To　construct　a　genetic　engineering　S.　tenebrarius　for　production　of　carbamoyltobramycin.　Recombinant　plasmid　pBK5　derivated　from　pKC1139　was　constructed　for　disrupting　gene　aprK.　pBK5　was　introduced　into　S.　tenebrariusTt-49　by　conjugation.　The　single　crossover　mutant　ST315　was　obtained　and　cultivated　for　several　rounds　of　sporulation　in　the　absence　of　erythromycin.　A　desired　double　crossover　mutant　ST316　was　achieved　by　PCR.　TLC　analysis　indicated　that　ST316　produced　high-yield　carbamoyltobramycin,　in　which　apramycin　biosynthesis　was　blocked　and　the　yield　of　carbamoyltobramycin　was　about　1　500　ug/mL.