KR102216450B1 - 글루타민 생산능을 향상시키는 형질전환용 재조합 벡터 및 이를 도입한 균주 - Google Patents
글루타민 생산능을 향상시키는 형질전환용 재조합 벡터 및 이를 도입한 균주 Download PDFInfo
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- KR102216450B1 KR102216450B1 KR1020200038628A KR20200038628A KR102216450B1 KR 102216450 B1 KR102216450 B1 KR 102216450B1 KR 1020200038628 A KR1020200038628 A KR 1020200038628A KR 20200038628 A KR20200038628 A KR 20200038628A KR 102216450 B1 KR102216450 B1 KR 102216450B1
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- South Korea
- Prior art keywords
- ala
- glna
- vector
- glu
- strain
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Abstract
본 발명은 서열번호 1의 아미노산 서열로 이루어진 글루타민합성효소를 암호화하는 염기서열을 포함하는 벡터로 형질전환됨으로서 글루타민 생산성이 향상된 균주에 대한 것이다.
Description
본 발명은 글루타민 생산능을 향상시키는 형질전환용 재조합 벡터 및 이를 도입한 균주에 관한 것이다.
세균의 글루타민(glutamine, GLN) 합성은 글루타민산(glutamate, GLU) 및 질소원로부터 글루타민합성효소(glutamine synthetase, GS)에 의해 합성된다.
GS의 활성은 글루타민합성효소 아데닐릴트랜스퍼라제(glutamine synthetase adenylyltransferase, ATase, glnE)에 의해 조절된다. ATase는 GS의 아데닐화(adenylylation)와 탈아데닐화(deadenylation)를 촉매하여 GS의 활성을 조절하며, 배지내 질소농도의 영향을 받는다. ATase의 활성은 PII(nitrogen regulatory protein P-II Gene, glnB)에 의해 조절된다. 도 1을 참조하면, 질소농도가 높으면 PII와 ATase의 활성화에 의해 GS의 활성이 저하되는 기전이 개시되어 있다. 따라서, 글루타민을 생산하기 위해서 질소원을 공급하면, 공급된 질소원에 의해 GS의 활성이 되먹임 저하(feedback inhibition)되어 글루타민 생산이 저하되는 문제가 있다.
따라서 글루타민 생산 효율을 증가시키기 위해서는 ATase에 의한 GS 의 되먹임 저하를 억제하는 것이 요구된다. 일본특허 JP4898441에 따르면 GS 활성 저하에 관여하는 glnB 및 glnE 유전자의 활성을 억제함으로서 글루타민 생산이 증가된 균주를 개시하고 있으나, 개선의 여지는 여전히 남아있다.
일 구체예에 따르면 서열번호 1의 아미노산 서열로 이루어진 글루타민합성효소(glutamine synthetase, GS)를 암호화하는 염기서열을 포함하는 벡터를 포함하는 글루타민 생산 균주를 제공한다.
일 양상에 따르면, 서열번호 1의 아미노산 서열로 이루어진 글루타민합성효소(glutamine synthetase, GS)를 암호화하는 염기서열을 포함하는 형질전환용 벡터를 제공한다.
상기 글루타민합성효소는 글루타메이트(glutamate)와 암모니아로부터 글루타민을 합성하는 효소이다. 상기 서열번호 1의 아미노산 서열로 이루어진 글루타민합성효소는 다른 서열로 이루어진 글루타민합성효소보다 ATase에 의해 활성이 억제되는 정도가 낮기 때문에 글루타민 생산성을 향상시킬 수 있다.
일 구체예에 따르면, 상기 글루타민합성효소를 암호화하는 염기서열은 서열번호 2의 염기서열로 이루어진 것일 수 있다.
일 구체예에 따르면, 상기 서열번호 1의 아미노산 서열로 이루어진 글루타민합성효소 및 상기 서열번호 2의 염기서열로 이루어진 glnA 유전자는 기탁번호 KFCC10694의 코리네박테리움 글루타미컴(Corynebacterium glutamicum) 균주에서 유래한 것일 수 있다. 일 실시예에 따르면, 상기 KFCC10694 유래 glnA 서열은 다른 코리네박테리움 글루타미컴 균주인 ATCC13032 유래 glnA 서열의 상동성을 비교한 결과, 염기서열 상동성이 88.2%, 이로부터 발현한 글루타민합성효소의 아미노산 서열 상동성이 93.7%에 불과한 것으로 확인되었다. 이러한 서열 차이로 인해 KFCC10694 유래 글루타민합성효소는 다른 서열로 이루어진 글루타민합성효소보다 ATase에 의해 활성이 억제되는 정도에서 차이가 있을 수 있다.
일 구체예에 따르면, 상기 형질전환용 벡터는 상기 글루타민합성효소를 암호화하는 염기서열과 작동가능하게 연결된 프로모터를 포함하는 것일 수 있다. 용어 "작동가능하게 연결되어 있는 (operably linked)"는 발현이 필요한 유전자와 이의 조절 서열이 서로 기능적으로 결합되어 유전자 발현을 가능케 하는 방식으로 연결된 것을 의미한다. 상기 프로모터에 의해 GS의 발현량이 증가하면 ATase에 의한 억제가 저해되어 글루타민 생산성이 향상될 수 있다. 상기 프로모터는 구성적(constitutive) 프로모터 또는 유도성(inducible) 프로모터일 수 있다. 예를 들면, 상기 구성적 프로모터는 PcspB, PaprE, P180, Psod, PdapA, PporB, PilvC, PL10, PL26, PI16, PI51, PH30, PH36일 수 있으며, 유도성 프로모터는 PlacUV5, Ptac, Ptrc, PpopB, PaceA/aceB, PgntP/gntK, PCJ1OX2, Ptac-M, PmalE1, PBAD일 수 있다. 일 실시예에 따르면, 상기 프로모터는 Psod(superoxide dismutase) 프로모터일 수 있다. 상기 형질전환용 벡터는 인핸서(enhancer)와 같은 발현 조절 서열을 포함할 수 있다.
일 구체예에 따르면, 상기 형질전환용 벡터는 상기 글루타민합성효소를 암호화하는 염기서열과 작동가능하게 연결된 전사종결인자 서열을 포함하는 것일 수 있다. 일 실시예에 따르면, 상기 전사종결인자 서열은 rrnBT1T2 서열일 수 있다.
상기 형질전환용 벡터는 선택 표지로서 항생제(예컨대, 네오마이신, 카베니실린, 카나마이신, 스펙티노마이신 또는 하이그로마이신 등) 내성 유전자(예컨대, 네오마이신 포스포트랜스퍼라아제(nptⅡ) 또는 하이그로마이신 포스포트랜스퍼라아제(hpt) 등)를 포함할 수 있다.
상기 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터, 바이러스 벡터 등을 포함하나 이에 한정되는 것은 아니다.
다른 양상에 의하면, 상기 형질전환용 벡터로 형질전환된 균주를 제공한다.
