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Due to the complex process and environmental pollution associated with the chemical production of <jats:sc>d<\/jats:sc>\u2010glucaric acid, bioconversion for its synthesis has garnered increasing attention in recent years. In this study, a novel cell factory was developed for the efficient production of <jats:sc>d<\/jats:sc>\u2010glucaric acid using <jats:sc>d<\/jats:sc>\u2010xylose and methanol. Mdh, Hps, Phi, Miox, Ino1, Suhb, and Udh were first co\u2010expressed in <jats:italic>E. coli<\/jats:italic> JM109 to construct the <jats:sc>d<\/jats:sc>\u2010glucaric acid synthesis pathway. The deletion of FrmRAB, RpiA, PfkA, and PfkB was then performed to block or weaken the endogenous competitive pathways. Next, adaptive evolution was carried out to improve cell growth and substrate utilization. With the purpose of further increasing the product titer, the NusA tag and myo\u2010inositol biosensor were introduced into engineered <jats:italic>E. coli<\/jats:italic> to enhance Miox expression. After medium optimization and fermentation process control, 3.0\u2009g\/L of <jats:sc>d<\/jats:sc>\u2010glucaric acid was finally obtained in the fed\u2010batch fermentation using modified Terrific Broth medium.<\/jats:p>","DOI":"10.1002\/bit.28974","type":"journal-article","created":{"date-parts":[[2025,3,19]],"date-time":"2025-03-19T01:10:31Z","timestamp":1742346631000},"page":"1472-1483","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Metabolic Engineering and Adaptive Evolution of <i>Escherichia coli<\/i> for Enhanced Conversion of D\u2011Xylose to D\u2010Glucaric Acid Mediated by Methanol"],"prefix":"10.1002","volume":"122","author":[{"given":"Wei\u2010Xiang","family":"Chen","sequence":"first","affiliation":[{"name":"College of Chemical Engineering Fujian Engineering Research Center of Advanced Manufacturing Technology for Fine Chemicals Fuzhou University Fuzhou P. R. 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