Systematic Engineering of Proteases in Saccharopolyspora Spinosa Reveals Synergistic Enhancement of Spinosad Biosynthesis via Substrate Flux Optimization

Natural product biosynthesis is tightly coupled to nitrogen allocation. However, scalable strategies to amplify pathway outputs remain limited. Here, atmospheric and room temperature plasma (ARTP) mutagenesis of Saccharopolyspora spinosa yielded a high‐performing strain, D184, which showed improved utilization of extracellular nitrogen sources together with enhanced spinosad production. To identify novel regulatory mechanisms linking nitrogen utilization with natural product biosynthetic efficiency, we established a combinatorial engineering workflow that integrates systematic protease genetic manipulation, multi‐omics analysis, and rational design. Using this workflow, the functional contributions of eight overexpressed high‐titer protease‐related targets were examined from a set of 21 protease‐related genes related to nitrogen utilization, which were categorized into three modules: membrane proteostasis ( htpX ), adenosine triphosphate (ATP)‐dependent proteolysis ( clpP ), and amino acid supply ( dap, pepP, metAP, alp ). Synergy modeling, coupled with cross‐module coordination, highlighted a representative triangular combination, pepP‐clpP‐htpX . Overexpressing this combination in D184 increased spinosad titer to 2318.87 mg/L in a nitrogen‐rich 50L bioreactor, corresponding to a 9.81 fold improvement over the wild type. Crucially, this study provided causal evidence that dynamic inter‐modular crosstalk governs nitrogen‐dependent metabolic flux in D184. These findings establish fundamental mechanistic insights into protease‐mediated regulation in S. spinosa and offer guidance for protease‐informed optimization of natural product biosynthesis.