일 구체예에 따르면, 상기 형질전환된 균주는 내생(native) glnA 유전자가 불활성화된 것일 수 있다. 용어 "내생(native)"는 미생물이 천연적으로 가지고 있는 유전자를 의미한다. 상기 불활성화(inactivation)은 해당 유전자의 전사, 해독, 유전자 산물의 활성의 손상을 초래하는 유전자 상의 변형을 의미하며, 프로모터의 불활성화도 포함될 수 있다. 이러한 유전자의 특이적 불활성화는 당 분야에서 확립된 방법을 통하여 수행할 수 있다. 예를 들면, 유전자의 결실 및 이형 서열(heterogenous sequence)의 삽입에 의한 유전자의 절단(truncation), 넌센스 돌연변이(nonsense mutation), 프레임쉬프트 돌연변이(frameshift mutation), 미스센스 돌연변이(missense mutation) 등에 의할 수 있다.
일 구체예에 따르면, 상기 형질전환된 균주는 내생(native) glnE 유전자가 불활성화된 것일 수 있다. 상기 내생 glnE 유전자의 발현이 저하 또는 상실되면 글루타민합성효소의 활성을 저해하는 ATase의 발현이 감소하므로 글루타민 생산능이 향상될 수 있다.
일 구체예에 따르면, 상기 형질전환된 균주는 코리네박테리움속 균주일 수 있으며, 예를 들면 기탁번호 KFCC10694로 기탁된 글루타미컴(Corynebacterium glutamicum) 균주일 수 있다.
상기 형질전환은 당해 분야에 공지된 바와 같이 숙주 세포에 따라 적합한 벡터 도입 기술을 선택할 수 있다. 예를 들면, 벡터 도입은 전기천공법 (electroporation), 열 충격 (heat-shock), 인산칼슘 (CaPO4) 침전, 염화칼슘 (CaCl2) 침전, 미세주입법 (microinjection), 폴리에틸렌글리콜 (PEG)법, DEAE-덱스트란법, 양이온 리포좀법, 초산 리튬-DMSO법, 또는 이들의 조합에 의해 수행될 수 있다.
또 다른 양상에 따르면, 상기 형질전환된 균주를 배양하는 단계를 포함하는 글루타민 생산방법을 제공한다.
상기 배양은 당업계에 알려진 적절한 배지와 배양조건에 따라 이루어질 수 있다. 또한 통상의 기술자라면 배지 및 배양 조건을 용이하게 조정하여 사용할 수 있다. 구체적으로, 상기 배지는 액체 배지일 수 있으나 이에 한정되는 것은 아니다. 배양 방법은 예를 들면, 회분식 배양 (batch culture), 연속식 배양 (continuous culture), 유가식 배양 (fed-batch culture) 또는 이들의 조합 배양을 포함할 수 있으나, 이에 한정되지 않는다.
상기 배지는 적절한 방식으로 특정 균주의 요건을 충족해야 하며, 통상의 기술자에 의해 적절하게 변형될 수 있다. 예를 들면, 코리네박테리아 균주에 대한 배양 배지는 공지된 문헌 (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981)을 참조할 수 있다. 또한 배지에 다양한 탄소원, 질소원 및 미량원소 성분을 포함할 수 있다. 사용될 수 있는 탄소원의 예를 들면 글루코스, 수크로스, 락토스, 프락토스, 말토스, 전분, 셀룰로스와 같은 당 및 탄수화물, 대두유, 해바라기유, 피마자유, 코코넛유 등과 같은 오일 및 지방, 팔미트산, 스테아린산, 리놀레산과 같은 지방산, 글리세롤, 에탄올과 같은 알코올, 아세트산과 같은 유기산이 포함될 수 있다. 이들 물질은 개별적으로 또는 혼합물로서 사용될 수 있다. 사용될 수 있는 질소원으로는 펩톤, 효모 추출물, 육즙, 맥아 추출물, 옥수수 침지액, 대두밀 및 요소 또는 무기 화합물이 있고, 예를 들면 황산 암모늄, 염화암모늄, 인산암모늄, 탄산암모늄 및 질산암모늄이 포함될 수 있다. 질소원 또한 개별적으로 또는 혼합물로서 사용할 수 있다. 사용될 수 있는 인의 공급원으로는 인산이수소칼륨 또는 인산수소이칼륨 또는 상응하는 나트륨-함유 염이 포함될 수 있다. 배양 배지는 성장에 필요한 황산마그네슘 또는 황산철과 같은 금속염을 함유할 수 있다. 그 외에, 아미노산 및 비타민과 같은 필수 성장 물질이 포함될 수 있다. 또한 배양 배지에 적절한 전구체들이 사용될 수 있다. 상기 배지 또는 개별 성분은 배양과정에서 배양액에 적절한 방식에 의해 회분식으로 또는 연속식으로 첨가될 수 있다.
배양 중에 수산화암모늄, 수산화칼륨, 암모니아, 인산 및 황산과 같은 화합물을 미생물 배양액에 적절한 방식으로 첨가하여 배양액의 pH를 조정할 수 있다. 또한, 배양 중에 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 추가적으로, 배양액의 호기 상태를 유지하기 위하여, 배양액 내로 산소 또는 산소-함유 기체 (예, 공기)를 주입할 수 있다. 배양액의 온도는 통상 20℃내지 45℃ 예를 들면 25℃ 내지 40℃일 수 있다. 배양기간은 원하는 양의 L-글루타민이 생성될 때까지 지속될 수 있으며, 예를 들면 5 내지 160 시간, 또는 10 내지 160시간일 수 있다.
상기 생산방법은 배양한 미생물 또는 배지로부터 L-글루타민을 회수하는 단계를 포함할 수 있다. 상기 L-글루타민을 회수하는 방법은 특별히 한정되는 것은 아니며, 배양방법에 따라 당해 분야에 공지된 적합한 방법을 이용하여 회수될 수 있다. 예를 들면 원심분리, 여과, 음이온 교환 크로마토그래피, 결정화, HPLC 등이 사용될 수 있다.
일 구체예에 따른 균주가 발현하는 글루타민합성효소는 ATase에 의한 되먹임 억제에 대한 영향이 낮으므로 글루타민 생산량을 현저히 증가시킬 수 있다.
도 1은 글루타민합성효소 활성의 조절 기전을 나타낸 것이다.
도 2는 일 실시예에 따른 pk19ms/△glnA 벡터를 나타낸 것이다.
도 3은 일 실시예에 따른 pk19ms/△glnE 벡터를 나타낸 것이다.
도 4는 일 실시예에 따른 pa'-glnA(ATCC13032) 벡터를 나타낸 것이다.
도 5는 일 실시예에 따른 pa'-glnA(KFCC10694) 벡터를 나타낸 것이다.
도 2는 일 실시예에 따른 pk19ms/△glnA 벡터를 나타낸 것이다.
도 3은 일 실시예에 따른 pk19ms/△glnE 벡터를 나타낸 것이다.
도 4는 일 실시예에 따른 pa'-glnA(ATCC13032) 벡터를 나타낸 것이다.
