaagca gaggatataa ggttggtgca ataaagaatg atgctcataa 3540
gttacagata gattacccag gaaaagacag ctttagattt acagaggcag gtgcggacaa 3600
tgttgttatt gcatcggata atacagtggc tatgataaaa aaagtaagtg gacccaaaag 3660
tatagaagaa ctactgttgc tttttcaaga tgtagatatt gtaatagtgg aaggcttcaa 3720
gggtaacgaa tttcctaaaa tagaagtata caggaaaaat acaagcaaat gtttacttta 3780
caaaaattct aaatataatt ttcaaaattt tgtagctatt gtaaccaatg aaaacttaat 3840
aactgatatt cctgtatttg atataaatga tacaaaaaaa atagctgatt ttattgagaa 3900
cgactttata ggaggtaaca aaaatggaga aaaccatgga acttcatgta gtcaaatatg 3960
acaaagaatg taaaaagaca gtagaggaat caactatttg tgaatatcct ttaaatgtat 4020
ttgtaaatgg tgaacatttg acagtactct tatgtacgcc tgaaaagctt aaggaattaa 4080
caataggttt cttgaccttt agaggtgtta taaaatctct agatgaaata aaatctctag 4140
agatagatga aaaaagtgga gcgtccaggg taactttgaa aaatagccaa tttaataaag 4200
agttgtattc aaagcaagtg cttcctataa catttaatga aaatgagaaa agtaagttct 4260
tttcgtatat tattgattcc atggaaatta gtataatcaa caatgataat gtttacattc 4320
atgtcgataa aatctatgat ctaatgatgg acaatcttgg atattccaag acgtttaaac 4380
tcactggagg aacacattgt gcagctcttt gtgatgaaga taaagtaata tctatttgtg 4440
aggatgtggc tagacacaat gctgtagaca agcttatagg tgaggcattt ataaaaaata 4500
tttatttaaa ggataaaata atatttgtga gcagcagagt atcttttgaa atggtatata 4560
aaattgctag gctaggggta cctataataa tatctaaatc tgcacctaca agtctttcta 4620
tagaatttgc aaaagcttta aatgttacat taattggatt tgtaagggga gaaagaatga 4680
atgtatatac aaatccacag agaataatat agtaatttct gttaacatat ttttgaatat 4740
aatcttaaaa aattaatcat atagttatat aaaaataata taatatttaa tgttaataat 4800
tagtccctaa ttaaatagga ctaagtttat attttaattt ttaaataagg taattaaaat 4860
aattttaaat taaatgtgtt atatgtttta aaattatttc ttaagtatag aggctcaaat 4920
ctttggttta gagctaatat cttattcctt ctaatatttt aagggggaaa tcaattataa 4980
tattcaaatg ggagggtgaa gtatttaatg ttaactaaac agcaaaatga agacctgtct 5040
ggacaagatg taattgaaaa atatcctaaa gagcagagat ttactcttgc tatactacag 5100
gatatacaga gaaagtacaa atatatacca agagaagcac tggagaattt agctaagtat 5160
ttggacacgc ctgtaagtag actgtatggt atggctactt tttataaggc attgagcctt 5220
actccaaaag gggaaaacat aataactgta tgtgatggaa ccgcttgcca tgttgctggt 5280
tctatggttg taatggatga acttgaaaag gcaataggaa ttaaaccagg tgaaactaca 5340
gaggatctca aattttcaat aaatacagtt aactgtatag gatgctgtgc aatagctcct 5400
gtcatgatga taaatgacaa atattatgga aatttaacac ctaaactggt tgaagaaatt 5460
cttagtgagt ataggagtga gagtgatgag tgataaaaaa actgtcaata tatgttgtgg 5520
gacaggttgt ttagctaaag gcagtatgga agtatatgaa gaaatgaagg cacaaatagc 5580
taaattaggg gcaaatgcag aagtaaatgt taaattaaaa gcaacaggtt gcgatggatt 5640
gtgtgagaaa ggtcctgtac tgaaaatata tccagatgac attgcatatt ttaaagttaa 5700
agtagaagat gtagaagacg tagtaaaaaa gacattgatg aatggggaaa taattgaaaa 5760
attattatat tttgagactg ctacaaaaca gagattaaga aatcataaag aaagtgaatt 5820
ttgtaaaaga caatacaaaa ttgctctcag aaatgttggt gaaatagatc caataagttt 5880
ggaagattat gttgaaagag gcggatacaa agctcttaaa aaagcaataa gcagcatgaa 5940
acctgaagat gtgcttgaag aaataacaaa atcaggtctt agaggaagag gtggagcagg 6000
attcccaaca ggacgtaaat ggaaaactgc tgcagatatt gatacatcac ctatatatgt 6060
agtatgtaat ggtgatgaag gagatcctgg agcatttatg gatagaagta taatggaggg 6120
agatcctaac agtgttatag aaggtatgac attatgtgct tatgcagtag gaggtacaaa 6180
tggatttgct tatataagag atgaatatgg acttgctgta gaaaatatgc agaaagctat 6240
