KR100731525B1 - opcA 유전자를 암호화하는 뉴클레오타이드 서열 - Google Patents
opcA 유전자를 암호화하는 뉴클레오타이드 서열 Download PDFInfo
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- KR100731525B1 KR100731525B1 KR1020017003075A KR20017003075A KR100731525B1 KR 100731525 B1 KR100731525 B1 KR 100731525B1 KR 1020017003075 A KR1020017003075 A KR 1020017003075A KR 20017003075 A KR20017003075 A KR 20017003075A KR 100731525 B1 KR100731525 B1 KR 100731525B1
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- 238000013519 translation Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 108010027345 wheylin-1 peptide Proteins 0.000 description 1
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Abstract
본 발명은 a) 서열 3 또는 서열 5 또는 서열 8 또는 서열 10에 따르는 아미노산 서열중 하나 이상을 포함하는 폴리펩타이드를 암호화하는 폴리뉴클레오타이드와 70% 이상의 정도로 동일한 폴리뉴클레오타이드, b) 서열 3 또는 서열 5 또는 서열 8 또는 서열 10에 따르는 아미노산 서열과 70% 이상의 정도로 동일한 아미노산 서열을 포함하는 폴리펩타이드를 암호화하는 폴리뉴클레오타이드, c) a) 또는 b)의 폴리뉴클레오타이드와 상보적인 폴리뉴클레오타이드, 또는 d) a), b) 또는 c)의 폴리뉴클레오타이드 서열중 15개 이상의 연속적인 뉴클레오타이드를 포함하는 폴리뉴클레오타이드로 이루어진 군으로부터 선택된 폴리뉴클레오타이드중 하나 이상을 포함하는, 코리네형 세균으로부터 분리된 폴리뉴클레오타이드 및, 특히 이미 L-아미노산을 생산하는 코리네형 미생물에서 a) opcA 유전자 외에, tal 유전자, tkt 유전자, zwf 유전자 또는 devB 유전자를 암호화하는 뉴클레오타이드 서열 하나 이상을 증폭시켜, 특히 과발현시키는 단계, b) 목적하는 L-아미노산을 배지중 또는 세균의 세포중에 농축시키는 단계, 및 c) L-아미노산을 분리시키는 단계를 수행함을 특징으로하는, L-아미노산을 발효적으로 제조하는 방법에 관한 것이다.
opcA 유전자, L-아미노산, L-라이신, 코리네형 미생물, 코리네박테리움 글루타미컴, zwf 유전자
Description
본 발명은 opcA 유전자를 암호화하는 뉴클레오타이드 서열, 및 opcA 유전자가 증폭되어 있는 코리네형 세균을 사용하여 아미노산, 특히 L-라이신을 발효적으로 제조하는 방법에 관한 것이다.
아미노산, 특히 L-라이신은 사람 의약 및 제약 산업, 특히 동물 영양 산업에 사용된다.
아미노산은 코리네형 세균, 특히, 코리네박테리움 글루타미컴 (Corynebacterium glutamicum)의 발효에 의해 생산된다. 아미노산의 중요성 때문에, 제조 방법을 개량하기위한 노력이 계속되고 있다. 생산 공정의 개량은 예를들어, 교반 및 산소 공급과 같은 발효 기술 수단, 또는 예를들어, 발효중 당 농도와 같은 영양 배지의 조성, 또는 예를 들어, 이온 교환 크로마토그래피에 의한 생산물 의 후처리, 또는 미생물 자체의 본질적인 생산 특성과 관련될 수 있다.
이들 미생물의 생산 특성은 돌연변이유발, 돌연변이주의 선별 및 선택 방법을 사용하여 개량한다. 이 방법으로 항대사물질에 대해 내성이거나 조절에 중요한 대사물질에 대해 영양요구성이며, 예를들어 L-라이신과 같은 L-아미노산을 생산하는 균주가 생산된다. 몇년간 재조합 DNA 기술을 또한 사용하여 아미노산을 생산하는 코리네박테리움 균주를 개량하였다.
상기 생합성에 있어서 펜토스 포스페이트 사이클의 중요성은 공지되어 있다.
따라서, 오이시(Oishi)와 아이다(Aida) (Agricultural and Biological Chemistry 29, 83-89 (1965))는 이미 브레비박테리움 암모니아게네스 (Brevibacterium ammoniagenes)의 "헥소스 모노포스페이트 폐쇄"에 대해 보고한 바 있다. 스기모토(Sugimoto)와 시오(Shio) (Agricultural and Biological Chemistry 51, 101-108 (1987))는 브레비박테리움 플라붐에 있어서 글루코스 6-포스페이트 데하이드로게나제의 조절에 대해 보고한 바 있다. 스기모토와 시오(Agricultural and Biological Chemistry 51, 1257-11263 (1987))는 브레비박테리움 플라붐에 있어서 글루코스 6-포스페이트 데하이드로게나제의 조절에 대해 보고한 바 있다.
JP-A-09224661에는 브레비박테리움 플라붐(Brevibacterium flavum) MJ-223 (FERM BP-1497)의, zwf로 명명된, 글루코스 6-포스페이트 데하이드로게나제 유전자의 뉴클레오타이드 서열이 기재되어 있다. JP-A-09224661에는 Met Val Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu로서 Zwf 폴리펩타이드의 N-말단 아미노산 서열이 기재되어 있다.
그러나, 이를 확인할 수 없었다.
발명의 목적
본 발명의 목적은 아미노산, 특히 L-라이신의 개량된 발효적 제조 방법의 새 로운 수단을 제공하는 것이다.
아미노산, 특히 L-라이신은 사람 의약, 제약 산업 및 특히 동물 영양 분야에 사용된다. 따라서, 아미노산, 특히 L-라이신의 생산을 위한 신규 개량 방법을 제공하는데 관심이 집중되어 있다.
L-라이신 또는 라이신이 이후 언급되는 경우, 염기 뿐만 아니라, 예를들어, 라이신 모노하이드로클로라이드 또는 라이신 설페이트와 같은 염도 의미하는 것이다.
본 발명은
a) 서열 3 또는 서열 5 또는 서열 8 또는 서열 10에 따르는 아미노산 서열중 하나 이상을 포함하는 폴리펩타이드를 암호화하는 폴리뉴클레오타이드와 70% 이상의 정도로 동일한 폴리뉴클레오타이드,
b) 서열 3 또는 서열 5 또는 서열 8 또는 서열 10에 따르는 아미노산 서열과 70% 이상의 정도로 동일한 아미노산 서열을 포함하는 폴리펩타이드를 암호화하는 폴리뉴클레오타이드,
c) a) 또는 b)의 폴리뉴클레오타이드와 상보적인 폴리뉴클레오타이드, 또는
d) a), b) 또는 c)의 폴리뉴클레오타이드 서열중 15개 이상의 연속적인 뉴클레오타이드를 포함하는 폴리뉴클레오타이드로 이루어진 군으로부터 선택된 폴리뉴클레오타이드 하나 이상을 포함하는, 코리네형 세균으로부터 분리된 폴리뉴클레오 타이드를 제공한다.
본 발명은 또한
(i) 서열 1, 서열 4, 서열 6, 서열 9로 이루어진 군으로부터 선택된 하나 이상의 뉴클레오타이드, 또는
(ii) 유전자 코드의 축퇴성 범위내에서 서열 (i)에 상응하는 하나 이상의 서열, 또는
(iii) 서열 (i) 또는 (ii)에 상보적인 서열과 하이브리드화되는 하나 이상의 서열, 및 임의로
(iv) (i)중 중성적 기능의 센스 돌연변이를 포함하는, 바람직하게 복제할 수 있는 DNA인, 청구의 범위 제1항에 청구된 바와 같은 폴리뉴클레오타이드를 제공한다.
본 발명은 또한
서열 4 또는 서열 9에 나타낸 바와 같은 뉴클레오타이드 서열을 포함하는, 청구의 범위 제4항에 청구된 바와 같은 폴리뉴클레오타이드,
서열 3, 서열 5, 서열 8 또는 서열 10에 나타낸 바와 같은 아미노산 서열중 하나 이상을 포함하는 폴리펩타이드를 암호화하는, 청구의 범위 제6항에 청구된 바와 같은 폴리뉴클레오타이드,
청구의 범위 제1항에 청구된 바와 같은 폴리뉴클레오타이드를 함유하는 벡터, 및
상기 벡터를 함유하는, 숙주 세포로서 작용하는 코리네형 세균을 제공한다.
본 발명은 또한 서열 4 또는 서열 9에 상응하는 폴리뉴클레오타이드 서열을 갖는 완전 유전자를 포함하는 상응하는 유전자 라이브러리를, 서열 4 또는 서열 9에 따르는 상기 언급된 폴리뉴클레오타이드의 서열 또는 이의 단편을 포함하는 프로브로 하이브리드화시킴으로써 선별하고, 상기 언급한 DNA 서열을 분리시킴으로써 수득할 수 있는 폴리뉴클레오타이드 서열을 실질적으로 포함하는 폴리뉴클레오타이드를 제공한다.
본 발명에 따르는 폴리뉴클레오타이드 서열은 OpcA 단백질을 암호화하는 전장의 cDNA를 분리하고 opcA 유전자 서열과 유사성이 높은 서열을 갖는 cDNA 또는 유전자를 분리하기위한 RNA, cDNA 및 DNA에 대한 하이브리드화 프로브로서 적합하다.
본 발명에 따르는 폴리뉴클레오타이드 서열은 또한 폴리머라제 연쇄 반응 (PCR)에 의한 OpcA 단백질을 암호화하는 유전자의 DNA 제조용 프라이머로서 적합하다.
프로브 또는 프라이머로서 제공되는 상기와 같은 올리고뉴클레오타이드는 30개 이상, 바람직하게는 20개 이상, 특히 바람직하게는 15개 이상의 연속적인 뉴클레오타이드를 포함한다. 뉴클레오타이드 길이가 40개 또는 50개인 올리고뉴클레오타이드가 또한 적합하다.
"분리된"은 이의 천연 환경으로부터 분리된 것을 의미한다.
"폴리뉴클레오타이드"는 일반적으로 폴리리보뉴클레오타이드 및 폴리데옥시리보뉴클레오타이드에 관한 것으로, 이들은 비개질된 RNA 또는 DNA 또는 개질된 RNA 또는 DNA일 수 있다.
"폴리펩타이드"는 펩타이드 결합을 통하여 결합된 아미노산 2개 이상을 포함하는 펩타이드 또는 단백질로 이해된다.
본 발명에 따르는 폴리펩타이드는 서열 3, 서열 5, 서열 8 또는 서열 10에 따르는 폴리펩타이드, 특히 OpcA 유전자 생성물의 생물학적 활성을 갖는 것들, 및 서열 3, 서열 5, 서열 8 또는 서열 10에 따르는 폴리펩타이드와 70% 이상의 정도로, 바람직하게는 서열 3, 서열 5, 서열 8 또는 서열 10에 따르는 폴리펩타이드와 80% 이상, 특히 90 내지 95% 이상의 정도로 동일하고, 상기 언급한 활성을 갖는 것들을 포함한다.
본 발명은 또한 글루코스 6-포스페이트 데하이드로게나제의 Zwf 서브-유니트를 형성하는 새로운 Zwf 단백질을 제공한다. 상기 해독 생성물의 아미노산 서열은 서열 2 및 서열 7에 나타낸다. 분리될 수 있는, 글루코스 6-포스페이트 데하이드로게나제의 Zwf 서브-유니트의 N-말단 아미노산 서열은 서열 11에 나타낸다.
본 발명은 또한 특히 이미 아미노산을 생산하며, opcA 유전자를 암호화하는 뉴클레오타이드 서열이 임의로 zwf 유전자와 함께 증폭, 특히 과-발현되어 있는 코리네형 세균을 사용하여, 아미노산, 특히 L-라이신을 발효적으로 제조하는 방법을 제공한다.
이와 관련하여 용어 "증폭"은 예를들어, 유전자 또는 유전자들의 카피수를 증가시키거나 강력한 프로모터를 사용하거나 활성이 높은 상응하는 효소 (단백질)를 암호화하는 유전자를 사용하거나 임의로 이들 수단을 병용함으로써 상응하는 DNA에 의해 암호화되는 미생물중의 효소 (단백질) 1종 이상의 세포내 활성이 증가 되는 것을 의미한다.
본 발명에서 제공되는 미생물은 글루코스, 슈크로스, 락토스, 프럭토스, 말토스, 당밀, 전분, 셀룰로스로부터 또는 글리세롤 및 에탄올로부터 L-아미노산, 특히 L-라이신을 제조할 수 있다. 이들은 대표적인 코리네형 세균, 특히 코리네박테리움 속의 세균일 수 있다. 코리네박테리움 속중에서, 특히 L-아미노산의 생산 능력에 대해 기술자들에게 공지된 코리네박테리움 글루타미컴 종을 언급할 수 있다.
코리네박테리움 속, 특히 코리네박테리움 글루타미컴 종중에서 적합한 균주는 예를들어, 공지된 야생형 균주인,
코리네박테리움 글루타미컴 ATCC13032,
코리네박테리움 아세토글루타미컴(Corynebacterium acetoglutamicum) ATCC15806,
코리네박테리움 아세토악시도필룸(Corynebacterium acetoacidophilum) ATCC13870,
코리네박테리움 써모아미노제네스(Corynebacterium thermoaminogenes) FERM BP-1539,
코리네박테리움 멜라세콜라(Corynebacterium melassecola) ATCC17965,
브레비박테리움 플라붐 ATCC14067,
브레비박테리움 락토퍼멘텀(Brevibacterium lactofermentum) ATCC13869 및
브레비박테리움 디바리카툼(Brevibacterium divaricatum) ATCC14020이고,
이들로부터 수득한 L-라이신 생산 돌연변이체 또는 균주인, 예를들어,
코리네박테리움 글루타미컴 FERM-P 1709,
브레비박테리움 플라붐 FERM-P 1708,
브레비박테리움 락토퍼멘텀 FERM-P 1712,
코리네박테리움 글루타미컴 FERM-P 6463,
코리네박테리움 글루타미컴 FERM-P 6464 및
코리네박테리움 글루타미컴 DSM 5715,
코리네박테리움 글루타미컴 DM58-1,
코리네박테리움 글루타미컴 DSM12866이고
이들로부터 수득한 L-트레오닌 생산 돌연변이체 또는 균주인, 예를들어,
코리네박테리움 글루타미컴 ATCC21649,
브리비박테리움 플라붐 BB69,
브레비박테리움 플라붐 DSM5399,
브레비박테리움 락토퍼멘텀 FERM-BP 269,
브레비박테리움 락토퍼멘텀 TBB-10이고,
이들로부터 수득한 L-이소류신-생산 돌연변이체 또는 균주인, 예를들어
코리네박테리움 글루타미컴 ATCC 14309,
코리네박테리움 글루타미컴 ATCC 14310,
코리네박테리움 글루타미컴 ATCC 14311,
코리네박테리움 글루타미컴 ATCC 15168,
코리네박테리움 암노니아게네스 ATCC 6871이고,
이들로부터 수득한 L-트립토판-생산 돌연변이체 또는 균주인, 예를들어
코리네박테리움 글루타미컴 ATCC 21850 및
코리네박테리움 글루타미컴 KY9218 (pKW9901)이다.
본 발명자들은 효소 글루코스 6-포스페이트 데하이드로게나제 (EC 2.7.1.11)의 OpcA 서브-유니트를 암호화하는 씨. 글루타미컴의 새로운 opcA 유전자를 분리하는데 성공하였다.
씨. 글루타미컴의 opcA 유전자 또는 기타 유전자를 분리하기위하여, 먼저 상기 미생물의 유전자 라이브러리를 이. 콜리중에 확립시킨다. 상기 유전자 라이브러리의 확립은 일반적으로 공지된 교과서 및 핸드북에 설명되어 있다. 예를들어, 다음 문헌을 언급할 수 있다: The textbook by Winnacker: Gene and Klone, Eine Einfuhrung in die Gentechnologie [Genes and Clones, An Introduction to Genetic Engineering] (Verlag Chemie, Weinheim, Germany, 1990) 또는 the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). 널리 공지된 유전자 라이브러리는 코하라 (Kohara) 등에 의해 λ-벡터에 확립된, 이. 콜리 K-12 균주 W3110 (Cell 50, 495-508 (1987))이다. 다른 문헌[참조: Bathe et al, Molecular and General Genetics, 252:255-265, 1996]에는 코스미드 벡터 SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164)의 원조하에 이. 콜리 K-12 균주 NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575)중에 확립된 씨. 글루타미컴 ATCC13032의 유전자 라이브러리가 설명되어 있다. 문헌[참조: Bormann et al., Molecular Microbiology 6(3), 317-326 (1992)]에는 또한 코스미드 pHC97 (Hohn and Collins, Gene 11, 291-298 (1980))을 사용한 씨. 글루타미컴 ATCC13032의 유전자 라이브러리가 설명되어 있다. 또한, 문헌[참조: O'Donohue, The Cloning and Molecular Analysis of Four Common Aromatic Amino Acid Biosynthetic Genes from Corynebacterium glutamicum. Ph.D. Thesis, National University of Ireland, Galway, 1997]에는 문헌[참조: Short et al., Nucleic Acids Research, 16:7583]에 기재된 λ Zap 발현 시스템을 사용한 씨. 글루타미컴 유전자의 클로닝이 설명되어 있다. 이. 콜리중에서 씨. 글루타미컴의 유전자 라이브러리를 제조하기 위하여, pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) 또는 pUC9 (Vieira et al., 1982, Gene, 19:259-268)과 같은 플라스미드를 또한 사용할 수 있다. 적합한 숙주는 특히 제한- 및 재조합-결함성인 이. 콜리 균주이다. 이들의 예는 균주 DH5αmcr로, 이는 문헌[참조: Grant et al.: Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649)]에 설명되어 있다. 이후, 코스미드의 도움으로 클로닝된 긴 DNA 단편을 서브-클로닝할 수 있고 이어서 예를 들어, 문헌[참조: Sanger et al: Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977)]에 기술된 바와 같이 서열 분석에 적합한 통상의 벡터중에서 서열분석될 수 있다.
