KR20230000995A - 신규한 폴리-4-하이드록시부티레이트 및 1,4-부탄다이올 생산방법 - Google Patents
신규한 폴리-4-하이드록시부티레이트 및 1,4-부탄다이올 생산방법 Download PDFInfo
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Abstract
본 출원은 신규한 폴리-4-하이드록시부티레이트 및/또는 1,4-부탄다이올 생산방법 및 폴리-4-하이드록시부티레이트 생산 경로를 이용하는 미생물에 관한 것이다.
Description
본 출원은 신규한 폴리-4-하이드록시부티레이트 및/또는 1,4-부탄다이올 생산방법 및 폴리-4-하이드록시부티레이트 생산 경로를 이용하는 미생물에 관한 것이다.
1,4-부탄다이올을 생산하기 위하여, 고효율 생산 미생물 및 발효공정기술 개발을 위한 다양한 연구들이 수행되고 있다. 미생물을 이용한 1,4-부탄다이올 생산은 대체로 4-히드록시부틸알데히드 생성 반응을 동반하나(US 9121042 B2), 4-히드록시부틸알데히드는 알데히드의 일종으로서 미생물에 유해한 단점이 있다.
또한, 현재까지 알려진 바이오 1,4-부탄다이올 생산 기술 중 하나로서, 1,4-부탄다이올 직접 발효로 포도당에서 발효를 통해 1,4-부탄다이올을 미생물에서 바로 생산하는 방법이나, 1,4-부탄다이올 자체의 미생물에 대한 독성으로 인해 생산성에 제약이 있다. 따라서, 효과적인 폴리-4-하이드록시부티레이트 및/또는 1,4-부탄다이올의 생산능 증가를 위한 연구가 여전히 필요한 실정이다.
본 출원의 하나의 목적은 1,4-부탄다이올 생산방법을 제공한다.
본 출원의 하나의 목적은 폴리-4-하이드록시부티레이트 및/또는 1,4-부탄다이올 생산용 미생물을 제공한다.
본 출원의 하나의 목적은 폴리-4-하이드록시부티레이트 생산방법을 제공한다.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 출원에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 출원에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 출원의 범주가 제한된다고 볼 수 없다. 또한, 본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
본 출원의 하나의 양태는 하기 (1) 내지 (5)를 포함하는 1,4-부탄다이올 생산방법을 제공한다:
(1) 숙시닐-coA (succinyl-coA; SuCoA)를 숙시네이트 세미알데하이드 (succinate semialdehyde; SSA)로 전환하는 단계;
(2) 숙시네이트 세미알데하이드 (succinate semialdehyde; SSA)를 4-하이드록시부티레이트(4-hydroxybutyrate; 4HB)로 전환하는 단계;
(3) 4-하이드록시부티레이트(4-hydroxybutyrate; 4HB)를 4-하이드록시부티릴 coA(4-hydroxybutyryl coA; 4HBCoA)로 전환하는 단계;
(4) 둘 이상의 4-하이드록시부티릴 coA(4-hydroxybutyryl coA; 4HBCoA)를 중합하여 폴리-4-하이드록시부티레이트(poly-4-hydroxybutyrate; P4HB)를 생산하는 단계; 및
(5) 폴리-4-하이드록시부티레이트를 1,4-부탄다이올로 분해시키는 단계.
상기 (1) 내지 (4)는 각각 숙시네이트 세미알데하이드 디하이드로게나제(succinate semialdehyde dehydrogenase), 4-하이드록시부티릭산 디하이드로게나제 (4-hydroxybutyric acid dehydrogenase), 4-하이드록시부티릴-coA 트랜스퍼라제 (4-hydroxybutyryl-CoA transferase), 및 폴리(3-하이드록시알카노에이트) 폴리머라제(Poly(3-hydroxyalkanoate) polymerase)로 이루어지는 군에서 선택되는 어느 하나 이상의 폴리펩티드; 상기 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 벡터, 또는 이들의 조합을 포함하는 미생물; 및 이의 배양물로 구성되는 군에서 선택되는 어느 하나 이상을 이용하는 것일 수 있으나, 이에 제한되지 않는다.
상기 숙시네이트 세미알데하이드 디하이드로게나제, 4-하이드록시부티릭산 디하이드로게나제, 4-하이드록시부티릴-coA 트랜스퍼라제, 및 폴리(3-하이드록시알카노에이트) 폴리머라제로 이루어진 군에서 선택되는 어느 하나 이상은 외래 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 또는 이를 포함하는 벡터를 포함하는 미생물에 의해 강화된 것일 수 있으나, 이에 제한되지 않는다.
포도당 발효로 직접 1,4-부탄다이올을 생산하는 경우에는 1,4-부탄다이올의 미생물에 대한 독성으로 인해 생산성에 제약이 있다. 반면, 본 출원에서 세포 친화적인 폴리-4-하이드록시부티레이트를 우선적으로 생산하므로 본 출원의 생산방법은 폴리-4-하이드록시부티레이트 생산성이 증가된 것일 수 있다. 또한, 본 출원은 폴리-4-하이드록시부티레이트 생산 단계에서 유전자를 도입하여 옥살로아세테이트를 이용한 환원형 TCA 경로를 활성화; 고생산성 발효공정; 및/또는 환원형 TCA 경로를 통해 발효에서 발생하는 이산화탄소를 재이용으로 폴리-4-하이드록시부티레이트 생산능이 증가된 것일 수 있다. 또한, 이와 같은 폴리-4-하이드록시부티레이트 생산성 증가에 따라 1,4-부탄다이올의 생산성이 추가로 증가된 것일 수 있다.
본 출원의 1,4-부탄다이올 생산방법은 TCA 경로(TCA cycle), 환원형 TCA 경로(reductive TCA cycle), 및 글리옥실레이트 경로(glyoxylate cycle)로 이루어진 군에서 선택되는 어느 하나 이상의 경로를 더 포함할 수 있다. 상기 경로는 숙시닐-coA로 전환하는 것일 수 있다.
일 구현 예로, 본 출원의 1,4-부탄다이올 생산방법은 TCA 경로를 포함하는 것일 수 있다. 이 때, 포도당 한 분자는 해당과정(glycolysis pathway)을 통해 생산된 피루베이트가 TCA 경로를 거쳐 숙시닐-coA로 전환될 수 있다.
상기 TCA 경로는 (a1) 피루베이트를 아세틸-coA로 전환하는 단계; (b1) 아세틸-coA 및 옥살로아세테이트를 시트레이트로 전환하는 단계; (c1) 시트레이트를 아이소시트레이트로 전환하는 단계; (d1) 아이소시트레이트를 α-케토글루타레이트로 전환하는 단계; (e1) α-케토글루타레이트를 숙시닐-coA로 전환하는 단계; 및 (f1) 피루베이트를 옥살로아세테이트로 전환하는 단계로 구성되는 군에서 선택되는 어느 하나 이상을 포함하는 것일 수 있다.
상기 (d1)은 아이소시트레이트에서 α-케토글루타레이트 및 이산화탄소로 전환되는 단계일 수 있고, 상기 (e1)은 α-케토글루타레이트에서 숙시닐-coA 및 이산화탄소로 전환되는 단계일 수 있다.
일 구현 예로, 상기 (a1) 내지 (f1)은 각각 피루베이트 디하이드로게나제(pyruvate dehydrogenase), 시트레이트 신타아제(citrate synthase), 아코니타제(aconitase), 아이소시트레이트 디하이드로게나제(isocitrate dehydrogenase), α-케토글루타레이트 디하이드로게나제(α-ketoglutarate dehydrogenase), 및 피루베이트 카복실라아제(pyruvate carboxylase)로 이루어지는 군에서 선택되는 어느 하나 이상의 폴리펩티드; 상기 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 벡터, 또는 이들의 조합을 포함하는 미생물; 및 이의 배양물로 구성되는 군에서 선택되는 어느 하나 이상을 이용하는 것일 수 있으나, 이에 제한되지 않는다.
상기 생산방법은 (g1) 포스포에놀피루베이트를 옥살로아세테이트로 전환하는 단계를 더 포함하는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 상기 (g1) 은 포스포에놀피루베이트 카복실라제(phosphoenolpyruvate carboxylase) 폴리펩티드; 상기 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 벡터, 또는 이들의 조합을 포함하는 미생물; 및 이의 배양물로 구성되는 군에서 선택되는 어느 하나 이상을 이용하는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 생산방법은 질소, 황, 인, 및 마그네슘으로 구성되는 군에서 선택되는 하나 이상의 제한 조건에서 포스포에놀피루베이트 카복실라제 유전자(ppc)의 전사가 저해받지 않는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 생산방법은 질소, 인, 황, 및 마그네슘으로 구성되는 군에서 선택되는 어느 하나 이상을 제한시키는 단계를 포함하는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 생산방법은 질소, 인, 황, 및 마그네슘으로 구성되는 군에서 선택되는 어느 하나 이상이 제한시키는 단계를 포함하더라도 상기 제한시키는 단계를 포함하지 않는 방법에 비해 폴리-4-하이드록시부티레이트 및/또는 1,4-부탄다이올이 생산이 감소되지 않는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 상기 전사는 프로모터에 의해 저해받지 않는 것일 수 있으나, 이에 제한되지 않는다. 상기 프로모터는 프로모터 활성을 가지는 서열번호 45로 표시되는 폴리뉴클레오티드일 수 있고, 상기 프로모터 활성을 가지는 서열번호 45로 표시되는 뉴클레오티드 서열의 목적 유전자는 포스포에놀피루베이트 카복실라제를 코딩하는 폴리뉴클레오티드일 수 있다. 질소제한 조건에서 ppc 의 전사가 저해받지 않는 프로모터를 사용할 경우 rTCA 경로가 강화되고 폴리-4-하이드록시부티레이트 및/또는 1,4-부탄다이올의 생산이 증가될 수 있다. 질소제한 조건에서 포스포에놀피루베이트 카복실라제 유전자(ppc)의 전사가 저해받지 않으면서 야생형 ppc 프로모터와 동등 이상의 활성을 갖는 프로모터를 사용할 경우, 폴리-4-하이드록시부티레이트 및/또는 1,4-부탄다이올의 생산에 효과적일 수 있다.
일 구현 예로, 상기 생산방법은 (g1) 단계가 강화된 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 1,4-부탄다이올 생산방법은 환원형 TCA 경로를 포함하는 것일 수 있다. 상기 환원형 TCA 경로를 통해 옥살로아세테이트는 산화적 TCA 경로에 포함된 카복시이탈(decarboxylation) 과정을 거치지 않아 부가적인 이산화탄소 발생 없이 말레이트(malate), 푸마레이트(fumarate), 및 숙시네이트(succinate)를 거쳐 숙시닐-coA로 전환될 수 있다.
상기 환원형 TCA 경로는 (a2) 옥살로아세테이트를 말레이트로 전환하는 단계; (b2) 말레이트를 푸마레이트로 전환하는 단계; (c2) 푸마레이트를 숙시네이트로 전환하는 단계; 및 (d2) 숙시네이트를 숙시닐-coA로 전환하는 단계로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 것일 수 있으나, 이에 제한되지 않는다.
본 출원의 생산방법은 상기 환원형 TCA 경로에 앞서, (e2) 포스포에놀피루베이트를 옥살로아세테이트로 전환하는 단계가 더 포함된 것일 수 있다. 상기 (e2)는 상기 (g1)와 동일할 수 있다.
일 구현 예로, 본 출원의 생산방법은 환원형 TCA 경로가 강화된 것일 수 있으며, 상기 환원형 TCA 경로 강화는 (e2) 포스포에놀피루베이트를 옥살로아세테이트로 전환하는 단계의 강화를 포함할 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 상기 환원형 TCA 경로는 하기 (I) 내지 (XII)로 이루어진 군에서 선택되는 어느 하나 이상에 의해 강화된 것일 수 있으나, 이에 제한되지 않는다:
(I) 피루베이트 키나아제(pyruvate kinase) 약화; (II) 포스포에놀피루베이트 카복실라제(PEP carboxylase) 강화; (III) 카보닉 안하이드라제(carbonic anhydrase) 강화; (IV) 시트레이트 신타아제(citrate synthase) 조절; (V) 피루베이트 카복실라제(pyruvate carboxylase) 강화; (VI) NAD+-의존성 말레이트 디하이드로게나제(NAD+-dependent malate dehydrogenase) 약화; (VII) NADP+-의존성 말레이트 디하이드로게나제(NADP+-dependent malate dehydrogenase) 약화; (VIII) 포스포글루코네이트 디하이드라타아제(phosphogluconate dehydratase) 약화; (IX) 2-케토-4-하이드록시글루타레이트:2-케토-3-디옥시글루코네이트 6-포스페이트 알돌레이즈(2-keto-4-hydroxyglutarate:2-keto-3-deoxygluconate 6-phosphate aldolase; KHG/KDPG aldolase) 약화; (X) 아스파테이트 아미노트랜스퍼라제(aspartate aminotransferase) 약화; (XI) 글루코스-특이적 PTS 효소 IIBC 컴포넌트(glucose-specific PTS enzyme IIBC component) 약화; 및 (XII) 바이카보네이트 트랜스포터(bicarbonate transporter) 강화.
일 구현 예로, 상기 포스포에놀피루베이트를 옥살로아세테이트로 전환하는 단계는 피루베이트 키나아제 약화 및 카보닉 안하이드라제 강화; 피루베이트 키나아제 약화 및 포스포에놀피루베이트 카복실라제 강화; 시트레이트 신타아제 조절 및 포스포에놀피루베이트 카복실라제 강화; 시트레이트 신타아제 조절 및 카보닉 안하이드라제 강화; 및 피루베이트 키나아제 약화, 포스포에놀피루베이트 카복실라제 강화, 및 피루베이트 카복실라제 강화에 의해 강화된 것일 수 있으며, 선택적으로 카보닉 안하이드라제 강화, NAD+-의존성 말레이트 디하이드로게나제 약화, NADP+-의존성 말레이트 디하이드로게나제 약화, 포스포글루코네이트 디하이드라타아제 약화, KHG/KDPG 알돌레이즈 약화, 아스파테이트 아미노트랜스퍼라제 약화, 글루코스-특이적 PTS 효소 IIBC 컴포넌트 약화; 및/또는 바이카보네이트 트랜스포터 강화로 구성되는 군에서 선택되는 어느 하나 이상에 의해 강화된 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 상기 환원형 TCA 경로는 (II) 포스포에놀피루베이트 카복실라제 강화; (VI) NAD+-의존성 말레이트 디하이드로게나제 약화;
(VII) NADP+-의존성 말레이트 디하이드로게나제 약화; 및/또는
(X) 아스파테이트 아미노트랜스퍼라제 약화를 포함하는 것일 수 있다.
일 구현 예로, 상기 환원형 TCA 경로는 (II) 포스포에놀피루베이트 카복실라제가 강화된 것일 수 있다.
일 구현 예로, 상기 환원형 TCA 경로는 (VI) NAD+-의존성 말레이트 디하이드로게나제 및 (VII) NADP+-의존성 말레이트 디하이드로게나제가 약화된 것일 수 있다.
일 구현 예로, 상기 환원형 TCA 경로는 (X) 아스파테이트 아미노트랜스퍼라제가 약화된 것일 수 있다.
일 구현 예로, 본 출원의 rTCA 경로를 통해 발효에서 발생되는 이산화탄소를 재이용하여 폴리-4-하이드록시부티레이트 및/또는 1,4-부탄다이올 생산 수율을 증가시키는 것일 수 있다.
일 구현 예로, 본 출원의 1,4-부탄다이올 생산방법은 글리옥실레이트 경로를 포함하는 것일 수 있다.
상기 글리옥실레이트 경로는 (a3) 아이소시트레이트를 글리옥실레이트(glyoxylate) 및 숙시네이트(succinate)로 전환하는 단계; (b3) 글리옥실레이트(glyoxylate) 및 아세틸-coA를 말레이트 및 coA로 전환하는 단계; (c3) 시트레이트를 아이소시트레이트로 전환하는 단계; (d3) 피루베이트를 옥살로아세테이트로 전환하는 단계; (e3) 포스포에놀피루베이트를 옥살로아세테이트로 전환하는 단계; (f3) 옥살로아세테이트 및 아세틸-coA를 시트레이트로 전환하는 단계; (g3) 말레이트를 푸마레이트로 전환하는 단계; (h3) 푸마레이트를 숙시네이트로 전환하는 단계; 및 (i3) 숙시네이트를 숙시닐-coA로 전환하는 단계로 구성되는 군에서 어느 하나 이상을 더 포함하는 것일 수 있으나, 이에 제한되지 않는다. 상기 (f3)은 (b1)과, 상기 (g3)은 (b2)와, 상기 (h3)은 (c2)와, 상기 (i3)은 상기 (d1)와 동일할 수 있다.
일 구현 예로, 상기 글리옥실레이트 경로는 (i) 시트레이트 신타아제(citrate synthase) 강화; (ii) 아이소시트레이트 디하이드로게나제(isocitrate dehydrogenase) 약화; (iii) 아이소시트레이트 리아제(isocitrate lyase) 강화; (iv) 아이소시트레이트 디하이드로게나제 키나아제/포스파테이즈(Isocitrate dehydrogenase kinase/phosphatase) 강화; (v) 말레이트 신타아제 G(malate synthase G) 강화; 및 (vi) 말레이트 신타아제 A(malate synthase A) 강화로 이루어진 군에서 선택되는 어느 하나 이상에 의한 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 상기 글리옥실레이트 경로를 포함하는 본 출원의 방법은 (j3) α-케토글루타레이트를 숙시닐-coA로 전환하는 단계 및/또는 (k3) 옥살로아세테이트를 말레이트로 전환하는 단계가 더 약화된 것일 수 있으나, 이에 제한되지 않는다.
상기 글리옥실레이트 경로의 생성물인 숙시네이트 및 말레이트는 모두 환원형 TCA 경로를 이용하여 숙시닐-coA로 전환될 수 있다.
본 출원에서, "숙시네이트 세미알데하이드 디하이드로게나제(succinate semialdehyde dehydrogenase)"는 숙시닐-coA (succinyl-coA; SuCoA)가 숙시네이트 세미알데하이드 (succinate semialdehyde; SSA)로 전환되는 반응을 촉매할 수 있는 효소이다. 상기 숙시네이트 세미알데하이드 디하이드로게나제는 SucD와 혼용될 수 있다.
일 구현 예로, 본 출원의 SucD 단백질은 클로스트리듐 클루이베리(Clostridium Kluyveri) 유래일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 SucD에 포함된다. 일 구현 예로, 본 출원의 SucD 단백질은 서열번호 1 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 숙시네이트 세미알데하이드 디하이드로게나제 유전자는 sucD, 숙시네이트 세미알데하이드 디하이드로게나제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 sucD 유전자는 예를 들면 서열번호 2의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "4-하이드록시부티릭산 디하이드로게나제 (4-hydroxybutyric acid dehydrogenase)"는 숙시네이트 세미알데하이드 (succinate semialdehyde; SSA)가 4-하이드록시부티레이트(4-hydroxybutyrate; 4HB)로 전환되는 반응을 촉매할 수 있는 효소이다. 상기 4-하이드록시부티릭산 디하이드로게나제는 숙시네이트 세미알데하이드 리덕타제(succinate semialdehyde reductase) 및 4HbD와 혼용될 수 있다.
일 구현 예로, 본 출원의 4HbD 단백질은 아라비돕시스 탈리아나(Arabidopsis thaliana) 유래일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 4HbD에 포함된다. 일 구현 예로, 본 출원의 4HbD 단백질은 서열번호 3 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 4-하이드록시부티릭산 디하이드로게나제 유전자는 4hbD, 4-하이드록시부티릭산 디하이드로게나제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 4hbD 유전자는 예를 들면 서열번호 4의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "4-하이드록시부티릴-coA 트랜스퍼라제 (4-hydroxybutyryl-CoA transferase)"는 4-하이드록시부티레이트(4-hydroxybutyrate; 4HB)가 4-하이드록시부티릴 coA(4-hydroxybutyryl coA; 4HBCoA)로 전환되는 반응을 촉매할 수 있는 효소이다. 상기 4-하이드록시부티릴-coA 트랜스퍼라제는 OrfZ와 혼용될 수 있다.
일 구현 예로, 본 출원의 OrfZ 단백질은 클로스트리듐 클루이베리(Clostridium Kluyveri) 유래일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 OrfZ에 포함된다. 일 구현 예로, 본 출원의 OrfZ 단백질은 서열번호 5 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 4-하이드록시부티릴-coA 트랜스퍼라제 유전자는 orfZ, 4-하이드록시부티릴-coA 트랜스퍼라제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 orfZ 유전자는 예를 들면 서열번호 6의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 상기 SucD, 4HbD, 및/또는 OrfZ 의 아미노산 및 유전자 서열은 US 9084467 B2에서 얻을 수 있으나, 이에 제한되지 않는다.
본 출원에서, "폴리(3-하이드록시알카노에이트) 폴리머라제(Poly(3-hydroxyalkanoate) polymerase)"는 둘 이상의 4-하이드록시부티릴 coA(4-hydroxybutyryl coA; 4HBCoA)를 폴리-4-하이드록시부티레이트(poly-4-hydroxybutyrate; P4HB)로 중합하는 반응을 촉매할 수 있는 효소이다. 상기 폴리(3-하이드록시알카노에이트) 폴리머라제는 PhaC와 혼용될 수 있다.
일 구현 예로, 본 출원의 PhaC 단백질은 다양한 미생물 유래일 수 있고, 구체적으로는 슈도모나스 푸티다(Pseudomonas putida) 또는 랄스토니아 유트로파(Ralstonia eutropha) 유래일 수 있으며, 이들 유래의 융합 단백질일 수 있고 이와 동일한 서열 또는 활성을 갖는 한 PhaC에 포함된다.
일 구현 예로, 본 출원의 PhaC 단백질은 서열번호 7 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
일 구현 예로, 상기 PhaC의 아미노산 서열 및 유전자 서열은 WO 2014058655 A1에서 얻을 수 있으나, 이에 제한되지 않는다.
본 출원에서, 폴리(3-하이드록시알카노에이트) 폴리머라제 유전자는 phaC, 폴리(3-하이드록시알카노에이트) 폴리머라제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 phaC 유전자는 예를 들면 서열번호 8의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, 피루베이트 디하이드로게나제(pyruvate dehydrogenase)는 피루베이트가 아세틸-coA로 전환되는 반응을 촉매할 수 있는 효소; 시트레이트 신타아제(citrate synthase)는 옥살로아세테이트 및 아세틸-coA를 축합하여 시트레이트를 생성하는 반응을 촉매할 수 있는 효소; 아코니타제(aconitase)는 시트레이트가 아이소시트레이트로 전환되는 반응을 촉매할 수 있는 효소; 아이소시트레이트 디하이드로게나제(isocitrate dehydrogenase)는 아이소시트레이트가 α-케토글루타레이트로 전환되는 반응을 촉매할 수 있는 효소; α-케토글루타레이트 디하이드로게나제(α-ketoglutarate dehydrogenase)는 α-케토글루타레이트가 숙시닐-coA로 전환되는 반응을 촉매할 수 있는 효소; 숙시닐-coA 신테타아제(succinyl-coA synthetase)는 숙시닐-coA가 숙시네이트로 전환되는 반응을 촉매할 수 있는 효소; 및 피루베이트 카복실라아제(pyruvate carboxylase)는 피루베이트가 옥살로아세테이트로 전환되는 반응을 촉매할 수 있는 효소를 의미한다.
상기 피루베이트 디하이드로게나제 내지 피루베이트 카복실라아제는 TCA 경로에 포함되는 효소일 수 있으며, 미생물 또는 생산방법에 내재적인 것일 수 있고, 또는 야생형 효소에 비해 강화된 것일 수 있다.
본 출원에서, "피루베이트 키나아제(pyruvate kinase)"는 포스포에놀피루베이트(phosphoenolpyruvate; PEP)가 피루베이트로 전환되는 반응을 촉매할 수 있는 효소이다. 상기 피루베이트 키나아제는 Pyk와 혼용될 수 있다.
일 구현 예로, 본 출원의 Pyk 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 Pyk에 포함된다.
일 구현 예로, 본 출원의 Pyk 단백질은 약화된 것일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 Pyk에 포함된다. 일 구현 예로, 본 출원의 Pyk 단백질은 서열번호 9, 서열번호 11 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 피루베이트 키나아제 유전자는 pykA, pykF, 및 피루베이트 키나아제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 pykA, pykF 유전자는 예를 들면 서열번호 10 또는 서열번호 12의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "포스포에놀피루베이트 카복실라제(PEP carboxylase)"는 포스포에놀피루베이트가 옥살로아세테이트로 전환되는 반응을 촉매하는 효소이다. 상기 포스포에놀피루베이트 카복실라제는 PPC와 혼용될 수 있다.
일 구현 예로, 본 출원의 PPC 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 PPC에 포함된다.
일 구현 예로, 본 출원의 PPC 단백질은 강화된 것일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 PPC에 포함된다. 일 구현 예로, 본 출원의 PPC 단백질은 서열번호 13 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 포스포에놀피루베이트 카복실라제 유전자는 ppc 및 포스포에놀피루베이트 카복실라제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 ppc 유전자는 예를 들면 서열번호 14 의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 미생물은 질소(nitrogen), 황(sulfur), 인(phosphorous), 및 마그네슘(magnesium)으로 구성되는 군에서 선택되는 어느 하나 이상의 영양소 제한 조건에서도 폴리-4-하이드록시부티레이트 생산능이 있고, 특히, 이의 증가된 생산능을 갖는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 미생물은 질소(nitrogen), 황(sulfur), 인(phosphorous), 및/또는 마그네슘(magnesium) 제한 조건에서 포스포에놀피루베이트 카복실라제 유전자(ppc)의 전사가 저해받지 않는 프로모터를 더 포함하는 것일 수 있다. 상기 프로모터는 프로모터 활성을 가지는 서열번호 45로 표시되는 폴리뉴클레오티드일 수 있고, 상기 프로모터 활성을 가지는 서열번호 45로 표시되는 뉴클레오티드 서열의 목적 유전자는 포스포에놀피루베이트 카복실라제를 코딩하는 폴리뉴클레오티드일 수 있다. 질소제한 조건에서 ppc 의 전사가 저해받지 않는 프로모터를 사용할 경우 폴리-4-하이드록시부티레이트 및/또는 1,4-부탄다이올의 생산이 증가될 수 있다. 기존 대장균의 ppc 발현은 질소제한 조건에서 저해를 받으나, 미생물은 질소제한 조건에서 포스포에놀피루베이트 카복실라제 유전자(ppc)의 전사가 저해받지 않으면서 야생형 ppc 프로모터와 동등 이상의 활성을 갖는 프로모터를 사용할 경우, 폴리-4-하이드록시부티레이트 및/또는 1,4-부탄다이올의 생산에 효과적일 수 있다.
본 출원에서, "카보닉 안하이드라제(carbonic anhydrase)"는 하이드로젠카보네이트(hydrogencarbonate)를 이산화탄소 및 물로 분해하는 것을 촉매할 수 있는 효소이다. 상기 카보닉 안하이드라제는 포스포에놀피루베이트 카복실라제의 보조 역할을 수행하는 효소일 수 있고, PPC의 원활한 성능발휘를 위해서는 중탄산염(Bicarbonate; HCO3-) 공급이 필요한데, 카보닉 안하이드라제는 이산화탄소로부터 HCO3-를 생산할 수 있는 효소일 수 있다. 상기 카보닉 안하이드라제는 EcaA와 혼용될 수 있다.
일 구현 예로, 본 출원의 EcaA 단백질은 노스톡 속(Nostoc sp.) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 EcaA에 포함된다.
일 구현 예로, 본 출원의 EcaA 단백질은 강화된 것일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 EcaA에 포함된다. 일 구현 예로, 본 출원의 EcaA 단백질은 서열번호 15 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 카보닉 안하이드라제 유전자는 ecaA 및 카보닉 안하이드라제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 ecaA 유전자는 예를 들면 서열번호 16의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "시트레이트 신타아제(citrate synthase)" 옥살로아세테이트 및 아세틸-coA를 축합하여 시트레이트를 생성하는 반응을 촉매할 수 있는 효소이다. 상기 시트레이트 신타아제는 GltA와 혼용될 수 있다.
일 구현 예로, 본 출원의 GltA 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 GltA에 포함된다.
일 구현 예로, 본 출원의 GltA 단백질은 조절된 것일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 GltA에 포함된다. 일 구현 예로, 본 출원의 GltA 단백질은 서열번호 17 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 시트레이트 신타아제 유전자는 gltA 및 시트레이트 신타아제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 gltA 유전자는 예를 들면 서열번호 18의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "피루베이트 카복실라제(pyruvate carboxylase)"는 포스포에놀피루베이트가 옥살로아세테이트로 전환되는 반응을 촉매할 수 있는 효소이다. 상기 피루베이트 카복실라제는 Pyc와 혼용될 수 있다.
일 구현 예로, 본 출원의 Pyc 단백질은 리조븀 에틀리(Rhizobium etli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 Pyc 에 포함된다.
일 구현 예로, 본 출원의 Pyc 단백질은 강화된 것일 수 있고, 외래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 Pyc에 포함된다. 일 구현 예로, 본 출원의 Pyc 단백질은 서열번호 19 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 피루베이트 카복실라제 유전자는 pyc 및 피루베이트 카복실라제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 pyc 유전자는 예를 들면 서열번호 20의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "NAD+-의존성 말레이트 디하이드로게나제(NAD+-dependent malate dehydrogenase)"는 말레이트를 피루베이트로 전환하는 반응을 촉매할 수 있는 효소이다. 상기 NAD+-의존성 말레이트 디하이드로게나제는 MaeA와 혼용될 수 있다.
일 구현 예로, 본 출원의 MaeA 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 MaeA에 포함된다.
일 구현 예로, 본 출원의 MaeA 단백질은 약화된 것일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 MaeA 에 포함된다. 일 구현 예로, 본 출원의 MaeA 단백질은 서열번호 21 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, NAD+-의존성 말레이트 디하이드로게나제 유전자는 maeA 및 NAD+-의존성 말레이트 디하이드로게나제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 maeA 유전자는 예를 들면 서열번호 22의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "NADP+-의존성 말레이트 디하이드로게나제(NADP+-dependent malate dehydrogenase)"는 말레이트를 피루베이트로 전환하는 반응을 촉매할 수 있는 효소이다. 상기 NADP+-의존성 말레이트 디하이드로게나제는 MaeB와 혼용될 수 있다.
일 구현 예로, 본 출원의 MaeB 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 MaeB에 포함된다.
일 구현 예로, 본 출원의 MaeB 단백질은 약화된 것일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 MaeB 에 포함된다. 일 구현 예로, 본 출원의 MaeB 단백질은 서열번호 23 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, NADP+-의존성 말레이트 디하이드로게나제 유전자는 maeB 및 NADP+-의존성 말레이트 디하이드로게나제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 maeB 유전자는 예를 들면 서열번호 24의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "포스포글루코네이트 디하이드라타아제(phosphogluconate dehydratase"는 6-포스포-D-글루코네이트(6-phospho-D-gluconate)가 2-디하이드로-3-디옥시-6-포스포-D-글루코네이트(2-dehydro-3-deoxy-6-phospho-D-gluconate)로 전환되는 반응을 촉매할 수 있는 효소이다. 상기 포스포글루코네이트 디하이드라타아제는 EDD와 혼용될 수 있다.
일 구현 예로, 본 출원의 EDD 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 EDD에 포함된다.
일 구현 예로, 본 출원의 EDD 단백질은 약화된 것일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 EDD 에 포함된다. 일 구현 예로, 본 출원의 EDD 단백질은 서열번호 25 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 포스포글루코네이트 디하이드라타아제 유전자는 edd 및 포스포글루코네이트 디하이드라타아제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 edd 유전자는 예를 들면 서열번호 26의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "2-케토-4-하이드록시글루타레이트:2-케토-3-디옥시글루코네이트 6-포스페이트 알돌레이즈(2-keto-4-hydroxyglutarate:2-keto-3-deoxygluconate 6-phosphate aldolase; KHG/KDPG aldolase)"는 4-하이드록시-2-옥소글루타레이트를 글리옥실레이트 및 피루베이트로의 전환을 촉매할 수 있는 효소이다. 상기 포스포글루코네이트 디하이드라타아제는 KHG/KDPG 알돌레이즈, Eda와 혼용될 수 있다.
일 구현 예로, 본 출원의 Eda 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 Eda에 포함된다.
일 구현 예로, 본 출원의 Eda 단백질은 약화된 것일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 Eda 에 포함된다. 일 구현 예로, 본 출원의 Eda 단백질은 서열번호 27 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, KHG/KDPG 알돌레이즈 유전자는 eda 및 KHG/KDPG 알돌레이즈를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 eda 유전자는 예를 들면 서열번호 28의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "아스파테이트 아미노트랜스퍼라제(aspartate aminotransferase)"는 2-옥소글루타레이트 및 아스파테이트를 글루타메이트 및 옥소아세테이트로 전환하는 반응을 촉매할 수 있는 효소이다. 상기 아스파테이트 아미노트랜스퍼라제는 AspC와 혼용될 수 있다.
일 구현 예로, 본 출원의 AspC 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 AspC에 포함된다.
일 구현 예로, 본 출원의 AspC 단백질은 약화된 것일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 AspC 에 포함된다. 일 구현 예로, 본 출원의 AspC 단백질은 서열번호 29 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 아스파테이트 아미노트랜스퍼라제 유전자는 aspC, 아스파테이트 아미노트랜스퍼라제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 aspC 유전자는 예를 들면 서열번호 30의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "글루코스-특이적 PTS 효소 IIBC 컴포넌트(glucose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system(PTS) enzyme IIBC component)"는 포도당 수송에 관여하는 효소인 PTS 의 부분이다. 일 구현 예로, 상기 글루코스-특이적 PTS 효소 IIBC 컴포넌트는 포스포에놀피루베이트 pool을 증가시켜, 환원형 TCA 경로를 강화시킬 수 있다. 상기 글루코스-특이적 PTS 효소 IIBC 컴포넌트는 IIBC와 혼용될 수 있다.
일 구현 예로, 본 출원의 IIBC 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 IIBC에 포함된다.
일 구현 예로, 본 출원의 IIBC 단백질은 약화된 것일 수 있고, 이와 동일한 서열 또는 활성을 갖는 한 IIBC에 포함된다. 일 구현 예로, 본 출원의 IIBC 단백질은 서열번호 31 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, 글루코스-특이적 PTS 효소 IIBC 컴포넌트 유전자는 ptsG, 글루코스-특이적 PTS 효소 IIBC 컴포넌트를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 ptsG 유전자는 예를 들면 서열번호 32의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "바이카보네이트 트랜스포터(bicarbonate transporter)"는 중탄산염(bicarbonate)을 수송하는 운반체로서, 이산화탄소의 세포 내 유입을 증가시킬 수 있다. 상기 바이카보네이트 트랜스포터 는 SbtA와 혼용될 수 있다.
일 구현 예로, 본 출원의 SbtA 단백질은 시네코시스티스 속(Synechocyctis sp.)유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 IIBC에 포함된다.
일 구현 예로, 본 출원의 SbtA 단백질은 강화된 것일 수 있고, 외래일 수 있으며, 시아노박테리아(Cyanobacteria) 유래일 수 있으나, 이와 동일한 서열 또는 활성을 갖는 한 SbtA에 포함된다. 일 구현 예로, 본 출원의 SbtA 단백질은 서열번호 33 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, SbtA 유전자는 sbtA, 바이카보네이트 트랜스포터를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 sbtA 유전자는 예를 들면 서열번호 34의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "아이소시트레이트 디하이드로게나제(isocitrate dehydrogenase)"는 아이소시트레이트를 2-옥소글루타레이트로 전환하는 반응을 촉매할 수 있는 효소이다. 상기 아이소시트레이트 디하이드로게나제는 Icd와 혼용될 수 있다.
일 구현 예로, 본 출원의 Icd 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 Icd에 포함된다.
일 구현 예로, 본 출원의 Icd 단백질은 약화된 것일 수 있으나, 이와 동일한 서열 또는 활성을 갖는 한 Icd에 포함된다. 일 구현 예로, 본 출원의 Icd 단백질은 서열번호 35 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, Icd 유전자는 icd, 아이소시트레이트 디하이드로게나제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 icd 유전자는 예를 들면 서열번호 36의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "아이소시트레이트 리아제(isocitrate lyase)"아이소시트레이트를 글리옥실레이트 및 숙시네이트로 전환하는 반응을 촉매할 수 있는 효소이다. 상기 아이소시트레이트 리아제는 AceA와 혼용될 수 있다.
일 구현 예로, 본 출원의 AceA 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 AceA에 포함된다.
일 구현 예로, 본 출원의 AceA 단백질은 강화된 것일 수 있으나, 이와 동일한 서열 또는 활성을 갖는 한 AceA에 포함된다. 일 구현 예로, 본 출원의 AceA 단백질은 서열번호 37 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, AceA 유전자는 aceA, 아이소시트레이트 리아제를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 aceA 유전자는 예를 들면 서열번호 38의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "아이소시트레이트 디하이드로게나제 키나아제/포스파테이즈(Isocitrate dehydrogenase kinase/phosphatase)"는 아이소시트레이트 디하이드로게나제를 인산화 또는 탈인산화를 촉매할 수 있는 효소이다. 일 구현 예로, 상기 아이소시트레이트 디하이드로게나제 키나아제/포스파테이즈는 Icd를 약화시킬 수 있다, 상기 아이소시트레이트 디하이드로게나제 키나아제/포스파테이즈는 AceK와 혼용될 수 있다.