taataaagca aaagatgaaa atttattagg taataatata ttaggaactg atttttcctt 6300
cgatatacag atagtaagag gtggaggagc ttttgtatgt ggtgaatcta ctgcacttat 6360
gtcgtctata gaaggtatgg taggtgaacc tagagctaaa tatatacaca ctacagaaaa 6420
aggattgtgg ggacaaccta cagttttaaa taatgtagaa acttgggcca atgtacctat 6480
aataattgaa aaaggcggag attggtatca tgctatagga actatggaga agagtaaggg 6540
aacaaaggta ttctcattag ttggaaaagt taagaatact ggacttgtag aagtacctat 6600
gggaactact cttagagaaa taatatatga tattggcggt ggagtattaa atgatagaaa 6660
gtttaaggca gttcaaatag gtggaccttc aggtggatgt ttaccagctg aatatttaga 6720
tttgccagta gattatgata ctttggttaa agcagattcc atgatgggtt caggcggaat 6780
gatcgtaatg gatgatagaa cctgtatggt agatgtaact agatattact tgagcttctt 6840
ggctgaagaa tcttgtggaa agtgtgtacc ttgtagagaa ggcgtaaaga ggatgcttga 6900
aatactcact gacatatgca atggtgatgg aaaagaagga gacatagaag agcttcttga 6960
aatatgttcc atgacaagca aggcatctct gtgcagtctt ggtaagagtg ctccaaatcc 7020
agtaattgct tctataagat attttagaga tgagtttgaa gagcatataa agaataagag 7080
atgtagagca ggagtttgta agaaacttac tacatttggt atagacgagg ataaatgtaa 7140
gggatgcgat atgtgtaaaa agaattgtcc agctgattgt ataacagggg aaattaagaa 7200
accacataca atagatgctg ataagtgctt gagatgcggt aactgcatga acatctgtaa 7260
gtttgatgct gttaaggttc tatagggagg tgaatgtaga tatgaaaatt acaatagatg 7320
gaaaagcttg tgaagctgaa aaaggagaat tcatattaca aatagcaaga agaaataaca 7380
tatatatacc tacattatgt cacagcgatg cattgcctgg gcttgctagc tgtagactat 7440
gtatagttaa agtagtagat aggggacgtg caaagatagt aacttcctgt atattccctg 7500
taagtaagga agtagaagtt ataactaatg acgatgaaat aaagagaatg agaaaaaaca 7560
tagttatgct tttaaaagta agatgccctg aaaataaaga ggtaaatgaa ttagctaaag 7620
cctttggagt agaggaaaag agagtaaaga ggttcaaatt ggatccagaa caaaattgtg 7680
ttttgtgcgg actttgtgca aaagcttgca aggaattagg tactggagca atttcaacag 7740
ttaatagggg tatgtataaa gaagtagcaa ctccatatca cgaatcttca ccggaatgta 7800
taggatgtgc ttcctgtgca aatgtttgtc caactaatgc aataaaagtt gtggataaag 7860
atggagaaag agaaatatgg ggcaaaaaat tcaagatggt taagtgtgat ttgtgcggag 7920
aatattttgc tacagaagaa catgtaaaat atgcttacaa taggcttgga aaagagcagc 7980
cagaaaaact tatgtgcagc agctgcaaga agaaaattac agccaaagat gtcaaaaata 8040
tttttgagaa cgtgtgaaaa ttaaaagagg ggtggtaatt taaatgaaac cagagtttaa 8100
ttcttttgta atagccgatc ctgacaagtg cataggctgt agatcttgtg agattgcctg 8160
tgctgcaaaa catagagaag atactcaagg aaaaaccatt ggaactatga ataataaagt 8220
tactccaagg ttattctttg ttaaaaataa aggaaatgta atgccagtac aatgcagaca 8280
ttgtgaggat gcaccatgtc taaatgcctg cccagttaat gctatagttg aaaaagatgg 8340
aagtatcatt ataaatgaaa gtgcatgtat aggatgccag acctgtacaa tagtatgtcc 8400
ggtaggtgct gtaagtttac tgcctagaac tcaaggtaaa gtagttacag gaggaattca 8460
ggttaaagta agagcagcag cttataaatg tgatttatgt aaggaagagg gaggagaacc 8520
tgcttgcgtc aaagaatgtc caaaagaggc cttgaggtta gtagatccta gagaagataa 8580
aaaagatcgt agtgtgaaag ctgctatgga actgttaaat ataaacgcaa atctctaatt 8640
tataagttat aaaaatccgg aaaagaaagg aatgttaaat atgccaacta gtacttctat 8700
gataaatata gatgaagaat tatgtacagg ctgcagacga tgtgcggatg tctgccctgt 8760
agatgctata gaaggtgaac agggtaaacc tcagaagata aatactgaaa agtgtgttat 8820
gtgcggacaa tgcattcaag tttgtaaagg ctatcaatct gtatacgacg atgttcctac 8880
tccagttagc aaaaggttat ttgatagagg attgttaaag gaagtagatg aaccattatt 8940
