KR20220168150A - 프룩토오스 대사 유전체가 돌연변이된 균주 - Google Patents
프룩토오스 대사 유전체가 돌연변이된 균주 Download PDFInfo
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- KR20220168150A KR20220168150A KR1020220067642A KR20220067642A KR20220168150A KR 20220168150 A KR20220168150 A KR 20220168150A KR 1020220067642 A KR1020220067642 A KR 1020220067642A KR 20220067642 A KR20220067642 A KR 20220067642A KR 20220168150 A KR20220168150 A KR 20220168150A
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- Prior art keywords
- ala
- gly
- leu
- val
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 71
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Abstract
본 발명은 프룩토오스 대사 유전체가 돌연변이된 균주에 관한 것으로, 본 발명에 따른 발현 카세트, 이를 포함하는 벡터를 통해 새로운 당 대사경로를 구축할 수 있고, 이를 통해 형질 전환된 돌연변이 또는 변이된 유전자를 포함하는 돌연변이 균주는 새로운 당 대사경로가 구축되어 있다.
Description
본 발명은 프룩토오스 대사 유전체가 돌연변이된 균주에 관한 것이다.
D-타가토스는 D-갈락토오스의 이성질체이며 과일, 우유, 치즈 등에 존재하는 천연 당류이다. D-타가토스는 다양한 건강 기능적 특성과 설탕과 매우 유사한 단맛을 가지고 있기 때문에 여러 제품 적용 시 건강과 맛을 동시에 만족시킬 수 있는 대체 감미료로 사용된다.
한편, 일반적으로 효소의 활성 및 구조적 안정성을 증진시키거나 새로운 기질에 대한 활성을 부여하는 등 원하는 목적에 부합하도록 효소의 특성을 변환시키는 개량기술로 분자진화 (directed evolution) 기술이 사용되고 있다. 이러한 기술을 수행하기 위한 변이주 라이브러리를 제조하기 위해 가장 일반적인 방법으로 많이 사용되는 것은 error-prone PCR 방법으로 PCR 수행시 DNA 중합효소의 에러발생율을 조절하여 무작위적으로 돌연변이를 도입하는 방법이다. 이렇게 만들어진 변이주들을 이용하여 단백질을 발현시킨 후, 활성이 좋은 변이주를 선별함으로써 우수한 활성을 갖는 개량 효소를 수득하게 되는데 원하는 효소의 특징과 목적에 맞는 효율적인 스크리닝 기술을 개발하는 것이 분자진화의 핵심기술이라 할 수 있다.
대한민국 공개특허 제10-2018-0074550호에서는 균주에 타 균주 유래의 효소를 도입하여 변형된 당 대사 경로를 갖는 재조합 균주 및 이를 통해 D-타가토스를 수득하는 가능성을 개시하고 있으나, 최근 원자재 값의 상승으로 인해 수익률이 떨어지게 되어, 신규한 대사경로를 통해 D-타가토스를 수득할 수 있는 균주의 개발 필요성이 높아지고 있다.
이에, 본 발명자들은 연구를 통해 변형된 당 대사 경로를 갖는 돌연변이 균주 및 이의 용도를 확인하여 본 발명을 완성하였다.
본 발명의 일 양상은 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자; 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자; 및 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자를 포함하는, 발현 카세트를 제공하는 것을 목적으로 한다.
본 발명의 다른 일 양상은 상기 발현 카세트를 포함하는 재조합 벡터를 제공하는 것을 목적으로 한다.
본 발명의 다른 일 양상은 상기 재조합 벡터로 형질 전환된 돌연변이 균주를 제공하는 것을 목적으로 한다.
본 발명의 다른 일 양상은 1) 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자; 2) 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자; 및 3) 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자 중 어느 하나 이상을 포함하는 돌연변이 균주를 제공하는 것을 목적으로 한다.
본 발명의 다른 일 양상은 상기 돌연변이 균주를 D-프룩토오스 혹은 D-타가토스를 포함하는 배지에서 배양하는 단계; 를 포함하는 D-프룩토오스 비대사성과 D-타가토스 대사능의 특징을 모두 갖는 균주를 생산하는 방법을 제공하는 것을 목적으로 한다.
본 발명의 일 양상은 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자; 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자; 및 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자를 포함하는, 발현 카세트를 제공한다.
상기 서열번호 1의 아미노산 서열은 야생형 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)의 아미노산 서열을 의미하고, 본 발명에서는 상기 서열번호 1의 야생형 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA) 아미노산 서열이 불활성화 되도록 변이되어 프룩토오스 이용성을 억제하고 타가토스 이용성을 개선할 수 있다.
상기 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)는 포도당신생합성 및 해당과정에서 역반응에서 프룩토오스 1,5-바이포스페이트 (FBP)를 형성하기 위하여 글리세랄데하이드 3-포스페이트 (G3P)와 디하이드록시아세톤 포스페이트 (DHAP 또는 glycerone-phosphate)의 알돌 축합을 촉매하는 기능을 한다.
본 발명의 일 구체예로, 상기 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)를 암호화하는 유전자는 서열번호 4를 포함하는 것일 수 있다.
