KR101835935B1 - L-아르기닌을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 l-아르기닌의 제조 방법 - Google Patents
L-아르기닌을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 l-아르기닌의 제조 방법 Download PDFInfo
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- KR101835935B1 KR101835935B1 KR1020150076331A KR20150076331A KR101835935B1 KR 101835935 B1 KR101835935 B1 KR 101835935B1 KR 1020150076331 A KR1020150076331 A KR 1020150076331A KR 20150076331 A KR20150076331 A KR 20150076331A KR 101835935 B1 KR101835935 B1 KR 101835935B1
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Abstract
본 발명은 L-아르기닌을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 L-아르기닌을 생산하는 방법에 관한 것이다.
Description
본 발명은 L-아르기닌을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 L-아르기닌을 생산하는 방법에 관한 것이다.
L-아르기닌은 아미노산류 강화제, 의약품 및 식품 등에 널리 이용되는 아미노산으로, 산업계에서는 L-아르기닌을 효율적으로 생산하는 방법의 개발이 요구되어 왔다.
종래에 알려져 있는 생물학적 발효법에 의한 L-아르기닌의 제조방법은 탄소원, 질소원으로부터 직접 L-아르기닌을 생산하는 방법으로서, 글루타민산(glutamate) 생산 균주인 브레비박테리움(Brevibacterium) 또는 코리네박테리움(Corynebacterium)속 미생물로부터 유도된 변이주를 이용하는 방법, 세포융합으로 생육 개선된 아미노산 생산 균주를 이용하는 방법 등이 보고되었다. 최근에는 아르기닌 생합성 오페론의 발현을 억제하는 유전자 argR을 불활성화시킨 유전자 재조합 균주를 이용하는 방법(미국 등록특허 US 7,160,705)과 아르기닌 오페론의 argF를 과발현(over-expression)시키는 방법(대한민국 등록특허 제10-0854234호) 등이 보고되어 있다. 특히 종래 아르기닌 생산에 있어서 아르기닌 오페론을 조절하는 argR의 결손이 중요한 요소로 생각되어 왔다.
코리네박테리움 미생물의 경우, 아르기닌 생합성에 관여하는 argCJBDFR 유전자가 오페론(operon)의 형태로 이루어져 있으며, 세포 내 아르기닌에 의해서 피드백 저해(feedback-inhibition)를 받는 것으로 현재까지 알려져 있어(Vehary Sakanyan, et al, Microbiology, 142:9-108, 1996), 고수율의 L-아르기닌을 생산하는 데에는 한계가 있는 것으로 알려져 있다.
이에, 본 발명자들은 L-아르기닌의 생산 수율을 높이고자 노력하였으며, 그 결과 기존에 중요한 요소로 알려진 아르기닌 리프레서(argR)의 결손없이 아르기닌 오페론 강화와 오르니틴 카바모일트랜스퍼라제 활성을 강화함으로써 L-아르기닌 생산 모균주에 비해 고수율로 L-아르기닌을 생산할 수 있음을 확인하고 본 발명을 완성하게 되었다.
본 발명의 목적은 L-아르기닌을 생산하는 코리네박테리움 속 미생물을 제공하는 것이다.
본 발명의 또 다른 목적은, 상기 코리네박테리움 속 미생물을 이용하여 L-아르기닌의 생산 방법을 제공하는 것이다.
상기의 목적을 달성하기 위한 하나의 양태로서, 본 발명은 아르기닌 오페론 및 오르니틴 카바모일트랜스퍼라제의 활성이 강화된 L-아르기닌을 생산하는 코리네박테리움 속 미생물을 제공한다.
본 발명에서 아르기닌 오페론은 L-아르기닌을 생합성하는 기작에 관여하는 효소들로 이루어진 오페론으로, 특히 L-아르기닌의 생합성의 고리형 단계를 구성하는 효소들로 이루어져 있다. 구체적으로, N-아세틸글루타밀포스페이트 환원효소(N-acetyl glutamylphosphate reductase, ArgC), 글루타메이트 N-아세틸트랜스퍼라제(glutamate N-acetyltransferase, ArgJ), N-아세틸글루타메이트 키나아제(N-acetylglutamate kinase, ArgB), 아세틸오르니틴 아미노트랜스퍼라제(acetylornithine aminotransferase, ArgD), 오르니틴 카바모일트랜스퍼라제(ornithine cabomoyltransferase, ArgF) 및 아르기닌 억제자(argine repressor, ArgR)로 구성되어 있으며, 상기 효소들은 L-아르기닌 생합성 과정의 연속적인 효소반응에 관여한다.
아르기닌 오페론을 구성하는 상기 효소들은 L-글루타메이트를 전구체로 하여 최종적으로 L-아르기닌을 생합성하는데 관여한다. 글루타메이트 N-아세틸트랜스퍼라제(ArgJ)는 L-글루타메이트(L-glutamate)를 전구체로 하여 N-아세틸글루타메이트(N-acetylglutamate)를 합성하며 argJ 유전자로 코딩되는 것일 수 있다. 이때의 아세틸기(acetyl group)는 N-아세틸오르니틴(N-acetylornithine)을 L-오르니틴(L-ornithine)으로 분해하여 얻는다. 코리네박테리움 속 미생물에서의 L-아르기닌 생합성은 이 글루타메이트 N-아세틸트랜스퍼라제가 리사이클링 반응을 한다고 알려져 있다.
생성된 N-아세틸글루타메이트(N-acetylglutamate)는 N-아세틸글루타메이트 키나아제(ArgB) 효소에 의해 N-아세틸글루타밀 포스페이트(N-acetylglutamyl phosphate)로 합성되며, 조효소로 ATP가 소모되어 ADP가 생성되고, argB 유전자로 코딩되는 것일 수 있다. 최종 산물인 L-아르기닌에 의해 피드백 저해를 받는다고 알려져 있어, L-아르기닌에 의한 피드백 저해를 해제하는 변이가 공지되어 있으며, 이를 활용하여 L-아르기닌 생산능을 향상 시킬 수 있다고 보고 되고 있다(등록특허 CN 102021154 및 Amino Acids. 2012 Jul;43(1):255-66. doi: 10.1007/s00726-011-1069-x. Epub 2011 Sep 8.).
N-아세틸글루타밀포스페이트 환원효소(ArgC)는 대장균이나 효모에서는 아세틸글루타메이트 세미알데히드 디하이드로게나제(acetylglutamate semialdehyde dehydrogenase)로도 불리기도 하며, argC 유전자로 코딩되는 것일 수 있다. 상기 효소에 의해 N-아세틸글루타밀 포스페이트(N-acetylglutamyl phosphate)를 N-아세틸글루타메이트 5-세미알데하이드(N-acetylglutamate 5-semialdehyde)로 전환한다. 조효소로는 NADPH가 사용되어 에너지를 제공한다. 생성된 N-아세틸글루타메이트 5-세미알데하이드(N-acetylglutamate 5-semialdehyde)는 아미노기 공여체로 L-글루타메이트를 사용하여 N-아세틸오르니틴(N-acetylornithine)으로 전환되며, 이 반응은 아세틸오르니틴 아미노트랜스퍼라제(ArgD) 효소에 의해 매개된다. 아세틸오르니틴 아미노트랜스퍼라제는 argD 유전자로 코딩되는 것일 수 있다. 그리고 전환된 N-아세틸오르니틴(N-acetylornithine)은 글루타메이트 N-아세틸트랜스퍼라제(ArgJ) 효소의 리사이클링 반응에 의해 아세틸기(acetyl group)을 L-글루타메이트에 전달하고 L-오르니틴(L-ornithine)으로 반응된다.
오르니틴 카바모일트랜스퍼라제(ArgF)는 일반적으로 오르니틴 트랜스카바모일라제로도 불리우며, argF 또는 argF2 유전자로 코딩되는 것일 수 있다. L-오르니틴(L-ornithine)과 카바모일 포스페이트(carbamoyl phosphate)를 결합하여 L-시트룰린(L-citrullline)를 생성하며, 인산기(phosphate)가 부반응산물로 생성된다. 생성된 L-시트룰린(L-citrullline)은 상기 특허에서 언급한 아르기닌 오페론과 분리되어 유전자에 존재하는 알지니노숙신산 신타아제(ArgG), 알지니노숙신산 라이에이즈(ArgH) 효소반응에 의해 최종적으로 L-아르기닌으로 합성된다. 총 8단계의 생합성 단계를 거쳐 L-아르기닌을 합성하게 되며, 본 발명에서는 아르기닌 오페론(argCJBDFR)의 활성을 강화함으로써 L-아르기닌 생산능 향상을 유도하였다.