tgcagcatat aataaaggtc aggcaaagag tgttaaagaa attttacaaa acaaagatgt 9000
atttaaaatt gtgcaatgtg cacctgctgt aagagttgct ataggagagg attttggaat 9060
gcctcttgga actttaagtg aaggaaaaat ggcagctgca ctcagaaaat taggatttga 9120
caaagtatat gatacaaact ttggtgcaga tcttactata atggaagaag gtagtgagtt 9180
actaaaaaga gtagctgaag gcggagtttt gccaatgttt acttcttgtt gtccagcatg 9240
ggtaaaatat gcagaacaaa catatccaga acttttacct catctttcaa gttgtaagtc 9300
tccaaatcag atggctggag ctatatttaa aacttatgga gcagagataa ataaggttaa 9360
tccggctaaa atttataatg tatctgttat gccatgtaca tgcaaggaat ttgaaagtga 9420
aagagaagaa atgcatgaca gtggacacag ggatgtagat gcagttataa ctacaaggga 9480
attagcacaa ctgttcaaag atgctgatat agattttaat actattgaag aagaacagtt 9540
tgatactcct cttggtatgt ataccggtgc aggaactata tttggtgcta caggtggagt 9600
tatggaagca gcacttagaa ctggatatga actttatact aaaaaaacta ttccaagtat 9660
agatcttact atggtaagag gtggagaagg ttttagaact gctgaagtag atttagggga 9720
tattagacta aaagtaggag tagtttccgg cttaaaaaat gtaaaagacg ttatggaatc 9780
agtaaaggca ggcaaatgtg atttgcactt tatagaggtt atgacctgtc ctcaaggatg 9840
tataagtggt ggaggacaac ctaaagttat acttgattca gataaagagg aagcttataa 9900
taataggaaa aagggactat ataatcatga cgctaatctt acttatagaa aatcacatga 9960
aaatccagaa ataaagaaaa tatatgatga gttcttagac aaaccattag gagctaagtc 10020
tcatgaatta ttgcacacta aatatatctc aagaaaaaag gagagttaat aaaatgaaga 10080
attgcctcgt agtagcagat cctaataaat gcataggatg taggacttgt gaagcagctt 10140
gtggtattgc acattcagga ggggactttt ttaacacaaa tgtatccaag attaatttta 10200
atcctcgctt aaatgtgata aaaactgcta aagtaagtgc tcctgttcaa tgcagacaat 10260
gcgaagatgc accttgtggt aaagcttgtc cagttaacgc tatttcaaat gaaaatggtt 10320
atgttagtgt aaataaagat gtatgtgttg gatgtaaaat ctgcatgtta gcttgtcctt 10380
ttggagctat tgaattagct tctcaatata gggatggaga agttgtagac caaaagggac 10440
ttaagatgag tgaggaaggt aatcctactg tgaatggaaa aggaagagtg gtagcaaata 10500
agtgtgatct ctgccaggat agggatggag ggcctgcttg tatagaagtt tgtcctacaa 10560
aatctctcaa actagttact tatgatgaca ataataatat agttgaaaaa aaagatgacg 10620
acgaacgtga agtaagctaa 10640
<210> 67
<211> 10633
<212> DNA
<213> Unknown
<220>
<223> Clostridium ragsdalei
<400> 67
atggataaaa aagttttaac tgtttgtcct tactgcggtg ctggttgtaa gttatacttg 60
catgtaaagg atggcaagat aattaaagca gagcctgcta atggtaggac aaatgaaggg 120
tccttgtgtt taaaaggacg atttggctgg gattttctaa atgatcctaa aatattgaca 180
tctagaatta aacatccaat gataagaaaa aatggagagt tagaagaggt aagttgggat 240
gaagctatta gttttacggc ttcaaaattg tcacaaataa aagagaaata tggacctgat 300
tctataatgg gaacaggatg tgctaggggt tctggaaatg aagcaaacta cgtaatgcaa 360
aagtttatga gggcggttat tggaaccaat aacgtagatc actgtgccag agtttgacat 420
gctccttctg tagccggtct ggcttacgtt ttaggaaatg gtgctatgtc aaatggtata 480
catgaaatag atgatacaga tttactactt atttttggat ataatggagc agcttcgcat 540
ccaatagttg ctaagagaat agttagggca aaacaaaaag gtgcaaaggt aatagttgta 600
gatccacgta taacagagtc tggtaggata gcagatttat ggctccctat aaaaaatgga 660
acaaatatgg ttcttgtaaa tacttttgcc aatatactta taaacaagca attttatgac 720
aaacaatatg tagaagatca tactgttggt tttgaagaat ataaatctat agttgaggat 780
tatacgcctg aatatgcaga aaaagttact ggtatacctg cagaggatat agtagaagct 840
atgaaaatgt actccagtgc taaaaatgct atgatattgt acggtatggg agtatgtcag 900
tttgctcaag ctgtagatgt agtaaaaggg ttagcttcaa tagctttatt aactggtaat 960