서열명 | Sequence | 서열번호 |
fbaA WT amino acid | MSKIFDFVKP GVITGDDVQK VFQVAKENNF ALPAVNCVGT DSINAVLETA AKVKAPVIVQ FSNGGASFIA GKGVKSDVPQ GAAILGAISG AHHVHQMAEH YGVPVILHTD HCAKKLLPWI DGLLDAGEKH FAATGKPLFS SHMIDLSEES LQENIEICSK YLERMSKIGM TLEIELGCTG GEEDGVDNSH MDASALYTQP EDVDYAYTEL SKISPRFTIA ASFGNVHGVY KPGNVVLTPT ILRDSQEYVS KKHNLPHNSL NFVFHGGSGS TAQEIKDSVS YGVVKMNIDT DTQWATWEGV LNYYKANEAY LQGQLGNPKG EDQPNKKYYD PRVWLRAGQT SMIARLEKAF QELNAIDVL |
1 |
fbaA WT gene | atgtctaaga tttttgattt cgtaaaacct ggcgtaatca ctggtgatga cgtacagaaa gttttccagg tagcaaaaga aaacaacttc gcactgccag cagtaaactg cgtcggtact gactccatca acgccgtact ggaaaccgct gctaaagtta aagcgccggt tatcgttcag ttctccaacg gtggtgcttc ctttatcgct ggtaaaggcg tgaaatctga cgttccgcag ggtgctgcta tcctgggcgc gatctctggt gcgcatcacg ttcaccagat ggctgaacat tatggtgttc cggttatcct gcacactgac cactgcgcga agaaactgct gccgtggatc gacggtctgt tggacgcggg tgaaaaacac ttcgcagcta ccggtaagcc gctgttctct tctcacatga tcgacctgtc tgaagaatct ctgcaagaga acatcgaaat ctgctctaaa tacctggagc gcatgtccaa aatcggcatg actctggaaa tcgaactggg ttgcaccggt ggtgaagaag acggcgtgga caacagccac atggacgctt ctgcactgta cacccagccg gaagacgttg attacgcata caccgaactg agcaaaatca gcccgcgttt caccatcgca gcgtccttcg gtaacgtaca cggtgtttac aagccgggta acgtggttct gactccgacc atcctgcgtg attctcagga atatgtttcc aagaaacaca acctgccgca caacagcctg aacttcgtat tccacggtgg ttccggttct actgctcagg aaatcaaaga ctccgtaagc tacggcgtag taaaaatgaa catcgatacc gatacccaat gggcaacctg ggaaggcgtt ctgaactact acaaagcgaa cgaagcttat ctgcagggtc agctgggtaa cccgaaaggc gaagatcagc cgaacaagaa atactacgat ccgcgcgtat ggctgcgtgc cggtcagact tcgatgatcg ctcgtctgga gaaagcattc caggaactga acgcgatcga cgttctgtaa |
4 |
상기 서열번호 2의 아미노산 서열은 야생형 포스포트랜스퍼라아제 시스템 G (PTS system glucose-specific EIICB component, ptsG)의 아미노산 서열을 의미하고, 본 발명에서는 상기 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환시켜 글루코오스 트랜스포터인 야생형 ptsG를 비인산화 (non-phosphorylation) 형태인 프룩토오스를 세포내로 유입할 수 있도록 한다.
상기 포스포트랜스퍼라아제 시스템 G (PTS system glucose-specific EIICB component, ptsG)는 주요한 탄수화물 활성 전달 시스템으로, 당기질의 인산화와 함께 세포막을 가로질러 들어오도록 하는 기능을 한다.
본 발명의 일 구체예로, 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자는 서열번호 5를 포함하는 것일 수 있다.
서열명 | Sequence | 서열번호 |
ptsG WT amino acid | MFKNAFANLQ K V GKSLMLPV SVLPIAGILL GVGSANFSWL PAVVSHVMAE AGGSVFANMP LIFAIGVALG FTNNDGVSAL AAVVAYGIMV KTMAVVAPLV LHLPAEEIAS KHLADTGVLG GIISGAIAAY MFNRFYRIKL PEYLGFFAGK RFVPIISGLA AIFTGVVLSF IWPPIGSAIQ TFSQWAAYQN PVVAFGIYGF IERCLVPFGL HHIWNVPFQM QIGEYTNAAG QVFHGDIPRY MAGDPTAGKL SGGFLFKMYG LPAAAIAIWH SAKPENRAKV GGIMISAALT SFLTGITEPI EFSFMFVAPI LYIIHAILAG LAFPICILLG MRDGTSFSHG LIDFIVLSGN SSKLWLFPIV GIGYAIVYYT IFRVLIKALD LKTPGREDAT EDAKATGTSE MAPALVAAFG GKENITNLDA CITRLRVSVA DVSKVDQAGL KKLGAAGVVV AGSGVQAIFG TKSDNLKTEM DEYIRNH |
2 |
ptsGV12F amino acid | MFKNAFANLQ K F GKSLMLPV SVLPIAGILL GVGSANFSWL PAVVSHVMAE AGGSVFANMP LIFAIGVALG FTNNDGVSAL AAVVAYGIMV KTMAVVAPLV LHLPAEEIAS KHLADTGVLG GIISGAIAAY MFNRFYRIKL PEYLGFFAGK RFVPIISGLA AIFTGVVLSF IWPPIGSAIQ TFSQWAAYQN PVVAFGIYGF IERCLVPFGL HHIWNVPFQM QIGEYTNAAG QVFHGDIPRY MAGDPTAGKL SGGFLFKMYG LPAAAIAIWH SAKPENRAKV GGIMISAALT SFLTGITEPI EFSFMFVAPI LYIIHAILAG LAFPICILLG MRDGTSFSHG LIDFIVLSGN SSKLWLFPIV GIGYAIVYYT IFRVLIKALD LKTPGREDAT EDAKATGTSE MAPALVAAFG GKENITNLDA CITRLRVSVA DVSKVDQAGL KKLGAAGVVV AGSGVQAIFG TKSDNLKTEM DEYIRNH |
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ptsG WT gene | atgtttaaga atgcatttgc taacctgcaa aag gtc ggta aatcgctgat gctgccggtatccgtactgc ctatcgcagg tattctgctg ggcgtcggtt ccgcgaattt cagctggctg cccgccgttg tatcgcatgt tatggcagaa gcaggcggtt ccgtctttgc aaacatgcca ctgatttttg cgatcggtgt cgccctcggc tttaccaata acgatggcgt atccgcgctg gccgcagttg ttgcctatgg catcatggtt aaaaccatgg ccgtggttgc gccactggta ctgcatttac ctgctgaaga aatcgcctct aaacacctgg cggatactgg cgtactcgga gggattatct ccggtgcgat cgcagcgtac atgtttaacc gtttctaccg tattaagctg cctgagtatc ttggcttctt tgccggtaaa cgctttgtgc cgatcatttc tggcctggct gccatcttta ctggcgttgt gctgtccttc atttggccgc cgattggttc tgcaatccag accttctctc agtgggctgc ttaccagaac ccggtagttg cgtttggcat ttacggtttc atcgaacgtt gcctggtacc gtttggtctg caccacatct ggaacgtacc tttccagatg cagattggtg aatacaccaa cgcagcaggt caggttttcc acggcgacat tccgcgttat atggcgggtg acccgactgc gggtaaactg tctggtggct tcctgttcaa aatgtacggt ctgccagctg ccgcaattgc tatctggcac tctgctaaac cagaaaaccg cgcgaaagtg