일 구현 예로, 본 출원의 AceK 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 AceK에 포함된다.
일 구현 예로, 본 출원의 AceK 단백질은 강화된 것일 수 있으나, 이와 동일한 서열 또는 활성을 갖는 한 AceK에 포함된다. 일 구현 예로, 본 출원의 AceK 단백질은 서열번호 39, 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, AceK 유전자는 aceK, 아이소시트레이트 디하이드로게나제 키나아제/포스파테이즈를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 aceK 유전자는 예를 들면 서열번호 40의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "말레이트 신타아제 G(malate synthase G)" 글리옥실레이트와 아세틸-coA를 말레이트로 전환하는 반응을 촉매할 수 있는 효소이다. 상기 말레이트 신타아제 G는 GlcB와 혼용될 수 있다.
일 구현 예로, 본 출원의 GlcB 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 GlcB에 포함된다.
일 구현 예로, 본 출원의 GlcB 단백질은 강화된 것일 수 있으나, 이와 동일한 서열 또는 활성을 갖는 한 GlcB에 포함된다. 일 구현 예로, 본 출원의 GlcB 단백질은 서열번호 41 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, GlcB 유전자는 glcB, 말레이트 신타아제 G를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 glcB 유전자는 예를 들면 서열번호 42의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
본 출원에서, "말레이트 신타아제 A(malate synthase A)"는 글리옥실레이트와 아세틸-coA를 말레이트로 전환하는 반응을 촉매할 수 있는 효소이다. 상기 말레이트 신타아제 A는 AceB와 혼용될 수 있다.
일 구현 예로, 본 출원의 AceB 단백질은 내재적이거나, 에스케리키아속, 에스케리키아 콜라이(Escherichia coli) 유래일 수 있으며, 이와 동일한 서열 또는 활성을 갖는 한 AceB에 포함된다.
일 구현 예로, 본 출원의 AceB 단백질은 강화된 것일 수 있으나, 이와 동일한 서열 또는 활성을 갖는 한 AceB에 포함된다. 일 구현 예로, 본 출원의 AceB 단백질은 서열번호 43 또는 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하거나, 가지거나, 이루어지거나, 상기 아미노산 서열로 필수적으로 이루어지는(essentially consisting of) 것일 수 있다.
본 출원에서, AceB 유전자는 aceB, 말레이트 신타아제 A를 코딩하는 폴리뉴클레오티드 등과 혼용될 수 있다. 상기 aceB 유전자는 예를 들면 서열번호 44의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
상기 효소들(예를 들어, SucD 등)의 아미노산 서열은 공지의 데이터 베이스인 NCBI의 GenBank 등 다양한 데이터 베이스에서 그 서열을 얻을 수 있으나, 이에 제한되지 않는다.
본 출원에서 '특정 서열번호로 기재된 아미노산 서열을 포함하는 폴리펩티드 또는 단백질', '특정 서열번호로 기재된 아미노산 서열로 이루어진 폴리펩티드 또는 단백질' 또는 '특정 서열번호로 기재된 아미노산 서열을 갖는 폴리펩티드 또는 단백질'라고 기재되어 있더라도, 해당 서열번호의 아미노산 서열로 이루어진 폴리펩티드와 동일 혹은 상응하는 활성을 가지는 경우라면, 일부 서열이 결실, 변형, 치환, 보존적 치환 또는 부가된 아미노산 서열을 갖는 단백질도 본 출원에서 사용될 수 있음은 자명하다. 예를 들어, 상기 아미노산 서열 N-말단, 내부, 그리고/또는 C-말단에 단백질의 기능을 변경하지 않는 서열 추가, 자연적으로 발생할 수 있는 돌연변이, 이의 잠재성 돌연변이 (silent mutation) 또는 보존적 치환을 가지는 경우이다.
구체적으로, 본 출원의 단백질은 특정 서열번호의 아미노산 서열을 포함하거나, 또는 특정 서열번호의 아미노산 서열과 적어도 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 또는 99% 상동성 또는 동일성을 가지는 아미노산 서열을 포함할 수 있다. 또한, 상기 상동성 또는 동일성을 가지며, 상기 단백질에 상응하는 효능을 나타내는 아미노산 서열이라면, 일부 서열이 결실, 변형, 치환 또는 부가된 아미노산 서열을 가지더라도 본 출원의 범위 내에 포함됨은 자명하다.
상기 "보존적 치환(conservative substitution)"은 한 아미노산을 유사한 구조적 및/또는 화학적 성질을 갖는 또 다른 아미노산으로 치환시키는 것을 의미한다. 이러한 아미노산 치환은 일반적으로 잔기의 극성, 전하, 용해도, 소수성, 친수성 및/또는 양친매성(amphipathic nature)에서의 유사성에 근거하여 발생할 수 있다. 통상적으로, 보존적 치환은 폴리펩티드의 활성에 거의 영향을 미치지 않거나 또는 영향을 미치지 않을 수 있다.
본 출원에서 용어, '상동성 (homology)' 또는 '동일성 (identity)'은 두 개의 주어진 아미노산 서열 또는 염기 서열 상호간 동일 또는 유사한 정도를 의미하며 백분율로 표시될 수 있다. 용어 상동성 및 동일성은 종종 상호교환적으로 이용될 수 있다.
보존된(conserved) 폴리뉴클레오티드 또는 폴리펩티드의 서열 상동성 또는 동일성은 표준 배열 알고리즘에 의해 결정되며, 사용되는 프로그램에 의해 확립된 디폴트 갭 페널티가 함께 이용될 수 있다. 실질적으로, 상동성을 갖거나(homologous) 또는 동일한(identical) 서열은 일반적으로 서열 전체 또는 전체-길이의 적어도 약 50%, 60%, 70%, 80% 또는 90%에 해당하는 일부분과 중간 또는 높은 엄격한 조건(stringent conditions)에서 하이브리드할 수 있다. 하이브리드화에는 폴리뉴클레오티드에서 일반 코돈 또는 코돈 축퇴성을 고려한 코돈을 함유하는 폴리뉴클레오티드와의 하이브리드화 역시 포함됨이 자명하다.
임의의 두 폴리뉴클레오티드 또는 폴리펩티드 서열이 상동성, 유사성 또는 동일성을 갖는지 여부는, 예를 들어, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444에서와 같은 디폴트 파라미터를 이용하여 "FASTA" 프로그램과 같은 공지의 컴퓨터 알고리즘을 이용하여 결정될 수 있다. 또는, EMBOSS 패키지의 니들만 프로그램(EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277)(버전 5.0.0 또는 이후 버전)에서 수행되는 바와 같은, 니들만-운치(Needleman-Wunsch) 알고리즘(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453)이 사용되어 결정될 수 있다(GCG 프로그램 패키지 (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994, 및 [CARILLO ETA/.](1988) SIAM J Applied Math 48: 1073을 포함한다). 예를 들어, 국립 생물공학 정보 데이터베이스 센터의 BLAST, 또는 ClustalW를 이용하여 상동성, 유사성 또는 동일성을 결정할 수 있다.
폴리뉴클레오티드 또는 폴리펩티드의 상동성, 유사성 또는 동일성은, 예를 들어, Smith and Waterman, Adv. Appl. Math (1981) 2:482 에 공지된 대로, 예를 들면, Needleman et al. (1970), J Mol Biol. 48:443과 같은 GAP 컴퓨터 프로그램을 이용하여 서열 정보를 비교함으로써 결정될 수 있다. 요약하면, GAP 프로그램은 두 서열 중 더 짧은 것에서의 기호의 전체 수로, 유사한 배열된 기호(즉, 뉴클레오티드 또는 아미노산)의 수를 나눈 값으로 정의할 수 있다. GAP 프로그램을 위한 디폴트 파라미터는 (1) 이진법 비교 매트릭스(동일성을 위해 1 그리고 비-동일성을 위해 0의 값을 함유함) 및 Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979)에 의해 개시된 대로, Gribskov et al(1986) Nucl. Acids Res. 14: 6745의 가중된 비교 매트릭스 (또는 EDNAFULL (NCBI NUC4.4의 EMBOSS 버전) 치환 매트릭스); (2) 각 갭을 위한 3.0의 페널티 및 각 갭에서 각 기호를 위한 추가의 0.10 페널티 (또는 갭 개방 패널티 10, 갭 연장 패널티 0.5); 및 (3) 말단 갭을 위한 무 페널티를 포함할 수 있다.
또한, 임의의 두 폴리뉴클레오티드 또는 폴리펩티드 서열이 상동성, 유사성 또는 동일성을 갖는지 여부는 정의된 엄격한 조건하에서 써던 혼성화 실험에 의해 서열을 비교함으로써 확인할 수 있으며, 정의되는 적절한 혼성화 조건은 해당 기술 범위 내이고, 당업자에게 잘 알려진 방법(예컨대, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York)으로 결정될 수 있다.
본 출원의 유전자(예를 들어, sucD)는 공지의 데이터 베이스인 NCBI의 GenBank 등 다양한 데이터 베이스에서 그 서열을 얻을 수 있으나, 이에 제한되지 않는다.
일 예로, 클로스트리듐 클루이베리 유래의 sucD 유전자는 서열번호 2; 아라비돕시스 탈리아마 유래의 4hbD유전자는 서열번호 4; 클로스트리듐 클루이베리 유래의 orfZ 유전자는 서열번호 6; 슈도모나스 푸티다/랄스토니아 유트로파 유래의 폴리(3-하이드록시알카노에이트) 폴리머라제 융합 단백질 phaC3/C1 유전자는 서열번호 8; 에스케리키아 콜라이 유래의 pykA, pykF, ppc, gltA, maeA, maeB. edd, eda, ptsG, icd, aceA, aceK, glcB, 및 aceB 유전자는 각각 서열번호 10, 12, 14, 18, 22, 24, 26, 28, 30, 32, 36. 38, 40, 42, 및 44; 노스톡 속 유래의 ecaA 유전자는 서열번호 16; 리조븀 에틀리 유래의 pyc 유전자는 서열번호 20; 및 시네코시스티스 속 유래의 sbtA 유전자는 서열번호 34의 염기서열을 포함하거나, 가지거나, 또는 이루어지는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 유전자는 에스케리키아 속 또는 코리네박테리움 속 미생물에 적합하도록 코돈 최적화된 것일 수 있으나, 이에 제한되지 않는다.
본 출원에서 용어, "폴리뉴클레오티드"는 뉴클레오티드 단위체(monomer)가 공유결합에 의해 길게 사슬모양으로 이어진 뉴클레오티드의 중합체(polymer)로 일정한 길이 이상의 DNA 가닥이다.
본 출원의 폴리뉴클레오티드 또는 유전자는 코돈의 축퇴성(degeneracy)으로 인하여 또는 특정한 폴리펩티드를 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 폴리펩티드의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩 영역에 다양한 변형이 이루어질 수 있다. 상기 폴리뉴클레오티드 또는 유전자는 예를 들면 서열번호 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 및/또는 44의 염기서열을 포함할 수 있으며, 이와 상동성 또는 동일성이 80% 이상, 90% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상인 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.
또한, 본 출원의 폴리뉴클레오티드 또는 유전자는 공지의 유전자 서열로부터 제조될 수 있는 프로브, 예를 들면, 본 출원의 염기 서열의 전체 또는 일부에 대한 상보 서열과 엄격한 조건 하에 하이드리드화하여, 본 출원의 아미노산 서열을 코딩하는 서열이라면 제한없이 포함될 수 있다. 상기 "엄격한 조건(stringent condition)"이란 폴리뉴클레오티드 간의 특이적 혼성화를 가능하게 하는 조건을 의미한다. 이러한 조건은 문헌(예컨대, J. Sambrook et al., 상동)에 구체적으로 기재되어 있다. 예를 들어, 상동성 또는 동일성이 높은 폴리뉴클레오티드끼리, 40% 이상, 구체적으로 90% 이상, 보다 구체적으로 95% 이상, 96% 이상, 97% 이상, 98% 이상, 더욱 구체적으로 99% 이상의 상동성 또는 동일성을 갖는 폴리뉴클레오티드끼리 하이브리드화하고, 그보다 상동성 또는 동일성이 낮은 폴리뉴클레오티드끼리 하이브리드화하지 않는 조건, 또는 통상의 써던 하이브리드화(southern hybridization)의 세척 조건인 60℃, 1ХSSC, 0.1% SDS, 구체적으로 60℃, 0.1ХSSC, 0.1% SDS, 보다 구체적으로 68℃, 0.1ХSSC, 0.1% SDS에 상당하는 염 농도 및 온도에서, 1회, 구체적으로 2회 내지 3회 세정하는 조건을 열거할 수 있다.
혼성화는 비록 혼성화의 엄격도에 따라 염기 간의 미스매치(mismatch)가 가능할지라도, 두 개의 핵산이 상보적 서열을 가질 것을 요구한다. 용어, "상보적"은 서로 혼성화가 가능한 뉴클레오티드 염기 간의 관계를 기술하는데 사용된다. 예를 들면, DNA에 관하여, 아데닌은 티민에 상보적이며 시토신은 구아닌에 상보적이다. 따라서, 본 출원의 폴리뉴클레오티드는 또한 실질적으로 유사한 핵산 서열뿐만 아니라 전체 서열에 상보적인 단리된 핵산 단편을 포함할 수 있다.
구체적으로, 상동성 또는 동일성을 가지는 폴리뉴클레오티드는 55 ℃의 Tm 값에서 혼성화 단계를 포함하는 혼성화 조건을 사용하고 상술한 조건을 사용하여 탐지할 수 있다. 또한, 상기 Tm 값은 60 ℃, 63 ℃ 또는 65 ℃일 수 있으나, 이에 제한되는 것은 아니고 그 목적에 따라 당업자에 의해 적절히 조절될 수 있다.
폴리뉴클레오티드를 혼성화하는 적절한 엄격도는 폴리뉴클레오티드의 길이 및 상보성 정도에 의존하고 변수는 해당기술분야에 잘 알려져 있다(Sambrook et al., supra, 9.50-9.51, 11.7-11.8 참조).
본 출원에서, 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 또는 이들의 조합을 포함하는 미생물; 및 이의 배양물로 구성되는 군에서 선택되는 어느 하나 이상을 이용하는 것은, 상기 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 또는 이들의 조합을 포함하는 미생물을 배양 및/또는 상기 배양된 미생물 또는 배지로부터 특정 물질(예를 들어, 숙시네이트 세미알데하이드, 4-하이드록시부티레이트, 또는 4-하이드록시부티릴 coA 등)을 회수하는 것일 수 있으나, 이에 제한되지 않는다.
본 출원의 생산방법에서의 "약화"는 특정한 경로 또는 단계의 약화일 수 있고, 상기 경로나 단계에 관여하는 효소의 약화일 수 있다. 상기 약화는 불활성화(inactivation), 결핍(deficiency), 하향조절(down-regulation), 감소(decrease), 저하(reduce), 감쇠(attenuation) 등의 용어와 혼용될 수 있다. 상기 효소의 약화는 상기 효소 폴리펩티드; 상기 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 벡터, 또는 이들의 조합을 포함하는 미생물; 및 이의 배양물로 구성되는 군에서 선택되는 어느 하나 이상을 감소, 결실, 또는 불활성화시키는 것일 수 있다. 본 출원의 생산방법이 미생물을 이용할 수 있다는 점에서, 본 출원의 생산방법에서의 "약화"는 "폴리펩티드 활성의 약화"도 포함한다.
본 출원의 생산방법에서의 "강화"는 특정한 경로 또는 단계의 강화일 수 있고, 상기 경로나 단계에 관여하는 효소의 강화일 수 있다. 상기 강화는 활성화(activation), 상향조절(up-regulation), 과발현(overexpression), 증가(increase) 등의 용어와 혼용될 수 있다. 상기 효소의 강화는 상기 효소 폴리펩티드; 상기 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 벡터, 또는 이들의 조합을 포함하는 미생물; 및 이의 배양물로 구성되는 군에서 선택되는 어느 하나 이상을 증가, 활성화, 또는 과발현시키는 것일 수 있다. 본 출원의 생산방법이 미생물을 이용할 수 있다는 점에서, 본 출원의 생산방법에서의 "강화"는 폴리펩티드 활성의 "강화"도 포함한다.
본 출원에서 용어, "조절"은 상기 "강화"및/또는 "약화"일 수 있으나, 이에 제한되지 않는다.
본 출원에서 상기 (5) 폴리-4-하이드록시부티레이트를 1,4-부탄다이올로 분해하는 단계는 화학적 방법을 이용하는 것일 수 있으며, 열분해, 수소화, 또는 이들의 조합을 이용하는 것일 수 있으나, 폴리-4-하이드록시부티레이트를 1,4-부탄다이올로 분해할 수 있는 한, 이에 제한되지 않는다.
본 출원의 다른 하나의 양태는 숙시네이트 세미알데하이드 디하이드로게나제, 4-하이드록시부티릭산 디하이드로게나제, 4-하이드록시부티릴-coA 트랜스퍼라제, 및 폴리(3-하이드록시알카노에이트) 폴리머라제 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 벡터, 또는 이들의 조합을 포함하는 미생물을 제공한다.
상기 숙시네이트 세미알데하이드 디하이드로게나제, 4-하이드록시부티릭산 디하이드로게나제, 4-하이드록시부티릴-coA 트랜스퍼라제, 폴리(3-하이드록시알카노에이트) 폴리머라제 폴리펩티드, 폴리뉴클레오티드, 벡터, 하기 1,4-부탄다이올, TCA 경로, 환원형 TCA 경로, 피루베이트 디하이드로게나제, 시트레이트 신타아제, 아코니타제, 아이소시트레이트 디하이드로게나제, α-케토글루타레이트 디하이드로게나제, 피루베이트 카복실라아제, (I) 내지 (XII), (i) 내지 (vi), 및 이와 관련된 효소들 등은 다른 양태에서 설명한 바와 같다.
본 출원에서 상기 미생물은 세포 친화적인 폴리-4-하이드록시부티레이트를 우선적으로 생산하는 것이므로 본 출원의 미생물은 폴리-4-하이드록시부티레이트 생산능이 증가된 것일 수 있고, 이에 따라 1,4-부탄다이올의 생산능이 추가로 증가된 것일 수 있다. 또한, 본 출원의 미생물은 폴리-4-하이드록시부티레이트 생산 단계에서 유전자를 도입하여 옥살로아세테이트를 이용한 환원형 TCA 경로 활성화; 고생산성 발효공정; 및/또는 환원형 TCA 경로를 통해 발효에서 발생하는 이산화탄소를 재이용으로 폴리-4-하이드록시부티레이트 생산능이 증가된 것일 수 있다.
일 구현 예로, 상기 미생물은 숙시네이트 세미알데하이드 디하이드로게나제, 4-하이드록시부티릭산 디하이드로게나제, 4-하이드록시부티릴-coA 트랜스퍼라제, 및 폴리(3-하이드록시알카노에이트) 폴리머라제로 구성되는 군에서 선택되는 어느 하나 이상의 폴리펩티드의 활성이 강화된 것일 수 있다:
일 구현 예로, 상기 숙시네이트 세미알데하이드 디하이드로게나제, 4-하이드록시부티릭산 디하이드로게나제, 4-하이드록시부티릴-coA 트랜스퍼라제, 및/또는 폴리(3-하이드록시알카노에이트) 폴리머라제를 코딩하는 유전자는 외래 도입된 것일 수 있으나, 이에 제한되지 않는다.
일 예로, 상기 숙시네이트 세미알데하이드 디하이드로게나제, 및/또는 4-하이드록시부티릴-coA 트랜스퍼라제를 코딩하는 유전자는 클로스트리디움 클루이베리 유래일 수 있고, 4-하이드록시부티릭산 디하이드로게나제를 코딩하는 유전자는 아라비돕시스 탈리아나(Arabidopsis thaliana) 유래일 수 있으나, 이에 제한되지 않는다.
일 예로, 상기 폴리(3-하이드록시알카노에이트) 폴리머라제를 코딩하는 유전자는 슈도모나스 푸티다(Pseudomonas putida) 또는 랄스토니아 유트로파(Ralstonia eutropha) 유래일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 상기 미생물은 TCA 경로를 포함하는 것일 수 있다.
일 구현 예로, 상기 미생물은 피루베이트 디하이드로게나제, 시트레이트 신타아제, 아코니타제, 아이소시트레이트 디하이드로게나제, α-케토글루타레이트 디하이드로게나제, 및 피루베이트 카복실라아제로 이루어지는 군에서 선택되는 어느 하나 이상의 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 또는 이들의 조합을 포함하는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 미생물은 환원형 TCA 경로를 포함하는 것일 수 있고, 강화된 것일 수 있다.
일 구현 예로, 본 출원의 미생물은 하기 (I) 내지 (XII)로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 것일 수 있으나, 이에 제한되지 않는다:
(I) 피루베이트 키나아제 약화;
(II) 포스포에놀피루베이트 카복실라제 강화;
(III) 카보닉 안하이드라제 강화;
(IV) 시트레이트 신타아제 조절;
(V) 피루베이트 카복실라제 강화;
(VI) NAD+-의존성 말레이트 디하이드로게나제 약화;
(VII) NADP+-의존성 말레이트 디하이드로게나제 약화;
(VIII) 포스포글루코네이트 디하이드라타아제 약화;
(IX) 2-케토-4-하이드록시글루타레이트 KDPG:2-케토-3-디옥시글루코네이트 6-포스페이트 알돌레이즈 약화;
(X) 아스파테이트 아미노트랜스퍼라제 약화;
(XI) 글루코스-특이적 PTS 효소 IIBC 컴포넌트 약화; 및
(XII) 바이카보네이트 트랜스포터 강화
일 구현 예로, 본 출원의 미생물은 (II) 포스포에놀피루베이트 카복실라제 강화;
(VI) NAD+-의존성 말레이트 디하이드로게나제 약화;
(VII) NADP+-의존성 말레이트 디하이드로게나제 약화; 및/또는
(X) 아스파테이트 아미노트렌스트랜스퍼라제 약화를 포함할 수 있다.
일 구현 예로, 본 출원의 미생물은 (II) 포스포에놀피루베이트 카복실라제 강화를 포함할 수 있다.
일 구현 예로, 본 출원의 미생물은 (VI) NAD+-의존성 말레이트 디하이드로게나제 약화 및 (VII) NADP+-의존성 말레이트 디하이드로게나제 약화를 포함할 수 있다.
일 구현 예로, 본 출원의 미생물은 (X) 아스파테이트 아미노트렌스트랜스퍼라제 약화를 포함할 수 있다.
일 구현 예로, 상기 피루베이트 카복실라제를 코딩하는 유전자는 외래 유전자일 수 있으며, 구체적으로 리조븀 에틀리(Rhizobium etli) 유래일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 상기 시트레이트 신타아제 조절은 유전자 변이에 의한 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 미생물은 글리옥실레이트 경로를 포함하는 것일 수 있으며, 강화된 것일 수 있다.
일 구현 예로, 본 출원의 미생물은 하기 (i) 내지 (vi)로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 것일 수 있으나, 이에 제한되지 않는다:
(i) 시트레이트 신타아제 강화;
(ii) 아이소시트레이트 디하이드로게나제 약화;
(iii) 아이소시트레이트 리아제 강화;
(iv) 아이소시트레이트 디하이드로게나제 키나아제/포스파테이즈 강화;
(v) 말레이트 신타아제 G 강화; 및
(vi) 말레이트 신타아제 A 강화.
상기 숙시네이트 세미알데하이드 디하이드로게나제 등은 다른 양태에서 설명한 바와 같다.
일 구현 예로, 본 출원의 미생물은 질소, 황, 인, 및 마그네슘으로 구성되는 군에서 선택되는 어느 하나 이상의 영양소 제한 조건에서도 폴리-4-하이드록시부티레이트 생산능이 있는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 미생물은 질소, 황, 인, 및 마그네슘으로 구성되는 군에서 선택되는 하나 이상의 제한 조건에서 포스포에놀피루베이트 카복실라제 유전자(ppc)의 전사가 저해받지 않는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원의 미생물은 질소, 인, 황, 및 마그네슘으로 구성되는 군에서 선택되는 어느 하나 이상의 영양소 제한 조건에서도 상기 영양소 제한이 없는 조건에 비해 폴리-4-하이드록시부티레이트 생산능이 유지되거나 감소되지 않는 것일 수 있으나, 이에 제한되지 않는다.
일 구현 예로, 본 출원에서 목적하는 폴리펩티드(예를 들어, SucD, PhaC, OrfZ)가 포함되거나 강화된 미생물은 목적하는 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드 또는 이를 포함하는 벡터를 포함하는 것일 수 있다.
본 출원의 벡터는 적합한 숙주 내에서 목적 폴리펩티드를 발현시킬 수 있도록 적합한 발현조절영역(또는 발현조절서열)에 작동 가능하게 연결된 상기 목적 폴리펩티드를 코딩하는 폴리뉴클레오티드의 염기서열을 포함하는 DNA 제조물을 포함할 수 있다. 상기 발현조절영역은 전사를 개시할 수 있는 프로모터, 그러한 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합부위를 코딩하는 서열, 및 전사 및 해독의 종결을 조절하는 서열을 포함할 수 있다. 벡터는 적당한 숙주세포 내로 형질전환된 후, 숙주 게놈과 무관하게 복제되거나 기능할 수 있으며, 게놈 그 자체에 통합될 수 있다.
본 출원에서 사용되는 벡터는 특별히 한정되지 않으며, 당업계에 알려진 임의의 벡터를 이용할 수 있다. 통상 사용되는 벡터의 예로는 천연 상태이거나 재조합된 상태의 플라스미드, 코스미드, 바이러스 및 박테리오파지를 들 수 있다. 예를 들어, 파지 벡터 또는 코스미드 벡터로서 pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, 및 Charon21A 등을 사용할 수 있으며, 플라스미드 벡터로서 pDZ계, pBR계, pUC계, pBluescriptII계, pGEM계, pTZ계, pCL계 및 pET계 등을 사용할 수 있다. 구체적으로는 pDZ, pDC, pDCM2(대한민국 공개특허공보 제10-2020-0136813호), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pIMR53 벡터 등을 사용할 수 있다.
일례로 세포 내 염색체 삽입용 벡터를 통해 목적 폴리펩티드를 코딩하는 폴리뉴클레오티드를 염색체 내로 삽입할 수 있다. 상기 폴리뉴클레오티드의 염색체 내로의 삽입은 당업계에 알려진 임의의 방법, 예를 들면, 상동재조합(homologous recombination)에 의하여 이루어질 수 있으나, 이에 한정되지는 않는다. 상기 염색체 삽입 여부를 확인하기 위한 선별 마커(selection marker)를 추가로 포함할 수 있다. 상기 선별 마커는 벡터로 형질전환된 세포를 선별, 즉 목적 핵산 분자의 삽입 여부를 확인하기 위한 것으로, 약물 내성, 영양 요구성, 세포 독성제에 대한 내성 또는 표면 폴리펩티드의 발현과 같은 선택가능 표현형을 부여하는 마커들이 사용될 수 있다. 선택제(selective agent)가 처리된 환경에서는 선별 마커를 발현하는 세포만 생존하거나 다른 표현 형질을 나타내므로, 형질전환된 세포를 선별할 수 있다.
본 출원에서 용어 "형질전환"은 표적 폴리펩티드를 코딩하는 폴리뉴클레오티드를 포함하는 벡터를 숙주세포 혹은 미생물 내에 도입하여 숙주세포 내에서 상기 폴리뉴클레오티드가 코딩하는 폴리펩티드가 발현할 수 있도록 하는 것을 의미한다. 형질전환된 폴리뉴클레오티드는 숙주세포 내에서 발현될 수 있기만 한다면, 숙주세포의 염색체 내에 삽입되어 위치하거나 염색체 외에 위치하거나 상관없이 이들 모두를 포함할 수 있다. 또한, 상기 폴리뉴클레오티드는 목적 폴리펩티드를 코딩하는 DNA 및/또는 RNA를 포함한다. 상기 폴리뉴클레오티드는 숙주세포 내로 도입되어 발현될 수 있는 것이면, 어떠한 형태로도 도입될 수 있다. 예를 들면, 상기 폴리뉴클레오티드는 자체적으로 발현되는데 필요한 모든 요소를 포함하는 유전자 구조체인 발현 카세트(expression cassette)의 형태로 숙주세포에 도입될 수 있다. 상기 발현 카세트는 통상 상기 폴리뉴클레오티드에 작동 가능하게 연결되어 있는 프로모터(promoter), 전사 종결신호, 리보좀 결합부위 및 번역 종결신호를 포함할 수 있다. 상기 발현 카세트는 자체 복제가 가능한 발현 벡터 형태일 수 있다. 또한, 상기 폴리뉴클레오티드는 그 자체의 형태로 숙주세포에 도입되어 숙주세포에서 발현에 필요한 서열과 작동 가능하게 연결되어 있는 것일 수도 있으며, 이에 제한되지 않는다.
또한, 상기에서 용어 "작동 가능하게 연결"된 것이란 본 출원의 목적 폴리펩티드를 코딩하는 폴리뉴클레오티드의 전사를 개시 및 매개하도록 하는 프로모터 서열과 상기 폴리뉴클레오티드 서열이 기능적으로 연결되어 있는 것을 의미한다.
일 구현 예로, 본 출원의 숙시네이트 세미알데하이드 디하이드로게나제, 4-하이드록시부티릭산 디하이드로게나제, 4-하이드록시부티릴-coA 트랜스퍼라제, 및 폴리(3-하이드록시알카노에이트) 폴리머라제 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 벡터, 또는 이들의 조합을 포함하는 미생물은 이들을 포함하지 않는 미생물에 비해 숙시네이트 세미알데하이드 디하이드로게나제, 4-하이드록시부티릭산 디하이드로게나제, 4-하이드록시부티릴-coA 트랜스퍼라제, 및 폴리(3-하이드록시알카노에이트) 폴리머라제 활성이 강화된 것일 수 있으나, 이에 제한되지 않는다.
본 출원에서 용어, "미생물" 또는 "균주"는 야생형 미생물이나 자연적 또는 인위적으로 유전적 변형이 일어난 미생물을 모두 포함하며, 외부 유전자가 삽입되거나 내재적 유전자의 활성이 강화되거는 등의 원인으로 인해서 특정 기작이 강화된 미생물일 수 있다.
일 구현 예로, 본 출원의 미생물은 에스케리키아 속(Genus Escherichia) 또는 코리네박테리움 속(Genus Corynebacterium)일 수 있으며, 구체적으로 에스케리키아 콜라이 (Escherichia coli) 또는 코리네박테리움 글리타미쿰 ( Corynebacterium glutamicum)일 수 있으나, 이에 제한되지 않는다.
본 출원의 미생물은 폴리-4-하이드록시부티레이트 생산용일 수 있으며, 이로부터 생산된 폴리-4-하이드록시부티레이트는 화학적 공정에 의해 1,4-부탄다이올을 생산하여, 본 출원의 미생물이 1,4-부탄다이올 생산에 이용될 수 있으나, 이에 제한되지 않는다.
본 출원의 미생물은 본 출원의 강화 또는 도입의 대상이 되는 폴리펩티드(예를 들어, SucD, 4HbD, OrfZ, PhaC, PPC, EcaA, Glta, Pyc, AceK, 및/또는 AceA 등), 이를 코딩하는 폴리뉴클레오티드, 또는 폴리뉴클레오티드를 포함하는 벡터 중 어느 하나 이상을 포함하는 미생물; 본 출원의 강화 또는 도입의 대상이 되는 폴리펩티드 또는 유전자를 발현하도록 변형된 미생물; 본 출원의 강화 또는 도입의 대상이 되는 폴리펩티드 또는 유전자를 발현하는 미생물; 본 출원의 폴리펩티드 또는 유전자를 활성을 갖는 미생물; 본 출원의 약화의 대상이 되는 폴리펩티드(예를 들어, Pyk, GltA, MaeA, MaeB, Edd, Eda, 및/또는 AspC 등)가 약화되도록 변형된 미생물; 및/또는 본 출원의 약화의 대상이 되는 폴리펩티드 또는 유전자 또는 이의 활성이 약화된 미생물 (예컨대, 재조합 균주)일 수 있으나, 이에 제한되지 않는다.
본 출원의 미생물은 자연적으로 본 출원의 폴리펩티드(예를 들어, SucD, MaeA 등), 1,4-부탄다이올 및/또는 폴리-4-하이드록시부티레이트 생산능을 가지고 있는 미생물; 또는 폴리펩티드, 1,4-부탄다이올 및/또는 폴리-4-하이드록시부티레이트 생산능이 없는 모균주에 본 출원의 강화 또는 도입의 대상이 되는 폴리펩티드, 유전자, 폴리뉴클레오티드, 또는 이를 포함하는 벡터가 도입되거나, 약화의 대상이 되는 유전자 또는 폴리뉴클레오티드 또는 이의 활성이 약화되어 폴리펩티드가 강화 또는 약화, 또는 1,4-부탄다이올 및/또는 폴리-4-하이드록시부티레이트 생산능이 강화되거나 부여된 미생물일 수 있으나 이에 제한되지 않는다.
일 예로, 본 출원의 미생물은 본 출원의 폴리펩티드, 유전자, 폴리뉴클레오티드, 또는 이를 포함하는 벡터로 형질전환(이에 따라, 강화, 약화, 도입 등)되어, 1,4-부탄다이올 및/또는 폴리-4-하이드록시부티레이트를 생산할 수 있거나 생산능이 증가된 미생물을 모두 포함할 수 있다.
예를 들어, 본 출원의 미생물은 천연의 야생형 미생물, 1,4-부탄다이올 및/또는 폴리-4-하이드록시부티레이트를 생산하는 미생물에 본 출원의 폴리펩티드 가 발현되거나 약화되어, 1,4-부탄다이올 및/또는 폴리-4-하이드록시부티레이트 생산능이 증가된 재조합 미생물일 수 있다. 상기 1,4-부탄다이올 및/또는 폴리-4-하이드록시부티레이트 생산능이 증가된 재조합 미생물은, 천연의 야생형 미생물 또는 본 출원의 폴리펩티드 비변형 미생물 (즉, 야생형 유전자를 포함하는 미생물, 본 출원의 유전자가 강화되거나 도입되지 않는 미생물, 또는 본 출원의 유전자가 약화되지 않은 미생물)에 비하여 1,4-부탄다이올 및/또는 폴리-4-하이드록시부티레이트 생산능이 증가된 미생물일 수 있으나, 이에 제한되는 것은 아니다.
일 예로, 상기 생산능이 증가된 재조합 미생물은 변이 전 모균주 또는 비변형 미생물의 1,4-부탄다이올 및/또는 폴리-4-하이드록시부티레이트 생산능에 비하여 약 0.001% 이상 또는 0.01% 이상 1,4-부탄다이올 및/또는 폴리-4-하이드록시부티레이트 생산능이 높아진 것일 수 있으나, 변이 전 모균주 또는 비변형 미생물의 생산능에 비해 +값의 증가량을 갖는 한, 이에 제한되지 않는다. 상기 용어 "약(about)"은 ±0.5, ±0.4, ±0.3, ±0.2, ±0.1 등을 모두 포함하는 범위로, 약 이란 용어 뒤에 나오는 수치와 동등하거나 유사한 범위의 수치를 모두 포함하나, 이에 제한되지 않는다.
본 출원에서 용어, "비변형 미생물"은 미생물에 자연적으로 발생할 수 있는 돌연변이를 포함하는 미생물을 제외하는 것이 아니며, 야생형 미생물 또는 천연형 미생물 자체이거나, 자연적 또는 인위적 요인에 의한 유전적 변이로 형질이 변화되기 전 미생물을 의미할 수 있다. 예를 들어, 상기 비변형 미생물은 본 명세서에 기재된 폴리펩티드가 발현되지 않거나, 약화되지 않거나, 도입되기 전의 미생물을 의미할 수 있다. 상기 "비변형 미생물"은 "변형 전 균주", "변형 전 미생물", "비변이 균주", "비변형 균주", "비변이 미생물" 또는 "기준 미생물"과 혼용될 수 있다.
본 출원의 미생물에서 폴리뉴클레오티드의 일부 또는 전체의 변형은 (a) 미생물 내 염색체 삽입용 벡터를 이용한 상동 재조합 또는 유전자가위 (engineered nuclease, e.g., CRISPR-Cas9)을 이용한 유전체 교정 및/또는 (b) 자외선 및 방사선 등과 같은 빛 및/또는 화학물질 처리에 의해 유도될 수 있으나 이에 제한되지 않는다. 상기 유전자 일부 또는 전체의 변형 방법에는 DNA 재조합 기술에 의한 방법이 포함될 수 있다. 예를 들면, 목적 유전자와 상동성이 있는 뉴클레오티드 서열을 포함하는 뉴클레오티드 서열 또는 벡터를 상기 미생물에 주입하여 상동 재조합(homologous recombination)이 일어나게 함으로써 유전자 일부 또는 전체의 결손이 이루어질 수 있다. 상기 주입되는 뉴클레오티드 서열 또는 벡터는 우성 선별 마커를 포함할 수 있으나, 이에 제한되는 것은 아니다.
