To enhance spinosad biosynthesis, we investigated the propionyl-CoA carboxylase (PCC) complex responsible for precursor synthesis. Through CRISPR interference (CRISPRi) and overexpression strategies, we constructed six engineered strains to systematically regulate the core PCC subunits encoded by pccA, pccB1, and pccB2. Upregulation of these genes enhanced PCC activity, improved energy and precursor utilization, and activated metabolic pathways, ultimately increasing spinosad production.