ggcggtatta tgatctccgc ggcgctgacc tcgttcctga ccggtatcac cgagccgatc gagttctcct tcatgttcgt tgcgccgatc ctgtacatca tccacgcgat tctggcaggc ctggcattcc caatctgtat tcttctgggg atgcgtgacg gtacgtcgtt ctcgcacggt ctgatcgact tcatcgttct gtctggtaac agcagcaaac tgtggctgtt cccgatcgtc ggtatcggtt atgcgattgt ttactacacc atcttccgcg tgctgattaa agcactggat ctgaaaacgc cgggtcgtga agacgcgact gaagatgcaa aagcgacagg taccagcgaa atggcaccgg ctctggttgc tgcatttggt ggtaaagaaa acattactaa cctcgacgca tgtattaccc gtctgcgcgt cagcgttgct gatgtgtcta aagtggatca ggccggcctg aagaaactgg gcgcagcggg cgtagtggtt gctggttctg gtgttcaggc gattttcggt actaaatccg ataacctgaa aaccgagatg gatgagtaca tccgtaacca ctaa |
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ptsGV12F gene | atgtttaaga atgcatttgc taacctgcaa aag tty ggta aatcgctgat gctgccggta tccgtactgc ctatcgcagg tattctgctg ggcgtcggtt ccgcgaattt cagctggctg cccgccgttg tatcgcatgt tatggcagaa gcaggcggtt ccgtctttgc aaacatgcca ctgatttttg cgatcggtgt cgccctcggc tttaccaata acgatggcgt atccgcgctg gccgcagttg ttgcctatgg catcatggtt aaaaccatgg ccgtggttgc gccactggta ctgcatttac ctgctgaaga aatcgcctct aaacacctgg cggatactgg cgtactcgga gggattatct ccggtgcgat cgcagcgtac atgtttaacc gtttctaccg tattaagctg cctgagtatc ttggcttctt tgccggtaaa cgctttgtgc cgatcatttc tggcctggct gccatcttta ctggcgttgt gctgtccttc atttggccgc cgattggttc tgcaatccag accttctctc agtgggctgc ttaccagaac ccggtagttg cgtttggcat ttacggtttc atcgaacgtt gcctggtacc gtttggtctg caccacatct ggaacgtacc tttccagatg cagattggtg aatacaccaa cgcagcaggt caggttttcc acggcgacat tccgcgttat atggcgggtg acccgactgc gggtaaactg tctggtggct tcctgttcaa aatgtacggt ctgccagctg ccgcaattgc tatctggcac tctgctaaac cagaaaaccg cgcgaaagtg ggcggtatta tgatctccgc ggcgctgacc tcgttcctga ccggtatcac cgagccgatc gagttctcct tcatgttcgt tgcgccgatc ctgtacatca tccacgcgat tctggcaggc ctggcattcc caatctgtat tcttctgggg atgcgtgacg gtacgtcgtt ctcgcacggt ctgatcgact tcatcgttct gtctggtaac agcagcaaac tgtggctgtt cccgatcgtc ggtatcggtt atgcgattgt ttactacacc atcttccgcg tgctgattaa agcactggat ctgaaaacgc cgggtcgtga agacgcgact gaagatgcaa aagcgacagg taccagcgaa atggcaccgg ctctggttgc tgcatttggt ggtaaagaaa acattactaa cctcgacgca tgtattaccc gtctgcgcgt cagcgttgct gatgtgtcta aagtggatca ggccggcctg aagaaactgg gcgcagcggg cgtagtggtt gctggttctg gtgttcaggc gattttcggt actaaatccg ataacctgaa aaccgagatg gatgagtaca tccgtaacca ctaa |
5 |
상기 서열번호 3의 아미노산 서열은 야생형 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)의 아미노산 서열을 의미하고, 본 발명의 일 구체예로 상기 서열번호 3의 야생형 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이되어 타가토스 알돌라아제 (kbaY)가 발현되도록 하는 것일 수 있다.
상기 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)는 N-아세틸 갈락토사민 이동 및 대사를 위한 aga 오페론의 억제 기전을 하는 기능을 가지는 것으로 예측된다.
본 발명의 일 구체예로, 상기 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)를 암호화하는 유전자는 서열번호 6을 포함하는 것일 수 있다.
서열명 | Sequence | 서열번호 |
agaR WT amino acid | MSNTDASGEK RVTGTSERRE QIIQRLRQQG SVQVNDLSAL YGVSTVTIRN DLAFLEKQGI AVRAYGGALI CDSTTPSVEP SVEDKSALNT AMKRSVAKAA VELIQPGHRV ILDSGTTTFE IARLMRKHTD VIAMTNGMNV ANALLEAEGV ELLMTGGHLR RQSQSFYGDQ AEQSLQNYHF DMLFLGVDAI DLERGVSTHN EDEARLNRRM CEVAERIIVV TDSSKFNRSS LHKIIDTQRI DMIIVDEGIP ADSLEGLRKA GVEVILVGE- |
3 |
agaR WT gene | atgagtaata ccgacgcttc aggtgagaag cgagtgacag gcaccagcga gcgacgagaa cagatcattc agcgtctgcg acagcaaggg agtgtgcagg ttaacgatct gtcggcattg tatggcgtat ctaccgtgac gatccgcaac gatctggcgt ttctggaaaa gcaggggatc gctgtgcgtg cctatggtgg cgcgttgatc tgcgatagca cgacgccgtc agtcgagcca tcagtggaag ataaaagcgc actgaacacc gcgatgaaac gcagcgttgc gaaagctgcc gttgagttga ttcagccagg tcatcgggtg atcctcgatt ccgggaccac cacttttgag attgctcgtc tgatgcgcaa gcacactgac gtaattgcga tgaccaacgg tatgaacgtg gctaatgctt tgctggaagc ggaaggcgtt gagctgctga tgaccggcgg gcatttgcgc cgtcagtcgc aatcttttta cggcgatcag gctgaacaat cgctgcaaaa ttaccacttc gatatgctgt ttcttggtgt agatgcgatc gatctggagc gcggcgtcag cacgcataat gaagatgaag cccgtttaaa ccgccggatg tgcgaagttg cggaacggat catcgtagtc accgattcca gtaagttcaa ccgctccagt ttacataaga tcattgatac tcaacgtatc gacatgatca ttgttgatga aggcattcct gcggatagtc tggaaggact gcgaaaggct ggggttgaag tgattctggt cggggagtga | 6 |
본 발명에 있어서, 상기 아미노산 또는 유전자 서열은 서열번호 1 내지 6 아미노산 또는 유전자 서열을 의미하는 것 뿐만 아니라, 적어도 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 또는 99% 이상의 상동성 또는 동일성을 가지는 아미노산 또는 유전자 서열을 가지는 것을 포함하는 것으로, 본 발명에 기재되어 있는 각 아미노산 또는 유전자 서열의 기능을 하는 경우라면 본 발명의 범위에 포함되는 것이다.