tttggaagac ctaatgtagg cataggacct gtaagaggcc agaacaatgt gcaaggtgcc 1020
tgcgatatgg gagcacttcc taatgtatac ccaggttatc aaagtgtaac tgacgatgca 1080
attagagaaa aatttgaaaa agcttgggga gttaaacttt caaacaaagt tggttatcac 1140
ctgacacgag ttcctgaatt aacgcttaaa gaggataaaa taaaagcata ttatataatg 1200
ggcgaagatc cagctcaaag tgatcctgat tctaatgaaa tgagggaaac acttgataaa 1260
atggaacttg taatagttca agatatattt atgaataaaa ctgcactcca tgcagatgta 1320
attttacctt ctacgtcttg gggagaacat gaaggagtct tcagttctgc tgatagagga 1380
ttccagagat ttagaaaagc tgtagaacct aagggcgatg ttaaaccaga ttgggagata 1440
atttcagaaa ttgcatgtgc tatgggttat gatatgcatt ataacaatac tgaggaaata 1500
tgggatgaac ttataaattt atgcccaaat ttcaaaggag caacttataa gagattggat 1560
gaattaggag gaattcaatg gccttgtcca tctgaagatc atccaggaac ttcttatctc 1620
tacaaaggaa ataaatttaa tacacctact ggaaaagcaa atttatttgc agcagaatgg 1680
agacctccta tagagaagac agatgaagaa tatccacttg ttctttctac agttagagaa 1740
gtagggcatt actccgtaag aacaatgaca ggaaactgta gggcactcca gcagttagct 1800
gatgaaccag gatatgtaca aattaatcca gtggatgcaa aggctaaaaa aataatagat 1860
ggtgagctta tgagagtaag ttcacgaaga ggttctgtag ttgcccgtgc acttgttact 1920
gaaagggcaa ataaaggagc agtttatatg acctatcaat ggtgggtagg tgcatgtaat 1980
gagcttacag ctaataattt agatccagta tcaaaaactc ctgaattaaa gtattgtgca 2040
gtgaaggtag aagctataga agatcagaaa gaagctgaaa agtttataaa agatcaatat 2100
gcttcaataa agaaaaagat gaatgtttaa tataaagatg gctaaaaaga ccttgattaa 2160
aaggtctttt agccaaaagc tttaaatcaa taggagttga tagactgtga tagacgtaca 2220
tgaagcattc aatatagtaa tgaataatac aaaactgctt aaaagtgaag atatgtccct 2280
gataaattct cttaacaggg tattggcaga ggatataagc tcaaaagata atcttccccc 2340
atttgacaaa tcctgtatgg atgggtatgc tttaaaaagt gaagatacta aggaaaaaat 2400
gtcaaaattt caaattaagg gaagcataaa ggcgggagat ttttctgata tagtattaaa 2460
aaatggtgaa gccataaaaa taatgacagg agctccagta ccaaaaggtg cagatgcaat 2520
tattcaaata gaaaaagtaa aagtagaagg aaaagaactt catgtattgg aaaaggtatc 2580
tcctggaacc aacatattta aaactggtga ggagataaaa attggtgatg ttgctttaaa 2640
aaagggaaaa atcgtaagac ctgcagaaat agggttattg gcatcactag gttatactaa 2700
aataaaatgc tacaaagtcc ctaaaattat aataataaat acaggggatg aacttataaa 2760
tatagatcaa aacttaatgc aaggtaaaat aagaaattgt aatgaataca cattaattgc 2820
ccttattaaa aatttaaatg cagaagttaa atcgtatggg ataataagag atgataagaa 2880
taaaattttt aatgctataa aaactgcatt tgaagaggga gatataatca taactactgg 2940
aggagcatct gtgggtgatt acgattttat agaagatgtt cttaaggaaa taggaacaga 3000
tataaaattt acttcggtag ctattaaacc aggaaaacca gttgtttttg caacttttaa 3060
agataaatta ttctttggac ttccaggaaa tccactttca gtaataaatt catttgaaag 3120
ttttgtagca ccatctatta aaaaaaatga ttggaagaga tgatgcattt cctgaagaat 3180
ttcctgtaac tttaaaagat gattttaagt caggaaaaga aagagactgt tatatgtatg 3240
tagatataaa aaaggaagat aaccattatt atgcttatga tgtaggaaga caagattcca 3300
atggcctttt tacacttact aagtcaaatg gcgtggttat catgaaaaag ggaactagta 3360
tagcaaaagc tggagatatt ttaaatggaa aatttatatt caaataagag gtcagtaatt 3420
tctattattt catctagttc aaattcaggt aagactacct taatagaagg aataataaga 3480
attttaaaaa gcagaggata taaggttggt gcaataaaga atgatgctca taagttacag 3540
atagattatc caggaaaaga cagctttaga tttacagagg caggtgcaga caatgttgtt 3600
attgcatcgg ataatacagt ggctatgata aaaaaagtaa gtggaccaaa aagtatagaa 3660
gaattgctgt tgctttttca ggatgtagat attgtaatag tggaaggttt taagggcaac 3720