이어서 상기 수득한 DNA 서열을 예를들어, 스타덴(Staden)의 프로그램(Nucleic Acids Research 14, 217-232 (1986)), 버틀러(Butler)의 GCG 프로그램 (Methods of Biochemical Analysis 39, 74-97 (1998), 피어슨(Pearson)과 림프만(Lipman)의 FASTA 알고리즘 (Proceedings of the National Academy of Sciences USA 85, 2444-2448 (1988)) 또는 알트슐(Altschul) 등의 BLAST의 알고리즘 (Nature Genetics 6, 119-129 (1994))과 같은 공지된 알고리즘 및/또는 서열 분석 프로그램으로 조사하고 공개적으로 입수할 수 있는 데이타 뱅크에 기재된 서열 엔트리와 비교할 수 있다. 뉴클레오타이드 서열에 대해 공개적으로 입수할 수 있는 데이타뱅크의 예로는 기관[European Molecular Biologies Laboratories (EMBL, Heidelberg, Germay)]의 데이터뱅크 또는 기관[National Center for Bioltechnology Information (NCBI, Bethesda, MD, USA)]의 데이터 뱅크가 있다.
본 발명은 opcA 유전자를 암호화하며 서열 1 및 서열 4로서 본 발명을 구성하는 씨. 글루타미컴의 신규 DNA 서열을 제공한다. 상응하는 단백질의 아미노산 서열이 또한 상기한 방법에 의해 본 DMA 서열로부터 유래되었다. OpcA 유전자 생성물에 대해 수득한 아미노산 서열은 서열 3 및 서열 5에 나타낸다. OpcA 유전자 생성물의 아미노산 서열로부터 분석된 분자량은 대략 34.7 킬로 달톤이다.
서열 1은 또한 zwf 유전자의 암호화 영역을 나타낸다. Zwf 유전자 생성물의 수득된 아미노산 서열은 서열 2에 나타낸다. Zwf 유전자 생성물의 아미노산 서열로부터 분석된 분자량은 대략 57.5 킬로 달톤이다.
상기한 방식으로 생성된 유전자 라이브러리는 또한 예를들어, zwf 유전자 (JP-A-09224661)와 같은 공지된 서열의 뉴클레오타이드 프로브와 하이브리드화시킴으로써 조사할 수 있다. 하이브리드화에서 포지티브 반응을 나타내는 클론의 클로닝된 DNA를 또한 서열분석하면 한편으로는 상기 사용된 프로브의 공지된 뉴클레오타이드 서열을 제공하고 다른 한편으로는 인접한 새로운 DNA 서열을 제공한다.
본 발명은 또한 opcA 유전자를 암호하하며 서열 6 및 서열 9로서 본 발명을 구성하는 씨. 글루타미컴의 신규 DNA 서열을 제공한다. 상응하는 단백질의 아미노산 서열이 또한 상기한 방법으로 본 DNA 서열로부터 유래되었다. OpcA 유전자 생성물의 수득된 아미노산 서열은 서열 8 및 서열 10에 나타낸다. OpcA 유전자 생성물의 아미노산 서열로부터 분석된 분자량은 대략 34.7 킬로 달톤이다.
서열 6은 또한 zwf 유전자의 암호화 영역을 나타낸다. Zwf 유전자 생성물의 수득된 아미노산 서열은 서열 7에 나타낸다. Zwf 유전자 생성물의 아미노산 서열로부터 분석된 분자량은 대략 57.5 킬로 달톤이다.
OpcA 단백질 및 Zwf 단백질의 아미노산 서열을 적어도 부분적으로 결정하기위한 다른 공정은 크로마토그래피법에 의해 글루코스 6-포스페이트 데하이드로게나제 효소 단백질을 균질하게 정제하는 것이다. 단백질 정제 및 제조에 대한 방법 및 지침은 예를들어 문헌[참조: the textbook by Schleifer and Wensink: Practical Methods in Molecular Biology(Springer Verlag, Berlin, Germany, 1981), the Handbook by Harris and Angal: Protein Purification Methods: A Practical Approach (IRL Press, Oxford, UK, 1989), the Textbook by Scopes: Protein Purification: Principles and Practice, 3rd ed. (Springer Verlag, New York, USA, 1993)] 및 일반적으로 공지된 교과서 및 핸드북에 설명되어 있다. 정제된 폴리펩타이드의 N-말단 아미노산 서열은 에드만(Edman) (Archives of Biochemistry 22, 475 (1949))에 의해 기술된 N-말단 서열분석 방법으로 결정할 수 있다. 단백질 서열분석을 위한 추가의 방법 및 지침은 예를들어 문헌[참조: Smith, Protein Sequencing Protocolls: Methods in Molecular Biology, Vol. 64 및 Vol. 112 (Humana Press, Totowa, NJ, USA, 1996) 및 Kamp et al.: Protein Structure Analysis: Preparation, Characterization, and Microsequencing (Springer Verlag, New York, NY, USA, 1997)]에 설명되어 있다.
이 방법으로 효소 글루코스 6-포스페이트 데하이드로게나제가 각 경우 분자량이 대략 30 kDa 및 대략 60 kDa인 2개의 서브-유니트로 이루어져 있음을 밝힐 수 있다. OpcA 서브-유니트 및 OpcA 단백질의 N-말단 아미노산 서열은 서열 12에 나타낸다. Zwf 서브-유니트 및 Zwf 단백질의 N-말단 아미노산 서열은 서열 11에 나타낸다.
유전자 코드의 축퇴성에 의해 서열 1, 서열 4, 서열 6 또는 서열 9로부터 비롯된 암호화 DNA 서열이 또한 본 발명을 구성한다. 동일한 방법으로, 서열 4 또는 서열 9 또는 서열 4의 일부 또는 서열 9의 일부와 하이브리드화되는 DNA 서열이 본 발명을 구성한다. 보존적 아미노산 치환, 예를들어, 단백질중 글라이신의 알라닌으로의 치환 또는 아스파르트산으로의 글루탐산으로의 치환이 또한 기술자들사이에서는 "센스 돌연변이"로 공지되어 있는데, 이는 단백질의 활성에 있어서 기본적인 변화를 일으키지 않는, 즉, 기능적으로 변함이 없다. 추가로 단백질의 N 및/또는 C 말단상에서의 변화는 이의 기능을 실질적으로 손상시킬 수 없거나 오히려 안정화시킬 수 있는 것으로 공지되어 있다. 이에 대한 정보는 다음 문헌[참조: Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), O'Regan et al. (Gene 77:237-251 (1989)), Sahin-Toch et al. (Protein Sciences 3:240-247 (1994)), Hochuli et al. (Bio/Technology 6:1321-1325 (1988))] 및 유전학 및 분자생물학의 공지된 교과서에 기재되어 있다. 대응하는 방법으로 서열 3, 서열 5, 서열 8 또는 서열 10으로부터 비롯된 아미노산 서열이 또한 본 발명을 구성한다.
최종적으로, 서열 4 또는 서열 9로부터 비롯된 프라이머를 사용한 폴리머라제 연쇄 반응 (PCR)에 의해 제조되는 DNA 서열이 본 발명을 구성한다. 상기와 같은 올리고뉴클레오타이드는 전형적으로 길이가 뉴클레오타이드 15개 이상이다.
당해 분야의 기술자들은 문헌[참조: the handbook "The DIG System User's Guide for Filter Hybridisation" from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) 및 Liebl et al. International Journal of Systematic Bacteriology (1991) 41:255-260]에서 하이브리드화에 의한 DNA 서열 확인에 대한 지침을 발견할 수 있다. 당해 분야의 숙련가들은 폴리머라제 연쇄 반응 (PCR)을 사용한 DNA 서열 증폭에 대한 지침을 문헌[참조: the handbook by Gait, Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) 및 Newton & Graham, PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994)]으로부터 발견할 수 있다.
본 발명자들은 opcA 유전자가, 임의로 zwf 유전자와 함께 과-발현된 후, 개선된 방식으로, 코리네형 세균이 아미노산, 특히 L-라이신을 생산한다는 것을 밝혔다.
과-발현시키기 위해, 상응하는 유전자의 카피수를 증가시킬 수 있거나, 구조 유전자의 프로모터 및 조절 영역 또는 리보솜 결합 부위 상류를 돌연변이시킬 수 있다. 구조 유전자의 상류가 도입된 발현 카세트가 동일하게 작용한다. 유도성 프로모터에 의해, 발효적 L-라이신 생산 과정에 있어서 발현을 추가로 증가시킬 수 있다. 발현은 또한 m-RNA의 수명을 연장시키는 방법에 의해 향상될 수 있다. 또한, 효소 단백질의 분해를 방지함으로써 효소 활성을 증가시킨다. 유전자 또는 유전자 작제물은 다양한 카피수의 플라스미드에 존재하거나, 염색체중에 통합되어 증폭될 수 있다. 달리, 배지의 조성 및 배양 공정을 변화시켜 당해 유전자를 또한 과-발현시킬 수 있다.
당해 분야의 기술자들은 이에 대한 지침을 문헌[참조: Martin et al. (Bio/Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), Eikmanns et al. (Gene 102, 93-98 (1991))유럽 특허 명세서 EPS 0 472 869, 미국 특허 제4,601,893호, Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991)), Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), 특허원 WO 96/15246, Malumbres et al. (Gene 134, 15-24 (1993)), 일본 공개 공보 JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), Makrides (Microbiological Reviews 60:512-538 (1996))] 및 유전학 및 분자생물학의 공지된 교재으로부터 발견할 수 있다,.
일례로, 본 발명에 따르는 opcA 유전자를 플라스미드를 사용하여 과-발현시킨다.
적합한 플라스미드는 코리네형 세균에서 복제되는 플라스미드이다. 수많은 공지된 플라스미드 벡터, 예를들면, pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) 또는 pHS2-1 (Sonnen et al., Gene 107:69-74 (1991))은 크립틱(cryptic) 플라스미드 pHM1519, pBL1 또는 pGA1를 기본으로 한다. 다른 플라스미드 벡터, 예를들어, pCG4 (US-A 4,489,160), 또는 pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), 또는 pAG1 (US-A 5,158,891)을 기본으로하는 것들을 동일한 방법으로 사용할 수 있다.
예를 들어, 도 2에 나타낸 이. 콜리 - 씨. 글루타미컴 셔틀 벡터 pEC-T18mob2를 사용한다. opcA 유전자와 zwf 유전자를 pEC-T18mob2의 SphI/SalI 절단 부위 영역중으로 도입시킨 후, 도 3에 나타낸 플라스미드 pECzwfopcA를 형성시킨다.
또한 추가로 적합한 플라스미드 벡터는 문헌[참조: Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994))]에 설명된 바와 같이 hom-thrB 오페론의 복제 또는 증폭시키기 위해 염색체중으로의 통합에 의한 유전자 증폭 과정에 의해 사용될 수 있는 것들이다. 이 방법으로, 완전한 유전자가 숙주 (전형적으로 이. 콜리)에서 복제될 수 있지만, 씨. 글루타미컴에서는 복제되지 않는 플라스미드 벡터중에 클로닝된다. 가능한 벡터의 예는 다음과 같다: pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob 또는 pK19mob (Schafer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; US-A 5,487,993), pCRRBlunt (Intitrogen, Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234:534-541 (1993)), pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173:4510-4516) 또는 pBGS8 (Spratt et al., 1986, Gene 41:337-342). 이어서 증폭시킬 유전자를 함유하는 플라스미드 벡터를 접합 또는 형질전환법에 의해 목적하는 씨. 글루타미컴 균주중으로 이동시킨다. 접합 방법은 예를들면, 문헌[참조: Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994))]에 기재되어 있다. 형질전환법은 예를들어, 문헌[참조: Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) 및 Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994))]에 기재되어 있다. "교차 (cross-over)"에 의해 상동성 재조합시킨 후, 생성된 균주는 당해 유전자를 2카피수 이상으로 함유한다.
또한, 아미노산, 특히 L-아미노산의 생산을 위해, opcA 유전자 이외에, 임의로 zwf 유전자와 함께, 특정 생합성 경로, 해당작용, 보충작용(anaplerotic), 펜토스 포스페이트 경로 또는 아미노산 수송에 관여하는 효소 1종 이상을 증폭시키거나 과-발현시키는 것이 유리할 수 있다.
따라서, 예를들어, L-라이신의 제조의 경우, 다음군으로 이루어진 군으로부터 선택된 유전자 1종 이상을 동시에 증폭, 특히 과-발현시키는 것이 유리할 수 있다:
● 디하이드로디피콜리네이트 신타제를 암호화하는 dapA 유전자 (EP-B 0 197 335),
● 피드백 내성 아스파르테이트 키나제를 암호화하는 lysC 유전자 (Kalinowski et al. (1990), Molecular and General Genetics 224:317-324),
● 글리세롤알데하이드 3-포스페이트 데하이드로게나제를 암호화하는 gap 유전자 (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
● 피루베이트 카복실라제를 암호화하는 pyc 유전자 (DE-A-198 31 609),
● 기관[European Molecular Biology Laboratories](EMBL, Heidelberg, Germany)의 데이타뱅크의 트랜스케톨라제를 암호화하는 tkt 유전자 (수탁번호 AB023377),
● 6-포스포글루코네이트 데하이드로게나제를 암호화하는 gnd 유전자 (JP-A-9-224662),
● 라이신 수송체를 암호화하는 lysE 유전자 (DE-A-195 48 222),
● zwal 유전자 (DE 199 59 328.0; DSM 13115), 또는
● 에놀라제를 암호화하는 eno 유전자 (DE: 199 41 478.5),
● 트랜스알돌라제를 암호화하는 tal 유전자 (DSM 13263).
또한, 아미노산, 특히 L-라이신의 생산을 위해, 임의로 zwf 유전자와 조합된 opcA 유전자를 증폭시키는 것 외에,
● 포스포에놀 피루베이트 카복시키나제를 암호화하는 pck 유전자 (DE 199 50 409.1 DSM 13047) 및/또는
● 글루코스 6-포스페이트 이소머라제를 암호화하는 pgi 유전자 (US 09/396,478, DSM 12969), 또는
● 피루베이트 옥시다제를 암호화하는 poxB 유전자 (DE 199 51 975.7; DSM 13114), 또는
● zwa2 유전자 (DE: 199 59 327.2; DSM 13113)을 동시에 감쇠시키는 것이 유리할 수 있다.
opcA 유전자를 과-발현시키는 것 외에 아미노산, 특히 L-라이신을 생산하기 위해, 바람직하지못한 부반응을 제거하는 것이 또한 유리할 수 있다 (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
본 발명에 따라 제조되는 미생물은 L-아미노산, 특히 L-라이신의 생산을 목적으로 연속적으로 또는 배치 공정(배치 배양) 또는 유가식 공정 (공급 공정) 또는 반복적인 유가식 공정(반복적 공급 공정)과 같이 비연속식으로 배양할 수 있다. 공지된 배양법에 대한 요약은 문헌[참조: Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) 또는 the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and Peripheral Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994))]에 설명되어 있다.
사용되는 배양 배지는 반드시 적합한 방식으로 특정 균주의 요구조건을 충족시켜야 한다. 다양한 미생물에 대한 배양 배지의 설명은 문헌[참조: the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981)]에 있다.
당 및 탄수화물 (예를들면, 글루코스, 슈크로스, 락토스, 프럭토스, 말토스, 당밀, 전분 및 셀룰로스), 오일 및 지방 (예를들면, 대두유, 해바라기유, 땅콩유 및 코코넛유), 지방산 (예를들면, 팔미트산, 스테아르산 및 리놀레산), 알코올 (예를들면, 글리세롤 및 에탄올), 및 유기산 (예를들면, 아세트산)이 탄소원으로서 사용될 수 있다. 이들 물질은 개별적으로 또는 혼합물로서 사용될 수 있다. 유기 질소-함유 화합물 (예를들면, 펩톤, 효모 추출액, 육즙, 맥아 추출액, 옥수수 침지액, 대두박분 및 우레아), 또는 무기 화합물 (예를들면, 황산암모늄, 염화암모늄, 인산암모늄, 탄산암모늄 및 질산암모늄)이 질소원으로서 사용될 수 있다. 질소원은 개별적으로 또는 혼합물로서 사용될 수 있다. 인산, 인산이수소칼륨 또는 인산수소이칼륨, 또는 상응하는 나트륨-함유염이 인 공급원으로서 사용될 수 있다. 배양 배지는 또한 성장에 필수적인 금속염, 예를들면 황산마그네슘 또는 황화철을 함유해야만 한다. 최종적으로, 상기 언급한 물질외에, 아미노산 및 비타민과 같은 필수 성장 물질이 사용될 수 있다. 이와는 별도로, 적합한 전구체를 배양 배지에 가할 수 있다. 상기 언급한 출발 물질은 1회분 형태로 배양물에 가할 수 있거나, 배양중 적절한 방식으로 공급할 수 있다.
배지의 pH를 조절하기위하여 염기성 화합물 (예, 수산화나트륨, 수산화칼륨, 암모니아 또는 암모니아수) 또는 산성 화합물 (예, 인산 또는 황산)을 적합한 방식으로 사용할 수 있다. 예를들어, 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 거품 형성을 방지할 수 있다. 예를들어, 항생제와 같은 적합한 선택적 작용을 갖는 물질을 배지에 가하여 플라스미드의 안정성을 유지할 수 있다. 산소 또는 예를들어, 공기와 같은 산소-함유 가스 혼합물을 배지에 도입하여 호기성 조건을 유지한다. 배양 온도는 통상 20 내지 45 ℃이며 바람직하게는 25 내지 40 ℃이다. 최대의 L-아미노산이 형성될 때 까지 배양을 계속한다. 이것은 통상 10 시간 내지 160 시간내에 성취된다.
L-아미노산 분석은 문헌[참조: Spackman et al., Analytical Chemistry, 30, (1958), 1190]에 설명된 바와 같이 음이온 교환 크로마토그래피에 이어서 닌하이드린 유도체화에 의해 수행할 수 있다.
다음 미생물이 부다페스트 조약에 따라서 기관[Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany)]에 기탁되었다:
● DSM 13264로서 코리네박테리움 글루타미컴 ATCC13032/pECzwfopcA.
서열 1은 또한 신규 devB 유전자를 함유한다. 아미노산, 특히 L-라이신의 발효적 제조를 위하여 본 발명에 따르는 방법을 사용한다.