본 출원에서 용어, 폴리펩티드 활성의 "약화"는 내재적 활성에 비하여 활성이 감소되거나 또는 활성이 없는 것을 모두 포함하는 개념이다. 상기 약화는 불활성화(inactivation), 결핍(deficiency), 하향조절(down-regulation), 감소(decrease), 저하(reduce), 감쇠(attenuation) 등의 용어와 혼용될 수 있다.
상기 약화는 상기 폴리펩티드를 코딩하는 폴리뉴클레오티드의 변이 등으로 폴리펩티드 자체의 활성이 본래 미생물이 가지고 있는 폴리펩티드의 활성에 비해 감소 또는 제거된 경우, 이를 코딩하는 폴리뉴클레오티드의 유전자의 발현 저해 또는 폴리펩티드로의 번역(translation) 저해 등으로 세포 내에서 전체적인 폴리펩티드 활성 정도 및/또는 농도(발현량)가 천연형 균주에 비하여 낮은 경우, 상기 폴리뉴클레오티드의 발현이 전혀 이루어지지 않은 경우, 및/또는 폴리뉴클레오티드의 발현이 되더라도 폴리펩티드의 활성이 없는 경우 역시 포함할 수 있다. 상기 "내재적 활성"은 자연적 또는 인위적 요인에 의한 유전적 변이로 형질이 변화하는 경우, 형질 변화 전 모균주, 야생형 또는 비변형 미생물이 본래 가지고 있던 특정 폴리펩티드의 활성을 의미한다. 이는 "변형 전 활성"과 혼용되어 사용될 수 있다. 폴리펩티드의 활성이 내재적 활성에 비하여 "불활성화, 결핍, 감소, 하향조절, 저하, 감쇠"한다는 것은, 형질 변화 전 모균주 또는 비변형 미생물이 본래 가지고 있던 특정 폴리펩티드의 활성에 비하여 낮아진 것을 의미한다.
이러한 폴리펩티드의 활성의 약화는, 당업계에 알려진 임의의 방법에 의하여 수행될 수 있으나 이로 제한되는 것은 아니며, 당해 분야에 잘 알려진 다양한 방법의 적용으로 달성될 수 있다(예컨대, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012 등).
구체적으로, 본 출원의 폴리펩티드 활성의 약화는
1) 폴리펩티드를 코딩하는 유전자 전체 또는 일부의 결손;
2) 폴리펩티드를 코딩하는 유전자의 발현이 감소하도록 발현조절영역(또는 발현조절서열)의 변형;
3) 폴리펩티드의 활성이 제거 또는 약화되도록 상기 폴리펩티드를 구성하는 아미노산 서열의 변형(예컨대, 아미노산 서열 상의 1 이상의 아미노산의 삭제/치환/부가);
4) 폴리펩티드의 활성이 제거 또는 약화되도록 상기 폴리펩티드를 코딩하는 유전자 서열의 변형 (예를 들어, 폴리펩티드의 활성이 제거 또는 약화되도록 변형된 폴리펩티드를 코딩하도록 상기 폴리펩티드 유전자의 핵산염기 서열 상의 1 이상의 핵산염기의 삭제/치환/부가);
5) 폴리펩티드를 코딩하는 유전자 전사체의 개시코돈 또는 5'-UTR 지역을 코딩하는 염기서열의 변형;
6) 폴리펩티드를 코딩하는 상기 유전자의 전사체에 상보적으로 결합하는 안티센스 올리고뉴클레오티드(예컨대, 안티센스 RNA)의 도입;
7) 리보솜(ribosome)의 부착이 불가능한 2차 구조물을 형성시키기 위하여 폴리펩티드를 코딩하는 유전자의 사인-달가르노(Shine-Dalgarno) 서열 앞단에 사인-달가르노 서열과 상보적인 서열의 부가;
8) 폴리펩티드를 코딩하는 유전자 서열의 ORF(open reading frame)의 3' 말단에 반대 방향으로 전사되는 프로모터의 부가(Reverse transcription engineering, RTE); 또는
9) 상기 1) 내지 8) 중 선택된 2 이상의 조합일 수 있으나, 이에, 특별히 제한되는 것은 아니다.
예컨대,
상기 1) 폴리펩티드를 코딩하는 상기 유전자 일부 또는 전체의 결손은, 염색체 내 내재적 목적 폴리펩티드를 코딩하는 폴리뉴클레오티드 전체의 제거, 일부 뉴클레오티드가 결실된 폴리뉴클레오티드로의 교체 또는 마커 유전자로 교체일 수 있다.
또한, 상기 2) 발현조절영역(또는 발현조절서열)의 변형은, 결실, 삽입, 비보존적 또는 보존적 치환 또는 이들의 조합으로 발현조절영역(또는 발현조절서열) 상의 변이 발생, 또는 더욱 약한 활성을 갖는 서열로의 교체일 수 있다. 상기 발현조절영역에는 프로모터, 오퍼레이터 서열, 리보좀 결합부위를 코딩하는 서열, 및 전사와 해독의 종결을 조절하는 서열을 포함하나, 이에 한정되는 것은 아니다.
또한, 상기 5) 폴리펩티드를 코딩하는 유전자 전사체의 개시코돈 또는 5'-UTR 지역을 코딩하는 염기서열 변형은, 예를 들면, 내재적 개시코돈에 비해 폴리펩티드 발현율이 더 낮은 다른 개시코돈을 코딩하는 염기서열로 치환하는 것일 수 있으나, 이에 제한되지 않는다.
또한, 상기 3) 및 4)의 아미노산 서열 또는 폴리뉴클레오티드 서열의 변형은 폴리펩티드의 활성을 약화하도록 상기 폴리펩티드의 아미노산 서열 또는 상기 폴리펩티드를 코딩하는 폴리뉴클레오티드 서열을 결실, 삽입, 비보존적 또는 보존적 치환 또는 이들의 조합으로 서열상의 변이 발생, 또는 더욱 약한 활성을 갖도록 개량된 아미노산 서열 또는 폴리뉴클레오티드 서열 또는 활성이 없도록 개량된 아미노산 서열 또는 폴리뉴클레오티드 서열로의 교체일 수 있으나, 이에 한정되는 것은 아니다. 예를 들면, 폴리뉴클레오티드 서열 내 변이를 도입하여 종결 코돈을 형성시킴으로써, 유전자의 발현을 저해하거나 약화시킬 수 있으나, 이에 제한되지 않는다.
상기 6) 폴리펩티드를 코딩하는 상기 유전자의 전사체에 상보적으로 결합하는 안티센스 올리고뉴클레오티드(예컨대, 안티센스 RNA)의 도입은 예를 들어 문헌 [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986]을 참고할 수 있다.
상기 7) 리보솜(ribosome)의 부착이 불가능한 2차 구조물을 형성시키기 위하여 폴리펩티드를 코딩하는 유전자의 사인-달가르노(Shine-Dalgarno) 서열 앞단에 사인-달가르노 서열과 상보적인 서열의 부가는 mRNA 번역을 불가능하게 하거나 속도를 저하시키는 것일 수 있다.
상기 8) 폴리펩티드를 코딩하는 유전자서열의 ORF(open reading frame)의 3' 말단에 반대 방향으로 전사되는 프로모터의 부가(Reverse transcription engineering, RTE)는 상기 폴리펩티드를 코딩하는 유전자의 전사체에 상보적인 안티센스 뉴클레오티드를 만들어 활성을 약화하는 것일 수 있다.
본 출원에서 용어, 폴리펩티드 활성의 "강화"는, 폴리펩티드의 활성이 내재적 활성에 비하여 증가되는 것을 의미한다. 상기 강화는 활성화(activation), 상향조절(up-regulation), 과발현(overexpression), 증가(increase) 등의 용어와 혼용될 수 있다. 여기서 활성화, 강화, 상향조절, 과발현, 증가는 본래 가지고 있지 않았던 활성을 나타내게 되는 것, 또는 내재적 활성 또는 변형 전 활성에 비하여 향상된 활성을 나타내게 되는 것을 모두 포함할 수 있다. 상기 "내재적 활성"은 자연적 또는 인위적 요인에 의한 유전적 변이로 형질이 변화하는 경우, 형질 변화 전 모균주 또는 비변형 미생물이 본래 가지고 있던 특정 폴리펩티드의 활성을 의미한다. 이는 "변형 전 활성"과 혼용되어 사용될 수 있다. 폴리펩티드의 활성이 내재적 활성에 비하여 "강화", "상향조절", "과발현" 또는 "증가"한다는 것은, 형질 변화 전 모균주 또는 비변형 미생물이 본래 가지고 있던 특정 폴리펩티드의 활성 및/또는 농도(발현량)에 비하여 향상된 것을 의미한다.
상기 강화는 외래의 폴리펩티드를 도입하거나, 내재적인 폴리펩티드의 활성 강화 및/또는 농도(발현량)를 통해 달성할 수 있다. 상기 폴리펩티드의 활성의 강화 여부는 해당 폴리펩티드의 활성 정도, 발현량 또는 해당 폴리펩티드로부터 배출되는 산물의 양의 증가로부터 확인할 수 있다.
상기 폴리펩티드의 활성의 강화는 당해 분야에 잘 알려진 다양한 방법의 적용이 가능하며, 목적 폴리펩티드의 활성을 변형전 미생물보다 강화시킬 수 있는 한, 제한되지 않는다. 구체적으로, 분자생물학의 일상적 방법인 당업계의 통상의 기술자에게 잘 알려진 유전자 공학 및/또는 단백질 공학을 이용한 것일 수 있으나, 이로 제한되지 않는다(예컨대, Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012 등).
구체적으로, 본 출원의 폴리펩티드 활성의 강화는
1) 폴리펩티드를 코딩하는 폴리뉴클레오티드의 세포 내 카피수 증가;
2) 폴리펩티드를 코딩하는 염색체상의 유전자 발현조절영역을 활성이 강력한 서열로 교체;
3) 폴리펩티드를 코딩하는 유전자 전사체의 개시코돈 또는 5'-UTR 지역을 코딩하는 염기서열의 변형;
4) 폴리펩티드 활성이 강화되도록 상기 폴리펩티드의 아미노산 서열의 변형;
5) 폴리펩티드 활성이 강화되도록 상기 폴리펩티드를 코딩하는 폴리뉴클레오티드 서열의 변형 (예를 들어, 폴리펩티드의 활성이 강화되도록 변형된 폴리펩티드를 코딩하도록 상기 폴리펩티드 유전자의 폴리뉴클레오티드 서열의 변형);
6) 폴리펩티드의 활성을 나타내는 외래 폴리펩티드 또는 이를 코딩하는 외래 폴리뉴클레오티드의 도입;
7) 폴리펩티드를 암호화하는 폴리뉴클레오티드의 코돈 최적화;
8) 폴리펩티드의 삼차구조를 분석하여 노출 부위를 선택하여 변형하거나 화학적으로 수식; 또는
9) 상기 1) 내지 8) 중 선택된 2 이상의 조합일 수 있으나, 이에, 특별히 제한되는 것은 아니다.
보다 구체적으로,
상기 1) 폴리펩티드를 코딩하는 폴리뉴클레오티드의 세포 내 카피수 증가는, 해당 폴리펩티드를 코딩하는 폴리뉴클레오티드가 작동가능하게 연결된, 숙주와 무관하게 복제되고 기능할 수 있는 벡터의 숙주세포 내로의 도입에 의해 달성되는 것일 수 있다. 또는, 해당 폴리펩티드를 코딩하는 폴리뉴클레오티드가 숙주세포 내의 염색체 내에 1 카피 또는 2 카피 이상 도입에 의해 달성되는 것일 수 있다. 상기 염색체 내에 도입은 숙주세포 내의 염색체 내로 상기 폴리뉴클레오티드를 삽입시킬 수 있는 벡터가 숙주세포 내에 도입됨으로써 수행될 수 있으나, 이에 제한되지 않는다. 상기 벡터는 전술한 바와 같다.
상기 2) 폴리펩티드를 코딩하는 염색체상의 유전자 발현조절영역(또는 발현조절서열)을 활성이 강력한 서열로 교체는, 예를 들면, 상기 발현조절영역의 활성을 더욱 강화하도록 결실, 삽입, 비보존적 또는 보존적 치환 또는 이들의 조합으로 서열상의 변이 발생, 또는 더욱 강한 활성을 가지는 서열로의 교체일 수 있다. 상기 발현조절영역은, 특별히 이에 제한되지 않으나 프로모터, 오퍼레이터 서열, 리보좀 결합 부위를 코딩하는 서열, 그리고 전사 및 해독의 종결을 조절하는 서열 등을 포함할 수 있다. 일 예로, 본래의 프로모터를 강력한 프로모터로 교체시키는 것일 수 있으나, 이에 제한되지 않는다.
공지된 강력한 프로모터의 예에는 CJ1 내지 CJ7 프로모터(미국등록특허 US 7662943 B2), lac 프로모터, trp 프로모터, trc 프로모터, tac 프로모터, 람다 파아지 PR 프로모터, PL 프로모터, tet 프로모터, gapA 프로모터, SPL7 프로모터, SPL13(sm3) 프로모터(미국등록특허 US 10584338 B2), O2 프로모터(미국등록특허 US 10273491 B2), tkt 프로모터, yccA 프로모터 등이 있으나, 이에 제한되지 않는다.
상기 3) 폴리펩티드를 코딩하는 유전자 전사체의 개시코돈 또는 5'-UTR 지역을 코딩하는 염기서열 변형은, 예를 들면, 내재적 개시코돈에 비해 폴리펩티드 발현율이 더 높은 다른 개시코돈을 코딩하는 염기 서열로 치환하는 것일 수 있으나, 이에 제한되지 않는다.
상기 4) 및 5)의 아미노산 서열 또는 폴리뉴클레오티드 서열의 변형은, 폴리펩티드의 활성을 강화하도록 상기 폴리펩티드의 아미노산 서열 또는 상기 폴리펩티드를 코딩하는 폴리뉴클레오티드 서열을 결실, 삽입, 비보존적 또는 보존적 치환 또는 이들의 조합으로 서열상의 변이 발생, 또는 더욱 강한 활성을 갖도록 개량된 아미노산 서열 또는 폴리뉴클레오티드 서열 또는 활성이 증가하도록 개량된 아미노산 서열 또는 폴리뉴클레오티드 서열로의 교체일 수 있으나, 이에 한정되는 것은 아니다. 상기 교체는 구체적으로 상동재조합에 의하여 폴리뉴클레오티드를 염색체내로 삽입함으로써 수행될 수 있으나, 이에 제한되지 않는다. 이때 사용되는 벡터는 염색체 삽입 여부를 확인하기 위한 선별 마커 (selection marker)를 추가로 포함할 수 있다. 상기 선별 마커는 전술한 바와 같다.
상기 6) 폴리펩티드의 활성을 나타내는 외래 폴리뉴클레오티드의 도입은, 상기 폴리펩티드와 동일/유사한 활성을 나타내는 폴리펩티드를 코딩하는 외래 폴리뉴클레오티드의 숙주세포 내 도입일 수 있다. 상기 외래 폴리뉴클레오티드는 상기 폴리펩티드와 동일/유사한 활성을 나타내는 한 그 유래나 서열에 제한이 없다. 상기 도입에 이용되는 방법은 공지된 형질전환 방법을 당업자가 적절히 선택하여 수행될 수 있으며, 숙주 세포 내에서 상기 도입된 폴리뉴클레오티드가 발현됨으로써 폴리펩티드가 생성되어 그 활성이 증가될 수 있다.
상기 7) 폴리펩티드를 암호화하는 폴리뉴클레오티드의 코돈 최적화는, 내재 폴리뉴클레오티드가 숙주세포 내에서 전사 또는 번역이 증가하도록 코돈 최적화한 것이거나, 또는 외래 폴리뉴클레오티드가 숙주세포 내에서 최적화된 전사, 번역이 이루어지도록 이의 코돈을 최적화한 것일 수 있다.
상기 8) 폴리펩티드의 삼차구조를 분석하여 노출 부위를 선택하여 변형하거나 화학적으로 수식하는 것은, 예를 들어 분석하고자 하는 폴리펩티드의 서열정보를 기지 단백질들의 서열정보가 저장된 데이터베이스와 비교함으로써 서열의 유사성 정도에 따라 주형 단백질 후보를 결정하고 이를 토대로 구조를 확인하여, 변형하거나 화학적으로 수식할 노출 부위를 선택하여 변형 또는 수식하는 것일 수 있다.
이와 같은 폴리펩티드 활성의 강화는, 상응하는 폴리펩티드의 활성 또는 농도 발현량이 야생형이나 변형 전 미생물 균주에서 발현된 폴리펩티드의 활성 또는 농도를 기준으로 하여 증가되거나, 해당 폴리펩티드로부터 생산되는 산물의 양의 증가되는 것일 수 있으나, 이에 제한되는 것은 아니다.
본 출원에서 용어, 폴리펩티드 활성의 "조절"은 상기 폴리펩티드 활성의 "강화"및/또는 폴리펩티드 활성의 "약화"일 수 있으나, 이에 제한되지 않는다. 일 구현 예로, GltA는 활성이 강화 또는 약화된 것일 수 있으나, 이에 제한되지 않는다.
본 출원의 또 다른 하나의 양태는 본 출원의 미생물을 배양하는 단계를 포함하는 폴리-4-하이드록시부티레이트(poly-4-hydroxybutyrate, poly(4-hydroxybutyrate); P4HB) 생산방법을 제공한다.
본 출원에서 용어 "폴리-4-하이드록시부티레이트"는 4-하이드록시부티레이트의 중합체로서 폴리에스터에 속하는 화합물이다. 상기 폴리-4-하이드록시부티레이트는 폴리-4-하이드록시부타노에이트(poly-4-hydroxybutanoate, P4HA)와 혼용될 수 있으며, 하기 화학식 1로 표시되는 것일 수 있으나, 이에 제한되지 않는다.
(상기 n은 1 이상의 정수)
본 출원에서, 용어 "배양"은 본 출원의 미생물을 적당히 조절된 환경 조건에서 생육시키는 것을 의미한다. 본 출원에서, 배양과정은 당업계에 알려진 적당한 배지와 배양조건에 따라 이루어질 수 있다. 이러한 배양 과정은 선택되는 균주에 따라 당업자가 용이하게 조정하여 사용할 수 있다. 구체적으로 상기 배양은 회분식, 연속식 및/또는 유가식일 수 있으나, 이에 제한되는 것은 아니다.
본 출원의 미생물을 적당한 탄소원, 질소원, 인원, 무기화합물, 아미노산 및/또는 비타민 등을 함유한 통상의 배지 내에서 호기성 조건 하에서 온도, pH 등을 조절하면서 배양할 수 있다.
본 출원의 배양에서 배양온도는 20 내지 35℃ 구체적으로는 25 내지 35℃, 28 내지 35℃를 유지할 수 있고, 약 10 내지 160 시간, 약 20 시간 내지 130 시간, 약 24 시간 내지 120 시간, 약 36 시간 내지 120 시간, 약 48시간 내지 120시간, 약 48 시간, 약 72 시간, 또는 약 120 시간 동안 배양할 수 있으나, 이에 한정되는 것은 아니다.
본 출원의 배양에 의하여 생산된 폴리-4-하이드록시부티레이트는 배지 중으로 분비되거나 미생물 내에 잔류할 수 있다.
본 출원의 폴리-4-하이드록시부티레이트 생산방법은, 본 출원의 미생물을 준비하는 단계, 상기 미생물을 배양하기 위한 배지를 준비하는 단계, 또는 이들의 조합(순서에 무관, in any order)을, 예를 들어, 상기 배양하는 단계 이전에, 추가로 포함할 수 있다.
본 출원의 폴리-4-하이드록시부티레이트 생산방법은, 상기 미생물 배양에 따른 배지(배양이 수행된 배지) 또는 본 출원의 미생물로부터 폴리-4-하이드록시부티레이트를 회수하는 단계를 추가로 포함할 수 있다. 상기 회수하는 단계는 상기 배양하는 단계 이후에 추가로 포함될 수 있다.
상기 회수는 본 출원의 미생물의 배양 방법, 예를 들어 회분식, 연속식 또는 유가식 배양 방법 등에 따라 당해 기술 분야에 공지된 적합한 방법을 이용하여 목적하는 폴리-4-하이드록시부티레이트를 수집(collect)하는 것일 수 있다. 예를 들어, 원심분리, 여과, 결정화 단백질 침전제에 의한 처리(염석법), 추출, 세포 파쇄, 초음파 파쇄, 한외여과, 투석법, 분자체 크로마토그래피(겔여과), 흡착크로마토그래피, 이온교환 크로마토그래피, 친화도 크로마토그래피 등의 각종 크로마토그래피, HPLC 또는 이들의 방법을 조합하여 사용될 수 있으며, 당해 분야에 공지된 적합한 방법을 이용하여 배지 또는 미생물로부터 목적하는 폴리-4-하이드록시부티레이트를 회수할 수 있다.
또한, 본 출원의 폴리-4-하이드록시부티레이트 생산방법은, 추가적으로 정제 단계를 포함할 수 있다. 상기 정제는 당해 기술분야에 공지된 적합한 방법을 이용하여, 수행할 수 있다. 일 예에서, 본 출원의 폴리-4-하이드록시부티레이트 생산방법이 회수 단계와 정제 단계를 모두 포함하는 경우, 상기 회수 단계와 정제 단계는 순서에 상관없이 이시적(또는 연속적)으로 수행되거나, 동시에 또는 하나의 단계로 통합되어 수행될 수 있으나, 이에 제한되는 것은 아니다.
본 출원의 또 다른 하나의 양태는 본 출원의 미생물을 배양하는 단계; 상기 미생물 또는 배지로부터 폴리-4-하이드록시부티레이트를 회수하는 단계; 및 폴리-4-하이드록시부티레이트를 1,4-부탄다이올로 분해하는 단계를 포함하는 1,4-부탄다이올 생산방법을 제공한다.
본 출원의 1,4-부탄다이올 생산방법은 본 출원의 미생물이 생산한 폴리-4-하이드록시부티레이트를 1,4-부탄다이올로 분해하는 단계를 추가로 포함하는 것일 수 있다. 본 출원의 1,4-부탄다이올 생산방법에 있어서, 상기 분해하는 단계는 상기 배양하는 단계 또는 상기 회수하는 단계 이후에 추가로 포함될 수 있다. 상기 분해하는 단계는 당해 기술분야에 공지된 적합한 방법을 이용하여 수행할 수 있다. 일 구현 예로, 상기 폴리-4-하이드록시부티레이트를 1,4-부탄다이올로 분해하는 단계는 열분해, 수소화, 또는 이들의 조합일 수 있다.
본 출원의 또 다른 하나의 양태는 본 출원의 미생물 또는 이의 배양물을 포함하는 폴리-4-하이드록시부티레이트 생산용 조성물을 제공한다.
상기 미생물, 폴리-4-하이드록시부티레이트 등은 다른 양태에서 설명한 바와 같다.
본 출원의 조성물은 통상 사용되는 임의의 적합한 부형제를 추가로 포함할 수 있으며, 이러한 부형제는, 예를 들어 보존제, 습윤제, 분산제, 현탁화제, 완충제, 안정화제 또는 등장화제 등일 수 있으나, 이에 한정되는 것은 아니다.
본 출원의 또 다른 하나의 양태는 본 출원의 미생물 또는 이의 배양물의 폴리-4-하이드록시부티레이트 생산 용도를 제공한다.
본 출원의 또 다른 하나의 양태는 본 출원의 미생물 또는 이의 배양물의 1,4-부탄다이올 생산 용도를 제공한다.
상기 미생물, 폴리-4-하이드록시부티레이트 등은 다른 양태에서 설명한 바와 같다.
본 출원의 방법 또는 미생물을 이용할 경우, 효과적인 폴리-4-하이드록시부티레이트 및 1,4-부탄다이올 생산이 가능하다.
도 1은 산화적 TCA 경로를 이용한 폴리-4-하이드록시부티레이트 및 1,4-부탄다이올 생산 경로를 나타내는 도이다.
도 2는 환원형 TCA 경로를 이용한 폴리-4-하이드록시부티레이트 및 1,4-부탄다이올 생산 경로를 나타내는 도이다.
도 3은 글리옥실레이트 경로를 이용한 폴리-4-하이드록시부티레이트 및 1,4-부탄다이올 생산 경로를 나타내는 도이다.
도 4 내지 도 6는 환원형 TCA 경로를 이용한 폴리-4-하이드록시부티레이트 및 1,4-부탄다이올 생산 경로에서 포스포에놀피루베이트-옥살레이트 경로 강화 방법을 나타내는 도이다.
도 7은 환원형 TCA 경로를 이용한 폴리-4-하이드록시부티레이트 생산 결과를 나타낸 도이다.
도 8은 글리옥실레이트 경로를 이용한 폴리-4-하이드록시부티레이트 생산 결과를 나타낸 도이다.
도 2는 환원형 TCA 경로를 이용한 폴리-4-하이드록시부티레이트 및 1,4-부탄다이올 생산 경로를 나타내는 도이다.
도 3은 글리옥실레이트 경로를 이용한 폴리-4-하이드록시부티레이트 및 1,4-부탄다이올 생산 경로를 나타내는 도이다.
도 4 내지 도 6는 환원형 TCA 경로를 이용한 폴리-4-하이드록시부티레이트 및 1,4-부탄다이올 생산 경로에서 포스포에놀피루베이트-옥살레이트 경로 강화 방법을 나타내는 도이다.
도 7은 환원형 TCA 경로를 이용한 폴리-4-하이드록시부티레이트 생산 결과를 나타낸 도이다.
도 8은 글리옥실레이트 경로를 이용한 폴리-4-하이드록시부티레이트 생산 결과를 나타낸 도이다.
이하 본 출원을 실시예에 의해 보다 상세하게 설명한다. 그러나 하기 실시예는 본 출원을 예시하기 위한 바람직한 실시양태에 불과한 것이며 따라서, 본 출원의 권리범위를 이에 한정하는 것으로 의도되지는 않는다. 한편, 본 명세서에 기재되지 않은 기술적인 사항들은 본 출원의 기술 분야 또는 유사 기술 분야에서 숙련된 통상의 기술자이면 충분히 이해하고 용이하게 실시할 수 있다.
실시예 1: 포스포에놀피루베이트 카복실라제 발현 조절 및 산화적 TCA(Oxidative TCA) 경로를 이용한 P4HB 생산
1-1:
산화적 TCA (Oxidative TCA; oTCA) 경로를 이용한 P4HB 생산 경로
대장균에서 포도당을 사용하여 P4HB 생산을 평가하였다. 포도당은 포스포트랜스퍼라제(Phosphotransferase) 시스템을 통해 하나의 포스포에놀피루베이트(Phosphoenolpyruvate; PEP)와 하나의 아세틸-coA 및 이산화탄소로 산화될 수 있다. PEP는 하나의 이산화탄소를 고정하며 옥살로아세테이트(oxaloacetate)로 포스포에놀피루베이트 카복실라제(PEP carboxylase; PPC)에 의해서 전환될 수 있다. 아세틸-coA와 옥살로아세테이트는 시트레이트(citrate)를 거쳐 2개의 이산화탄소와 하나의 숙시닐-CoA(succinyl-coA)로 산화될 수 있다. 폴리-4-하이드록시부티레이트(P4HB)는 이 TCA 경로의 중간 물질인 숙시닐-CoA로부터 생산이 가능할 수 있다. 숙시닐-CoA로부터 P4HB의 생산은 NADH 혹은 NADPH 의존적인 숙시네이트 세미알데하이드 디하이드로게나제(succinate semialdehyde dehydrogenase; SucD)에 의해서 숙시닐 세미알데하이드(succinyl semialdehyde)로 전환된다. 숙시닐 세미알데하이드는 NADH 의존적인 4-하이드록시부티릭산 디하이드로게나제 (4-hydroxybutyric acid dehydrogenase; 4HbD)에 의해서 4-하이드록시부티레이트(4-hydroxybutyrate; 4HB)로 전환되며 이는 다시 4-하이드록시부티릴-coA 트랜스퍼라제(4-hydroxybutyryl-CoA transferase; OrfZ)에 의해서 4-하이드록시부티릴-coA(4-hydrxybutyril-CoA)로 전환될 수 있다. 최종적으로, 폴리(3-하이드록시알카노에이트) 폴리머라제(Poly(3-hydroxyalkanoate) polymerase; PhaC)에 의해서 4-하이드록시부티릴-coA(4-hydrxybutyril-CoA)로부터 폴리-4-하이드록시부티레이트(poly-4-hydroxybutyrate; P4HB)가 생성될 수 있도록 하였다.
전술한 SucD, 4HbD, OrfZ, PhaC, PPC 및 이들의 유전자 서열은 서열번호 1 내지 서열번호 8, 서열번호 13, 및 서열번호 14에 나타내었다.
1-2:
포스포에놀피루베이트 카복실라제 발현 조절용 프로모터 제작
균체 성장에 필수적인 질소원을 제한함으로써 균체 성장을 억제시키고 탄소 흐름을 목표 물질인 P4HB로 유도시킴으로써 이의 생산성을 증가시키고자 하였다. 다만, 대장균의 포스포에놀피루베이트 카복실라제(PEP carboxylase)를 코딩하는 ppc 유전자의 프로모터가 질소 제한 조건에서 조절인자(Nac, DNA-binding transcriptional dual regulator)에 의해 발현이 저해되므로, 이를 극복하기 위한 프로모터를 제작하고자 하였다.
이를 위하여, 야생의 프로모터(Pn)와 동등 또는 그 이상의 발현 세기를 가지며 조절인자 Nac의 영향을 받지 않는 프로모터를 제작하였으며, Pn 프로모터 서열은 다음과 같다.
5'- TCGCAGCATTTGACGTCACCGCTTTTACGTGGCTTTATAAAAGACGACGAAAAGCAAAGCCCGAGCATATTCGCGCCAATGCGACGTGAAGGATACAGGGCTATCAAACGATAAGATGGGGTGTCTGGGGTAAT-3' (서열번호 60)
그 결과, 하기 표 1에 나타난 바와 같이, Pn과 동일한 세기의 프로모터 1종(Psynk1) 및 약 15배 강한 프로모터 1종(PsynK2)을 확인하였다.
| 프로모터 종류 | GFP/OD |
| Pn | 2.2 ± 0.3 |
| PsynK1 | 2.6 ± 0.4 |
| PsynK2 | 32.2 ± 2.2 |
배지는 포도당을 탄소원으로 하는 M9-최소 배지를 사용하였고, 48시간, 800rpm, 37℃조건으로 96웰 플레이트에서 배양 후 흡광도를 측정하였다.
1-3: P4HB 생산 균주 제작
실시예 1-2에서 제작한 프로모터 3종을 실시예 1-1의 P4HB 생합성 경로가 구축되어 있는 미생물 균주에 도입하여 P4HB 생산 균주인 다음과 같은 미생물 3종을 제작하였다. 하기 SucD, 4HbD, OrfZ, PhaC, PPC 및 이들의 유전자 서열은 서열번호 1 내지 서열번호 8, 서열번호 13, 및 서열번호 14에 나타내었다.
| 균주 | 대표 유전자형 |
| #1 | Pn-ppc, PuspA-sucD*-4hbD*-phaC* |
| #2 | PsynK2-ppc, PuspA-sucD*-4hbD*-phaC* |
| #3 | PsynK1-ppc, PuspA-sucD*-4hbD*-phaC* |
목표 유전자의 발현을 위해 사용된 프로모터들은 PuspA, PsynK1, 및 PsynK2 가 있으며 이들에 관한 유전자 정보는 ecocyc.org 와 parts.igem.org 에서 수득할 수 있다. 사용된 프로모터 서열은 다음과 같다.
- PsynK1 (5'-tttacagctagctcagtcctaggtattatgctagc-3'); 서열번호 45
- PsynK2 (5'-ctgacagctagctcagtcctaggtataatgctagc-3'); 서열번호 46
- PuspA (5'-AACCACTATCAATATATTCATGTCGAAAATTTGTTTATCTAACGAGTAAGCAAGGCGGATTGACGGATCATCCGGGTCGCTATAAGGTATAGTTCGCAGGACGCGGGTGACGTAACGGCACAAGAAACG-3'); 서열번호 47
전술한 P4HB 생합성 경로가 구축되어 있는 미생물 제작에는 범용적으로 사용되는 기술을 적용하였다 (대표문헌: Sambrook et al., Molecular cloning: A Laboratory Manual, Thrid Ed, Cold Spring Harbor Laboratory, New York (2001)). 구체적으로, 목적 유전자는 공지의 데이터베이스에서 유전자 정보를 수득하고 이를 위한 프라이머를 제작하여, PCR(polymerase chain reaction)을 통해 대상 유전자를 증폭함으로써 원하는 벡터(pCL)에 리가아제를 사용하여 도입하였다. 또는, 화학적으로 합성하는 방법도 사용하였으며, 이는 대장균에서 발현이 원활이 이루어 지도록 코돈 최적화가 필요할 경우에 사용하였다. 목표 유전자의 발현을 위해서는 프로모터와 터미네이터를 PCR를 통해 대상 유전자에 연결하였다. 이렇게 제작된 벡터를 대상 균주에 도입하기 위해서, 일반적인 열충격(Heat shock) 기술을 사용하였다. 더 높은 효율이 필요할 경우는 전기천공(Electroporation) 기술을 사용하였다.
유전자 발현 외에 염색체 상에서 특정 유전자의 약화/결실은 올리고머를 사용하였고, 일반적으로 알려진 Red/ET recombineering 을 이용하였으며, 관련 기술은 Datsenko and Wanner (Proc. Natl. Acad. Sci, USA, 2000, 97,6640-6645)에 보고된 기술을 사용하였다. 결실에 사용된 올리고머는 다음과 같다.
pykF-Left (서열번호 48)
GAAAGCAAGTTTCTCCCATCCTTCTCAACTTAAAGACTAAGACTGTCATG
pykF-right (서열번호 49)
GATATACAAATTAATTCACAAAAGCAATATTACAGGACGTGAACAGATGC
pykA-Left (서열번호 50)
TTTCATGTTCAAGCAACACCTGGTTGTTTCAGTCAACGGAGTATTACATG
pykA-right (서열번호 51)
TGGCGTTTTCGCCGCATCCGGCAACGTACTTACTCTACCGTTAAAATACG
maeB-left (서열번호 52)
TTCAGGGTAAGCGTGAGAGTTAAAAAAAATTACAGCGGTTGGGTTTGCGC
maeB-right (서열번호 53)
TTGCCCACACACTTTATTTGTGAACGTTACGTGAAAGGAACAACCAAATG
maeA-left (서열번호 54)
CCCGGTAGCCTTCACTACCGGGCGCAGGCTTAGATGGAGGTACGGCGGTA
maeA-right (서열번호 55)
GGCCGACGCCCTGGCGGTAAAGCAAAGACGATAAAAGCCCCCCAGGGATG
aspC-left (서열번호 56)
TTTTCAGCGGGCTTCATTGTTTTTAATGCTTACAGCACTGCCACAATCGC
aspC-right (서열번호 57)
TACCCTGATAGCGGACTTCCCTTCTGTAACCATAATGGAACCTCGTCATG
Icd-left (서열번호 58)
AACGTGGTGGCAGACGAGCAAACCAGTAGCGCTCGAAGGAGAGGTGAATG
Icd-right (서열번호 59)
CCCGTTAATAAATTTAACAAACTACGGCATTACATGTTTTCGATGATCGC
1-4: P4HB 생산 균주의 P4HB 생산능 평가
실시예 1-3에서 제작한 균주 3종의 P4HB 생산 테스트를 진행하기 위해 플라스크 실험을 수행하였다. 배양 조건은 48시간, 230rpm, 및 30℃ 이며, 배지는 이전 공개된 자료를 바탕으로 제작하여 사용하였다 (미국 특허 번호 9,084,467 B2). 구체적으로, 1xE2 최소 배지에 1x 미량원소(Trace salt solution)를 첨가하여 배지를 제조하였으며, 이의 탄소:질소 비율 (C/N ratio)을 30:1로 조절하여 사용하였다.
P4HB 분석 조건은 공개된 문헌(미국 특허 번호 9,084,467 B2)을 참조하여 설정하였다. 간략히 설명하면, 전술한 바와 같이 균주 3종의 배양물을 각각 1 mL 채취하여 4,000rpm에서 균체를 회수하였다. 회수한 균주를 동결건조한 후, 샘플의 부탄올 용해(Butanolysis)를 진행하기 위한 제제(Dioxane 용액에 99.9% 부탄올 및 4N HCl를 첨가한 제제)를 첨가하고 93 ℃에서 6시간동안 열처리 하였다. 열처리한 용액을 600 rpm에서 상분리시키고, 유기상(Organic phase)을 채취하여 가스 크로마토그래피(GC) 분석하였다. 4-하이드록시부티레이트(4HB)의 표준 시약은 10% γ -부티로락톤(γ-butyrolactone)을 사용하여 제조하였다.
위와 같은 조건을 사용하여 분석을 진행한 결과, 하기 표 3에 나타나는 바와 같이, 질소제한을 받지 않은 프로모터인 PsynK1을 포함한 균주에서 Pn을 포함한 균주보다 P4HB 농도가 22% 향상된 것을 확인하였다.
| 균주 | P4HB 농도 (g/L) |
| #1 | 14.13 ± 0.05 |
| #2 | 14.81 ± 0.10 |
| #3 | 17.29 ± 0.02 |
한편, 표 3으로부터 지나치게 강한 프로모터를 사용할 경우 (PsynK2) 질소 제한 인자에 영향을 받지 않더라도 P4HB 생산성이 향상되지 않는 것을 추가로 확인하였다.
