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Due to the lack of high\u2010throughput screening methods, existing genetically engineered bacteria have been developed by simply optimizing the expression of L\u2010cysteine\u2010related genes one by one. To overcome this limitation, in this study, a biosensor\u2010based approach for multilevel biosynthetic pathway optimization of L\u2010cysteine from the DecR regulator variant of <jats:italic>Escherichia coli<\/jats:italic> was applied. Through protein engineering, we obtained the DecR<jats:sup>N29Y\/C81E\/M90Q\/M99E<\/jats:sup> variant\u2010based biosensor with improved specificity and an 8.71\u2010fold increase in dynamic range. Using the developed biosensor, we performed high\u2010throughput screening of the constructed promoter and RBS combination library, and successfully obtained the optimized strain, which resulted in a 6.29\u2010fold increase in L\u2010cysteine production. Molecular dynamics (MD)\u00a0simulations and electrophoretic mobility shift analysis (EMSA)\u00a0showed that the N29Y\/C81E\/M90Q\/M99E variant had enhanced induction activity. This enhancement may be due to the increased binding of the variant to DNA in the presence of L\u2010cysteine, which enhances transcriptional activation. Overall, our biosensor\u2010based strategy provides a promising approach for optimizing biosynthetic pathways at multiple levels. The successful implementation of this strategy demonstrates its potential for screening improved recombinant strains.<\/jats:p>","DOI":"10.1002\/bit.28716","type":"journal-article","created":{"date-parts":[[2024,4,18]],"date-time":"2024-04-18T03:50:56Z","timestamp":1713412256000},"page":"2133-2146","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Engineering of the Lrp\/AsnC\u2010type transcriptional regulator DecR as a genetically encoded biosensor for multilevel optimization of L\u2010cysteine biosynthesis pathway in <i>Escherichia coli<\/i>"],"prefix":"10.1002","volume":"121","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0889-0357","authenticated-orcid":false,"given":"Zhiyou","family":"Zhou","sequence":"first","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering East China University of Science and Technology Shanghai 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