첨부된 서열:
다음 서열이 서열 프로토콜의 형태로 첨부된다:
서열 : 설명
1 : 코리네박테리움 글루타미컴 ATCC13032로부터 분리된 DNA 서열
2 : 서열 1로부터 유래된 Zwf 단백질의 아미노산 서열
3 : 서열 1로부터 유래된 OpcA 단백질의 아미노산 서열
4 : 서열 1로부터 획득한 ATCC13032의 opcA 유전자의 DNA 서열
5 : 서열 4로부터 유래된 OpcA 단백질의 아미노산 서열
6 : 코리네박테리움 글루타미컴 ASO19로부터 분리된 DNA 서열
7 : 서열 6으로부터 유래된 Zwf 단백질의 아미노산 서열
8 : 서열 6으로부터 유래된 OpcA 단백질의 아미노산 서열
9 : 서열 6로부터 획득한 ASO19의 opcA 유전자의 DNA 서열
10 : 서열 9로부터 유래된 OpcA 단백질의 아미노산 서열
11 : 분리될 수 있는 ATCC13032로부터 글루코스 6-포스페이트 데하이드로게나제의 Zwf 단백질의 N-말단의 아미노산 서열
12 : ATCC13032로부터, 분리될 수 있는, 글루코스 6-포스페이트 데하이드로게나제의 OpcA 단백질의 N-말단의 아미노산 서열
하기의 도면이 첨부된다:
도 1: 플라스미드 pBOB102의 지도
도 2: 플라스미드 pEC-T18mob2의 지도
도 3: 플라스미드 pECzwfopcA의 지도
사용되는 약어는 다음과 같은 의미를 갖는다:
도 1:
Neo r: 네오마이신/카나마이신 내성
ColEl ori: 플라스미드 ColE1의 복제 오리진
CMV: 사이토메갈로바이러스 프로모터
lacP: lac 오페론의 프로모터
lacZ: β-갈락토시다제 유전자의 5'-말단 (lacZa 유전자 단편)
SV40 3' 스플라이스: 시미안 바이러스 40의 3' 스플라이스 부위
SV40 폴리A: 시미안 바이러스 40의 폴리아데닐화 부위
fl(-)ori: 필라멘터스파아지 f1의 복제 오리진
SV40 ori: 시미안 바이러스 40의 복제 오리진
도 2 및 3:
Tet: 테트라사이클린에 대해 내성인 유전자
oriV: 이. 콜리의 플라스미드-암호화된 복제 오리진
RP4mob: 플라스미드를 이동시키기위한 mob 영역
rep: 씨. 글루타미컴 플라스미드 pGA1으로부터의 플라스미드-암호화된 복제 오리진
per: PGA1으로부터의 카피수를 조절하기위한 유전자
lacZ-알파: β-갈락토시다제 유전자의 lacZα 유전자 단편 (N-말단)
lacZ알파': lacZα 유전자 단편의 5'-말단
'lacZ알파: lacZα 유전자 단편의 3'-말단
zwf: zwf 유전자
opcA: opcA 유전자
추가의 약어:
ApaI: 제한 효소 ApaI의 절단 부위
BamHI: 제한 효소 BamHI의 절단 부위
ClaI: 제한 효소 ClaI의 절단 부위
EcoRI: 제한 효소 EcoRI의 절단 부위
HindIII: 제한 효소 HindIII의 절단 부위
MstII: 제한 효소 MstII의 절단 부위
NheI: 제한 효소 NheI의 절단 부위
NsiI: 제한 효소 NsiI의 절단 부위
SacI: 제한 효소 SacI의 절단 부위
SalI: 제한 효소 SalI의 절단 부위
SpeI: 제한 효소 SpeI의 절단 부위
SphI: 제한 효소 SphI의 절단 부위
SspI: 제한 효소 SspI의 절단 부위
XbaI: 제한 효소 XbaI의 절단 부위
다음 실시예는 본 발명을 추가로 설명한다. (달리 언급되지 않는한) 문헌[참조: Sambrook et al., (Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbour Laboratories, USA)]에 기술된 분자생물학 기술, 예를들면, 플라스 미드 DNA 분리, 제한 효소 처리, 연결, 이. 콜리의 표준 형질전환법 등을 사용한다.
실시예 1
코리네박테리움 글루타미컴 균주 ASO19의 유전자 라이브러리의 작제
코리네박테리움 글루타미컴 균주 ASO19의 DNA 라이브러리(Yoshihama et al., Journal of Bacteriology 162, 591-597 (1985))를 λ Zap 발현™시스템 (Short et al., (1988) Nucleic Acids Research, 16:7583-7600)을 사용하여 문헌[참조: O'Donohue (O'Donohue, M. (1997). The Cloning and Molecular Analysis of Four Common Aromatic Amino Acid Biosynthetic Genes from Corynebacterium glutamicum. Ph D. Thesis, National University of Ireland, Galway.)]에 설명된 바와 같이 작제한다. λ Zap 발현™키트를 Stratagene (Stratagene, 11011 North Torrey Pines Rd., La Jolla, California 92037.)으로부터 구입하여 제조업자의 지침에 따라 사용한다. ASO19-DNA를 제한 효소 Sau3A로 분해시켜 BamHI 처리되고 탈인산화된 λ Zap 발현™ 암에 연결시킨다.
실시예 2
opcA 및 zwf 유전자의 클로닝 및 서열분석
1. zwf 프로브의 작제
zwf 유전자 내부의, 방사성표지된 올리고뉴클레오타이드를 사용하여 상기한 ASO19 λ Zap 발현™ 라이브러리를 탐지한다. zwf 유전자 내부의 축퇴성 PCR 프라이머를 사용하여 상기 올리고뉴클레오타이드를 생산한다. zwf DNA 단편의 PCR 증폭용으로 디자인된 상기 축퇴성 뉴클레오타이드 프라이머는 다음과 같다:
zwf1: 5' ATY GAY CAC TAY YTS GGY AAR GA 3'
zwf2: 5' RAA WGG MAC RCC YKS CCA 3'
여기서 R=A+G; Y=C+T; W=A+T; M=A+C; S=G+C; K=T+G.
생성된 PCR 생성물의 평가된 크기는 대략 480 bp이다.
최적의 PCR 조건은 다음과 같이 결정되었다:
35 사이클
94 ℃ 1분
60 ℃ 1분
72 ℃ 30초
2.5-3.5 mM MgCl2
100-150 ng ASO19 게놈성 DNA
생성된 PCR 생성물의 서열 분석 결과 상기 생성물은 zwf 유전자의 내부 영역인 것으로 확인되었다. 서열 분석은 통상적인 정배향 및 역배향 프라이머, 및 제조회사[Pharmacia Biotech (St. Albans, Herts, UK)]로부터의 T7 서열분석 키트를 사용하여 수행한다.
2. 클로닝
ASO19 λ Zap 발현™ 라이브러리의 서열분석은 λ Zap 발현™ 시스템 프로토콜 (Stratagene, 11011 North Torrey Pines Rd., La Jolla, California 92037.)에 따라서 수행한다. 이어서 써던 블롯 분석을 분리된 클론에 대해 수행한다. DNA의 써던 전달은 문헌[참조: Schleicher and Schuell protocols manual]에 기재된 바와 같이 막으로서 Nytran™ ("Nytran, Modified Nylon-66 Membrane Filters" (March 1987), Schleicher and Schuell, Dassel, Germany)을 사용한다. 상기한 바와 동일한 프라이머와 최적의 PCR 조건을 사용하여 합성된 이본쇄 DNA 단편을 제조회사[Amersham Life Science (Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, UK)]로부터의 멀티프라임(Multiprime)™ DNA 표지화 키트를 사용하여 제조업자의 지침에 따라서 α-32P-dCTP로 방사선 표지시킨다. 예비 하이브리드화, 하이브리드화 및 세척 조건은 문헌[참조: Schleicher and Schuell protocols manual]에 기재되어 있다. 문헌[참조: the Handbook of Sambrook et al.]에 개략되어 있는 공정에 따라서 AgFa Curix RPIL 필름을 사용하여 자가방사선사진촬영을 수행한다. 따라서 수개의 zwf 클론이 확인되었다. 플라스미드 DNA를 상기 클론중 하나로부터 분리시켜 pBOB102로 명명하고 (도 1) 추가 분석을 위하여 선택한다.
3. 서열분석
생거(Sanger et al.)의 생거 디데옥시 쇄 종결법 (Proceedings of the National Academy of Sciences USA 74, 5463-5467 (1977))을 사용하여 pBOB102의 클로닝된 삽입체를 서열분석한다. 제조회사[Pharmacia Biotech (St. Albans, Herts, UK)]로부터 T7 서열분석 키트 및 α-35S-dCTP를 사용하는 방법을 적용한다. 샘플을 3 내지 8시간 동안 TBE 완충액중 6% 폴리아크릴아미드/우레아 겔상에서 50 mA의 일정한 전류로, 파마시아 클로닝 및 서열분석 지침 매뉴얼 ("T7 서열분석™ Kit", ref. XY-010-00-19, Pharmacia Biotech, 1994)에 따라서 전기영동한다. 초기 서열 분석은 제조회사[Pharmacia Biotech]로부터 구입한 통상적인 정배향 및 M13 역배향 프라이머를 사용하여 수행한다:
통상적인 정배향 프라이머: 5' GTA ATA CGA CTC ACT ATA GGG C 3'
M13 역배향 프라이머: 5' GGA AAC AGC TAT GAC CAT G 3'
이어서 수득한 서열로부터 내부 프라이머를 디자인하는데, 이는 전체 opcA 유전자를 추론할 수 있도록한다. 내부 프라이머의 서열은 다음과 같다:
내부 프라이머 1: 5' TCA ACC CTG AGT CCA CC 3'
내부 프라이머 2: 5' CTG ACC ACG AGC GGA GG 3'
내부 프라이머 3: 5' ATG GTG ATC TGG ACG TG 3'
내부 프라이머 4: 5' CTG GCG ACT TGG CTC GA 3'
내부 프라이머 5: 5' CTT CCG GAT ACC ACC ACC 3'
이어서 수득한 서열을 DNA Strider 프로그램 [Marck (1988), Nucleic Acids Research 16: 182901836), version 1.0]을 사용하여 애플 맥킨토시 컴퓨터상에서 분석한다. 이 프로그램은 제한 부위 사용, 개방 판독 프레임 분석 및 코돈 사용 결정과 같은 분석을 가능하게 한다. 수득한 DNA 서열과 EMBL 및 Genbank 데이타베이스의 DNA 서열간의 조사는 BLAST 프로그램(Altschul et al., (1997) Nucleic Acids Research, 25: 3389-3402)을 사용하여 수행한다. DNA와 단백질 서열은 Clustal V 및 Clustal W 프로그램 (Higgins and Sharp, 1988 Gene 73:237-244)을 사용하여 정렬한다.
상기 수득한 서열을 서열 6에 나타낸다. 수득한 뉴클레오타이드 서열의 분석으로 957개 염기쌍의 개방 판독 프레임이 밝혀졌으며 이는 opcA 유전자로 명명된다. 이는 서열 8 및 서열 10에 나타낸 319개 아미노산의 단백질을 암호화한다. zwf 유전자의 암호화 영역을 또한 서열 6에 나타낸다. 514개 아미노산으로 구성된 Zwf-단백질의 아미노산 서열을 서열 7에 나타낸다.
실시예 3
코리네박테리움 글루타미컴 ATCC 13032로부터 게놈성 코스미드 유전자 라이브러리의 제조
코리네박테리움 글루타미컴 ATCC 13032로부터 염색체 DNA를 문헌 (Tauch et al., 1995, Plasmid 33:168-179)에 기재된 바와 같이 분리하여 제한 효소 Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02)로 부분적으로 절단한다. 상기 DNA 단편을 새우 알칼린 포스파타제 (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250)으로 탈인산화시킨다. Stratagene (La Jolla, USA, Product Description SuperCos Vector Kit, Code no. 251301)로부터 입수한, 코스미드 벡터 SuperCos1 DNA(Wahl et al. (1987) Proceedings of the National Academy of Sciences, USA 84:2160-2164)를 제한 효소 XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Code no. 27-0948-02)로 절단하고 새우 알칼린 포스파타제로 마찬가지로 탈인산화시킨다. 이어서 코스미드-DNA를 제한 효소 BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04)로 절단한다. 이 방법으로 처리한 코스미드-DNA를 상기 처리한 ATCC13032-DNA와 혼합하고 배치를 T4 DNA 리가제[Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code No. 27-0870-04]로 처리한다. 기가팩(Gigapack) II XL 팩킹 추출액 (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217)을 사용하여 파아지중에 연결 혼합물을 팩킹시킨다. 이. 콜리 균주 NM554 (Raleigh et al. 1988, Nucleic Acid Research. 16:1563-1575)를 감염시키기위하여 상기 세포를 10 mM MgSO4 중에 넣고 상기 파아지 현탁액 분취량과 혼합한다. 코스미드 라이브러리의 감염 및 적정은 문헌[참조: Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor]에 기재된 바와 같이 수행하고, 암피실린 100 ㎍/㎖를 함유하는 LB-한천 (Lennox, 1955, Virology, 1:190)상에 세포를 도말한다. 37 ℃에서 밤새 배양시킨 후 각각의 재조합 클론을 선택한다.
실시예 4
ATCC 13032의 opcA 및 zwf 유전자의 분리 및 서열분석
각각의 콜로니의 코스미드 DNA를 퀴아프렙 스핀 미니프렙 키트(Qiaprep Spin Miniprep Kit) (Product No. 27106, Qiagen, Hilden, Germany)를 사용하여 제조업자의 지침에 따라서 분리하고 제한 효소 Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sua3AI, Product No. 27-0913-02)로 부분적으로 절단한다. DNA 단편을 새우 알칼린 포스파타제 (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250)로 탈인산화시킨다. 겔 전기영동하여 분리시킨 후 크기 범위가 1500 내지 2000 bp인 코스미드 단편을 QiaExII 겔 추출 키트 (Product No. 20021, Qiagen, Hilden, Germany)를 사용하여 분리한다. 인비트로겐(Invitrogen)로부터 입수한 서열분석 벡터 pZero-1(Groningen, Holland, Product Description Zero Background Cloning Kit, Product No. K2500-01)의 DNA를 제한 효소 BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04)로 절단한다. 서열분석 벡터 pZero-1중에 상기 코스미드 단편을 문헌[참조: Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor]에 기재된 바와 같이 연결시키고, DNA 혼합물은 T4-리가제 (Pharmacia Biotech, Freibrug, Germany)로 밤새 항온처리 한다. 상기 연결 혼합물을 이. 콜리 균주 DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649)중으로 전기천공시키고 (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) 제오신 50 ㎍/㎖를 함유하는 LB-한천 (Lennox, 1955, Virology, 1:190)상에 도말한다. 재조합 클론의 플라스미드는 Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany)으로 제조한다. 서열분석은 짐머만 등 (Zimmermann et al. 1990, Nucleic Acids Research, 18:1067)에 따른 변법으로 생거 등의 디데옥시 쇄 종결법 (Sanger et al., 1977, Proceedings of the National Academies of Sciences U.S.A., 74:5463-5467)에 따라 수행한다. 제조회사[PE Applied Biosystems]로부터의 RR 로다민 터미네이터 사이클 서열분석 키트(RR dRhodamin Terminator Cycle Sequencing Kit) (Product No. 403044, Weiterstadt, Germany)를 사용한다. 겔 전기영동에 의한 분리 및 서열분석 반응의 분석은 "로티포레시스(Rotiphoresis) NF 아크릴아미드/비스아크릴아미드" 겔 (29:1) (Product No. A124.1, Roth, Karlsruhe, Germany)중에서 제조회사[PE Applied Biosystems (Weiterstadt, Germany)]로부터의 "ABI Prism 377" 서열분석기를 사용하여 수행한다.
이어서 수득한 초기 서열 데이타를 스타덴(Staden) 프로그램 팩키지 (1986, Nucleic Acids Research, 14:217-231) 버젼 97-0를 사용하여 프로세싱한다. pZero1 유도체의 각각의 서열을 연속 콘티그중으로 어셈블링한다. 암호화 영역의 컴퓨터-보조 분석은 프로그램 XNIP (Staden, 1986, Nucleic Acids Research, 14:217-231)로 수행한다. 추가 분석은 "BLAST 서치 프로그램" (Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402)으로, 기관["National Center for Biotechnology Information" (NCBI, Bethesda, MD, USA)]의 비-중복 데이타 뱅크에 대해 수행한다.
수득한 뉴클레오타이드 서열은 서열 1에 나타낸다. 뉴클레오타이드 서열의 분석으로 957개 염기쌍의 암호화 영역이 밝혀졌으며, 이를 opcA 유전자로 명명한다. 종결 코돈을 포함하는 상기 opcA 유전자를 서열 4에 나타낸다. 상기 opcA 유전자는 서열 3 및 서열 5에 나타낸 319개 아미노산의 단백질을 암호화한다.
실시예 5
코리네박테리움 글루타미컴 ATCC13032의 글루코스-6-포스페이트 데하이드로게나제의 정제 및 N-말단 서열분석
1. 균주 ATCC 13032의 배양
글루코스-6-포스페이트 데하이드로게나제의 정제를 위하여 코리네박테리움 글루타미컴 ATCC 13032를 Labfors 발효 시스템 (Infors AG, Bottmingen, Switzerland)으로 30 ℃에서 최소 배지에서 호기적으로 증식시킨다. 전배양물 (BactoR Brain Heart Infusion medium, Difco Laboratories, Detroit, USA)를 15시간 동안 30 ℃에서 배양하여 2.5ℓ 최소 배지의 접종을 위해 사용한다. 배지는 다음 성분을 함유한다 (ℓ당 함량): 20 g (NH4)2SO4; 1 g KH2PO
4; 1 g K2HPO4; 0.25 g MgSO4 x 7H2O; 10 ㎎ CaCl2; 0.2 ㎎ 비오틴; 30 ㎎ 프로토카테추산; 1 ㎎ FeSO4 x 7H2O; 1 ㎎ MnSO4 x H2O; 0.1 ㎎ ZnSO4 x 7H2O; 0.02 ㎎ CuSO4; 0.002 ㎎ NiCl2 x 6H2O; 1.2 g HCl; 0.2 g 폴리프로필렌 글리콜; 75 ㎎ 트리트리플렉스 II 및 100 g 글루코스. 발효중 수산화나트륨을 연속적으로 가하여 pH값을 7.0에서 일정하게 유지한다. 후기 대수 증식기에서 세포를 수거한다. Avanti J-25 원심분리기와 벡크만(Beckman)의 JA10 로터 (Fullerton, USA)를 사용하여 6400 g에서 15분간 4 ℃에서 원심분리시키고 10 mM MgCl2을 함유하는 100 mM TRIS-HCl pH 7.5에서 세척한 후 침강물을 사용할 때 까지 -20 ℃에서 보관한다.
