Metabolic Engineering and Adaptive Evolution of Escherichia coli for Enhanced Conversion of D‑Xylose to D‐Glucaric Acid Mediated by Methanol

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.