Model‐Guided Systematic Metabolic Engineering for Enhanced Spinosad Biosynthesis in Saccharopolyspora spinosa NHF132

Abstract Spinosad (a mixture of spinosyns A and D) is a macrocyclic lactone green bioinsecticide produced by Saccharopolyspora spinosa . It is known for its high efficiency, low toxicity, and broad‐spectrum activity. Although numerous strategies have been employed to enhance spinosad production, intricate regulation of secondary metabolism and inefficient genetic manipulation impede systematic and comprehensive metabolic engineering in this spinosad‐producing strain. In this study, a genome‐scale metabolic model (GEM) for Sa. spinosa NHF132 is developed to dissect the intricate secondary metabolic pathways of spinosad biosynthesis, analyzing interactions among precursors, key enzymes, and competing or bypass pathways. Guided by the model, the impact of rhamnose precursor overexpression, gene cluster amplification, short‐chain acyl‐CoA enhancement, and chassis optimization on spinosad production is systematically evaluated. By integrating these metabolic engineering strategies, engineered strain NHF132‐BAC‐ SP43 ‐NCM achieved a spinosad titer of 1816.8 mg L −1 , a 553.3% increase over the starting strain, with substantial improvements in yield and product proportion. The model‐driven framework for metabolic engineering of complex secondary metabolites in actinomycetes substantially increased spinosad production and offered valuable insights for other complex natural products.