2. 효소 정제
붕해 시스템 (Disintegrator S, BIOmatic, Rodgau-Hainhausen, Germany)에서 세포를 파쇄시킨다. 세포를 먼저 100 mM TRIS-HCl, 10 mM MgCl2, 0.75 mM DTT 및 수종의 프로테아제 억제제의 혼합물 (complete™, Roche, Mannheim, Germany)로 이루어진 pH 7.5 완충액중에 현탁시킨다. 세포 습윤 중량 대 전체 현탁액 중량비를 0.3으로 조정한다. 전체 현탁액 용적 100 ㎖ 당 직경이 0.1 내지 0.25 ㎜인 유리 비드 (Fisher scientific, Dusseldorf, Germany) 100 ㎖ 첨가후, 5000 rpm에서 12분간 세포를 파쇄한다. 파쇄중 온도 상승은 빙냉시켜 방지한다. 유리 비드를 제거한 후 L8-70M 원심분리기와 벡크만의 Ti45 로터 (Fullerton, USA)를 사용하여 235000 g에서 90분간 4 ℃에서 초원심분리 단계를 수행한다. 상등액을 글루코스-6-포스페이트 데하이드로게나제의 정제용 조 추출액으로 사용한다. 모든 정제 단계를 벡크만의 Biosys2000 시스템 (Fullerton, USA)으로 수행한다.
조 추출액을 XK 50/30 칼럼 (Pharmacia, Freiburg, Germany)에 적용시키는데, 이 칼럼은 Fractogel EMD DEAE-650(S) 물질 (Merck, Darmstadt)를 함유한다. 전체 베드 용적은 500 ㎖이다. 상기 칼럼을 먼저 30 mM MgCl2 및 0.75 mM DTT를 함유하는 50 mM TRIS-HCl pH 7.5로 평형화시킨다. 조 추출액 적용후 칼럼을 144 mM KCl을 함유하는 상기와 동일한 완충액으로 세척한다. 95분내에 144 mM에서 320 mM 까지의 선형 KCl 구배로 용출시킨다. 유속은 7.4 ㎖/분이다. 활성 분획을 모으고 1500 g, 4 ℃에서 Varifuge 3.0R 원심분리기 (Heraeus, Hanau, Germany)를 사용하여 centriprepR농축기 (Amicon, Beverly, USA)에서 농축시킨다. 30 mM MgCl2 및 0.75 mM DTT를 함유하는 50 mM TRIS-HCl pH 7.5로 희석시켜 KCl 농도를 40 mM로 조정한다. 이후 부분적으로 정제된 글루코스-6-포스페이트 데하이드로게나제를 XK26/20 칼럼 (Pharmacia, Freiburg, Germany)에 적용하는데, 이 칼럼에는 레드-세파로스(Red-Sepharose) CL6B (Pharmacia, Freiburg, Germany) 65 ㎖가 충전되어 있다. 상기 칼럼을 30 mM MgCl2 및 0.75 mM DTT를 함유하는 50 mM TRIS-HCl pH 7.5로 평형화시킨다. 590분내에 0 내지 800 mM KCl의 선형 농도 구배로 0.87 ㎖/분의 유속에서 용출시킨다.
활성 글루코스-6-포스페이트 데하이드로게나제 분획을 모은후, KCl 농도를 상기와 동일한 방법으로 10 mM로 감소시킨다. 이후 상기 용액을 XK16/20 칼럼 (Pharmacia, Freisburg, Germany)에 적용하는데, 이 칼럼은 2',5'-ADP-세파로스 매트릭스 (Pharmacia, Freiburg, Germany) 20 ㎖를 함유한다. 상기 칼럼을 레드-세파로스 CL6B 칼럼과 동일한 완충액으로 평형화시킨다. 0 내지 2 mM NADP 선형 농도 구배로 용출시킨다. 활성 글루코스-6-포스페이트 데하이드로게나제-분획을 모아 겔 여과 칼럼에 적용한다.
겔 여과를 위하여 직경이 1.6 ㎝이고 베드 용적이 114 ㎖인 슈퍼덱스(Superdex) G200pg 칼럼 (Pharmacia, Freiburg, Germany)을 사용한다. 1 ㎖/분의 유속에서 30 mM MgCl2, 200 mM KCl 및 0.75 mM DTT를 함유하는 50 mM TRIS-HCl pH 7.5를 사용하여 용출시킨다. 활성 분획을 모아 centriprepR 30 농축기 (Amicon, Beverly, USA)중에서 한외여과에 의해 농축시킨다. 상기 정제된 글루코스-6-포스페이트 데하이드로게나제 용액에 50% (v/v) 글리세롤을 첨가한 후 -20 ℃에서 보관한다.
전체 정제 공정중 글루코스-6-포스페이트-데하이드로게나제 활성과 단백질 농도를 측정한다.
글루코스-6-포스페이트-데하이드로게나제-활성 측정용 검정 시스템은 50 mM TRIS-HCl pH 7.5, 10 mM MgCl2, 1mM NADP 및 200 mM 칼륨 글루타메이트를 함유한다. 4 mM 글루코스-6-포스페이트를 가하여 반응을 개시하고 NADPH가 형성된 다음 30 ℃, 340 ㎚에서의 흡광도 증가량을 측정한다. 단백질 농도는 Coomassie Brilliant Blue 염색 (Stoscheck, Methods in Enzymology 182, 50-68 (1990)) 후 분광계로 측정한다. 단백질 표준물로서 소 혈청 알부민을 사용한다. 모든 측정은 UV-160A 광도계 (Shimadzu, Kyoto, Japan)를 사용하여 수행한다.
글루코스-6-포스페이트 데하이드로게나제의 순도는 라엠리(Laemmli) 방법 (Laemmli, U.K., Nature 227, 680-685 (1970))에 따른 변성 비연속적 SDS-겔전기영동에 의해 시험한다. 2',5'-ADP 세파로스 리간드 친화성 물질을 사용한 3차 정제 단계후, 분자량이 약 60 kDa 및 30 kDa인 2종의 상이한 단백질이 수득될 수 있다. 이들 2종의 단백질은 겔 여과 크로마토그래피로 분리할 수 없다. 이 제제의 비활성은 213 U/㎎ 단백질인 것으로 측정되었다.
3. N-말단 서열분석
상기 정제된 글루코스-6-P 데하이드로게나제의 N-말단 서열분석은 에드만(Edman)의 방법(Edman and Begg, European Journal of Biochemistry 1, 80-91 (1967)에 따라서 ProciseR단백질 서열분석 시스템 (Applied Biosystems, Foster City, USA)을 사용하여 수행한다.
60 kDa 단백질의 경우 다음 N-말단 서열이 수득되었다: Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Trp Xaa Asn Pro Leu Arg Asp. 이는 또한 서열 11에 나타낸다.
30 kDa 단백질의 경우 다음 N-말단 서열이 수득되었다: Met Ile Phe Xaa Leu Pro Asp Xaa Xaa Xaa Gln Gln Ile Ser Lys. 이는 또한 서열 12에 나타낸다.
실시예 6
pGEM T-벡터중으로 zwf 및 opcA 유전자의 클로닝
PCR을 사용하여 씨. 글루타미컴 ATCC 13032의 전체 zwf 및 opcA 유전자와 플랭킹 상류 및 다운스트림 영역을 함유하는 DNA 단편을 증폭시킨다. PCR 반응은 서열 1 및 서열 6으로부터 디자인된 올리고뉴클레오타이드 프라이머를 사용하여 수행한다. 게놈성 DNA를 코리네박테리움 글루타미컴 ATCC13032로부터 히리(Heery)와 두니칸(Dunican) (Applied and Environmental Microbiology. 59:791-799 (1993))에 따라 분리시켜 주형으로 사용한다. 사용되는 프라이머는 다음과 같다:
zwf 정배향 프라이머: 5' AGA ATC AGC ACG CTG CAT CAG 3'
opcA 역배향 프라이머: 5' AGT ATG GTG CGC GTA CTA 3'
PCR 파라미터는 다음과 같다:
35회 사이클
95 ℃ 3분
94 ℃ 1분
47 ℃ 1분
72 ℃ 45초
2.5 mM MgCl2
대략 150-200 ng DNA 주형.
수득한 PCR 생성물을 제조회사[Promega Corp.]로부터 구입한 상업적으로 입수가능한 pGEM-T 벡터 (pGEM-T Easy Vector System 1, cat. no. A1360, Promega UK, Southampton)중으로 숙주로서 이. 콜리 균주 JM109 (Yanisch-Perron et al., Gene 33:103-119 (1985))를 사용하여 클로닝시킨다.
실시예 7
셔틀 벡터 pEC-T18mob2의 제조
이. 콜리 - 씨. 글루타미컴 셔틀 벡터 pEC-T18mob2를 선행 기술에 따라서 작제한다.
상기 벡터는 복제 효능인자 per를 포함한 플라스미드 pGA1의 복제 영역 rep (US-A-5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), 플라스미드 pAG1의 테트라사이클린 내성-부여 tetA(Z) 유전자 (US-A-5,158,891; gene library entry at the National Center for Biotechnology Information (NCBI, Bethesda, JD, USA), 수탁번호: AF121000), 플라스미드 pMB1의 복제 영역 oriV (Sutcliffe, Cold Spring Harbor Symposium on Quantitative Biology 43, 77-90 (1979)), lac 프로모터와 다중 클로닝 부위 ( mcs)를 포함한 lacZα 유전자 단편 (Norrander et al. Gene 26, 101-106 (1983)) 및 플라스미드 RP4의 mob 영역 (Simon et al., 1983) Bio/Technology 1:784-791)을 함유한다.
상기 작제된 벡터를 이. 콜리 균주 DH5α (Hanahan, In: DNA cloning. A practical approach. Vol I. IRL-Press, Oxford, Washington DC, USA)에 형질전환시킨다. 상기 형질전환 배치를 5 ㎎/ℓ 테트라사이클린이 보충된 LB 한천 (Sambrook et al., Molecular cloning: a Laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)상에 도말하여 플라스미드-함유 세포를 선택한다. 제조회사[Qiagen]로부터의 퀴아프렙 스핀 미니프렙 키트(QIAprep Spin Minipre Kit)를 사용하여 형질전환체로부터 플라스미드 DNA를 분리하고 제한 효소 EcoRI 및 HindIII로 절단하여 조사한 다음 아가로스 겔 전기영동시킨다 (0.8%).
상기 플라스미드를 pEC-T18mob2로 명명하고 도 2에 나타낸다. 이는 균주 에스케리치아 콜리 K-12 균주 DH5α/pEC-T18mob2의 형태로 기관[Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany)]에 DSM 13244으로서 기탁되었다.
실시예 8
코리네박테리움 글루타미컴중 글루코스-6-포스페이트 데하이드로게나제의 발현
이어서 전체 zwf 및 opcA 유전자를 SphI/SalI 단편상에서 이들 유전자를 함유하는 pGEM T-벡터로부터 (실시예 6 참조) 분리시켜 이. 콜리 - 씨. 글루타미컴 셔틀 벡터 pEC-T18mob2 (실시예 7 및 도 2 참조)의 lacZα SphI/SalI 부위중으로 클로닝시킨다. 이러한 셔틀 벡터는 2개의 SphI 부위를 함유한다. lacZα의 다중 클로닝 부위내에 첫번째 부위가 위치하며 테트라사이클린 내성을 부여하는 유전자내에 두번째 부위가 위치한다. 따라서 테트라사이클린 (Sigma-Aldrich, PO Box 2424, Wimborne, Dorset BH21 7YR, UK) (5 ㎎/ℓ)이 선별압으로서 사용되어 온전한 테트라사이클린 내성 유전자를 함유하는 클론만이 증식한다. 상기 새로운 작제물을 pECzwfopcA로 표시한다 (도 3). SacI (Boehringer Mannheim GmbH, Germany)를 사용한 제한 효소 분석으로 pEC-T18mob2의 lacZα 유전자중 즉, lac 프로모터의 다운스트림에 있는 zwf와 opcA 유전자의 정확한 배향을 밝힌다. 코리네박테리움 글루타미컴 ATCC13032 (American Type Culture Collection, Manasas, VA, USA)를 상기 작제물로 형질전환시키고 전기적 형질전환체를 이소프로필-티오갈락토피라노시드 (IPTG), 5-브로모-4-클로로-3-인돌릴-갈락토피라노시드 (XGAL) 및 테트라사이클린이 각각 1 mM, 0.02% 및 5 ㎎/ℓ의 농도로 보충된 루리아(Luria) 한천상에서 선별한다. 한천 플레이트를 48시간 동안 30 ℃에서 배양시킨다. 신속한 플라스미드 제조는 문헌[참조: O'Gara and Dunican, (Applied and Environmental Microbiology 61: 4477-4479 (1995))]에 기재된 바와 같이 수행하고, Sac I 절단은 요구되는 클론이 존재함을 확증시켜준다. 상기 클론중 하나를 ATCC13032/pECzwfopcA로 명명한다.
실시예 9
증폭된 opcA 유전자를 갖는 아미노산 생산자의 제조
L-라이신-생산 균주 코리네박테리움 글루타미컴 DSM5715는 EP-B-0435132에 설명되어 있으며 L-트레오닌-생산 균주 브레비박테리움 플라붐 DSM5399는 EP-B-0385940에 설명되어 있다. 두 균주 모두 부다페스트 조약에 따라서 기관[Baunschweig (Germany)의 Deutsche Sammlung fur Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures)]에 기탁되어 있다.
상기 균주 DSM5715와 DSM5399를 플라스미드 pECzwfopcA (실시예 8)로 문헌[참조: Liebl et al., (FEMS Microbiology Letters, 53:299-303 (1989))]에 기재된 전기천공 방법을 사용하여 형질전환시킨다. 18.5 g/ℓ 뇌-심장 주입 육즙, 0.5M 솔비톨, 5g/l 박토-트립톤, 2.5g/l 박토-효모 추출물, 5g/l NaCl 및 18 g/ℓ 박토-한천를 포함하며, 5 ㎎/ℓ 테트라사이클린이 보충되어 있는, LBHIS 한천상에서 형질전환체를 선별한다. 2일간 33 ℃에서 배양한다.
이 방법으로 수득한 균주를 DSM5715/pECzwfopcA 및 DSM5399/pECzwfopcA로 명명한다.
실시예 10
L-트레오닌의 제조
실시예 9에서 수득한 씨. 글루타미컴 균주 DSM5399/pECzwfopcA를 트레오닌 생산에 적합한 영양 배지중에서 배양하여 배양 상등액중의 트레오닌 함량을 측정한다.
이를 위하여, 상기 균주를 먼저 24 시간 동안 33 ℃에서 상응하는 항생제를 함유한 한천 플레이트(테트라사이클린 (5 ㎎/ℓ)을 함유하는 뇌-심장 한천)상에서 배양시킨다. 상기 한천 평판 배양으로부터 출발하여, 전배양물을 접종한다 (100ml 들이 원추형 플라스크 배지 10 ㎖). 완전 배지 Cg III를 전배양용 배지로 사용한다.
배지 Cg III:
NaCl 2.5 g/ℓ
박토-펩톤 10 g/ℓ
박토-효모 추출액 10 g/ℓ
글루코스 (별도로 오토클레이빙시킴) 2% (w/v)
pH는 pH 7.4로 조정한다.
테트라사이클린 (5 ㎎/ℓ)을 상기 배지에 가한다. 전배양물을 진탕기상에서 16 시간 동안 33 ℃에서 240 rpm으로 배양시킨다. 본배양물의 초기 OD (660 ㎚)가 0.1이 되도록 상기 전배양물을 본배양물에 접종한다. 상기 배지 MM을 본배양용으로 사용한다.
배지 MM:
CSL (옥수수 침지액) 5 g/ℓ
MOPS (모르폴리노프로판설폰산) 20 g/ℓ
글루코스 (별도로 오토클레이빙시킴) 50 g/ℓ
(NH4)2SO4 25 g/ℓ
KH2PO4 0.1 g/ℓ
MgSO4·7H2O 1.0 g/ℓ
CaCl2·2H2O 10 ㎎/ℓ
FeSO4·7H2O 10 ㎎/ℓ
MnSO4·H2O 5.0 ㎎/ℓ
바이오틴 (멸균 여과한 것) 0.3 ㎎/ℓ
티아민·HCl (멸균 여과한 것) 0.2 ㎎/ℓ
L-류신 (멸균 여과한 것) 0.1 g/ℓ
CaCO3 25 g/ℓ
CSL, MOPS 및 염 용액은 암모니아수로 pH 7로 조정하여 오토클레이빙시킨다. 멸균 물질 및 비타민 용액 뿐만 아니라 건식 오토클레이빙시킨 CaCO3를 가한다.
배양은 배플이 존재하는 100 ㎖ 들이 원추형 플라스크중에서 100ml 용적으로 수행한다. 테트라사이클린 (5 ㎎/ℓ)을 가한다. 배양을 33 ℃에서 80% 대기 습도 에서 수행한다.
72시간후, OD를 660 ㎚의 측정 파장에서 Biomek 1000(Beckmann Instruments GmbH, Munich)을 사용하여 측정한다. 형성된 트레오닌의 양은 이온 교환 크로마토그래피 및 후-칼럼 유도체화에 따른 닌하이드린 검출에 의한 제조회사[Eppendorf-BioTronik (Hamburg, Germany)]로부터의 아미노산 분석기로 측정한다.
시험 결과를 표 1에 나타낸다.
균주 | OD | L-트레오닌 g/ℓ |
DSM5399 | 12.3 | 0.74 |
DSM5399/pECzwfopcA | 9.9 | 1.0 |
실시예 11
L-라이신의 제조
실시예 9에서 수득한 씨. 글루타미컴 균주 DSM5715/pECzwfopcA를 라이신 생산에 적합한 영양 배지중에서 배양하여 배양 상등액중의 라이신 함량을 측정한다.
이를 위하여, 상기 균주를 먼저 24 시간 동안 33 ℃에서 상응하는 항생제를 갖는 한천 플레이트 (테트라사이클린 (5 ㎎/ℓ)을 함유하는 뇌-심장 한천)상에서 배양시킨다. 상기 한천 평판 배양으로부터 출발하는, 전배양물을 접종한다 (원추형 플라스크 100 ㎖중 배지 10 ㎖). 완전 배지 Cg III를 전배양용 배지로서 사용한다.
배지 Cg III:
NaCl 2.5 g/ℓ
박토-펩톤 10 g/ℓ
박토-효모 추출물 10 g/ℓ
글루코스 (별도로 오토클레이빙시킴) 2% (w/v)
pH는 pH 7.4로 조정한다.
테트라사이클린 (5 ㎎/ℓ)을 상기 배지에 가한다. 전배양물을 진탕기상에서 16 시간 동안 33 ℃에서 240 rpm으로 배양시킨다. 본배양물의 초기 OD (660 ㎚)가 0.1이 되도록 상기 전배양물을 본배양물에 접종한다. 상기 배지 MM을 본배양용으로 사용한다.
