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Kharel, Madan Kumar,Basnet, Devi Bahadur,Lee, Hei Chan,Liou, Kwangkyoung,Woo, Jin Suk,Kim, Byung-Gee,Sohng, Jae Kyung Elsevier 2004 FEMS microbiology letters Vol.230 No.2
<P><B>Abstract</B></P><P>The biosynthetic gene cluster for tobramycin, a 2-deoxystreptamine-containing aminoglycoside antibiotic, was isolated from <I>Streptomyces tenebrarius</I> ATCC 17920. A genomic library of <I>S. tenebrarius</I> was constructed, and a cosmid, pST51, was isolated by the probes based on the core regions of 2-deoxy-<I>scyllo</I>-inosose (DOI) synthase, and <SMALL>L</SMALL>-glutamine:DOI aminotransferase and <SMALL>L</SMALL>-glutamine:<I>scyllo</I>-inosose aminotransferase. Sequencing of 33.9 kb revealed 24 open reading frames (ORFs) including putative tobramycin biosynthetic genes. We demonstrated that one of these ORFs, <I>tbmA</I>, encodes DOI synthase by in vitro enzyme assay of the purified protein. The catalytic residues of TbmA and dehydroquinate synthase were studied by homology modeling. The gene cluster found is likely to be involved in the biosynthesis of tobramycin.</P>
Kharel, Madan Kumar,Lee, Hei Chan,Sohng, Jae Kyung,Liou, Kwangkyoung 한국공업화학회 2002 Journal of Industrial and Engineering Chemistry Vol.8 No.5
Different medium components were screened to improve the productivity of the novel bioactive compound, cystocin from the Streptomyces sp. GC0001. Plackett and Burman statistical design was employed to screen the effective components. Finally, response surface methodology based on three factors Box-Behnken design was applied to optimize the limiting variables such as soytone, glucose and magnesium sulfate concentration. The antibiotic yield was increased accordingly with the concentration of soytone and glucose. Magnesium sulfate has vital role in productivity besides the other carbon and nitrogen sources. Pharmamedia retained the strongest negative effect for the production of antibiotic and the effect due to sucrose and calcium carbonate was minor. The optimal concentrations of medium components for the cystocin production are determined as; soytone (50 g/L), glucose (40 g/L) and magnesium sulfate (30 g/L).
Identification of 2-Deoxy-scyllo-inosose Synthase in Aminoglycoside Producer Streptomyces
KHAREL, MADAN KUMAR,SUBBA, BIMALA,LEE, HEI CHAN,LIOU, KWANGKYOUNG,WOO, JIN SUK,KIM, DONG HWAN,MOON, YOUNG-HO,SOHNG, JAE KYUNG 한국미생물 · 생명공학회 2003 Journal of microbiology and biotechnology Vol.13 No.5
Although most of the DOS containing aminoglycosides are produced by Streptomyces, very little information is available about their biosynthesis. In the present paper. we report a method to isolate DO1 synthase, a key enzyme in the biosynthesis of DOS, from aminoglycoside producer Streptomyces. PCR primers based on conserved region of DO1 synthases were specific and reliable for the isolation of the biosynthetic genes of DOS containing aminoglycosides or the screening of the aminoglycoside producers. The use of DO1 synthase as a probe could save both time and cost of cloning aminoglycoside biosynthetic genes,
An Approach to Clone the Biosynthetic Genes of 2-deoxystreptamine Containing Aminoglycosides
Subba, Bimala,Kharel, Madan Kumar,Woo, Jin Suk,Lee, Hei Chan,Liou, Kwangkyoung,Sohng, Jae Kyung 한국공업화학회 2003 응용화학 Vol.7 No.1
Despite the most of the 2-deoxystreptamiue (2-DOS) containing aminoglycosids are produced from Streptomyces, very little information existe about their biosynthetic genrs; cloming and characterization. 2-deoxy-scyllo0inosose synthaes (2-DOIS) was osolated from S. kanamyceticus and expressed in heterologous host. We have isolated 2-DOS biosynthetic genes; 2-deoxy-scyllo-inosose synthase, 2-deoxy-scyllo-inosose aminotransferase and aminoglyccoside resistance ribosomal methylases, by PCR experiment using the primers designed from the conserved region of respective genes. The sequencing of PCR amplified DNA fragments reveled their good homology with btrC and btrB of Bacillus cirulans, the sole 2-DOIS gene exists in the database until now, and btrB of Bacillus circulans the sole 2-DOIS gene exists in the database until now, and with other ami noglycodide resistance ribosomal methylases. As a goal for isolation of biosynthetic gene cluster of tobramycin, a cosmid, pST51 was isolated and sequencing of this cosmid is under progress. The PCR primers are specific and reliable to isolate the biosymthetic genes of 2-DOS containing aminoglycosides or for screening the aminoglycoside producers. The use of 2-DOIS as probe can save both time and cost for cloning aminoglycoside biosynthetic genes.
Subba, Bimala,Kharel, Madan Kumar,Liou, Kwangkyoung,Lee, Hei-Chan,Sohng, Jae Kyung 한국공업화학회 2004 응용화학 Vol.8 No.1
Genomic libraries of S. ribosidificus and S. fradiae were constructed for sorting out the biosynthetic genes for 4, 5 disubstitued-2-deoxystreptamine (DOI) containing amino glycosides antibiotics. Screening over 10,000 colonies of each library by the conserved region-based probes revealed 6 positive cosmids. Sequencing about 40kb-region reveled 25 and 29 open reading frames (ORFs) putatively involved in ribostamycin and neomycin biosynthesis. The genetic organization of these gene clusters is in good agreement with the previously reported butirosin biosynthetic genes cluster. Furthermore acetylation by RbmJ of several amino glycosides was also conformed by in vitro enzyme assay of the purified protein.
Subba, Bimala,Kharel, Madan Kumar,Lee, Hei Chan,Liou, Kwangkyoung,Kim, Byung-Gee,Sohng, Jae Kyung Korean Society for Molecular Biology 2005 Molecules and cells Vol.20 No.1
<P>A cluster of genes for ribostamycin (Rbm) biosynthesis was isolated from Streptomyces ribosidificus ATCC 21294. Sequencing of 31.892 kb of the genomic DNA of S. ribosidificus revealed 26 open reading frames (ORFs) encoding putative Rbm biosynthetic genes as well as resistance and other genes. One of ten putative Rbm biosynthetic genes, rbmA, was expressed in S. lividans TK24, and shown to encode 2-deoxy-scyllo-inosose (DOI) synthase. Acetylation of various aminoglycoside-aminocyclitol (AmAcs) by RbmI confirmed it to be an aminoglycoside 3-N-acetyltransferase. Comparison of the genetic control of ribostamycin and butirosin biosynthesis pointed to a common biosynthetic route for these compounds, despite the considerable differences between them in genetic organization.</P>