gaatttccta aaatagaagt atacaggaaa aatgcaagta aatgtttact ctacaaaaat 3780
tctgaatata attttcaaaa ttttgtagct attgtaacca atgaaaattt aataactgat 3840
attcctgtat ttgatataaa tgatacaaaa aaagtagctg attttattga aagcaacttt 3900
ataggaggta acaaaaatgg agaaaaccat ggaacttcat gtagtcaaat atgacaaaga 3960
atgtaaaaag acagtggagg aatcaactat ttgcgaatat cctttaaatg tatttgtaaa 4020
tggtgaacat ttgacagtgc tcttatgtac tcctgaaaag cttaaggaat taacaatagg 4080
tttcttggtc tttaaaggta ttataaaatc tctagatgaa ataaaatctt tagagataga 4140
tgaaaaaagt ggagcgtcca gggtaatctt gaaaaataga caatttaata aagagttgta 4200
ttcaaagcaa gtacttccta caacatttaa tgaaaatgaa aaaagtaaat tcttttcata 4260
tattattgat tccatgaaaa ttagtctaat caataatgat aatgtttaca tacatgttga 4320
taaaatctat ggcttaatga tggacaatct tggatattcc aagactttta aactcactgg 4380
aggagcacat tgtgcagctc tttgtgatga agataaagta atatctattt gtgaggatgt 4440
ggccagacac aatgctgtag acaagcttat aggtgaggca tttataaaaa atatttgttt 4500
aaaagataaa ataatatttg tgagtagcag ggtatctttt gaaatggtat ataaaattgc 4560
tagacttggg gtacctataa taatatctaa atctgcacct acaaatcttt ctatagaatt 4620
tgcaaaagct ttaaatgtta cattaattgg atttgtaagg ggagaaagaa tgaatgtata 4680
tacaaatcca cagagaataa tatagtaatt tctgataata catttttgaa tataatctta 4740
aaaaattagt catatagtta tataaaaata atataatatt taatgttgat aattagttct 4800
taattaaata ggactaaatt tatattttaa tttttaaata aggtaattaa aataatttta 4860
aattaaatat gttatatgtt ttaaaattat ttcttaagca tagaggctca aatctttgat 4920
ttagagctaa tatcttattc cttctaatat tttaaggggg aaatcaatta taatattcaa 4980
atgggagggt gaagtattta atgttaacta aacagcaaaa tgaagacctg tctggacaag 5040
atgtaattga aaaatatcct aaagagcaga gatttactct tgctatacta caggatatac 5100
agagaaagta caaatatata cccagagaag cactggagaa tttagctaag tatttggaca 5160
cgcctgtaag tagactgtat ggtatggcta ctttttataa ggcattgagc cttactccaa 5220
aaggggaaaa cataataact gtatgtgatg gaaccgcttg ccatgttgct ggttctatgg 5280
ttgtaatgga tgaacttgaa aaggcaatag gaattaaacc aggtgaaact acagaagatc 5340
ttaaattttc aataaataca gttaactgta taggatgctg tgcaatagct cctgtcatga 5400
tgataaatga caaatatttt ggaaatttaa cacctaaact ggttgaagaa attcttagtg 5460
agtataggag tgaaagccat gagtgataaa aaaattgtca atatatgttg tggaacaggt 5520
tgcttagcta aaggcagcaa ggaagtatat gaagaaatga aggcacaaat agctaaatta 5580
ggggcaaatg cagaagtaaa tgttaaatta aaagcaacag gttgcgatgg attgtgtgag 5640
aaaggtcctg tactgaaaat atatccagat gacattgcat attttaaagt taaagtagaa 5700
gatgtagaag acgtagtaaa aaagacattg atgaatgggg aaataattga aaaattatta 5760
tattttgaaa ctgctacaaa acagagatta agaaatcata aagaaagtga attttgtaaa 5820
agacaataca aaattgctct cagaaatgtt ggtgaaatag atccaataag tttggaagat 5880
tatgttgaaa gaggcggata taaagctctt aaaaaagcaa taagcagcat gaaacctgaa 5940
gatgtgcttg aagaaataac aaaatcaggt cttagaggaa gaggtggagc aggattccca 6000
acaggacgta aatggaaaac tgctgcagat attgatacat cacctatata tgtagtatgc 6060
aatggtgatg agggagatcc tggagcattt atggatagaa gtataatgga gggagatcct 6120
aacagtgtta tagaaggtat gacattgtgt gcctatgcag taggaggtac aaacggcttt 6180
gcttatataa gagatgaata tggacttgct gtagaaaata tgcagaaagc tattaataaa 6240
gcaaaagatg aaaatttatt aggtaataat atattaggaa ctgacttttc cttcgatata 6300
cagatagtaa gaggtggagg agcttttgta tgtggtgagt ctactgcact tatgtcatct 6360
atagaaggta tggtaggaga acctagagct aaatatatac acactacaga aaaaggattg 6420
tggggacaac ctactgtttt aaataatgta gaaacttggg ccaatgtacc tataataatt 6480