실시예 2: 환원형 TCA (rTCA) 경로를 이용한 P4HB 생산
포스포에놀피루베이트 카복실라제의 카복실화 반응으로 고정된 이산화탄소를 환원형 TCA 경로를 이용하여 P4HB 생산 경로로 유도하고자 하였다. 환원형 TCA 경로를 통한 P4HB 생산에서 포스포에놀피루베이트 카복실라제 효소를 통해 포스포에놀피루베이트로부터 생성된 옥살로아세테이트는 산화적(oxidative) TCA 경로에 포함된 탈카복실화(decarboxylation) 과정을 거치지 않아 이산화탄소발생 없이 말레이트(malate), 푸마레이트(fumarate), 및 숙시네이트(succinate)를 거쳐 숙시닐-CoA까지 환원될 수 있다. 생성된 숙시닐-CoA는 실시예 1-1의 경로와 동일한 방법을 거쳐 P4HB로 전환될 수 있다.
효율적인 rTCA경로 활성화를 위해 피루베이트 키나아제(pyruvate kinase) 유전자 (pykFA)가 제거된 균주(하기 균주번호 4)를 제작하였다. 제작한 균주 3종은 하기 표 4에 나타내었으며, 하기 균주번호 3은 전술한 표 2의 균주번호 3과 동일하다. 하기 SucD, 4HbD, OrfZ, PhaC, PPC 및 이들의 유전자 서열은 서열번호 1 내지 서열번호 8, 서열번호 13, 및 서열번호 14에 나타내었다. 또한, 하기 MaeAB, AspC, PykFA, 및 이들의 유전자 서열은 서열번호 9 내지 서열번호 12, 서열번호 21 내지 서열번호 24, 서열번호 29, 서열번호 30에 나타내었다.
| 균주 | 대표 유전자 형 |
| #3 | PsynK1-ppc, PuspA-sucD*-4hbD*-phaC* |
| #4 | ΔpykFA PsynK1-ppc, PuspA-sucD*-4hbD*-phaC* |
| #5 | ΔmaeAB ΔaspC ΔpykFA PsynK1-ppc, PupA-sucD*-4hbD*-phaC* |
피루베이트 키나아제를 제거할 경우 PEP에서 피루베이트로 향하는 탄소흐름을 제어하여 PEP의 세포 내 농도를 보존하고, 이를 바탕으로 PEP 카복실라제 경로 활성화를 촉진할 수 있다. 이를 통해 rTCA 경로에 필수적인 옥살로아세테이트의 세포 내 농도를 높일 수 있음을 시사한다.
추가적으로, 확보된 옥살로아세테이트와 말레이트 등의 rTCA 경로상의 경쟁경로를 제거하기 위해 아미노트랜스퍼라제(aspartate aminotransferase; AspC) 와 말레이트 디하이드로게나제(malic enzyme; MaeAB)를 추가로 제거한 균주 또한 제작(상기 균주번호 5)하여 rTCA기반 P4HB 생산 평가를 진행하였다.
상기 표 4의 균주를 대상으로 실시예 1-4와 동일한 방법으로 P4HB 생산성 평가를 하였으며, rTCA 경로가 활성화 될 경우 균체 성장이 향상되었고, 도 7에 나타나는 바와 같이, rTCA 경로가 도입된 균주(균주번호 4 및 5)에서 rTCA 경로가 도입되지 않은 균주(균주번호 3)에 비해 P4HB 수율이 약 28%에서 최대 43%까지 향상된 것을 확인하였다. rTCA 경로를 이용할 경우 기존 48 wt% 수준의 P4HB 이론적 최대 수율을 58 wt%까지 향상시킬 수 있었다.
실시예 3: 글리옥실레이트 경로를 이용한 P4HB 생산
글리옥실레이트 경로(Glyoxylate cycle)를 이용하여 P4HB 생산을 함으로써 2-옥소글루타레이트(2-oxoglutartate)의 탈카복실화(decarboxylation) 경로를 우회하여 이산화탄소로의 탄소 손실을 줄여 결과적으로 P4HB의 생산 수율을 증가시키고자 하였다.
이 경로의 활성화를 위해서 TCA 경로 상의 아이소시트레이트 디하이드로게나제(isocitrate dehydrogenase; icd)를 제거하여 탄소흐름을 강제적으로 글리옥실레이트 경로로 유도시켰다. 글리옥실레이트 경로의 활성화를 위해 대장균 유래의 아이소시트레이트 리아제(isocitrate lyase; aceA) 및 말레이트 신타아제(malate synthase; aceB)를 도입하되 도입되는 미생물 내의 기존 야생형 프로모터를 합성 프로모터로 교체하여 포도당 기반 카타볼릭 억제(Catabolic repression)을 받지 않도록 하였다. 제작한 균주 2종은 하기 표 5에 나타내었다.또한, 하기 Icd, AceBA, 및 이의 유전자 서열은 서열번호 35, 서열번호 36, 서열번호 38, 서열번호 39, 서열번호 43, 및 서열번호 44에 나타내었다.
| 균주 | 대표 유전자 형 |
| #6 | △icd PsynK1-ppc, PuspA-sucD*-4hbD*-phaC* |
| #7 | △icd PsynK1-ppc, PuspA-sucD*-4hbD*-phaC*, pCL-PsynK1-aceBA |
이 때, 글리옥실레이트 경로의 생성물인 숙시네이트와 말레이트 모두 환원형 TCA 경로를 이용하여 숙시닐-CoA로 전환될 수 있다. 생성된 숙시닐-CoA는 위 실시예 1-1의 P4HB 생성 경로를 통해 P4HB로 전환 될 수 있다. 이렇게 제작된 균주의 발효 시, 글루탐산(glutamate)을 외부에서 보강해 주기 위해 Monosodium glutamate (MSG) 50mM을 첨가하였다. 이렇게 제작된 균주를 포도당 배지에 배양하여 PHA 생산을 확인한 결과, 도 8에 나타난 바와 같이, 2.7g/L의 PHA가 생산됨(균주번호 7 GLU+MSG)을 확인하였다. 글리옥실레이트 경로가 도입 되지 않은 균주(균주번호 6)에서 PHA 생산은 0.9g/L에 그쳤으며 글리옥실레이트 경로가 도입되어 있더라도 포도당이 없을 경우(균주번호 7 MSG) PHA는 생산은 관측되지 않았다. 본 방법을 통해 기존의 이론적 최대 수율 48 wt% 에서 58wt%까지 향상시킬 수 있었다.
이상의 설명으로부터, 본 출원이 속하는 기술분야의 당업자는 본 출원이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 출원의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 출원의 범위에 포함되는 것으로 해석되어야 한다.
<110> CJ CheilJedang Corporation
<120> Novel method for preparing poly-4-hydroxybutyrate or
1,4-butanediol
<130> KPA211025-KR-P1
<150> KR 10-2021-0083270
<151> 2021-06-25
<160> 60
<170> KoPatentIn 3.0
<210> 1
<211> 453
<212> PRT
<213> Artificial Sequence
<220>
<223> SucD
<400> 1
Met Ser Asn Glu Val Ser Ile Lys Glu Leu Ile Glu Lys Ala Lys Val
1 5 10 15
Ala Gln Lys Lys Leu Glu Ala Tyr Ser Gln Glu Gln Val Asp Val Leu
20 25 30
Val Lys Ala Leu Gly Lys Val Val Tyr Asp Asn Ala Glu Met Phe Ala
35 40 45
Lys Glu Ala Val Glu Glu Thr Glu Met Gly Val Tyr Glu Asp Lys Val
50 55 60
Ala Lys Cys His Leu Lys Ser Gly Ala Ile Trp Asn His Ile Lys Asp
65 70 75 80
Lys Lys Thr Val Gly Ile Ile Lys Glu Glu Pro Glu Arg Ala Leu Val
85 90 95
Tyr Val Ala Lys Pro Lys Gly Val Val Ala Ala Thr Thr Pro Ile Thr
100 105 110
Asn Pro Val Val Thr Pro Met Cys Asn Ala Met Ala Ala Ile Lys Gly
115 120 125
Arg Asn Thr Ile Ile Val Ala Pro His Pro Lys Ala Lys Lys Val Ser
130 135 140
Ala His Thr Val Glu Leu Met Asn Ala Glu Leu Lys Lys Leu Gly Ala
145 150 155 160
Pro Glu Asn Ile Ile Gln Ile Val Glu Ala Pro Ser Arg Glu Ala Ala
165 170 175
Lys Glu Leu Met Glu Ser Ala Asp Val Val Ile Ala Thr Gly Gly Ala
180 185 190
Gly Arg Val Lys Ala Ala Tyr Ser Ser Gly Arg Pro Ala Tyr Gly Val
195 200 205
Gly Pro Gly Asn Ser Gln Val Ile Val Asp Lys Gly Tyr Asp Tyr Asn
210 215 220
Lys Ala Ala Gln Asp Ile Ile Thr Gly Arg Lys Tyr Asp Asn Gly Ile
225 230 235 240
Ile Cys Ser Ser Glu Gln Ser Val Ile Ala Pro Ala Glu Asp Tyr Asp
245 250 255
Lys Val Ile Ala Ala Phe Val Glu Asn Gly Ala Phe Tyr Val Glu Asp
260 265 270
Glu Glu Thr Val Glu Lys Phe Arg Ser Thr Leu Phe Lys Asp Gly Lys
275 280 285
Ile Asn Ser Lys Ile Ile Gly Lys Ser Val Gln Ile Ile Ala Asp Leu
290 295 300
Ala Gly Val Lys Val Pro Glu Gly Thr Lys Val Ile Val Leu Lys Gly
305 310 315 320
Lys Gly Ala Gly Glu Lys Asp Val Leu Cys Lys Glu Lys Met Cys Pro
325 330 335
Val Leu Val Ala Leu Lys Tyr Asp Thr Phe Glu Glu Ala Val Glu Ile
340 345 350
Ala Met Ala Asn Tyr Met Tyr Glu Gly Ala Gly His Thr Ala Gly Ile
355 360 365
His Ser Asp Asn Asp Glu Asn Ile Arg Tyr Ala Gly Thr Val Leu Pro
370 375 380
Ile Ser Arg Leu Val Val Asn Gln Pro Ala Thr Thr Ala Gly Gly Ser
385 390 395 400
Phe Asn Asn Gly Phe Asn Pro Thr Thr Thr Leu Gly Cys Gly Ser Trp
405 410 415
Gly Arg Asn Ser Ile Ser Glu Asn Leu Thr Tyr Glu His Leu Ile Asn
420 425 430
Val Ser Arg Ile Gly Tyr Phe Asn Lys Glu Ala Lys Val Pro Ser Tyr
435 440 445
Glu Glu Ile Trp Gly
450
<210> 2
<211> 1362
<212> DNA
<213> Artificial Sequence
<220>
<223> sucD
<400> 2
atgtccaacg aggttagcat taaggagctg attgagaagg cgaaagtggc gcagaaaaag 60
ctggaagcgt atagccaaga gcaagttgac gttctggtca aggcgctggg taaagttgtg 120
tacgacaacg ccgagatgtt cgcgaaagag gcggtggagg aaaccgagat gggtgtttac 180
gaggataaag tggctaaatg tcatctgaaa tctggtgcaa tctggaatca cattaaagat 240
aagaaaaccg ttggtattat caaggaagaa ccggagcgtg cgctggtgta cgtcgcgaag 300
cctaaaggtg ttgtggcggc gacgacccct atcaccaatc ctgtggttac cccgatgtgt 360
aacgcgatgg cagcaattaa aggtcgcaac accatcattg tcgccccgca tccgaaggcg 420
aagaaggtga gcgcgcacac cgtggagctg atgaatgcag aactgaaaaa gttgggtgcg 480
ccggaaaaca ttatccagat cgttgaagcc ccaagccgtg aagcagccaa ggagttgatg 540
gagagcgcag acgtggttat cgccacgggt ggcgcaggcc gtgttaaagc agcgtactcc 600
tccggccgtc cggcatacgg tgtcggtccg ggcaattctc aggtcattgt cgataagggt 660
tacgattata acaaagctgc ccaggacatc attaccggcc gcaagtatga caacggtatc 720
atttgcagct ctgagcagag cgtgatcgca ccggcggagg actacgacaa ggtcatcgcg 780
gctttcgtcg agaatggcgc gttctatgtc gaggatgagg aaactgtgga gaaattccgt 840
agcacgctgt tcaaggatgg caagatcaat agcaaaatca tcggtaaatc cgtgcagatc 900
atcgctgacc tggctggtgt caaggtgccg gaaggcacca aggtgatcgt gttgaagggc 960
aagggtgccg gtgaaaagga cgttctgtgc aaggagaaaa tgtgcccggt cctggttgcc 1020
ctgaaatatg acacctttga ggaggcggtc gagatcgcga tggccaacta tatgtacgag 1080
ggtgcgggcc ataccgccgg tatccacagc gataacgacg agaatatccg ctacgcgggt 1140
acggtgctgc caatcagccg tctggttgtc aaccagccag caactacggc cggtggtagc 1200
tttaacaatg gttttaatcc gaccaccacc ttgggctgcg gtagctgggg ccgtaactcc 1260
attagcgaga acctgacgta tgagcatctg attaatgtca gccgtattgg ctatttcaat 1320
aaggaggcaa aagttcctag ctacgaggag atctggggtt aa 1362
<210> 3
<211> 289
<212> PRT
<213> Artificial Sequence
<220>
<223> 4HbD
<400> 3
Met Glu Val Gly Phe Leu Gly Leu Gly Ile Met Gly Lys Ala Met Ser
1 5 10 15
Met Asn Leu Leu Lys Asn Gly Phe Lys Val Thr Val Trp Asn Arg Thr
20 25 30
Leu Ser Lys Cys Asp Glu Leu Val Glu His Gly Ala Ser Val Cys Glu
35 40 45
Ser Pro Ala Glu Val Ile Lys Lys Cys Lys Tyr Thr Ile Ala Met Leu
50 55 60
Ser Asp Pro Cys Ala Ala Leu Ser Val Val Phe Asp Lys Gly Gly Val
65 70 75 80
Leu Glu Gln Ile Cys Glu Gly Lys Gly Tyr Ile Asp Met Ser Thr Val
85 90 95
Asp Ala Glu Thr Ser Leu Lys Ile Asn Glu Ala Ile Thr Gly Lys Gly
100 105 110
Gly Arg Phe Val Glu Gly Pro Val Ser Gly Ser Lys Lys Pro Ala Glu
115 120 125
Asp Gly Gln Leu Ile Ile Leu Ala Ala Gly Asp Lys Ala Leu Phe Glu
130 135 140
Glu Ser Ile Pro Ala Phe Asp Val Leu Gly Lys Arg Ser Phe Tyr Leu
145 150 155 160
Gly Gln Val Gly Asn Gly Ala Lys Met Lys Leu Ile Val Asn Met Ile
165 170 175
Met Gly Ser Met Met Asn Ala Phe Ser Glu Gly Leu Val Leu Ala Asp
180 185 190
Lys Ser Gly Leu Ser Ser Asp Thr Leu Leu Asp Ile Leu Asp Leu Gly
195 200 205
Ala Met Thr Asn Pro Met Phe Lys Gly Lys Gly Pro Ser Met Thr Lys
210 215 220
Ser Ser Tyr Pro Pro Ala Phe Pro Leu Lys His Gln Gln Lys Asp Met
225 230 235 240
Arg Leu Ala Leu Ala Leu Gly Asp Glu Asn Ala Val Ser Met Pro Val
245 250 255
Ala Ala Ala Ala Asn Glu Ala Phe Lys Lys Ala Arg Ser Leu Gly Leu
260 265 270
Gly Asp Leu Asp Phe Ser Ala Val Ile Glu Ala Val Lys Phe Ser Arg
275 280 285
Glu
<210> 4
<211> 870
<212> DNA
<213> Artificial Sequence
<220>
<223> 4hbD
<400> 4
atggaagtag gttttctggg tctgggcatt atgggtaaag ctatgtccat gaacctgctg 60
aaaaacggtt tcaaagttac cgtgtggaac cgcactctgt ctaaatgtga tgaactggtt 120
gaacacggtg caagcgtgtg cgagtctccg gctgaggtga tcaagaaatg caaatacacg 180
atcgcgatgc tgagcgatcc gtgtgcagct ctgtctgttg ttttcgataa aggcggtgtt 240
ctggaacaga tctgcgaggg taagggctac atcgacatgt ctaccgtcga cgcggaaact 300
agcctgaaaa ttaacgaagc gatcacgggc aaaggtggcc gttttgtaga aggtcctgtt 360
agcggttcca aaaagccggc agaagacggc cagctgatca tcctggcagc aggcgacaaa 420
gcactgttcg aggaatccat cccggccttt gatgtactgg gcaaacgttc cttttatctg 480
ggtcaggtgg gtaacggtgc gaaaatgaaa ctgattgtta acatgatcat gggttctatg 540
atgaacgcgt ttagcgaagg tctggtactg gcagataaaa gcggtctgtc tagcgacacg 600
ctgctggata ttctggatct gggtgctatg acgaatccga tgttcaaagg caaaggtccg 660
tccatgacta aatccagcta cccaccggct ttcccgctga aacaccagca gaaagacatg 720
cgtctggctc tggctctggg cgacgaaaac gctgttagca tgccggtcgc tgcggctgcg 780
aacgaagcct tcaagaaagc ccgtagcctg ggcctgggcg atctggactt ttctgctgtt 840
atcgaagcgg taaaattctc tcgtgaataa 870
<210> 5
<211> 429
<212> PRT
<213> Artificial Sequence
<220>
<223> OrfZ
<400> 5
Met Glu Trp Glu Glu Ile Tyr Lys Glu Lys Leu Val Thr Ala Glu Lys
1 5 10 15
Ala Val Ser Lys Ile Glu Asn His Ser Arg Val Val Phe Ala His Ala
20 25 30
Val Gly Glu Pro Val Asp Leu Val Asn Ala Leu Val Lys Asn Lys Asp
35 40 45
Asn Tyr Ile Gly Leu Glu Ile Val His Met Val Ala Met Gly Lys Gly
50 55 60
Glu Tyr Thr Lys Glu Gly Met Gln Arg His Phe Arg His Asn Ala Leu
65 70 75 80
Phe Val Gly Gly Cys Thr Arg Asp Ala Val Asn Ser Gly Arg Ala Asp
85 90 95
Tyr Thr Pro Cys Phe Phe Tyr Glu Val Pro Ser Leu Phe Lys Glu Lys
100 105 110
Arg Leu Pro Val Asp Val Ala Leu Ile Gln Val Ser Glu Pro Asp Lys
115 120 125
Tyr Gly Tyr Cys Ser Phe Gly Val Ser Asn Asp Tyr Thr Lys Pro Ala
130 135 140
Ala Glu Ser Ala Lys Leu Val Ile Ala Glu Val Asn Lys Asn Met Pro
145 150 155 160
Arg Thr Leu Gly Asp Ser Phe Ile His Val Ser Asp Ile Asp Tyr Ile
165 170 175
Val Glu Ala Ser His Pro Leu Leu Glu Leu Gln Pro Pro Lys Leu Gly
180 185 190
Asp Val Glu Lys Ala Ile Gly Glu Asn Cys Ala Ser Leu Ile Glu Asp
195 200 205
Gly Ala Thr Leu Gln Leu Gly Ile Gly Ala Ile Pro Asp Ala Val Leu
210 215 220
Leu Phe Leu Lys Asn Lys Lys Asn Leu Gly Ile His Ser Glu Met Ile
225 230 235 240
Ser Asp Gly Val Met Glu Leu Val Lys Ala Gly Val Ile Asn Asn Lys
245 250 255
Lys Lys Thr Leu His Pro Gly Lys Ile Val Val Thr Phe Leu Met Gly
260 265 270
Thr Lys Lys Leu Tyr Asp Phe Val Asn Asn Asn Pro Met Val Glu Thr
275 280 285
Tyr Ser Val Asp Tyr Val Asn Asn Pro Leu Val Ile Met Lys Asn Asp
290 295 300
Asn Met Val Ser Ile Asn Ser Cys Val Gln Val Asp Leu Met Gly Gln
305 310 315 320
Val Cys Ser Glu Ser Ile Gly Leu Lys Gln Ile Ser Gly Val Gly Gly
325 330 335
Gln Val Asp Phe Ile Arg Gly Ala Asn Leu Ser Lys Gly Gly Lys Ala
340 345 350
Ile Ile Ala Ile Pro Ser Thr Ala Gly Lys Gly Lys Val Ser Arg Ile
355 360 365
Thr Pro Leu Leu Asp Thr Gly Ala Ala Val Thr Thr Ser Arg Asn Glu
370 375 380
Val Asp Tyr Val Val Thr Glu Tyr Gly Val Ala His Leu Lys Gly Lys
385 390 395 400
Thr Leu Arg Asn Arg Ala Arg Ala Leu Ile Asn Ile Ala His Pro Lys
405 410 415
Phe Arg Glu Ser Leu Met Asn Glu Phe Lys Lys Arg Phe
420 425
<210> 6
<211> 1290
<212> DNA
<213> Artificial Sequence
<220>
<223> orfZ
<400> 6
atggagtggg aagagatata taaagagaaa ctggtaactg cagaaaaagc tgtttcaaaa 60
atagaaaacc atagcagggt agtttttgca catgcagtag gagaacccgt agatttagta 120
aatgcactag ttaaaaataa ggataattat ataggactag aaatagttca catggtagct 180
atgggcaaag gtgaatatac aaaagagggt atgcaaagac attttagaca taatgcttta 240
tttgtaggcg gatgtactag agatgcagta aattcaggaa gagcagatta tacaccttgt 300
tttttctatg aagtgccaag tttgtttaaa gaaaaacgtt tgcctgtaga tgtagcactt 360
attcaggtaa gtgagccaga taaatatggc tactgcagtt ttggagtttc caatgactat 420
accaagccag cagcagaaag tgctaagctt gtaattgcag aagtgaataa aaacatgcca 480
agaactcttg gagattcttt tatacatgta tcagatattg attatatagt ggaagcttca 540
cacccattgt tagaattgca gcctcctaaa ttgggagatg tagaaaaagc cataggagaa 600
aactgtgcat ctttaattga agatggagct actcttcagc ttggaatagg tgctatacca 660
gatgcggtac ttttattctt aaagaacaaa aagaatttag gaatacattc tgagatgata 720
tcagatggtg tgatggaact ggtgaaggca ggggttatca ataacaagaa aaagaccctc 780
catccaggca aaatagttgt aacattttta atgggaacaa aaaaattata tgattttgta 840
aacaataatc caatggtaga aacttattct gtagattatg taaataatcc actggtaatt 900
atgaaaaatg acaatatggt ttcaataaat tcttgtgttc aagtagactt aatgggacaa 960
gtatgttctg aaagtatagg attgaaacag ataagtggag tgggaggcca ggtagatttt 1020
attagaggag ctaatctatc aaagggtgga aaggctatta tagctatacc ttccacagct 1080
ggaaaaggaa aagtttcaag aataactcca cttctagata ctggtgctgc agttacaact 1140
tctagaaatg aagtagatta tgtagttact gaatatggtg ttgctcatct taagggcaaa 1200
actttaagaa atagggcaag agctctaata aatatcgctc atccaaaatt cagagaatca 1260
ttaatgaatg aatttaaaaa gagattttag 1290
<210> 7
<211> 606
<212> PRT
<213> Artificial Sequence
<220>
<223> PhaC
<400> 7
Met Thr Arg Arg Arg Phe His Met Ser Asn Lys Asn Asn Asp Glu Leu
1 5 10 15
Ala Thr Gly Lys Gly Ala Ala Ala Ser Ser Thr Glu Gly Lys Ser Gln
20 25 30
Pro Phe Lys Phe Pro Pro Gly Pro Leu Asp Pro Ala Thr Trp Leu Glu
35 40 45
Trp Ser Arg Gln Trp Gln Gly Pro Glu Gly Asn Gly Gly Thr Val Pro
50 55 60
Gly Gly Phe Pro Gly Phe Glu Ala Phe Ala Ala Ser Pro Leu Ala Gly
65 70 75 80
Val Lys Ile Asp Pro Ala Gln Leu Ala Glu Ile Gln Gln Arg Tyr Met
85 90 95
Arg Asp Phe Thr Glu Leu Trp Arg Gly Leu Ala Gly Gly Asp Thr Glu
100 105 110
Ser Ala Gly Lys Leu His Asp Arg Arg Phe Ala Ser Glu Ala Trp His
115 120 125
Lys Asn Ala Pro Tyr Arg Tyr Thr Ala Ala Phe Tyr Leu Leu Asn Ala
130 135 140
Arg Ala Leu Thr Glu Leu Ala Asp Ala Val Glu Ala Asp Pro Lys Thr
145 150 155 160
Arg Gln Arg Ile Arg Phe Ala Val Ser Gln Trp Val Asp Ala Met Ser
165 170 175
Pro Ala Asn Phe Leu Ala Thr Asn Pro Asp Ala Gln Asn Arg Leu Ile
180 185 190
Glu Ser Arg Gly Glu Ser Leu Arg Ala Gly Met Arg Asn Met Leu Glu
195 200 205
Asp Leu Thr Arg Gly Lys Ile Ser Gln Thr Asp Glu Thr Ala Phe Glu
210 215 220
Val Gly Arg Asn Met Ala Val Thr Glu Gly Ala Val Val Phe Glu Asn
225 230 235 240
Glu Phe Phe Gln Leu Leu Gln Tyr Lys Pro Leu Thr Asp Lys Val Tyr
245 250 255
Thr Arg Pro Leu Leu Leu Val Pro Pro Cys Ile Asn Lys Phe Tyr Ile
260 265 270
Leu Asp Leu Gln Pro Glu Gly Ser Leu Val Arg Tyr Ala Val Glu Gln
275 280 285
Gly His Thr Val Phe Leu Val Ser Trp Arg Asn Pro Asp Ala Ser Met
290 295 300
Ala Gly Cys Thr Trp Asp Asp Tyr Ile Glu Asn Ala Ala Ile Arg Ala
305 310 315 320
Ile Glu Val Val Arg Asp Ile Ser Gly Gln Asp Lys Ile Asn Thr Leu
325 330 335
Gly Phe Cys Val Gly Gly Thr Ile Ile Ser Thr Ala Leu Ala Val Leu
340 345 350
Ala Ala Arg Gly Glu His Pro Val Ala Ser Leu Thr Leu Leu Thr Thr
355 360 365
Leu Leu Asp Phe Thr Asp Thr Gly Ile Leu Asp Val Phe Val Asp Glu
370 375 380
Pro His Val Gln Leu Arg Glu Ala Thr Leu Gly Gly Ala Ser Gly Gly
385 390 395 400
Leu Leu Arg Gly Val Glu Leu Ala Asn Thr Phe Ser Phe Leu Arg Pro
405 410 415
Asn Asp Leu Val Trp Asn Tyr Val Val Asp Asn Tyr Leu Lys Gly Asn
420 425 430
Thr Pro Val Pro Phe Asp Leu Leu Phe Trp Asn Gly Asp Ala Thr Asn
435 440 445
Leu Pro Gly Pro Trp Tyr Cys Trp Tyr Leu Arg His Thr Tyr Leu Gln
450 455 460
Asn Glu Leu Lys Glu Pro Gly Lys Leu Thr Val Cys Asn Glu Pro Val
465 470 475 480
Asp Leu Gly Ala Ile Asn Val Pro Thr Tyr Ile Tyr Gly Ser Arg Glu
485 490 495
Asp His Ile Val Pro Trp Thr Ala Ala Tyr Ala Ser Thr Ala Leu Leu
500 505 510
Lys Asn Asp Leu Arg Phe Val Leu Gly Ala Ser Gly His Ile Ala Gly
515 520 525
Val Ile Asn Pro Pro Ala Lys Lys Lys Arg Ser His Trp Thr Asn Asp
530 535 540
Ala Leu Pro Glu Ser Ala Gln Asp Trp Leu Ala Gly Ala Glu Glu His
545 550 555 560
His Gly Ser Trp Trp Pro Asp Trp Met Thr Trp Leu Gly Lys Gln Ala
565 570 575
Gly Ala Lys Arg Ala Ala Pro Thr Glu Tyr Gly Ser Lys Arg Tyr Ala
580 585 590
Ala Ile Glu Pro Ala Pro Gly Arg Tyr Val Lys Ala Lys Ala
595 600 605
<210> 8
<211> 1821
<212> DNA
<213> Artificial Sequence
<220>
<223> phaC
<400> 8
atgactagaa ggaggtttca tatgagtaac aagaacaacg atgagctggc gacgggtaaa 60
ggtgctgctg catcttctac tgaaggtaaa tctcagccgt ttaaattccc accgggtccg 120
ctggacccgg ccacttggct ggaatggagc cgtcagtggc aaggtccgga gggcaatggc 180
ggtaccgtgc cgggtggctt tccgggtttc gaagcgttcg cggcgtcccc gctggcgggc 240
gtgaaaatcg acccggctca gctggcagag atccagcagc gttatatgcg tgatttcacc 300
gagctgtggc gtggtctggc aggcggtgac accgagagcg ctggcaaact gcatgaccgt 360
cgcttcgcgt ccgaagcgtg gcacaaaaac gcgccgtatc gctatactgc ggcattttac 420
ctgctgaacg cacgtgcact gacggaactg gctgatgcag tagaagcgga tccgaaaacc 480
cgtcagcgta tccgttttgc ggtttcccag tgggtagatg ctatgagccc ggctaacttc 540
ctggccacca acccggacgc tcagaaccgt ctgatcgaga gccgtggtga aagcctgcgt 600
gccggcatgc gcaatatgct ggaagatctg acccgcggta aaatttccca aaccgatgag 660
actgccttcg aagtaggccg taacatggca gttaccgaag gtgctgtggt attcgaaaac 720
gagttcttcc agctgctgca gtacaaacct ctgactgaca aagtatacac ccgtccgctg 780
ctgctggtac cgccgtgcat taacaagttc tatattctgg acctgcagcc ggaaggttct 840
ctggtccgtt acgcagtcga acagggtcac actgtattcc tggtgagctg gcgcaatcca 900
gacgctagca tggctggctg tacctgggat gactatattg aaaacgcggc tatccgcgcc 960
atcgaggttg tgcgtgatat cagcggtcag gacaagatca acaccctggg cttttgtgtt 1020
ggtggcacga tcatctccac tgccctggcg gtcctggccg cccgtggtga gcacccggtg 1080
gcctctctga ccctgctgac taccctgctg gacttcaccg atactggtat cctggatgtt 1140
ttcgtggacg agccacacgt tcagctgcgt gaggcgactc tgggcggcgc cagcggcggt 1200
ctgctgcgtg gtgtcgagct ggccaatacc ttttccttcc tgcgcccgaa cgacctggtt 1260
tggaactacg ttgttgacaa ctatctgaaa ggcaacaccc cggtaccttt cgatctgctg 1320
ttctggaacg gtgatgcaac caacctgcct ggtccatggt actgttggta cctgcgtcat 1380
acttacctgc agaacgaact gaaagagccg ggcaaactga ccgtgtgtaa cgaacctgtg 1440
gacctgggcg cgattaacgt tcctacttac atctacggtt cccgtgaaga tcacatcgta 1500
ccgtggaccg cggcttacgc cagcaccgcg ctgctgaaga acgatctgcg tttcgtactg 1560
ggcgcatccg gccatatcgc aggtgtgatc aaccctcctg caaagaaaaa gcgttctcat 1620
tggaccaacg acgcgctgcc agaatccgcg caggattggc tggcaggtgc tgaggaacac 1680
catggttcct ggtggccgga ttggatgacc tggctgggta aacaagccgg tgcaaaacgt 1740
gcagctccaa ctgaatatgg tagcaagcgt tatgctgcaa tcgagccagc gccaggccgt 1800
tacgttaaag cgaaagcata a 1821
<210> 9
<211> 480
<212> PRT
<213> Artificial Sequence
<220>
<223> PykA
<400> 9
Met Ser Arg Arg Leu Arg Arg Thr Lys Ile Val Thr Thr Leu Gly Pro
1 5 10 15
Ala Thr Asp Arg Asp Asn Asn Leu Glu Lys Val Ile Ala Ala Gly Ala
20 25 30
Asn Val Val Arg Met Asn Phe Ser His Gly Ser Pro Glu Asp His Lys
35 40 45
Met Arg Ala Asp Lys Val Arg Glu Ile Ala Ala Lys Leu Gly Arg His
50 55 60
Val Ala Ile Leu Gly Asp Leu Gln Gly Pro Lys Ile Arg Val Ser Thr
65 70 75 80
Phe Lys Glu Gly Lys Val Phe Leu Asn Ile Gly Asp Lys Phe Leu Leu
85 90 95
Asp Ala Asn Leu Gly Lys Gly Glu Gly Asp Lys Glu Lys Val Gly Ile
100 105 110
Asp Tyr Lys Gly Leu Pro Ala Asp Val Val Pro Gly Asp Ile Leu Leu
115 120 125
Leu Asp Asp Gly Arg Val Gln Leu Lys Val Leu Glu Val Gln Gly Met
130 135 140
Lys Val Phe Thr Glu Val Thr Val Gly Gly Pro Leu Ser Asn Asn Lys
145 150 155 160
Gly Ile Asn Lys Leu Gly Gly Gly Leu Ser Ala Glu Ala Leu Thr Glu
165 170 175
Lys Asp Lys Ala Asp Ile Lys Thr Ala Ala Leu Ile Gly Val Asp Tyr
180 185 190
Leu Ala Val Ser Phe Pro Arg Cys Gly Glu Asp Leu Asn Tyr Ala Arg
195 200 205
Arg Leu Ala Arg Asp Ala Gly Cys Asp Ala Lys Ile Val Ala Lys Val
210 215 220
Glu Arg Ala Glu Ala Val Cys Ser Gln Asp Ala Met Asp Asp Ile Ile
225 230 235 240
Leu Ala Ser Asp Val Val Met Val Ala Arg Gly Asp Leu Gly Val Glu
245 250 255
Ile Gly Asp Pro Glu Leu Val Gly Ile Gln Lys Ala Leu Ile Arg Arg
260 265 270
Ala Arg Gln Leu Asn Arg Ala Val Ile Thr Ala Thr Gln Met Met Glu
275 280 285
Ser Met Ile Thr Asn Pro Met Pro Thr Arg Ala Glu Val Met Asp Val
290 295 300
Ala Asn Ala Val Leu Asp Gly Thr Asp Ala Val Met Leu Ser Ala Glu
305 310 315 320
Thr Ala Ala Gly Gln Tyr Pro Ser Glu Thr Val Ala Ala Met Ala Arg
325 330 335
Val Cys Leu Gly Ala Glu Lys Ile Pro Ser Ile Asn Val Ser Lys His
340 345 350
Arg Leu Asp Val Gln Phe Asp Asn Val Glu Glu Ala Ile Ala Met Ser
355 360 365
Ala Met Tyr Ala Ala Asn His Leu Lys Gly Val Thr Ala Ile Ile Thr
370 375 380
Met Thr Glu Ser Gly Arg Thr Ala Leu Met Thr Ser Arg Ile Ser Ser
385 390 395 400
Gly Leu Pro Ile Phe Ala Met Ser Arg His Glu Arg Thr Leu Asn Leu
405 410 415
Thr Ala Leu Tyr Arg Gly Val Thr Pro Val His Phe Asp Ser Ala Asn
420 425 430
Asp Gly Val Ala Ala Ala Ser Glu Ala Val Asn Leu Leu Arg Asp Lys
435 440 445
Gly Tyr Leu Met Ser Gly Asp Leu Val Ile Val Thr Gln Gly Asp Val
450 455 460
Met Ser Thr Val Gly Ser Thr Asn Thr Thr Arg Ile Leu Thr Val Glu
465 470 475 480
<210> 10
<211> 1443
<212> DNA
<213> Artificial Sequence
<220>
<223> pykA
<400> 10
atgtccagaa ggcttcgcag aacaaaaatc gttaccacgt taggcccagc aacagatcgc 60
gataataatc ttgaaaaagt tatcgcggcg ggtgccaacg ttgtacgtat gaacttttct 120
cacggctcgc ctgaagatca caaaatgcgc gcggataaag ttcgtgagat tgccgcaaaa 180
ctggggcgtc atgtggctat tctgggtgac ctccaggggc ccaaaatccg tgtatccacc 240
tttaaagaag gcaaagtttt cctcaatatt ggggataaat tcctgctcga cgccaacctg 300
ggtaaaggtg aaggcgacaa agaaaaagtc ggtatcgact acaaaggcct gcctgctgac 360
gtcgtgcctg gtgacatcct gctgctggac gatggtcgcg tccagttaaa agtactggaa 420
gttcagggca tgaaagtgtt caccgaagtc accgtcggtg gtcccctctc caacaataaa 480
ggtatcaaca aacttggcgg cggtttgtcg gctgaagcgc tgaccgaaaa agacaaagca 540
gacattaaga ctgcggcgtt gattggcgta gattacctgg ctgtctcctt cccacgctgt 600
ggcgaagatc tgaactatgc ccgtcgcctg gcacgcgatg caggatgtga tgcgaaaatt 660
gttgccaagg ttgaacgtgc ggaagccgtt tgcagccagg atgcaatgga tgacatcatc 720
ctcgcctctg acgtggtaat ggttgcacgt ggcgacctcg gtgtggaaat tggcgacccg 780
gaactggtcg gcattcagaa agcgttgatc cgtcgtgcgc gtcagctaaa ccgagcggta 840
atcacggcga cccagatgat ggagtcaatg attactaacc cgatgccgac gcgtgcagaa 900
gtcatggacg tagcaaacgc cgttctggat ggtactgacg ctgtgatgct gtctgcagaa 960
actgccgctg ggcagtatcc gtcagaaacc gttgcagcca tggcgcgcgt ttgcctgggt 1020
gcggaaaaaa tcccgagcat caacgtttct aaacaccgtc tggacgttca gttcgacaat 1080
gtggaagaag ctattgccat gtcagcaatg tacgcagcta accacctgaa aggcgttacg 1140
gcgatcatca ccatgaccga atcgggtcgt accgcgctga tgacctcccg tatcagctct 1200