상기 아르기닌 오페론을 구성하는 효소는 상기 활성을 가지기만 하면 유래와 상관없이 본원의 발명의 범위내에 포함되나, 구체적으로 상기 효소는 코리네박테리움 속 유래의 단백질일 수 있다. 더욱 구체적으로 글루타메이트 N-아세틸트랜스퍼라제(ArgJ)는 서열번호 19의 아미노산 서열을 포함할 수 있으며, 또는 이와 최소한 70%, 구체적으로 80%, 더욱 구체적으로 90% 이상의 상동성을 가지는 아미노산 서열을 포함할 수 있다. N-아세틸글루타메이트 키나아제(ArgB)는 서열번호 21의 아미노산 서열을 포함할 수 있으며, 또는 이와 최소한 70%, 구체적으로 80%, 더욱 구체적으로 90% 이상의 상동성을 가지는 아미노산 서열을 포함할 수 있다. 또한 당해 효소의 경우 아르기닌에 의해 피드백 저해를 해제하기 위해 당업계에 공지된 변이를 도입할 수 있다. N-아세틸글루타밀포스페이트 환원효소(ArgC)는 서열번호 23의 아미노산 서열을 포함할 수 있으며, 또는 이와 최소한 70%, 구체적으로 80%, 더욱 구체적으로 90% 이상의 상동성을 가지는 아미노산 서열을 포함할 수 있다. 아세틸오르니틴 아미노트랜스퍼라제(ArgD)는 서열번호 25의 아미노산 서열을 포함할 수 있으며, 또는 이와 최소한 70%, 구체적으로 80%, 더욱 구체적으로 90% 이상의 상동성을 가지는 아미노산 서열을 포함할 수 있다. 오르니틴 카바모일트랜스퍼라제(ArgF)는 서열번호 1 또는 서열번호 3의 아미노산 서열을 포함하는 것 또는 상기 서열번호 1 또는 서열번호 3의 아미노산 서열과 최소한 70%의 상동성을 가지는 아미노산 서열을 포함하는 것일 수 있다. 구체적으로는 상기 서열번호 1 또는 3의 아미노산 서열과 최소한 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 이상의 아미노산 서열 상동성을 나타내는 아미노산 서열을 포함하는 것일 수 있다.
본 발명에서 용어, "상동성(homology)"은 두 비교 대상인 아미노산 서열 또는 염기 서열의 유사한 정도를 나타내기 위한 것으로서, 이러한 상동성은 두 서열을 육안으로 비교하여 결정할 수도 있으나, 비교대상이 되는 서열을 나란히 배열하여 상동성 정도를 분석해 주는 생물정보 알고리즘(bioinformatic algorithm)을 사용하여 결정할 수 있다. 상기 두 개의 아미노산 서열 사이의 상동성은 백분율로 표시할 수 있다. 유용한 자동화된 알고리즘은 Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA)의 GAP, BESTFIT, FASTA와 TFASTA 컴퓨터 소프트웨어 모듈에서 이용가능하다. 다른 유용한 배열에 대한 알고리즘과 상동성 결정은 FASTP, BLAST, BLAST2,PSIBLAST와 CLUSTAL W를 포함하는 소프트웨어에서 자동화되어 있다.
본 발명에서 상기 아르기닌 오페론 활성의 강화는 아르기닌 오페론에 존재하는 효소들 중 하나 이상의 효소의 활성이 강화되는 것을 의미하나, argR 유전자를 단독으로 강화하는 경우는 포함되지 않는다. 예를 들어, 아르기닌 오페론 활성의 강화는 아르기닌 오페론에 존재하는 하나의 효소에 대한 프로모터를 강화하는 것을 통하여 오페론에 존재하는 모든 효소의 활성이 강화되는 것일 수 있으며, 구체적으로 N-아세틸글루타밀포스페이트 환원효소(argC)에 대한 프로모터 강화하는 것을 통하여 오페론 전체 활성이 강화되는 것일 수 있다. 또한, 아르기닌 오페론을 구성하는 효소들 중 하나 이상의 효소를 암호화하는 유전자의 발현을 증가시키는 것 역시 본원에서 아르기닌 오페론 활성의 강화에 해당할 수 있다.
본 발명의 용어, 활성의 '강화'는 특정 단백질 활성을 가지고 있지 않은 미생물에 그 단백질의 활성을 부여하거나, 그 단백질의 활성을 보유하고 있는 미생물에서 세포 내 활성을 증가시키는 등으로, 상기 단백질의 세포 내 활성을 내재적 활성에 비해 증가시키는 것을 의미한다. 상기 내재적 활성이란 여기서 내재적 활성이란 코리네박테리아 속 미생물이 천연의 상태로 가지고 있거나 변형 전 상태로 가지고 있는 효소의 활성상태를 의미한다.
상기 효소의 활성을 강화 또는 증가시키는 방법은 당해 분야에서 잘 알려진 다양한 방법의 적용이 가능하다. 그 방법의 예는, 하기 예로 제한되는 것은 아니지만, 해당 효소들을 암호화하는 염기서열을 포함하는 폴리뉴클레오타이드를 추가로 염색체에 삽입하는 방법 또는 상기 폴리뉴클레오타이드를 벡터 시스템에 도입하는 방법 등에 의하여 효소들을 암호화하는 염기서열의 카피수를 증가시키는 방법, 효소들의 프로모터를 강한 프로모터로 교체하는 방법, 구체적으로 프로모터에 변이를 도입하는 방법이 포함될 수 있으며 유전자 변이에 의해 활성이 강한 효소로 변이시키는 방법 등이 있다.
구체적인 예로, 본 발명에서는 아르기닌 오페론에 존재하는 효소의 프로모터를 변이 또는 치환을 통해 내재적 프로모터에 비하여 강한 프로모터로 변이시킬 수 있다. 상기 내재적 효소의 프로모터 대신 염기치환 변이를 갖는 개량형 프로모터 또는 이종 프로모터가 연결될 수 있는데, 상기 이종 프로모터의 예로는 pcj7 프로모터(대한민국 등록특허 제10-0620092호), lysCP1 프로모터(대한민국 등록특허 제10-0930203호), EF-Tu 프로모터, groEL 프로모터, aceA 프로모터, aceB 프로모터 등이 있으나 이에 한정되는 것은 아니다.
본 발명의 용어, "프로모터"는 폴리머라제에 대한 결합 부위를 포함하고 프로모터 하위 유전자의 mRNA로의 전사 개시 활성을 가지는, 암호화 영역의 상위(upstream)의 비해독된 핵산서열, 즉, 폴리머라제가 결합하여 유전자의 전사를 개시하도록 하는 DNA 영역을 말하며, mRNA 전사 개시부위의 5' 부위에 위치한다.
삭제
본 발명에서 상기 오르니틴 카바모일트랜스퍼라제 활성을 강화시키는 방법은 당해 분야에서 잘 알려진 다양한 방법의 적용이 가능하며, 이는 상기에서 기술된 바와 같다. 구체적으로 오르니틴 카바모일트랜스퍼라제를 암호화하는 폴리뉴클레오타이드를 포함하는 발현 벡터를 균주에 형질전환하는 것을 통해 이루어지는 것일 수 있으나, 이에 한정되는 것은 아니다.
삭제
본 발명의 용어 "형질전환"은 DNA를 숙주내로 도입하여 DNA가 염색체외 인자로서 또는 염색체 통합완성에 의해 복제가능하게 되는 것을 의미한다. 구체적으로 본 발명의 형질전환체는 상기 DNA를 포함하는 벡터가 숙주 세포 내로 형질전환된 후, 벡터 내의 프로모터 활성을 갖는 핵산 분자 서열이 숙주 세포 게놈 상의 내생적(endogeneous) 목적 유전자의 프로모터 부위의 서열과 상동 재조합을 일으키며 염색체 내로 삽입되거나, 플라스미드 형태로 보유할 수 있다.
본 발명의 벡터를 형질전환시키는 방법은 핵산을 세포 내로 도입하는 어떤 방법도 포함되며, 숙주세포에 따라 당 분야에서 공지된 바와 같이 적합한 표준 기술을 선택하여 수행할 수 있다. 예를 들어, 전기천공법(electroporation), 인산칼슘(CaPO4) 침전, 염화칼슘(CaCl2) 침전, 미세주입법(microinjection), 폴리에틸렌글리콜(PEG)법, DEAE-덱스트란법, 양이온 리포좀법, 및 초산 리튬-DMSO법 등이 사용될 수 있으나, 이에 한정되지 않는다.
본 발명의 용어, "코리네박테리움 속 미생물(Corynebacterium sp.)"은 L-아르기닌 생산능을 갖는 모든 코리네박테리움 속 균주를 포함할 수 있으며, 그 예로 코리네박테리움 글루타미쿰(Corynebacterium glutamicum), 코리네박테리움 암모니아게네스(Corynebacterium ammoniagenes), 코리네박테리움 써모아미노게네스(Corynebacterium thermoaminogenes), 브레비박테리움 플라붐(Brevibacterium flavum) 또는 브레비박테리움 락토페르멘툼(Brevibacterium fermentum) 등이 사용될 수 있으나, 이에 제한되지는 않는다. 구체적으로는 코리네박테리움 글루타미쿰(Corynebacterium glutamicum)을 사용할 수 있지만, 이들 예에 한정되는 것은 아니다.
본 발명의 또 하나의 양태로서, 본 발명은 상기 L-아르기닌을 생산하는 코리네박테리움 속 미생물을 적절한 배양 배지에서 배양하는 단계를 포함하는 L-아르기닌을 생산하는 방법을 제공한다.
본 발명에서 미생물의 배양은 널리 공지된 방법에 따라서 수행될 수 있고, 배양 온도, 배양 시간 및 배지의 pH 등의 조건은 적절하게 조절될 수 있다. 이들 공지된 배양 방법은 문헌[Chmiel; Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik(Gustav Fischer Verlag, Stuttgart, 1991), 및 Storhas; Bioreaktoren und periphere Einrichtungen(Vieweg Verlag, Braunschweig / Wiesbaden, 1994)]에 상세히 기술되어 있다. 또한, 배양 방법에는 회분식 배양(batch culture), 연속식 배양(continuous culture) 및 유가식 배양(fed-batch culture)이 포함되며, 구체적으로는 배치 공정 또는 주입 배치 또는 반복 주입 배치 공정(fed batch or repeated fed batch process)에서 연속식으로 배양할 수 있으나, 이에 제한되는 것은 아니다.