본 발명의 "불활성화"는 효소, 전사 인자, 수송 단백질 등 단백질을 암호화하는 유전자의 발현이 천연형 균주, 야생형 균주 또는 변형 전의 균주에 비하여 전혀 발현이 되지 않는 경우이거나 발현이 되더라도 그 활성이 없는 경우를 의미한다. 본 발명에 있어서 상기 불활성화는 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) 등 유전자의 전사가 이루어지지 않거나 전사가 이루어지더라도 목적하는 단백질 (아미노산)의 기능을 하지 못하는 것을 의미한다.
본 발명의 일 예시로 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자 및/또는 서열번호 3의 agar 억제 유전자 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자를 포함하는 경우 상기 두 효소가 발현 또는 기능하지 못하도록 변이된 유전자를 포함하는 것일 수 있고, 상기 두 유전자를 결손하여 두 효소의 발현이 이루어지지 않도록 하는 것일 수 있다.
본 발명의 일 구체예로는 유전자를 결손하는 것을 의미한다.
본 발명의 일 양상은 상기 발현 카세트를 포함하는 재조합 벡터를 제공한다.
본 발명에서 사용되는 용어, "재조합 벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 진핵세포에서 이용 가능한 프로모터, 인핸서, 종결신호 및 폴리아데닐레이션 신호는 공지되어 있다.
본 발명에서 사용되는 용어, "작동가능하게 연결된"은 유전자 발현 조절 서열과 다른 뉴클레오티드 서열사이의 기능적인 결합을 의미한다. 상기 유전자 발현 조절 서열은 복제원점(replication origin), 프로모터 및 전사 종결 서열(terminator) 등으로 이루어진 군으로부터 선택되는 1종 이상일 수 있다. 전사 종결 서열은 폴리아데닐화 서열(pA)일 수 있으며, 복제 원점은 f1 복제원점, SV40 복제원점, pMB1 복제원점, 아데노 복제원점, AAV 복제원점 또는 BBV 복제원점 등일 수 있으나, 이에 한정되는 것은 아니다.
본 발명의 일 구체예에 따른 재조합 벡터는 플라스미드 벡터, 코즈미드 벡터 및 박테리오파아지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터 및 아데노-연관 바이러스 벡터와 같은 바이러스 벡터로 이루어진 군으로부터 선택되는 것일 수 있다. 재조합 발현벡터로 사용될 수 있는 벡터는 당업계에서 사용되는 플라스미드(예를 들어, pcDNA 시리즈, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX 시리즈, pET 시리즈, pUC19 등), 파지(예를 들어, λgt4λB, λ-Charon, λΔz1, M13 등) 또는 바이러스 벡터(예를 들어, 아데노-연관 바이러스(AAV) 벡터 등) 등을 기본으로 하여 제작될 수 있으나, 이에 한정되는 것은 아니다.
본 발명의 재조합 벡터는 하나 이상의 선택성 마커를 더 포함할 수 있다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질주입된 세포를 비형질주입 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 예를 들어, 글리포세이트(glyphosate), 글루포시네이트암모늄(glufosinate ammonium) 또는 포스피노트리신(phosphinothricin)과 같은 제초제 저항성 유전자, 암피실린(ampicillin), 카나 마이신(kanamycin), G418, 블레오마이신(Bleomycin), 하이그로마이신(hygromycin), 클로람페니콜 (chloramphenicol)과 같은 항생제 내성 유전자일 수 있으나, 이에 한정되는 것은 아니다.
본 발명의 재조합 벡터의 제작은 당해 기술 분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용하여 수행될 수 있다.
본 발명의 다른 일 양상은 상기 재조합 벡터로 형질 전환된 돌연변이 균주를 제공한다.
상기 재조합 벡터로 형질전환하는 방법은 당업계에 널리 알려진 삽입 방법을 사용할 수 있다. 상기 운반 방법은 예를 들어, 숙주 세포가 원핵 세포인 경우, CaCl2 방법 또는 전기 천공 방법 등을 사용할 수 있고, 숙주 세포가 진핵 세포인 경우에는, 미세 주입법, 칼슘 포스페이트 침전법, 전기 천공법, 리포좀-매개 형질감염법, 열충격 및 유전자 밤바드먼트 등을 사용할 수 있으나, 이에 한정하지는 않는다.
또한, 본 발명의 다른 일 양상은 1) 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자; 2) 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자; 및 3) 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자 중 어느 하나 이상을 포함하는 돌연변이 균주 를 제공한다.
상기 돌연변이 균주는 전술한 재조합 벡터로 형질 전환된 돌연변이 균주외에도 다양한 방법의 돌연변이를 통해 전술한 1) 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자; 2) 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자; 및 3) 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자 중 어느 하나 이상의 유전자 돌연변이 서열을 포함하게 된 것일 수 있다.
상기 돌연변이 균주에 포함되는 유전자 서열은 1) 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자; 2) 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자; 및 3) 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자 중 어느 하나의 유전자 서열을 포함하는 것일 수 있고,
1) 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자; 및 2) 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자를 포함하거나, 1) 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자; 및 3) 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자를 포함하거나, 2) 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자; 및 3) 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자 을 포함하는 것일 수 있으며,
1) 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자; 2) 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자; 및 3) 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자를 포함하는 것일 수도 있다.
