d-Glucaric　acid　is　a　value-added　dicarboxylic　acid　that　can　be　utilized　in　the　chemical,　food,　and　pharmaceutical　industries.　Due　to　the　complex　process　and　environmental　pollution　associated　with　the　chemical　production　of　d-glucaric　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　d-glucaric　acid　using　d-xylose　and　methanol.　Mdh,　Hps,　Phi,　Miox,　Ino1,　Suhb,　and　Udh　were　first　co-expressed　in　E.　coli　JM109　to　construct　the　d-glucaric　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-inositol　biosensor　were　introduced　into　engineered　E.　coli　to　enhance　Miox　expression.　After　medium　optimization　and　fermentation　process　control,　3.0　g/L　of　d-glucaric　acid　was　finally　obtained　in　the　fed-batch　fermentation　using　modified　Terrific　Broth　medium.　©　2025　Wiley　Periodicals　LLC.