Pristinamycin, produced by Streptomyces pristinaespiralis, which is a streptogramin-like antibiotic consisting of two chemically unrelated components: pristinamycin I (PI) and pristinamycin II (PII), shows potent activity against many antibiotic-resistant pathogens. However, so far pristinamycin production titers are still quite low, particularly those of PI. In this study, we constructed a PI single component producing strain by deleting the PII biosynthetic genes (snaE1 and snaE2). Then, two metabolic engineering approaches, including deletion of the repressor gene papR3 and chromosomal integration of an extra copy of the PI biosynthetic gene cluster (BGC), were employed to improve PI production. The final engineered strain ΔPIIΔpapR3/PI produced a maximum PI level of 132 mg/L, with an approximately 2.4-fold higher than that of the parental strain S. pristinaespiralis HCCB10218. Considering that the PI biosynthetic genes are clustered in two main regions in the 210 kb “supercluster” containing the PI and PII biosynthetic genes as well as a cryptic polyketide BGC, these two regions were cloned separately and then were successfully assembled into the PI BGC by the transformation-associated recombination (TAR) system. Collectively, the metabolic engineering approaches employed is very efficient for strain improvement in order to enhance PI titer.