상기 돌연변이 균주의 숙주는 당업계에 공지된 어떠한 숙주를 이용할 수 있으며, 원핵 세포로는, 예를 들어, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움, 세라티아 마르세슨스 및 다양한 슈도모나스 종과 같은 장내균과 균주 등이 있으며, 진핵 세포에 형질 전환시키는 경우에는 숙주 세포로서, 효모(Saccharomyce cerevisiae), 곤충 세포, 식물 세포 및 동물 세포, 예를 들어, SP2/0, CHO(Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN 및 MDCK 세포주 등이 이용될 수 있고, 구체적으로 상기 돌연변이 균주의 숙주는 대장균일 수 있다.
본 발명의 일 구체예로 상기 돌연변이 균주는 D-타가토스 대사능을 가질 수 있다. 구체적으로, 상기 재조합 균주는 전술한 발현카세트 또는 돌연변이를 포함하고 있기 때문에 D-타가토스 대사능을 가질 수 있다. 상기 D-타가토스 대사능은 D-타가토스를 에너지원으로 활용하는 것을 의미한다.
본 발명의 다른 일 양상은 상기 돌연변이 균주를 D-타가토스를 포함하는 배지에서 배양하는 단계; 를 포함하는 D-타가토스 대사능을 갖는 균주를 생산하는 방법을 제공한다.
상기 배양하는 단계는 상기 돌연변이 균주를 D-타가토스를 포함하는 배지에서 배양하는 단계로서, 상기 배지는 돌연변이 균주를 배양하기 위해 공지의 성분을 함유할 수 있다.
본 발명에 따른 발현 카세트, 이를 포함하는 벡터를 통해 새로운 당 대사경로를 구축할 수 있고, 이를 통해 형질 전환된 돌연변이 균주 또는 변이된 유전자를 포함하는 돌연변이 균주는 새로운 당 대사경로가 구축되어있다.
도 1은 본 발명의 돌연변이 균주를 제조하는 과정을 나타낸 도면이다.
도 2는 본 발명의 발현 벡터 또는 변이들을 통해 새로이 구축된 당대사 경로를 표시한 것이다.
도 3은 본 발명의 agaR 변이에 따른 오페론 발현기전을 나타낸 것이다.
도 4는 돌연변이가 포함된 균주의 활성을 확인한 것이다.
도 5는 본 발명의 균주의 배양 양상을 나타낸 것으로, 좌측은 탄소원에 따른 균주 배양 양상을 나타낸 것이고, 우측은 프룩토오스 에피머화 효소와 균주를 함께 배양하였을 때 배양 양상을 나타낸 것이다.
도 2는 본 발명의 발현 벡터 또는 변이들을 통해 새로이 구축된 당대사 경로를 표시한 것이다.
도 3은 본 발명의 agaR 변이에 따른 오페론 발현기전을 나타낸 것이다.
도 4는 돌연변이가 포함된 균주의 활성을 확인한 것이다.
도 5는 본 발명의 균주의 배양 양상을 나타낸 것으로, 좌측은 탄소원에 따른 균주 배양 양상을 나타낸 것이고, 우측은 프룩토오스 에피머화 효소와 균주를 함께 배양하였을 때 배양 양상을 나타낸 것이다.
이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
실시예 1: 프룩토스 비대사성과 타가토스 대사능의 특징을 가진 돌연변이 균주의 제조
대장균은 타가토스를 프룩토스와 공통된 phosphotransferase system (PTS)을 통해 대사하기 때문에, 프룩토스 비대사 균주를 만들기위해 프룩토스 주요 PTS에 포함되어 있는 transporter (fruAB) 혹은 kinase (fruK) 유전자를 선정하면 타가토스 또한 이용하지 못하게 되기 때문에, 타가토스와 프룩토스 대사가 겹치지 않으면서 프룩토스 이용성에 중요한 역할을 하는 유전자를 선별하고자 하였다. 따라서 transporter와 kinase에 의해 phosphorylation 되고 난 후 대사체를 glycolysis 회로로 진입하게 하는 핵심 유전자인 aldolase fbaA를 삭제하여 프룩토스 이용성이 지연된 균주를 만들고자 하였다 (도 1, 2).
B.licheniformis 유래 gatY 유전자를 E.coli 에 형질전환시킨 후 타가토스 배지에서 적응진화 시킨 균주로부터 gatY 유전자를 curing 시켰다. Curing 시킨 균주를 타가토스 배지에서 다시 적응진화 시킨 후 전장유전체 분석을 통해 agaR 부분이 실활되어있음을 확인하였다 (도 3).
실험예 1: 돌연변이 균주의 활성 확인
fbaA 유전자 삭제와 ptsGV12F 유전자 돌연변이, agaR 유전자 삭제 균주의 경우 야생형 대장균 대비 프룩토스 대사능은 감소하고 타가토스 이용성을 획득하는 것을 확인하였다.
agaR유전자를 삭제시킬 경우 대장과 야생형 균주를 세가지 당영양원 배지(Glc(글루코스), Fru(프록토스), Tag(타가토스))에서 배양하여 유전자 발현 정도를 qRT-PCR을 통해 mRNA level 분석을 시행하였다. 그 결과 도 4와 같이, agaR 유전자가 삭제되는 경유 kbaY tagatose-1,6 bisphosphate aldolase가 과발현되어 타가토스 이용성이 용이해짐을 확인하였다.
실험예 2: 돌연변이 균주의 성장 확인
프룩토스 대사 유전체가 변현된 균주를 프룩토스와 타가토스 배지 내 배양 시킨 결과 프룩토스에 대한 기질 이용성은 감소하고 타가토스 이용성은 획득한 것을 확인 하였다.
그 결과, 도 5에서 확인되는 바와 같이, 본 균주에 프룩토스 에피머화 효소를 도입시키고 프룩토스 배지 내에서 성장시켰을 때 효소 활성 의존적으로 성장하는 것을 확인하였다.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.
