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D-glucaric acid is an important bio-based building block of polymers and is a high value-added chemical that can be used in a variety of applications. In the present study, the <i>Udh</i> target genes from <i>Pseudomonas putida</i> and <i>Pseudomonas syringae</i> were used together to construct the expression vector pETDuet-2 × Udh. The transformants of BL21 (DE3) with vector pETDuet-2 × Udh were applied to produce glucaric acid from glucuronic acid. After optimizing the induction conditions, the highest Udh expression was achieved when 0.4 mmol·L⁻¹ isopropyl-β-d–thiogalactoside (IPTG) was added to the cell cultures at an OD₆₀₀ value of 0.6 followed by culturing at 26 °C for 6 h. The production of glucaric acid substantially reached 5.24 ± 0.015 g·L⁻¹ in fed-batch cultures in a 30 L tank. In the present study, a new system for glucaric acid production was established, which was more economic and friendly to the environment.