Abstract: In order to improve the yield of butenyl-spinosyns produced by Saccharopolyspora pogona, we used ribosome engineering technology to screen the paromomycin resistant mutants. In 10×MIC paromomycin concentration, we isolated 54 spontaneous paromomycin resistant mutants. By a preliminary study on metabolites, we found that the mutants had significant differences in synthesis ability of butenyl-spinosyn. There were 6 mutants in which the yields were higher than the wild strain, about 20% were positive and 45% were negative mutants. The mutant P-7 has the largest increase rate of butenyl-spinosyns yield, which improved 2.2-fold compared with the wild strain. DNA sequence analysis showed that two point mutations occurred in the conserved regions of rpsL gene encoding ribosomal protein S12, the 314th by C mutation for A, the corresponding proline mutated to glutamine. The 320th by C mutation for T, the corresponding alanine mutated to valine.