배지 MM:
CSL (옥수수 침지액) 5 g/ℓ
MOPS (모르폴리노프로판설폰산) 20 g/ℓ
글루코스 (별도로 오토클레이빙시킴) 58 g/ℓ
(NH4)2SO4) 25 g/ℓ
KH2PO4 0.1 g/ℓ
MgSO4·7H2O 1.0 g/ℓ
CaCl2·2H2O 10 ㎎/ℓ
FeSO4·7H2O 10 ㎎/ℓ
MnSO4·H2O 5.0 ㎎/ℓ
바이오틴 (멸균 여과한 것) 0.3 ㎎/ℓ
티아민·HCl (멸균 여과한 것) 0.2 ㎎/ℓ
L-류신 (멸균 여과한 것) 0.1 g/ℓ
CaCO3 25 g/ℓ
CSL, MOPS 및 염 용액은 암모니아수로 pH 7로 조정하여 오토클레이빙시킨다. 멸균 물질 및 비타민 용액 뿐만 아니라 건식 오토클레이빙시킨 CaCO3를 가한다.
배양은 배플이 존재하는 100 ㎖ 들이 원추형 플라스크중에서 배플을 사용하여 10ml 용적으로 수행한다. 테트라사이클린 (5 ㎎/ℓ)을 가한다. 배양을 33 ℃에서 80% 대기 습도에서 수행한다.
72시간후, OD를 660 ㎚의 측정 파장에서 Biomek 1000(Beckmann Instruments GmbH, Munchen)을 사용하여 측정한다. 형성된 라이신의 양은 이온 교환 크로마토그래피 및 후-칼럼 유도체화에 따른 닌하이드린 검출에 의한 제조회사[Eppendorf-BioTronik (Hamburg, Germany)]로부터의 아미노산 분석기로 측정한다.
시험 결과를 표 2에 나타낸다.
균주 | OD | L-라이신 HCl g/ℓ |
DSM5715 | 10.8 | 16.0 |
DSM5715/pECzwfopcA | 8.1 | 17.1 |
원본(제출용)
0-1 0-1-1 | PCT/RO/134 서식(EASY) 기탁된 미생물 또는 기타 생물학적 재료에 관한 사항(PCT 규칙 13 bis) 작성 버젼 | PCT-EASY 버젼 2.90(2000년 3월 8일 갱신) |
0-2 | 국제출원번호 | |
0-3 | 출원인 또는 대리인의 참조번호 | 990213 BT |
1 1-1 1-2 | 아래에 기재된 사항은 당해 면 및 행에서 언급된 기탁된 미생물 또는 기타 생물학적 재료에 관한 것이다: 면 행 | 18 24-30 |
1-3 1-3-1 1-3-2 1-3-3 1-3-4 | 기탁의 표시 기탁기관의 명칭 기탁기관의 주소 기탁일 수탁번호 | DSMZ-도이체 삼룽 폰 미크로오르가니즈멘 운트 젤쿨투렌 게엠베하 독일 데-38124 브라운 슈베이크 마쉐로더 벡 1베 2000년 1월 26일(2000. 1. 26.) DSMZ 13264 |
1-4 | 추가 사항 | 없음 |
1-5 | 기재 사항이 적용되는 지정국 | 모든 지정국 |
1-6 | 별도로 제공되는 사항 이들 사항은 후에 국제사무국에 제출될 것이다. | 없음 |
수리기관용 | ||
0-4 | 본 서식은 국제출원과 함께 접수되었다: (예 또는 아니오) | 예 |
0-4-1 | 확인관 | (서명) |
국제사무국용 | ||
0-5 | 본 서식은 국제사무국에 의해 접수되었다: | |
0-5-1 | 확인관 |
<110> Degussa-Huls Aktiengesellschaft
Forschungszentrum Juelich GmbH
National University of Ireland
<120> Nucleotide sequences which code for the opcA gene
<130> 990213 BT
<150> US 60/142,915
<151> 1999-07-09
<150> US 09/531,267
<151> 2000-03-20
<160> 12
<170> KopatentIn 1.71
<210> 1
<211> 6995
<212> DNA
<213> Corynebacterium glutamicum ATCC13032
<220>
<221> CDS
<222> (3658)..(5202)
<223> zwf
<220>
<221> CDS
<222> (5217)..(6173)
<223> opcA
<400> 1
cacatttgaa ccacagttgg ttataaaatg ggttcaacat cactatggtt agaggtgttg 60
acgggtcaga ttaagcaaag actactttcg gggtagatca cctttgccaa atttgaacca 120
attaacctaa gtcgtagatc tgatcatcgg atctaacgaa aacgaaccaa aactttggtc 180
ccggtttaac ccaggaagga ttgaccacct tgacgctgtc acctgaactt caggcgctca 240
ctgtacgcaa ttacccctct gattggtccg atgtggacac caaggctgta gacactgttc 300
gtgtcctcgc tgcagacgct gtagaaaact gtggctccgg ccacccaggc accgcaatga 360
gcctggctcc ccttgcatac accttgtacc agcgggttat gaacgtagat ccacaggaca 420
ccaactgggc aggccgtgac cgcttcgttc tttcttgtgg ccactcctct ttgacccagt 480
acatccagct ttacttgggt ggattcggcc ttgagatgga tgacctgaag gctctgcgca 540
cctgggattc cttgacccca ggacaccctg agtaccgcca caccaagggc gttgagatca 600
ccactggccc tcttggccag ggtcttgcat ctgcagttgg tatggccatg gctgctcgtc 660
gtgagcgtgg cctattcgac ccaaccgctg ctgagggcga atccccattc gaccaccaca 720
tctacgtcat tgcttctgat ggtgacctgc aggaaggtgt cacctctgag gcatcctcca 780
tcgctggcac ccagcagctg ggcaacctca tcgtgttctg ggatgacaac cgcatctcca 840
tcgaagacaa cactgagatc gctttcaacg aggacgttgt tgctcgttac aaggcttacg 900
gctggcagac cattgaggtt gaggctggcg aggacgttgc agcaatcgaa gctgcagtgg 960
ctgaggctaa gaaggacacc aagcgaccta ccttcatccg cgttcgcacc atcatcggct 1020
tcccagctcc aactatgatg aacaccggtg ctgtgcacgg tgctgctctt ggcgcagctg 1080
aggttgcagc aaccaagact gagcttggat tcgatcctga ggctcacttc gcgatcgacg 1140
atgaggttat cgctcacacc cgctccctcg cagagcgcgc tgcacagaag aaggctgcat 1200
ggcaggtcaa gttcgatgag tgggcagctg ccaaccctga gaacaaggct ctgttcgatc 1260
gcctgaactc ccgtgagctt ccagcgggct acgctgacga gctcccaaca tgggatgcag 1320
atgagaaggg cgtcgcaact cgtaaggctt ccgaggctgc acttcaggca ctgggcaaga 1380
cccttcctga gctgtggggc ggttccgctg acctcgcagg ttccaacaac accgtgatca 1440
agggctcccc ttccttcggc cctgagtcca tctccaccga gacctggtct gctgagcctt 1500
acggccgtaa cctgcacttc ggtatccgtg agcacgctat gggatccatc ctcaacggca 1560
tttccctcca cggtggcacc cgcccatacg gcggaacctt cctcatcttc tccgactaca 1620
tgcgtcctgc agttcgtctt gcagctctca tggagaccga cgcttactac gtctggaccc 1680
acgactccat cggtctgggc gaagatggcc caacccacca gcctgttgaa accttggctg 1740
cactgcgcgc catcccaggt ctgtccgtcc tgcgtcctgc agatgcgaac gagaccgccc 1800
aggcttgggc tgcagcactt gagtacaagg aaggccctaa gggtcttgca ctgacccgcc 1860
agaacgttcc tgttctggaa ggcaccaagg agaaggctgc tgaaggcgtt cgccgcggtg 1920
gctacgtcct ggttgagggt tccaaggaaa ccccagatgt gatcctcatg ggctccggct 1980
ccgaggttca gcttgcagtt aacgctgcga aggctctgga agctgagggc gttgcagctc 2040
gcgttgtttc cgttccttgc atggattggt tccaggagca ggacgcagag tacatcgagt 2100
ccgttctgcc tgcagctgtg accgctcgtg tgtctgttga agctggcatc gcaatgcctt 2160
ggtaccgctt cttgggcacc cagggccgtg ctgtctccct tgagcacttc ggtgcttctg 2220
cggattacca gaccctgttt gagaagttcg gcatcaccac cgatgcagtc gtggcagcgg 2280
ccaaggactc cattaacggt taattgccct gctgttttta gcttcaaccc ggggcaatat 2340
gattctccgg aattttattg ccccgggttg ttgttgttaa tcggtacaaa gggtcttaag 2400
cacatccctt acttgcctgc tctccttgag cacagttcaa gaacaattct tttaaggaaa 2460
atttagtttc atgtctcaca ttgatgatct tgcacagctc ggcacttcca cttggctcga 2520
cgacctctcc cgcgagcgca ttacttccgg caatctcagc caggttattg aggaaaagtc 2580
tgtagtcggt gtcaccacca acccagctat tttcgcagca gcaatgtcca agggcgattc 2640
ctacgacgct cagatcgcag agctcaaggc cgctggcgca tctgttgacc aggctgttta 2700
cgccatgagc atcgacgacg ttcgcaatgc ttgtgatctg ttcaccggca tcttcgagtc 2760
ctccaacggc tacgacggcc gcgtgtccat cgaggttgac ccacgtatct ctgctgaccg 2820
cgacgcaacc ctggctcagg ccaaggagct gtgggcaaag gttgatcgtc caaacgtcat 2880
gatcaagatc cctgcaaccc caggttcttt gccagcaatc accgacgctt tggctgaggg 2940
catcagcgtt aacgtcacct tgatcttctc cgttgctcgc taccgcgagg tcatcgctgc 3000
gttcatcgag ggcatcaagc aggctgctgc aaacggccac gacgtctcca agatccactc 3060
tgtggcttcc ttcttcgtct cccgcgtcga cgttgagatc gacaagcgcc tcgaggcaat 3120
cggatccgat gaggctttgg ctctgcgcgg caaggcaggc gttgccaacg ctcagcgcgc 3180
ttacgctgtg tacaaggagc ttttcgacgc cgccgagctg cctgaaggtg ccaacactca 3240
gcgcccactg tgggcatcca ccggcgtgaa gaaccctgcg tacgctgcaa ctctttacgt 3300
ttccgagctg gctggtccaa acaccgtcaa caccatgcca gaaggcacca tcgacgcggt 3360
tctggagcag ggcaacctgc acggtgacac cctgtccaac tccgcggcag aagctgacgc 3420
tgtgttctcc cagcttgagg ctctgggcgt tgacttggca gatgtcttcc aggtcctgga 3480
gaccgagggt gtggacaagt tcgttgcttc ttggagcgaa ctgcttgagt ccatggaagc 3540
tcgcctgaag tagaatcagc acgctgcatc agtaacggcg acatgaaatc gaattagttc 3600
gatcttatgt ggccgttaca catctttcat taaagaaagg atcgtgacac taccatc 3657
gtg agc aca aac acg acc ccc tcc agc tgg aca aac cca ctg cgc gac 3705
Val Ser Thr Asn Thr Thr Pro Ser Ser Trp Thr Asn Pro Leu Arg Asp
1 5 10 15
ccg cag gat aaa cga ctc ccc cgc atc gct ggc cct tcc ggc atg gtg 3753
Pro Gln Asp Lys Arg Leu Pro Arg Ile Ala Gly Pro Ser Gly Met Val
20 25 30
atc ttc ggt gtc act ggc gac ttg gct cga aag aag ctg ctc ccc gcc 3801
Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu Pro Ala
35 40 45
att tat gat cta gca aac cgc gga ttg ctg ccc cca gga ttc tcg ttg 3849
Ile Tyr Asp Leu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe Ser Leu
50 55 60
gta ggt tac ggc cgc cgc gaa tgg tcc aaa gaa gac ttt gaa aaa tac 3897
Val Gly Tyr Gly Arg Arg Glu Trp Ser Lys Glu Asp Phe Glu Lys Tyr
65 70 75 80
gta cgc gat gcc gca agt gct ggt gct cgt acg gaa ttc cgt gaa aat 3945
Val Arg Asp Ala Ala Ser Ala Gly Ala Arg Thr Glu Phe Arg Glu Asn
85 90 95
gtt tgg gag cgc ctc gcc gag ggt atg gaa ttt gtt cgc ggc aac ttt 3993
Val Trp Glu Arg Leu Ala Glu Gly Met Glu Phe Val Arg Gly Asn Phe
100 105 110
gat gat gat gca gct ttc gac aac ctc gct gca aca ctc aag cgc atc 4041
Asp Asp Asp Ala Ala Phe Asp Asn Leu Ala Ala Thr Leu Lys Arg Ile
115 120 125
gac aaa acc cgc ggc acc gcc ggc aac tgg gct tac tac ctg tcc att 4089
Asp Lys Thr Arg Gly Thr Ala Gly Asn Trp Ala Tyr Tyr Leu Ser Ile
130 135 140
cca cca gat tcc ttc aca gcg gtc tgc cac cag ctg gag cgt tcc ggc 4137
Pro Pro Asp Ser Phe Thr Ala Val Cys His Gln Leu Glu Arg Ser Gly
145 150 155 160
atg gct gaa tcc acc gaa gaa gca tgg cgc cgc gtg atc atc gag aag 4185
Met Ala Glu Ser Thr Glu Glu Ala Trp Arg Arg Val Ile Ile Glu Lys
165 170 175
cct ttc ggc cac aac ctc gaa tcc gca cac gag ctc aac cag ctg gtc 4233
Pro Phe Gly His Asn Leu Glu Ser Ala His Glu Leu Asn Gln Leu Val
180 185 190
aac gca gtc ttc cca gaa tct tct gtg ttc cgc atc gac cac tat ttg 4281
Asn Ala Val Phe Pro Glu Ser Ser Val Phe Arg Ile Asp His Tyr Leu
195 200 205
ggc aag gaa aca gtt caa aac atc ctg gct ctg cgt ttt gct aac cag 4329
Gly Lys Glu Thr Val Gln Asn Ile Leu Ala Leu Arg Phe Ala Asn Gln
210 215 220
ctg ttt gag cca ctg tgg aac tcc aac tac gtt gac cac gtc cag atc 4377
Leu Phe Glu Pro Leu Trp Asn Ser Asn Tyr Val Asp His Val Gln Ile
225 230 235 240
acc atg gct gaa gat att ggc ttg ggt gga cgt gct ggt tac tac gac 4425
Thr Met Ala Glu Asp Ile Gly Leu Gly Gly Arg Ala Gly Tyr Tyr Asp
245 250 255
ggc atc ggc gca gcc cgc gac gtc atc cag aac cac ctg atc cag ctc 4473
Gly Ile Gly Ala Ala Arg Asp Val Ile Gln Asn His Leu Ile Gln Leu
260 265 270
ttg gct ctg gtt gcc atg gaa gaa cca att tct ttc gtg cca gcg cag 4521
Leu Ala Leu Val Ala Met Glu Glu Pro Ile Ser Phe Val Pro Ala Gln
275 280 285
ctg cag gca gaa aag atc aag gtg ctc tct gcg aca aag ccg tgc tac 4569
Leu Gln Ala Glu Lys Ile Lys Val Leu Ser Ala Thr Lys Pro Cys Tyr
290 295 300
cca ttg gat aaa acc tcc gct cgt ggt cag tac gct gcc ggt tgg cag 4617
Pro Leu Asp Lys Thr Ser Ala Arg Gly Gln Tyr Ala Ala Gly Trp Gln
305 310 315 320
ggc tct gag tta gtc aag gga ctt cgc gaa gaa gat ggc ttc aac cct 4665
Gly Ser Glu Leu Val Lys Gly Leu Arg Glu Glu Asp Gly Phe Asn Pro
325 330 335
gag tcc acc act gag act ttt gcg gct tgt acc tta gag atc acg tct 4713
Glu Ser Thr Thr Glu Thr Phe Ala Ala Cys Thr Leu Glu Ile Thr Ser
340 345 350
cgt cgc tgg gct ggt gtg ccg ttc tac ctg cgc acc ggt aag cgt ctt 4761
Arg Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg Thr Gly Lys Arg Leu
355 360 365
ggt cgc cgt gtt act gag att gcc gtg gtg ttt aaa gac gca cca cac 4809
Gly Arg Arg Val Thr Glu Ile Ala Val Val Phe Lys Asp Ala Pro His
370 375 380
cag cct ttc gac ggc gac atg act gta tcc ctt ggc caa aac gcc atc 4857
Gln Pro Phe Asp Gly Asp Met Thr Val Ser Leu Gly Gln Asn Ala Ile
385 390 395 400
gtg att cgc gtg cag cct gat gaa ggt gtg ctc atc cgc ttc ggt tcc 4905
Val Ile Arg Val Gln Pro Asp Glu Gly Val Leu Ile Arg Phe Gly Ser
405 410 415
aag gtt cca ggt tct gcc atg gaa gtc cgt gac gtc aac atg gac ttc 4953
Lys Val Pro Gly Ser Ala Met Glu Val Arg Asp Val Asn Met Asp Phe
420 425 430
tcc tac tca gaa tcc ttc act gaa gaa tca cct gaa gca tac gag cgc 5001
Ser Tyr Ser Glu Ser Phe Thr Glu Glu Ser Pro Glu Ala Tyr Glu Arg
435 440 445
ctc att ttg gat gcg ctg tta gat gaa tcc agc ctc ttc cct acc aac 5049
Leu Ile Leu Asp Ala Leu Leu Asp Glu Ser Ser Leu Phe Pro Thr Asn
450 455 460
gag gaa gtg gaa ctg agc tgg aag att ctg gat cca att ctt gaa gca 5097
Glu Glu Val Glu Leu Ser Trp Lys Ile Leu Asp Pro Ile Leu Glu Ala
465 470 475 480
tgg gat gcc gat gga gaa cca gag gat tac cca gcg ggt acg tgg ggt 5145
Trp Asp Ala Asp Gly Glu Pro Glu Asp Tyr Pro Ala Gly Thr Trp Gly
485 490 495
cca aag agc gct gat gaa atg ctt tcc cgc aac ggt cac acc tgg cgc 5193
Pro Lys Ser Ala Asp Glu Met Leu Ser Arg Asn Gly His Thr Trp Arg
500 505 510
agg cca taa tttaggggca aaaa atg atc ttt gaa ctt ccg gat acc acc 5243
Arg Pro Met Ile Phe Glu Leu Pro Asp Thr Thr
515 520
acc cag caa att tcc aag acc cta act cga ctg cgt gaa tcg ggc acc 5291
Thr Gln Gln Ile Ser Lys Thr Leu Thr Arg Leu Arg Glu Ser Gly Thr
525 530 535 540
cag gtc acc acc ggc cga gtg ctc acc ctc atc gtg gtc act gac tcc 5339
Gln Val Thr Thr Gly Arg Val Leu Thr Leu Ile Val Val Thr Asp Ser
545 550 555
gaa agc gat gtc gct gca gtt acc gag tcc acc aat gaa gcc tcg cgc 5387
Glu Ser Asp Val Ala Ala Val Thr Glu Ser Thr Asn Glu Ala Ser Arg
