KR101152876B1 - 구아노신-이노신 키나아제의 추가 발현에 의한 재조합 대장균에서 gdp-푸코즈의 생산방법 - Google Patents
구아노신-이노신 키나아제의 추가 발현에 의한 재조합 대장균에서 gdp-푸코즈의 생산방법 Download PDFInfo
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Abstract
대장균 내에 존재하는 대표적인 NADPH 재생산효소인 글루코오스-6-포스페이트 데하이드로지나아제(glucose-6-phosphate dehydrogenase; G6PDH)와 ManB, ManC, Gmd 및 WcaG를 동시발현한 재조합 대장균을 이용하여, pH-스탯(pH-stat) 방식의 유가식 배양에서 최대 235.2±3.3mg/L의 GDP-푸코즈를 생산하였는데, 이는 G6PDH를 추가발현하지 않은 재조합 대장균에 비해 21% 향상한 결과이다.
또한, 구아노신-이노신 키나아제(guanosine-inosine kinase; Gsk)를 추가발현한 재조합 대장균을 이용하여, pH-스탯 방식의 유가식 배양에서 최대 305.5±5.3mg/L의 GDP-푸코즈를 생산하였는데, 이는 Gsk를 추가발현하지 않은 재조합대장균보다 58% 향상된 결과이다.
Description
도 2는 대장균의 개괄적인 탄소대사경로 및 NADPH 재생산에 관련된 효소들을 보여준다.
도 3은 대장균에서 GDP-L-푸코즈의 생합성 경로와 구아노신 누클레오타이드의 생합성 경로를 보여준다.
도 4는 본 실험에서 사용된 플라스미드를 보여준다.
도 5는 GDP-푸코즈 생합성 효소들을 발현하는 균주인 이 콜라이(E. coli) BL21 star(DE3)/pmBCGW의 pH-스탯 방식의 유가식 배양 결과이다. 건조균체량(●), 포도당 농도(□), 아세트산 농도(■), GDP-푸코즈 농도(▲) 및 IPTG 첨가에 의한 GDP-푸코즈 생합성 효소의 발현유도(화살표).
도 6은 GDP-푸코즈 생합성 효소들과 G6PDH를 동시발현하는 균주인 이 콜라이(E. coli) BL21 star(DE3)/pmBCGW+pMWzwf의 pH-스탯 방식의 유가식 배양 결과이다. 건조균체량(●), 포도당 농도(□), 아세트산 농도(■), GDP-푸코즈 농도(▲) 및 IPTG 첨가에 의한 GDP-푸코즈 생합성 효소의 발현유도(화살표).
도 7은 GDP-푸코즈 생합성 효소들과 GuaA 및 GuaB를 동시발현하는 균주인 이 콜라이(E. coli) BL21 star(DE3)/pmBCGW+pHguaBA의 pH-스탯 방식의 유가식 배양 결과이다. 건조균체량(●), 포도당 농도(□), 아세트산 농도(■), GDP-푸코즈 농도(▲) 및 IPTG 첨가에 의한 GDP-푸코즈 생합성 효소의 발현유도 (화살표).
도 8은 GDP-푸코즈 생합성 효소들과 GuaC를 동시발현하는 균주인 이 콜라이(E. coli) BL21 star(DE3)/pmBCGW+pHguaC의 pH-스탯 방식의 유가식 배양 결과이다. 건조균체량(●), 포도당 농도(□), 아세트산 농도(■), GDP-푸코즈 농도(▲) 및 IPTG 첨가에 의한 GDP-푸코즈 생합성 효소의 발현유도(화살표).
도 9는 GDP-푸코즈 생합성 효소들과 Gsk를 동시발현하는 균주인 이 콜라이(E. coli) BL21 star(DE3)/pmBCGW+pHgsk의 pH-스탯 방식의 유가식 배양 결과이다. 건조균체량(●), 포도당 농도(□), 아세트산 농도(■), GDP-푸코즈 농도(▲) 및 IPTG 첨가에 의한 GDP-푸코즈 생합성 효소의 발현유도(화살표).
도 10은 재조합 대장균의 세포 내 구아노신 누클레오타이드 농도 분석결과이다. (a) 이 콜라이(E. coli) BL21 star(DE3)/pACYCDuet-1, (b) 이 콜라이(E. coli) BL21 star(DE3)/pHgsk.