사용되는 배양 배지는 특정한 균주의 요구 조건을 적절하게 충족시켜야 한다. 다양한 미생물에 대한 배양 배지는 공지되어 있다(예를 들면, "Manual of Methods for General Bacteriology" from American Society for Bacteriology (Washington D.C., USA, 1981)). 배지 내 탄소 공급원은 당 및 탄수화물(예: 글루코오스, 슈크로오스, 락토오스, 프럭토오스, 말토오스, 몰라세, 전분 및 셀룰로오스), 유지 및 지방(예: 대두유, 해바라기씨유, 땅콩유 및 코코넛유), 지방산(예: 팔미트산, 스테아르산 및 리놀레산), 알콜(예: 글리세롤 및 에탄올) 및 유기산(예: 아세트산) 등을 이용할 수 있다. 이들 물질은 개별적으로 또는 혼합물로서 사용될 수 있으나, 이에 한정되지 않는다. 질소 공급원은 질소-함유 유기 화합물(예: 펩톤, 효모 추출액, 육즙, 맥아 추출액, 옥수수 침지액, 대두 박분 및 우레아), 또는 무기 화합물(예: 황산암모늄, 염화암모늄, 인산암모늄, 탄산암모늄 및 질산암모늄)을 이용할 수 있으며, 이들 물질 또한 개별적으로 또는 혼합물로서 사용될 수 있으나, 이에 한정되지 않는다. 인 공급원으로서 인산이수소칼륨 또는 인산수소이칼륨 또는 상응하는 나트륨 함유 염을 이용할 수 있으나, 이에 한정되지 않는다. 배양 배지는 성장에 필수적인 금속염(예: 황산마그네슘 또는 황산철)을 함유할 수 있으며, 아미노산 및 비타민과 같은 필수 성장-촉진 물질을 상기 언급한 물질 외에 사용할 수 있다. 또한, 적합한 전구체를 상기 배양 배지에 추가로 가할 수 있다. 상기 공급 물질은 배양배지에 한번에 모두 가하거나, 배양 중 적절하게 공급할 수 있다.
배양 배지의 pH는 염기성 화합물(예: 수산화나트륨, 수산화칼륨 또는 암모니아) 또는 산성 화합물(예: 인산 또는 황산)을 적절히 사용하여 조절할 수 있다. 발포는 지방산 폴리글리콜 에스테르와 같은 거포제를 사용하여 조절할 수 있다. 산소 또는 산소-함유 가스 혼합물, 예를 들어 공기를 배양배지 중으로 도입시켜 호기성 조건을 유지시킬 수 있다. 배양 온도는 20 내지 45℃, 구체적으로는 25 내지 40℃일 수 있다. 배양은 원하는 L-아미노산의 생성량이 최대로 얻어질 때까지 계속될 수 있으며, 구체적으로 배양시간은 10 내지 160 시간일 수 있다. L-아르기닌은 배양 배지 중으로 배출되거나, 세포 중에 포함되어 있을 수 있다.
한편, 본 발명의 상기한 미생물을 배양하는 단계를 포함하는 L-아르기닌의 생산 방법은 상기 배양하는 단계에서 생성되는 L-아르기닌을 회수하는 단계를 추가로 포함할 수 있다. 즉, 본 발명의 L-아르기닌의 생산 방법은 본 발명의 코리네박테리움 속 미생물을 배양 배지에서 배양하는 단계; 및 상기 배양 배지 및 미생물에서 L-아르기닌을 회수하는 단계를 포함하는 L-아르기닌을 생산하는 방법일 수 있다. 아르기닌을 회수하는 단계는 당업계에 널리 알려져 있는 아르기닌 회수 방법을 이용하여 세포 또는 배양 배지로부터 아르기닌을 분리해내는 것일 수 있다. L-아르기닌 회수 방법의 예로서, 원심분리, 여과, 추출, 분무, 건조, 증방, 침전, 결정화, 전기영동, 분별용해, 크로마토그래피(예를 들면, 이온 교환 크로마토그래피, 친화성, 소수성, 크기배제 및 HPLC) 등의 방법이 있으나, 이들 예에 한정되는 것은 아니다.
본 발명에 따른 아르기닌 오페론 및 오르니틴 카바모일트랜스퍼라제(ArgF 또는 ArgF2)의 활성이 강화된 L-아르기닌을 생산하는 코리네박테리움 속 미생물을 이용하여 고수율로 L-아르기닌을 생산할 수 있다. 또한, 상기 고수율로 생산된 L-아르기닌은 인간 의약 및 약학산업에 유용하게 사용될 수 있을 것이다.
이하, 본 발명을 하기 실시예에 의해 보다 상세하게 설명한다. 단, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것일 뿐 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다.
실시예
1: 아르기닌
오페론
강화 벡터 제작
미생물의 염색체 상에서 아르기닌 오페론을 강화하기 위하여, N-아세틸글루타밀포스페이트 환원효소(N-acetylglutamyl phosphate reductase, ArgC)의 자가 프로모터를 결손하고 다른 프로모터로 치환하는 벡터를 제작하였다. 치환 프로모터로는 강한 발현 유도 활성을 가지는 lysCP1(대한민국 등록특허 제10-0930203호, 서열번호 18)를 이용하였다.
먼저, 야생형 코리네박테리움 글루타미쿰(기탁번호 ATCC13869) 균주의 염색체 DNA를 주형으로 서열번호 13 (SF_pargC_PR_pDC infusion 프라이머; 5'-CGAGCTCGGTACCCGGGCAAAGAATACGGCTTCCTTGGC-3') 및 서열번호 14 (SR_pargC_PR_XbaI-XhoI-BamHI infusion/제한효소 프라이머; 5'-CTGGATCCTCGAGTCTAGAGACGGGTTAGACATGCAAAA-3') 프라이머쌍과 서열번호 15 (SF_pargC_PR_SpeI-ScaI-BamHI infusion/제한효소 프라이머; 5'-GACTCGAGGATCCAGTACTAGTATGATAATCAAGGTTGCAAT-3') 및 서열번호 16 (SR_pargC_PR_pDC infusion 프라이머; 5'-TGCAGGTCGACTCTAGGGTAACGCCTTCTTTCAAAG-3') 프라이머 쌍을 이용하여 1차 PCR(polymerase Chain Reaction)로 DNA 단편을 증폭하였다. 구체적인 PCR 조건은 다음과 같다. PCR 기기(Bio-rad C1000 thermal cycler)와 Pfu 폴리머라제(Pfu polymerase, macrogen)를 이용하여 95 ℃에서 10분간 변성, 55 ℃에서 30초간 어닐링, 72 ℃에서 1분간 중합하는 사이클을 28번 반복하였다.
이렇게 수득한 1차 PCR 단편은 fragment DNA purification kit(geneall)을 이용하여 정제하고, 미리 XmaⅠ-XbaⅠ 제한효소로 절단하여 준비한 pD 벡터와 혼합하여 3개의 DNA 단편을 연결하였다. 상기 DNA 단편들의 연결은 In-fusion Cloning Kit (Clontech)를 사용하여 50 ℃에서 10분 반응을 통해 pD-RargC_PR 벡터를 완성하였다.
치환 프로모터의 삽입은 pDZ-lysCP1 (대한민국 등록특허 제10-0930203호)를 주형으로 서열번호 5(SF_PlysCP1_XhoI-XbaI infusion 프라이머; 5'-CCGTCTCTAGACTCGAGCCATCTTTTGGGGTGCGG-3') 및 서열번호 6 (SR_PlysCP1_SpeI infusion 프라이머; 5'-TTGATTATCATACTAGTCTTTGTGCACCTTTCGAT-3')의 프라이머쌍을 이용하여 lysCP1 프로모터를 증폭하고, 이를 XhoⅠ-SpeⅠ 제한효소 처리된 pD-PargC_PR 벡터와 혼합하여 연결하였다. PCR 및 In-fusion 방법은 상기와 동일하며, 이를 통하여 최종적으로 pD-PargC::lysCP1 벡터를 완성하였다.
실시예
2: 오르니틴
카바모일트랜스퍼라제의
강화 발현벡터 제작
아르기닌 생합성 효소 중 하나인 오르니틴 카바모일트랜스퍼라제를 강화하기 위해 재조합 발현벡터를 제작하였다. p117-cj7-GFP(대한민국 등록특허 제10-0620092호)를 기반 벡터로 이용하였으며, 상기 기반 벡터에서 GFP를 코딩하는 염기서열을 EcoRⅤ-XbaⅠ 제한효소로 처리하여 제거한 후 야생형 코리네박테리움 글루타미쿰 ATCC13869 유래 argF와 대한민국 등록특허 제10-0830290호의 argF2를 삽입하였다.
상기 argF 유전자는 야생형 코리네박테리움 글루타미쿰(기탁번호 ATCC13869) 균주의 염색체 DNA를 주형으로 서열번호 7(SF_argF_EcoRV infusion 프라이머; 5'-ACGAAAGGAAACACTCGATATCATGACTTCACAACCACAGGT-3') 및 서열번호 8(SR_argF_XbaI infusion 프라이머; 5'-GCCAAAACAGCTCTAGATTACCTCGGCTGGTGGGCCA-3') 프라이머 쌍을 이용하여 PCR을 통해 DNA 단편을 증폭하였다. Pfu 폴리머라제를 이용하였으며, 95 ℃에서 10분간 변성, 55 ℃에서 30초간 어닐링, 72 ℃에서 2분간 중합하는 사이클을 28번 반복하는 조건으로 진행하였다. 이렇게 얻어진 PCR 단편을 정제하고 EcoRⅤ-XbaⅠ 제한효소 처리된 p117-cj7-GFP와 혼합하여, In-fusion Cloning 방법으로 연결하여 p117-Pcj7-argF 재조합 발현벡터를 완성하였다.