도 5는 일 실시예에 따른 pa'-glnA(KFCC10694) 벡터를 나타낸 것이다.
이하 하나 이상의 구체예를 실시예를 통해 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
실시예 1: KFCC-10694 염색체 상의 glnA, glnE 유전자 결손용 벡터 제작
벡터 제작에 사용한 재료는 Wizard Genomic DNA Purification Kit (Promega, USA), PrimeSTAR Max DNA Polymerase (Takara, Japan), DNA ligation kit (Takara, Japan), HindIII, BamHI(NEB, England)를 사용하였다.
1-1. glnA 결실용 벡터 제작
KFCC-10694 균주(Corynebacterium glutamicum MWM-891020)의 염색체 DNA를 주형으로 프라이머 1 및 프라이머 2로 PCR을 실시하여 KFCC-10694의 glnA의 left arm 증폭산물을 얻었다. 마찬가지로 프라이머 3 및 프라이머 4의 조합으로 PCR을 실시하여 glnA의 right arm 증폭 산물을 얻었다.
상기 glnA의 left arm과 right arm 증폭 산물을 프라이머 1 및 프라이머 4 조합으로 크로스오버 PCR을 실시하여 left arm과 right arm이 결합된 증폭산물을 얻고, pK19mobSacB 벡터의 BamH I 위치에 삽입하였다. 제작된 glnA 결실용 벡터는 pK19ms/△glnA로 명명하였다. (도 1 참조)
1-2. glnE 결실용 벡터 제작
상기 glnA 결실용 벡터와 동일한 방법으로, 프라이머 5와 프라이머 6의 조합으로 PCR을 실시하여 KFCC-10694의 glnE의 left arm 증폭 산물을 얻고, 프라이머 7과 프라이머 8의 조합으로 PCR을 실시하여 glnE의 right arm 증폭 산물을 얻었다.
상기 glnE의 left arm과 right arm 증폭 산물을 프라이머 5와 프라이머 8의 조합을 이용한 크로스오버 PCR로 증폭하여 left arm과 right arm이 결합된 산물을 얻고, 이를 pK19mobSacB 벡터의 HindIII위치에 삽입하였다. 제작된 glnE 결실용 벡터는 pK19ms/△glnE로 명명하였다. (도 2 참조)
상기 KFCC-10694의 glnA, KFCC-10694의 glnE, 프라이머 1 내지 8의 염기서열에 대한 정보는 하기 표 1에 개시되어 있다.
프라이머 | 서열목록 | 서열 (5’→3’) |
Primer 1 | 5 | gggatccatacccaagatggcatgg |
Primer 2 | 6 | gctggaggatttagatttggtgactcctcattgaca |
Primer 3 | 7 | tgtcaatgaggagtcaccaaatctaaatcctccagc |
Primer 4 | 8 | gggatcccttaaaaagcttttcgac |
Primer 5 | 9 | ggaagcttcttgacctgcatgatctcg |
Primer 6 | 10 | gccaatcgagaacgcatgcccactactttacggtca |
Primer 7 | 11 | atgaccgtaaagtagtgggcatgcgttctcgattggc |
Primer 8 | 12 | ggaagctttacaccaaccacaactgc |
실시예 2: glnA 과발현 벡터 2종 제작
ATCC13032의 glnA 유전자 DNA를 주형으로 프라이머 9 및 프라이머 10 조합으로 PCR을 실시하여 glnA(AT) 증폭산물을 얻었다.
이와 별개로, KFCC10694의 glnA 유전자 DNA를 주형으로 프라이머 11 및 프라이머 12를 조합으로 PCR을 실시하여 glnA(KF)증폭산물을 얻었다.
상기 glnA(AT)와 glnA(KF)의 상동성을 확인한 결과, 염기서열 상동성이 88.2%, 아미노산 서열 상동성이 93.7%에 불과한 것으로 확인되었다. glnA(AT)와 glnA(KF)의 아미노산 서열과 염기서열은 하기 표 2에 기재되어 있다.
목록 | 서열목록 | 서열정보 |
KFCC10694 GS 아미노산 |
1 | vafntpeeivkfikdenvefvdvrftdvpgteqhfsipaalfdeeaieeglafdgssirgfttidesdmnllpdlgtatidpfrkaktlnikffvhdpftreafsrdprnvarkaeqylastgiadtcnfgaeaefylfdsvrystdinssfyhvdtnegwwnrgretnldgtpnlgaknrvkggyfpvapydqtveirddmvnylsnagfqlerfhhevgggqqeinyrfntmlhaaddiqtfkyiikntahlhgktatfmpkplagdngsgmhahqslwkdgkplfhdesgyaglsdiaryyiggilhhagavlaftnptlnsyhrlvpgfeapinlvysqrnrsaavripitgsnpkakriefrapdpsgnpyfgfaammmagldgiknriephapvdkdlyelppeeaasipqaptsleaslkalqedsdfltesdvftedliesyiqykydneitpvrlrptpqefemyfdc |
KFCC10694 glnA 염기 |
2 | gtggcgtttaataccccggaagaaatagtcaagttcatcaaagacgagaacgtcgaattcgtagacgttcgcttcaccgatgtaccaggaactgaacagcacttcagcatcccagccgccctcttcgatgaagaggccatcgaagaaggcctagcattcgacggatcctcgatccgcggattcaccaccatcgatgagtctgacatgaacctcctaccagacctcggaactgccaccattgacccgttccgcaaggccaagactctgaacatcaagttcttcgttcatgatccattcacccgcgaagctttctcccgcgacccacgcaatgtggcacgcaaggcagagcagtacctcgcctccaccggcattgcagatacctgcaacttcggcgcagaagccgagttctacctctttgattcagtccgctactccaccgatattaactccagcttctaccacgttgataccaatgaaggctggtggaaccgtggccgggaaaccaaccttgatggcaccccaaaccttggcgccaagaaccgtgtcaagggcggatacttccctgttgcaccatatgaccaaaccgtggaaatccgcgatgatatggtcaactacctctcaaacgctggtttccaacttgagcgtttccaccacgaggtcggcggtggacagcaggagatcaactaccgcttcaacaccatgctgcacgcggctgatgatattcagacattcaagtacatcatcaagaacaccgctcacctccacggcaagaccgcaacctttatgcctaagccactggctggcgacaacggctctggaatgcacgcacaccagtccctatggaaggacggcaagccactcttccacgatgagtccggttacgcaggcctatctgacatcgcccgctactacattggtggcatcctgcaccacgcaggtgcagtattggcgttcaccaacccaaccctgaactcctaccaccgtttagttcctggcttcgaggcgccaatcaacttggtgtactcccagcgcaaccgctctgctgctgtacgtatcccaatcaccggatccaacccaaaggcaaagcgcatcgagttccgcgctccggacccatcaggcaacccatacttcggcttcgctgccatgatgatggctggccttgacggcatcaagaaccgcatcgagccacacgcaccagtggataaggatctctacgagcttccaccagaggaagctgcctccatcccacaggctccaacctcccttgaagcttcattgaaggctcttcaggaagattccgacttcctcaccgagtctgatgtcttcaccgaagatctcatcgagtcctacatccagtacaagtacgacaacgagatcaccccagtccgtttgcgcccaactcctcaagagttcgaaatgtacttcgactgctaa |
ATCC13032 GS 아미노산 |