gaaaaaggtg gagattggta tcatgctata ggaactatgg agaagagtaa gggaacaaaa 6540
gtattctcat tagttggaaa agttaagaat actggacttg tagaagtacc tatgggaact 6600
actcttagag aaataatata tgatattggc ggtggagtat taaacgacag aaagtttaag 6660
gcagttcaaa taggtggacc ttcaggagga tgtttaccat ctgaatattt agacttgcca 6720
gtagattatg atactttggt taaagcggat tctatgatgg gttccggcgg aatgatcgta 6780
atggatgata gaacctgtat ggtagatgta actagatatt acttgagttt cttagctgaa 6840
gaatcttgtg gaaagtgtgt accttgtaga gaaggcgtaa agagaatgct tgaaatactc 6900
actgatatat gcaatggtga tggaaaagaa ggagacatag aagagcttct tgaaatatgt 6960
tccatgacaa gcaaggcatc tctgtgcagt cttggtaaga gtgctccaaa tccagtaaaa 7020
gcagctataa gatattttag agatgaattt gaagaacata taaagaataa gagatgtaga 7080
gcaggagttt gtaagaaact tactacattt ggtatagatc aagataaatg taagggatgc 7140
gatatgtgta aaaagaattg tccagctgat tgtataacag gggaaattaa gaaaccacat 7200
acaatagatg ctgataagtg cttgagatgc ggtaactgca tgaacatctg taagtttgat 7260
gctgttaagg ttttataggg aggtgaatgt agatatgaaa attacaatag atggaaaagc 7320
ttgtgaagct gaaaaaggag aattcatatt acaaatagca agaagaaata atatatatat 7380
acctacactg tgccacagtg atgcattgcc tgggcttgct agctgtagac tgtgtatagt 7440
taaagtagta gataggggac gtgcaaagat agtaacttcc tgtatattcc ctgtaagtaa 7500
ggaagtagaa gttataacta atgacgatga aataaagaga atgagaaaaa acatagttat 7560
gcttttaaaa gtaagatgcc ctgaaaataa agaagtaaat gaattagcta aagcctttgg 7620
agtagaggaa aagagagtaa agaggttcaa attggatcca gaacaaaatt gtgttttgtg 7680
cggactttgt gcaaaagctt gcaaggaatt aggtactgga gcaatctcaa cagttaatag 7740
gggtatgtat aaagaagtag caactccata tcacgaatct tcaccagaat gtataggatg 7800
tgcttcctgt gcaaatgttt gtccaactaa tgcaataaaa gttgtggata aagatggaga 7860
aagagaaata tggggcaaaa aattcaagat ggttaaatgt gatttgtgcg gagaatattt 7920
tgctacagaa gaacacgtaa aatatgctta caataggctt ggaaaagagc agccagaaaa 7980
gcttatgtgc agcagctgca agaagaaagt tacagccaaa gatgtcaaaa atatttttga 8040
gaacgtgtga aaattaaaag aggggtggta atttaaatga aaccagagtt taattctttt 8100
gtaatagccg atcctgacaa gtgcataggc tgtagatctt gtgagattgc ctgtgctgca 8160
aaacatagag aagatactca aggaaaaact attggaacta tgaataataa agttactcca 8220
aggttattct ttgttaaaaa taaaggaaat gtaatgccgg tacaatgcag acattgtgag 8280
gatgcaccat gtctaaatgc ctgcccagtt aatgctatag ttgaaaaaga tggaagtatc 8340
attataaatg aaagtgcatg tatagggtgt cagacctgta caatagtatg tccggtaggt 8400
gctgtaagtt tacttcctag aactcaaggt aaagtagtta caggaggaat tcaggttaaa 8460
gtaagagcag cagcttataa atgtgattta tgtaaggaag agggaggaga acctgcctgc 8520
gtcaaagaat gtccaaaaga ggccttaagg ttagtagatc ctagagaaga taaaaaagat 8580
cgtagtgtga aagctgctat ggaactgtta aatataaacg caaatctcta atttataagt 8640
tataaaaatc cggaaaagaa aggaatgtta aatatgccaa ctagtacttc tatgataaat 8700
atagatgaag aattatgtac aggctgcaga cgatgtgcgg atgtctgccc tgtagatgct 8760
atagaaggtg aacagggtaa acctcagaag ataaatactg aaaagtgtgt tatgtgcgga 8820
caatgcattc aagtttgtaa aggctatcaa tctgtatacg atgatattcc tactccagtt 8880
agcaaaaggt tatttgatag aggattgtta aaggaagtag atgaaccatt atttgcagca 8940
tataataaag gtcaggcaaa gagagttaaa gaaattttac aaaacaaaga tgtatttaaa 9000
attgtgcaat gtgcacctgc tgtaagagtt gctataggag aggattttgg aatgcctctt 9060
ggaactttaa gtgaaggaaa aatggcagct gcactcagaa aattaggatt tgacaaagta 9120
tatgatacaa actttggtgc agatcttact ataatggaag aaggtagtga gttactaaaa 9180
