Bottromycin with the biological activity against Gram-positive bacteria and mycoplasma was isolated from Streptomyces bottropensis.It exhibited activities against methicillin sensitive Staphylococcus aureus (MRSA) and Vancomycin-Resistant Enterococcus (VRE).Cosmid 4E11 carrying the whole biosynthetic gene cluster of bottromycin was obtained by constructing genomic library and PCR screening,and then transformed into Streptomyces coelicolor M145 by intergeneric conjugation.The recombinant strain M145/4E11 was obtained.The results of fermentation showed that the M145/4E11 could produce bottromycin.Based on the Red/ET recombination system and technology,the native promoters of btmA and btmD were replaced by erythromycin promoter (ermE*P1) in M145/4E11,and bottromycin yield of the mutant was increased successfully.