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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.
Bimala subba,Lee, Hei Chan,Sohng, Jae Kyung 한국공업화학회 2004 응용화학 Vol.8 No.2
A genomic library of S. ribosidificus was constructed for sorting out the biosynthetic genes for 4,5-disubstituted 2-deoxystreptamine (DOI) containing amino glycosides antibiotics ribostamycin. Screening over 10, 000 colonies of the library by the conserved region based probes revealed 6 positive cosmids. Sequencing about 32 kb regions reveled 27 open reading frames (ORFs) putatively involved in ribostamycin biosynthesis. The genetic organization of these gene clusters is in good agreement with the previously reported butirosin biosynthetic genes cluster. Out of 10 putative Rbm biosynthetic genes, the rbmA was expressed in S. lividans TK24 and was shown to encode 2-deoxy-scylloinosose (DOI) synthase. Furthermore, acetylations of various aminoglycosideaminocyclitol (AmAcs) with RbmI concluded the rbml as an aminoglycoside 3-N-acetyltrasferase.
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>
송재경,Bimala Subba,Madan Kumar Kharel,Hei Chan Lee,류광경,김병기 한국분자세포생물학회 2005 Molecules and cells Vol.20 No.1
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 aminoglycosideaminocyclitol (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.
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,
Keshav Kumar Nepal,오태진,Bimala Subba,유진철,송재경 한국분자세포생물학회 2009 Molecules and cells Vol.27 No.1
Amino acid homology analysis predicted that rbmD, a putative glycosyltransferase from Streptomyces ribosidificus ATCC 21294, has the highest homology with neoD in neomycin biosynthesis. S. fradiae BS1, in which the production of neomycin was abolished, was generated by disruption of the neoD gene in the neomycin producer S. fradiae. The restoration of neomycin by self complementation suggested that there was no polar effect in the mutant. In addition, S. fradiae BS6 was created with complementation by rbmD in S. fradiae BS1, and secondary metabolite analysis by ESI/MS, LC/MS and MS/MS showed the restoration of neomycin production in S. fradiae BS6. These gene inactivation and complementation studies suggested that, like neoD, rbmD functions as a 2-N-acetlyglucosaminyltransferase and demonstrated the potential for the generation of novel aminoglycoside antibiotics using glycosyltransferases in vivo.