<110> Industry-Academic Cooperation Foundation, Yonsei University
<120> Fructose Metabolic Genes Mutated Strains
<130> PN210160-P1
<150> KR 10-2021-0076939
<151> 2021-06-14
<160> 6
<170> KoPatentIn 3.0
<210> 1
<211> 359
<212> PRT
<213> Artificial Sequence
<220>
<223> fbaA WT amino acid
<400> 1
Met Ser Lys Ile Phe Asp Phe Val Lys Pro Gly Val Ile Thr Gly Asp
1 5 10 15
Asp Val Gln Lys Val Phe Gln Val Ala Lys Glu Asn Asn Phe Ala Leu
20 25 30
Pro Ala Val Asn Cys Val Gly Thr Asp Ser Ile Asn Ala Val Leu Glu
35 40 45
Thr Ala Ala Lys Val Lys Ala Pro Val Ile Val Gln Phe Ser Asn Gly
50 55 60
Gly Ala Ser Phe Ile Ala Gly Lys Gly Val Lys Ser Asp Val Pro Gln
65 70 75 80
Gly Ala Ala Ile Leu Gly Ala Ile Ser Gly Ala His His Val His Gln
85 90 95
Met Ala Glu His Tyr Gly Val Pro Val Ile Leu His Thr Asp His Cys
100 105 110
Ala Lys Lys Leu Leu Pro Trp Ile Asp Gly Leu Leu Asp Ala Gly Glu
115 120 125
Lys His Phe Ala Ala Thr Gly Lys Pro Leu Phe Ser Ser His Met Ile
130 135 140
Asp Leu Ser Glu Glu Ser Leu Gln Glu Asn Ile Glu Ile Cys Ser Lys
145 150 155 160
Tyr Leu Glu Arg Met Ser Lys Ile Gly Met Thr Leu Glu Ile Glu Leu
165 170 175
Gly Cys Thr Gly Gly Glu Glu Asp Gly Val Asp Asn Ser His Met Asp
180 185 190
Ala Ser Ala Leu Tyr Thr Gln Pro Glu Asp Val Asp Tyr Ala Tyr Thr
195 200 205
Glu Leu Ser Lys Ile Ser Pro Arg Phe Thr Ile Ala Ala Ser Phe Gly
210 215 220
Asn Val His Gly Val Tyr Lys Pro Gly Asn Val Val Leu Thr Pro Thr
225 230 235 240
Ile Leu Arg Asp Ser Gln Glu Tyr Val Ser Lys Lys His Asn Leu Pro
245 250 255
His Asn Ser Leu Asn Phe Val Phe His Gly Gly Ser Gly Ser Thr Ala
260 265 270
Gln Glu Ile Lys Asp Ser Val Ser Tyr Gly Val Val Lys Met Asn Ile
275 280 285
Asp Thr Asp Thr Gln Trp Ala Thr Trp Glu Gly Val Leu Asn Tyr Tyr
290 295 300
Lys Ala Asn Glu Ala Tyr Leu Gln Gly Gln Leu Gly Asn Pro Lys Gly
305 310 315 320
Glu Asp Gln Pro Asn Lys Lys Tyr Tyr Asp Pro Arg Val Trp Leu Arg
325 330 335
Ala Gly Gln Thr Ser Met Ile Ala Arg Leu Glu Lys Ala Phe Gln Glu
340 345 350
Leu Asn Ala Ile Asp Val Leu
355
<210> 2
<211> 477
<212> PRT
<213> Artificial Sequence
<220>
<223> ptsG WT amino acid
<400> 2
Met Phe Lys Asn Ala Phe Ala Asn Leu Gln Lys Val Gly Lys Ser Leu
1 5 10 15
Met Leu Pro Val Ser Val Leu Pro Ile Ala Gly Ile Leu Leu Gly Val
20 25 30
Gly Ser Ala Asn Phe Ser Trp Leu Pro Ala Val Val Ser His Val Met
35 40 45
Ala Glu Ala Gly Gly Ser Val Phe Ala Asn Met Pro Leu Ile Phe Ala
50 55 60
Ile Gly Val Ala Leu Gly Phe Thr Asn Asn Asp Gly Val Ser Ala Leu
65 70 75 80
Ala Ala Val Val Ala Tyr Gly Ile Met Val Lys Thr Met Ala Val Val
85 90 95
Ala Pro Leu Val Leu His Leu Pro Ala Glu Glu Ile Ala Ser Lys His
100 105 110
Leu Ala Asp Thr Gly Val Leu Gly Gly Ile Ile Ser Gly Ala Ile Ala
115 120 125
Ala Tyr Met Phe Asn Arg Phe Tyr Arg Ile Lys Leu Pro Glu Tyr Leu
130 135 140
Gly Phe Phe Ala Gly Lys Arg Phe Val Pro Ile Ile Ser Gly Leu Ala
145 150 155 160
Ala Ile Phe Thr Gly Val Val Leu Ser Phe Ile Trp Pro Pro Ile Gly
165 170 175
Ser Ala Ile Gln Thr Phe Ser Gln Trp Ala Ala Tyr Gln Asn Pro Val
180 185 190
Val Ala Phe Gly Ile Tyr Gly Phe Ile Glu Arg Cys Leu Val Pro Phe
195 200 205
Gly Leu His His Ile Trp Asn Val Pro Phe Gln Met Gln Ile Gly Glu
210 215 220
Tyr Thr Asn Ala Ala Gly Gln Val Phe His Gly Asp Ile Pro Arg Tyr
225 230 235 240
Met Ala Gly Asp Pro Thr Ala Gly Lys Leu Ser Gly Gly Phe Leu Phe
245 250 255
Lys Met Tyr Gly Leu Pro Ala Ala Ala Ile Ala Ile Trp His Ser Ala
260 265 270
Lys Pro Glu Asn Arg Ala Lys Val Gly Gly Ile Met Ile Ser Ala Ala
275 280 285
Leu Thr Ser Phe Leu Thr Gly Ile Thr Glu Pro Ile Glu Phe Ser Phe
290 295 300
Met Phe Val Ala Pro Ile Leu Tyr Ile Ile His Ala Ile Leu Ala Gly
305 310 315 320
Leu Ala Phe Pro Ile Cys Ile Leu Leu Gly Met Arg Asp Gly Thr Ser