3 | vafetpeeivkfikdenvefvdvrftdlpgteqhfsipaasfdadtieeglafdgssirgfttidesdmnllpdlgtatldpfrkaktlnvkffvhdpftreafsrdprnvarkaeqylastgiadtcnfgaeaefylfdsvrystemnsgfyevdteegwwnrgketnldgtpnlgaknrvkggyfpvapydqtvdvrddmvrnlaasgfalerfhhevgggqqeinyrfntmlhaaddiqtfkyiikntarlhgkaatfmpkplagdngsgmhahqslwkdgkplfhdesgyaglsdiaryyiggilhhagavlaftnatlnsyhrlvpgfeapinlvysqrnrsaavripitgsnpkakriefrapdpsgnpylgfaammmagldgiknriephapvdkdlyelppeeaasipqaptsleaslkalqedtdfltesdvftedlieayiqykydneispvrlrptpqefelyfdc |
ATCC13032 glnA 염기 |
4 | gtggcgtttgaaaccccggaagaaattgtcaagttcatcaaggatgaaaacgtcgagttcgttgacgttcgattcaccgaccttcccggcaccgagcagcacttcagcatcccagctgccagcttcgatgcagatacaatcgaagaaggtctcgcattcgacggatcctcgatccgtggcttcaccacgatcgacgaatctgacatgaatctcctgccagacctcggaacggccacccttgatccattccgcaaggcaaagaccctgaacgttaagttcttcgttcacgatcctttcacccgcgaggcattctcccgcgacccacgcaacgtggcacgcaaggcagagcagtacctggcatccaccggcattgcagacacctgcaacttcggcgccgaggctgagttctacctcttcgactccgttcgctactccaccgagatgaactccggcttctacgaagtagataccgaagaaggctggtggaaccgtggcaaggaaaccaacctcgacggcaccccaaacctgggcgcaaagaaccgcgtcaagggtggctacttcccagtagcaccatacgaccaaaccgttgacgtgcgcgatgacatggttcgcaacctcgcagcttccggcttcgctcttgagcgtttccaccacgaagtcggtggcggacagcaggaaatcaactaccgcttcaacaccatgctccacgcggcagatgatatccagaccttcaagtacatcatcaagaacaccgctcgcctccacggcaaggctgcaaccttcatgcctaagccactggctggcgacaacggttccggcatgcacgctcaccagtccctctggaaggacggcaagccactcttccacgatgagtccggctacgcaggcctgtccgacatcgcccgctactacatcggcggcatcctgcaccacgcaggcgctgttctggcgttcaccaacgcaaccctgaactcctaccaccgtctggttccaggcttcgaggctccaatcaacctggtgtactcacagcgcaaccgttccgctgctgtccgtatcccaatcaccggatccaacccgaaggcaaagcgcatcgaattccgcgctccagacccatcaggcaacccatacctgggctttgcagcgatgatgatggccggcctcgacggcatcaagaaccgcatcgagccacacgctccagtggacaaggacctctacgaactaccaccagaggaagctgcatccattccacaggcaccaacctccctggaagcatccctgaaggcactgcaggaagacaccgacttcctcaccgagtctgacgtcttcaccgaggatctcatcgaggcgtacatccagtacaagtacgacaacgagatctccccagttcgcctgcgcccaaccccgcaggaattcgaattgtacttcgactgctaa |
sod 프로모터는 ATCC13032 염색체 DNA를 주형으로 프라이머 13(forward)과 프라이머 14(backward)의 조합으로 증폭한 제 1 sod 프로모터 증폭산물, 프라이머 13(forward)과 프라이머 15(backward)의 조합으로 증폭한 제 2 sod 프로모터 증폭산물을 제작하였다. 역방향 프라이머를 프라이머 14 및 15로 구별하여 사용한 이유는 glnA 서열 차이 때문이며, sod 프로모터 서열은 동일하다.(하기 표 3의 gggtaaaaaatcctttcg 참조)
rrnBT1T2 전사종결인자(터미네이터) 서열은 대장균 DH5a 염색체 DNA를 주형으로 하여 프라이머 16(forward)과 프라이머 17(backward)의 조합으로 증폭한 제 1 rrnBT1T2 증폭산물; 및 프라이머 18(forward) 및 프라이머 17(backward)의 조합으로 증폭한 제 2 rrnBT1T2 증폭산물을 얻었다. 정방향 프라이머를 프라이머 16 및 18로 구별하여 사용한 이유는 glnA 서열 차이 때문이며, 전사종결인자의 서열은 동일하다. (하기 표 3의 agaatttgcctggcggca 참조)
프라이머 13과 프라이머 17의 조합을 이용한 크로스오버 PCR을 실시하여 상기 제 1 sod 프로모터 증폭산물, glnA(AT), 및 제 1 rrnBT1T2 터미네이터 증폭산물을 E. Coli-Corynebacterium 셔틀벡터인 pa'벡터 내 BamHI 위치에 삽입하여 pa'-glnA(AT) 벡터를 제작하였다. (도 4 참조)
마찬가지로, 프라이머 13과 프라이머 17의 조합을 이용한 크로스오버 PCR을 실시하여 상기 제 2 sod 프로모터 증폭산물, glnA(KF), 및 제 2 rrnBT1T2 터미네이터 증폭산물을 E. Coli-Corynebacterium 셔틀벡터인 pa'벡터 내 BamHI 위치에 삽입하여 pa'-glnA(KF) 벡터를 제작하였다. (도 5 참조)
실시예 2에서 사용된 실험 재료는 PrimeSTAR Max DNA Polymerase (Takara, Japan), DNA ligation kit (Takara, Japan), BamHI(NEB, England)이다.
상기 프라이머 9 내지 18의 염기서열은 하기 표 3에 개시되어 있다.
프라이머 | 서열목록 | 서열 (5’→3’) |
Primer 9 | 13 | cgaaaggattttttacccgtggcgtttgaaaccccg |
Primer 10 | 14 | tgccgccaggcaaattctttagcagtcgaagtacaa |
Primer 11 | 15 | cgaaaggattttttacccgtggcgtttaataccccgg |
Primer 12 | 16 | ctgccgccaggcaaattctttagcagtcgaagtacat |
Primer 13 | 17 | gggatccagctgccaattattccggg |
Primer 14 | 18 | cggggtttcaaacgccac gggtaaaaaatcctttcg |
Primer 15 | 19 | ccggggtattaaacgccac gggtaaaaaatcctttcg |
Primer 16 | 20 | ttgtacttcgactgctaa agaatttgcctggcggca |
Primer 17 | 21 | gggatccttcgttttatttgatgcc |
Primer 18 | 22 | atgtacttcgactgctaa agaatttgcctggcggcag |
실시예 3:
glnA 유전자 및/또는 glnE유전자가 결손된 KFCC10694 균주 제작
컴피턴트 세포로 만들어진 KFCC10694 균주에 상기 실시예 1에서 제작된 pK19ms/△glnA 벡터를 전기충격요법(electroporation)으로 도입하고, 2YT KM AGAR 배지에 도말 후, 30℃ 배양기에서 4일간 배양하여 콜로니를 얻었다. 1차 상동재조합이 유도된 콜로니 중 형질전환이 확인된 콜로니를 2YT액체배지에서 12시간 배양후 2YT Sucrose GM AGAR배지에 도말하고 2차 상동재조합을 통해 항생제 마커를 제거하였다. 선별된 콜로니를 PCR 및 서열분석을 통해 glnA 유전자가 의도대로 제거 되었는지를 최종확인 하였다. 상기 과정을 통해 제작된 glnA 유전자가 결손된 균주를 D10694A로 명명하였다.
