Analysis Of Genes Involved In Carbamoyltobramycin Biosynthesis

Streptomyces tenebrarius H6 produces three kinds of antibiotics: ampramycin, carbamoyltobramycin and some carbamoylkanamycinB. To study the biosynthetic pathway of these antibiotics, the tbmA has been disrupted. First, the 863bp inside fragment of tbmA gene was obtained by PCR, the gene disruption vector (pSPU112) was constructed and transferred into S.tenebrarius H6 by conjugation. Recombinant S.tenebrarius H6(pSPU112)was obtained. Southern blot experiment showed that the tbmA was correctly disrupted.With producing apramycin, the disruptant no longer produces carbamoyltobramycin, the result indicates that the tbmA is not related to apramycin biosynthesis but closely related to carbamoyltobramycin biosynthesis.To study the biosynthetic gene cluster of carbamoyltobramycin, the gene disruption experiment of 50kb regions cloned by Li tian bo has been done,which indicated the approximate linkage regions related to carbamoyltobramycin biosynthesis.The relationship between 50kb regions cloned by Li tian bo and 13.8kb putative carbamoyltobramycin biosynthesis gene cluster has been studied by Southern blot experiment, the result indicates that the two fragments are independent. We deduced that the segment uncloned in the rigt side of 50kb regions cloned by Li tian bo still contained the carbamoyltobramycin biosynthetic genes.Restriction map and sequence analysis of G fragment showed that it cantains a new ORF and a incomplete ORF. By analysing with BLASTX, no homology was found, so that the new ORF was supposed to be an unknown new gene, designated as tobH. Choosing the inside fragment of tobH,the gene disruption vectors (pSPU119) was constructed and transferred into S.tenebrarius H6 by conjugation. At last we got the gene disruptant H6(pSPU119). Southern blot experiment showed that the tobH was correctly disrupted. The disruptant no longer produces carbamoyltobramycin, so we could conclude that tobH is closely related to carbamoyltobramycin biosynthesis.