Synthetic biology can fully promote the efficient bio-manufacturing of target natural products in the aspects of efficient chassis cell construction, active natural product mining and metabolic pathway optimization. However, in the study of synthetic biology of Saccharopolyspora spinosa, due to the limited understanding of the important functional genes regulating spinosyn biosynthesis and their action mechanisms, it is difficult to construct efficient cell factories to significantly increase the spinosyn production through the “design-build-test-learn” strategy. In order to solve this problem, an excellent chassis strain CW-12 with high spinosyn production, fast growth rate and strong extracellular glucose uptake ability was obtained by artificial mutagenesis, and comparative proteomic analysis was performed. The results showed that the enhancement of metabolic pathways related to intracellular carbon metabolism, fatty acid metabolism, amino acid biosynthesis, and TCA cycle was an important reason for the improvement of spinosyn biosynthesis of mutant CW-12, and 668 up-regulated proteins were screened. Subsequently, we selected 3-hydroxyacyl-CoA dehydrogenase (SS_2202) and acetyl-CoA acetyltransferase (SS_2203) to analyze their action mechanisms, confirming that their overexpression can effectively promote the spinosyn biosynthesis. This study has certain guiding significance on how to explore the important functional genes regulating spinosyn biosynthesis and the analysis of their action mechanisms, and lays an important foundation for the subsequent construction of spinosyn efficient biosynthesis cell factories through synthetic biology strategies.