동일한 방법으로, 상기 D10694A균주에 상기 실시예 1에서 제작한 pK19ms/△glnE벡터를 사용하여 glnA와 glnE유전자가 동시에 결손된 균주를 제작하였다. 본 실험에 사용된 배지에는 glutamine을 100mg/L의 농도로 첨가하여 사용하였다. 상기 과정을 통해 제작된 glnA 및 glnE 유전자가 모두 결손된 균주를 D10694AE로 명명하였다.
상기 실시예 3에서 사용된 실험 재료는 2YT AGAR (Tryptone 16g/L, Yeast extract 10g/L, NaCl 5g/L, AGAR 1.5%), 2YT KM AGAR (2YT AGAR, Kanamycine 15mg/L), 2YT Sucrose GM AGAR(2YT AGAR, Sucrose 100g/L, Glutamine 100mg/L), electrophorator(BIO-RAD, USA) 이다.
실시예 4: pa'-glnA(AT) 벡터 또는 pa'-glnA(KF) 벡터 도입 균주 제작
실시예 2에서 제작된 pa'-glnA(ATCC13032) 벡터, pa'-glnA(KFCC10694) 벡터를 상기 실시예 3에서 제작한 D10694A 및 D10694AE 균주에 전기충격요법 (electroporation)으로 도입하였다. 벡터를 도입한 균주들을 2YT KM AGAR 배지에 도말하고 30℃ 배양기에서 3일간 배양하여 콜로니를 얻었다. 상기 과정을 통해 제작된 균주들을 D10694A/pa-glnA(AT), D10694A/pa-glnA(KF), D10694AE/pa-glnA(AT), D10694AE/pa-glnA(KF) 로 명명하였다.
실시예 4에서 사용된 실험 재료는 2YT AGAR (Tryptone 16g/L, Yeast extract 10g/L, NaCl 5g/L, AGAR 1.5%), 2YT KM AGAR (2YT AGAR, Kanamycine 15mg/L), Electrophorator(BIO-RAD, USA)이다.
실시예 5: 실시예 4에서 제작된 균주의 글루타민 생산성 확인
종배지 20ml를 500ml 삼각 플라스크에 분주하여 통상의 방법에 따라 가압 멸균한 후 균주를 접종하여 30℃에서 24 시간 동안 진탕배양한 것을 종균배양액으로 하였다. 생산배지 100ml를 500ml 삼각 플라스크에 분주하여 통상의 방법에 따라 가압 멸균한 후 미리 준비한 종균배양액 100ml를 접종하여 30℃에서 72시간 진탕 배양 하였다. D10694A와 D10694AE균주에 대한 생산성 비교에는 배지에 100mg/L의 글루타민을 첨가하였다. 배양 종료액에서의 L-글루타민 함량의 결정은 통상의 HPLC법으로 정량하였다.
글루타민 생산성 실험결과는 하기 표 4에 기재되어 있다.
균주명 | OD(1/100) | △(L-GLN(%)) |
KFCC10694 | 0.256 | 3.56 |
D10694A (glnA 결손) | 0.135 | n.d. |
D10694AE (glnA 및 glnE 결손) | 0.185 | n.d. |
D10694A/Pa-glnA(AT) | 0.138 | 1.97 |
D10694A/Pa-glnA(KF) | 0.202 | 5.24 |
D10694AE/Pa-glnA(AT) | 0.209 | 3.88 |
D10694AE/Pa-glnA(KF) | 0.217 | 5.31 |
상기 표 3의 실험결과에 따르면, D10694A 균주 및 D10694AE는 glnA(GS 발현 유전자)가 결손되어 L-글루타민이 생산되지 않았다.
상기 표 3의 D10694A/Pa-glnA(AT)와 D10694A/Pa-glnA(KF)의 글루타민 생산을 살펴보면, D10694A/Pa-glnA(AT)는 모주인 KFCC10694보다 L-글루타민 생산성이 감소한 반면 D10694A/Pa-glnA(KF)는 모주인 KFCC10694보다 생산성이 향상되었다. 양균주는 동일한 sod 프로모터를 도입하였음에도 불구하고, glnA(AT)보다 glnA(KF)를 도입한 경우 생산성이 현저히 높았다. 이는 glnA(AT)와 glnA(KF)는 되먹임 억제 정도에서 차이가 있음을 시사한다.
ATase가 불활성화되어 되먹임 억제를 받지 않는 D10694AE/Pa-glnA(AT)와 D10694AE/Pa-glnA(KF)의 글루타민 생산을 살펴보면, D10694AE/Pa-glnA(AT) 균주는 D10694A/Pa-glnA(AT)보다 생산성이 약 1.91% 향상되었으므로 ATase 활성 여부에 따른 차이가 크다는 것을 알 수 있었다. 그러나, D10694AE/Pa-glnA(KF) 균주는 D10694A/Pa-glnA(KF)보다 생산성이 0.07% 향상되었으나 그 정도는 크지 않았으므로 ATase 활성 여부에 따른 차이가 크지 않다는 것을 알 수 있었다. 이는 glnA(KF)는 ATase에 의한 되먹임 억제의 영향이 적다는 것을 의미한다.
보다 자세히 살펴보면, D10694A/Pa-glnA(KF) 균주는 모주 대비 약 47% 증가된 생산성을 나타내었으며, D10694AE/Pa-glnA(KF) 균주는 모주 대비 약 49% 증가된 생산성을 나타내었다. 여기서 주목할 것은, D10694A/Pa-glnA(KF)와 D10694AE/Pa-glnA(KF)의 생산성 차이가 모주 대비 2%에 불과하여 크지 않았다는 점이다. D10694AE/Pa-glnA(KF)는 D10694A/Pa-glnA(KF)보다 GS의 되먹임 저해에 관여하는 glnE유전자를 추가적으로 결손시켰음에도 불구하고, 모주 대비 단 2%의 생산성 향상을 나타내었으므로, ATase 활성의 유무에 따른 글루타민 생산성 차이가 크지 않은 것이다. 이는 glnA(KF) 도입에 의한 생산성 증가는 단순히 sod 프로모터의 기여에 의한 것만 아니라 glnA(KF)의 되먹임 저해에 대한 저항이 기여하였음을 시사한다.
