Abstract: Ansamitoci n P-3（AP-3） which belonging to ansamycins has high antitumor activity. To further improve the AP-3 productivity in Actinomycetes, the technology of genome shuffling was employed. Firstly, the conditions of protoplasts preparation, treatment and fusion of A. pretiosum were optimized. And then the highyielding strains L-40, L-117, L-506 and L-522 mutated by ARTP（atmospheric room temperature plasma） were used as the starting parental strains for genome rearrangement. After three rounds of genome rearrangement, a mutant strain G3-96 was screened by high throughput screening. Its AP-3 yield in shaking flask fermentation reached to 335.9 mg/L, which was 47.5% and 93.8% higher than that of par-ental strain L40 and wild-type strain.The experimental results showed that genome rearrangement was a fine stra-tegy for increasing the yield of production and also provided good chassis strains for the fermentation to produce AP-3.