상기 argF2 유전자는 야생형 코리네박테리움 글루타미쿰(기탁번호 ATCC 13032)의 염색체 DNA를 주형으로 서열번호 9(SF_argF2_EcoRV infusion 프라이머; 5'-ACGAAAGGAAACACTCGATATCATGGCCAGAAAACATCTGCT-3') 및 서열번호 10(SR_argF2_XbaI infusion 프라이머; 5'-GCCAAAACAGCTCTAGACTACGCATTGATCGACCGAG-3') 프라이머쌍과 Pfu 폴리머라제(Pfu polymerase, macrogen)를 이용하여 95 ℃에서 10분간 변성, 55 ℃에서 30초간 어닐링, 72 ℃에서 2분간 중합하는 사이클을 28번 반복하여 수득하였다. 상기 얻어진 PCR 단편을 정제하고 EcoRⅤ-XbaⅠ 제한효소 처리된 p117-cj7-GFP와 혼합하여, In-fusion Cloning Kit를 이용하여 연결하였다. 상기 과정을 통하여 최종적으로 p117-Pcj7-argF2 재조합 발현벡터를 완성하였다.
추가적으로 argF 및 argF2 유전자를 동시 발현하는 재조합 발현벡터를 제작하였다. 상기 제작된 p117-Pcj7-argF 발현벡터를 기반으로 NotI 제한효소를 처리한 후 p117-Pcj7-argF2를 삽입하였다. 구체적으로 p117-Pcj7-argF2 재조합 플라스미드를 주형으로 서열번호 11 (SF_Pcj7_argF2_NotI infusion 프라이머; 5'-CCTTTTTGCGGCGGCCGCAGAAACATCCCAGCGCTACT-3') 및 서열번호 12 (SR_argF2_NotI infusion 프라이머; 5'-CACCGCGGTGGCGGCCGCCGCAAAAAGGCCATCCGTCA-3') 프라이머를 Pfu 폴리머라제를 이용하여 95 ℃에서 10분간 변성, 55 ℃에서 30초간 어닐링, 72 ℃에서 2.5분간 중합하는 사이클을 28번 반복하여 수득하였다. 상기 얻어진 PCR 단편을 정제하고 NotⅠ 제한효소 처리된 p117-Pcj7-argF 와 혼합하여, In-fusion Cloning Kit를 이용하여 연결하였다. 상기 과정을 통하여 최종적으로 p117-Pcj7-argF/Pcj7-argF2 재조합 발현벡터를 완성하였다.
실시예
3: 재조합 벡터 삽입 균주 제작
3-1. 아르기닌
오페론
강화 벡터 삽입
코리네박테리움의 염색체에 아르기닌 오페론의 자가 프로모터를 치환하기 위해 기존 아르기닌 생산균주에 상기 실시예 1에서 제작한 pD-PargC::lysCP1 재조합 벡터를 형질전환시켜 재조합 벡터 삽입 균주를 제작하였다. 구체적으로 기존 아르기닌 생산균주인 KCCM10741P (대한민국 등록특허 제10-07916590호) 및 ATCC21831에 상기 실시예 1에서 제작한 pD-PargC::lysCP1 재조합 벡터를 형질전환시켜, 모균주가 가지고 있는 자가 프로모터 서열과 상기 벡터상의 프로모터 서열을 상동 재조합을 통해 치환시킴으로써, 염색체 내로 lysCP1 프로모터 서열을 삽입시켰다.
형질전환은 1차적으로 전기펄스법(Appl Microbiol Biotechnol. 1999 Oct;52(4):541-5)을 이용하여 재조합 벡터를 KCCM10741P 및 ATCC21831에 삽입시켰으며, 상동성 서열의 재조합에 의해 염색체 상에 삽입된 균주는 카나마이신(kanamycin) 25 mg/L 를 함유한 배지에서 선별하였다. 선별된 1차 균주는 다시 2차 교차(cross-over)를 거쳐, lysCP1 프로모터로 치환되고 벡터가 제거된 균주를 선정하였다. 최종 형질전환된 균주의 프로모터 치환여부는 서열번호 5 및 서열번호 6 프라이머쌍을 사용하여 PCR을 수행함으로써 확인하였고 이 균주를 KCCM10741P_ΔPargC::lysCP1 및 ATCC21831_ΔPargC::lysCP1으로 명명하였다.
3-2. 오르니틴
카바모일트랜스퍼라제
강화 벡터 삽입
상기 실시예 2에서 제작한 p117-Pcj7-argF, p117-Pcj7-argF2, p117-Pcj7-argF/Pcj7-argF2 재조합 발현벡터를 전기펄스법을 이용하여 상기 균주 KCCM10741P_ΔPargC::lysCP1 및 ATCC21831_ΔPargC::lysCP1에 삽입하였으며, 카나마이신 (kanamycin) 25 mg/L를 함유한 배지에서 선별하여 최종적으로 argF, argF2, argF/argF2를 추가발현하는 균주를 제작하였다. 상기 균주를 각각 KCCM10741P_ΔPargC::lysCP1_Pcj7-argF, KCCM10741P_ΔPargC::lysCP1_Pcj7-argF2, KCCM10741P_ΔPargC::lysCP1_Pcj7-argF/Pcj7-argF2, ATCC21831_ΔPargC::lysCP1_Pcj7-argF, ATCC21831_ΔPargC::lysCP1_Pcj7-argF2 및 ATCC21831_ΔPargC::lysCP1_Pcj7-argF/Pcj7-argF2 로 명명하고, 이 중 KCCM10741P_ΔPargC::lysCP1_Pcj7-argF2를 CA06-2044로 재명명하여 부다페스트 조약 하에 2013년 12월 9일자로 한국미생물보존센터(KCCM)에 기탁하여 기탁번호 KCCM11498P를 부여 받았다.
실시예
4: 제작 균주의 평가
상기 실시예 3에서 제작한 아르기닌 생산균주인 코리네박테리움 글루타미쿰 KCCM10741P_ΔPargC::lysCP1, KCCM10741P_ΔPargC::lysCP1_Pcj7-argF, KCCM10741P_ΔPargC::lysCP1_Pcj7-argF2, KCCM10741P_ΔPargC::lysCP1_Pcj7-argF/Pcj7-argF2, ATCC21831_ΔPargC::lysCP1, ATCC21831_ΔPargC::lysCP1_Pcj7-argF, ATCC21831_ΔPargC::lysCP1_Pcj7-argF2 및 ATCC21831_ΔPargC::lysCP1_Pcj7-argF/Pcj7-argF2를 이용하여 아르기닌 오페론과 오르니틴 카바모일트랜스퍼라제의 활성강화가 아르기닌 생산능에 미치는 영향을 파악하기 위하여 하기와 같은 방법으로 배양하였다. 이 때 대조군으로서는 모균주인 코리네박테리움 글루타미쿰 KCCM10741P 및 ATCC21831을 사용하였으며, 생산 배지 [포도당 6%, 황산암모늄 3%, 제1인산칼륨 0.1%, 황산마그네슘7수염 0.2%, CSL(옥수수 침지액) 1.5%, NaCl 1%, 이스트 익스트렉트 0.5%, 비오틴 100ug/L, pH7.2] 25 ml을 넣은 250ml 코너-바플 플라스크에 1백금이의 균주를 접종하고 30 ℃에서 48시간 동안 200 rpm으로 배양하여 생산하였다. 배양종료 후 HPLC로 L-아르기닌의 생산량을 측정하였으며, 그 결과는 하기 표 1과 같았다.
균주 | OD | 아르기닌 농도 (g/L) |
오르니틴 농도 (g/L) |
KCCM10741P | 91 | 3.0 | 0.2 |
KCCM10741P _ΔPargC::lysCP1 | 72 | 2.2 | 1.9 |
KCCM10741P _ΔPargC::lysCP1_Pcj7-argF | 69 | 4.3 | 0.2 |
KCCM10741P _ΔPargC::lysCP1_Pcj7-argF2 | 70 | 4.1 | 0.5 |
KCCM10741P _ΔPargC::lysCP1_Pcj7-argF/Pcj7-argF2 | 69 | 4.5 | 0.2 |
ATCC21831 | 102 | 4.2 | 0.3 |
ATCC21831_ΔPargC::lysCP1 | 86 | 3.2 | 2.9 |
ATCC21831_ΔPargC::lysCP1_Pcj7-argF | 86 | 5.5 | 0.3 |
ATCC21831_ΔPargC::lysCP1_Pcj7-argF2 | 88 | 5.3 | 0.6 |
ATCC21831_ΔPargC::lysCP1_Pcj7-argF/Pcj7-argF2 | 85 | 5.6 | 0.3 |
상기 표 1에서 나타낸 바와 같이, 아르기닌 오페론과 오르니틴 카마모일트랜스퍼라제를 암호화하는 유전자가 동시 강화된 균주는 대조군 대비 아르기닌 생산능이 최대 50% 향상됨을 확인하였다. 또한, 아르기닌 오페론 단독 강화(KCCM10741P _ΔPargC::lysCP1 및 ATCC21831_ΔPargC::lysCP1) 에서 보이는 아르기닌 농도 감소 및 오르니틴 농도 증가는 argF, argF2 또는 argF 및 argF2를 도입함으로써 해결되었으며, 결과적으로 아르기닌 농도 증가의 결과를 보여주고 있다.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.
