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However, scalable strategies to amplify pathway outputs remain limited. Here, atmospheric and room temperature plasma (ARTP) mutagenesis of\n                    <jats:italic>Saccharopolyspora spinosa<\/jats:italic>\n                    yielded a high\u2010performing 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\u2010omics analysis, and rational design. Using this workflow, the functional contributions of eight overexpressed high\u2010titer protease\u2010related targets were examined from a set of 21 protease\u2010related genes related to nitrogen utilization, which were categorized into three modules: membrane proteostasis (\n                    <jats:italic>htpX<\/jats:italic>\n                    ), adenosine triphosphate (ATP)\u2010dependent proteolysis (\n                    <jats:italic>clpP<\/jats:italic>\n                    ), and amino acid supply (\n                    <jats:italic>dap, pepP, metAP, alp<\/jats:italic>\n                    ). Synergy modeling, coupled with cross\u2010module coordination, highlighted a representative triangular combination,\n                    <jats:italic>pepP\u2010clpP\u2010htpX<\/jats:italic>\n                    . Overexpressing this combination in D184 increased spinosad titer to 2318.87\u00a0mg\/L in a nitrogen\u2010rich 50L bioreactor, corresponding to a 9.81 fold improvement over the wild type. Crucially, this study provided causal evidence that dynamic inter\u2010modular crosstalk governs nitrogen\u2010dependent metabolic flux in D184. These findings establish fundamental mechanistic insights into protease\u2010mediated regulation in\n                    <jats:italic>S. spinosa<\/jats:italic>\n                    and offer guidance for protease\u2010informed optimization of natural product biosynthesis.\n                  <\/jats:p>","DOI":"10.1002\/advs.202522638","type":"journal-article","created":{"date-parts":[[2026,1,31]],"date-time":"2026-01-31T12:32:53Z","timestamp":1769862773000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Systematic Engineering of Proteases in\n                    <i>Saccharopolyspora Spinosa<\/i>\n                    Reveals Synergistic Enhancement of Spinosad Biosynthesis via Substrate Flux Optimization"],"prefix":"10.1002","volume":"13","author":[{"given":"Duo","family":"Jin","sequence":"first","affiliation":[{"name":"Hunan Provincial Key Laboratory of Microbial Molecular Biology College of Life Science Hunan 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Microbial Molecular Biology College of Life Science Hunan Normal University  Changsha China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zirui","family":"Dai","sequence":"additional","affiliation":[{"name":"Hunan Provincial Key Laboratory of Microbial Molecular Biology College of Life Science Hunan Normal University  Changsha China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xirong","family":"Liu","sequence":"additional","affiliation":[{"name":"Hunan Norchem Pharmaceutical Co., Ltd  Changsha Hunan China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zirong","family":"Zhu","sequence":"additional","affiliation":[{"name":"Hunan Provincial Key Laboratory of Microbial Molecular Biology College of Life Science Hunan Normal University  Changsha China"},{"name":"Key Laboratory for Matter Microstructure and Function of Hunan Province Key Laboratory of Low\u2010dimensional Quantum Structures and Quantum Control School of Physics and Electronics Hunan Normal University  Changsha China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jie","family":"Rang","sequence":"additional","affiliation":[{"name":"Hunan Provincial Key Laboratory of Microbial Molecular Biology College of Life Science Hunan Normal University  Changsha China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8443-2916","authenticated-orcid":false,"given":"Liqiu","family":"Xia","sequence":"additional","affiliation":[{"name":"Hunan Provincial Key Laboratory of Microbial Molecular Biology College of Life Science Hunan Normal University  Changsha 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