ggtctgccaa ttttcgccat gtcgcgccat gaacgtacgc tgaacctgac tgctctctat 1260
cgtggcgtta cgccggtgca ctttgatagc gctaatgacg gcgtagcagc tgccagcgaa 1320
gcggttaatc tgctgcgcga taaaggttac ttgatgtctg gtgacctggt gattgtcacc 1380
cagggcgacg tgatgagtac cgtgggttct actaatacca cgcgtatttt aacggtagag 1440
taa 1443
<210> 11
<211> 470
<212> PRT
<213> Artificial Sequence
<220>
<223> PykF
<400> 11
Met Lys Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Lys Thr Glu Ser
1 5 10 15
Glu Glu Met Leu Ala Lys Met Leu Asp Ala Gly Met Asn Val Met Arg
20 25 30
Leu Asn Phe Ser His Gly Asp Tyr Ala Glu His Gly Gln Arg Ile Gln
35 40 45
Asn Leu Arg Asn Val Met Ser Lys Thr Gly Lys Thr Ala Ala Ile Leu
50 55 60
Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr Met Lys Leu Glu Gly Gly
65 70 75 80
Asn Asp Val Ser Leu Lys Ala Gly Gln Thr Phe Thr Phe Thr Thr Asp
85 90 95
Lys Ser Val Ile Gly Asn Ser Glu Met Val Ala Val Thr Tyr Glu Gly
100 105 110
Phe Thr Thr Asp Leu Ser Val Gly Asn Thr Val Leu Val Asp Asp Gly
115 120 125
Leu Ile Gly Met Glu Val Thr Ala Ile Glu Gly Asn Lys Val Ile Cys
130 135 140
Lys Val Leu Asn Asn Gly Asp Leu Gly Glu Asn Lys Gly Val Asn Leu
145 150 155 160
Pro Gly Val Ser Ile Ala Leu Pro Ala Leu Ala Glu Lys Asp Lys Gln
165 170 175
Asp Leu Ile Phe Gly Cys Glu Gln Gly Val Asp Phe Val Ala Ala Ser
180 185 190
Phe Ile Arg Lys Arg Ser Asp Val Ile Glu Ile Arg Glu His Leu Lys
195 200 205
Ala His Gly Gly Glu Asn Ile His Ile Ile Ser Lys Ile Glu Asn Gln
210 215 220
Glu Gly Leu Asn Asn Phe Asp Glu Ile Leu Glu Ala Ser Asp Gly Ile
225 230 235 240
Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Val Glu Glu Val
245 250 255
Ile Phe Ala Gln Lys Met Met Ile Glu Lys Cys Ile Arg Ala Arg Lys
260 265 270
Val Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Ile Lys Asn Pro
275 280 285
Arg Pro Thr Arg Ala Glu Ala Gly Asp Val Ala Asn Ala Ile Leu Asp
290 295 300
Gly Thr Asp Ala Val Met Leu Ser Gly Glu Ser Ala Lys Gly Lys Tyr
305 310 315 320
Pro Leu Glu Ala Val Ser Ile Met Ala Thr Ile Cys Glu Arg Thr Asp
325 330 335
Arg Val Met Asn Ser Arg Leu Glu Phe Asn Asn Asp Asn Arg Lys Leu
340 345 350
Arg Ile Thr Glu Ala Val Cys Arg Gly Ala Val Glu Thr Ala Glu Lys
355 360 365
Leu Asp Ala Pro Leu Ile Val Val Ala Thr Gln Gly Gly Lys Ser Ala
370 375 380
Arg Ala Val Arg Lys Tyr Phe Pro Asp Ala Thr Ile Leu Ala Leu Thr
385 390 395 400
Thr Asn Glu Lys Thr Ala His Gln Leu Val Leu Ser Lys Gly Val Val
405 410 415
Pro Gln Leu Val Lys Glu Ile Thr Ser Thr Asp Asp Phe Tyr Arg Leu
420 425 430
Gly Lys Glu Leu Ala Leu Gln Ser Gly Leu Ala His Lys Gly Asp Val
435 440 445
Val Val Met Val Ser Gly Ala Leu Val Pro Ser Gly Thr Thr Asn Thr
450 455 460
Ala Ser Val His Val Leu
465 470
<210> 12
<211> 1413
<212> DNA
<213> Artificial Sequence
<220>
<223> pykF
<400> 12
atgaaaaaga ccaaaattgt ttgcaccatc ggaccgaaaa ccgaatctga agagatgtta 60
gctaaaatgc tggacgctgg catgaacgtt atgcgtctga acttctctca tggtgactat 120
gcagaacacg gtcagcgcat tcagaatctg cgcaacgtga tgagcaaaac tggtaaaacc 180
gccgctatcc tgcttgatac caaaggtccg gaaatccgca ccatgaaact ggaaggcggt 240
aacgacgttt ctctgaaagc tggtcagacc tttactttca ccactgataa atctgttatc 300
ggcaacagcg aaatggttgc ggtaacgtat gaaggtttca ctactgacct gtctgttggc 360
aacaccgtac tggttgacga tggtctgatc ggtatggaag ttaccgccat tgaaggtaac 420
aaagttatct gtaaagtgct gaacaacggt gacctgggcg aaaacaaagg tgtgaacctg 480
cctggcgttt ccattgctct gccagcactg gctgaaaaag acaaacagga cctgatcttt 540
ggttgcgaac aaggcgtaga ctttgttgct gcttccttta ttcgtaagcg ttctgacgtt 600
atcgaaatcc gtgagcacct gaaagcgcac ggcggcgaaa acatccacat catctccaaa 660
atcgaaaacc aggaaggcct caacaacttc gacgaaatcc tcgaagcctc tgacggcatc 720
atggttgcgc gtggcgacct gggtgtagaa atcccggtag aagaagttat cttcgcccag 780
aagatgatga tcgaaaaatg tatccgtgca cgtaaagtcg ttatcactgc gacccagatg 840
ctggattcca tgatcaaaaa cccacgcccg actcgcgcag aagccggtga cgttgcaaac 900
gccatcctcg acggtactga cgcagtgatg ctgtctggtg aatccgcaaa aggtaaatac 960
ccgctggaag cggtttctat catggcgacc atctgcgaac gtaccgaccg cgtgatgaac 1020
agccgtctcg agttcaacaa tgacaaccgt aaactgcgca ttaccgaagc ggtatgccgt 1080
ggtgccgttg aaactgctga aaaactggat gctccgctga tcgtggttgc tactcagggc 1140
ggtaaatctg ctcgcgcagt acgtaaatac ttcccggatg ccaccatcct ggcactgacc 1200
accaacgaaa aaacggctca tcagttggta ctgagcaaag gcgttgtgcc gcagcttgtt 1260
aaagagatca cttctactga tgatttctac cgtctgggta aagaactggc tctgcagagc 1320
ggtctggcac acaaaggtga cgttgtagtt atggtttctg gtgcactggt accgagcggc 1380
actactaaca ccgcatctgt tcacgtcctg taa 1413
<210> 13
<211> 883
<212> PRT
<213> Artificial Sequence
<220>
<223> PPC
<400> 13
Met Asn Glu Gln Tyr Ser Ala Leu Arg Ser Asn Val Ser Met Leu Gly
1 5 10 15
Lys Val Leu Gly Glu Thr Ile Lys Asp Ala Leu Gly Glu His Ile Leu
20 25 30
Glu Arg Val Glu Thr Ile Arg Lys Leu Ser Lys Ser Ser Arg Ala Gly
35 40 45
Asn Asp Ala Asn Arg Gln Glu Leu Leu Thr Thr Leu Gln Asn Leu Ser
50 55 60
Asn Asp Glu Leu Leu Pro Val Ala Arg Ala Phe Ser Gln Phe Leu Asn
65 70 75 80
Leu Ala Asn Thr Ala Glu Gln Tyr His Ser Ile Ser Pro Lys Gly Glu
85 90 95
Ala Ala Ser Asn Pro Glu Val Ile Ala Arg Thr Leu Arg Lys Leu Lys
100 105 110
Asn Gln Pro Glu Leu Ser Glu Asp Thr Ile Lys Lys Ala Val Glu Ser
115 120 125
Leu Ser Leu Glu Leu Val Leu Thr Ala His Pro Thr Glu Ile Thr Arg
130 135 140
Arg Thr Leu Ile His Lys Met Val Glu Val Asn Ala Cys Leu Lys Gln
145 150 155 160
Leu Asp Asn Lys Asp Ile Ala Asp Tyr Glu His Asn Gln Leu Met Arg
165 170 175
Arg Leu Arg Gln Leu Ile Ala Gln Ser Trp His Thr Asp Glu Ile Arg
180 185 190
Lys Leu Arg Pro Ser Pro Val Asp Glu Ala Lys Trp Gly Phe Ala Val
195 200 205
Val Glu Asn Ser Leu Trp Gln Gly Val Pro Asn Tyr Leu Arg Glu Leu
210 215 220
Asn Glu Gln Leu Glu Glu Asn Leu Gly Tyr Lys Leu Pro Val Glu Phe
225 230 235 240
Val Pro Val Arg Phe Thr Ser Trp Met Gly Gly Asp Arg Asp Gly Asn
245 250 255
Pro Asn Val Thr Ala Asp Ile Thr Arg His Val Leu Leu Leu Ser Arg
260 265 270
Trp Lys Ala Thr Asp Leu Phe Leu Lys Asp Ile Gln Val Leu Val Ser
275 280 285
Glu Leu Ser Met Val Glu Ala Thr Pro Glu Leu Leu Ala Leu Val Gly
290 295 300
Glu Glu Gly Ala Ala Glu Pro Tyr Arg Tyr Leu Met Lys Asn Leu Arg
305 310 315 320
Ser Arg Leu Met Ala Thr Gln Ala Trp Leu Glu Ala Arg Leu Lys Gly
325 330 335
Glu Glu Leu Pro Lys Pro Glu Gly Leu Leu Thr Gln Asn Glu Glu Leu
340 345 350
Trp Glu Pro Leu Tyr Ala Cys Tyr Gln Ser Leu Gln Ala Cys Gly Met
355 360 365
Gly Ile Ile Ala Asn Gly Asp Leu Leu Asp Thr Leu Arg Arg Val Lys
370 375 380
Cys Phe Gly Val Pro Leu Val Arg Ile Asp Ile Arg Gln Glu Ser Thr
385 390 395 400
Arg His Thr Glu Ala Leu Gly Glu Leu Thr Arg Tyr Leu Gly Ile Gly
405 410 415
Asp Tyr Glu Ser Trp Ser Glu Ala Asp Lys Gln Ala Phe Leu Ile Arg
420 425 430
Glu Leu Asn Ser Lys Arg Pro Leu Leu Pro Arg Asn Trp Gln Pro Ser
435 440 445
Ala Glu Thr Arg Glu Val Leu Asp Thr Cys Gln Val Ile Ala Glu Ala
450 455 460
Pro Gln Gly Ser Ile Ala Ala Tyr Val Ile Ser Met Ala Lys Thr Pro
465 470 475 480
Ser Asp Val Leu Ala Val His Leu Leu Leu Lys Glu Ala Gly Ile Gly
485 490 495
Phe Ala Met Pro Val Ala Pro Leu Phe Glu Thr Leu Asp Asp Leu Asn
500 505 510
Asn Ala Asn Asp Val Met Thr Gln Leu Leu Asn Ile Asp Trp Tyr Arg
515 520 525
Gly Leu Ile Gln Gly Lys Gln Met Val Met Ile Gly Tyr Ser Asp Ser
530 535 540
Ala Lys Asp Ala Gly Val Met Ala Ala Ser Trp Ala Gln Tyr Gln Ala
545 550 555 560
Gln Asp Ala Leu Ile Lys Thr Cys Glu Lys Ala Gly Ile Glu Leu Thr
565 570 575
Leu Phe His Gly Arg Gly Gly Ser Ile Gly Arg Gly Gly Ala Pro Ala
580 585 590
His Ala Ala Leu Leu Ser Gln Pro Pro Gly Ser Leu Lys Gly Gly Leu
595 600 605
Arg Val Thr Glu Gln Gly Glu Met Ile Arg Phe Lys Tyr Gly Leu Pro
610 615 620
Glu Ile Thr Val Ser Ser Leu Ser Leu Tyr Thr Gly Ala Ile Leu Glu
625 630 635 640
Ala Asn Leu Leu Pro Pro Pro Glu Pro Lys Glu Ser Trp Arg Arg Ile
645 650 655
Met Asp Glu Leu Ser Val Ile Ser Cys Asp Val Tyr Arg Gly Tyr Val
660 665 670
Arg Glu Asn Lys Asp Phe Val Pro Tyr Phe Arg Ser Ala Thr Pro Glu
675 680 685
Gln Glu Leu Gly Lys Leu Pro Leu Gly Ser Arg Pro Ala Lys Arg Arg
690 695 700
Pro Thr Gly Gly Val Glu Ser Leu Arg Ala Ile Pro Trp Ile Phe Ala
705 710 715 720
Trp Thr Gln Asn Arg Leu Met Leu Pro Ala Trp Leu Gly Ala Gly Thr
725 730 735
Ala Leu Gln Lys Val Val Glu Asp Gly Lys Gln Ser Glu Leu Glu Ala
740 745 750
Met Cys Arg Asp Trp Pro Phe Phe Ser Thr Arg Leu Gly Met Leu Glu
755 760 765
Met Val Phe Ala Lys Ala Asp Leu Trp Leu Ala Glu Tyr Tyr Asp Gln
770 775 780
Arg Leu Val Asp Lys Ala Leu Trp Pro Leu Gly Lys Glu Leu Arg Asn
785 790 795 800
Leu Gln Glu Glu Asp Ile Lys Val Val Leu Ala Ile Ala Asn Asp Ser
805 810 815
His Leu Met Ala Asp Leu Pro Trp Ile Ala Glu Ser Ile Gln Leu Arg
820 825 830
Asn Ile Tyr Thr Asp Pro Leu Asn Val Leu Gln Ala Glu Leu Leu His
835 840 845
Arg Ser Arg Gln Ala Glu Lys Glu Gly Gln Glu Pro Asp Pro Arg Val
850 855 860
Glu Gln Ala Leu Met Val Thr Ile Ala Gly Ile Ala Ala Gly Met Arg
865 870 875 880
Asn Thr Gly
<210> 14
<211> 2652
<212> DNA
<213> Artificial Sequence
<220>
<223> ppc
<400> 14
atgaacgaac aatattccgc attgcgtagt aatgtcagta tgctcggcaa agtgctggga 60
gaaaccatca aggatgcgtt gggagaacac attcttgaac gcgtagaaac tatccgtaag 120
ttgtcgaaat cttcacgcgc tggcaatgat gctaaccgcc aggagttgct caccacctta 180
caaaatttgt cgaacgacga gctgctgccc gttgcgcgtg cgtttagtca gttcctgaac 240
ctggccaaca ccgccgagca ataccacagc atttcgccga aaggcgaagc tgccagcaac 300
ccggaagtga tcgcccgcac cctgcgtaaa ctgaaaaacc agccggaact gagcgaagac 360
accatcaaaa aagcagtgga atcgctgtcg ctggaactgg tcctcacggc tcacccaacc 420
gaaattaccc gtcgtacact gatccacaaa atggtggaag tgaacgcctg tttaaaacag 480
ctcgataaca aagatatcgc tgactacgaa cacaaccagc tgatgcgtcg cctgcgccag 540
ttgatcgccc agtcatggca taccgatgaa atccgtaagc tgcgtccaag cccggtagat 600
gaagccaaat ggggctttgc cgtagtggaa aacagcctgt ggcaaggcgt accaaattac 660
ctgcgcgaac tgaacgaaca actggaagag aacctcggct acaaactgcc cgtcgaattt 720
gttccggtcc gttttacttc gtggatgggc ggcgaccgcg acggcaaccc gaacgtcact 780
gccgatatca cccgccacgt cctgctactc agccgctgga aagccaccga tttgttcctg 840
aaagatattc aggtgctggt ttctgaactg tcgatggttg aagcgacccc tgaactgctg 900
gcgctggttg gcgaagaagg tgccgcagaa ccgtatcgct atctgatgaa aaacctgcgt 960
tctcgcctga tggcgacaca ggcatggctg gaagcgcgcc tgaaaggcga agaactgcca 1020
aaaccagaag gcctgctgac acaaaacgaa gaactgtggg aaccgctcta cgcttgctac 1080
cagtcacttc aggcgtgtgg catgggtatt atcgccaacg gcgatctgct cgacaccctg 1140
cgccgcgtga aatgtttcgg cgtaccgctg gtccgtattg atatccgtca ggagagcacg 1200
cgtcataccg aagcgctggg cgagctgacc cgctacctcg gtatcggcga ctacgaaagc 1260
tggtcagagg ccgacaaaca ggcgttcctg atccgcgaac tgaactccaa acgtccgctt 1320
ctgccgcgca actggcaacc aagcgccgaa acgcgcgaag tgctcgatac ctgccaggtg 1380
attgccgaag caccgcaagg ctccattgcc gcctacgtga tctcgatggc gaaaacgccg 1440
tccgacgtac tggctgtcca cctgctgctg aaagaagcgg gtatcgggtt tgcgatgccg 1500
gttgctccgc tgtttgaaac cctcgatgat ctgaacaacg ccaacgatgt catgacccag 1560
ctgctcaata ttgactggta tcgtggcctg attcagggca aacagatggt gatgattggc 1620
tattccgact cagcaaaaga tgcgggagtg atggcagctt cctgggcgca atatcaggca 1680
caggatgcat taatcaaaac ctgcgaaaaa gcgggtattg agctgacgtt gttccacggt 1740
cgcggcggtt ccattggtcg cggcggcgca cctgctcatg cggcgctgct gtcacaaccg 1800
ccaggaagcc tgaaaggcgg cctgcgcgta accgaacagg gcgagatgat ccgctttaaa 1860
tatggtctgc cagaaatcac cgtcagcagc ctgtcgcttt ataccggggc gattctggaa 1920
gccaacctgc tgccaccgcc ggagccgaaa gagagctggc gtcgcattat ggatgaactg 1980
tcagtcatct cctgcgatgt ctaccgcggc tacgtacgtg aaaacaaaga ttttgtgcct 2040
tacttccgct ccgctacgcc ggaacaagaa ctgggcaaac tgccgttggg ttcacgtccg 2100
gcgaaacgtc gcccaaccgg cggcgtcgag tcactacgcg ccattccgtg gatcttcgcc 2160
tggacgcaaa accgtctgat gctccccgcc tggctgggtg caggtacggc gctgcaaaaa 2220
gtggtcgaag acggcaaaca gagcgagctg gaggctatgt gccgcgattg gccattcttc 2280
tcgacgcgtc tcggcatgct ggagatggtc ttcgccaaag cagacctgtg gctggcggaa 2340
tactatgacc aacgcctggt agacaaagca ctgtggccgt taggtaaaga gttacgcaac 2400
ctgcaagaag aagacatcaa agtggtgctg gcgattgcca acgattccca tctgatggcc 2460
gatctgccgt ggattgcaga gtctattcag ctacggaata tttacaccga cccgctgaac 2520
gtattgcagg ccgagttgct gcaccgctcc cgccaggcag aaaaagaagg ccaggaaccg 2580
gatcctcgcg tcgaacaagc gttaatggtc actattgccg ggattgcggc aggtatgcgt 2640
aataccggct aa 2652
<210> 15
<211> 264
<212> PRT
<213> Artificial Sequence
<220>
<223> EcaA
<400> 15
Met Ser Ser Thr Leu Tyr Arg Arg Gln Leu Leu Lys Leu Leu Gly Met
1 5 10 15
Ser Val Leu Gly Thr Ser Phe Ser Ser Cys Val Thr Ser Pro Ala Arg
20 25 30
Ala Lys Thr Val Asn Trp Gly Tyr Ile Gly Lys Val Gly Pro Glu His
35 40 45
Trp Gly Glu Leu Ser Pro Asp Phe Ala Leu Cys Gln Ile Gly Arg Lys
50 55 60
Gln Thr Pro Ile Asp Leu Gln Ile Ala Asp Val Lys Asp Val His Ser
65 70 75 80
Ser Ser Gln Asp Leu Leu Val Thr Asn Tyr Gln Pro Thr Ala Leu His
85 90 95
Leu Ile Asn Asn Gly Lys Thr Val Gln Val Asn Tyr Gln Pro Gly Ser
100 105 110
Tyr Leu Lys Tyr Ala His Gln Lys Phe Glu Leu Leu Gln Phe His Phe
115 120 125
His His Phe Ser Glu His Arg Val Asp Gly Lys Leu Tyr Asp Met Glu
130 135 140
Leu His Leu Val His Arg Ser Lys Ser Gly Asp Leu Ala Val Met Gly
145 150 155 160
Ile Phe Leu Gln Ala Gly Ala Phe Asn Pro Thr Leu Gln Ile Ile Trp
165 170 175
Asp Ala Thr Pro Gln Asn Gln Gly Thr Asp Lys Arg Ile Glu Asp Ile
180 185 190
Asn Ile Asp Ala Ser Gln Phe Leu Pro Ala Gln His Arg Phe Phe Thr
195 200 205
Tyr Ser Gly Ser Leu Thr Thr Pro Pro Cys Ser Glu Asn Val Leu Trp
210 215 220
Cys Val Met Ala Thr Pro Ile Glu Ala Ser Pro Ala Gln Ile Ala Lys
225 230 235 240
Phe Ser Gln Met Phe Pro Gln Asn Ala Arg Pro Val Gln Pro Leu Asn
245 250 255
Asp Arg Leu Val Ile Glu Ala Ile
260
<210> 16
<211> 795
<212> DNA
<213> Artificial Sequence
<220>
<223> ecaA
<400> 16
atgtcttcta ccctgtaccg tcgtcagctg ctgaaactgc tgggtatgtc tgttctgggt 60
acctctttct cttcttgcgt tacctctccg gctcgtgcta aaaccgttaa ctggggttac 120
atcggtaaag ttggtccgga acactggggt gaactgtctc cggacttcgc tctgtgccag 180
atcggtcgta aacagacccc gatcgacctg caaatcgctg acgttaaaga cgttcactct 240
tcttctcagg acctgctggt taccaactac cagccgaccg ctctgcacct gatcaacaac 300
ggtaaaaccg ttcaggttaa ctaccagccg ggttcttacc tgaaatacgc tcaccagaaa 360
ttcgaactgc tgcaattcca cttccaccac ttctctgaac accgtgttga cggtaaactg 420
tacgacatgg aactgcacct ggttcaccgt tctaaatctg gtgacctggc tgttatgggt 480
atcttcctgc aagctggtgc tttcaacccg accctgcaaa tcatctggga cgctaccccg 540
cagaaccagg gtaccgacaa acgtatcgaa gacatcaaca tcgacgcttc tcagttcctg 600
ccggctcagc accgtttctt cacctactct ggttctctga ccaccccgcc gtgctctgaa 660
aacgttctgt ggtgcgttat ggctaccccg atcgaagcta gtccggctca gatcgctaaa 720
ttctctcaga tgttcccgca gaacgctcgt ccggttcagc cgctgaacga ccgtctggtt 780
atcgaagcta tctaa 795
<210> 17
<211> 427
<212> PRT
<213> Artificial Sequence
<220>
<223> GltA
<400> 17
Met Ala Asp Thr Lys Ala Lys Leu Thr Leu Asn Gly Asp Thr Ala Val
1 5 10 15
Glu Leu Asp Val Leu Lys Gly Thr Leu Gly Gln Asp Val Ile Asp Ile
20 25 30
Arg Thr Leu Gly Ser Lys Gly Val Phe Thr Phe Asp Pro Gly Phe Thr
35 40 45
Ser Thr Ala Ser Cys Glu Ser Lys Ile Thr Phe Ile Asp Gly Asp Glu
50 55 60
Gly Ile Leu Leu His Arg Gly Phe Pro Ile Asp Gln Leu Ala Thr Asp
65 70 75 80
Ser Asn Tyr Leu Glu Val Cys Tyr Ile Leu Leu Asn Gly Glu Lys Pro
85 90 95
Thr Gln Glu Gln Tyr Asp Glu Phe Lys Thr Thr Val Thr Arg His Thr
100 105 110
Met Ile His Glu Gln Ile Thr Arg Leu Phe His Ala Phe Arg Arg Asp
115 120 125
Ser His Pro Met Ala Val Met Cys Gly Ile Thr Gly Ala Leu Ala Ala
130 135 140
Phe Tyr His Asp Ser Leu Asp Val Asn Asn Pro Arg His Arg Glu Ile
145 150 155 160
Ala Ala Phe Arg Leu Leu Ser Lys Met Pro Thr Met Ala Ala Met Cys
165 170 175
Tyr Lys Tyr Ser Ile Gly Gln Pro Phe Val Tyr Pro Arg Asn Asp Leu
180 185 190
Ser Tyr Ala Gly Asn Phe Leu Asn Met Met Phe Ser Thr Pro Cys Glu
195 200 205
Pro Tyr Glu Val Asn Pro Ile Leu Glu Arg Ala Met Asp Arg Ile Leu
210 215 220
Ile Leu His Ala Asp His Glu Gln Asn Ala Ser Thr Ser Thr Val Arg
225 230 235 240
Thr Ala Gly Ser Ser Gly Ala Asn Pro Phe Ala Cys Ile Ala Ala Gly
245 250 255
Ile Ala Ser Leu Trp Gly Pro Ala His Gly Gly Ala Asn Glu Ala Ala
260 265 270
Leu Lys Met Leu Glu Glu Ile Ser Ser Val Lys His Ile Pro Glu Phe
275 280 285
Val Arg Arg Ala Lys Asp Lys Asn Asp Ser Phe Arg Leu Met Gly Phe
290 295 300
Gly His Arg Val Tyr Lys Asn Tyr Asp Pro Arg Ala Thr Val Met Arg
305 310 315 320
Glu Thr Cys His Glu Val Leu Lys Glu Leu Gly Thr Lys Asp Asp Leu
325 330 335
Leu Glu Val Ala Met Glu Leu Glu Asn Ile Ala Leu Asn Asp Pro Tyr
340 345 350
Phe Ile Glu Lys Lys Leu Tyr Pro Asn Val Asp Phe Tyr Ser Gly Ile
355 360 365
Ile Leu Lys Ala Met Gly Ile Pro Ser Ser Met Phe Thr Val Ile Phe
370 375 380
Ala Met Ala Arg Thr Val Gly Trp Ile Ala His Trp Ser Glu Met His
385 390 395 400
Ser Asp Gly Met Lys Ile Ala Arg Pro Arg Gln Leu Tyr Thr Gly Tyr
405 410 415
Glu Lys Arg Asp Phe Lys Ser Asp Ile Lys Arg
420 425
<210> 18
<211> 1284
<212> DNA
<213> Artificial Sequence
<220>
<223> gltA
<400> 18
atggctgata caaaagcaaa actcaccctc aacggggata cagctgttga actggatgtg 60
ctgaaaggca cgctgggtca agatgttatt gatatccgta ctctcggttc aaaaggtgtg 120
ttcacctttg acccaggctt cacttcaacc gcatcctgcg aatctaaaat tacttttatt 180
gatggtgatg aaggtatttt gctgcaccgc ggtttcccga tcgatcagct ggcgaccgat 240
tctaactacc tggaagtttg ttacatcctg ctgaatggtg aaaaaccgac tcaggaacag 300
tatgacgaat ttaaaactac ggtgacccgt cataccatga tccacgagca gattacccgt 360
ctgttccatg ctttccgtcg cgactcgcat ccaatggcag tcatgtgtgg tattaccggc 420
gcgctggcgg cgttctatca cgactcgctg gatgttaaca atcctcgtca ccgtgaaatt 480
gccgcgttcc gcctgctgtc gaaaatgccg accatggccg cgatgtgtta caagtattcc 540
attggtcagc catttgttta cccgcgcaac gatctctcct acgccggtaa cttcctgaat 600
atgatgttct ccacgccgtg cgaaccgtat gaagttaatc cgattctgga acgtgctatg 660
gaccgtattc tgatcctgca cgctgaccat gaacagaacg cctctacctc caccgtgcgt 720
accgctggct cttcgggtgc gaacccgttt gcctgtatcg cagcaggtat tgcttcactg 780
tggggacctg cgcacggcgg tgctaacgaa gcggcgctga aaatgctgga agaaatcagc 840
tccgttaaac acattccgga atttgttcgt cgtgcgaaag acaaaaatga ttctttccgc 900
ctgatgggct tcggtcaccg cgtgtacaaa aattacgacc cgcgcgccac cgtaatgcgt 960
gaaacctgcc atgaagtgct gaaagagctg ggcacgaagg atgacctgct ggaagtggct 1020
atggagctgg aaaacatcgc gctgaacgac ccgtacttta tcgagaagaa actgtacccg 1080
aacgtcgatt tctactctgg tatcatcctg aaagcgatgg gtattccgtc ttccatgttc 1140
accgtcattt tcgcaatggc acgtaccgtt ggctggatcg cccactggag cgaaatgcac 1200
agtgacggta tgaagattgc ccgtccgcgt cagctgtata caggatatga aaaacgcgac 1260
tttaaaagcg atatcaagcg ttaa 1284
<210> 19
<211> 1154
<212> PRT
<213> Artificial Sequence
<220>
<223> Pyc
<400> 19
Met Pro Ile Ser Lys Ile Leu Val Ala Asn Arg Ser Glu Ile Ala Ile
1 5 10 15
Arg Val Phe Arg Ala Ala Asn Glu Leu Gly Ile Lys Thr Val Ala Ile
20 25 30
Trp Ala Glu Glu Asp Lys Leu Ala Leu His Arg Phe Lys Ala Asp Glu
35 40 45
Ser Tyr Gln Val Gly Arg Gly Pro His Leu Ala Arg Asp Leu Gly Pro
50 55 60
Ile Glu Ser Tyr Leu Ser Ile Asp Glu Val Ile Arg Val Ala Lys Leu
65 70 75 80
Ser Gly Ala Asp Ala Ile His Pro Gly Tyr Gly Leu Leu Ser Glu Ser
85 90 95
Pro Glu Phe Val Asp Ala Cys Asn Lys Ala Gly Ile Ile Phe Ile Gly
100 105 110
Pro Lys Ala Asp Thr Met Arg Gln Leu Gly Asn Lys Val Ala Ala Arg
115 120 125
Asn Leu Ala Ile Ser Val Gly Val Pro Val Val Pro Ala Thr Glu Pro
130 135 140
Leu Pro Asp Asp Met Ala Glu Val Ala Lys Met Ala Glu Glu Ile Gly
145 150 155 160
Tyr Pro Val Met Leu Lys Ala Ser Trp Gly Gly Gly Gly Arg Gly Met
165 170 175
Arg Ala Ile Arg Asp Pro Lys Asp Leu Ala Arg Glu Val Thr Glu Ala
180 185 190
Lys Arg Glu Ala Met Ala Ala Phe Gly Lys Asp Glu Val Tyr Leu Glu
195 200 205
Lys Leu Val Glu Arg Ala Arg His Val Glu Ser Gln Val Leu Gly Asp
210 215 220
Thr His Gly Asn Val Val His Leu Phe Glu Arg Asp Cys Ser Ile Gln
225 230 235 240
Arg Arg Asn Gln Lys Val Val Glu Arg Ala Pro Ala Pro Tyr Leu Ser
245 250 255
Glu Ala Gln Arg Gln Glu Leu Ala Ala Tyr Ser Leu Lys Ile Ala Ala
260 265 270
Ala Thr Asn Tyr Ile Gly Ala Gly Thr Val Glu Tyr Leu Met Asp Ala
275 280 285
Asp Thr Gly Lys Phe Tyr Phe Ile Glu Val Asn Pro Arg Ile Gln Val
290 295 300
Glu His Thr Val Thr Glu Val Val Thr Gly Ile Asp Ile Val Lys Ala
305 310 315 320
Gln Ile His Ile Leu Asp Gly Ala Ala Ile Gly Thr Pro Glu Ser Gly
325 330 335
Val Pro Thr Gln Ala Asp Ile Arg Leu Asn Gly His Ala Leu Gln Cys
340 345 350
Arg Ile Thr Thr Glu Asp Pro Glu His Asn Phe Ile Pro Asp Tyr Gly
355 360 365
Arg Ile Thr Ala Tyr Arg Ser Ala Ser Gly Phe Gly Ile Arg Leu Asp
370 375 380
Gly Gly Thr Ser Tyr Ser Gly Ala Ile Ile Thr Arg Tyr Tyr Asp Pro
385 390 395 400
Leu Leu Val Lys Val Thr Ala Trp Ala Pro Asn Pro Ser Glu Ala Ile
405 410 415
Ser Arg Met Asp Arg Ala Leu Arg Glu Phe Arg Ile Arg Gly Val Ala
420 425 430
Thr Asn Leu Thr Phe Leu Glu Ala Ile Ile Gly His Pro Lys Phe Arg
435 440 445
Asp Asn Ser Tyr Thr Thr Arg Phe Ile Asp Thr Thr Pro Glu Leu Phe
450 455 460
Gln Gln Val Lys Arg Gln Asp Arg Ala Thr Lys Leu Leu Thr Tyr Leu
465 470 475 480
Ala Asp Val Thr Val Asn Gly His Pro Glu Ala Lys Asp Arg Pro Lys
485 490 495
Pro Ile Ala Asn Ala Ala Lys Pro Val Val Pro Tyr Ala Asn Gly Asn
500 505 510
Gly Val Lys Asp Gly Thr Lys Gln Leu Leu Asp Arg Leu Gly Pro Lys
515 520 525
Lys Phe Gly Glu Trp Met Arg Asn Glu Lys Arg Val Leu Leu Thr Asp
530 535 540
Thr Thr Met Arg Asp Gly His Gln Ser Leu Leu Ala Thr Arg Met Arg
545 550 555 560
Thr Tyr Asp Ile Ala Arg Ile Ala Gly Thr Tyr Ala His Ala Leu Pro
565 570 575
Asn Leu Leu Ser Leu Glu Cys Trp Gly Gly Ala Thr Phe Asp Val Ser
580 585 590
Met Arg Phe Leu Thr Glu Asp Pro Trp Glu Arg Leu Ala Leu Ile Arg
595 600 605
Glu Gly Ala Pro Asn Leu Leu Leu Gln Met Leu Leu Arg Gly Ala Asn
610 615 620
Gly Val Gly Tyr Thr Asn Tyr Pro Asp Asn Val Val Lys Tyr Phe Val
625 630 635 640
Arg Gln Ala Ala Arg Gly Gly Ile Asp Leu Phe Arg Val Phe Asp Cys
645 650 655
Leu Asn Trp Val Glu Asn Met Arg Val Ser Met Asp Ala Ile Ala Glu
660 665 670
Glu Asn Lys Leu Cys Glu Ala Ala Ile Cys Tyr Thr Gly Asp Ile Leu
675 680 685
Asn Ser Ala Arg Pro Lys Tyr Asp Leu Lys Tyr Tyr Thr Glu Leu Ala
690 695 700
Val Glu Leu Glu Lys Ala Gly Ala His Ile Ile Ala Val Lys Asp Met
705 710 715 720
Ala Gly Leu Leu Lys Pro Ala Ala Ala Lys Val Leu Phe Lys Ala Leu
725 730 735
Arg Glu Ala Thr Gly Leu Pro Ile His Phe His Thr His Asp Thr Ser
740 745 750
Gly Ile Ala Ala Ala Thr Val Leu Ala Ala Val Glu Ala Gly Val Asp
755 760 765
Ala Val Asp Ala Ala Met Asp Ala Leu Ser Gly Asn Thr Ser Gln Pro
770 775 780
Cys Leu Gly Ser Ile Val Glu Ala Leu Ser Gly Ser Glu Arg Asp Pro
785 790 795 800
Gly Leu Asp Pro Glu Trp Ile Arg Arg Ile Ser Phe Tyr Trp Glu Ala
805 810 815
Val Arg Asn Gln Tyr Ala Ala Phe Glu Ser Asp Leu Lys Gly Pro Ala
820 825 830
Ser Glu Val Tyr Leu His Glu Met Pro Gly Gly Gln Phe Thr Asn Leu
835 840 845
Lys Glu Gln Ala Arg Ser Leu Gly Leu Glu Thr Arg Trp His Gln Val
850 855 860
Ala Gln Ala Tyr Ala Asp Ala Asn Gln Met Phe Gly Asp Ile Val Lys
865 870 875 880
Val Thr Pro Ser Ser Lys Val Val Gly Asp Met Ala Leu Met Met Val
885 890 895
Ser Gln Asp Leu Thr Val Ala Asp Val Val Ser Pro Glu Arg Glu Val
900 905 910