<110> CJ CheilJedang Corporation
<120> A microorganism of genus corynebacterium having an ability to produce
L-arginine and a method for producing L-arginine using the same
<130> KPA140146-KR-P1
<150> KR10-2014-0137794
<151> 2014-10-13
<160> 26
<170> KopatentIn 2.0
<210> 1
<211> 319
<212> PRT
<213> Corynebacterium glutamicum ArgF
<400> 1
Met Thr Ser Gln Pro Gln Val Arg His Phe Leu Ala Asp Asp Asp Leu
1 5 10 15
Thr Pro Ala Glu Gln Ala Glu Val Leu Thr Leu Ala Ala Lys Leu Lys
20 25 30
Ala Ala Pro Phe Ser Glu Arg Pro Leu Glu Gly Pro Lys Ser Val Ala
35 40 45
Val Leu Phe Asp Lys Thr Ser Thr Arg Thr Arg Phe Ser Phe Asp Ala
50 55 60
Gly Ile Ala His Leu Gly Gly His Ala Ile Val Val Asp Ser Gly Ser
65 70 75 80
Ser Gln Met Gly Lys Gly Glu Thr Leu Gln Asp Thr Ala Ala Val Leu
85 90 95
Ser Arg Tyr Val Glu Ala Ile Val Trp Arg Thr Tyr Ala His Ser Asn
100 105 110
Phe His Ala Met Ala Glu Thr Ser Thr Val Pro Leu Val Asn Ser Leu
115 120 125
Ser Asp Asp Leu His Pro Cys Gln Ile Leu Ala Asp Leu Gln Thr Ile
130 135 140
Val Glu Asn Leu Ser Pro Glu Glu Gly Pro Ala Gly Leu Lys Gly Lys
145 150 155 160
Lys Ala Val Tyr Leu Gly Asp Gly Asp Asn Asn Met Ala Asn Ser Tyr
165 170 175
Met Ile Gly Phe Ala Thr Ala Gly Met Asp Ile Ser Ile Ile Ala Pro
180 185 190
Glu Gly Phe Gln Pro Arg Ala Glu Phe Val Glu Arg Ala Glu Lys Arg
195 200 205
Gly Gln Glu Thr Gly Ala Lys Val Val Val Thr Asp Ser Leu Asp Glu
210 215 220
Val Ala Gly Ala Asp Val Val Ile Thr Asp Thr Trp Val Ser Met Gly
225 230 235 240
Met Glu Asn Asp Gly Ile Asp Arg Thr Thr Pro Phe Val Pro Tyr Gln
245 250 255
Val Asn Asp Glu Val Met Ala Lys Ala Asn Asp Gly Ala Ile Phe Leu
260 265 270
His Cys Leu Pro Ala Tyr Arg Gly Lys Glu Val Ala Ala Ser Val Ile
275 280 285
Asp Gly Pro Ala Ser Lys Val Phe Asp Glu Ala Glu Asn Arg Leu His
290 295 300
Ala Gln Lys Ala Leu Leu Val Trp Leu Leu Ala His Gln Pro Arg
305 310 315
<210> 2
<211> 960
<212> DNA
<213> Corynebacterium glutamicum argF
<400> 2
atgacttcac aaccacaggt tcgccatttc ctggctgatg atgatctcac ccctgcagag 60
caggcagagg ttttgaccct agccgcaaag ctcaaggcag cgccgttttc ggagcgtcca 120
ctcgagggac caaagtccgt tgcagttctt tttgataaga cttcaactcg tactcgcttc 180
tccttcgacg cgggcatcgc tcatttgggt ggacatgcca tcgtcgtgga ttccggcagc 240
tcacagatgg gtaagggcga gaccctgcag gacaccgcag ctgtattgtc ccgctacgtg 300
gaagcaattg tgtggcgcac ctacgcacac agcaatttcc acgccatggc ggagacgtcc 360
actgtgccac tggtgaactc cttgtccgat gatctgcacc catgccagat tctggctgat 420
ctgcagacca tcgtggaaaa cctcagccct gaagaaggcc cagcaggcct taagggtaag 480
aaggctgtgt acctgggcga tggcgacaac aacatggcca actcctacat gattggcttt 540
gccaccgcgg gcatggatat ctccatcatc gctcctgaag ggttccagcc tcgtgcggaa 600
ttcgtggagc gcgcggaaaa gcgtggccag gaaaccggcg cgaaggttgt tgtcaccgac 660
agcctcgacg aggttgccgg cgccgatgtt gtcatcaccg atacctgggt atccatgggt 720
atggaaaacg acggcatcga tcgcaccaca cctttcgttc cctaccaggt caacgatgag 780
gtcatggcga aagctaacga cggcgccatc ttcctgcact gccttcctgc ctaccgcggc 840
aaagaagtgg cagcctccgt gattgatgga ccagcgtcca aagttttcga tgaagcagaa 900
aaccgcctcc acgctcagaa agcactgctg gtgtggctgc tggcccacca gccgaggtaa 960
960
<210> 3
<211> 274
<212> PRT
<213> Corynebacterium glutamicum ArgF2
<400> 3
Met Ala Arg Lys His Leu Leu Ser Leu Ala Asp Trp Asn Arg Gly Glu
1 5 10 15
Leu Glu Ala Leu Phe Glu Leu Ala Glu Gln Tyr Glu Ala Gly Gly Gly
20 25 30
Pro Arg Phe Asp Gly Ala Ala Ala Met Phe Phe Pro Pro Thr Ser Leu
35 40 45
Arg Thr Arg Leu Ser Phe Glu Arg Gly Ala Thr Ala Met Gly Leu Gln
50 55 60
Pro Ile Thr Phe Pro Ser Asp Ser Leu Asp Lys Asp Glu Asp Leu Val
65 70 75 80
Asp Val Val Gly Tyr Leu Ser Gln Trp Ala Asp Val Val Val Val Arg
85 90 95
His Pro Gln Leu Thr Ala Leu Gln Arg Leu Ala Ser Ala Asp Ala Ala
100 105 110
Pro Val Ile Asn Ala Met Thr Ser Glu Asn His Pro Cys Glu Val Leu
115 120 125
Ser Asp Leu Tyr Ala Leu Ser Arg His His Asp Ile Ser Ala Leu Arg
130 135 140
Tyr Leu Phe Val Gly Gly Asp Gly Asn Ile Ala Arg Ala Trp Trp Glu
145 150 155 160
Ala Ala Gln Ala Phe Gly Leu Glu Met Arg Gln Ser Cys Pro Glu Glu
165 170 175
Leu Arg Val Val Gly Met Pro Trp Glu Glu Asn Leu Pro His Ala Ile
180 185 190
Ala Ser Ala Asp Val Val Leu Thr Asp Gly Pro Gly Arg His Ala Glu
195 200 205
Leu Leu Glu Pro Tyr Arg Val Thr Ala Ala Leu Leu Asp Arg Ala Pro
210 215 220
Arg Gly Val Arg Leu Ala Pro Cys Pro Pro Phe Ile Arg Gly Arg Glu
225 230 235 240
Val Ser Ala Asp Ala Ile Glu His Pro Ala Phe Val Gly Tyr Ser Phe
245 250 255
Lys Arg His Leu Met Pro Val Gln Gln Ala Ile Leu Ala Arg Ser Ile
260 265 270
Asn Ala
<210> 4
<211> 825
<212> DNA
<213> Corynebacterium glutamicum argF2
<400> 4
atggccagaa aacatctgct ctccctggca gactggaaca gaggcgagct tgaggcatta 60
ttcgagctcg cggagcagta tgaagctggc ggtgggccac gattcgatgg tgccgcggcg 120
atgttcttcc cgccgacgag tttgcgtaca cggctctcat tcgagcgtgg ggcaacggca 180
atgggactcc agccgatcac gttcccgtca gacagcctgg acaaggacga agatctcgtc 240
gacgtcgtcg gctatctctc gcagtgggct gatgtcgttg tcgtccgaca cccgcaattg 300
acggcgcttc agcggttggc gtcagcggat gcagcgcccg tgatcaacgc gatgacgagt 360
gagaaccatc cgtgcgaagt cctctcggac ttgtatgcgc tgtctcgtca ccatgacatt 420
tcggccctgc ggtacctgtt tgtcggtggc gatggcaaca tcgccagggc ctggtgggag 480
gcggcccaag cgttcggcct cgagatgcgg cagagttgtc ctgaagagct gcgtgtcgtc 540
gggatgccgt gggaggagaa cctgccgcat gcaattgcat cagcggatgt cgtgctgacg 600
gatgggccag gtagacatgc ggagttactc gagccgtatc gtgtgaccgc tgcgttgttg 660
gatcgcgcgc cccgtggagt gcggctcgcg ccctgcccgc cgttcatccg cgggcgcgaa 720
gtgagcgccg atgcgatcga gcatccggcg ttcgtcgggt actcgttcaa gcgtcatctc 780
atgccggttc agcaggcgat tctggctcgg tcgatcaatg cgtag 825
<210> 5
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> SF_PlysCP1_XhoI-XbaI primer
<400> 5
ccgtctctag actcgagcca tcttttgggg tgcgg 35
<210> 6
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> SR_PlysCP1_SpeI primer
<400> 6
ttgattatca tactagtctt tgtgcacctt tcgat 35
<210> 7
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> SF_argF_EcoRV primer
<400> 7
acgaaaggaa acactcgata tcatgacttc acaaccacag gt 42
<210> 8
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> SR_argF_XbaI primer
<400> 8
gccaaaacag ctctagatta cctcggctgg tgggcca 37
<210> 9
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> SF_argF2_EcoRV primer
<400> 9
acgaaaggaa acactcgata tcatggccag aaaacatctg ct 42
<210> 10