<110> Daesang Corporation
<120> Recombinant vector for transformation to improve glutamine
production capacity and recombinant strain which introduced the
same
<130> PN200066
<160> 22
<170> KoPatentIn 3.0
<210> 1
<211> 477
<212> PRT
<213> Artificial Sequence
<220>
<223> KFCC10694 GS
<400> 1
Val Ala Phe Asn Thr Pro Glu Glu Ile Val Lys Phe Ile Lys Asp Glu
1 5 10 15
Asn Val Glu Phe Val Asp Val Arg Phe Thr Asp Val Pro Gly Thr Glu
20 25 30
Gln His Phe Ser Ile Pro Ala Ala Leu Phe Asp Glu Glu Ala Ile Glu
35 40 45
Glu Gly Leu Ala Phe Asp Gly Ser Ser Ile Arg Gly Phe Thr Thr Ile
50 55 60
Asp Glu Ser Asp Met Asn Leu Leu Pro Asp Leu Gly Thr Ala Thr Ile
65 70 75 80
Asp Pro Phe Arg Lys Ala Lys Thr Leu Asn Ile Lys Phe Phe Val His
85 90 95
Asp Pro Phe Thr Arg Glu Ala Phe Ser Arg Asp Pro Arg Asn Val Ala
100 105 110
Arg Lys Ala Glu Gln Tyr Leu Ala Ser Thr Gly Ile Ala Asp Thr Cys
115 120 125
Asn Phe Gly Ala Glu Ala Glu Phe Tyr Leu Phe Asp Ser Val Arg Tyr
130 135 140
Ser Thr Asp Ile Asn Ser Ser Phe Tyr His Val Asp Thr Asn Glu Gly
145 150 155 160
Trp Trp Asn Arg Gly Arg Glu Thr Asn Leu Asp Gly Thr Pro Asn Leu
165 170 175
Gly Ala Lys Asn Arg Val Lys Gly Gly Tyr Phe Pro Val Ala Pro Tyr
180 185 190
Asp Gln Thr Val Glu Ile Arg Asp Asp Met Val Asn Tyr Leu Ser Asn
195 200 205
Ala Gly Phe Gln Leu Glu Arg Phe His His Glu Val Gly Gly Gly Gln
210 215 220
Gln Glu Ile Asn Tyr Arg Phe Asn Thr Met Leu His Ala Ala Asp Asp
225 230 235 240
Ile Gln Thr Phe Lys Tyr Ile Ile Lys Asn Thr Ala His Leu His Gly
245 250 255
Lys Thr Ala Thr Phe Met Pro Lys Pro Leu Ala Gly Asp Asn Gly Ser
260 265 270
Gly Met His Ala His Gln Ser Leu Trp Lys Asp Gly Lys Pro Leu Phe
275 280 285
His Asp Glu Ser Gly Tyr Ala Gly Leu Ser Asp Ile Ala Arg Tyr Tyr
290 295 300
Ile Gly Gly Ile Leu His His Ala Gly Ala Val Leu Ala Phe Thr Asn
305 310 315 320
Pro Thr Leu Asn Ser Tyr His Arg Leu Val Pro Gly Phe Glu Ala Pro
325 330 335
Ile Asn Leu Val Tyr Ser Gln Arg Asn Arg Ser Ala Ala Val Arg Ile
340 345 350
Pro Ile Thr Gly Ser Asn Pro Lys Ala Lys Arg Ile Glu Phe Arg Ala
355 360 365
Pro Asp Pro Ser Gly Asn Pro Tyr Phe Gly Phe Ala Ala Met Met Met
370 375 380
Ala Gly Leu Asp Gly Ile Lys Asn Arg Ile Glu Pro His Ala Pro Val
385 390 395 400
Asp Lys Asp Leu Tyr Glu Leu Pro Pro Glu Glu Ala Ala Ser Ile Pro
405 410 415
Gln Ala Pro Thr Ser Leu Glu Ala Ser Leu Lys Ala Leu Gln Glu Asp
420 425 430
Ser Asp Phe Leu Thr Glu Ser Asp Val Phe Thr Glu Asp Leu Ile Glu
435 440 445
Ser Tyr Ile Gln Tyr Lys Tyr Asp Asn Glu Ile Thr Pro Val Arg Leu
450 455 460
Arg Pro Thr Pro Gln Glu Phe Glu Met Tyr Phe Asp Cys
465 470 475
<210> 2
<211> 1434
<212> DNA
<213> Artificial Sequence
<220>
<223> KFCC10694 glnA
<400> 2
gtggcgttta ataccccgga agaaatagtc aagttcatca aagacgagaa cgtcgaattc 60
gtagacgttc gcttcaccga tgtaccagga actgaacagc acttcagcat cccagccgcc 120
ctcttcgatg aagaggccat cgaagaaggc ctagcattcg acggatcctc gatccgcgga 180
ttcaccacca tcgatgagtc tgacatgaac ctcctaccag acctcggaac tgccaccatt 240
gacccgttcc gcaaggccaa gactctgaac atcaagttct tcgttcatga tccattcacc 300
cgcgaagctt tctcccgcga cccacgcaat gtggcacgca aggcagagca gtacctcgcc 360
tccaccggca ttgcagatac ctgcaacttc ggcgcagaag ccgagttcta cctctttgat 420
tcagtccgct actccaccga tattaactcc agcttctacc acgttgatac caatgaaggc 480
tggtggaacc gtggccggga aaccaacctt gatggcaccc caaaccttgg cgccaagaac 540
cgtgtcaagg gcggatactt ccctgttgca ccatatgacc aaaccgtgga aatccgcgat 600
gatatggtca actacctctc aaacgctggt ttccaacttg agcgtttcca ccacgaggtc 660
ggcggtggac agcaggagat caactaccgc ttcaacacca tgctgcacgc ggctgatgat 720
attcagacat tcaagtacat catcaagaac accgctcacc tccacggcaa gaccgcaacc 780
tttatgccta agccactggc tggcgacaac ggctctggaa tgcacgcaca ccagtcccta 840
tggaaggacg gcaagccact cttccacgat gagtccggtt acgcaggcct atctgacatc 900
gcccgctact acattggtgg catcctgcac cacgcaggtg cagtattggc gttcaccaac 960
ccaaccctga actcctacca ccgtttagtt cctggcttcg aggcgccaat caacttggtg 1020
tactcccagc gcaaccgctc tgctgctgta cgtatcccaa tcaccggatc caacccaaag 1080
gcaaagcgca tcgagttccg cgctccggac ccatcaggca acccatactt cggcttcgct 1140
gccatgatga tggctggcct tgacggcatc aagaaccgca tcgagccaca cgcaccagtg 1200
gataaggatc tctacgagct tccaccagag gaagctgcct ccatcccaca ggctccaacc 1260
tcccttgaag cttcattgaa ggctcttcag gaagattccg acttcctcac cgagtctgat 1320
gtcttcaccg aagatctcat cgagtcctac atccagtaca agtacgacaa cgagatcacc 1380
ccagtccgtt tgcgcccaac tcctcaagag ttcgaaatgt acttcgactg ctaa 1434
<210> 3
<211> 477
<212> PRT
<213> Artificial Sequence
<220>
<223> ATCC13032 GS
<400> 3
Val Ala Phe Glu Thr Pro Glu Glu Ile Val Lys Phe Ile Lys Asp Glu
1 5 10 15
Asn Val Glu Phe Val Asp Val Arg Phe Thr Asp Leu Pro Gly Thr Glu
20 25 30
