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Here, we reported a paradigm for the design, mechanistic elucidation, and optimization of the novel biosensor to detect cadaverine, an important polymer monomer. We identified a diamine\u2010responsive promoter P\n                    <jats:sub>pyrF<\/jats:sub>\n                    via transcriptomic analysis for biosensor construction. Further investigation suggested that Ihf\u03b2 plays a key role in P\n                    <jats:sub>pyrF<\/jats:sub>\n                    activity regulation, while cadaverine and structurally related amines may activate P\n                    <jats:sub>pyrF<\/jats:sub>\n                    through modulation of Ihf\u03b2\u2010DNA interactions. Stepwise engineering was then performed to enhance biosensor performance, including mutagenesis of the promoter DNA sequence and the Ihf\u03b2 sequence, as well as RBS tuning, resulting in more than a 2.33\u2010fold increase in fold induction. Guided by the biosensor, we implemented a multi\u2010level engineering strategy that spanned tuning a specific exporter, mining optimized pathways from SCRaMbLE libraries, and genome\u2010wide identification of beneficial gene targets. These iterative efforts improved cadaverine titer by 44% to a record 114.2\u00a0g\/L in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    , concurrently uncovering the critical roles of biofilm formation and carbon flux rewiring for overproduction. Our study establishes a biosensor\u2010driven framework for optimizing complex pathways and uncovering novel targets in strain engineering.\n                  <\/jats:p>","DOI":"10.1002\/advs.77720","type":"journal-article","created":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T18:00:31Z","timestamp":1790532031000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Design of a Novel Diamine Biosensor to Guide the Engineering of\n                    <i>Escherichia coli<\/i>\n                    for Cadaverine Overproduction"],"prefix":"10.1002","author":[{"given":"Jia","family":"Feng","sequence":"first","affiliation":[{"name":"State Key Laboratory of Materials\u2010Oriented Chemical Engineering College of Biotechnology and Pharmaceutical Engineering National Engineering Research Center for Biotechnology Nanjing Tech University  Nanjing Jiangsu 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Chemical Engineering College of Biotechnology and Pharmaceutical Engineering National Engineering Research Center for Biotechnology Nanjing Tech University  Nanjing Jiangsu China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Liao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Materials\u2010Oriented Chemical Engineering College of Biotechnology and Pharmaceutical Engineering National Engineering Research Center for Biotechnology Nanjing Tech University  Nanjing Jiangsu China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siyuan","family":"Gao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Materials\u2010Oriented Chemical Engineering College of Biotechnology and Pharmaceutical Engineering National Engineering Research Center for Biotechnology Nanjing Tech University  Nanjing Jiangsu 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