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BACKGROUND D-Pantothenate (DPA) is an important functional chemical that has been widely applied in healthcare, cosmetics, animal food and feed industries. METHODS AND RESULTS In this study, a high-yield DPA-producing strain was constructed by metabolic engineering strategies with targeting metabolic driving and by-products minimization. The metabolic driving force of push and pull was firstly obtained to improve the production of DPA via enrichment of precursor pool and synthetic pathway, accumulating 4.29 g L-1 DPA in shake flask fermentation. To eliminate the metabolic pressure on DPA production, an amino throttling system was proposed and successfully attenuated the synthesis of four competitive amino acids by a single-step regulation of gdhA. Further minimization of acetate was carried out by pta deletion, and utilization of β-alanine was improved via enhancing its uptake system with producing 5.78 g L-1 DPA. Finally, the engineered strain produced 66.39 g L-1 DPA with β-alanine addition in fermentor under fed-batch fermentation. CONCLUSION This study paved a foundation for the industrial production of DPA. This article is protected by copyright. All rights reserved.