Klebsiella pneumoniae 에서 트립토판 생산조절 변이주의 분리 및 그 특성

지연태,이세영 한국유전학회 1986 Genes & Genomics Vol.8 No.4

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Klebsiella pneumoniae에서 트립토판 생산증대를 위한 숙주개발 및 재조합 trp plasmid의 발현

지연태,홍광원,박장현,이세영 한국미생물학회 1987 미생물학회지 Vol.25 No.1

In order to increase the production of tryptophan by maximizing expression of recombinant trp plasmid, Klebsiella pneumoniae KC 105(pheA tyrA trpE trpR tyrR) was genetically modified. KC 107, inosine monophospate(IMP) auxotroph from KC 105 and KC 108, histidine(His) auxotroph from KC 107 were also derived respectively to increase phosphoribosylpyrophosphate(PRPP) production which is required for tryptophan biosynthesis. From KC 107 phosphoribosylpyrophosphate consumption which is required for tryptophan biosynthesis. From KC 107 and KC 108, KC 109 and KC 110, both arginine auxotrophs were derived respectively. To investigate the expression of recombinant trp plasmid in the selected K. pneumoniae mutants, the auxotrophic mutants were transformed with recombinant trp plasmids pSC 101-$trpE^{FBR}$, pSC 101-trpL(.DELTA.att) $trpE^{FBR}$ (pSC 101-trp-AF). Amount of tryptophan produced and activities of tryptophan synthase of $trpR^{-}$ mutant (KC 100) and $tyrR^{-}$ mutnat(KC 105) containing recombinant plasmid pSC 101-trp operon were increased by 30-40% as compared with KC 99(pheA tyrA trpE) containing recombinant plasmid pSC 101-trp operon. Activities of tryptophan synthase and production of tryptophan of KC 108 ($His^{-}$) and KC 109($Arg^{-}$) containing recombinant plasmid pSC 101-trp operon were increase by two-fold as compared with KC 107 containing pSC 101-trp operon.

Escherichia coli K - 12의 trpL ( Δatt ) trpE 변이주 구성과 그의 특성

지연태,이세영 ( Youn Tae Chi,Se Yong Lee ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.3

In order to use as gene source for the cloning of trp operon, the trp operon with attenuator region in the leader sequence deleted and trpE gene mutated to feedback inhibition resistance was constructed and its properties were characterized. When 10^(-3)M tryptophan was added to the reaction mixture of anthranilate synthetase assay, activity of enzyme preparation from the trpL (Δatt) trpEFBR was unchanged while that of enzyme preparation from wild type strain was reduced approximately 50 %. The isolated trpL (Δatt)trpE^(FBR)trpR- (LC113) mutant showed 10-fold increase in anthranilate synthetase activity and 20-fold increase in tryptophan synthetase activity over wild type strain when grown in the presence of 10-3M tryptophan. Deletion of attenualor region in the leader sequence was confirmed by the fact that the anthranilate synthetase activity of this mutant decreased much less than the trpR- parental strain by the addition of 10^(-3) M tryptophan in the growth medium.

Plasmid RP4 :: Mucts 61 에 의한 E . coli K - 12 trpL ( Δatt ) trpE 유전자의 in vivo Cloning

지연태,김병문,이세영 ( Youn Tae Chi,Byung Moon Kim,Se Yong Lee ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.3

