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접합전달을 이용한 Streptomyces natalensis ATCC27448의 형질전환 최적화 및 attB-site의 특성연구
이강무,최선욱,박해룡,황용일,Lee Kang-Mu,Choi Sun-Uk,Park Hae-Ryong,Hwang Yong-Il 한국미생물학회 2005 미생물학회지 Vol.41 No.2
상업적으로 중요한 macrolide계 항진균 학생물질인 natamycin을 생산하는 Streptomyces natalensis ATCC27448의 환자 유전학적인 연구를 위해 대장균으로부터 S. natalensis로 plasmid DNA를 직접 도입하는 형질전환법을 확립하였다. 이러한 S. natalensis의 형질전환은 oriT와 attP 단편을 가지고 있는, ${\Phi}C31$ 유래의 integration 벡터인 pSET152를 이용하여 Escherichia coli ET12567/pUZ28002을 DNA 공여체(donor)로 이용한 접합전달법(conjugal transfer)을 사용하여 확립하였다. 접합전달의 가장 높은 효율은 10 mM의 $MgCl_2$를 포함한 MS 배지에서, $6.25\times10^8$의 E. coli 공여체와 열처리를 하지 않은 S. natalensis의 포자를 사용하여 얻어졌다. 또 얻어진 접합전달체 (exconjugant)에 대하여 southern blot hybridization과 벡터가 삽입된 염색체부분의 염기서열분석을 통해 attB site와 pseudo-attB site를 확인하다. attB site의 경우에는 다른 방선균들처럼 S. natalensis 염색체의 pirin 상동체를 코드하는 ORF내에 존재하였으나 pseudo-attB site는 염색체내 다른 site (GenBank accession no. $YP\_117731$)에 존재하였고 그 염기서열은 attB 염기서열과 차이를 나타내었다. Streptomyces natalensis ATCC27448 produces natamycin, a commercially important macrolide antifungal antibiotic. For molecular genetic study of S. natalensis, we have developed a system for introducing DNA into S. natalensis via conjugal transfer from Escherichia coli. An effective transformation procedure for S. natalensis was established based on transconjugation from E, coli ET12567/pUZ8002 using a ${\Phi}C31$-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 10 mM $MgCl_2$ using $6.25\times10^8$ of E.coli donor cells without heat treatment of spores. In addition, southern blot analysis of exconjugants and the sequence of plasmids containing DNA flanking the insertion sites from the chromosome revealed that S. natalensis contains a single ${\Phi}C31$ attB site and at least a secondary or pseudo attB site. Similar to the case of various Streptomyces species, a single ${\Phi}C31$ attB site of S. natalensis is present within an ORF encoding a pirin-homolog, but a pseudo-attB site is present within a distinct site (GenBank accession no. $YP\_117731$) and also its sequence deviates from the consensus sequences of attB sequence.
CaCO3 가 Bacillus subtilis 에 의한 청국장메주 발효에 미치는 영향
이강무(Kang Moo Lee),이시경(Si Kyung Lee),주현규(Hyung Kyu Joo) 한국응용생명화학회 1994 Applied Biological Chemistry (Appl Biol Chem) Vol.37 No.6
This study was carried out to investigate the effect of CaCO₃ on the Chungkook-jang Meju fermentation. B. subtilis was cultured on the Meju added 0, 0.01, 0.1, 1% CaCO₃, respectively, and the chemical composition, protease activity, amino acid and vitamin B complex were examined with fermentation time. The inner temperature of the CaCO₃ treatments during fermentation was increased as compared with the control group. Titratable acidity in Meju decreased as CaCO₃ cocentration increased. And protease activity and amino-nitrogen content were however high in same order. The content of amino acid was on the increase in every treatments, it made no difference between CaCO₃, treatments and the control. Vitamin B complex content in the CaCO₃ treatments was increased than in the control. The 0.01% added treatment showed the highest amount of vitamin B complex content in all the treatments.