Neomycin, an aminoglycoside antibiotic, is widely used in the livestock husbandry due to its higher antimicrobial activity and availability of feed additives in animals. However, its production yield is relatively low and cannot meet the needs of developing market and clinical application. Here, the entire natural neo cluster was cloned from Streptomyces fradiae CGMCC 4.576 by φBT1 integrase-mediated site-specific recombination. Then, the rational reconstruction of the neo cluster was performed by using λ-Red-mediated PCR targeting for improving neomycin production. In order to coordinate with this attempt, the supplementation of suitable precursors was carried out. The constructed recombinant strain Sf/pKCZ03 has multi-copy of the neo cluster modified by disrupting the negative regulatory gene neoI and replacing the native promoter of the neoE-D with P