In order to provide the trp gene source for the construction of recombinant multi-copy plasmid, and to study the expression of E. coli trp operon in the Aerobacter aerogenes 62-1, the regulation-free tryptophan operon of E. coli trpL (dart) trpEFBR, was cloned by in viva cloning technique using hybrid plasmid RP₄: : Mucts 61. When the chromosomal trpL (datt) trpE^(FBR) gene of E. coli LC110 was conjugally transferred by the plasmid RP₄: :Mucts 61 to E. coli LC501 and A. aerogenes 62-1, transfer frequencies were 3. 4×10^(-8) and 1. 2×10^(-9), respectively, Two E. coli transeonjugants, KUB101 and KUB102, and A. aerogeney transconjugant, AKB101, were isolated and characterized. In order to confirm the parental genetic character of the trp operon in the transconjugants, the activities of tryptophan synthetase and anthranilate synthetase were assayed. Transconjugants KUB102 and AKB101 had resistance to attenuator control and feedback inhibition by the tryptophan. However, KUB101 had resistance to feedback inhibition, but sensitive to attenuator control by the tryptophan, Drug resistance and Trp+ phenotype of the transconjugants were stable in the both complete and minimal medium containing ampicillin (100 ㎍). kanamycin (25 ㎍), and tetracycline (50 ㎍). The plasmid DNA of the predicted size (RP₄::Mucts 61-trp) was identified from the transconjugants by the agarose gel electrophoresis. On the basis of the results, it was concluded that the specific E. coli trp operon could be conveniently cloned in vivo in both E. coli and A. aerogenes 62-1 using hybrid plasmid RP₄: : Mucts 61 and that E. coli K-12 trp gene could be normally expressed in the A. aerogenes 62-1.

Klebsiella Pneumoniae 의 L - Serine diaminase 와 Tryptophan Aminotransferase 결실변이가 E . Coli trp Operon 발현에 미치는 영향

지연태,김익영,이세영 ( Youn Tae Chi,Ick Young Kim,Se Yong Lee ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.1

As a part of the host development for a amplified recombinant E. coli trp operon (R_6K::Mu-trpL(△att)trpE^(FBR)) for the production of tryptophan, the 5-methyltryptophan-resistan mutation (5-MT^r), tryptophan aminotransferase-deficient mutation (tat^-) and L-serine deaminase-deficient mutation (ssd^-) were introduced into the K. pneumoniae host cell and their effects on the expression of the trp operon and production of tryptophan were examined. The 5-MT^s mutation derepressed the expression of the recombinant E. coli trp operon and both tat^- and ssd^- mutation increased the activities of anthranilate synthetase and tryptophan synthetase as well as the producation of tryptophan in K. pneumoniae.

대장균의 arop 변이주 구성과 그의 Tryptophan Transport System

지연태,안병우,이세영 한국유전학회 1983 Genes & Genomics Vol.5 No.3

N/A

Effects of $tat^-$ and $ssd^-$ Mutation on the Expression of a Recombinant trp Plasmid and Tryptophan Production in Klebsiella pnenmoniae

지연태,김익영,이세영,Chi, Youn-Tae,Kim, Ick-Young,Lee, Se-Yong 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.1

트립토판 생산성 증대를 위한 균주 개발의 일환으로 숙주 박테리아인 Klebsiella pneumoniae의 5-methyltryptophan 내성주(5-$MT^r$), tryptophan aminotransferase 결손주($tat^-$) 및 L-serine deaminase의 결손주($ssd^-$)를 얻고, 또한 이들 변이가 증폭된 재조합 E. coli trp operon ($R_6K::Mu-trpL({\Delta}tt)trpE^{FBR}$)의 발현에 어떻게 영향을 미치는지 조사하였다. K. pneumoniae에서 5-$MT^r$, $tat^-$, $ssd^-$ 돌연변이는 재초합 E. coli trp operon의 발현을 증가시켰으며, trp operon의 최종산물인 트립토판의 생산성도 증가시켰다. As a part of the host development for a amplified recombinant E. coli trp operon ($R_6K::Mu-trpL({\Delta}att)trpE^{FBR}$) for the production of tryptophan, the 5-methyltryptophan-resistan mutation (5-$MT^r$), tryptophan aminotransferase-deficient mutation ($tat^-$) and L-serine deaminase-deficient mutation ($ssd^-$) were introduced into the K. pneumoniae host cell and their effects on the expression of the try operon and production of tryptophan were examined. The 5-$MT^r$ mutation derepressed the expression of the recombinant E. coli trp operon and both $tat^-$ and $ssd^-$ mutation increased the activities of anthranilate synthetase and tryptophan synthetase as well as the producation of tryptophan in K. pneumoniae.