Ser Phe Pro Glu Ser Val Val Ser Met Leu Lys Gly Asp Leu Gly Gln
915 920 925
Pro Pro Ser Gly Trp Pro Ala Ala Leu Gln Lys Lys Ala Leu Lys Gly
930 935 940
Glu Lys Pro Tyr Thr Val Arg Pro Gly Ser Leu Leu Lys Glu Ala Asp
945 950 955 960
Leu Asp Ala Glu Arg Lys Val Ile Glu Thr Lys Leu Glu Arg Glu Val
965 970 975
Ser Asp Phe Glu Phe Ala Ser Tyr Leu Met Tyr Pro Lys Val Phe Thr
980 985 990
Asp Phe Ala Leu Ala Ser Asp Thr Tyr Gly Pro Val Ser Val Leu Pro
995 1000 1005
Thr Pro Ala Tyr Phe Tyr Gly Leu Ala Asp Gly Asp Glu Leu Phe Ala
1010 1015 1020
Asp Ile Glu Lys Gly Lys Thr Leu Val Ile Val Asn Gln Ala Met Ser
1025 1030 1035 1040
Ala Thr Asp Ser Gln Gly Met Val Thr Val Phe Phe Glu Leu Asn Gly
1045 1050 1055
Gln Pro Arg Arg Ile Lys Val Pro Asp Arg Ala His Gly Ala Thr Gly
1060 1065 1070
Ala Ala Val Arg Arg Lys Ala Glu Pro Gly Asn Gly Ala His Val Gly
1075 1080 1085
Ala Pro Met Pro Gly Val Ile Ser Arg Val Phe Ala Ser Ser Gly Gln
1090 1095 1100
Ala Val Ser Ala Gly Asp Val Leu Val Ser Ile Glu Ala Met Lys Met
1105 1110 1115 1120
Glu Thr Ala Ile His Ala Glu Lys Asp Gly Thr Ile Ala Glu Ile Leu
1125 1130 1135
Val Lys Ala Gly Asp Gln Ile Asp Ala Lys Asp Leu Leu Val Val Tyr
1140 1145 1150
Ala Gly
<210> 20
<211> 3465
<212> DNA
<213> Artificial Sequence
<220>
<223> pyc
<400> 20
ttgcccattt ccaagatact cgttgccaat cgctctgaaa tagccattcg cgtgttccgc 60
gcggccaacg agcttggaat aaaaacggtg gcgatctggg cggaagagga caagctggcg 120
ctgcaccgct tcaaggcaga cgaaagctat caggtcggcc gcggcccgca tcttgctcgc 180
gacctcggac cgatcgagag ctatctgtcg atcgatgagg tgatccgcgt cgccaagctt 240
tccggcgccg acgccattca ccccggctac ggcctcttgt cggaaagccc ggaattcgtc 300
gatgcctgca acaaggccgg catcatcttc atcggcccga aggccgatac gatgcgccag 360
ctcggcaaca aggtggcggc gcgcaatctg gcgatctcgg tcggcgtgcc cgtcgtgccg 420
gcaaccgaac cgttgccgga cgatatggcc gaagtggcga agatggccga agagatcggt 480
tatcccgtca tgctgaaggc ttcctggggc ggtggcggcc gcggcatgcg cgcaatccgc 540
gatccgaagg atctcgcccg cgaggtgact gaggccaagc gcgaggcgat ggcggccttc 600
ggcaaggacg aagtctatct ggaaaagctg gtcgagcgtg cccgccatgt cgaaagccag 660
gtcctcggcg acacacacgg caatgtcgtg catctcttcg agcgtgactg ctcgatccag 720
cgccgcaacc agaaggtcgt cgagcgcgcg ccggcgccct atctttccga ggcgcagcgc 780
caggagctcg ccgcctattc gctgaagatc gcagcggcga ccaactatat cggcgccggc 840
accgtcgaat atctgatgga tgccgatacc ggaaaattct acttcatcga ggtcaatccg 900
cggatccagg tcgagcatac ggtgaccgaa gtggtcaccg gcatcgatat cgtcaaggcg 960
cagatccata tcctcgacgg ggctgcgatc ggcacgccgg aatcgggcgt tcccactcag 1020
gctgacatcc gtctcaacgg ccatgcgctg cagtgccgca tcaccacgga agatccggag 1080
cacaacttca ttccggatta cggccgcatc accgcttatc gctcggcttc cggcttcggc 1140
atccgtctcg acggcggcac ctcctattcc ggggcgatca tcacccgcta ttatgatccg 1200
ctgctcgtca aggtgacggc ctgggcgccg aacccgtccg aagcgatttc ccgcatggac 1260
cgggcgctgc gcgaattccg catccgcggc gtcgccacca acctgacctt cctcgaagcg 1320
atcatcggcc acccgaaatt ccgcgacaac agctacacca cccgcttcat cgacacgacg 1380
ccggagctgt tccagcaggt caagcgccag gaccgcgcga cgaagctctt gacctatctc 1440
gccgacgtca ccgtcaacgg ccatcccgag gccaaggacc ggccgaagcc gattgccaac 1500
gccgccaagc cggtggtgcc ctatgccaac ggcaatggcg tcaaggacgg caccaagcag 1560
ctgctcgaca ggctcggccc gaagaaattc ggcgaatgga tgcgcaatga gaagcgcgtg 1620
cttttgaccg atacgacaat gcgcgacggc catcagtcgc tgcttgccac ccgcatgcgc 1680
acctatgaca tcgccaggat cgccggcacc tatgcgcatg cgctgccaaa ccttctgtcg 1740
ctcgaatgct ggggcggcgc caccttcgac gtctccatgc gcttcctgac cgaagatccg 1800
tgggagcggc tggcgctgat ccgcgagggc gcgccgaacc tgctcctgca gatgctgctg 1860
cgcggcgcca acggcgtcgg ctacaccaac tatcccgaca atgtcgtcaa atatttcgtc 1920
cgccaggcgg ccagaggcgg catcgatctc ttccgcgtct tcgactgcct gaactgggtc 1980
gagaacatgc gggtatcaat ggatgcgatc gccgaggaga acaaactctg cgaggcggcg 2040
atctgctata ccggcgatat cctcaattcc gcccgcccga aatacgacct gaaatactat 2100
accgagcttg cggtcgagct cgaaaaggcc ggcgcccata tcatcgcggt caaggatatg 2160
gcgggtctgt tgaagccggc ggcagcgaag gtgctgttca aggcgctgcg cgaggcgacc 2220
ggcctgccga tccacttcca cacccatgac acctcgggca ttgcggccgc caccgttctt 2280
gccgccgtcg aagccggtgt cgatgccgtc gatgcggcga tggatgcgct ttccggcaat 2340
acttcgcagc cctgtctcgg ctcgatcgtc gaggcgctct ccggctccga gcgtgatccc 2400
ggcctcgatc cggaatggat ccgccgcatc tccttctatt gggaagcggt gcgcaatcag 2460
tatgccgcct tcgaaagcga cctcaagggg ccggcctcgg aagtctatct gcacgaaatg 2520
ccgggcggcc agttcaccaa cctcaaagag caggcccgtt cgctgggact ggaaactcgc 2580
tggcaccagg tggcgcaggc ctatgccgac gccaaccaga tgttcggtga tatcgtcaag 2640
gtgacgccct cttccaaggt ggtcggcgac atggcgctga tgatggtcag ccaggacctg 2700
acggtcgccg acgtcgtcag cccagagcgc gaagtctcct tcccggaatc ggtggtctcg 2760
atgctgaagg gcgatctcgg ccagccgccg tcgggatggc cggcagcgct gcagaagaag 2820
gcactgaagg gcgaaaagcc ctatacggtg cgtcccggct cgctgctgaa ggaagccgat 2880
ctcgatgccg agcgcaaggt catcgagacg aagctggaac gcgaggtcag cgacttcgag 2940
ttcgcctcat acctgatgta tccgaaggtc ttcaccgact ttgcgctcgc ctccgatacc 3000
tatgggccgg tctcggtgtt gccgacgccg gcttatttct acgggctggc cgatggcgac 3060
gagctgtttg ccgatatcga aaagggcaag accctcgtca tcgtcaatca ggcgatgagc 3120
gccaccgaca gccagggcat ggtcaccgtc ttcttcgagc tcaacggcca gccgcggcgc 3180
atcaaggtgc cggaccgggc gcatggggcg acgggggctg ccgtgcgccg caaggccgag 3240
cccggcaatg gtgcccatgt cggcgcgccg atgcccggcg tcatcagccg cgtcttcgcc 3300
tcatcaggcc aggccgtcag cgctggtgac gtgctcgtct ccatcgaggc gatgaagatg 3360
gaaaccgcga tccatgcgga aaaggacgga acgattgcgg aaatcctcgt caaggccggc 3420
gaccagatcg atgccaagga cctgcttgtc gtctatgccg gttga 3465
<210> 21
<211> 565
<212> PRT
<213> Artificial Sequence
<220>
<223> MaeA
<400> 21
Met Glu Pro Lys Thr Lys Lys Gln Arg Ser Leu Tyr Ile Pro Tyr Ala
1 5 10 15
Gly Pro Val Leu Leu Glu Phe Pro Leu Leu Asn Lys Gly Ser Ala Phe
20 25 30
Ser Met Glu Glu Arg Arg Asn Phe Asn Leu Leu Gly Leu Leu Pro Glu
35 40 45
Val Val Glu Thr Ile Glu Glu Gln Ala Glu Arg Ala Trp Ile Gln Tyr
50 55 60
Gln Gly Phe Lys Thr Glu Ile Asp Lys His Ile Tyr Leu Arg Asn Ile
65 70 75 80
Gln Asp Thr Asn Glu Thr Leu Phe Tyr Arg Leu Val Asn Asn His Leu
85 90 95
Asp Glu Met Met Pro Val Ile Tyr Thr Pro Thr Val Gly Ala Ala Cys
100 105 110
Glu Arg Phe Ser Glu Ile Tyr Arg Arg Ser Arg Gly Val Phe Ile Ser
115 120 125
Tyr Gln Asn Arg His Asn Met Asp Asp Ile Leu Gln Asn Val Pro Asn
130 135 140
His Asn Ile Lys Val Ile Val Val Thr Asp Gly Glu Arg Ile Leu Gly
145 150 155 160
Leu Gly Asp Gln Gly Ile Gly Gly Met Gly Ile Pro Ile Gly Lys Leu
165 170 175
Ser Leu Tyr Thr Ala Cys Gly Gly Ile Ser Pro Ala Tyr Thr Leu Pro
180 185 190
Val Val Leu Asp Val Gly Thr Asn Asn Gln Gln Leu Leu Asn Asp Pro
195 200 205
Leu Tyr Met Gly Trp Arg Asn Pro Arg Ile Thr Asp Asp Glu Tyr Tyr
210 215 220
Glu Phe Val Asp Glu Phe Ile Gln Ala Val Lys Gln Arg Trp Pro Asp
225 230 235 240
Val Leu Leu Gln Phe Glu Asp Phe Ala Gln Lys Asn Ala Met Pro Leu
245 250 255
Leu Asn Arg Tyr Arg Asn Glu Ile Cys Ser Phe Asn Asp Asp Ile Gln
260 265 270
Gly Thr Ala Ala Val Thr Val Gly Thr Leu Ile Ala Ala Ser Arg Ala
275 280 285
Ala Gly Gly Gln Leu Ser Glu Lys Lys Ile Val Phe Leu Gly Ala Gly
290 295 300
Ser Ala Gly Cys Gly Ile Ala Glu Met Ile Ile Ser Gln Thr Gln Arg
305 310 315 320
Glu Gly Leu Ser Glu Glu Ala Ala Arg Gln Lys Val Phe Met Val Asp
325 330 335
Arg Phe Gly Leu Leu Thr Asp Lys Met Pro Asn Leu Leu Pro Phe Gln
340 345 350
Thr Lys Leu Val Gln Lys Arg Glu Asn Leu Ser Asp Trp Asp Thr Asp
355 360 365
Ser Asp Val Leu Ser Leu Leu Asp Val Val Arg Asn Val Lys Pro Asp
370 375 380
Ile Leu Ile Gly Val Ser Gly Gln Thr Gly Leu Phe Thr Glu Glu Ile
385 390 395 400
Ile Arg Glu Met His Lys His Cys Pro Arg Pro Ile Val Met Pro Leu
405 410 415
Ser Asn Pro Thr Ser Arg Val Glu Ala Thr Pro Gln Asp Ile Ile Ala
420 425 430
Trp Thr Glu Gly Asn Ala Leu Val Ala Thr Gly Ser Pro Phe Asn Pro
435 440 445
Val Val Trp Lys Asp Lys Ile Tyr Pro Ile Ala Gln Cys Asn Asn Ala
450 455 460
Phe Ile Phe Pro Gly Ile Gly Leu Gly Val Ile Ala Ser Gly Ala Ser
465 470 475 480
Arg Ile Thr Asp Glu Met Leu Met Ser Ala Ser Glu Thr Leu Ala Gln
485 490 495
Tyr Ser Pro Leu Val Leu Asn Gly Glu Gly Met Val Leu Pro Glu Leu
500 505 510
Lys Asp Ile Gln Lys Val Ser Arg Ala Ile Ala Phe Ala Val Gly Lys
515 520 525
Met Ala Gln Gln Gln Gly Val Ala Val Lys Thr Ser Ala Glu Ala Leu
530 535 540
Gln Gln Ala Ile Asp Asp Asn Phe Trp Gln Ala Glu Tyr Arg Asp Tyr
545 550 555 560
Arg Arg Thr Ser Ile
565
<210> 22
<211> 1698
<212> DNA
<213> Artificial Sequence
<220>
<223> maeA
<400> 22
atggaaccaa aaacaaaaaa acagcgttcg ctttatatcc cttacgctgg ccctgtactg 60
ctggaatttc cgttgttgaa taaaggcagt gccttcagca tggaagaacg ccgtaacttc 120
aacctgctgg ggttactgcc ggaagtggtc gaaaccatcg aagaacaagc ggaacgagca 180
tggatccagt atcagggatt caaaaccgaa atcgacaaac acatctacct gcgtaacatc 240
caggacacta acgaaaccct cttctaccgt ctggtaaaca atcatcttga tgagatgatg 300
cctgttattt ataccccaac cgtcggcgca gcctgtgagc gtttttctga gatctaccgc 360
cgttcacgcg gcgtgtttat ctcttaccag aaccggcaca atatggacga tattctgcaa 420
aacgtgccga accataatat taaagtgatt gtggtgactg acggtgaacg cattctgggg 480
cttggtgacc agggcatcgg cgggatgggc attccgatcg gtaaactgtc gctctatacc 540
gcctgtggcg gcatcagccc ggcgtatacc cttccggtgg tgctggatgt cggaacgaac 600
aaccaacagc tgcttaacga tccgctgtat atgggctggc gtaatccgcg tatcactgac 660
gacgaatact atgaattcgt tgatgaattt atccaggctg tgaaacaacg ctggccagac 720
gtgctgttgc agtttgaaga ctttgctcaa aaaaatgcga tgccgttact taaccgctat 780
cgcaatgaaa tttgttcttt taacgatgac attcagggca ctgcggcggt aacagtcggc 840
acactgatcg cagcaagccg cgcggcaggt ggtcagttaa gcgagaaaaa aatcgtcttc 900
cttggcgcag gttcagcggg atgcggcatt gccgaaatga tcatctccca gacccagcgc 960
gaaggattaa gcgaggaagc ggcgcggcag aaagtcttta tggtcgatcg ctttggcttg 1020
ctgactgaca agatgccgaa cctgctgcct ttccagacca aactggtgca gaagcgcgaa 1080
aacctcagtg actgggatac cgacagcgat gtgctgtcac tgctggatgt ggtgcgcaat 1140
gtaaaaccag atattctgat tggcgtctca ggacagaccg ggctgtttac ggaagagatc 1200
atccgtgaga tgcataaaca ctgtccgcgt ccgatcgtga tgccgctgtc taacccgacg 1260
tcacgcgtgg aagccacacc gcaggacatt atcgcctgga ccgaaggtaa cgcgctggtc 1320
gccacgggca gcccgtttaa tccagtggta tggaaagata aaatctaccc tatcgcccag 1380
tgtaacaacg cctttatttt cccgggcatc ggcctgggtg ttattgcttc cggcgcgtca 1440
cgtatcaccg atgagatgct gatgtcggca agtgaaacgc tggcgcagta ttcaccattg 1500
gtgctgaacg gcgaaggtat ggtactgccg gaactgaaag atattcagaa agtctcccgc 1560
gcaattgcgt ttgcggttgg caaaatggcg cagcagcaag gcgtggcggt gaaaacctct 1620
gccgaagccc tgcaacaggc cattgacgat aatttctggc aagccgaata ccgcgactac 1680
cgccgtacct ccatctaa 1698
<210> 23
<211> 759
<212> PRT
<213> Artificial Sequence
<220>
<223> MaeB
<400> 23
Met Asp Asp Gln Leu Lys Gln Ser Ala Leu Asp Phe His Glu Phe Pro
1 5 10 15
Val Pro Gly Lys Ile Gln Val Ser Pro Thr Lys Pro Leu Ala Thr Gln
20 25 30
Arg Asp Leu Ala Leu Ala Tyr Ser Pro Gly Val Ala Ala Pro Cys Leu
35 40 45
Glu Ile Glu Lys Asp Pro Leu Lys Ala Tyr Lys Tyr Thr Ala Arg Gly
50 55 60
Asn Leu Val Ala Val Ile Ser Asn Gly Thr Ala Val Leu Gly Leu Gly
65 70 75 80
Asn Ile Gly Ala Leu Ala Gly Lys Pro Val Met Glu Gly Lys Gly Val
85 90 95
Leu Phe Lys Lys Phe Ala Gly Ile Asp Val Phe Asp Ile Glu Val Asp
100 105 110
Glu Leu Asp Pro Asp Lys Phe Ile Glu Val Val Ala Ala Leu Glu Pro
115 120 125
Thr Phe Gly Gly Ile Asn Leu Glu Asp Ile Lys Ala Pro Glu Cys Phe
130 135 140
Tyr Ile Glu Gln Lys Leu Arg Glu Arg Met Asn Ile Pro Val Phe His
145 150 155 160
Asp Asp Gln His Gly Thr Ala Ile Ile Ser Thr Ala Ala Ile Leu Asn
165 170 175
Gly Leu Arg Val Val Glu Lys Asn Ile Ser Asp Val Arg Met Val Val
180 185 190
Ser Gly Ala Gly Ala Ala Ala Ile Ala Cys Met Asn Leu Leu Val Ala
195 200 205
Leu Gly Leu Gln Lys His Asn Ile Val Val Cys Asp Ser Lys Gly Val
210 215 220
Ile Tyr Gln Gly Arg Glu Pro Asn Met Ala Glu Thr Lys Ala Ala Tyr
225 230 235 240
Ala Val Val Asp Asp Gly Lys Arg Thr Leu Asp Asp Val Ile Glu Gly
245 250 255
Ala Asp Ile Phe Leu Gly Cys Ser Gly Pro Lys Val Leu Thr Gln Glu
260 265 270
Met Val Lys Lys Met Ala Arg Ala Pro Met Ile Leu Ala Leu Ala Asn
275 280 285
Pro Glu Pro Glu Ile Leu Pro Pro Leu Ala Lys Glu Val Arg Pro Asp
290 295 300
Ala Ile Ile Cys Thr Gly Arg Ser Asp Tyr Pro Asn Gln Val Asn Asn
305 310 315 320
Val Leu Cys Phe Pro Phe Ile Phe Arg Gly Ala Leu Asp Val Gly Ala
325 330 335
Thr Ala Ile Asn Glu Glu Met Lys Leu Ala Ala Val Arg Ala Ile Ala
340 345 350
Glu Leu Ala His Ala Glu Gln Ser Glu Val Val Ala Ser Ala Tyr Gly
355 360 365
Asp Gln Asp Leu Ser Phe Gly Pro Glu Tyr Ile Ile Pro Lys Pro Phe
370 375 380
Asp Pro Arg Leu Ile Val Lys Ile Ala Pro Ala Val Ala Lys Ala Ala
385 390 395 400
Met Glu Ser Gly Val Ala Thr Arg Pro Ile Ala Asp Phe Asp Val Tyr
405 410 415
Ile Asp Lys Leu Thr Glu Phe Val Tyr Lys Thr Asn Leu Phe Met Lys
420 425 430
Pro Ile Phe Ser Gln Ala Arg Lys Ala Pro Lys Arg Val Val Leu Pro
435 440 445
Glu Gly Glu Glu Ala Arg Val Leu His Ala Thr Gln Glu Leu Val Thr
450 455 460
Leu Gly Leu Ala Lys Pro Ile Leu Ile Gly Arg Pro Asn Val Ile Glu
465 470 475 480
Met Arg Ile Gln Lys Leu Gly Leu Gln Ile Lys Ala Gly Val Asp Phe
485 490 495
Glu Ile Val Asn Asn Glu Ser Asp Pro Arg Phe Lys Glu Tyr Trp Thr
500 505 510
Glu Tyr Phe Gln Ile Met Lys Arg Arg Gly Val Thr Gln Glu Gln Ala
515 520 525
Gln Arg Ala Leu Ile Ser Asn Pro Thr Val Ile Gly Ala Ile Met Val
530 535 540
Gln Arg Gly Glu Ala Asp Ala Met Ile Cys Gly Thr Val Gly Asp Tyr
545 550 555 560
His Glu His Phe Ser Val Val Lys Asn Val Phe Gly Tyr Arg Asp Gly
565 570 575
Val His Thr Ala Gly Ala Met Asn Ala Leu Leu Leu Pro Ser Gly Asn
580 585 590
Thr Phe Ile Ala Asp Thr Tyr Val Asn Asp Glu Pro Asp Ala Glu Glu
595 600 605
Leu Ala Glu Ile Thr Leu Met Ala Ala Glu Thr Val Arg Arg Phe Gly
610 615 620
Ile Glu Pro Arg Val Ala Leu Leu Ser His Ser Asn Phe Gly Ser Ser
625 630 635 640
Asp Cys Pro Ser Ser Ser Lys Met Arg Gln Ala Leu Glu Leu Val Arg
645 650 655
Glu Arg Ala Pro Glu Leu Met Ile Asp Gly Glu Met His Gly Asp Ala
660 665 670
Ala Leu Val Glu Ala Ile Arg Asn Asp Arg Met Pro Asp Ser Ser Leu
675 680 685
Lys Gly Ser Ala Asn Ile Leu Val Met Pro Asn Met Glu Ala Ala Arg
690 695 700
Ile Ser Tyr Asn Leu Leu Arg Val Ser Ser Ser Glu Gly Val Thr Val
705 710 715 720
Gly Pro Val Leu Met Gly Val Ala Lys Pro Val His Val Leu Thr Pro
725 730 735
Ile Ala Ser Val Arg Arg Ile Val Asn Met Val Ala Leu Ala Val Val
740 745 750
Glu Ala Gln Thr Gln Pro Leu
755
<210> 24
<211> 2280
<212> DNA
<213> Artificial Sequence
<220>
<223> maeB
<400> 24
atggatgacc agttaaaaca aagtgcactt gatttccatg aatttccagt tccagggaaa 60
atccaggttt ctccaaccaa gcctctggca acacagcgcg atctggcgct ggcctactca 120
ccaggcgttg ccgcaccttg tcttgaaatc gaaaaagacc cgttaaaagc ctacaaatat 180
accgcccgag gtaacctggt ggcggtgatc tctaacggta cggcggtgct ggggttaggc 240
aacattggcg cgctggcagg caaaccggtg atggaaggca agggcgttct gtttaagaaa 300
ttcgccggga ttgatgtatt tgacattgaa gttgacgaac tcgacccgga caaatttatt 360
gaagttgtcg ccgcgctcga accaaccttc ggcggcatca acctcgaaga cattaaagcg 420
ccagaatgtt tctatattga acagaaactg cgcgagcgga tgaatattcc ggtattccac 480
gacgatcagc acggcacggc aattatcagc actgccgcca tcctcaacgg cttgcgcgtg 540
gtggagaaaa acatctccga cgtgcggatg gtggtttccg gcgcgggtgc cgcagcaatc 600
gcctgtatga acctgctggt agcgctgggt ctgcaaaaac ataacatcgt ggtttgcgat 660
tcaaaaggcg ttatctatca gggccgtgag ccaaacatgg cggaaaccaa agccgcatat 720
gcggtggtgg atgacggcaa acgtaccctc gatgatgtga ttgaaggcgc ggatattttc 780
ctgggctgtt ccggcccgaa agtgctgacc caggaaatgg tgaagaaaat ggctcgtgcg 840
ccaatgatcc tggcgctggc gaacccggaa ccggaaattc tgccgccgct ggcgaaagaa 900
gtgcgtccgg atgccatcat ttgcaccggt cgttctgact atccgaacca ggtgaacaac 960
gtcctgtgct tcccgttcat cttccgtggc gcgctggacg ttggcgcaac cgccatcaac 1020
gaagagatga aactggcggc ggtacgtgcg attgcagaac tcgcccatgc ggaacagagc 1080
gaagtggtgg cttcagcgta tggcgatcag gatctgagct ttggtccgga atacatcatt 1140
ccaaaaccgt ttgatccgcg cttgatcgtt aagatcgctc ctgcggtcgc taaagccgcg 1200
atggagtcgg gcgtggcgac tcgtccgatt gctgatttcg acgtctacat cgacaagctg 1260
actgagttcg tttacaaaac caacctgttt atgaagccga ttttctccca ggctcgcaaa 1320
gcgccgaagc gcgttgttct gccggaaggg gaagaggcgc gcgttctgca tgccactcag 1380
gaactggtaa cgctgggact ggcgaaaccg atccttatcg gtcgtccgaa cgtgatcgaa 1440
atgcgcattc agaaactggg cttgcagatc aaagcgggcg ttgattttga gatcgtcaat 1500
aacgaatccg atccgcgctt taaagagtac tggaccgaat acttccagat catgaagcgt 1560
cgcggcgtca ctcaggaaca ggcgcagcgg gcgctgatca gtaacccgac agtgatcggc 1620
gcgatcatgg ttcagcgtgg ggaagccgat gcaatgattt gcggtacggt gggtgattat 1680
catgaacatt ttagcgtggt gaaaaatgtc tttggttatc gcgatggcgt tcacaccgca 1740
ggtgccatga acgcgctgct gctgccgagt ggtaacacct ttattgccga tacatatgtt 1800
aatgatgaac cggatgcaga agagctggcg gagatcacct tgatggcggc agaaactgtc 1860
cgtcgttttg gtattgagcc gcgcgttgct ttgttgtcgc actccaactt tggttcttct 1920
gactgcccgt cgtcgagcaa aatgcgtcag gcgctggaac tggtcaggga acgtgcacca 1980
gaactgatga ttgatggtga aatgcacggc gatgcagcgc tggtggaagc gattcgcaac 2040
gaccgtatgc cggacagctc tttgaaaggt tccgccaata ttctggtgat gccgaacatg 2100
gaagctgccc gcattagtta caacttactg cgtgtttcca gctcggaagg tgtgactgtc 2160
ggcccggtgc tgatgggtgt ggcgaaaccg gttcacgtgt taacgccgat cgcatcggtg 2220
cgtcgtatcg tcaacatggt ggcgctggcc gtggtagaag cgcaaaccca accgctgtaa 2280
2280
<210> 25
<211> 603
<212> PRT
<213> Artificial Sequence
<220>
<223> Edd
<400> 25
Met Asn Pro Gln Leu Leu Arg Val Thr Asn Arg Ile Ile Glu Arg Ser
1 5 10 15
Arg Glu Thr Arg Ser Ala Tyr Leu Ala Arg Ile Glu Gln Ala Lys Thr
20 25 30
Ser Thr Val His Arg Ser Gln Leu Ala Cys Gly Asn Leu Ala His Gly
35 40 45
Phe Ala Ala Cys Gln Pro Glu Asp Lys Ala Ser Leu Lys Ser Met Leu
50 55 60
Arg Asn Asn Ile Ala Ile Ile Thr Ser Tyr Asn Asp Met Leu Ser Ala
65 70 75 80
His Gln Pro Tyr Glu His Tyr Pro Glu Ile Ile Arg Lys Ala Leu His
85 90 95
Glu Ala Asn Ala Val Gly Gln Val Ala Gly Gly Val Pro Ala Met Cys
100 105 110
Asp Gly Val Thr Gln Gly Gln Asp Gly Met Glu Leu Ser Leu Leu Ser
115 120 125
Arg Glu Val Ile Ala Met Ser Ala Ala Val Gly Leu Ser His Asn Met
130 135 140
Phe Asp Gly Ala Leu Phe Leu Gly Val Cys Asp Lys Ile Val Pro Gly
145 150 155 160
Leu Thr Met Ala Ala Leu Ser Phe Gly His Leu Pro Ala Val Phe Val
165 170 175
Pro Ser Gly Pro Met Ala Ser Gly Leu Pro Asn Lys Glu Lys Val Arg
180 185 190
Ile Arg Gln Leu Tyr Ala Glu Gly Lys Val Asp Arg Met Ala Leu Leu
195 200 205
Glu Ser Glu Ala Ala Ser Tyr His Ala Pro Gly Thr Cys Thr Phe Tyr
210 215 220
Gly Thr Ala Asn Thr Asn Gln Met Val Val Glu Phe Met Gly Met Gln
225 230 235 240
Leu Pro Gly Ser Ser Phe Val His Pro Asp Ser Pro Leu Arg Asp Ala
245 250 255
Leu Thr Ala Ala Ala Ala Arg Gln Val Thr Arg Met Thr Gly Asn Gly
260 265 270
Asn Glu Trp Met Pro Ile Gly Lys Met Ile Asp Glu Lys Val Val Val
275 280 285
Asn Gly Ile Val Ala Leu Leu Ala Thr Gly Gly Ser Thr Asn His Thr
290 295 300
Met His Leu Val Ala Met Ala Arg Ala Ala Gly Ile Gln Ile Asn Trp
305 310 315 320
Asp Asp Phe Ser Asp Leu Ser Asp Val Val Pro Leu Met Ala Arg Leu
325 330 335
Tyr Pro Asn Gly Pro Ala Asp Ile Asn His Phe Gln Ala Ala Gly Gly
340 345 350
Val Pro Val Leu Val Arg Glu Leu Leu Lys Ala Gly Leu Leu His Glu
355 360 365
Asp Val Asn Thr Val Ala Gly Phe Gly Leu Ser Arg Tyr Thr Leu Glu
370 375 380
Pro Trp Leu Asn Asn Gly Glu Leu Asp Trp Arg Glu Gly Ala Glu Lys
385 390 395 400
Ser Leu Asp Ser Asn Val Ile Ala Ser Phe Glu Gln Pro Phe Ser His
405 410 415
His Gly Gly Thr Lys Val Leu Ser Gly Asn Leu Gly Arg Ala Val Met
420 425 430
Lys Thr Ser Ala Val Pro Val Glu Asn Gln Val Ile Glu Ala Pro Ala
435 440 445
Val Val Phe Glu Ser Gln His Asp Val Met Pro Ala Phe Glu Ala Gly
450 455 460
Leu Leu Asp Arg Asp Cys Val Val Val Val Arg His Gln Gly Pro Lys
465 470 475 480
Ala Asn Gly Met Pro Glu Leu His Lys Leu Met Pro Pro Leu Gly Val
485 490 495
Leu Leu Asp Arg Cys Phe Lys Ile Ala Leu Val Thr Asp Gly Arg Leu
500 505 510
Ser Gly Ala Ser Gly Lys Val Pro Ser Ala Ile His Val Thr Pro Glu
515 520 525
Ala Tyr Asp Gly Gly Leu Leu Ala Lys Val Arg Asp Gly Asp Ile Ile
530 535 540
Arg Val Asn Gly Gln Thr Gly Glu Leu Thr Leu Leu Val Asp Glu Ala
545 550 555 560
Glu Leu Ala Ala Arg Glu Pro His Ile Pro Asp Leu Ser Ala Ser Arg
565 570 575
Val Gly Thr Gly Arg Glu Leu Phe Ser Ala Leu Arg Glu Lys Leu Ser
580 585 590
Gly Ala Glu Gln Gly Ala Thr Cys Ile Thr Phe
595 600
<210> 26
<211> 1812
<212> DNA
<213> Artificial Sequence
<220>
<223> edd
<400> 26
atgaatccac aattgttacg cgtaacaaat cgaatcattg aacgttcgcg cgagactcgc 60
tctgcttatc tcgcccggat agaacaagcg aaaacttcga ccgttcatcg ttcgcagttg 120
gcatgcggta acctggcaca cggtttcgct gcctgccagc cagaagacaa agcctctttg 180
aaaagcatgt tgcgtaacaa tatcgccatc atcacctcct ataacgacat gctctccgcg 240
caccagcctt atgaacacta tccagaaatc attcgtaaag ccctgcatga agcgaatgcg 300
gttggtcagg ttgcgggcgg tgttccggcg atgtgtgatg gtgtcaccca ggggcaggat 360
ggaatggaat tgtcgctgct aagccgcgaa gtgatagcga tgtctgcggc ggtggggctg 420
tcccataaca tgtttgatgg tgctctgttc ctcggtgtgt gcgacaagat tgtcccgggt 480
ctgacgatgg cagccctgtc gtttggtcat ttgcctgcgg tgtttgtgcc gtctggaccg 540
atggcaagcg gtttgccaaa taaagaaaaa gtgcgtattc gccagcttta tgccgaaggt 600
aaagtggacc gcatggcctt actggagtca gaagccgcgt cttaccatgc gccgggaaca 660
tgtactttct acggtactgc caacaccaac cagatggtgg tggagtttat ggggatgcag 720
ttgccaggct cttcttttgt tcatccggat tctccgctgc gcgatgcttt gaccgccgca 780
gctgcgcgtc aggttacacg catgaccggt aatggtaatg aatggatgcc gatcggtaag 840
atgatcgatg agaaagtggt ggtgaacggt atcgttgcac tgctggcgac cggtggttcc 900
actaaccaca ccatgcacct ggtggcgatg gcgcgcgcgg ccggtattca gattaactgg 960
gatgacttct ctgacctttc tgatgttgta ccgctgatgg cacgtctcta cccgaacggt 1020
ccggccgata ttaaccactt ccaggcggca ggtggcgtac cggttctggt gcgtgaactg 1080
ctcaaagcag gcctgctgca tgaagatgtc aatacggtgg caggttttgg tctgtctcgt 1140
tatacccttg aaccatggct gaataatggt gaactggact ggcgggaagg ggcggaaaaa 1200
tcactcgaca gcaatgtgat cgcttccttc gaacaacctt tctctcatca tggtgggaca 1260
aaagtgttaa gcggtaacct gggccgtgcg gttatgaaaa cctctgccgt gccggttgag 1320
aaccaggtga ttgaagcgcc agcggttgtt tttgaaagcc agcatgacgt tatgccggcc 1380
tttgaagcgg gtttgctgga ccgcgattgt gtcgttgttg tccgtcatca ggggccaaaa 1440
gcgaacggaa tgccagaatt acataaactc atgccgccac ttggtgtatt attggaccgg 1500
tgtttcaaaa ttgcgttagt taccgatgga cgactctccg gcgcttcagg taaagtgccg 1560
tcagctatcc acgtaacacc agaagcctac gatggcgggc tgctggcaaa agtgcgcgac 1620
ggggacatca ttcgtgtgaa tggacagaca ggcgaactga cgctgctggt agacgaagcg 1680
gaactggctg ctcgcgaacc gcacattcct gacctgagcg cgtcacgcgt gggaacagga 1740
cgtgaattat tcagcgcctt gcgtgaaaaa ctgtccggtg ccgaacaggg cgcaacctgt 1800
atcacttttt aa 1812
<210> 27
<211> 213
<212> PRT
<213> Artificial Sequence
<220>
<223> Eda
<400> 27
Met Lys Asn Trp Lys Thr Ser Ala Glu Ser Ile Leu Thr Thr Gly Pro
1 5 10 15
Val Val Pro Val Ile Val Val Lys Lys Leu Glu His Ala Val Pro Met
20 25 30
Ala Lys Ala Leu Val Ala Gly Gly Val Arg Val Leu Glu Val Thr Leu
35 40 45
Arg Thr Glu Cys Ala Val Asp Ala Ile Arg Ala Ile Ala Lys Glu Val
50 55 60
Pro Glu Ala Ile Val Gly Ala Gly Thr Val Leu Asn Pro Gln Gln Leu
65 70 75 80
Ala Glu Val Thr Glu Ala Gly Ala Gln Phe Ala Ile Ser Pro Gly Leu
85 90 95