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> SR_argF2_XbaI primer
<400> 10
gccaaaacag ctctagacta cgcattgatc gaccgag 37
<210> 11
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> SF_Pcj7_argF2_NotI primer
<400> 11
cctttttgcg gcggccgcag aaacatccca gcgctact 38
<210> 12
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> SR_argF2_NotI primer
<400> 12
caccgcggtg gcggccgccg caaaaaggcc atccgtca 38
<210> 13
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> SF_pargC_PR_pDC primer
<400> 13
cgagctcggt acccgggcaa agaatacggc ttccttggc 39
<210> 14
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> SR_pargC_PR_XbaI-XhoI-BamHI primer
<400> 14
ctggatcctc gagtctagag acgggttaga catgcaaaa 39
<210> 15
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> SF_pargC_PR_SpeI-ScaI-BamHI primer
<400> 15
gactcgagga tccagtacta gtatgataat caaggttgca at 42
<210> 16
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> SR_pargC_PR_pDC primer
<400> 16
tgcaggtcga ctctagggta acgccttctt tcaaag 36
<210> 17
<211> 317
<212> DNA
<213> Corynebacterium glutamicum cj7 promoter
<400> 17
agaaacatcc cagcgctact aatagggagc gttgaccttc cttccacgga ccggtaatcg 60
gagtgcctaa aaccgcatgc ggcttaggct ccaagatagg ttctgcgcgg ccgggtaatg 120
catcttcttt agcaacaagt tgaggggtag gtgcaaataa gaacgacata gaaatcgtct 180
cctttctgtt tttaatcaac atacaccacc acctaaaaat tccccgacca gcaagttcac 240
agtattcggg cacaatatcg ttgccaaaat attgtttcgg aatatcatgg gatacgtacc 300
caacgaaagg aaacact 317
<210> 18
<211> 353
<212> DNA
<213> Corynebacterium glutamicum lysCP1 promoter
<400> 18
ccatcttttg gggtgcggag cgcgatccgg tgtctgacca cggtgcccca tgcgattgtt 60
aatgccgatg ctagggcgaa aagcacggcg agcagattgc tttgcacttg attcagggta 120
gttgactaaa gagttgctcg cgaagtagca cctgtcactt ttgtctcaaa tattaaatcg 180
aatatcaata tatggtctgt ttattggaac gcgtcccagt ggctgagacg catccgctaa 240
agccccagga accctgtgca gaaagaaaac actcctctgg ctaggtagac acagtttatt 300
gtggtagagt tgagcgggta actgtcagca cgtagatcga aaggtgcaca aag 353
<210> 19
<211> 388
<212> PRT
<213> Corynebacterium glutamicum ArgJ
<400> 19
Met Ala Lys Lys Gly Ile Thr Ala Pro Lys Gly Phe Val Ala Ser Ala
1 5 10 15
Thr Thr Ala Gly Ile Lys Ala Ser Gly Asn Pro Asp Met Ala Leu Val
20 25 30
Val Asn Gln Gly Pro Glu Phe Ser Ala Ala Ala Val Phe Thr Arg Asn
35 40 45
Arg Val Phe Ala Ala Pro Val Lys Val Ser Arg Glu Asn Val Ala Asp
50 55 60
Gly Gln Ile Arg Ala Val Leu Tyr Asn Ala Gly Asn Ala Asn Ala Cys
65 70 75 80
Asn Gly Leu Gln Gly Glu Lys Asp Ala Arg Glu Ser Val Ser His Leu
85 90 95
Ala Gln Asn Leu Gly Leu Glu Asp Ser Asp Ile Gly Val Cys Ser Thr
100 105 110
Gly Leu Ile Gly Glu Leu Leu Pro Met Asp Lys Leu Asn Thr Gly Ile
115 120 125
Asp Gln Leu Thr Ala Glu Gly Ala Leu Gly Asp Asn Gly Ala Ala Ala
130 135 140
Ala Lys Ala Ile Met Thr Thr Asp Thr Val Asp Lys Glu Thr Val Val
145 150 155 160
Phe Ala Asp Gly Trp Thr Val Gly Gly Met Gly Lys Gly Val Gly Met
165 170 175
Met Ala Pro Ser Leu Ala Thr Met Leu Val Cys Leu Thr Thr Asp Ala
180 185 190
Ser Val Thr Gln Glu Met Ala Gln Ile Ala Leu Ala Asn Ala Thr Ala
195 200 205
Val Thr Phe Asp Thr Leu Asp Ile Asp Gly Ser Thr Ser Thr Asn Asp
210 215 220
Thr Val Phe Leu Leu Ala Ser Gly Ala Ser Gly Ile Thr Pro Thr Gln
225 230 235 240
Asp Glu Leu Asn Asp Ala Val Tyr Ala Ala Cys Ser Asp Ile Ala Ala
245 250 255
Lys Leu Gln Ala Asp Ala Glu Gly Val Thr Lys Arg Val Ala Val Thr
260 265 270
Val Val Gly Thr Thr Asn Asn Glu Gln Ala Ile Asn Ala Ala Arg Thr
275 280 285
Val Ala Arg Asp Asn Leu Phe Lys Cys Ala Met Phe Gly Ser Asp Pro
290 295 300
Asn Trp Gly Arg Val Leu Ala Ala Val Gly Met Ala Asp Ala Asp Met
305 310 315 320
Glu Pro Glu Lys Ile Ser Val Phe Phe Asn Asp Gln Ala Val Cys Leu
325 330 335
Asp Ser Thr Gly Ala Pro Gly Ala Arg Glu Val Asp Leu Ser Gly Ala
340 345 350
Asp Ile Asp Val Arg Ile Asp Leu Gly Thr Ser Gly Glu Gly Gln Ala
355 360 365
Thr Val Arg Thr Thr Asp Leu Ser Phe Ser Tyr Val Glu Ile Asn Ser
370 375 380
Ala Tyr Ser Ser
385
<210> 20
<211> 1167
<212> DNA
<213> Corynebacterium glutamicum argJ
<400> 20
atggccaaaa aaggcattac cgcgccgaaa ggcttcgttg cttctgcaac gaccgcgggt 60
attaaagctt ctggcaatcc tgacatggcg ttggtggtta accagggtcc agagttttcc 120
gcagcggccg tgtttacacg caaccgagtt ttcgcagcgc ctgtgaaggt gagccgggag 180
aacgttgctg atggccagat cagggctgtt ttgtacaacg ctggtaatgc taatgcgtgt 240
aatggtctgc agggtgagaa ggatgctcgt gagtctgttt ctcatctagc tcaaaatttg 300
ggcttggagg attccgatat tggtgtgtgt tccactggtc ttattggtga gttgcttccg 360
atggataagc tcaatacagg tattgatcag ctgaccgctg agggcgcttt gggtgacaat 420
ggtgcagctg ctgccaaggc gatcatgacc actgacacgg tggataagga aaccgtcgtg 480
tttgctgatg gttggactgt cggcggaatg ggcaagggcg tgggcatgat ggcgccgtct 540
cttgccacca tgctggtctg cttgaccact gatgcatccg ttactcagga aatggctcag 600
attgcgctgg ctaatgctac ggccgttacg tttgacaccc tggatattga tggatcaacc 660
tccaccaatg acaccgtgtt cctgctggca tctggcgcta gcggaatcac cccaactcag 720
gatgaactca acgatgcggt gtacgcagct tgttctgata tcgcagcgaa gcttcaggct 780
gatgcagagg gggtgaccaa gcgcgttgct gtgacagtgg tgggaaccac caacaacgag 840
caggcgatca atgcggctcg cacggttgct cgtgacaatt tgttcaagtg cgcaatgttt 900
ggatctgatc caaactgggg tcgcgtgttg gctgcagtcg gcatggctga tgctgatatg 960
gaaccagaga agatttctgt gttcttcaat gatcaagcag tatgccttga ttccactggc 1020
gctcctggtg ctcgtgaggt ggatctttcc ggcgctgaca ttgatgtccg aattgatttg 1080
ggcaccagtg gggaaggcca ggcaacagtt cgaaccactg acctgagctt ctcctacgtg 1140
gagatcaact ccgcgtacag ctcttaa 1167
<210> 21
<211> 317
<212> PRT
<213> Corynebacterium glutamicum ArgB
<400> 21
Met Asn Asp Leu Ile Lys Asp Leu Gly Ser Glu Val Arg Ala Asn Val
1 5 10 15
Leu Ala Glu Ala Leu Pro Trp Leu Gln His Phe Arg Asp Lys Ile Val
20 25 30
Val Val Lys Tyr Gly Gly Asn Ala Met Val Asp Asp Asp Leu Lys Ala
35 40 45
Ala Phe Ala Ala Asp Met Val Phe Leu Arg Thr Val Gly Ala Lys Pro
50 55 60
Val Val Val His Gly Gly Gly Pro Gln Ile Ser Glu Met Leu Asn Arg
65 70 75 80
Val Gly Leu Gln Gly Glu Phe Lys Gly Gly Phe Arg Val Thr Thr Pro
85 90 95
Glu Val Met Asp Ile Val Arg Met Val Leu Phe Gly Gln Val Gly Arg
100 105 110
Asp Leu Val Gly Leu Ile Asn Ser His Gly Pro Tyr Ala Val Gly Thr
115 120 125
Ser Gly Glu Asp Ala Gly Leu Phe Thr Ala Gln Lys Arg Met Val Asn
130 135 140
Ile Asp Gly Val Pro Thr Asp Ile Gly Leu Val Gly Asp Ile Ile Asn
145 150 155 160