Plasmid $RP_4$::Mucts 61에 의한E. coli K-12 trpL (${\Delta}att$) $trpE^{FBR}$ 유전자의 in vivo Cloning

지연태,김병문,이세영,Chi, Youn-Tae,Kim, Byung-Moon,Lee, Se-Yong 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.3

박테리아의 multi-copy plasmid vector에 사용될 trp gene source로서의 이용과 Aerobacter aerogenes에서의 E. coli trp operon 발현에 관한 정보를 연구할 목적으로 E. coli K-12의 $trpL({\Delta}att)trpE^{FBR}$ 유전자를 hybrid plasmid $RP_4$::Mucts 61을 이용하여 E. coli LC501과 A. aerogenes 62-1에 각각 in vivo cloning 하였다. E. coli LC110의 염색체 $trpL({\Delta}att)trpE^{FBR}$ 유전자를 hybrid plasmid $RP_4$::Mucts 61 에 의해 E. coli LC501과 A. aerogenes 62-1에 도입하였을 때의 transfer frequency는 각각 $3.4{\times}10^{-8}$과 $1.2{\times}10^{-9}$이었고, 이 때 E. coli transconjugant KUB101과 KUB102 그리 고 A. aerogenes 62-1의 transconjugant AKB101을 분리 하였다. 이들 transconjugant들에서 tryptophan synthetase와 anthranilate synthetase 활성을 측정하여 공여 E. coli trp operon의 유전적인 특성들이 그대로 유지되는지를 확인하였다. transconjugant KUB102와 AKB101은 공여주에서와 마찬가지로 트립토판에 의한 attenuator control과 feedback inhibition을 모두 받지 않았고, KUB101은 트립토판에 의 한 attenuator control은 받았으나 feedback inhibition은 받지 않았다. ampicillin($100\;{\mu}g/ml$), kanamycin($25\;{\mu}g/ml$) 그리고 tetracycline($50\;{\mu}g/ml$)이 각각 포함된 최소배지와 완전배지에서 transconjugant들의 항생제 저항성과 $Trp^+$ 형질은 안정하였으며 예상한 크기의 plasmid DNA ($RP_4$::Mucts 61-trp)가 transconjugant들에서 agarose 전기영동으로 확인되었다. 이상의 결과로부터 E. coli trp operon을 간편하게 E. coli와 A. aerogenes에서 in vivo cloning할 수 있고 E. coli trp operon이 A. aerogenes에서 정상적으로 발현될 수 있음을 확인하였다. In order to provide the trp gene source for the construction of recombinant multi-copy plasmid, and to study the expression of E. coli trp operon in the Aerobacter aerogenes 62-1, the regulation-free tryptophan operon of E. coli trpL (${\Delta}att$) $trpE^{FBR}$, was cloned by in vivo cloning technique using hybrid plasmid $RP_4$::Mucts 61. When the chromosomal trpL (${\Delta}att$) $trpE^{FBR}$ gene of E. coli LC110 was conjugally transferred by the plasmid $RP_4$::Mucts 61 to E. coli LC501 and A. aerogenes 62-1, transfer frequencies were $3.4{\times}10^{-8}$ and $1.2{\times}10^{-9}$, respectively. Two E. coli transconjugants, KUB101 and KUB102, and A. aerogenes transconjugant, AKB101, were isolated and characterized. In order to confirm the parental genetic character of the trp operon in the transconjugants, the activities of tryptophan synthetase and anthranilate synthetase were assayed. Transconjugants KUB102 and AKB101 had resistance to attenuator control and feedback inhibition by the tryptophan. However, KUB101 had resistance to feedback inhibition, but sensitive to attenuator control by the tryptophan. Drug resistance and $Trp^+$ phenotype of the transconjugants were stable in the both complete and minimal medium containing ampicillin ($100\;{\mu}g$), kanamycin ($25\;{\mu}g$), and tetracycline ($50\;{\mu}g$). The plasmid DNA of the predicted size ($RP_4$::Mucts 61-trp) was identified from the transconjugants by the agarose gel electrophoresis. On the basis of the results, it was concluded that the specific E. coli trp operon could be conveniently cloned in vivo in both E. coli and A. aerogenes 62-1 using hybrid plasmid $RP_4$::Mucts 61 and that E. coli K-12 trp gene could be normally expressed in the A. aerogenes 62-1.

