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β-Alanine is a naturally occurring β-amino acid that has been widely applied in the life and health field. Although microbial fermentation is a promising method for industrial production of β-alanine, an efficient microbial cell factory is still lacking. In this study, a new metabolically engineered Escherichia coli strain for β-alanine production was developed through a series of introduction, deletion, and overexpression of genes involved in its biosynthesis pathway. First, the L-aspartate a-decarboxylase gene, BtADC, from Bacillus tequilensis, with higher catalytic activity to produce β-alanine from aspartate, was constitutively expressed in E. coli, leading to an increased production of β-alanine up to 2.76 g/L. Second, three native aspartate kinase genes, akI, akII, and akIII, were knocked out to promote the production of β-alanine to a higher concentration of 4.43 g/L by preventing from bypass loss of aspartate. To increase the amount of aspartate, the native AspC gene was replaced with PaeAspDH, a L-aspartate dehydrogenase gene from Pseudomonas aeruginosa, accompanied with the overexpression of the native AspA gene, to further improve the production level of β-alanine to 9.27 g/L. Last, increased biosynthesis of oxaloacetic acid (OAA) was achieved by a combination of overexpression of the native PPC, introduction of CgPC, a pyruvate decarboxylase from Corynebacterium glutamicum, and deletion of ldhA, pflB, pta, and adhE in E. coli, to further enhance the production of β-alanine. Finally, the engineered E. coli strain produced 43.12 g/L β-alanine in fed-batch fermentation. Our study will lay a solid foundation for the promising application of β-alanine in the life and health field. KEY POINTS: • Overexpression of BtADC resulted in substantial accumulation of β-alanine. • The native AspC was replaced with PaeAspDH to catalyze the transamination of OAA. • Deletion of gluDH prevented from losing carbon flux in TCA recycle. • A 43.12-g/L β-alanine production in fed-batch fermentation was achieved. Graphical abstract.