Abstract: In order to breed a mutant strain for high production of apramycin，carbamoyltobramycin biosynthesis genes tobS1-C were delected from Streptomyces tenebrarius through homologous recombination.Gene deletion vector pSH6 was constructed and introduced into S.tenebrarius Tt49 through conjugal transfer.tobS1-C genes in the chromosome were displaced by deletion allele on the plasmid via double crossover.Then the mutant S.tenebrarius T103（△tobS1-C）was proved to be genetically stable.Shaking flask experiments and TLC analysis showed that the mutant strain only produced apramycin.