Cloning And Function Analysis Of Genes Involved In Antibiotics Biosynthesis In Streptomyces Tenebrarius H6

Streptomyces tenebrarius H6 produces three kinds of antibiotics: apramycin, carbamoyltobramycin and some carbamoylkanamycinB. To study the biosynthetic genes of these antibiotics, the total DNA of S.tenebrarius H6 , partially digested with Sau3Al to yield fragments of 6-15kb ,was ligated with vector pIJ702.The ligation mixture was used to transform S.lividans TK24 protoplasts. At last two of the transformants were used for further study. One showed high resiatance to apramycin, and its plasmid pSPU41 contained a 8.2kb insert, the other clone showed high resiatance to tobramycin, and its plasmid pSPU51 contained a 10.6kb insert.Restriction map and sequence analysis of pSPU41 showed that part of the sequence overlapped with the previously cloned DNA fragment in our lab. The 2828bp fragment, downstream of apramycin resistant gene ,cantains two new ORFs. By analysing with BLASTX, no homology was found, so that the two new ORFs were supposed to be unknown new genes, designated as aprF and aprG respectively. Choosing the inside fragment of aprF and aprG ,two gene disruption vectors (pSPU55-2 and pSPU58-1) were constructed and transferred into S.tenebrarius H6 by conjugation respectively. At last we got two gene disruptants: H6(pSPU55-2) and H6(pSPU58-1). Southern blot experiments showed that the aprF and aprG were correctly disrupted respectively. The disruptants no longer produced apramycin, so we can concluded that aprF and aprG were closely related to apramycin biosynthesis.After analyzing of the restriction map and partially sequencing of the pSPU51,we found three ORFs(ORF1,ORF2,ORF3) .Blast analysis showed...