Val Asp Ala Ser Ser Leu Met Asp Ile Ile Glu Ala Gly Arg Ile Pro
165 170 175
Val Val Ser Thr Ile Ala Pro Gly Glu Asp Gly Gln Ile Tyr Asn Ile
180 185 190
Asn Ala Asp Thr Ala Ala Gly Ala Leu Ala Ala Ala Ile Gly Ala Glu
195 200 205
Arg Leu Leu Val Leu Thr Asn Val Glu Gly Leu Tyr Thr Asp Trp Pro
210 215 220
Asp Lys Ser Ser Leu Val Ser Lys Ile Lys Ala Thr Glu Leu Glu Ala
225 230 235 240
Ile Leu Pro Gly Leu Asp Ser Gly Met Ile Pro Lys Met Glu Ser Cys
245 250 255
Leu Asn Ala Val Arg Gly Gly Val Ser Ala Ala His Val Ile Asp Gly
260 265 270
Arg Ile Ala His Ser Val Leu Leu Glu Leu Leu Thr Met Gly Gly Ile
275 280 285
Gly Thr Met Val Leu Pro Asp Val Phe Asp Arg Glu Asn Tyr Pro Glu
290 295 300
Gly Thr Val Phe Arg Lys Asp Asp Lys Asp Gly Glu Leu
305 310 315
<210> 22
<211> 954
<212> DNA
<213> Corynebacterium glutamicum argB
<400> 22
atgaatgact tgatcaaaga tttaggctct gaggtgcgcg caaatgtcct cgctgaggcg 60
ttgccatggt tgcagcattt ccgcgacaag attgttgtcg tgaaatatgg cggaaacgcc 120
atggtggatg atgatctcaa ggctgctttt gctgccgaca tggtcttctt gcgcaccgtg 180
ggcgcaaaac cagtggtggt gcacggtggt ggacctcaga tttctgagat gctaaaccgt 240
gtgggtctcc agggcgagtt caagggtggt ttccgtgtga ccactcctga ggtcatggac 300
attgtgcgca tggtgctctt tggtcaggtc ggtcgcgatt tagttggttt gatcaactct 360
catggccctt acgctgtggg aacctccggt gaggatgccg gcctgtttac cgcgcagaag 420
cgcatggtca acatcgatgg cgtacccact gatattggtt tggtcggaga catcattaat 480
gtcgatgcct cttccttgat ggatatcatc gaggccggtc gcattcctgt ggtctctacg 540
attgctccag gcgaagacgg ccagatttac aacatcaacg ccgataccgc agcgggtgct 600
ttggctgcag cgattggtgc agaacgcctg ctggttctca ccaatgtgga aggtctgtac 660
accgattggc ctgataagag ctcactggtg tccaagatca aggccaccga gctggaggcc 720
attcttccgg gacttgattc cggcatgatt ccaaagatgg agtcttgctt gaatgcggtg 780
cgtgggggag taagcgctgc tcatgtcatt gacggccgca tcgcgcactc ggtgttgctg 840
gagcttttga ccatgggtgg aattggcacg atggtgctgc cggatgtttt tgatcgggag 900
aattatccgg aaggcaccgt ttttagaaaa gacgacaagg atggggaact gtaa 954
<210> 23
<211> 347
<212> PRT
<213> Corynebacterium glutamicum ArgC
<400> 23
Met Thr Ile Lys Val Ala Ile Ala Gly Ala Ser Gly Tyr Ala Gly Gly
1 5 10 15
Glu Ile Leu Arg Leu Leu Leu Gly His Pro Ala Tyr Ala Ser Gly Glu
20 25 30
Leu Glu Ile Gly Ala Leu Thr Ala Ala Ser Thr Ala Gly Ser Thr Leu
35 40 45
Gly Glu Leu Met Pro His Ile Pro Gln Leu Ala Asp Arg Val Ile Gln
50 55 60
Asp Thr Thr Ala Glu Thr Leu Ala Gly His Asp Val Val Phe Leu Gly
65 70 75 80
Leu Pro His Gly Phe Ser Ala Glu Ile Ala Leu Gln Leu Gly Pro Asp
85 90 95
Val Thr Val Ile Asp Cys Ala Ala Asp Phe Arg Leu Gln Asn Ala Ala
100 105 110
Asp Trp Glu Lys Phe Tyr Gly Ser Glu His Gln Gly Thr Trp Pro Tyr
115 120 125
Gly Ile Pro Glu Ile Pro Gly His Arg Glu Ala Leu Arg Gly Ala Lys
130 135 140
Arg Val Ala Val Pro Gly Cys Phe Pro Thr Gly Ala Thr Leu Ala Leu
145 150 155 160
Leu Pro Ala Val Gln Ala Gly Leu Ile Glu Pro Asp Val Ser Val Val
165 170 175
Ser Ile Thr Gly Val Ser Gly Ala Gly Lys Lys Ala Ser Val Ala Leu
180 185 190
Leu Gly Ser Glu Thr Met Gly Ser Leu Lys Ala Tyr Asn Thr Ser Gly
195 200 205
Lys His Arg His Thr Pro Glu Ile Ala Gln Asn Leu Gly Glu Val Ser
210 215 220
Asp Lys Pro Val Lys Val Ser Phe Thr Pro Val Leu Ala Pro Leu Pro
225 230 235 240
Arg Gly Ile Leu Thr Thr Ala Thr Ala Pro Leu Lys Glu Gly Val Thr
245 250 255
Ala Glu Gln Ala Arg Ala Val Tyr Glu Glu Phe Tyr Ala Gln Glu Thr
260 265 270
Phe Val His Val Leu Pro Glu Gly Ala Gln Pro Gln Thr Gln Ala Val
275 280 285
Leu Gly Ser Asn Met Cys His Val Gln Val Glu Ile Asp Glu Glu Ala
290 295 300
Gly Lys Val Leu Val Thr Ser Ala Ile Asp Asn Leu Thr Lys Gly Thr
305 310 315 320
Ala Gly Ala Ala Val Gln Cys Met Asn Leu Ser Val Gly Phe Asp Glu
325 330 335
Ala Ala Gly Leu Pro Gln Val Gly Val Ala Pro
340 345
<210> 24
<211> 1044
<212> DNA
<213> Corynebacterium glutamicum argC
<400> 24
atgacaatca aggttgcaat cgcaggagcc agtggatatg ccggcggaga aatccttcgt 60
ctccttttag gccatccagc ttatgcatct ggtgaactag aaatcggagc actcaccgcg 120
gcatcaaccg caggcagcac gctcggtgaa ttgatgccac acattccgca gttggcggat 180
cgtgttattc aagacaccac agctgaaact ctagccggtc atgatgtcgt atttctagga 240
cttccacacg gattctctgc agaaattgca cttcagctcg gaccagatgt cacagtgatt 300
gactgtgcag ctgactttcg tctgcaaaat gctgcagatt gggagaagtt ctacggctca 360
gagcaccagg gaacatggcc ttatggcatt ccagaaatac caggacaccg cgaggctctt 420
cgtggtgcta agcgtgtagc agtgccagga tgtttcccaa ccggtgcaac cttggctctt 480
cttcctgcgg ttcaagcggg acttatcgag ccagatgttt ccgtagtgtc catcaccggc 540
gtatcaggtg caggtaagaa agcatctgtt gcactacttg gctcggaaac catgggttca 600
ctcaaggcgt acaacacctc cggaaagcac cgccacaccc cggaaattgc ccagaacctc 660
ggcgaagtca gcgacaagcc agtcaaggtg agcttcaccc cagtgcttgc accgttacct 720
cgcggaattc tcaccactgc aaccgcacct ttgaaagaag gcgttaccgc agagcaggct 780
cgcgcagtat atgaagagtt ctatgcacag gaaaccttcg tgcatgttct tccagaaggt 840
gcacagccac aaacccaagc agttcttggc tccaacatgt gccacgtgca ggtagaaatt 900
gatgaggaag caggcaaagt ccttgttacc tccgcaatcg ataacctcac caagggaact 960
gccggcgccg ctgttcagtg catgaactta agcgttggct ttgatgaggc agcaggcctg 1020
ccacaggtcg gcgtcgcacc ttaa 1044
<210> 25
<211> 391
<212> PRT
<213> Corynebacterium glutamicum ArgD
<400> 25
Met Ser Thr Leu Glu Thr Trp Pro Gln Val Ile Ile Asn Thr Tyr Gly
1 5 10 15
Thr Pro Pro Val Glu Leu Val Ser Gly Lys Gly Ala Thr Val Thr Asp
20 25 30
Asp Gln Gly Lys Val Tyr Ile Asp Leu Leu Ala Gly Ile Ala Val Asn
35 40 45
Ala Leu Gly His Ala His Pro Ala Ile Ile Glu Ala Val Thr Asn Gln
50 55 60
Ile Gly Gln Leu Gly His Val Ser Asn Leu Phe Ala Ser Arg Pro Val
65 70 75 80
Val Glu Val Ala Glu Glu Leu Ile Lys Arg Phe Ser Leu Asp Asp Ala
85 90 95
Thr Leu Ala Ala Gln Thr Arg Val Phe Phe Cys Asn Ser Gly Ala Glu
100 105 110
Ala Asn Glu Ala Ala Phe Lys Ile Ala Arg Leu Thr Gly Arg Ser Arg
115 120 125
Ile Leu Ala Ala Val His Gly Phe His Gly Arg Thr Met Gly Ser Leu
130 135 140
Ala Leu Thr Gly Gln Pro Asp Lys Arg Glu Ala Phe Leu Pro Met Pro
145 150 155 160
Ser Gly Val Glu Phe Tyr Pro Tyr Gly Asp Thr Asp Tyr Leu Arg Lys
165 170 175
Met Val Glu Thr Asn Pro Thr Asp Val Ala Ala Ile Phe Leu Glu Pro
180 185 190
Ile Gln Gly Glu Thr Gly Val Val Pro Ala Pro Glu Gly Phe Leu Lys
195 200 205
Ala Val Arg Glu Leu Cys Asp Glu Tyr Gly Ile Leu Met Ile Thr Asp
210 215 220
Glu Val Gln Thr Gly Val Gly Arg Thr Gly Asp Phe Phe Ala His Gln
225 230 235 240