도 11은 본 발명의 실시예 1 및 2에서 수행한 대조군 및 실험군의 pH-스탯 방식에 의한 유가식 배양 결과의 요약이다.
PCR 프라이머 | 서열(5'->3') | 비고 |
manC_F (NcoI) | ACATGCCATGGATGGCGCAGTCGAAACTCTAT | manC와 manB는 클로스터(cluster)로 나란히 존재하기 때문에 함께 PCR 됨 |
manB_R (EcoRI) | ACCGGAATTCTTACTCGTTCAGCAACGTCAG | |
zwf_F (NcoI) | CATGCCATGGCGGTAACGCAAACAGC | |
zwf_R (EcoRI) | ACCGGAATTCTTACTCAAACTCATTCCAGGAAC | |
guaBA_F (NdeI) | GGAATTCCATATGCTACGTATCGCTAAAGAA | guaB와 guaA는 클로스터(cluster)로 나란히 존재하기 때문에 함께 PCR 됨 |
guaBA_R (XhoI) | CCGCTCGAGTCATTCCCACTCAATGGTAGC | |
guaC_F | ACATGCCATGGGCATGCGTATTGAAGAAGATCTG | |
guaC_R (SacI) | AAAACTGCAGTTACAGGTTGTTGAAGATGCG | |
gsk_F (NcoI) | ACATGCCATGGGCATGAAATTTCCCGGTAAACGT | |
gsk_R (SacI) | AAAACTGCAGTTAACGATCCCAGTAAGACTC |
유전자 | 비고 | |
pETDuet-1 | - | 2개의 T7 프로모터 및 MCS, pBR322 replicon, Ampr |
pACYCDuet-1 | - | 2개의 T7 프로모터 및 MCS, p15A replicon, Cmr |
pmBCGW | manB , manC , gmd , wcaG | pETDuet-1 + gmd - wcaG (NdeI/XhoI) + manB - manC (NcoI/SacI) |
pMWzwf | zwf | pACYCDuet-1 + zwf (NcoI/EcoRI) |
pHguaBA | guaA , guaB | pACYCDuet-1 + guaA - guaB (NdeI/XhoI) |
pHguaC | guaC | pACYCDuet-1 + guaC (NcoI/SacI) |
pHgsk | gsk | pACYCDuet-1 + gsk (NcoI/SacI) |
Claims (4)
- 대장균을 배양함으로써 GDP-푸코즈를 생산하는 방법에 있어서,
대장균으로,
GDP-D-만노오스-4,6-데하이드라타아제, GDP-4-케토-6-데옥시만노오스 3,5-에피머라아제-4-리덕타아제, 포스포만노뮤타아제, 만노오스-1-포스페이트 구아닐트랜스퍼라아제 및 구아노신-이노신 키나아제가 과발현되도록 재조합된 대장균을 사용하는 것을 특징으로 하는 GDP-푸코즈의 생산방법
- 제1항에 있어서,
상기 과발현은,
유도 프로모터에 의해 조절되는 것을 특징으로 하는 GDP-푸코즈의 생산방법
- 제1항 또는 제2항에 있어서,
상기 배양은,
pH-스탯 방식의 유가식 배양인 것을 특징으로 하는 GDP-푸코즈의 생산방법
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KR20010050030A (ko) * | 1999-08-10 | 2001-06-15 | 히라타 다다시 | Gdp-푸코즈의 제조방법 |
US20020058313A1 (en) | 2000-09-26 | 2002-05-16 | Risto Renkonen | Use of recombinant enzymes for preparing GDP-L-fucose and fucosylated glycans |
KR20020062361A (ko) * | 1999-12-21 | 2002-07-25 | 교와 핫꼬 고교 가부시끼가이샤 | 개변된 α1,2-푸코오스 전이효소 유전자 및α1,2-푸코오스 전이효소와 푸코오스 함유 당질의 제조법 |
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KR20010050030A (ko) * | 1999-08-10 | 2001-06-15 | 히라타 다다시 | Gdp-푸코즈의 제조방법 |
KR20020062361A (ko) * | 1999-12-21 | 2002-07-25 | 교와 핫꼬 고교 가부시끼가이샤 | 개변된 α1,2-푸코오스 전이효소 유전자 및α1,2-푸코오스 전이효소와 푸코오스 함유 당질의 제조법 |
US20020058313A1 (en) | 2000-09-26 | 2002-05-16 | Risto Renkonen | Use of recombinant enzymes for preparing GDP-L-fucose and fucosylated glycans |
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