560 565 570
gag cac cca tct cgc gtg atc att ttg gtg gtt ggc gat aaa act gca 5435
Glu His Pro Ser Arg Val Ile Ile Leu Val Val Gly Asp Lys Thr Ala
575 580 585
gaa aac aaa gtt gac gca gaa gtc cgt atc ggt ggc gac gct ggt gct 5483
Glu Asn Lys Val Asp Ala Glu Val Arg Ile Gly Gly Asp Ala Gly Ala
590 595 600
tcc gag atg atc atc atg cat ctc aac gga cct gtc gct gac aag ctc 5531
Ser Glu Met Ile Ile Met His Leu Asn Gly Pro Val Ala Asp Lys Leu
605 610 615 620
cag tat gtc gtc aca cca ctg ttg ctt cct gac acc ccc atc gtt gct 5579
Gln Tyr Val Val Thr Pro Leu Leu Leu Pro Asp Thr Pro Ile Val Ala
625 630 635
tgg tgg cca ggt gaa tca cca aag aat cct tcc cag gac cca att gga 5627
Trp Trp Pro Gly Glu Ser Pro Lys Asn Pro Ser Gln Asp Pro Ile Gly
640 645 650
cgc atc gca caa cga cgc atc act gat gct ttg tac gac cgt gat gac 5675
Arg Ile Ala Gln Arg Arg Ile Thr Asp Ala Leu Tyr Asp Arg Asp Asp
655 660 665
gca cta gaa gat cgt gtt gag aac tat cac cca ggt gat acc gac atg 5723
Ala Leu Glu Asp Arg Val Glu Asn Tyr His Pro Gly Asp Thr Asp Met
670 675 680
acg tgg gcg cgc ctt acc cag tgg cgg gga ctt gtt gcc tcc tca ttg 5771
Thr Trp Ala Arg Leu Thr Gln Trp Arg Gly Leu Val Ala Ser Ser Leu
685 690 695 700
gat cac cca cca cac agc gaa atc act tcc gtg agg ctg acc ggt gca 5819
Asp His Pro Pro His Ser Glu Ile Thr Ser Val Arg Leu Thr Gly Ala
705 710 715
agc ggc agt acc tcg gtg gat ttg gct gca ggc tgg ttg gcg cgg agg 5867
Ser Gly Ser Thr Ser Val Asp Leu Ala Ala Gly Trp Leu Ala Arg Arg
720 725 730
ctg aaa gtg cct gtg atc cgc gag gtg aca gat gct ccc acc gtg cca 5915
Leu Lys Val Pro Val Ile Arg Glu Val Thr Asp Ala Pro Thr Val Pro
735 740 745
acc gat gag ttt ggt act cca ctg ctg gct atc cag cgc ctg gag atc 5963
Thr Asp Glu Phe Gly Thr Pro Leu Leu Ala Ile Gln Arg Leu Glu Ile
750 755 760
gtt cgc acc acc ggc tcg atc atc atc acc atc tat gac gct cat acc 6011
Val Arg Thr Thr Gly Ser Ile Ile Ile Thr Ile Tyr Asp Ala His Thr
765 770 775 780
ctt cag gta gag atg ccg gaa tcc ggc aat gcc cca tcg ctg gtg gct 6059
Leu Gln Val Glu Met Pro Glu Ser Gly Asn Ala Pro Ser Leu Val Ala
785 790 795
att ggt cgt cga agt gag tcc gac tgc ttg tct gag gag ctt cgc cac 6107
Ile Gly Arg Arg Ser Glu Ser Asp Cys Leu Ser Glu Glu Leu Arg His
800 805 810
atg gat cca gat ttg ggc tac cag cac gca cta tcc ggc ttg tcc agc 6155
Met Asp Pro Asp Leu Gly Tyr Gln His Ala Leu Ser Gly Leu Ser Ser
815 820 825
gtc aag ctg gaa acc gtc taaggagaaa tacaacacta tggttgatgt 6203
Val Lys Leu Glu Thr Val
830
agtacgcgca cgcgatactg aagatttggt tgcacaggct gcctccaaat tcattgaggt 6263
tgttgaagca gcaactgcca ataatggcac cgcacaggta gtgctcaccg gtggtggcgc 6323
cggcatcaag ttgctggaaa agctcagcgt tgatgcggct gaccttgcct gggatcgcat 6383
tcatgtgttc ttcggcgatg agcgcaatgt ccctgtcagt gattctgagt ccaatgaggg 6443
ccaggctcgt gaggcactgt tgtccaaggt ttctatccct gaagccaaca ttcacggata 6503
tggtctcggc gacgtagatc ttgcagaggc agcccgcgct tacgaagctg tgttggatga 6563
attcgcacca aacggctttg atcttcacct gctcggcatg ggtggcgaag gccatatcaa 6623
ctccctgttc cctcacaccg atgcagtcaa ggaatcctcc gcaaaggtca tcgcggtgtt 6683
tgattcccct aagcctcctt cagagcgtgc aactctaacc cttcctgcgg ttcactccgc 6743
aaagcgcgtg tggttgctgg tttctggtgc ggagaaggct gaggcagctg cggcgatcgt 6803
caacggtgag cctgctgttg agtggcctgc tgctggagct accggatctg aggaaacggt 6863
attgttcttg gctgatgatg ctgcaggaaa tctctaagca gcgccagctc taacaagaag 6923
ctttaacaag aagctctaac gaaaagcact aacaaactaa tccgggtgcg aaccttcatc 6983
tgaatcgatg ga 6995
<210> 2
<211> 514
<212> PRT
<213> Corynebacterium glutamicum ATCC13032
<400> 2
Val Ser Thr Asn Thr Thr Pro Ser Ser Trp Thr Asn Pro Leu Arg Asp
1 5 10 15
Pro Gln Asp Lys Arg Leu Pro Arg Ile Ala Gly Pro Ser Gly Met Val
20 25 30
Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu Pro Ala
35 40 45
Ile Tyr Asp Leu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe Ser Leu
50 55 60
Val Gly Tyr Gly Arg Arg Glu Trp Ser Lys Glu Asp Phe Glu Lys Tyr
65 70 75 80
Val Arg Asp Ala Ala Ser Ala Gly Ala Arg Thr Glu Phe Arg Glu Asn
85 90 95
Val Trp Glu Arg Leu Ala Glu Gly Met Glu Phe Val Arg Gly Asn Phe
100 105 110
Asp Asp Asp Ala Ala Phe Asp Asn Leu Ala Ala Thr Leu Lys Arg Ile
115 120 125
Asp Lys Thr Arg Gly Thr Ala Gly Asn Trp Ala Tyr Tyr Leu Ser Ile
130 135 140
Pro Pro Asp Ser Phe Thr Ala Val Cys His Gln Leu Glu Arg Ser Gly
145 150 155 160
Met Ala Glu Ser Thr Glu Glu Ala Trp Arg Arg Val Ile Ile Glu Lys
165 170 175
Pro Phe Gly His Asn Leu Glu Ser Ala His Glu Leu Asn Gln Leu Val
180 185 190
Asn Ala Val Phe Pro Glu Ser Ser Val Phe Arg Ile Asp His Tyr Leu
195 200 205
Gly Lys Glu Thr Val Gln Asn Ile Leu Ala Leu Arg Phe Ala Asn Gln
210 215 220
Leu Phe Glu Pro Leu Trp Asn Ser Asn Tyr Val Asp His Val Gln Ile
225 230 235 240
Thr Met Ala Glu Asp Ile Gly Leu Gly Gly Arg Ala Gly Tyr Tyr Asp
245 250 255
Gly Ile Gly Ala Ala Arg Asp Val Ile Gln Asn His Leu Ile Gln Leu
260 265 270
Leu Ala Leu Val Ala Met Glu Glu Pro Ile Ser Phe Val Pro Ala Gln
275 280 285
Leu Gln Ala Glu Lys Ile Lys Val Leu Ser Ala Thr Lys Pro Cys Tyr
290 295 300
Pro Leu Asp Lys Thr Ser Ala Arg Gly Gln Tyr Ala Ala Gly Trp Gln
305 310 315 320
Gly Ser Glu Leu Val Lys Gly Leu Arg Glu Glu Asp Gly Phe Asn Pro
325 330 335
Glu Ser Thr Thr Glu Thr Phe Ala Ala Cys Thr Leu Glu Ile Thr Ser
340 345 350
Arg Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg Thr Gly Lys Arg Leu
355 360 365
Gly Arg Arg Val Thr Glu Ile Ala Val Val Phe Lys Asp Ala Pro His
370 375 380
Gln Pro Phe Asp Gly Asp Met Thr Val Ser Leu Gly Gln Asn Ala Ile
385 390 395 400
Val Ile Arg Val Gln Pro Asp Glu Gly Val Leu Ile Arg Phe Gly Ser
405 410 415
Lys Val Pro Gly Ser Ala Met Glu Val Arg Asp Val Asn Met Asp Phe
420 425 430
Ser Tyr Ser Glu Ser Phe Thr Glu Glu Ser Pro Glu Ala Tyr Glu Arg
435 440 445
Leu Ile Leu Asp Ala Leu Leu Asp Glu Ser Ser Leu Phe Pro Thr Asn
450 455 460
Glu Glu Val Glu Leu Ser Trp Lys Ile Leu Asp Pro Ile Leu Glu Ala
465 470 475 480
Trp Asp Ala Asp Gly Glu Pro Glu Asp Tyr Pro Ala Gly Thr Trp Gly
485 490 495
Pro Lys Ser Ala Asp Glu Met Leu Ser Arg Asn Gly His Thr Trp Arg
500 505 510
Arg Pro
<210> 3
<211> 319
<212> PRT
<213> Corynebacterium glutamicum ATCC13032
<400> 3
Met Ile Phe Glu Leu Pro Asp Thr Thr Thr Gln Gln Ile Ser Lys Thr
1 5 10 15
Leu Thr Arg Leu Arg Glu Ser Gly Thr Gln Val Thr Thr Gly Arg Val
20 25 30
Leu Thr Leu Ile Val Val Thr Asp Ser Glu Ser Asp Val Ala Ala Val
35 40 45
Thr Glu Ser Thr Asn Glu Ala Ser Arg Glu His Pro Ser Arg Val Ile
50 55 60
Ile Leu Val Val Gly Asp Lys Thr Ala Glu Asn Lys Val Asp Ala Glu
65 70 75 80
Val Arg Ile Gly Gly Asp Ala Gly Ala Ser Glu Met Ile Ile Met His
85 90 95
Leu Asn Gly Pro Val Ala Asp Lys Leu Gln Tyr Val Val Thr Pro Leu
100 105 110
Leu Leu Pro Asp Thr Pro Ile Val Ala Trp Trp Pro Gly Glu Ser Pro
115 120 125
Lys Asn Pro Ser Gln Asp Pro Ile Gly Arg Ile Ala Gln Arg Arg Ile
130 135 140
Thr Asp Ala Leu Tyr Asp Arg Asp Asp Ala Leu Glu Asp Arg Val Glu
145 150 155 160
Asn Tyr His Pro Gly Asp Thr Asp Met Thr Trp Ala Arg Leu Thr Gln
165 170 175
Trp Arg Gly Leu Val Ala Ser Ser Leu Asp His Pro Pro His Ser Glu
180 185 190
Ile Thr Ser Val Arg Leu Thr Gly Ala Ser Gly Ser Thr Ser Val Asp
195 200 205
Leu Ala Ala Gly Trp Leu Ala Arg Arg Leu Lys Val Pro Val Ile Arg
210 215 220
Glu Val Thr Asp Ala Pro Thr Val Pro Thr Asp Glu Phe Gly Thr Pro
225 230 235 240
Leu Leu Ala Ile Gln Arg Leu Glu Ile Val Arg Thr Thr Gly Ser Ile
245 250 255
Ile Ile Thr Ile Tyr Asp Ala His Thr Leu Gln Val Glu Met Pro Glu
260 265 270
Ser Gly Asn Ala Pro Ser Leu Val Ala Ile Gly Arg Arg Ser Glu Ser
275 280 285
Asp Cys Leu Ser Glu Glu Leu Arg His Met Asp Pro Asp Leu Gly Tyr
290 295 300
Gln His Ala Leu Ser Gly Leu Ser Ser Val Lys Leu Glu Thr Val
305 310 315
<210> 4
<211> 960
<212> DNA
<213> Corynebacterium glutamicum ATCC13032
<220>
<221> CDS
<222> (1)..(957)
<223> opcA
<400> 4
atg atc ttt gaa ctt ccg gat acc acc acc cag caa att tcc aag acc 48
Met Ile Phe Glu Leu Pro Asp Thr Thr Thr Gln Gln Ile Ser Lys Thr
1 5 10 15
cta act cga ctg cgt gaa tcg ggc acc cag gtc acc acc ggc cga gtg 96
Leu Thr Arg Leu Arg Glu Ser Gly Thr Gln Val Thr Thr Gly Arg Val
20 25 30
ctc acc ctc atc gtg gtc act gac tcc gaa agc gat gtc gct gca gtt 144
Leu Thr Leu Ile Val Val Thr Asp Ser Glu Ser Asp Val Ala Ala Val
35 40 45
acc gag tcc acc aat gaa gcc tcg cgc gag cac cca tct cgc gtg atc 192
Thr Glu Ser Thr Asn Glu Ala Ser Arg Glu His Pro Ser Arg Val Ile
50 55 60
att ttg gtg gtt ggc gat aaa act gca gaa aac aaa gtt gac gca gaa 240
Ile Leu Val Val Gly Asp Lys Thr Ala Glu Asn Lys Val Asp Ala Glu
65 70 75 80
gtc cgt atc ggt ggc gac gct ggt gct tcc gag atg atc atc atg cat 288
Val Arg Ile Gly Gly Asp Ala Gly Ala Ser Glu Met Ile Ile Met His
85 90 95
ctc aac gga cct gtc gct gac aag ctc cag tat gtc gtc aca cca ctg 336
Leu Asn Gly Pro Val Ala Asp Lys Leu Gln Tyr Val Val Thr Pro Leu
100 105 110
ttg ctt cct gac acc ccc atc gtt gct tgg tgg cca ggt gaa tca cca 384
Leu Leu Pro Asp Thr Pro Ile Val Ala Trp Trp Pro Gly Glu Ser Pro
115 120 125
aag aat cct tcc cag gac cca att gga cgc atc gca caa cga cgc atc 432
Lys Asn Pro Ser Gln Asp Pro Ile Gly Arg Ile Ala Gln Arg Arg Ile
130 135 140
act gat gct ttg tac gac cgt gat gac gca cta gaa gat cgt gtt gag 480
Thr Asp Ala Leu Tyr Asp Arg Asp Asp Ala Leu Glu Asp Arg Val Glu
145 150 155 160
aac tat cac cca ggt gat acc gac atg acg tgg gcg cgc ctt acc cag 528
Asn Tyr His Pro Gly Asp Thr Asp Met Thr Trp Ala Arg Leu Thr Gln
165 170 175
tgg cgg gga ctt gtt gcc tcc tca ttg gat cac cca cca cac agc gaa 576
Trp Arg Gly Leu Val Ala Ser Ser Leu Asp His Pro Pro His Ser Glu
180 185 190
atc act tcc gtg agg ctg acc ggt gca agc ggc agt acc tcg gtg gat 624
Ile Thr Ser Val Arg Leu Thr Gly Ala Ser Gly Ser Thr Ser Val Asp
195 200 205
ttg gct gca ggc tgg ttg gcg cgg agg ctg aaa gtg cct gtg atc cgc 672
Leu Ala Ala Gly Trp Leu Ala Arg Arg Leu Lys Val Pro Val Ile Arg
210 215 220
gag gtg aca gat gct ccc acc gtg cca acc gat gag ttt ggt act cca 720
Glu Val Thr Asp Ala Pro Thr Val Pro Thr Asp Glu Phe Gly Thr Pro
225 230 235 240
ctg ctg gct atc cag cgc ctg gag atc gtt cgc acc acc ggc tcg atc 768
Leu Leu Ala Ile Gln Arg Leu Glu Ile Val Arg Thr Thr Gly Ser Ile
245 250 255
atc atc acc atc tat gac gct cat acc ctt cag gta gag atg ccg gaa 816
Ile Ile Thr Ile Tyr Asp Ala His Thr Leu Gln Val Glu Met Pro Glu
260 265 270
tcc ggc aat gcc cca tcg ctg gtg gct att ggt cgt cga agt gag tcc 864
Ser Gly Asn Ala Pro Ser Leu Val Ala Ile Gly Arg Arg Ser Glu Ser
275 280 285
gac tgc ttg tct gag gag ctt cgc cac atg gat cca gat ttg ggc tac 912
Asp Cys Leu Ser Glu Glu Leu Arg His Met Asp Pro Asp Leu Gly Tyr
290 295 300
cag cac gca cta tcc ggc ttg tcc agc gtc aag ctg gaa acc gtc taa 960
Gln His Ala Leu Ser Gly Leu Ser Ser Val Lys Leu Glu Thr Val
305 310 315
<210> 5
<211> 319
<212> PRT
<213> Corynebacterium glutamicum ATCC13032
<400> 5
Met Ile Phe Glu Leu Pro Asp Thr Thr Thr Gln Gln Ile Ser Lys Thr
1 5 10 15
Leu Thr Arg Leu Arg Glu Ser Gly Thr Gln Val Thr Thr Gly Arg Val
20 25 30
Leu Thr Leu Ile Val Val Thr Asp Ser Glu Ser Asp Val Ala Ala Val
35 40 45
Thr Glu Ser Thr Asn Glu Ala Ser Arg Glu His Pro Ser Arg Val Ile
50 55 60
Ile Leu Val Val Gly Asp Lys Thr Ala Glu Asn Lys Val Asp Ala Glu
65 70 75 80
Val Arg Ile Gly Gly Asp Ala Gly Ala Ser Glu Met Ile Ile Met His
85 90 95
Leu Asn Gly Pro Val Ala Asp Lys Leu Gln Tyr Val Val Thr Pro Leu
100 105 110
Leu Leu Pro Asp Thr Pro Ile Val Ala Trp Trp Pro Gly Glu Ser Pro
115 120 125
Lys Asn Pro Ser Gln Asp Pro Ile Gly Arg Ile Ala Gln Arg Arg Ile
130 135 140
Thr Asp Ala Leu Tyr Asp Arg Asp Asp Ala Leu Glu Asp Arg Val Glu
145 150 155 160
Asn Tyr His Pro Gly Asp Thr Asp Met Thr Trp Ala Arg Leu Thr Gln
165 170 175