agagtagctg aaggtggagt tttgccaatg tttacttctt gttgtccagc atgggtaaaa 9240
tatgcagaac aaacatatcc agaactttta cctcatcttt caagttgtaa gtctccaaat 9300
cagatggctg gagctatatt taaaacttat ggagcagaga taaataaggt taatccggct 9360
aaaatttata atgtatctgt tatgccatgt acatgcaagg aatttgaaag tgaaagagaa 9420
gaaatgcatg acagtggaca cagagatgta gatgcagtta taactacaag ggaattagca 9480
caactgttca aagatgctga tatagatttt aatactattg aagaagaaca gtttgatact 9540
cctcttggta tgtatactgg tgcaggaact atatttggtg ctacaggtgg agttatggaa 9600
gcagcactta gaactggata tgaactttat actaaaaaaa ctattccaag tatagatctt 9660
actatggtaa gaggtggaga aggttttaga actgctgaag tagatttagg agatattaga 9720
ctaaaagtag gagtagtttc cggcttaaaa aatgtaaaag atgttatgga atcagtaaag 9780
gcaggtaaat gtgatttaca ctttatagag gttatgactt gtcctcaagg atgtataagt 9840
ggtggaggac aacctaaggt tatacttgat tcagataaag aagaagctta taataatagg 9900
aaaaagggac tatataatca tgacgctaat cttacttata gaaaatcaca tgaaaatcca 9960
gaaataaaga aaatatatga tgagttctta gacaaaccat taggagctaa gtctcatgaa 10020
ttattgcaca ctaaatatat ctcaagaaaa aaggagagtt aataaaatga agaattgcct 10080
cgtagtagca gatcctaata aatgcatagg atgtaggact tgtgaagcag cttgtggtat 10140
tgcacattca ggaggggact tttttaatac aaatgtatcc aaaattaatt ttaatcctcg 10200
cttaaatgtg ataaaaactg ctaaagtaag tgctcctgtt caatgcagac aatgcgaaga 10260
tgcaccttgt ggtaaagctt gtccagttaa cgctatttca aatgaaaatg gttatgttag 10320
tgtagataaa gatgtatgtg ttggatgtaa aatctgcatg ttagcttgtc cttttggagc 10380
tattgaatta gcttctcaat ataaggatgg agaagttgta gaccaaaagg gacttaagat 10440
gagtgaggaa ggtaatccta ctgtaaatgg aaaaggaaga gtggtagcaa ataagtgtga 10500
tctttgccag gatagggatg gaggacctgc ttgcatagaa gtttgtccta caaaatctct 10560
caaattagtt acttatgatg acaataataa tatagttgaa aaaaaagatg acgacgaacg 10620
tgaagtaggc taa 10633
Claims (46)
- 하나 이상의 외인성 NADPH-의존적 효소를 발현하도록 개조되고,
상기 NADPH-의존적 효소는 수소화효소, 포르메이트 탈수소화효소, 메틸렌-THF-탈수소화효소, 하이드록시메틸글루타릴-CoA(HMG-CoA) 환원효소, 3-하이드록시부티릴-CoA 탈수화효소(아세토아세틸 CoA 환원효소로도 알려짐), 트랜스-2-에노일-CoA 환원효소, NADH/NADPH 상호-의존적 효소, 또는 NADH/NADPH 이분성 효소이고,
모체 미생물에 비해 증가된 NADPH 활용도를 갖는, 재조합 클로스트리듐(Clostridium) 미생물. - 청구항 1에 있어서, 상기 수소화효소는 이분성 NADP Fe-유일 수소화효소인, 미생물.
- 청구항 1에 있어서, 상기 포르메이트 탈수소화효소는 이분성 NADP 포르메이트 탈수소화효소인, 미생물.
- 청구항 1에 있어서, 상기 3-하이드록시부티릴-CoA 탈수화효소는 phaB(EC 1.1.1.36)인, 미생물.
- 청구항 4에 있어서, 상기 미생물이 외인성 3-하이드록시부티릴-CoA 탈수화효소 phaJ(EC 4.2.1.119)를 더 포함하는, 미생물.
- 청구항 4에 있어서, 상기 미생물이 외인성 NADH-의존적 3-하이드록시부티릴-CoA 탈수화효소 hbd(EC 1.1.1.157)를 더 포함하는, 미생물.
- 청구항 1에 있어서, 상기 트랜스-2-에노일-CoA 환원효소는 크로토닐-CoA 환원효소(부티릴-CoA 탈수소화효소로도 알려짐)인, 미생물.
- 청구항 7에 있어서, 상기 크로토닐-CoA 환원효소는 ccr(EC 1.3.1.86) 또는 ccrRs (EC 1.3.1.85)인, 미생물.
- 청구항 8에 있어서, 상기 미생물이 외인성 NADH-의존 크로토닐-CoA 환원효소(부티릴-CoA 탈수소화효소로도 알려짐)ter(EC 1.3.1.44)를 더 포함하는, 미생물.
- 청구항 1에 있어서, 상기 미생물이 모체 미생물에 비해 하나 이상의 발효 생성물의 증가된 생산을 갖는, 미생물.
- 청구항 1에 있어서, 상기 NADPH-의존적 효소의 NADH-의존적 이소형이 모체 미생물에 비해 약화 또는 녹아웃(knocked out)된, 미생물.
- 청구항 1에 있어서, 상기 미생물이 일산화탄소영양 박테리움인, 미생물.
- 청구항 1에 있어서, 상기 미생물이 클로스트리듐 오토에타노게눔(Clostridium autoethanogenum), 클로스트리듐 륭달리이(Clostridium ljungdahlii), 클로스트리듐 라그스달레이(Clostridium ragsdalei), 클로스트리듐 카복시디보란스(Clostridium carboxidivorans), 클로스트리듐 드라케이(Clostridium drakei), 클로스트리듐 스카톨로게네스(Clostridium scatologenes), 클로스트리듐 아세티쿰(Clostridium aceticum), 클로스트리듐 포르미코아세티쿰(Clostridium formicoaceticum), 및 클로스트리듐 마그눔(Clostridium magnum) 으로 이루어진 군으로부터 선택되는 모체 미생물로부터 유래되는, 미생물.
