In order to enhance pristinamycin production, six homologous ptr genes from high pristinamycin-producing strains of Streptomyces pristinaespiralis were selected for DNA shuffling, and the reason for the altered activities of the shuffled ptr gene was speculated by sequence alignment. The highest pristinamycin yield of 0.12 g/L was achieved with a sixfold increase in strain sps16 obtained by DNA shuffling when compared to ancestral strain ATCC 25486. Sequence analysis of the ptr gene variant from the sps16 strain indicated that five mutations (H16P, N63D, T75P, Q107R, and P435A) were introduced into the gene, two of them (N63D and T75P) located in the second of the 14 transmembrane segments (TMS). Prediction of the secondary structure of the gene product indicated that mutations at the N-terminus resulted in the shortening of the corresponding α-helix, while the mutation at the C-terminus lengthened the helix. In conclusion, combination of DNA shuffling with genome shuffling is an effective breeding strategy for increasing the antibiotic yield by directed evolution of target genes.