Pristinamycin, which is a streptogramin antibiotic produced by Streptomyces pristinaespiralis, contains two chemically unrelated compounds, pristinamycin I (PI) and pristinamycin II (PII). Semi-synthetic derivatives of PI and PII have been approved for use in human medicine to treat a broad range of drug-resistant pathogens. In this study, we design and implement a combinatorial metabolic engineering strategy for improving PII production. First, an extra copy of the PII biosynthetic gene cluster, which was assembled using a modified Gibson assembly method for cloning large DNA fragments with high GC contents, was introduced into a high-producing strain S. pristinaespiralis HCCB10218. This duplication of the PII biosynthetic gene cluster resulted in a maximum increase in PII titer by 45%. Second, all seven cluster-situated regulatory genes (from papR1 to papR6 and spbR) were systematically manipulated. Higher PII titers were achieved by deleting either one of the two repressor genes papR3 or papR5 in combination with overexpression of both activator genes papR4 and papR6, and the resulting strains ∆papR3+R4R6 and ∆papR5+R4R6 showed maximum increases in PII production by 99% and 75%, respectively. A combination of the above two different approaches was employed. Integration of the assembled PII gene cluster (BAC-F1F15) into ∆papR5+R4R6 led to the highest PII titer improvement, which was approximately 1.5-fold higher than the parental strain. By adding the macroreticular resin, which can separate pristinamycin in situ and thereby lessen end-product feedback inhibition and toxic effects, PII titers of the final engineered strain ∆papR5+R4R6/BAC-F1F15 reached 1.13 and 1.16 g/L in the Erlenmeyer flask and 5-L bioreactor, respectively, with 5.13- and 5.26-fold improvements over the parental strain. Taken together, this combinatorial strategy is an efficient method to optimize PII biosynthesis of S. pristinaespiralis and may be extended to other industrially used streptomycetes for strain improvement.