Trp Arg Gly Leu Val Ala Ser Ser Leu Asp His Pro Pro His Ser Glu
180 185 190
Ile Thr Ser Val Arg Leu Thr Gly Ala Ser Gly Ser Thr Ser Val Asp
195 200 205
Leu Ala Ala Gly Trp Leu Ala Arg Arg Leu Lys Val Pro Val Ile Arg
210 215 220
Glu Val Thr Asp Ala Pro Thr Val Pro Thr Asp Glu Phe Gly Thr Pro
225 230 235 240
Leu Leu Ala Ile Gln Arg Leu Glu Ile Val Arg Thr Thr Gly Ser Ile
245 250 255
Ile Ile Thr Ile Tyr Asp Ala His Thr Leu Gln Val Glu Met Pro Glu
260 265 270
Ser Gly Asn Ala Pro Ser Leu Val Ala Ile Gly Arg Arg Ser Glu Ser
275 280 285
Asp Cys Leu Ser Glu Glu Leu Arg His Met Asp Pro Asp Leu Gly Tyr
290 295 300
Gln His Ala Leu Ser Gly Leu Ser Ser Val Lys Leu Glu Thr Val
305 310 315
<210> 6
<211> 3038
<212> DNA
<213> Corynebacterium glutamicum AS019
<220>
<221> CDS
<222> (115)..(1659)
<223> zwf
<220>
<221> CDS
<222> (1672)..(2628)
<223> opcA
<400> 6
cctgaagtag aatcagcacg ctgcatcagt aacggcgaca tgaaatcgaa ttagttcgat 60
cttatgtggc cgttacacat ctttcattaa agaaaggatc gtgacactac catc gtg 117
Val
1
agc aca aac acg acc ccc tcc agc tgg aca aac cca ctg cgc gac ccg 165
Ser Thr Asn Thr Thr Pro Ser Ser Trp Thr Asn Pro Leu Arg Asp Pro
5 10 15
cag gat aaa cga ctc ccc cgc atc gct ggc cct tcc ggc atg gtg atc 213
Gln Asp Lys Arg Leu Pro Arg Ile Ala Gly Pro Ser Gly Met Val Ile
20 25 30
ttc ggt gtc act ggc gac ttg gct cga aag aag ctg ctc ccc gcc att 261
Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu Pro Ala Ile
35 40 45
tat gat cta gca aac cgc gga ttg ctg ccc cca gga ttc tcg ttg gta 309
Tyr Asp Leu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe Ser Leu Val
50 55 60 65
ggt tac ggc cgc cgc gaa tgg tcc aaa gaa gac ttt gaa aaa tac gta 357
Gly Tyr Gly Arg Arg Glu Trp Ser Lys Glu Asp Phe Glu Lys Tyr Val
70 75 80
cgc gat gcc gca agt gct ggt gct cgt acg gaa ttc cgt gaa aat gtt 405
Arg Asp Ala Ala Ser Ala Gly Ala Arg Thr Glu Phe Arg Glu Asn Val
85 90 95
tgg gag cgc ctc gcc gag ggt atg gaa ttt gtt cgc ggc aac ttt gat 453
Trp Glu Arg Leu Ala Glu Gly Met Glu Phe Val Arg Gly Asn Phe Asp
100 105 110
gat gat gca gct ttc gac aac ctc gct gca aca ctc aag cgc atc gac 501
Asp Asp Ala Ala Phe Asp Asn Leu Ala Ala Thr Leu Lys Arg Ile Asp
115 120 125
aaa acc cgc ggc acc gcc ggc aac tgg gct tac tac ctg tcc att cca 549
Lys Thr Arg Gly Thr Ala Gly Asn Trp Ala Tyr Tyr Leu Ser Ile Pro
130 135 140 145
cca gat tcc ttc aca gcg gtc tgc cac cag ctg gag cgt tcc ggc atg 597
Pro Asp Ser Phe Thr Ala Val Cys His Gln Leu Glu Arg Ser Gly Met
150 155 160
gct gaa tcc acc gaa gaa gca tgg cgc cgc gtg atc atc gag aag cct 645
Ala Glu Ser Thr Glu Glu Ala Trp Arg Arg Val Ile Ile Glu Lys Pro
165 170 175
ttc ggc cac aac ctc gaa tcc gca cac gag ctc aac cag ctg gtc aac 693
Phe Gly His Asn Leu Glu Ser Ala His Glu Leu Asn Gln Leu Val Asn
180 185 190
gca gtc ttc cca gaa tct tct gtg ttc cgc atc gac cac tat ttg ggc 741
Ala Val Phe Pro Glu Ser Ser Val Phe Arg Ile Asp His Tyr Leu Gly
195 200 205
aag gaa aca gtt caa aac atc ctg gct ctg cgt ttt gct aac cag ctg 789
Lys Glu Thr Val Gln Asn Ile Leu Ala Leu Arg Phe Ala Asn Gln Leu
210 215 220 225
ttt gag cca ctg tgg aac tcc aac tac gtt gac cac gtc cag atc acc 837
Phe Glu Pro Leu Trp Asn Ser Asn Tyr Val Asp His Val Gln Ile Thr
230 235 240
atg gct gaa gat att ggc ttg ggt gga cgt gct ggt tac tac gac ggc 885
Met Ala Glu Asp Ile Gly Leu Gly Gly Arg Ala Gly Tyr Tyr Asp Gly
245 250 255
atc ggc gca ccg cgc gac gtc atc cag aac cac ctg atc cag ctc ttg 933
Ile Gly Ala Pro Arg Asp Val Ile Gln Asn His Leu Ile Gln Leu Leu
260 265 270
gct ctg gtt gcc atg gaa gaa cca att tct ttc gtg cca gcg gca cgg 981
Ala Leu Val Ala Met Glu Glu Pro Ile Ser Phe Val Pro Ala Ala Arg
275 280 285
cag gca gaa aag atc aag gtg ctc tct gcg aca aag ccg tgc tac cca 1029
Gln Ala Glu Lys Ile Lys Val Leu Ser Ala Thr Lys Pro Cys Tyr Pro
290 295 300 305
ttg gat aaa acc tcc gct cgt ggt cag tac gct gcc ggt tgg cag ggc 1077
Leu Asp Lys Thr Ser Ala Arg Gly Gln Tyr Ala Ala Gly Trp Gln Gly
310 315 320
tct gag tta gtc aag gga ctt cgc gaa gaa gat ggc ttc aac cct gag 1125
Ser Glu Leu Val Lys Gly Leu Arg Glu Glu Asp Gly Phe Asn Pro Glu
325 330 335
tcc acc act gag act ttt gcg gct tgt acc tta gag atc acg tct cgt 1173
Ser Thr Thr Glu Thr Phe Ala Ala Cys Thr Leu Glu Ile Thr Ser Arg
340 345 350
cgc tgg gct ggt gtg ccg ttc tac ctg cgc acc ggt aag cgt ctt ggt 1221
Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg Thr Gly Lys Arg Leu Gly
355 360 365
cgc cgt gtt act gag att gcc gtg gtg ttt aaa gac gca cca cac cag 1269
Arg Arg Val Thr Glu Ile Ala Val Val Phe Lys Asp Ala Pro His Gln
370 375 380 385
cct ttc gac ggc gac atg act gta tcc ctt ggc caa aac gcc atc gtg 1317
Pro Phe Asp Gly Asp Met Thr Val Ser Leu Gly Gln Asn Ala Ile Val
390 395 400
att cgc gtg cag cct gat gaa ggt gtg ctc atc cgc ttc ggt tcc aag 1365
Ile Arg Val Gln Pro Asp Glu Gly Val Leu Ile Arg Phe Gly Ser Lys
405 410 415
gtt cca ggt tct gcc atg gaa gtc cgt gac gtc aac atg gac ttc tcc 1413
Val Pro Gly Ser Ala Met Glu Val Arg Asp Val Asn Met Asp Phe Ser
420 425 430
tac tca gaa tcc ttc act gaa gaa tca cct gaa gca tac gag cgc ctc 1461
Tyr Ser Glu Ser Phe Thr Glu Glu Ser Pro Glu Ala Tyr Glu Arg Leu
435 440 445
att ttg gat gcg ctg tta gat gaa tcc agc ctc ttc cct acc aac gag 1509
Ile Leu Asp Ala Leu Leu Asp Glu Ser Ser Leu Phe Pro Thr Asn Glu
450 455 460 465
gaa gtg gaa ctg agc tgg aag att ctg gat cca att ctt gaa gca tgg 1557
Glu Val Glu Leu Ser Trp Lys Ile Leu Asp Pro Ile Leu Glu Ala Trp
470 475 480
gat gcc gat gga gaa cca gag gat tac cca gcg ggt acg tgg ggt cca 1605
Asp Ala Asp Gly Glu Pro Glu Asp Tyr Pro Ala Gly Thr Trp Gly Pro
485 490 495
aag agc gct gat gaa atg ctt tcc cgc aac ggt cac acc tgg cgc agg 1653
Lys Ser Ala Asp Glu Met Leu Ser Arg Asn Gly His Thr Trp Arg Arg
500 505 510
cca taa tttaggggca aa atg atc ttt gaa ctt ccg gat acc acc acc cag 1704
Pro Met Ile Phe Glu Leu Pro Asp Thr Thr Thr Gln
515 520 525
caa att tcc aag acc cta act cga ctg cgt gaa tcg ggc acc cag gtc 1752
Gln Ile Ser Lys Thr Leu Thr Arg Leu Arg Glu Ser Gly Thr Gln Val
530 535 540
acc acc ggc cga gtg ctc acc ctc atc gtg gtc act gac tcc gaa agc 1800
Thr Thr Gly Arg Val Leu Thr Leu Ile Val Val Thr Asp Ser Glu Ser
545 550 555
gat gtc gct gca gtt acc gag tcc acc aat gaa gcc tcg cgc gag cac 1848
Asp Val Ala Ala Val Thr Glu Ser Thr Asn Glu Ala Ser Arg Glu His
560 565 570
cca tct cgc gtg atc att ttg gtg gtt ggc gat aaa act gca gaa aac 1896
Pro Ser Arg Val Ile Ile Leu Val Val Gly Asp Lys Thr Ala Glu Asn
575 580 585 590
aaa gtt gac gca gaa gtc cgt atc ggt ggc gac gct ggt gct tcc gag 1944
Lys Val Asp Ala Glu Val Arg Ile Gly Gly Asp Ala Gly Ala Ser Glu
595 600 605
atg atc atc atg cat ctc aac gga cct gtc gct gac aag ctc cag tat 1992
Met Ile Ile Met His Leu Asn Gly Pro Val Ala Asp Lys Leu Gln Tyr
610 615 620
gtc gtc aca cca ctg ttg ctt cct gac acc ccc atc gtt gct tgg tgg 2040
Val Val Thr Pro Leu Leu Leu Pro Asp Thr Pro Ile Val Ala Trp Trp
625 630 635
cca ggt gaa tca cca aag aat cct tcc cag gac cca att gga cgc atc 2088
Pro Gly Glu Ser Pro Lys Asn Pro Ser Gln Asp Pro Ile Gly Arg Ile
640 645 650
gca caa cga cgc atc act gat gct ttg tac gac cgt gat gac gca cta 2136
Ala Gln Arg Arg Ile Thr Asp Ala Leu Tyr Asp Arg Asp Asp Ala Leu
655 660 665 670
gaa gat cgt gtt gag aac tat cac cca ggt gat acc gac atg acg tgg 2184
Glu Asp Arg Val Glu Asn Tyr His Pro Gly Asp Thr Asp Met Thr Trp
675 680 685
gcg cgc ctt acc cag tgg cgg gga ctt gtt gcc tcc tca ttg gat cac 2232
Ala Arg Leu Thr Gln Trp Arg Gly Leu Val Ala Ser Ser Leu Asp His
690 695 700
cca cca cac agc gaa atc act tcc gtg agg ctg acc ggt gca agc ggc 2280
Pro Pro His Ser Glu Ile Thr Ser Val Arg Leu Thr Gly Ala Ser Gly
705 710 715
agt acc tcg gtg gat ttg gct gca ggc tgg ttg gcg cgg agg ctg aaa 2328
Ser Thr Ser Val Asp Leu Ala Ala Gly Trp Leu Ala Arg Arg Leu Lys
720 725 730
gtg cct gtg atc cgc gag gtg aca gat gct ccc acc gtg cca acc gat 2376
Val Pro Val Ile Arg Glu Val Thr Asp Ala Pro Thr Val Pro Thr Asp
735 740 745 750
gag ttt ggt act cca ctg ctg gct atc cag cgc ctg gag atc gtt cgc 2424
Glu Phe Gly Thr Pro Leu Leu Ala Ile Gln Arg Leu Glu Ile Val Arg
755 760 765
acc acc ggc tcg atc atc atc acc atc tat gac gct cat acc ctt cag 2472
Thr Thr Gly Ser Ile Ile Ile Thr Ile Tyr Asp Ala His Thr Leu Gln
770 775 780
gta gag atg ccg gaa tcc ggc aat gcc cca tcg ctg gtg gct att ggt 2520
Val Glu Met Pro Glu Ser Gly Asn Ala Pro Ser Leu Val Ala Ile Gly
785 790 795
cgt cga agt gag tcc gac tgc ttg tct gag gag ctt cgc cac atg gat 2568
Arg Arg Ser Glu Ser Asp Cys Leu Ser Glu Glu Leu Arg His Met Asp
800 805 810
cca gat ttg ggc tac cag cac gca cta tcc ggc ttg tcc agc gtc aag 2616
Pro Asp Leu Gly Tyr Gln His Ala Leu Ser Gly Leu Ser Ser Val Lys
815 820 825 830
ctg gaa acc gtc taaggagaaa tacaacacta tggttgatgt agtacgcgca 2668
Leu Glu Thr Val
cgcatactga agatttggtt gcacaggctg cctccaaatt cattgaggtt gttgaagcag 2728
caactgccaa taatggcacc gcacaggtag tgctcaccgg tggtggcgcc ggcatcaagt 2788
tgctggaaaa gctcagcgtt gatgcggctg accttgcctg ggatcgcatt catgtgttct 2848
tcggcgatga gcgcaatgtc cctgtcagtg attctgagtc caatgagggc caggctcgtg 2908
aggcactgtt gtccaaggtt tctatccctg aagccaacat tcacggatat ggtctcggcg 2968
acgtagatct tgcagaggca gcccgcgctt acgaagctgt gttggatgaa ttcgcaccaa 3028
acggctttga 3038
<210> 7
<211> 514
<212> PRT
<213> Corynebacterium glutamicum AS019
<400> 7
Val Ser Thr Asn Thr Thr Pro Ser Ser Trp Thr Asn Pro Leu Arg Asp
1 5 10 15
Pro Gln Asp Lys Arg Leu Pro Arg Ile Ala Gly Pro Ser Gly Met Val
20 25 30
Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu Pro Ala
35 40 45
Ile Tyr Asp Leu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe Ser Leu
50 55 60
Val Gly Tyr Gly Arg Arg Glu Trp Ser Lys Glu Asp Phe Glu Lys Tyr
65 70 75 80
Val Arg Asp Ala Ala Ser Ala Gly Ala Arg Thr Glu Phe Arg Glu Asn
85 90 95
Val Trp Glu Arg Leu Ala Glu Gly Met Glu Phe Val Arg Gly Asn Phe
100 105 110
Asp Asp Asp Ala Ala Phe Asp Asn Leu Ala Ala Thr Leu Lys Arg Ile
115 120 125
Asp Lys Thr Arg Gly Thr Ala Gly Asn Trp Ala Tyr Tyr Leu Ser Ile
130 135 140
Pro Pro Asp Ser Phe Thr Ala Val Cys His Gln Leu Glu Arg Ser Gly
145 150 155 160
Met Ala Glu Ser Thr Glu Glu Ala Trp Arg Arg Val Ile Ile Glu Lys
165 170 175
Pro Phe Gly His Asn Leu Glu Ser Ala His Glu Leu Asn Gln Leu Val
180 185 190
Asn Ala Val Phe Pro Glu Ser Ser Val Phe Arg Ile Asp His Tyr Leu
195 200 205
Gly Lys Glu Thr Val Gln Asn Ile Leu Ala Leu Arg Phe Ala Asn Gln
210 215 220
Leu Phe Glu Pro Leu Trp Asn Ser Asn Tyr Val Asp His Val Gln Ile
225 230 235 240
Thr Met Ala Glu Asp Ile Gly Leu Gly Gly Arg Ala Gly Tyr Tyr Asp
245 250 255
Gly Ile Gly Ala Pro Arg Asp Val Ile Gln Asn His Leu Ile Gln Leu
260 265 270
Leu Ala Leu Val Ala Met Glu Glu Pro Ile Ser Phe Val Pro Ala Ala
275 280 285
Arg Gln Ala Glu Lys Ile Lys Val Leu Ser Ala Thr Lys Pro Cys Tyr
290 295 300
Pro Leu Asp Lys Thr Ser Ala Arg Gly Gln Tyr Ala Ala Gly Trp Gln
305 310 315 320
Gly Ser Glu Leu Val Lys Gly Leu Arg Glu Glu Asp Gly Phe Asn Pro
325 330 335
Glu Ser Thr Thr Glu Thr Phe Ala Ala Cys Thr Leu Glu Ile Thr Ser
340 345 350
Arg Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg Thr Gly Lys Arg Leu
355 360 365
Gly Arg Arg Val Thr Glu Ile Ala Val Val Phe Lys Asp Ala Pro His
370 375 380
Gln Pro Phe Asp Gly Asp Met Thr Val Ser Leu Gly Gln Asn Ala Ile
385 390 395 400
Val Ile Arg Val Gln Pro Asp Glu Gly Val Leu Ile Arg Phe Gly Ser
405 410 415
Lys Val Pro Gly Ser Ala Met Glu Val Arg Asp Val Asn Met Asp Phe
420 425 430
Ser Tyr Ser Glu Ser Phe Thr Glu Glu Ser Pro Glu Ala Tyr Glu Arg
435 440 445
Leu Ile Leu Asp Ala Leu Leu Asp Glu Ser Ser Leu Phe Pro Thr Asn
450 455 460
Glu Glu Val Glu Leu Ser Trp Lys Ile Leu Asp Pro Ile Leu Glu Ala
