Rerouting Fluxes of the Central Carbon Metabolism and Relieving Mechanism-Based Inactivation of l-Aspartate-α-decarboxylase for Fermentative Production of β-Alanine in Escherichia coli

β-Alanine, with the amino group at the β-position, is an important platform chemical that has been widely applied in pharmaceuticals and feed and food additives. However, the current modest titer and productivity, increased fermentation cost, and complicated operation are the challenges for producing β-alanine by microbial fermentation. In this study, a high-yield β-alanine-producing strain was constructed by combining metabolic engineering, protein engineering, and fed-batch bioprocess optimization strategies. First, an aspartate-α-decarboxylase from Bacillus subtilis was introduced in Escherichia coli W3110 to construct an initial β-alanine-producing strain. Production of β-alanine was obviously increased to 4.36 g/L via improving the metabolic flux and reducing carbon loss by rerouting fluxes of the central carbon metabolism. To further increase β-alanine production, mechanism-based inactivation of aspartate-α-decarboxylase was relieved by rational design to maintain the productivity at a high level in β-alanine fed-batch fermentation. Finally, fed-batch bioprocess optimization strategies were used to improve β-alanine production to 85.18 g/L with 0.24 g/g glucose yield and 1.05 g/L/h productivity in fed-batch fermentation. These strategies can be effectively used in the construction of engineered strains for β-alanine and production of its derivatives, and the final engineered strain was a valuable microbial cell factory that can be used for the industrial production of β-alanine.