Abstract: 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.