식품 · 생화학 : Klebsiella pneumoniae 에서 재조합 E . coli trp operon plasmid 의 발현과 트립토판 생산

지연태,이수영,이세영 한국토양비료학회 1987 한국토양비료학회 학술발표회 Vol.1987 No.38

Escherichia coli K-12의 $trpL({\Delta}att)trpE^{FBR}$ 변이주 구성과 그의 특성

지연태,이세영,Chi, Youn-Tae,Lee, Se-Yong 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.3

재조합 trp operon plasmid의 trp gene source 개발을 위해 trpE, D gene 산물인 anthranilate synthetase가 feedback inhibition을 받지 않고 또한 leader region내에 있는 attenuator site에서 attenuator control을 받지 않는 $trpL(\Delta{att})trpE^{FBR}$ 변이주를 얻고 그 특성을 조사하였다. 조효소 반응액에 트립토판을 $10^{-3}M$ 첨가하였을 때 anthranilate synthetase의 활성은 야생균주에서는 50% 감소한 반면, 변이주 LC1l3에서는 거의 변화가 없었다. LC113 변이주는 트립토판이 $10^{-3}M$ 존재하는 repression조건하에서 야생균주에 비해서 anthranilate synthetase 활성이 10배 증가하였고 tryptophan synthetase 활성은 20배 증가하였다. Donor 균주인 LC112 ($trpR^-trpE^{FBR}$)나 변이주인 LC113 [$trpR^-trpL({\Delta}att)trpE^{FBR}$ 균주는 모두 $trpR^-$인데, 최소 배지중에 트립토판을 $10^{-3}M$ 첨가한 상태에서 LC112 균주는 anthranilate synthetase 활성이 25% 감소된 반면, 변이 주인 LC113 균주는 anthranilate synthetase 활성이 약간만 감소 하였다. 이는 LC112 균주는 attenuator control을 받고, LC113 균주는 attenuator control을 받지 않음을 시사한다. 그러므로, 새로 구성된 변이주는$trpL({\Delta}att)trpE^{FBR}$의 유전형질을 갖는 변이주이며 재조합 trp operon plasmid의 trp gene source로서 유용하리라 생각된다. In order to use as gene source for the cloning of trp operon, the trp operon with attenuator region in the leader sequence deleted and trpE gene mutated to feedback inhibition resistance was constructed and its properties were characterized. When $10^{-3}M$ tryptophan was added to the reaction mixture of anthranilate synthetase assay, activity of enzyme preparation from the $trpL({\Delta}att)trpE^{FBR}$ was unchanged while that of enzyme preparation from wild type strain was reduced approximately 50%. The isolated $trpL({\Delta}att)trpE^{FBR}trpR^-$ (LC113) mutant showed 10-fold increase in anthranilate synthetase activity and 20-fold increase in tryptophan synthetase activity over wild type strain when grown in the presence of $10^{-3}M$ tryptophan. Deletion of attenualor region in the leader sequence was confirmed by the fact that the anthranilate synthetase activity of this mutant decreased much less than the $trpR^-$ parental strain by the addition of $10^{-3}M$ tryptophan in the growth medium.