325 330 335
Phe Ser His Gly Leu Ile Asp Phe Ile Val Leu Ser Gly Asn Ser Ser
340 345 350
Lys Leu Trp Leu Phe Pro Ile Val Gly Ile Gly Tyr Ala Ile Val Tyr
355 360 365
Tyr Thr Ile Phe Arg Val Leu Ile Lys Ala Leu Asp Leu Lys Thr Pro
370 375 380
Gly Arg Glu Asp Ala Thr Glu Asp Ala Lys Ala Thr Gly Thr Ser Glu
385 390 395 400
Met Ala Pro Ala Leu Val Ala Ala Phe Gly Gly Lys Glu Asn Ile Thr
405 410 415
Asn Leu Asp Ala Cys Ile Thr Arg Leu Arg Val Ser Val Ala Asp Val
420 425 430
Ser Lys Val Asp Gln Ala Gly Leu Lys Lys Leu Gly Ala Ala Gly Val
435 440 445
Val Val Ala Gly Ser Gly Val Gln Ala Ile Phe Gly Thr Lys Ser Asp
450 455 460
Asn Leu Lys Thr Glu Met Asp Glu Tyr Ile Arg Asn His
465 470 475
<210> 3
<211> 269
<212> PRT
<213> Artificial Sequence
<220>
<223> agaR WT amino acid
<400> 3
Met Ser Asn Thr Asp Ala Ser Gly Glu Lys Arg Val Thr Gly Thr Ser
1 5 10 15
Glu Arg Arg Glu Gln Ile Ile Gln Arg Leu Arg Gln Gln Gly Ser Val
20 25 30
Gln Val Asn Asp Leu Ser Ala Leu Tyr Gly Val Ser Thr Val Thr Ile
35 40 45
Arg Asn Asp Leu Ala Phe Leu Glu Lys Gln Gly Ile Ala Val Arg Ala
50 55 60
Tyr Gly Gly Ala Leu Ile Cys Asp Ser Thr Thr Pro Ser Val Glu Pro
65 70 75 80
Ser Val Glu Asp Lys Ser Ala Leu Asn Thr Ala Met Lys Arg Ser Val
85 90 95
Ala Lys Ala Ala Val Glu Leu Ile Gln Pro Gly His Arg Val Ile Leu
100 105 110
Asp Ser Gly Thr Thr Thr Phe Glu Ile Ala Arg Leu Met Arg Lys His
115 120 125
Thr Asp Val Ile Ala Met Thr Asn Gly Met Asn Val Ala Asn Ala Leu
130 135 140
Leu Glu Ala Glu Gly Val Glu Leu Leu Met Thr Gly Gly His Leu Arg
145 150 155 160
Arg Gln Ser Gln Ser Phe Tyr Gly Asp Gln Ala Glu Gln Ser Leu Gln
165 170 175
Asn Tyr His Phe Asp Met Leu Phe Leu Gly Val Asp Ala Ile Asp Leu
180 185 190
Glu Arg Gly Val Ser Thr His Asn Glu Asp Glu Ala Arg Leu Asn Arg
195 200 205
Arg Met Cys Glu Val Ala Glu Arg Ile Ile Val Val Thr Asp Ser Ser
210 215 220
Lys Phe Asn Arg Ser Ser Leu His Lys Ile Ile Asp Thr Gln Arg Ile
225 230 235 240
Asp Met Ile Ile Val Asp Glu Gly Ile Pro Ala Asp Ser Leu Glu Gly
245 250 255
Leu Arg Lys Ala Gly Val Glu Val Ile Leu Val Gly Glu
260 265
<210> 4
<211> 1080
<212> DNA
<213> Artificial Sequence
<220>
<223> fbaA WT gene
<400> 4
atgtctaaga tttttgattt cgtaaaacct ggcgtaatca ctggtgatga cgtacagaaa 60
gttttccagg tagcaaaaga aaacaacttc gcactgccag cagtaaactg cgtcggtact 120
gactccatca acgccgtact ggaaaccgct gctaaagtta aagcgccggt tatcgttcag 180
ttctccaacg gtggtgcttc ctttatcgct ggtaaaggcg tgaaatctga cgttccgcag 240
ggtgctgcta tcctgggcgc gatctctggt gcgcatcacg ttcaccagat ggctgaacat 300
tatggtgttc cggttatcct gcacactgac cactgcgcga agaaactgct gccgtggatc 360
gacggtctgt tggacgcggg tgaaaaacac ttcgcagcta ccggtaagcc gctgttctct 420
tctcacatga tcgacctgtc tgaagaatct ctgcaagaga acatcgaaat ctgctctaaa 480
tacctggagc gcatgtccaa aatcggcatg actctggaaa tcgaactggg ttgcaccggt 540
ggtgaagaag acggcgtgga caacagccac atggacgctt ctgcactgta cacccagccg 600
gaagacgttg attacgcata caccgaactg agcaaaatca gcccgcgttt caccatcgca 660
gcgtccttcg gtaacgtaca cggtgtttac aagccgggta acgtggttct gactccgacc 720
atcctgcgtg attctcagga atatgtttcc aagaaacaca acctgccgca caacagcctg 780
aacttcgtat tccacggtgg ttccggttct actgctcagg aaatcaaaga ctccgtaagc 840
tacggcgtag taaaaatgaa catcgatacc gatacccaat gggcaacctg ggaaggcgtt 900
ctgaactact acaaagcgaa cgaagcttat ctgcagggtc agctgggtaa cccgaaaggc 960
gaagatcagc cgaacaagaa atactacgat ccgcgcgtat ggctgcgtgc cggtcagact 1020
tcgatgatcg ctcgtctgga gaaagcattc caggaactga acgcgatcga cgttctgtaa 1080
1080
<210> 5
<211> 1434
<212> DNA
<213> Artificial Sequence
<220>
<223> ptsGV12F gene
<400> 5
atgtttaaga atgcatttgc taacctgcaa aagttyggta aatcgctgat gctgccggta 60
tccgtactgc ctatcgcagg tattctgctg ggcgtcggtt ccgcgaattt cagctggctg 120
