Abstract:By construction of small fragments genome bank, several different fragments from chromosomal DNA of streptomyces production strain HS007 were cloned into cloning vector pSP-SIM. According to the sequencing results, five of them, which were 3- to 7- kb in size with single restriction sites in the middle, were cut down and re-cloned into bacte-ria-streptomyces shuttle vector pHJL400. Subsequently, the marker cassette, composed of special labeling sequence, oriT, 2 FLP recognition target sites and apramycin resistance gene, was inserted into the single restriction sites to create re-combinant plasmids pHJL02AFOH, pHJL07AFOH, pHJL08AFOH, pHJL10AFOH and pHJL12AFOH. These recombinant plasmids were then transformed into target production strain HS007 by conjugal transfer method, and corresponding marked mutants named 02-72, 07-44, 08-02, 10-81 and 12-58 were screened. Two of these mutants, 02-72 and 12-58, did not show changes in fermentation ability, whereas the others lost partially or completely fermentation ability. The apra-mycin resistance gene and oriT, flanked by two FLP recognition target sites, were then removed by FLP-mediated deletion from recombinant plasmid pSP02AFOH to give pSP02F, whereas the special labeling sequence was still reserved in pSP02F because of being located out of the two FLP recognition target sites. Finally, replacement plasmid pGH02FH was constructed by replacing the vector part of pSP02F with bacteria-streptomyces shuttle vector pGH112 and transformed into mutant 02-72. By selecting apramycin sensitive colonies, marked mutant 02-72-36, whose chromosome was inserted by special labeling sequence without apramycin resistance gene, was screened. Fermentation confirmed that its production ability was not reduced. Such genome labeling technique might be used in other strains of streptomyces to protect the property marks.