Gln His Phe Ser Ile Pro Ala Ala Ser Phe Asp Ala Asp Thr Ile Glu
35 40 45
Glu Gly Leu Ala Phe Asp Gly Ser Ser Ile Arg Gly Phe Thr Thr Ile
50 55 60
Asp Glu Ser Asp Met Asn Leu Leu Pro Asp Leu Gly Thr Ala Thr Leu
65 70 75 80
Asp Pro Phe Arg Lys Ala Lys Thr Leu Asn Val Lys Phe Phe Val His
85 90 95
Asp Pro Phe Thr Arg Glu Ala Phe Ser Arg Asp Pro Arg Asn Val Ala
100 105 110
Arg Lys Ala Glu Gln Tyr Leu Ala Ser Thr Gly Ile Ala Asp Thr Cys
115 120 125
Asn Phe Gly Ala Glu Ala Glu Phe Tyr Leu Phe Asp Ser Val Arg Tyr
130 135 140
Ser Thr Glu Met Asn Ser Gly Phe Tyr Glu Val Asp Thr Glu Glu Gly
145 150 155 160
Trp Trp Asn Arg Gly Lys Glu Thr Asn Leu Asp Gly Thr Pro Asn Leu
165 170 175
Gly Ala Lys Asn Arg Val Lys Gly Gly Tyr Phe Pro Val Ala Pro Tyr
180 185 190
Asp Gln Thr Val Asp Val Arg Asp Asp Met Val Arg Asn Leu Ala Ala
195 200 205
Ser Gly Phe Ala Leu Glu Arg Phe His His Glu Val Gly Gly Gly Gln
210 215 220
Gln Glu Ile Asn Tyr Arg Phe Asn Thr Met Leu His Ala Ala Asp Asp
225 230 235 240
Ile Gln Thr Phe Lys Tyr Ile Ile Lys Asn Thr Ala Arg Leu His Gly
245 250 255
Lys Ala Ala Thr Phe Met Pro Lys Pro Leu Ala Gly Asp Asn Gly Ser
260 265 270
Gly Met His Ala His Gln Ser Leu Trp Lys Asp Gly Lys Pro Leu Phe
275 280 285
His Asp Glu Ser Gly Tyr Ala Gly Leu Ser Asp Ile Ala Arg Tyr Tyr
290 295 300
Ile Gly Gly Ile Leu His His Ala Gly Ala Val Leu Ala Phe Thr Asn
305 310 315 320
Ala Thr Leu Asn Ser Tyr His Arg Leu Val Pro Gly Phe Glu Ala Pro
325 330 335
Ile Asn Leu Val Tyr Ser Gln Arg Asn Arg Ser Ala Ala Val Arg Ile
340 345 350
Pro Ile Thr Gly Ser Asn Pro Lys Ala Lys Arg Ile Glu Phe Arg Ala
355 360 365
Pro Asp Pro Ser Gly Asn Pro Tyr Leu Gly Phe Ala Ala Met Met Met
370 375 380
Ala Gly Leu Asp Gly Ile Lys Asn Arg Ile Glu Pro His Ala Pro Val
385 390 395 400
Asp Lys Asp Leu Tyr Glu Leu Pro Pro Glu Glu Ala Ala Ser Ile Pro
405 410 415
Gln Ala Pro Thr Ser Leu Glu Ala Ser Leu Lys Ala Leu Gln Glu Asp
420 425 430
Thr Asp Phe Leu Thr Glu Ser Asp Val Phe Thr Glu Asp Leu Ile Glu
435 440 445
Ala Tyr Ile Gln Tyr Lys Tyr Asp Asn Glu Ile Ser Pro Val Arg Leu
450 455 460
Arg Pro Thr Pro Gln Glu Phe Glu Leu Tyr Phe Asp Cys
465 470 475
<210> 4
<211> 1434
<212> DNA
<213> Artificial Sequence
<220>
<223> ATCC13032 glnA
<400> 4
gtggcgtttg aaaccccgga agaaattgtc aagttcatca aggatgaaaa cgtcgagttc 60
gttgacgttc gattcaccga ccttcccggc accgagcagc acttcagcat cccagctgcc 120
agcttcgatg cagatacaat cgaagaaggt ctcgcattcg acggatcctc gatccgtggc 180
ttcaccacga tcgacgaatc tgacatgaat ctcctgccag acctcggaac ggccaccctt 240
gatccattcc gcaaggcaaa gaccctgaac gttaagttct tcgttcacga tcctttcacc 300
cgcgaggcat tctcccgcga cccacgcaac gtggcacgca aggcagagca gtacctggca 360
tccaccggca ttgcagacac ctgcaacttc ggcgccgagg ctgagttcta cctcttcgac 420
tccgttcgct actccaccga gatgaactcc ggcttctacg aagtagatac cgaagaaggc 480
tggtggaacc gtggcaagga aaccaacctc gacggcaccc caaacctggg cgcaaagaac 540
cgcgtcaagg gtggctactt cccagtagca ccatacgacc aaaccgttga cgtgcgcgat 600
gacatggttc gcaacctcgc agcttccggc ttcgctcttg agcgtttcca ccacgaagtc 660
ggtggcggac agcaggaaat caactaccgc ttcaacacca tgctccacgc ggcagatgat 720
atccagacct tcaagtacat catcaagaac accgctcgcc tccacggcaa ggctgcaacc 780
ttcatgccta agccactggc tggcgacaac ggttccggca tgcacgctca ccagtccctc 840
tggaaggacg gcaagccact cttccacgat gagtccggct acgcaggcct gtccgacatc 900
gcccgctact acatcggcgg catcctgcac cacgcaggcg ctgttctggc gttcaccaac 960
gcaaccctga actcctacca ccgtctggtt ccaggcttcg aggctccaat caacctggtg 1020
tactcacagc gcaaccgttc cgctgctgtc cgtatcccaa tcaccggatc caacccgaag 1080
gcaaagcgca tcgaattccg cgctccagac ccatcaggca acccatacct gggctttgca 1140
gcgatgatga tggccggcct cgacggcatc aagaaccgca tcgagccaca cgctccagtg 1200
gacaaggacc tctacgaact accaccagag gaagctgcat ccattccaca ggcaccaacc 1260
tccctggaag catccctgaa ggcactgcag gaagacaccg acttcctcac cgagtctgac 1320
gtcttcaccg aggatctcat cgaggcgtac atccagtaca agtacgacaa cgagatctcc 1380
ccagttcgcc tgcgcccaac cccgcaggaa ttcgaattgt acttcgactg ctaa 1434
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 1
<400> 5
gggatccata cccaagatgg catgg 25
<210> 6
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 2
<400> 6
gctggaggat ttagatttgg tgactcctca ttgaca 36
<210> 7
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 3
<400> 7
tgtcaatgag gagtcaccaa atctaaatcc tccagc 36
<210> 8
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 4
<400> 8
gggatccctt aaaaagcttt tcgac 25
<210> 9
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 5
<400> 9
ggaagcttct tgacctgcat gatctcg 27
<210> 10
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 6
<400> 10
gccaatcgag aacgcatgcc cactacttta cggtca 36
<210> 11
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 7