cccgccgttg tatcgcatgt tatggcagaa gcaggcggtt ccgtctttgc aaacatgcca 180
ctgatttttg cgatcggtgt cgccctcggc tttaccaata acgatggcgt atccgcgctg 240
gccgcagttg ttgcctatgg catcatggtt aaaaccatgg ccgtggttgc gccactggta 300
ctgcatttac ctgctgaaga aatcgcctct aaacacctgg cggatactgg cgtactcgga 360
gggattatct ccggtgcgat cgcagcgtac atgtttaacc gtttctaccg tattaagctg 420
cctgagtatc ttggcttctt tgccggtaaa cgctttgtgc cgatcatttc tggcctggct 480
gccatcttta ctggcgttgt gctgtccttc atttggccgc cgattggttc tgcaatccag 540
accttctctc agtgggctgc ttaccagaac ccggtagttg cgtttggcat ttacggtttc 600
atcgaacgtt gcctggtacc gtttggtctg caccacatct ggaacgtacc tttccagatg 660
cagattggtg aatacaccaa cgcagcaggt caggttttcc acggcgacat tccgcgttat 720
atggcgggtg acccgactgc gggtaaactg tctggtggct tcctgttcaa aatgtacggt 780
ctgccagctg ccgcaattgc tatctggcac tctgctaaac cagaaaaccg cgcgaaagtg 840
ggcggtatta tgatctccgc ggcgctgacc tcgttcctga ccggtatcac cgagccgatc 900
gagttctcct tcatgttcgt tgcgccgatc ctgtacatca tccacgcgat tctggcaggc 960
ctggcattcc caatctgtat tcttctgggg atgcgtgacg gtacgtcgtt ctcgcacggt 1020
ctgatcgact tcatcgttct gtctggtaac agcagcaaac tgtggctgtt cccgatcgtc 1080
ggtatcggtt atgcgattgt ttactacacc atcttccgcg tgctgattaa agcactggat 1140
ctgaaaacgc cgggtcgtga agacgcgact gaagatgcaa aagcgacagg taccagcgaa 1200
atggcaccgg ctctggttgc tgcatttggt ggtaaagaaa acattactaa cctcgacgca 1260
tgtattaccc gtctgcgcgt cagcgttgct gatgtgtcta aagtggatca ggccggcctg 1320
aagaaactgg gcgcagcggg cgtagtggtt gctggttctg gtgttcaggc gattttcggt 1380
actaaatccg ataacctgaa aaccgagatg gatgagtaca tccgtaacca ctaa 1434
<210> 6
<211> 810
<212> DNA
<213> Artificial Sequence
<220>
<223> agaR WT gene
<400> 6
atgagtaata ccgacgcttc aggtgagaag cgagtgacag gcaccagcga gcgacgagaa 60
cagatcattc agcgtctgcg acagcaaggg agtgtgcagg ttaacgatct gtcggcattg 120
tatggcgtat ctaccgtgac gatccgcaac gatctggcgt ttctggaaaa gcaggggatc 180
gctgtgcgtg cctatggtgg cgcgttgatc tgcgatagca cgacgccgtc agtcgagcca 240
tcagtggaag ataaaagcgc actgaacacc gcgatgaaac gcagcgttgc gaaagctgcc 300
gttgagttga ttcagccagg tcatcgggtg atcctcgatt ccgggaccac cacttttgag 360
attgctcgtc tgatgcgcaa gcacactgac gtaattgcga tgaccaacgg tatgaacgtg 420
gctaatgctt tgctggaagc ggaaggcgtt gagctgctga tgaccggcgg gcatttgcgc 480
cgtcagtcgc aatcttttta cggcgatcag gctgaacaat cgctgcaaaa ttaccacttc 540
gatatgctgt ttcttggtgt agatgcgatc gatctggagc gcggcgtcag cacgcataat 600
gaagatgaag cccgtttaaa ccgccggatg tgcgaagttg cggaacggat catcgtagtc 660
accgattcca gtaagttcaa ccgctccagt ttacataaga tcattgatac tcaacgtatc 720
gacatgatca ttgttgatga aggcattcct gcggatagtc tggaaggact gcgaaaggct 780
ggggttgaag tgattctggt cggggagtga 810
Claims (10)
- 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자;
서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자; 및
서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자를 포함하는, 발현 카세트.
- 제 1항에 있어서,
상기 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자는 서열번호 5인 발현 카세트.
- 제 1항에 있어서,
상기 aga 오페론 전사 억제제의 불활성화는 타가토스 알돌라아제 (kbaY)가 발현되도록 하는 것인 발현 카세트.
- 제 1항에 있어서,
상기 불활성화는 유전자를 결손하는 것인 발현 카세트.
- 제 1항의 발현 카세트를 포함하는 재조합 벡터.
- 제5항의 재조합 벡터로 형질 전환된 돌연변이 균주.
- 1) 서열번호 1의 프룩토오스-바이포스페이트 알돌라아제 (Fructose-bisphosphate aldolase class 2, fbaA)가 불활성화 되도록 변이된 유전자;
2) 서열번호 2의 아미노산 12번째 발린 (V)이 페닐알라닌 (F)로 치환된 신규한 포스포트랜스퍼라아제 시스템 G를 암호화하는 유전자; 및
3) 서열번호 3의 aga 오페론 전사 억제제 (Putative aga operon transcriptional repressor, agaR)가 불활성화 되도록 변이된 유전자 중 어느 하나 이상을 포함하는 돌연변이 균주.
- 제6항 또는 제7항에 있어서,
상기 재조합 균주는 대장균인 돌연변이 균주.
- 제6항 또는 제7항에 있어서,
상기 재조합 균주는 D-프룩토오스 대사능을 상실 혹은 감소되고 D-타가토스 대사능을 갖는 돌연변이 균주.
- 제6항 또는 제7항의 돌연변이 균주를 D-프룩토오스 혹은 D-타가토스를 포함하는 배지에서 배양하는 단계; 를 포함하는 D-프룩토오스 비대사성과 D-타가토스 대사능의 특징을 모두 갖는 균주를 생산하는 방법.
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