Thr Glu Pro Leu Leu Lys Ala Ala Thr Glu Gly Thr Ile Pro Leu Ile
100 105 110
Pro Gly Ile Ser Thr Val Ser Glu Leu Met Leu Gly Met Asp Tyr Gly
115 120 125
Leu Lys Glu Phe Lys Phe Phe Pro Ala Glu Ala Asn Gly Gly Val Lys
130 135 140
Ala Leu Gln Ala Ile Ala Gly Pro Phe Ser Gln Val Arg Phe Cys Pro
145 150 155 160
Thr Gly Gly Ile Ser Pro Ala Asn Tyr Arg Asp Tyr Leu Ala Leu Lys
165 170 175
Ser Val Leu Cys Ile Gly Gly Ser Trp Leu Val Pro Ala Asp Ala Leu
180 185 190
Glu Ala Gly Asp Tyr Asp Arg Ile Thr Lys Leu Ala Arg Glu Ala Val
195 200 205
Glu Gly Ala Lys Leu
210
<210> 28
<211> 642
<212> DNA
<213> Artificial Sequence
<220>
<223> eda
<400> 28
atgaaaaact ggaaaacaag tgcagaatca atcctgacca ccggcccggt tgtaccggtt 60
atcgtggtaa aaaaactgga acacgcggtg ccgatggcaa aagcgttggt tgctggtggg 120
gtgcgcgttc tggaagtgac tctgcgtacc gagtgtgcag ttgacgctat ccgtgctatc 180
gccaaagaag tgcctgaagc gattgtgggt gccggtacgg tgctgaatcc acagcagctg 240
gcagaagtca ctgaagcggg tgcacagttc gcaattagcc cgggtctgac cgagccgctg 300
ctgaaagctg ctaccgaagg gactattcct ctgattccgg ggatcagcac tgtttccgaa 360
ctgatgctgg gtatggacta cggtttgaaa gagttcaaat tcttcccggc tgaagctaac 420
ggcggcgtga aagccctgca ggcgatcgcg ggtccgttct cccaggtccg tttctgcccg 480
acgggtggta tttctccggc taactaccgt gactacctgg cgctgaaaag cgtgctgtgc 540
atcggtggtt cctggctggt tccggcagat gcgctggaag cgggcgatta cgaccgcatt 600
actaagctgg cgcgtgaagc tgtagaaggc gctaagctgt aa 642
<210> 29
<211> 396
<212> PRT
<213> Artificial Sequence
<220>
<223> AspC
<400> 29
Met Phe Glu Asn Ile Thr Ala Ala Pro Ala Asp Pro Ile Leu Gly Leu
1 5 10 15
Ala Asp Leu Phe Arg Ala Asp Glu Arg Pro Gly Lys Ile Asn Leu Gly
20 25 30
Ile Gly Val Tyr Lys Asp Glu Thr Gly Lys Thr Pro Val Leu Thr Ser
35 40 45
Val Lys Lys Ala Glu Gln Tyr Leu Leu Glu Asn Glu Thr Thr Lys Asn
50 55 60
Tyr Leu Gly Ile Asp Gly Ile Pro Glu Phe Gly Arg Cys Thr Gln Glu
65 70 75 80
Leu Leu Phe Gly Lys Gly Ser Ala Leu Ile Asn Asp Lys Arg Ala Arg
85 90 95
Thr Ala Gln Thr Pro Gly Gly Thr Gly Ala Leu Arg Val Ala Ala Asp
100 105 110
Phe Leu Ala Lys Asn Thr Ser Val Lys Arg Val Trp Val Ser Asn Pro
115 120 125
Ser Trp Pro Asn His Lys Ser Val Phe Asn Ser Ala Gly Leu Glu Val
130 135 140
Arg Glu Tyr Ala Tyr Tyr Asp Ala Glu Asn His Thr Leu Asp Phe Asp
145 150 155 160
Ala Leu Ile Asn Ser Leu Asn Glu Ala Gln Ala Gly Asp Val Val Leu
165 170 175
Phe His Gly Cys Cys His Asn Pro Thr Gly Ile Asp Pro Thr Leu Glu
180 185 190
Gln Trp Gln Thr Leu Ala Gln Leu Ser Val Glu Lys Gly Trp Leu Pro
195 200 205
Leu Phe Asp Phe Ala Tyr Gln Gly Phe Ala Arg Gly Leu Glu Glu Asp
210 215 220
Ala Glu Gly Leu Arg Ala Phe Ala Ala Met His Lys Glu Leu Ile Val
225 230 235 240
Ala Ser Ser Tyr Ser Lys Asn Phe Gly Leu Tyr Asn Glu Arg Val Gly
245 250 255
Ala Cys Thr Leu Val Ala Ala Asp Ser Glu Thr Val Asp Arg Ala Phe
260 265 270
Ser Gln Met Lys Ala Ala Ile Arg Ala Asn Tyr Ser Asn Pro Pro Ala
275 280 285
His Gly Ala Ser Val Val Ala Thr Ile Leu Ser Asn Asp Ala Leu Arg
290 295 300
Ala Ile Trp Glu Gln Glu Leu Thr Asp Met Arg Gln Arg Ile Gln Arg
305 310 315 320
Met Arg Gln Leu Phe Val Asn Thr Leu Gln Glu Lys Gly Ala Asn Arg
325 330 335
Asp Phe Ser Phe Ile Ile Lys Gln Asn Gly Met Phe Ser Phe Ser Gly
340 345 350
Leu Thr Lys Glu Gln Val Leu Arg Leu Arg Glu Glu Phe Gly Val Tyr
355 360 365
Ala Val Ala Ser Gly Arg Val Asn Val Ala Gly Met Thr Pro Asp Asn
370 375 380
Met Ala Pro Leu Cys Glu Ala Ile Val Ala Val Leu
385 390 395
<210> 30
<211> 1191
<212> DNA
<213> Artificial Sequence
<220>
<223> aspC
<400> 30
atgtttgaga acattaccgc cgctcctgcc gacccgattc tgggcctggc cgatctgttt 60
cgtgccgatg aacgtcccgg caaaattaac ctcgggattg gtgtctataa agatgagacg 120
ggcaaaaccc cggtactgac cagcgtgaaa aaggctgaac agtatctgct cgaaaatgaa 180
accaccaaaa attacctcgg cattgacggc atccctgaat ttggtcgctg cactcaggaa 240
ctgctgtttg gtaaaggtag cgccctgatc aatgacaaac gtgctcgcac ggcacagact 300
ccggggggca ctggcgcact acgcgtggct gccgatttcc tggcaaaaaa taccagcgtt 360
aagcgtgtgt gggtgagcaa cccaagctgg ccgaaccata agagcgtctt taactctgca 420
ggtctggaag ttcgtgaata cgcttattat gatgcggaaa atcacactct tgacttcgat 480
gcactgatta acagcctgaa tgaagctcag gctggcgacg tagtgctgtt ccatggctgc 540
tgccataacc caaccggtat cgaccctacg ctggaacaat ggcaaacact ggcacaactc 600
tccgttgaga aaggctggtt accgctgttt gacttcgctt accagggttt tgcccgtggt 660
ctggaagaag atgctgaagg actgcgcgct ttcgcggcta tgcataaaga gctgattgtt 720
gccagttcct actctaaaaa ctttggcctg tacaacgagc gtgttggcgc ttgtactctg 780
gttgctgccg acagtgaaac cgttgatcgc gcattcagcc aaatgaaagc ggcgattcgc 840
gctaactact ctaacccacc agcacacggc gcttctgttg ttgccaccat cctgagcaac 900
gatgcgttac gtgcgatttg ggaacaagag ctgactgata tgcgccagcg tattcagcgt 960
atgcgtcagt tgttcgtcaa tacgctgcag gaaaaaggcg caaaccgcga cttcagcttt 1020
atcatcaaac agaacggcat gttctccttc agtggcctga caaaagaaca agtgctgcgt 1080
ctgcgcgaag agtttggcgt atatgcggtt gcttctggtc gcgtaaatgt ggccgggatg 1140
acaccagata acatggctcc gctgtgcgaa gcgattgtgg cagtgctgta a 1191
<210> 31
<211> 477
<212> PRT
<213> Artificial Sequence
<220>
<223> PtsG
<400> 31
Met Phe Lys Asn Ala Phe Ala Asn Leu Gln Lys Val Gly Lys Ser Leu
1 5 10 15
Met Leu Pro Val Ser Val Leu Pro Ile Ala Gly Ile Leu Leu Gly Val
20 25 30
Gly Ser Ala Asn Phe Ser Trp Leu Pro Ala Val Val Ser His Val Met
35 40 45
Ala Glu Ala Gly Gly Ser Val Phe Ala Asn Met Pro Leu Ile Phe Ala
50 55 60
Ile Gly Val Ala Leu Gly Phe Thr Asn Asn Asp Gly Val Ser Ala Leu
65 70 75 80
Ala Ala Val Val Ala Tyr Gly Ile Met Val Lys Thr Met Ala Val Val
85 90 95
Ala Pro Leu Val Leu His Leu Pro Ala Glu Glu Ile Ala Ser Lys His
100 105 110
Leu Ala Asp Thr Gly Val Leu Gly Gly Ile Ile Ser Gly Ala Ile Ala
115 120 125
Ala Tyr Met Phe Asn Arg Phe Tyr Arg Ile Lys Leu Pro Glu Tyr Leu
130 135 140
Gly Phe Phe Ala Gly Lys Arg Phe Val Pro Ile Ile Ser Gly Leu Ala
145 150 155 160
Ala Ile Phe Thr Gly Val Val Leu Ser Phe Ile Trp Pro Pro Ile Gly
165 170 175
Ser Ala Ile Gln Thr Phe Ser Gln Trp Ala Ala Tyr Gln Asn Pro Val
180 185 190
Val Ala Phe Gly Ile Tyr Gly Phe Ile Glu Arg Cys Leu Val Pro Phe
195 200 205
Gly Leu His His Ile Trp Asn Val Pro Phe Gln Met Gln Ile Gly Glu
210 215 220
Tyr Thr Asn Ala Ala Gly Gln Val Phe His Gly Asp Ile Pro Arg Tyr
225 230 235 240
Met Ala Gly Asp Pro Thr Ala Gly Lys Leu Ser Gly Gly Phe Leu Phe
245 250 255
Lys Met Tyr Gly Leu Pro Ala Ala Ala Ile Ala Ile Trp His Ser Ala
260 265 270
Lys Pro Glu Asn Arg Ala Lys Val Gly Gly Ile Met Ile Ser Ala Ala
275 280 285
Leu Thr Ser Phe Leu Thr Gly Ile Thr Glu Pro Ile Glu Phe Ser Phe
290 295 300
Met Phe Val Ala Pro Ile Leu Tyr Ile Ile His Ala Ile Leu Ala Gly
305 310 315 320
Leu Ala Phe Pro Ile Cys Ile Leu Leu Gly Met Arg Asp Gly Thr Ser
325 330 335
Phe Ser His Gly Leu Ile Asp Phe Ile Val Leu Ser Gly Asn Ser Ser
340 345 350
Lys Leu Trp Leu Phe Pro Ile Val Gly Ile Gly Tyr Ala Ile Val Tyr
355 360 365
Tyr Thr Ile Phe Arg Val Leu Ile Lys Ala Leu Asp Leu Lys Thr Pro
370 375 380
Gly Arg Glu Asp Ala Thr Glu Asp Ala Lys Ala Thr Gly Thr Ser Glu
385 390 395 400
Met Ala Pro Ala Leu Val Ala Ala Phe Gly Gly Lys Glu Asn Ile Thr
405 410 415
Asn Leu Asp Ala Cys Ile Thr Arg Leu Arg Val Ser Val Ala Asp Val
420 425 430
Ser Lys Val Asp Gln Ala Gly Leu Lys Lys Leu Gly Ala Ala Gly Val
435 440 445
Val Val Ala Gly Ser Gly Val Gln Ala Ile Phe Gly Thr Lys Ser Asp
450 455 460
Asn Leu Lys Thr Glu Met Asp Glu Tyr Ile Arg Asn His
465 470 475
<210> 32
<211> 1434
<212> DNA
<213> Artificial Sequence
<220>
<223> ptsG
<400> 32
atgtttaaga atgcatttgc taacctgcaa aaggtcggta aatcgctgat gctgccggta 60
tccgtactgc ctatcgcagg tattctgctg ggcgtcggtt ccgcgaattt cagctggctg 120
cccgccgttg tatcgcatgt tatggcagaa gcaggcggtt ccgtctttgc aaacatgcca 180
ctgatttttg cgatcggtgt cgccctcggc tttaccaata acgatggcgt atccgcgctg 240
gccgcagttg ttgcctatgg catcatggtt aaaaccatgg ccgtggttgc gccactggta 300
ctgcatttac ctgctgaaga aatcgcctct aaacacctgg cggatactgg cgtactcgga 360
gggattatct ccggtgcgat cgcagcgtac atgtttaacc gtttctaccg tattaagctg 420
cctgagtatc ttggcttctt tgccggtaaa cgctttgtgc cgatcatttc tggcctggct 480
gccatcttta ctggcgttgt gctgtccttc atttggccgc cgattggttc tgcaatccag 540
accttctctc agtgggctgc ttaccagaac ccggtagttg cgtttggcat ttacggtttc 600
atcgaacgtt gcctggtacc gtttggtctg caccacatct ggaacgtacc tttccagatg 660
cagattggtg aatacaccaa cgcagcaggt caggttttcc acggcgacat tccgcgttat 720
atggcgggtg acccgactgc gggtaaactg tctggtggct tcctgttcaa aatgtacggt 780
ctgccagctg ccgcaattgc tatctggcac tctgctaaac cagaaaaccg cgcgaaagtg 840
ggcggtatta tgatctccgc ggcgctgacc tcgttcctga ccggtatcac cgagccgatc 900
gagttctcct tcatgttcgt tgcgccgatc ctgtacatca tccacgcgat tctggcaggc 960
ctggcattcc caatctgtat tcttctgggg atgcgtgacg gtacgtcgtt ctcgcacggt 1020
ctgatcgact tcatcgttct gtctggtaac agcagcaaac tgtggctgtt cccgatcgtc 1080
ggtatcggtt atgcgattgt ttactacacc atcttccgcg tgctgattaa agcactggat 1140
ctgaaaacgc cgggtcgtga agacgcgact gaagatgcaa aagcgacagg taccagcgaa 1200
atggcaccgg ctctggttgc tgcatttggt ggtaaagaaa acattactaa cctcgacgca 1260
tgtattaccc gtctgcgcgt cagcgttgct gatgtgtcta aagtggatca ggccggcctg 1320
aagaaactgg gcgcagcggg cgtagtggtt gctggttctg gtgttcaggc gattttcggt 1380
actaaatccg ataacctgaa aaccgagatg gatgagtaca tccgtaacca ctaa 1434
<210> 33
<211> 373
<212> PRT
<213> Artificial Sequence
<220>
<223> SbtA
<400> 33
Met Asp Phe Leu Ser His Phe Leu Thr Asp Phe Val Gly Gln Leu Gln
1 5 10 15
Ser Pro Thr Leu Ala Phe Leu Ile Gly Gly Met Val Ile Ala Ala Leu
20 25 30
Gly Thr Gln Leu Val Ile Pro Glu Ala Ile Ser Thr Ile Ile Val Phe
35 40 45
Met Leu Leu Thr Lys Ile Gly Leu Thr Gly Gly Met Ala Ile Arg Asn
50 55 60
Ser Asn Leu Thr Glu Met Leu Leu Pro Met Ile Phe Ser Val Ile Leu
65 70 75 80
Gly Ile Leu Ile Val Phe Ile Ala Arg Phe Thr Leu Ala Lys Leu Pro
85 90 95
Asn Val Lys Thr Val Asp Ala Leu Ala Thr Gly Gly Leu Phe Gly Ala
100 105 110
Val Ser Gly Ser Thr Met Ala Ala Ala Leu Thr Thr Leu Glu Glu Ser
115 120 125
Lys Ile Ser Tyr Glu Ala Trp Ala Gly Ala Leu Tyr Pro Phe Met Asp
130 135 140
Ile Pro Ala Leu Val Thr Ala Ile Val Val Ala Asn Ile Tyr Leu Asn
145 150 155 160
Lys Lys Lys Arg Arg Ala Ala Ala Ala Ile Glu Gly Ser Leu Ser Lys
165 170 175
Gln Pro Val Ala Ala Gly Asp Tyr Gly Asp Gln Gln Asp Tyr Pro Arg
180 185 190
Thr Arg Gln Glu Tyr Leu Ser Gln Gln Glu Pro Glu Asp Asn Arg Val
195 200 205
Lys Ile Trp Pro Ile Ile Glu Glu Ser Leu Gln Gly Pro Ala Leu Ser
210 215 220
Ala Met Leu Leu Gly Leu Ala Leu Gly Ile Phe Thr Lys Pro Glu Ser
225 230 235 240
Val Tyr Glu Gly Phe Tyr Asp Pro Leu Phe Arg Gly Leu Leu Ser Ile
245 250 255
Leu Met Leu Ile Met Gly Met Glu Ala Trp Ser Arg Ile Gly Glu Leu
260 265 270
Arg Lys Val Ala Gln Trp Tyr Val Val Tyr Ser Leu Val Ala Pro Ile
275 280 285
Val His Gly Phe Ile Ala Phe Gly Leu Gly Met Ile Ala His Tyr Thr
290 295 300
Thr Gly Phe Ser Leu Gly Gly Val Val Val Leu Ala Val Ile Ala Ala
305 310 315 320
Ser Ser Ser Asp Ile Ser Gly Pro Pro Thr Leu Arg Ala Gly Ile Pro
325 330 335
Ser Ala Asn Pro Ser Ala Tyr Ile Gly Ala Ser Thr Ala Ile Gly Thr
340 345 350
Pro Ile Ala Ile Gly Val Cys Ile Pro Leu Phe Ile Gly Leu Ala Gln
355 360 365
Thr Leu Gly Ala Gly
370
<210> 34
<211> 1122
<212> DNA
<213> Artificial Sequence
<220>
<223> sbtA
<400> 34
atggacttcc tgtctcactt cctgaccgac ttcgttggtc agctgcaatc tccgaccctg 60
gctttcctga tcggtggtat ggttatcgct gctctgggta cccagctggt tatcccggaa 120
gctatctcta ccatcatcgt tttcatgctg ctgaccaaaa tcggtctgac cggtggtatg 180
gctatccgta actctaacct gaccgaaatg ctgctgccga tgatcttctc tgttatcctg 240
ggtatcctga tcgttttcat cgctcgtttc accctggcta aactgccgaa cgttaaaacc 300
gttgacgctc tggctaccgg tggtctgttc ggtgctgttt ctggttctac catggctgct 360
gctctgacca ccctggaaga atctaaaatc tcttacgaag cgtgggctgg tgctctgtac 420
ccgttcatgg acatcccggc tctggttacc gctatcgttg ttgctaacat ctacctgaac 480
aaaaaaaaac gtcgtgctgc tgctgctatc gaaggttctc tgtctaaaca gccggttgct 540
gctggtgact acggtgacca gcaggactac ccgcgtaccc gtcaggaata cctgtctcag 600
caggaaccgg aagacaaccg tgttaaaatc tggccgatca tcgaagaatc tctgcaaggt 660
ccggctctgt ctgctatgct gctgggtctg gctctgggta tcttcaccaa accggaatct 720
gtttacgaag gtttctacga cccgctgttc cgtggtctgc tgtctatcct gatgctgatc 780
atgggtatgg aagcgtggtc tcgtatcggt gaactgcgta aagttgctca gtggtacgtt 840
gtttactctc tggttgctcc gatcgttcac ggtttcatcg ctttcggtct gggtatgatc 900
gctcactaca ccaccggttt ctctctgggt ggtgttgttg ttctggctgt tatcgctgct 960
tcttcttctg acatctctgg tccgccgacc ctgcgtgctg gtatcccgtc tgctaacccg 1020
tctgcttaca tcggtgcttc taccgctatc ggtaccccga tcgctatcgg tgtttgcatc 1080
ccgctgttca tcggtctggc tcagaccctg ggtgctggtt aa 1122
<210> 35
<211> 416
<212> PRT
<213> Artificial Sequence
<220>
<223> Icd
<400> 35
Met Glu Ser Lys Val Val Val Pro Ala Gln Gly Lys Lys Ile Thr Leu
1 5 10 15
Gln Asn Gly Lys Leu Asn Val Pro Glu Asn Pro Ile Ile Pro Tyr Ile
20 25 30
Glu Gly Asp Gly Ile Gly Val Asp Val Thr Pro Ala Met Leu Lys Val
35 40 45
Val Asp Ala Ala Val Glu Lys Ala Tyr Lys Gly Glu Arg Lys Ile Ser
50 55 60
Trp Met Glu Ile Tyr Thr Gly Glu Lys Ser Thr Gln Val Tyr Gly Gln
65 70 75 80
Asp Val Trp Leu Pro Ala Glu Thr Leu Asp Leu Ile Arg Glu Tyr Arg
85 90 95
Val Ala Ile Lys Gly Pro Leu Thr Thr Pro Val Gly Gly Gly Ile Arg
100 105 110
Ser Leu Asn Val Ala Leu Arg Gln Glu Leu Asp Leu Tyr Ile Cys Leu
115 120 125
Arg Pro Val Arg Tyr Tyr Gln Gly Thr Pro Ser Pro Val Lys His Pro
130 135 140
Glu Leu Thr Asp Met Val Ile Phe Arg Glu Asn Ser Glu Asp Ile Tyr
145 150 155 160
Ala Gly Ile Glu Trp Lys Ala Asp Ser Ala Asp Ala Glu Lys Val Ile
165 170 175
Lys Phe Leu Arg Glu Glu Met Gly Val Lys Lys Ile Arg Phe Pro Glu
180 185 190
His Cys Gly Ile Gly Ile Lys Pro Cys Ser Glu Glu Gly Thr Lys Arg
195 200 205
Leu Val Arg Ala Ala Ile Glu Tyr Ala Ile Ala Asn Asp Arg Asp Ser
210 215 220
Val Thr Leu Val His Lys Gly Asn Ile Met Lys Phe Thr Glu Gly Ala
225 230 235 240
Phe Lys Asp Trp Gly Tyr Gln Leu Ala Arg Glu Glu Phe Gly Gly Glu
245 250 255
Leu Ile Asp Gly Gly Pro Trp Leu Lys Val Lys Asn Pro Asn Thr Gly
260 265 270
Lys Glu Ile Val Ile Lys Asp Val Ile Ala Asp Ala Phe Leu Gln Gln
275 280 285
Ile Leu Leu Arg Pro Ala Glu Tyr Asp Val Ile Ala Cys Met Asn Leu
290 295 300
Asn Gly Asp Tyr Ile Ser Asp Ala Leu Ala Ala Gln Val Gly Gly Ile
305 310 315 320
Gly Ile Ala Pro Gly Ala Asn Ile Gly Asp Glu Cys Ala Leu Phe Glu
325 330 335
Ala Thr His Gly Thr Ala Pro Lys Tyr Ala Gly Gln Asp Lys Val Asn
340 345 350
Pro Gly Ser Ile Ile Leu Ser Ala Glu Met Met Leu Arg His Met Gly
355 360 365
Trp Thr Glu Ala Ala Asp Leu Ile Val Lys Gly Met Glu Gly Ala Ile
370 375 380
Asn Ala Lys Thr Val Thr Tyr Asp Phe Glu Arg Leu Met Asp Gly Ala
385 390 395 400
Lys Leu Leu Lys Cys Ser Glu Phe Gly Asp Ala Ile Ile Glu Asn Met
405 410 415
<210> 36
<211> 1251
<212> DNA
<213> Artificial Sequence
<220>
<223> icd
<400> 36
atggaaagta aagtagttgt tccggcacaa ggcaagaaga tcaccctgca aaacggcaaa 60
ctcaacgttc ctgaaaatcc gattatccct tacattgaag gtgatggaat cggtgtagat 120
gtaaccccag ccatgctgaa agtggtcgac gctgcagtcg agaaagccta taaaggcgag 180
cgtaaaatct cctggatgga aatttacacc ggtgaaaaat ccacacaggt ttatggtcag 240
gacgtctggc tgcctgctga aactcttgat ctgattcgtg aatatcgcgt tgccattaaa 300
ggtccgctga ccactccggt tggtggcggt attcgctctc tgaacgttgc cctgcgccag 360
gaactggatc tctacatctg cctgcgtccg gtacgttact atcagggcac tccaagcccg 420
gttaaacacc ctgaactgac cgatatggtt atcttccgtg aaaactcgga agacatttat 480
gcgggtatcg aatggaaagc agactctgcc gacgccgaga aagtgattaa attcctgcgt 540
gaagagatgg gggtgaagaa aattcgcttc ccggaacatt gtggtatcgg tattaagccg 600
tgttcggaag aaggcaccaa acgtctggtt cgtgcagcga tcgaatacgc aattgctaac 660
gatcgtgact ctgtgactct ggtgcacaaa ggcaacatca tgaagttcac cgaaggagcg 720
tttaaagact ggggctacca gctggcgcgt gaagagtttg gcggtgaact gatcgacggt 780
ggcccgtggc tgaaagttaa aaacccgaac actggcaaag agatcgtcat taaagacgtg 840
attgctgatg cattcctgca acagatcctg ctgcgtccgg ctgaatatga tgttatcgcc 900
tgtatgaacc tgaacggtga ctacatttct gacgccctgg cagcgcaggt tggcggtatc 960
ggtatcgccc ctggtgcaaa catcggtgac gaatgcgccc tgtttgaagc cacccacggt 1020
actgcgccga aatatgccgg tcaggacaaa gtaaatcctg gctctattat tctctccgct 1080
gagatgatgc tgcgccacat gggttggacc gaagcggctg acttaattgt taaaggtatg 1140
gaaggcgcaa tcaacgcgaa aaccgtaacc tatgacttcg agcgtctgat ggatggcgct 1200
aaactgctga aatgttcaga gtttggtgac gcgatcatcg aaaacatgta a 1251
<210> 37
<211> 434
<212> PRT
<213> Artificial Sequence
<220>
<223> AceA
<400> 37
Met Lys Thr Arg Thr Gln Gln Ile Glu Glu Leu Gln Lys Glu Trp Thr
1 5 10 15
Gln Pro Arg Trp Glu Gly Ile Thr Arg Pro Tyr Ser Ala Glu Asp Val
20 25 30
Val Lys Leu Arg Gly Ser Val Asn Pro Glu Cys Thr Leu Ala Gln Leu
35 40 45
Gly Ala Ala Lys Met Trp Arg Leu Leu His Gly Glu Ser Lys Lys Gly
50 55 60
Tyr Ile Asn Ser Leu Gly Ala Leu Thr Gly Gly Gln Ala Leu Gln Gln
65 70 75 80
Ala Lys Ala Gly Ile Glu Ala Val Tyr Leu Ser Gly Trp Gln Val Ala
85 90 95
Ala Asp Ala Asn Leu Ala Ala Ser Met Tyr Pro Asp Gln Ser Leu Tyr
100 105 110
Pro Ala Asn Ser Val Pro Ala Val Val Glu Arg Ile Asn Asn Thr Phe
115 120 125
Arg Arg Ala Asp Gln Ile Gln Trp Ser Ala Gly Ile Glu Pro Gly Asp
130 135 140
Pro Arg Tyr Val Asp Tyr Phe Leu Pro Ile Val Ala Asp Ala Glu Ala
145 150 155 160
Gly Phe Gly Gly Val Leu Asn Ala Phe Glu Leu Met Lys Ala Met Ile
165 170 175
Glu Ala Gly Ala Ala Ala Val His Phe Glu Asp Gln Leu Ala Ser Val
180 185 190
Lys Lys Cys Gly His Met Gly Gly Lys Val Leu Val Pro Thr Gln Glu
195 200 205
Ala Ile Gln Lys Leu Val Ala Ala Arg Leu Ala Ala Asp Val Thr Gly
210 215 220
Val Pro Thr Leu Leu Val Ala Arg Thr Asp Ala Asp Ala Ala Asp Leu
225 230 235 240
Ile Thr Ser Asp Cys Asp Pro Tyr Asp Ser Glu Phe Ile Thr Gly Glu
245 250 255
Arg Thr Ser Glu Gly Phe Phe Arg Thr His Ala Gly Ile Glu Gln Ala
260 265 270
Ile Ser Arg Gly Leu Ala Tyr Ala Pro Tyr Ala Asp Leu Val Trp Cys
275 280 285
Glu Thr Ser Thr Pro Asp Leu Glu Leu Ala Arg Arg Phe Ala Gln Ala
290 295 300
Ile His Ala Lys Tyr Pro Gly Lys Leu Leu Ala Tyr Asn Cys Ser Pro
305 310 315 320
Ser Phe Asn Trp Gln Lys Asn Leu Asp Asp Lys Thr Ile Ala Ser Phe
325 330 335
Gln Gln Gln Leu Ser Asp Met Gly Tyr Lys Phe Gln Phe Ile Thr Leu
340 345 350
Ala Gly Ile His Ser Met Trp Phe Asn Met Phe Asp Leu Ala Asn Ala
355 360 365
Tyr Ala Gln Gly Glu Gly Met Lys His Tyr Val Glu Lys Val Gln Gln
370 375 380
Pro Glu Phe Ala Ala Ala Lys Asp Gly Tyr Thr Phe Val Ser His Gln
385 390 395 400
Gln Glu Val Gly Thr Gly Tyr Phe Asp Lys Val Thr Thr Ile Ile Gln
405 410 415
Gly Gly Thr Ser Ser Val Thr Ala Leu Thr Gly Ser Thr Glu Glu Ser
420 425 430
Gln Phe
<210> 38
<211> 1305
<212> DNA
<213> Artificial Sequence
<220>
<223> aceA
<400> 38
atgaaaaccc gtacacaaca aattgaagaa ttacagaaag agtggactca accgcgttgg 60
gaaggcatta ctcgcccata cagtgcggaa gatgtggtga aattacgcgg ttcagtcaat 120
cctgaatgca cgctggcgca actgggcgca gcgaaaatgt ggcgtctgct gcacggtgag 180
tcgaaaaaag gctacatcaa cagcctcggc gcactgactg gcggtcaggc gctgcaacag 240
gcgaaagcgg gtattgaagc agtctatctg tcgggatggc aggtagcggc ggacgctaac 300
ctggcggcca gcatgtatcc ggatcagtcg ctctatccgg caaactcggt gccagctgtg 360
gtggagcgga tcaacaacac cttccgtcgt gccgatcaga tccaatggtc cgcgggcatt 420
gagccgggcg atccgcgcta tgtcgattac ttcctgccga tcgttgccga tgcggaagcc 480
ggttttggcg gtgtcctgaa tgcctttgaa ctgatgaaag cgatgattga agccggtgca 540
gcggcagttc acttcgaaga tcagctggcg tcagtgaaga aatgcggtca catgggcggc 600
aaagttttag tgccaactca ggaagctatt cagaaactgg tcgcggcgcg tctggcagct 660
gacgtgacgg gcgttccaac cctgctggtt gcccgtaccg atgctgatgc ggcggatctg 720
atcacctccg attgcgaccc gtatgacagc gaatttatta ccggcgagcg taccagtgaa 780
ggcttcttcc gtactcatgc gggcattgag caagcgatca gccgtggcct ggcgtatgcg 840
ccatatgctg acctggtctg gtgtgaaacc tccacgccgg atctggaact ggcgcgtcgc 900
tttgcacaag ctatccacgc gaaatatccg ggcaaactgc tggcttataa ctgctcgccg 960
tcgttcaact ggcagaaaaa cctcgacgac aaaactattg ccagcttcca gcagcagctg 1020
tcggatatgg gctacaagtt ccagttcatc accctggcag gtatccacag catgtggttc 1080
aacatgtttg acctggcaaa cgcctatgcc cagggcgagg gtatgaagca ctacgttgag 1140
aaagtgcagc agccggaatt tgccgccgcg aaagatggct ataccttcgt atctcaccag 1200
caggaagtgg gtacaggtta cttcgataaa gtgacgacta ttattcaggg cggcacgtct 1260
tcagtcaccg cgctgaccgg ctccactgaa gaatcgcagt tctaa 1305
<210> 39
<211> 578
<212> PRT
<213> Artificial Sequence
<220>
<223> AceK
<400> 39
Met Pro Arg Gly Leu Glu Leu Leu Ile Ala Gln Thr Ile Leu Gln Gly
1 5 10 15
Phe Asp Ala Gln Tyr Gly Arg Phe Leu Glu Val Thr Ser Gly Ala Gln
20 25 30
Gln Arg Phe Glu Gln Ala Asp Trp His Ala Val Gln Gln Ala Met Lys
35 40 45
Asn Arg Ile His Leu Tyr Asp His His Val Gly Leu Val Val Glu Gln
50 55 60
Leu Arg Cys Ile Thr Asn Gly Gln Ser Thr Asp Ala Ala Phe Leu Leu
65 70 75 80
Arg Val Lys Glu His Tyr Thr Arg Leu Leu Pro Asp Tyr Pro Arg Phe
85 90 95
Glu Ile Ala Glu Ser Phe Phe Asn Ser Val Tyr Cys Arg Leu Phe Asp
100 105 110
His Arg Ser Leu Thr Pro Glu Arg Leu Phe Ile Phe Ser Ser Gln Pro
115 120 125
Glu Arg Arg Phe Arg Thr Ile Pro Arg Pro Leu Ala Lys Asp Phe His
130 135 140
Pro Asp His Gly Trp Glu Ser Leu Leu Met Arg Val Ile Ser Asp Leu
145 150 155 160
Pro Leu Arg Leu Arg Trp Gln Asn Lys Ser Arg Asp Ile His Tyr Ile
165 170 175
Ile Arg His Leu Thr Glu Thr Leu Gly Thr Asp Asn Leu Ala Glu Ser
180 185 190
His Leu Gln Val Ala Asn Glu Leu Phe Tyr Arg Asn Lys Ala Ala Trp
195 200 205
Leu Val Gly Lys Leu Ile Thr Pro Ser Gly Thr Leu Pro Phe Leu Leu
210 215 220
Pro Ile His Gln Thr Asp Asp Gly Glu Leu Phe Ile Asp Thr Cys Leu
225 230 235 240
Thr Thr Thr Ala Glu Ala Ser Ile Val Phe Gly Phe Ala Arg Ser Tyr
245 250 255
Phe Met Val Tyr Ala Pro Leu Pro Ala Ala Leu Val Glu Trp Leu Arg
260 265 270
Glu Ile Leu Pro Gly Lys Thr Thr Ala Glu Leu Tyr Met Ala Ile Gly
275 280 285
Cys Gln Lys His Ala Lys Thr Glu Ser Tyr Arg Glu Tyr Leu Val Tyr
290 295 300
Leu Gln Gly Cys Asn Glu Gln Phe Ile Glu Ala Pro Gly Ile Arg Gly
305 310 315 320
Met Val Met Leu Val Phe Thr Leu Pro Gly Phe Asp Arg Val Phe Lys
325 330 335
Val Ile Lys Asp Arg Phe Ala Pro Gln Lys Glu Met Ser Ala Ala His
340 345 350
Val Arg Ala Cys Tyr Gln Leu Val Lys Glu His Asp Arg Val Gly Arg
355 360 365
Met Ala Asp Thr Gln Glu Phe Glu Asn Phe Val Leu Glu Lys Arg His
370 375 380
Ile Ser Pro Ala Leu Met Glu Leu Leu Leu Gln Glu Ala Ala Glu Lys
385 390 395 400
Ile Thr Asp Leu Gly Glu Gln Ile Val Ile Arg His Leu Tyr Ile Glu
405 410 415
Arg Arg Met Val Pro Leu Asn Ile Trp Leu Glu Gln Val Glu Gly Gln
420 425 430
Gln Leu Arg Asp Ala Ile Glu Glu Tyr Gly Asn Ala Ile Arg Gln Leu
435 440 445
Ala Ala Ala Asn Ile Phe Pro Gly Asp Met Leu Phe Lys Asn Phe Gly
450 455 460
Val Thr Arg His Gly Arg Val Val Phe Tyr Asp Tyr Asp Glu Ile Cys
465 470 475 480
Tyr Met Thr Glu Val Asn Phe Arg Asp Ile Pro Pro Pro Arg Tyr Pro
485 490 495
Glu Asp Glu Leu Ala Ser Glu Pro Trp Tyr Ser Val Ser Pro Gly Asp
500 505 510
Val Phe Pro Glu Glu Phe Arg His Trp Leu Cys Ala Asp Pro Arg Ile
515 520 525
Gly Pro Leu Phe Glu Glu Met His Ala Asp Leu Phe Arg Ala Asp Tyr
530 535 540
Trp Arg Ala Leu Gln Asn Arg Ile Arg Glu Gly His Val Glu Asp Val
545 550 555 560
Tyr Ala Tyr Arg Arg Arg Gln Arg Phe Ser Val Arg Tyr Gly Glu Met
565 570 575
Leu Phe
<210> 40