- 청구항 13에 있어서, 상기 클로스트리듐 오토에타노게눔은 DSMZ 수탁번호 DSM23693 하에 기탁된 클로스트리듐 오토에타노게눔인, 미생물.
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Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201361758441P | 2013-01-30 | 2013-01-30 | |
| US61/758,441 | 2013-01-30 | ||
| US201361828675P | 2013-05-29 | 2013-05-29 | |
| US61/828,675 | 2013-05-29 | ||
| PCT/US2014/013712 WO2014120852A2 (en) | 2013-01-30 | 2014-01-30 | Recombinant microorganisms comprising nadph dependent enzymes and methods of production thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20150110806A KR20150110806A (ko) | 2015-10-02 |
| KR102290728B1 true KR102290728B1 (ko) | 2021-08-19 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020157023535A Active KR102290728B1 (ko) | 2013-01-30 | 2014-01-30 | Nadph 의존적 효소를 포함하는 재조합 미생물 및 이의 생산방법 |
Country Status (11)
| Country | Link |
|---|---|
| US (1) | US9422565B2 (ko) |
| EP (1) | EP2951286B1 (ko) |
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| GB201507509D0 (en) * | 2015-04-30 | 2015-06-17 | Inside Biometrics Ltd | Electrochemical Test Device |
| KR102390716B1 (ko) * | 2015-05-27 | 2022-04-26 | 란자테크 엔지, 인크. | 코리스메이트-유도된 산물의 생산을 위한 유전적으로 조작된 미생물들 |
| CA2993327A1 (en) * | 2015-07-23 | 2017-01-26 | The Trustees Of Dartmouth College | Thermophilic microorganisms for conversion of lignocellulosic biomass to ethanol |
| CN106635936A (zh) * | 2015-11-04 | 2017-05-10 | 中国科学院青岛生物能源与过程研究所 | 一种联产琥珀酸和异戊二烯的基因工程菌及其构建方法 |
| US10358661B2 (en) * | 2015-12-28 | 2019-07-23 | Lanzatech New Zealand Limited | Microorganism with modified hydrogenase activity |
| CN108603178A (zh) * | 2016-01-06 | 2018-09-28 | 株式会社西化学 | Co水合酶以及使用其产生甲酸的方法 |
| GB201707034D0 (en) | 2017-05-03 | 2017-06-14 | Gradley Michell Lorraine | Modified microorganisms and methods for production of useful products |
| CN114437981A (zh) * | 2022-02-15 | 2022-05-06 | 科盛环保科技股份有限公司 | 一种快速培养硫自养反硝化细菌的发酵方法 |
| JP2024013695A (ja) | 2022-07-21 | 2024-02-01 | Tdk株式会社 | 磁気センサ、回転角検知装置、及び回転角検知装置を用いたブレーキシステム |
| WO2024191356A1 (en) * | 2023-03-14 | 2024-09-19 | Vidyasirimedhi Institute Of Science And Technology | A recombinant cell for generating energy-rich metabolites and a method for the same |
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| US20110236941A1 (en) * | 2010-10-22 | 2011-09-29 | Lanzatech New Zealand Limited | Recombinant microorganism and methods of production thereof |
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| UA72220C2 (uk) | 1998-09-08 | 2005-02-15 | Байоенджініерінг Рісорсиз, Інк. | Незмішувана з водою суміш розчинник/співрозчинник для екстрагування оцтової кислоти, спосіб одержання оцтової кислоти (варіанти), спосіб анаеробного мікробного бродіння для одержання оцтової кислоти (варіанти), модифікований розчинник та спосіб його одержання |
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| WO2009036095A1 (en) * | 2007-09-10 | 2009-03-19 | Joule Biotechnologies, Inc. | Engineered light-harvesting organisms |
| JP2011160673A (ja) * | 2010-02-04 | 2011-08-25 | Fujirebio Inc | 水素ガスの生産方法及びそのための微生物 |
| US20110236941A1 (en) * | 2010-10-22 | 2011-09-29 | Lanzatech New Zealand Limited | Recombinant microorganism and methods of production thereof |
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| CN104955938A (zh) | 2015-09-30 |
| CA2899587C (en) | 2019-09-17 |
| CN104955938B (zh) | 2018-03-02 |
| US9422565B2 (en) | 2016-08-23 |
| EP2951286B1 (en) | 2023-09-13 |
| JP2016508718A (ja) | 2016-03-24 |
| AU2014212462A1 (en) | 2015-08-20 |
| EP2951286A4 (en) | 2016-08-10 |
| KR20150110806A (ko) | 2015-10-02 |
| WO2014120852A3 (en) | 2015-02-05 |
| JP2020096602A (ja) | 2020-06-25 |
| CA2899587A1 (en) | 2014-08-07 |
| FI2951286T3 (fi) | 2023-11-20 |
| EP2951286A2 (en) | 2015-12-09 |
| JP7525541B2 (ja) | 2024-07-30 |
| JP2022116003A (ja) | 2022-08-09 |
| ES2961674T3 (es) | 2024-03-13 |
| AU2014212462B2 (en) | 2019-01-31 |
| PT2951286T (pt) | 2023-11-02 |
| US20140212976A1 (en) | 2014-07-31 |
| WO2014120852A2 (en) | 2014-08-07 |
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