<400> 11
atgaccgtaa agtagtgggc atgcgttctc gattggc 37
<210> 12
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 8
<400> 12
ggaagcttta caccaaccac aactgc 26
<210> 13
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 9
<400> 13
cgaaaggatt ttttacccgt ggcgtttgaa accccg 36
<210> 14
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 10
<400> 14
tgccgccagg caaattcttt agcagtcgaa gtacaa 36
<210> 15
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 11
<400> 15
cgaaaggatt ttttacccgt ggcgtttaat accccgg 37
<210> 16
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 12
<400> 16
ctgccgccag gcaaattctt tagcagtcga agtacat 37
<210> 17
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 13
<400> 17
gggatccagc tgccaattat tccggg 26
<210> 18
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 14
<400> 18
cggggtttca aacgccacgg gtaaaaaatc ctttcg 36
<210> 19
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 15
<400> 19
ccggggtatt aaacgccacg ggtaaaaaat cctttcg 37
<210> 20
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 16
<400> 20
ttgtacttcg actgctaaag aatttgcctg gcggca 36
<210> 21
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 17
<400> 21
gggatccttc gttttatttg atgcc 25
<210> 22
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 18
<400> 22
atgtacttcg actgctaaag aatttgcctg gcggcag 37
Claims (8)
- 서열번호 1의 아미노산 서열로 이루어진 글루타민합성효소를 암호화하는 염기서열을 포함하는 형질전환용 벡터.
- 제1항에 있어서,
상기 글루타민합성효소를 암호화하는 염기서열은 서열번호 2의 염기서열로 이루어진 것인,
형질전환용 벡터. - 제1항에 있어서,
상기 형질전환용 벡터는 상기 글루타민합성효소를 암호화하는 염기서열과 작동가능하게 연결된 프로모터를 포함하는 것인,
형질전환용 벡터. - 제1항에 있어서,
상기 형질전환용 벡터는 상기 글루타민합성효소를 암호화하는 염기서열과 작동가능하게 연결된 전사종결인자 서열을 포함하는 것인,
형질전환용 벡터. - 제1항의 형질전환용 벡터로 형질전환된 균주.
- 제5항에 있어서,
상기 균주는 내생 glnE 유전자의 발현이 불활성화된 것인,
균주. - 제5항에 있어서,
상기 균주는 코리네박테리움 글루타미컴(Corynebacterium glutamicum)인,
균주. - 제5항의 균주를 배양하는 단계를 포함하는,
글루타민 생산 방법.
Priority Applications (6)
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KR1020200038628A KR102216450B1 (ko) | 2020-03-30 | 2020-03-30 | 글루타민 생산능을 향상시키는 형질전환용 재조합 벡터 및 이를 도입한 균주 |
EP21780977.1A EP4130255A4 (en) | 2020-03-30 | 2021-03-24 | RECOMBINANT TRANSFORMATION VECTOR TO IMPROVE GLUTAMINE PRODUCTIVITY AND STRAIN THEREFOR |
US17/916,331 US20230220430A1 (en) | 2020-03-30 | 2021-03-24 | Recombinant vector for transformation improving glutamine productivity, and strain employing same |
PCT/KR2021/003633 WO2021201489A1 (ko) | 2020-03-30 | 2021-03-24 | 글루타민 생산능을 향상시키는 형질전환용 재조합 벡터 및 이를 도입한 균주 |
JP2022560115A JP7415041B2 (ja) | 2020-03-30 | 2021-03-24 | グルタミン生産能を向上させる形質転換用組換えベクターおよびこれを導入した菌株 |
CN202180026206.XA CN115349012B (zh) | 2020-03-30 | 2021-03-24 | 用于进行提高谷氨酰胺生产力的转化的重组载体和使用其的菌株 |
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KR1020200038628A KR102216450B1 (ko) | 2020-03-30 | 2020-03-30 | 글루타민 생산능을 향상시키는 형질전환용 재조합 벡터 및 이를 도입한 균주 |
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US (1) | US20230220430A1 (ko) |
EP (1) | EP4130255A4 (ko) |
JP (1) | JP7415041B2 (ko) |
KR (1) | KR102216450B1 (ko) |
CN (1) | CN115349012B (ko) |
WO (1) | WO2021201489A1 (ko) |
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WO2021201489A1 (ko) * | 2020-03-30 | 2021-10-07 | 대상 주식회사 | 글루타민 생산능을 향상시키는 형질전환용 재조합 벡터 및 이를 도입한 균주 |
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EP1783203B1 (en) * | 2004-06-25 | 2010-01-20 | Kyowa Hakko Bio Co., Ltd. | Process for producing substances |
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KR100834991B1 (ko) * | 1999-06-25 | 2008-06-05 | 백광산업 주식회사 | 대사 경로 단백질을 코딩하는 코리네박테리움 글루타미쿰유전자 |
JP4560998B2 (ja) * | 2001-02-05 | 2010-10-13 | 味の素株式会社 | 発酵法によるl−グルタミンの製造法及びl−グルタミン生産菌 |
CN106635946A (zh) | 2016-12-29 | 2017-05-10 | 廊坊梅花生物技术开发有限公司 | 一种谷氨酸棒杆菌及其构建方法与应用 |
KR102216450B1 (ko) * | 2020-03-30 | 2021-02-17 | 대상 주식회사 | 글루타민 생산능을 향상시키는 형질전환용 재조합 벡터 및 이를 도입한 균주 |
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- 2021-03-24 EP EP21780977.1A patent/EP4130255A4/en active Pending
- 2021-03-24 CN CN202180026206.XA patent/CN115349012B/zh active Active
- 2021-03-24 US US17/916,331 patent/US20230220430A1/en active Pending
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EP1424397B1 (en) * | 2002-11-26 | 2011-01-19 | Ajinomoto Co., Inc. | Method for producing L-glutamine and L-glutamine producing bacterium |
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WO2021201489A1 (ko) | 2021-10-07 |
EP4130255A4 (en) | 2024-04-24 |
JP7415041B2 (ja) | 2024-01-16 |
CN115349012A (zh) | 2022-11-15 |
CN115349012B (zh) | 2023-07-18 |
US20230220430A1 (en) | 2023-07-13 |
JP2023521310A (ja) | 2023-05-24 |
EP4130255A1 (en) | 2023-02-08 |
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