<211> 1737
<212> DNA
<213> Artificial Sequence
<220>
<223> aceK
<400> 40
atgccgcgtg gcctggaatt attgattgct caaaccattt tgcaaggctt cgatgctcag 60
tatggtcgat tcctcgaagt gacctccggt gcgcagcagc gtttcgaaca ggccgactgg 120
catgctgtcc agcaggcgat gaaaaaccgt atccatcttt acgatcatca cgttggtctg 180
gtcgtggagc aactgcgctg cattactaac ggccaaagta cggacgcggc atttttacta 240
cgtgttaaag agcattacac ccggctgttg ccggattacc cgcgcttcga gattgcggag 300
agctttttta actccgtgta ctgtcggtta tttgaccacc gctcgcttac tcccgagcgg 360
ctttttatct ttagctctca gccagagcgc cgctttcgta ccattccccg cccgctggcg 420
aaagactttc accccgatca cggctgggaa tctctactga tgcgcgttat cagcgaccta 480
ccgctgcgcc tgcgctggca gaataaaagc cgtgacatcc attacattat tcgccatctg 540
acggaaacgc tggggacaga caacctcgcg gaaagtcatt tacaggtggc gaacgaactg 600
ttttaccgca ataaagccgc ctggctggta ggcaaactga tcacaccttc cggcacattg 660
ccatttttgc tgccgatcca ccagacggac gacggcgagt tatttattga tacctgcctg 720
acgacgaccg ccgaagcgag cattgttttt ggctttgcgc gttcttattt tatggtttat 780
gcgccgctgc ccgcagcgct ggtcgagtgg ctacgggaaa ttctgccagg taaaaccacc 840
gctgaattgt atatggctat cggctgccag aagcacgcca aaaccgaaag ctaccgcgaa 900
tatctcgttt atctacaggg ctgtaatgag cagttcattg aagcgccggg tattcgtgga 960
atggtgatgt tggtgtttac gctgccgggc tttgatcggg tattcaaagt catcaaagac 1020
aggttcgcgc cgcagaaaga gatgtctgcc gctcacgttc gtgcctgcta tcaactggtg 1080
aaagagcacg atcgcgtggg ccgaatggcg gacacccagg agtttgaaaa ctttgtgctg 1140
gagaagcggc atatttcccc ggcattaatg gaattactgc ttcaggaagc agcggaaaaa 1200
atcaccgatc tcggcgaaca aattgtgatt cgccatcttt atattgagcg gcggatggtg 1260
ccgctcaata tctggctgga acaagtggaa ggtcagcagt tgcgcgacgc cattgaagaa 1320
tacggtaacg ctattcgcca gcttgccgct gctaacattt tccctggcga catgctgttt 1380
aaaaacttcg gtgtcacccg tcacgggcgt gtggtttttt atgattacga tgaaatttgc 1440
tacatgacgg aagtgaattt ccgcgacatc ccgccgccgc gctatccgga agacgaactt 1500
gccagcgaac cgtggtacag cgtctcgccg ggcgatgttt tcccggaaga gtttcgccac 1560
tggctatgcg ccgacccgcg tattggtccg ctgtttgaag agatgcacgc cgacctgttc 1620
cgcgctgatt actggcgcgc actacaaaac cgcatacgtg aagggcatgt ggaagatgtt 1680
tatgcgtatc ggcgcaggca aagatttagc gtacggtatg gggagatgct tttttga 1737
<210> 41
<211> 723
<212> PRT
<213> Artificial Sequence
<220>
<223> GlcB
<400> 41
Met Ser Gln Thr Ile Thr Gln Ser Arg Leu Arg Ile Asp Ala Asn Phe
1 5 10 15
Lys Arg Phe Val Asp Glu Glu Val Leu Pro Gly Thr Gly Leu Asp Ala
20 25 30
Ala Ala Phe Trp Arg Asn Phe Asp Glu Ile Val His Asp Leu Ala Pro
35 40 45
Glu Asn Arg Gln Leu Leu Ala Glu Arg Asp Arg Ile Gln Ala Ala Leu
50 55 60
Asp Glu Trp His Arg Ser Asn Pro Gly Pro Val Lys Asp Lys Ala Ala
65 70 75 80
Tyr Lys Ser Phe Leu Arg Glu Leu Gly Tyr Leu Val Pro Gln Pro Glu
85 90 95
Arg Val Thr Val Glu Thr Thr Gly Ile Asp Ser Glu Ile Thr Ser Gln
100 105 110
Ala Gly Pro Gln Leu Val Val Pro Ala Met Asn Ala Arg Tyr Ala Leu
115 120 125
Asn Ala Ala Asn Ala Arg Trp Gly Ser Leu Tyr Asp Ala Leu Tyr Gly
130 135 140
Ser Asp Ile Ile Pro Gln Glu Gly Ala Met Val Ser Gly Tyr Asp Pro
145 150 155 160
Gln Arg Gly Glu Gln Val Ile Ala Trp Val Arg Arg Phe Leu Asp Glu
165 170 175
Ser Leu Pro Leu Glu Asn Gly Ser Tyr Gln Asp Val Val Ala Phe Lys
180 185 190
Val Val Asp Lys Gln Leu Arg Ile Gln Leu Lys Asn Gly Lys Glu Thr
195 200 205
Thr Leu Arg Thr Pro Ala Gln Phe Val Gly Tyr Arg Gly Asp Ala Ala
210 215 220
Ala Pro Thr Cys Ile Leu Leu Lys Asn Asn Gly Leu His Ile Glu Leu
225 230 235 240
Gln Ile Asp Ala Asn Gly Arg Ile Gly Lys Asp Asp Pro Ala His Ile
245 250 255
Asn Asp Val Ile Val Glu Ala Ala Ile Ser Thr Ile Leu Asp Cys Glu
260 265 270
Asp Ser Val Ala Ala Val Asp Ala Glu Asp Lys Ile Leu Leu Tyr Arg
275 280 285
Asn Leu Leu Gly Leu Met Gln Gly Thr Leu Gln Glu Lys Met Glu Lys
290 295 300
Asn Gly Arg Gln Ile Val Arg Lys Leu Asn Asp Asp Arg His Tyr Thr
305 310 315 320
Ala Ala Asp Gly Ser Glu Ile Ser Leu His Gly Arg Ser Leu Leu Phe
325 330 335
Ile Arg Asn Val Gly His Leu Met Thr Ile Pro Val Ile Trp Asp Ser
340 345 350
Glu Gly Asn Glu Ile Pro Glu Gly Ile Leu Asp Gly Val Met Thr Gly
355 360 365
Ala Ile Ala Leu Tyr Asp Leu Lys Val Gln Lys Asn Ser Arg Thr Gly
370 375 380
Ser Val Tyr Ile Val Lys Pro Lys Met His Gly Pro Gln Glu Val Ala
385 390 395 400
Phe Ala Asn Lys Leu Phe Thr Arg Ile Glu Thr Met Leu Gly Met Ala
405 410 415
Pro Asn Thr Leu Lys Met Gly Ile Met Asp Glu Glu Arg Arg Thr Ser
420 425 430
Leu Asn Leu Arg Ser Cys Ile Ala Gln Ala Arg Asn Arg Val Ala Phe
435 440 445
Ile Asn Thr Gly Phe Leu Asp Arg Thr Gly Asp Glu Met His Ser Val
450 455 460
Met Glu Ala Gly Pro Met Leu Arg Lys Asn Gln Met Lys Ser Thr Pro
465 470 475 480
Trp Ile Lys Ala Tyr Glu Arg Asn Asn Val Leu Ser Gly Leu Phe Cys
485 490 495
Gly Leu Arg Gly Lys Ala Gln Ile Gly Lys Gly Met Trp Ala Met Pro
500 505 510
Asp Leu Met Ala Asp Met Tyr Ser Gln Lys Gly Asp Gln Leu Arg Ala
515 520 525
Gly Ala Asn Thr Ala Trp Val Pro Ser Pro Thr Ala Ala Thr Leu His
530 535 540
Ala Leu His Tyr His Gln Thr Asn Val Gln Ser Val Gln Ala Asn Ile
545 550 555 560
Ala Gln Thr Glu Phe Asn Ala Glu Phe Glu Pro Leu Leu Asp Asp Leu
565 570 575
Leu Thr Ile Pro Val Ala Glu Asn Ala Asn Trp Ser Ala Gln Glu Ile
580 585 590
Gln Gln Glu Leu Asp Asn Asn Val Gln Gly Ile Leu Gly Tyr Val Val
595 600 605
Arg Trp Val Glu Gln Gly Ile Gly Cys Ser Lys Val Pro Asp Ile His
610 615 620
Asn Val Ala Leu Met Glu Asp Arg Ala Thr Leu Arg Ile Ser Ser Gln
625 630 635 640
His Ile Ala Asn Trp Leu Arg His Gly Ile Leu Thr Lys Glu Gln Val
645 650 655
Gln Ala Ser Leu Glu Asn Met Ala Lys Val Val Asp Gln Gln Asn Ala
660 665 670
Gly Asp Pro Ala Tyr Arg Pro Met Ala Gly Asn Phe Ala Asn Ser Cys
675 680 685
Ala Phe Lys Ala Ala Ser Asp Leu Ile Phe Leu Gly Val Lys Gln Pro
690 695 700
Asn Gly Tyr Thr Glu Pro Leu Leu His Ala Trp Arg Leu Arg Glu Lys
705 710 715 720
Glu Ser His
<210> 42
<211> 2172
<212> DNA
<213> Artificial Sequence
<220>
<223> glcB
<400> 42
atgagtcaaa ccataaccca gagccgttta cgcattgacg ccaattttaa acgttttgtg 60
gatgaagaag ttttaccggg aacagggctg gacgctgcgg cgttctggcg caattttgat 120
gagatcgttc atgatctggc accagaaaat cgtcagttgc tggcagaacg cgatcgcatt 180
caggcagcgc ttgatgagtg gcatcgcagc aatccggggc cggtaaaaga taaagcggcc 240
tataaatctt tcctgcgtga actgggctac ctggtgccgc aaccggagcg cgtgacggtg 300
gaaaccacgg gcattgacag cgaaatcacc agccaggcgg ggccgcagct ggtggttccg 360
gcaatgaacg cccgctacgc gctgaacgcg gcgaacgctc gctggggctc actgtacgat 420
gcgttatacg gcagcgacat catcccgcag gaaggggcga tggtcagcgg ctacgatccg 480
caacgcggtg agcaggttat cgcctgggtt cggcgtttcc tcgatgaatc tctaccgctg 540
gaaaacggca gctatcagga tgtggtggcg tttaaggtgg ttgataaaca attacgcatc 600
cagttgaaaa atggtaaaga aaccacgtta cgtactccag cacagtttgt cggttaccgt 660
ggcgatgccg ctgcgccgac ctgcattttg ctgaaaaata acggcctgca tattgagctg 720
caaatcgatg ccaatgggcg gattggcaaa gacgatccgg cgcacatcaa cgatgttatc 780
gtcgaagctg ctatcagtac cattctcgac tgcgaagatt cggtcgcggc ggttgatgcg 840
gaagataaaa tcctgctgta ccgcaacctg ctgggcctga tgcaggggac tctgcaagag 900
aaaatggaga aaaacggtcg gcaaatcgtg cgtaaactga atgacgatcg tcattacacc 960
gccgccgatg gctctgaaat ttctctgcac ggacgctcgc tgctgtttat ccgcaacgtg 1020
ggtcatttga tgaccattcc tgtgatttgg gacagcgaag gcaatgaaat cccggaaggc 1080
attcttgatg gcgtcatgac tggcgcgatt gccctctatg atttaaaagt gcagaaaaac 1140
tcgcgcactg gcagcgtcta tattgtgaaa ccgaaaatgc acggtccgca ggaagtggcg 1200
ttcgccaaca aactgtttac ccgcattgag acaatgctcg gtatggcacc gaataccctg 1260
aaaatgggca ttatggatga agaacgtcgg acctcgctga acttgcgtag ctgtatcgct 1320
caggcgcgca accgcgtggc gttcatcaat accggtttcc tcgaccgtac cggcgatgaa 1380
atgcattcgg tgatggaagc tggcccgatg ctgcgtaaaa atcagatgaa atcgacgcct 1440
tggatcaaag cctacgagcg taataacgtg ctttccggtc tgttctgtgg gctgcgcggt 1500
aaagcgcaaa ttggtaaagg catgtgggca atgccggacc tgatggcaga catgtacagc 1560
cagaagggcg accaactgcg tgccggggca aacacagcct gggttccgtc accaaccgct 1620
gctacgctcc atgcgctgca ctaccaccaa accaacgtac agagcgtaca agccaacatt 1680
gcccagaccg agttcaatgc tgaatttgaa ccgctgctgg acgatctgct gactattccg 1740
gttgctgaaa acgctaactg gtcggcgcaa gagatccaac aagagctgga taacaacgtg 1800
caggggattc tggggtacgt ggtgcgctgg gtggagcagg ggattggttg ttcaaaagtg 1860
ccggatattc acaatgtggc gttgatggaa gaccgcgcaa cgctgcgtat ctccagccag 1920
catatcgcca actggttacg tcacggtatt ctgaccaaag aacaggtgca ggcgtcgctg 1980
gagaatatgg cgaaagtggt tgatcagcaa aacgctggcg atccggctta tcgtccgatg 2040
gcggggaatt tcgctaactc gtgtgctttt aaagctgcca gcgatttaat cttcctcggc 2100
gtgaaacagc caaacggcta taccgaaccg ttattacacg cctggcgttt acgcgaaaaa 2160
gaaagtcatt aa 2172
<210> 43
<211> 533
<212> PRT
<213> Artificial Sequence
<220>
<223> AceB
<400> 43
Met Thr Glu Gln Ala Thr Thr Thr Asp Glu Leu Ala Phe Thr Arg Pro
1 5 10 15
Tyr Gly Glu Gln Glu Lys Gln Ile Leu Thr Ala Glu Ala Val Glu Phe
20 25 30
Leu Thr Glu Leu Val Thr His Phe Thr Pro Gln Arg Asn Lys Leu Leu
35 40 45
Ala Ala Arg Ile Gln Gln Gln Gln Asp Ile Asp Asn Gly Thr Leu Pro
50 55 60
Asp Phe Ile Ser Glu Thr Ala Ser Ile Arg Asp Ala Asp Trp Lys Ile
65 70 75 80
Arg Gly Ile Pro Ala Asp Leu Glu Asp Arg Arg Val Glu Ile Thr Gly
85 90 95
Pro Val Glu Arg Lys Met Val Ile Asn Ala Leu Asn Ala Asn Val Lys
100 105 110
Val Phe Met Ala Asp Phe Glu Asp Ser Leu Ala Pro Asp Trp Asn Lys
115 120 125
Val Ile Asp Gly Gln Ile Asn Leu Arg Asp Ala Val Asn Gly Thr Ile
130 135 140
Ser Tyr Thr Asn Glu Ala Gly Lys Ile Tyr Gln Leu Lys Pro Asn Pro
145 150 155 160
Ala Val Leu Ile Cys Arg Val Arg Gly Leu His Leu Pro Glu Lys His
165 170 175
Val Thr Trp Arg Gly Glu Ala Ile Pro Gly Ser Leu Phe Asp Phe Ala
180 185 190
Leu Tyr Phe Phe His Asn Tyr Gln Ala Leu Leu Ala Lys Gly Ser Gly
195 200 205
Pro Tyr Phe Tyr Leu Pro Lys Thr Gln Ser Trp Gln Glu Ala Ala Trp
210 215 220
Trp Ser Glu Val Phe Ser Tyr Ala Glu Asp Arg Phe Asn Leu Pro Arg
225 230 235 240
Gly Thr Ile Lys Ala Thr Leu Leu Ile Glu Thr Leu Pro Ala Val Phe
245 250 255
Gln Met Asp Glu Ile Leu His Ala Leu Arg Asp His Ile Val Gly Leu
260 265 270
Asn Cys Gly Arg Trp Asp Tyr Ile Phe Ser Tyr Ile Lys Thr Leu Lys
275 280 285
Asn Tyr Pro Asp Arg Val Leu Pro Asp Arg Gln Ala Val Thr Met Asp
290 295 300
Lys Pro Phe Leu Asn Ala Tyr Ser Arg Leu Leu Ile Lys Thr Cys His
305 310 315 320
Lys Arg Gly Ala Phe Ala Met Gly Gly Met Ala Ala Phe Ile Pro Ser
325 330 335
Lys Asp Glu Glu His Asn Asn Gln Val Leu Asn Lys Val Lys Ala Asp
340 345 350
Lys Ser Leu Glu Ala Asn Asn Gly His Asp Gly Thr Trp Ile Ala His
355 360 365
Pro Gly Leu Ala Asp Thr Ala Met Ala Val Phe Asn Asp Ile Leu Gly
370 375 380
Ser Arg Lys Asn Gln Leu Glu Val Met Arg Glu Gln Asp Ala Pro Ile
385 390 395 400
Thr Ala Asp Gln Leu Leu Ala Pro Cys Asp Gly Glu Arg Thr Glu Glu
405 410 415
Gly Met Arg Ala Asn Ile Arg Val Ala Val Gln Tyr Ile Glu Ala Trp
420 425 430
Ile Ser Gly Asn Gly Cys Val Pro Ile Tyr Gly Leu Met Glu Asp Ala
435 440 445
Ala Thr Ala Glu Ile Ser Arg Thr Ser Ile Trp Gln Trp Ile His His
450 455 460
Gln Lys Thr Leu Ser Asn Gly Lys Pro Val Thr Lys Ala Leu Phe Arg
465 470 475 480
Gln Met Leu Gly Glu Glu Met Lys Val Ile Ala Ser Glu Leu Gly Glu
485 490 495
Glu Arg Phe Ser Gln Gly Arg Phe Asp Asp Ala Ala Arg Leu Met Glu
500 505 510
Gln Ile Thr Thr Ser Asp Glu Leu Ile Asp Phe Leu Thr Leu Pro Gly
515 520 525
Tyr Arg Leu Leu Ala
530
<210> 44
<211> 1602
<212> DNA
<213> Artificial Sequence
<220>
<223> aceB
<400> 44
atgactgaac aggcaacaac aaccgatgaa ctggctttca caaggccgta tggcgagcag 60
gagaagcaaa ttcttactgc cgaagcggta gaatttctga ctgagctggt gacgcatttt 120
acgccacaac gcaataaact tctggcagcg cgcattcagc agcagcaaga tattgataac 180
ggaacgttgc ctgattttat ttcggaaaca gcttccattc gcgatgctga ttggaaaatt 240
cgcgggattc ctgcggactt agaagaccgc cgcgtagaga taactggccc ggtagagcgc 300
aagatggtga tcaacgcgct caacgccaat gtgaaagtct ttatggccga tttcgaagat 360
tcactggcac cagactggaa caaagtgatc gacgggcaaa ttaacctgcg tgatgcggtt 420
aacggcacca tcagttacac caatgaagca ggcaaaattt accagctcaa gcccaatcca 480
gcggttttga tttgtcgggt acgcggtctg cacttgccgg aaaaacatgt cacctggcgt 540
ggtgaggcaa tccccggcag cctgtttgat tttgcgctct atttcttcca caactatcag 600
gcactgttgg caaagggcag tggtccctat ttctatctgc cgaaaaccca gtcctggcag 660
gaagcggcct ggtggagcga agtcttcagc tatgcagaag atcgctttaa tctgccgcgc 720
ggcaccatca aggcgacgtt gctgattgaa acgctgcccg ccgtgttcca gatggatgaa 780
atccttcacg cgctgcgtga ccatattgtt ggtctgaact gcggtcgttg ggattacatc 840
ttcagctata tcaaaacgtt gaaaaactat cccgatcgcg tcctgccaga cagacaggca 900
gtgacgatgg ataaaccatt cctgaatgct tactcacgcc tgttgattaa aacctgccat 960
aaacgcggtg cttttgcgat gggcggcatg gcggcgttta ttccgagcaa agatgaagag 1020
cacaataacc aggtgctcaa caaagtaaaa gcggataaat cgctggaagc caataacggt 1080
cacgatggca catggatcgc tcacccaggc cttgcggaca cggcaatggc ggtattcaac 1140
gacattctcg gctcccgtaa aaatcagctt gaagtgatgc gcgaacaaga cgcgccgatt 1200
actgccgatc agctgctggc accttgtgat ggtgaacgca ccgaagaagg tatgcgcgcc 1260
aacattcgcg tggctgtgca gtacatcgaa gcgtggatct ctggcaacgg ctgtgtgccg 1320
atttatggcc tgatggaaga tgcggcgacg gctgaaattt cccgtacctc gatctggcag 1380
tggatccatc atcaaaaaac gttgagcaat ggcaaaccgg tgaccaaagc cttgttccgc 1440
cagatgctgg gcgaagagat gaaagtcatt gccagcgaac tgggcgaaga acgtttctcc 1500
caggggcgtt ttgacgatgc cgcacgcttg atggaacaga tcaccacttc cgatgagtta 1560
attgatttcc tgaccctgcc aggctaccgc ctgttagcgt aa 1602
<210> 45
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> PsynK1-ppc promoter
<400> 45
tttacagcta gctcagtcct aggtattatg ctagc 35
<210> 46
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> PsynK2
<400> 46
ctgacagcta gctcagtcct aggtataatg ctagc 35
<210> 47
<211> 129
<212> DNA
<213> Artificial Sequence
<220>
<223> PuspA
<400> 47
aaccactatc aatatattca tgtcgaaaat ttgtttatct aacgagtaag caaggcggat 60
tgacggatca tccgggtcgc tataaggtat agttcgcagg acgcgggtga cgtaacggca 120
caagaaacg 129
<210> 48
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> pykF-Left
<400> 48
gaaagcaagt ttctcccatc cttctcaact taaagactaa gactgtcatg 50
<210> 49
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> pykF-right
<400> 49
gatatacaaa ttaattcaca aaagcaatat tacaggacgt gaacagatgc 50
<210> 50
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> pykA-Left
<400> 50
tttcatgttc aagcaacacc tggttgtttc agtcaacgga gtattacatg 50
<210> 51
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> pykA-right
<400> 51
tggcgttttc gccgcatccg gcaacgtact tactctaccg ttaaaatacg 50
<210> 52
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> maeB-left
<400> 52
ttcagggtaa gcgtgagagt taaaaaaaat tacagcggtt gggtttgcgc 50
<210> 53
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> maeB-right
<400> 53
ttgcccacac actttatttg tgaacgttac gtgaaaggaa caaccaaatg 50
<210> 54
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> maeA-left
<400> 54
cccggtagcc ttcactaccg ggcgcaggct tagatggagg tacggcggta 50
<210> 55
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> maeA-right
<400> 55
ggccgacgcc ctggcggtaa agcaaagacg ataaaagccc cccagggatg 50
<210> 56
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> aspC-left
<400> 56
ttttcagcgg gcttcattgt ttttaatgct tacagcactg ccacaatcgc 50
<210> 57
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> aspC-right
<400> 57
taccctgata gcggacttcc cttctgtaac cataatggaa cctcgtcatg 50
<210> 58
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Icd-left
<400> 58
aacgtggtgg cagacgagca aaccagtagc gctcgaagga gaggtgaatg 50
<210> 59
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Icd-right
<400> 59
cccgttaata aatttaacaa actacggcat tacatgtttt cgatgatcgc 50
<210> 60
<211> 134
<212> DNA
<213> Artificial Sequence
<220>
<223> Ppc promoter native
<400> 60
tcgcagcatt tgacgtcacc gcttttacgt ggctttataa aagacgacga aaagcaaagc 60
ccgagcatat tcgcgccaat gcgacgtgaa ggatacaggg ctatcaaacg ataagatggg 120
gtgtctgggg taat 134
Claims (36)
- (1) 숙시닐-coA (succinyl-coA; SuCoA)를 숙시네이트 세미알데하이드 (succinate semialdehyde; SSA)로 전환하는 단계;
(2) 숙시네이트 세미알데하이드 (succinate semialdehyde; SSA)를 4-하이드록시부티레이트(4-hydroxybutyrate; 4HB)로 전환하는 단계;
(3) 4-하이드록시부티레이트(4-hydroxybutyrate; 4HB)를 4-하이드록시부티릴 coA(4-hydroxybutyryl coA; 4HBCoA)로 전환하는 단계;
(4) 둘 이상의 4-하이드록시부티릴 coA(4-hydroxybutyryl coA; 4HBCoA)를 중합하여 폴리-4-하이드록시부티레이트(poly-4-hydroxybutyrate; P4HB)를 생산하는 단계; 및
(5) 폴리-4-하이드록시부티레이트를 1,4-부탄다이올로 분해하는 단계를 포함하는 1,4-부탄다이올 생산방법.
- 제1항에 있어서, 상기 (1) 내지 (4)는 숙시네이트 세미알데하이드 디하이드로게나제(succinate semialdehyde dehydrogenase), 4-하이드록시부티릭산 디하이드로게나제 (4-hydroxybutyric acid dehydrogenase), 4-하이드록시부티릴-coA 트랜스퍼라제 (4-hydroxybutyryl-CoA transferase), 및 폴리(3-하이드록시알카노에이트) 폴리머라제(Poly(3-hydroxyalkanoate) polymerase)로 이루어지는 군에서 선택되는 어느 하나 이상의 폴리펩티드; 상기 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 또는 이들의 조합을 포함하는 미생물; 및 이의 배양물로 구성되는 군에서 선택되는 어느 하나 이상을 이용하는 것인, 생산방법.
- 제1항에 있어서, 상기 생산방법은 TCA 경로를 더 포함하는 것인, 생산방법.
- 제3항에 있어서, 상기 TCA 경로는
(a1) 피루베이트를 아세틸-coA로 전환하는 단계;
(b1) 아세틸-coA 및 옥살로아세테이트를 시트레이트로 전환하는 단계;
(c1) 시트레이트를 아이소시트레이트로 전환하는 단계;
(d1) 아이소시트레이트를 α-케토글루타레이트로 전환하는 단계;
(e1) α-케토글루타레이트를 숙시닐-coA로 전환하는 단계; 및
(f1) 피루베이트를 옥살로아세테이트로 전환하는 단계로 구성되는 군에서 선택되는 어느 하나 이상을 포함하는 것인, 생산방법.
- 제1항에 있어서, 상기 생산방법은 (g1) 포스포에놀피루베이트를 옥살로아세테이트로 전환하는 단계를 더 포함하는 것인, 생산방법.
- 제5항에 있어서, 상기 (g1) 은 포스포에놀피루베이트 카복실라제(phosphoenolpyruvate carboxylase) 폴리펩티드; 상기 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 또는 이들의 조합을 포함하는 미생물; 및 이의 배양물로 구성되는 군에서 선택되는 어느 하나 이상을 이용하는 것인, 생산방법.
- 제1항에 있어서, 상기 생산방법은 (g1) 포스포에놀피루베이트를 옥살로아세테이트로 전환하는 단계가 강화된 것인, 생산방법.
- 제1항에 있어서, 상기 생산방법은 환원형 TCA 경로(reductive TCA cycle)를 더 포함하는 것인, 생산방법.
- 제8항에 있어서, 상기 환원형 TCA 경로는 (a2) 옥살로아세테이트를 말레이트로 전환하는 단계;
(b2) 말레이트를 푸마레이트로 전환하는 단계;
(c2) 푸마레이트를 숙시네이트로 전환하는 단계; 및
(d2) 숙시네이트를 숙시닐-coA로 전환하는 단계로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 것인, 생산방법.
- 제8항에 있어서, 상기 생산방법은 (e2) 포스포에놀피루베이트에서 피루베이트로 전환하는 단계가 약화된 것인, 생산방법.
- 제8항에 있어서, 상기 환원형 TCA 경로는 하기 (I) 내지 (XII)로 이루어진 군에서 선택되는 어느 하나 이상에 의해 강화된 것인, 생산방법:
(I) 피루베이트 키나아제(pyruvate kinase) 약화;
(II) 포스포에놀피루베이트 카복실라제(PEP carboxylase) 강화;
(III) 카보닉 안하이드라제(carbonic anhydrase) 강화;
(IV) 시트레이트 신타아제(citrate synthase) 조절;
(V) 피루베이트 카복실라제(pyruvate carboxylase) 강화;
(VI) NAD+-의존성 말레이트 디하이드로게나제(NAD+-dependent malate dehydrogenase) 약화;
(VII) NADP+-의존성 말레이트 디하이드로게나제(NADP+-dependent malate dehydrogenase) 약화;
(VIII) 포스포글루코네이트 디하이드라타아제(phosphogluconate dehydratase) 약화;
(IX) 2-케토-4-하이드록시글루타레이트 KDPG:2-케토-3-디옥시글루코네이트 6-포스페이트 알돌레이즈(2-keto-4-hydroxyglutarate:2-keto-3-deoxygluconate 6-phosphate; KHG/KDPG aldolase) 약화;
(X) 아스파테이트 아미노트랜스퍼라제(aspartate aminotransferase) 약화;
(XI) 글루코스-특이적 PTS 효소 IIBC 컴포넌트(glucose-specific PTS enzyme IIBC component) 약화; 및
(XII) 바이카보네이트 트랜스포터(bicarbonate transporter) 강화.
- 제1항에 있어서, 상기 생산방법은 글리옥실레이트 경로를 더 포함하는 것인, 생산방법.
- 제12항에 있어서, 상기 글리옥실레이트 경로는
(a3) 아이소시트레이트를 글리옥실레이트 및 숙시네이트로 전환하는 단계;
(b3) 글리옥실레이트 및 아세틸-coA를 말레이트 및 coA로 전환하는 단계;
(c3) 시트레이트를 아이소시트레이트로 전환하는 단계;
(d3) 피루베이트를 옥살로아세테이트로 전환하는 단계;
(e3) 포스포에놀피루베이트를 옥살로아세테이트로 전환하는 단계;
(f3) 옥살로아세테이트를 시트레이트로 전환하는 단계;
(g3) 말레이트를 푸마레이트로 전환하는 단계;
(h3) 푸마레이트를 숙시네이트로 전환하는 단계; 및
(i3) 숙시네이트를 숙시닐-coA로 전환하는 단계로 구성되는 군에서 어느 하나 이상을 더 포함하는 것인, 생산방법.
- 제13항에 있어서, (j3) α-케토글루타레이트를 숙시닐-coA로 전환하는 단계가 약화된 것인, 생산방법.
- 제13항에 있어서, (k3) 옥살로아세테이트를 말레이트로 전환하는 단계가 약화된 것인, 생산방법.
- 제13항에 있어서, 상기 글리옥실레이트 경로는 (i) 내지 (v)로 이루어진 군에서 선택되는 어느 하나 이상에 의해 강화된 것인, 생산방법:
(i) 시트레이트 신타아제(citrate synthase) 강화;
(ii) 아이소시트레이트 디하이드로게나제(isocitrate dehydrogenase) 약화;
(iii) 아이소시트레이트 리아제(isocitrate lyase) 강화;
(iv) 아이소시트레이트 디하이드로게나제 키나아제/포스파테이즈(Isocitrate dehydrogenase kinase/phosphatase) 강화;
(v) 말레이트 신타아제 G(malate synthase G) 강화; 및
(vi) 말레이트 신타아제 A(malate synthase A) 강화.
- 숙시네이트 세미알데하이드 디하이드로게나제, 4-하이드록시부티릭산 디하이드로게나제, 4-하이드록시부티릴-coA 트랜스퍼라제, 및 폴리(3-하이드록시알카노에이트) 폴리머라제 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 또는 이들의 조합을 포함하는 미생물.
- 제17항에 있어서, 상기 숙시네이트 세미알데하이드 디하이드로게나제, 4-하이드록시부티릭산 디하이드로게나제, 4-하이드록시부티릴-coA 트랜스퍼라제, 및 폴리(3-하이드록시알카노에이트) 폴리머라제로 구성되는 군에서 선택되는 어느 하나 이상의 폴리펩티드는 외래 도입된 것인, 미생물.
- 제17항에 있어서, 상기 숙시네이트 세미알데하이드 디하이드로게나제 및 4-하이드록시부티릴-coA 트랜스퍼라제 폴리펩티드는 클로스트리듐 클루이베리(Clostridium kluyveri) 유래이고, 상기 4-하이드록시부티릭산 디하이드로게나제 폴리펩티드는 아라비돕시스 탈리아나(Arabidopsis thaliana) 유래이며, 상기 폴리(3-하이드록시알카노에이트) 폴리머라제는 슈도모나스 푸티다(Pseudomonas putida) 또는 랄스토니아 유트로파(Ralstonia eutropha) 유래인 것인, 미생물.
- 제17항에 있어서, 상기 미생물은 TCA 경로를 포함하는 것인, 미생물.
- 제17항에 있어서, 상기 미생물은 피루베이트 디하이드로게나제, 시트레이트 신타아제, 아코니타제, 아이소시트레이트 디하이드로게나제, α-케토글루타레이트 디하이드로게나제, 및 피루베이트 카복실라아제로 이루어지는 군에서 선택되는 어느 하나 이상의 폴리펩티드, 이를 코딩하는 폴리뉴클레오티드, 또는 이들의 조합을 포함하는 것인, 미생물.
- 제17항에 있어서, 상기 미생물은 환원형 TCA 경로를 포함하는 것인, 미생물.
- 제17항에 있어서, 상기 미생물은 하기 (I) 내지 (XII)로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 것인, 미생물:
(I) 피루베이트 키나아제 약화;
(II) 포스포에놀피루베이트 카복실라제 강화;
(III) 카보닉 안하이드라제 강화;
(IV) 시트레이트 신타아제 조절;
(V) 피루베이트 카복실라제 강화;
(VI) NAD+-의존성 말레이트 디하이드로게나제 약화;
(VII) NADP+-의존성 말레이트 디하이드로게나제 약화;
(VIII) 포스포글루코네이트 디하이드라타아제 약화;
(IX) 2-케토-4-하이드록시글루타레이트 KDPG:2-케토-3-디옥시글루코네이트 6-포스페이트 알돌레이즈 약화;
(X) 아스파테이트 아미노트랜스퍼라제 약화;
(XI) 글루코스-특이적 PTS 효소 IIBC 컴포넌트 약화; 및
(XII) 바이카보네이트 트랜스포터 강화.
- 제17항에 있어서, 상기 미생물은 글리옥실레이트 경로를 포함하는 것인, 미생물.
- 제17항에 있어서, 상기 미생물은 하기 (i) 내지 (vi)로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 것인, 미생물:
(i) 시트레이트 신타아제 강화;
(ii) 아이소시트레이트 디하이드로게나제 약화;
(iii) 아이소시트레이트 리아제 강화;
(iv) 아이소시트레이트 디하이드로게나제 키나아제/포스파테이즈 강화;
(v) 말레이트 신타아제 G 강화; 및
(vi) 말레이트 신타아제 A 강화.
- 제17항에 있어서, 상기 미생물은 1,4-부탄다이올 생산에 이용되는 것인, 미생물.
- 제17항에 있어서, 상기 미생물은 폴리-4-하이드록시부티레이트 생산용인 것인, 미생물.
- 제17항에 있어서, 상기 미생물은 코리네박테리움 속 또는 에스케리키아 속인 것인, 미생물.
- 제17항에 있어서, 상기 미생물은 질소, 황, 인, 및 마그네슘으로 구성되는 군에서 선택되는 어느 하나 이상의 영양소 제한 조건에서도 폴리-4-하이드록시부티레이트 생산능이 있는 것인, 미생물.
- 제17항에 있어서, 상기 미생물은 프로모터 활성을 가지는 서열번호 45로 표시되는 뉴클레오티드 서열을 포함하는 미생물.
- 제30항에 있어서, 상기 프로모터 활성을 가지는 서열번호 45로 표시되는 뉴클레오티드 서열의 목적 유전자는 포스포에놀피루베이트 카복실라제를 코딩하는 폴리뉴클레오티드인 것인, 미생물.
- 제17항 내지 제31항 중 어느 한 항의 미생물을 배양하는 단계를 포함하는 폴리-4-하이드록시부티레이트 생산방법.
- 제32항에 있어서, 상기 생산방법은 상기 미생물 또는 배지로부터 폴리-4-하이드록시부티레이트를 회수하는 단계를 포함하는, 폴리-4-하이드록시부티레이트 생산방법.
- 제32항에 있어서, 상기 미생물을 배양하는 단계는 질소, 황, 인, 및 마그네슘으로 구성되는 군에서 선택되는 어느 하나 이상의 영양소를 제한한 배지에서 미생물을 배양하는 단계를 포함하는 것인, 생산방법.
- 제17항 내지 제31항 중 어느 한 항의 미생물을 배양하는 단계;
상기 미생물 또는 배지로부터 폴리-4-하이드록시부티레이트를 회수하는 단계; 및
폴리-4-하이드록시부티레이트를 1,4-부탄다이올로 분해하는 단계를 포함하는 1,4-부탄다이올 생산방법.
- 제35항에 있어서, 상기 폴리-4-하이드록시부티레이트를 1,4-부탄다이올로 분해하는 단계는 열분해, 수소화, 또는 이들의 조합인 것인, 1,4-부탄다이올 생산방법.
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| KR101632642B1 (ko) | 2015-01-29 | 2016-06-22 | 씨제이제일제당 주식회사 | 신규한 프로모터 및 그의 용도 |
| ES2907694T3 (es) | 2016-08-31 | 2022-04-26 | Cj Cheiljedang Corp | Nuevo promotor y uso del mismo |
| KR102278000B1 (ko) | 2020-03-17 | 2021-07-15 | 씨제이제일제당 주식회사 | 프리페네이트 디하이드라타아제 활성 강화를 통한 l-트립토판을 생산하는 방법 |
-
2022
- 2022-06-24 KR KR1020220077824A patent/KR20230000995A/ko unknown
- 2022-06-24 CN CN202280057841.9A patent/CN117916385A/zh active Pending
- 2022-06-24 WO PCT/KR2022/009082 patent/WO2022270991A1/ko active Application Filing
- 2022-06-24 EP EP22828838.7A patent/EP4349994A1/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| WO2022270991A1 (ko) | 2022-12-29 |
| CN117916385A (zh) | 2024-04-19 |
| EP4349994A1 (en) | 2024-04-10 |
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