465 470 475 480
Trp Asp Ala Asp Gly Glu Pro Glu Asp Tyr Pro Ala Gly Thr Trp Gly
485 490 495
Pro Lys Ser Ala Asp Glu Met Leu Ser Arg Asn Gly His Thr Trp Arg
500 505 510
Arg Pro
<210> 8
<211> 319
<212> PRT
<213> Corynebacterium glutamicum AS019
<400> 8
Met Ile Phe Glu Leu Pro Asp Thr Thr Thr Gln Gln Ile Ser Lys Thr
1 5 10 15
Leu Thr Arg Leu Arg Glu Ser Gly Thr Gln Val Thr Thr Gly Arg Val
20 25 30
Leu Thr Leu Ile Val Val Thr Asp Ser Glu Ser Asp Val Ala Ala Val
35 40 45
Thr Glu Ser Thr Asn Glu Ala Ser Arg Glu His Pro Ser Arg Val Ile
50 55 60
Ile Leu Val Val Gly Asp Lys Thr Ala Glu Asn Lys Val Asp Ala Glu
65 70 75 80
Val Arg Ile Gly Gly Asp Ala Gly Ala Ser Glu Met Ile Ile Met His
85 90 95
Leu Asn Gly Pro Val Ala Asp Lys Leu Gln Tyr Val Val Thr Pro Leu
100 105 110
Leu Leu Pro Asp Thr Pro Ile Val Ala Trp Trp Pro Gly Glu Ser Pro
115 120 125
Lys Asn Pro Ser Gln Asp Pro Ile Gly Arg Ile Ala Gln Arg Arg Ile
130 135 140
Thr Asp Ala Leu Tyr Asp Arg Asp Asp Ala Leu Glu Asp Arg Val Glu
145 150 155 160
Asn Tyr His Pro Gly Asp Thr Asp Met Thr Trp Ala Arg Leu Thr Gln
165 170 175
Trp Arg Gly Leu Val Ala Ser Ser Leu Asp His Pro Pro His Ser Glu
180 185 190
Ile Thr Ser Val Arg Leu Thr Gly Ala Ser Gly Ser Thr Ser Val Asp
195 200 205
Leu Ala Ala Gly Trp Leu Ala Arg Arg Leu Lys Val Pro Val Ile Arg
210 215 220
Glu Val Thr Asp Ala Pro Thr Val Pro Thr Asp Glu Phe Gly Thr Pro
225 230 235 240
Leu Leu Ala Ile Gln Arg Leu Glu Ile Val Arg Thr Thr Gly Ser Ile
245 250 255
Ile Ile Thr Ile Tyr Asp Ala His Thr Leu Gln Val Glu Met Pro Glu
260 265 270
Ser Gly Asn Ala Pro Ser Leu Val Ala Ile Gly Arg Arg Ser Glu Ser
275 280 285
Asp Cys Leu Ser Glu Glu Leu Arg His Met Asp Pro Asp Leu Gly Tyr
290 295 300
Gln His Ala Leu Ser Gly Leu Ser Ser Val Lys Leu Glu Thr Val
305 310 315
<210> 9
<211> 960
<212> DNA
<213> Corynebacterium glutamicum AS019
<220>
<221> CDS
<222> (1)..(957)
<223> opcA
<400> 9
atg atc ttt gaa ctt ccg gat acc acc acc cag caa att tcc aag acc 48
Met Ile Phe Glu Leu Pro Asp Thr Thr Thr Gln Gln Ile Ser Lys Thr
1 5 10 15
cta act cga ctg cgt gaa tcg ggc acc cag gtc acc acc ggc cga gtg 96
Leu Thr Arg Leu Arg Glu Ser Gly Thr Gln Val Thr Thr Gly Arg Val
20 25 30
ctc acc ctc atc gtg gtc act gac tcc gaa agc gat gtc gct gca gtt 144
Leu Thr Leu Ile Val Val Thr Asp Ser Glu Ser Asp Val Ala Ala Val
35 40 45
acc gag tcc acc aat gaa gcc tcg cgc gag cac cca tct cgc gtg atc 192
Thr Glu Ser Thr Asn Glu Ala Ser Arg Glu His Pro Ser Arg Val Ile
50 55 60
att ttg gtg gtt ggc gat aaa act gca gaa aac aaa gtt gac gca gaa 240
Ile Leu Val Val Gly Asp Lys Thr Ala Glu Asn Lys Val Asp Ala Glu
65 70 75 80
gtc cgt atc ggt ggc gac gct ggt gct tcc gag atg atc atc atg cat 288
Val Arg Ile Gly Gly Asp Ala Gly Ala Ser Glu Met Ile Ile Met His
85 90 95
ctc aac gga cct gtc gct gac aag ctc cag tat gtc gtc aca cca ctg 336
Leu Asn Gly Pro Val Ala Asp Lys Leu Gln Tyr Val Val Thr Pro Leu
100 105 110
ttg ctt cct gac acc ccc atc gtt gct tgg tgg cca ggt gaa tca cca 384
Leu Leu Pro Asp Thr Pro Ile Val Ala Trp Trp Pro Gly Glu Ser Pro
115 120 125
aag aat cct tcc cag gac cca att gga cgc atc gca caa cga cgc atc 432
Lys Asn Pro Ser Gln Asp Pro Ile Gly Arg Ile Ala Gln Arg Arg Ile
130 135 140
act gat gct ttg tac gac cgt gat gac gca cta gaa gat cgt gtt gag 480
Thr Asp Ala Leu Tyr Asp Arg Asp Asp Ala Leu Glu Asp Arg Val Glu
145 150 155 160
aac tat cac cca ggt gat acc gac atg acg tgg gcg cgc ctt acc cag 528
Asn Tyr His Pro Gly Asp Thr Asp Met Thr Trp Ala Arg Leu Thr Gln
165 170 175
tgg cgg gga ctt gtt gcc tcc tca ttg gat cac cca cca cac agc gaa 576
Trp Arg Gly Leu Val Ala Ser Ser Leu Asp His Pro Pro His Ser Glu
180 185 190
atc act tcc gtg agg ctg acc ggt gca agc ggc agt acc tcg gtg gat 624
Ile Thr Ser Val Arg Leu Thr Gly Ala Ser Gly Ser Thr Ser Val Asp
195 200 205
ttg gct gca ggc tgg ttg gcg cgg agg ctg aaa gtg cct gtg atc cgc 672
Leu Ala Ala Gly Trp Leu Ala Arg Arg Leu Lys Val Pro Val Ile Arg
210 215 220
gag gtg aca gat gct ccc acc gtg cca acc gat gag ttt ggt act cca 720
Glu Val Thr Asp Ala Pro Thr Val Pro Thr Asp Glu Phe Gly Thr Pro
225 230 235 240
ctg ctg gct atc cag cgc ctg gag atc gtt cgc acc acc ggc tcg atc 768
Leu Leu Ala Ile Gln Arg Leu Glu Ile Val Arg Thr Thr Gly Ser Ile
245 250 255
atc atc acc atc tat gac gct cat acc ctt cag gta gag atg ccg gaa 816
Ile Ile Thr Ile Tyr Asp Ala His Thr Leu Gln Val Glu Met Pro Glu
260 265 270
tcc ggc aat gcc cca tcg ctg gtg gct att ggt cgt cga agt gag tcc 864
Ser Gly Asn Ala Pro Ser Leu Val Ala Ile Gly Arg Arg Ser Glu Ser
275 280 285
gac tgc ttg tct gag gag ctt cgc cac atg gat cca gat ttg ggc tac 912
Asp Cys Leu Ser Glu Glu Leu Arg His Met Asp Pro Asp Leu Gly Tyr
290 295 300
cag cac gca cta tcc ggc ttg tcc agc gtc aag ctg gaa acc gtc taa 960
Gln His Ala Leu Ser Gly Leu Ser Ser Val Lys Leu Glu Thr Val
305 310 315
<210> 10
<211> 319
<212> PRT
<213> Corynebacterium glutamicum AS019
<400> 10
Met Ile Phe Glu Leu Pro Asp Thr Thr Thr Gln Gln Ile Ser Lys Thr
1 5 10 15
Leu Thr Arg Leu Arg Glu Ser Gly Thr Gln Val Thr Thr Gly Arg Val
20 25 30
Leu Thr Leu Ile Val Val Thr Asp Ser Glu Ser Asp Val Ala Ala Val
35 40 45
Thr Glu Ser Thr Asn Glu Ala Ser Arg Glu His Pro Ser Arg Val Ile
50 55 60
Ile Leu Val Val Gly Asp Lys Thr Ala Glu Asn Lys Val Asp Ala Glu
65 70 75 80
Val Arg Ile Gly Gly Asp Ala Gly Ala Ser Glu Met Ile Ile Met His
85 90 95
Leu Asn Gly Pro Val Ala Asp Lys Leu Gln Tyr Val Val Thr Pro Leu
100 105 110
Leu Leu Pro Asp Thr Pro Ile Val Ala Trp Trp Pro Gly Glu Ser Pro
115 120 125
Lys Asn Pro Ser Gln Asp Pro Ile Gly Arg Ile Ala Gln Arg Arg Ile
130 135 140
Thr Asp Ala Leu Tyr Asp Arg Asp Asp Ala Leu Glu Asp Arg Val Glu
145 150 155 160
Asn Tyr His Pro Gly Asp Thr Asp Met Thr Trp Ala Arg Leu Thr Gln
165 170 175
Trp Arg Gly Leu Val Ala Ser Ser Leu Asp His Pro Pro His Ser Glu
180 185 190
Ile Thr Ser Val Arg Leu Thr Gly Ala Ser Gly Ser Thr Ser Val Asp
195 200 205
Leu Ala Ala Gly Trp Leu Ala Arg Arg Leu Lys Val Pro Val Ile Arg
210 215 220
Glu Val Thr Asp Ala Pro Thr Val Pro Thr Asp Glu Phe Gly Thr Pro
225 230 235 240
Leu Leu Ala Ile Gln Arg Leu Glu Ile Val Arg Thr Thr Gly Ser Ile
245 250 255
Ile Ile Thr Ile Tyr Asp Ala His Thr Leu Gln Val Glu Met Pro Glu
260 265 270
Ser Gly Asn Ala Pro Ser Leu Val Ala Ile Gly Arg Arg Ser Glu Ser
275 280 285
Asp Cys Leu Ser Glu Glu Leu Arg His Met Asp Pro Asp Leu Gly Tyr
290 295 300
Gln His Ala Leu Ser Gly Leu Ser Ser Val Lys Leu Glu Thr Val
305 310 315
<210> 11
<211> 15
<212> PRT
<213> Corynebacterium glutamicum ATCC13032
<400> 11
Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Trp Xaa Asn Pro Leu Arg Asp
1 5 10 15
<210> 12
<211> 15
<212> PRT
<213> Corynebacterium glutamicum ATCC13032
<400> 12
Met Ile Phe Xaa Leu Pro Asp Xaa Xaa Xaa Gln Gln Ile Ser Lys
1 5 10 15
Claims (14)
- a) 서열 3에 따르는 아미노산 서열을 암호화하는 폴리뉴클레오타이드 및b ) a)의 폴리뉴클레오타이드와 상보적인 폴리뉴클레오타이드로 이루어진 군으로부터 선택된 폴리뉴클레오타이드 서열을 포함하는, 코리네형 세균으로부터 분리된 폴리뉴클레오타이드.
- 제1항에 있어서, 코리네형 세균중에서 복제할 수 있으며 추가로 유전자 tal, tkt, zwf 및 devB로 이루어진 그룹 중에서 선택되는 하나 이상의 유전자를 암호화하는 뉴클레오타이드 서열을 포함하는 재조합 DNA인 폴리뉴클레오타이드.
- 제1항에 있어서, RNA인 폴리뉴클레오타이드.
- 제1항에 있어서, 서열 4에 나타낸 뉴클레오타이드 서열을 포함하는 폴리뉴클레오타이드.
- 삭제
- 삭제
- 제1항에 따른 복제할 수 있는 DNA를 도입시킴으로써 형질전환시킨 코리네형 미생물.
- a) 제1항에 따른 폴리뉴클레오타이드의 opcA 유전자 외에, 서열 2에 따른 아미노산 서열을 암호화하는 폴리뉴클레오티드의 zwf 유전자 및 임의로 tkt 유전자 또는 devB 유전자중 하나 이상을 포함하는 플라스미드 벡터로 형질전환되어 상기 유전자가 과발현되는, 목적하는 L-아미노산을 생산하는 세균을 발효시키는 단계,b) 배지중 또는 세균의 세포중 및 농축된 세균 세포중에서 목적하는 생성물을 농축시키는 단계, 및c) 목적하는 L-아미노산을 분리시키는 단계를 수행함을 특징으로 하는, L-아미노산의 제조 방법.
- 제8항에 있어서, 언급된 유전자 외에, 펜토스 포스페이트 사이클의 하나 이상의 추가 유전자(들)이 과-발현되는 방법.
- 삭제
- 제8항에 있어서, 라이신을 제조하기 위하여, 제1항에 따른 폴리뉴클레오타이드의 opcA 유전자 외에,11.1 디하이드로디피콜리네이트 신타제를 암호화하는 dapA 유전자,11.2 피드백 내성 아스파르테이트 키나제를 암호화하는 lysC 유전자,11.3 글리세롤알데하이드 3-포스페이트 데하이드로게나제를 암호화하는 gap 유전자,11.4 피루베이트 카복실라제를 암호화하는 pyc 유전자,11.5 트랜스케톨라제를 암호화하는 tkt 유전자,11.6 6-포스포글루코네이트 데하이드로게나제를 암호화하는 gnd 유전자,11.7 라이신 수송체를 암호화하는 lysE 유전자,11.8 zwa1 유전자,11.9 에놀라제를 암호화하는 eno 유전자,11.10 트랜스알돌라제를 암호화하는 tal 유전자 및11.11 zwf 유전자로 이루어진 군으로부터 선택된 하나 이상의 유전자가 동시에 증폭된 세균을 발효시키는 방법.
- 제8항에 있어서, L-라이신을 제조하기 위하여,12.1 포스포에놀 피루베이트 카복시키나제를 암호화하는 pck 유전자,12.2 글루코스 6-포스페이트 이소머라제를 암호화하는 pgi 유전자,12.3 피루베이트 옥시다제를 암호화하는 poxB 유전자 또는12.4 zwa2 유전자로 이루어진 군으로부터 선택된 하나 이상의 유전자가 동시에 감쇠되어 있는 세균을 발효시키는 방법.
- 폴리머라제 연쇄 반응에 의해 opcA 유전자에 상응하는 효과를 나타내는 유전자의 DNA 제조를 위한, 제1항에 따른 폴리뉴클레오타이드 서열의 15개 이상의 연속 뉴클레오타이드로 이루어진 프라이머.
- 제1항에 따른 폴리뉴클레오타이드 서열의 15개 이상의 연속 뉴클레오타이드로 이루어진 하이브리드화 프로브.
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US09/531,267 | 2000-03-20 | ||
PCT/EP2000/006300 WO2001004322A1 (en) | 1999-07-09 | 2000-07-05 | Nucleotide sequences which code for the opca gene |
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US7267967B1 (en) * | 1997-10-04 | 2007-09-11 | Forschungszentrum Julich Gmbh | Nucleic acid encoding pyruvate carboxylase from coryneform glutamicum |
US6822084B1 (en) * | 1999-06-25 | 2004-11-23 | Basf Aktiengesellschaft | Corynebacterium glutamicum genes encoding stress, resistance and tolerance proteins |
US7270984B1 (en) * | 1999-06-25 | 2007-09-18 | Basf Aktiengesellschaft | Polynucleotides encoding a 6-phosphogluconolactonase polypeptide from corynebacterium glutamicum |
US6797509B1 (en) * | 1999-07-09 | 2004-09-28 | Degussa-Huls Ag | Nucleotide sequences which code for the tal gene |
US20060014259A9 (en) * | 1999-07-09 | 2006-01-19 | Kevin Burke | Process for the preparation of L-amino acids with amplification of the zwf gene |
DE19947791A1 (de) * | 1999-10-05 | 2001-04-12 | Degussa | Neue für das eno-Gen codierende Nukleotidsequenzen |
US6713289B2 (en) | 1999-10-05 | 2004-03-30 | Degussa Ag | Nucleotide sequences which code for the eno gene |
US20050112733A1 (en) * | 2000-03-20 | 2005-05-26 | Degussa Ag | Process for the preparation of L-amino acids with amplification of the zwf gene |
ATE292687T1 (de) * | 2000-03-20 | 2005-04-15 | Degussa | Verfahren zur fermentativen herstellung von l- aminosäuren unter verwendung eines amplifizierten gnd-gens |
WO2001098472A1 (fr) * | 2000-06-21 | 2001-12-27 | Kyowa Hakko Kogyo Co., Ltd | Nouvelle glucose-6-phosphate deshydrogenase |
US20030017554A1 (en) * | 2000-11-15 | 2003-01-23 | Mechthild Rieping | Process for the fermentative preparation of L-amino acids using strains of the enterobacteriaceae family |
DE10154180A1 (de) * | 2001-11-05 | 2003-05-15 | Basf Ag | gene die für genetische Stabilitäts-, genexpressions-und Faltungsproteine codieren |
DE10155505A1 (de) * | 2001-11-13 | 2003-05-22 | Basf Ag | Gene die für Glucose-6-Phosphat-Dehydrogenase Proteine codieren |
DE10210527A1 (de) | 2002-03-09 | 2003-09-18 | Degussa | Allele des aceA-Gens aus coryneformen Bakterien |
US20070092951A1 (en) | 2005-03-24 | 2007-04-26 | Degussa Ag | Alleles of the zwf gene from coryneform bacteria |
DE102005023829A1 (de) | 2005-05-24 | 2006-11-30 | Degussa Ag | Allele des opcA-Gens aus coryneformen Bakterien |
US8647642B2 (en) | 2008-09-18 | 2014-02-11 | Aviex Technologies, Llc | Live bacterial vaccines resistant to carbon dioxide (CO2), acidic PH and/or osmolarity for viral infection prophylaxis or treatment |
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CN104845923B (zh) * | 2014-02-14 | 2018-03-23 | 中国科学院微生物研究所 | 生产l‑组氨酸的方法及其专用重组菌 |
US11129906B1 (en) | 2016-12-07 | 2021-09-28 | David Gordon Bermudes | Chimeric protein toxins for expression by therapeutic bacteria |
US11180535B1 (en) | 2016-12-07 | 2021-11-23 | David Gordon Bermudes | Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria |
WO2019182413A1 (ko) | 2018-03-23 | 2019-09-26 | 씨제이제일제당 (주) | L-아미노산을 포함하는 과립 및 이의 제조방법 |
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