His Asp Gly Val Val Pro Asp Val Val Thr Met Ala Lys Gly Leu Gly
245 250 255
Gly Gly Leu Pro Ile Gly Ala Cys Leu Ala Thr Gly Arg Ala Ala Glu
260 265 270
Leu Met Thr Pro Gly Lys His Gly Thr Thr Phe Gly Gly Asn Pro Val
275 280 285
Ala Cys Ala Ala Ala Lys Ala Val Leu Ser Val Val Asp Asp Ala Phe
290 295 300
Cys Ala Glu Val Thr Arg Lys Gly Glu Leu Phe Lys Glu Leu Leu Ala
305 310 315 320
Lys Val Asp Gly Val Val Asp Val Arg Gly Arg Gly Leu Met Leu Gly
325 330 335
Val Val Leu Glu Arg Asp Val Ala Lys Gln Ala Val Leu Asp Gly Phe
340 345 350
Lys His Gly Val Ile Leu Asn Ala Pro Ala Asp Asn Ile Ile Arg Leu
355 360 365
Thr Pro Pro Leu Val Ile Thr Asp Glu Glu Ile Ala Asp Ala Val Lys
370 375 380
Ala Ile Ala Glu Thr Ile Ala
385 390
<210> 26
<211> 1176
<212> DNA
<213> Corynebacterium glutamicum argD
<400> 26
atgagcacgc tggaaacttg gccacaggtc attattaata cgtacggcac cccaccagtt 60
gagctggtgt ccggcaaggg cgcaaccgtc accgatgacc agggcaaagt ctacatcgac 120
ttgctcgcgg gcatcgcagt caacgcgttg ggccacgccc acccggcgat catcgaggcg 180
gtcaccaacc agatcggcca acttggtcac gtctcaaact tgttcgcatc caggcccgtc 240
gtcgaggtcg ccgaggagct catcaagcgt ttttcgcttg acgacgccac cctcgccgcg 300
caaacccggg ttttcttctg caactcgggc gccgaagcaa acgaggctgc tttcaagatt 360
gcacgcttga ctggtcgttc ccggattctg gctgcagttc atggtttcca cggccgcacc 420
atgggttccc tcgcgctgac tggccagcca gacaagcgtg aagcattcct gccaatgcca 480
agcggtgtgg agttctaccc ttacggcgac accgattact tgcgcaaaat ggtagaaacc 540
aacccaacgg atgtggctgc tatcttcctc gagccaatcc agggtgaaac gggcgttgtt 600
ccagcacctg aaggattcct caaggcagtg cgcgagctgt gcgatgagta cggcatcttg 660
atgatcaccg atgaagtcca gactggcgtt ggccgtaccg gcgatttctt tgcacatcag 720
cacgatggcg ttgttcccga tgtggtgacc atggccaagg gacttggcgg cggtcttccc 780
atcggtgctt gtttggccac tggccgtgca gctgaattga tgaccccagg caagcacggc 840
accactttcg gtggcaaccc agttgcttgt gcagctgcca aggcagtgct gtctgttgtc 900
gatgacgctt tctgcgcaga agttacccgc aagggcgagc tgttcaagga acttcttgcc 960
aaggttgacg gcgttgtaga cgtccgtggc aggggcttga tgttgggcgt ggtgctggag 1020
cgcgacgtcg caaagcaagc tgttcttgat ggttttaagc acggcgttat tttgaatgca 1080
ccggcggaca acattatccg tttgaccccg ccgctggtga tcaccgacga agaaatcgca 1140
gacgcagtca aggctattgc cgagacaatc gcataa 1176
Claims (4)
- 아르기닌 억제자 (ArgR)를 포함하는 아르기닌 오페론 및 오르니틴 카바모일트랜스퍼라제의 활성이 강화된, L-아르기닌을 생산하는 코리네박테리움 속 미생물.
- 제1항에 있어서, 상기 오르니틴 카마모일트랜스퍼라제는 서열번호 1 또는 서열번호 3의 아미노산 서열인, L-아르기닌을 생산하는 코리네박테리움 속 미생물.
- 제1항에 있어서, 상기 코리네박테리움 속 미생물은 코리네박테리움 글루타미쿰인 것을 특징으로 하는 L-아르기닌을 생산하는 코리네박테리움 속 미생물.
- 제1항 내지 제3항 중 어느 한 항의 코리네박테리움 속 미생물을 배양 배지에서 배양하는 단계; 및 상기 배양 배지 또는 미생물에서 L-아르기닌을 회수하는 단계를 포함하는 L-아르기닌을 생산하는 방법.
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TW104133479A TWI583790B (zh) | 2014-10-13 | 2015-10-13 | 具有產生l-精胺酸能力的棒狀桿菌屬微生物及使用該微生物產生l-精胺酸的方法 |
RU2016147322A RU2671106C1 (ru) | 2014-10-13 | 2015-10-13 | Микроорганизм рода Corynebacterium для продуцирования L-аргинина и способ получения L-аргинина с использованием этого микроорганизма |
PCT/KR2015/010768 WO2016060437A1 (ko) | 2014-10-13 | 2015-10-13 | L-아르기닌을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 l-아르기닌의 제조 방법 |
US15/515,551 US10626426B2 (en) | 2014-10-13 | 2015-10-13 | Microorganism of genus Corynebacterium having an ability to produce L-arginine and a method for producing L-arginine using the same |
ES15850829T ES2724000T3 (es) | 2014-10-13 | 2015-10-13 | Microorganismo del género Corynebacterium para producir L-arginina y método de producción de L-arginina utilizando el mismo |
PL15850829T PL3153573T3 (pl) | 2014-10-13 | 2015-10-13 | Mikroorganizm z rodzaju Corynebacterium do wytwarzania L-argininy i sposoby wytwarzania L-argininy przy jego zastosowaniu |
CN201580055711.1A CN107002027B (zh) | 2014-10-13 | 2015-10-13 | 具有生产l-精氨酸能力的棒状杆菌属微生物和使用其生产l-精氨酸的方法 |
HUE15850829A HUE042618T2 (hu) | 2014-10-13 | 2015-10-13 | Corynebacterium nemzetséghez tartozó mikroorganizmus L-arginin termelésére és eljárás L-arginin termelésére annak alkalmazásával |
BR112016029730-0A BR112016029730B1 (pt) | 2014-10-13 | 2015-10-13 | Microrganismo do gênero corynebacterium que tem a aptidão de produzir l-arginina e um método para a produção de l-arginina utilizando o mesmo |
JP2016575080A JP6476212B2 (ja) | 2014-10-13 | 2015-10-13 | L−アルギニンを生産するコリネバクテリウム属の微生物及びそれを用いたl−アルギニンの製造方法 |
DK15850829.1T DK3153573T3 (da) | 2014-10-13 | 2015-10-13 | Mikroorganisme af slægten Corynebacterium til fremstilling af L-arginin og fremgangsmåde til fremstilling af L-arginin under anvendelse af samme |
CA2951019A CA2951019C (en) | 2014-10-13 | 2015-10-13 | A microorganism of genus corynebacterium having an ability to produce l-arginine and a method for producing l-arginine using the same |
AU2015331160A AU2015331160B2 (en) | 2014-10-13 | 2015-10-13 | A Microorganism Of Genus Corynebacterium Having An Ability To Produce L-Arginine And A Method For Producing L-Arginine Using The Same |
EP15850829.1A EP3153573B1 (en) | 2014-10-13 | 2015-10-13 | Microorganism of genus corynebacterium for producing l-arginine, and l-arginine production method using same |
MYPI2017000069A MY188962A (en) | 2014-10-13 | 2015-10-13 | A microorganism of genus corynebacterium having an ability to produce l-arginine and an method for producing l-arginine using the same |
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KR101947945B1 (ko) * | 2018-01-25 | 2019-02-13 | 씨제이제일제당 (주) | L-아미노산을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 l-아미노산의 생산방법 |
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KR102134418B1 (ko) * | 2019-06-17 | 2020-07-16 | 씨제이제일제당 주식회사 | L-타이로신을 생산하는 미생물 및 이를 이용한 l-타이로신 생산 방법 |
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TW201619374A (zh) | 2016-06-01 |
EP3153573B1 (en) | 2019-04-03 |
BR112016029730B1 (pt) | 2023-11-14 |
RU2671106C1 (ru) | 2018-10-29 |
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JP6476212B2 (ja) | 2019-02-27 |
CN107002027A (zh) | 2017-08-01 |
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AU2015331160B2 (en) | 2018-08-02 |
CA2951019A1 (en) | 2016-04-21 |
EP3153573A4 (en) | 2017-12-06 |
ES2724000T3 (es) | 2019-09-05 |
CN107002027B (zh) | 2021-05-28 |
CA2951019C (en) | 2021-11-16 |
PL3153573T3 (pl) | 2019-08-30 |
MY188962A (en) | 2022-01-14 |
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