Fine and combinatorial regulation of key metabolic pathway for enhanced β‐alanine biosynthesis with non‐inducible <i>Escherichia coli</i>

β‐Alanine is the only β‐amino acid in nature and one of the most important three‐carbon chemicals. This work was aimed to construct a non‐inducible β‐alanine producer with enhanced metabolic flux towards β‐alanine biosynthesis in Escherichia coli . First of all, the assembled E. coli endogenous promoters and 5′‐untranslated regions (PUTR) were screened to finely regulate the combinatorial expression of genes panD BS and aspB CG for an optimal flux match between two key pathways. Subsequently, additional copies of key genes ( panD BS K104S and ppc ) were chromosomally introduced into the host A1. On these bases, dynamical regulation of the gene thrA was performed to reduce the carbon flux directed in the competitive pathway. Finally, the β‐alanine titer reached 10.25 g/L by strain A14‐R15, 361.7% higher than that of the original strain. Under fed‐batch fermentation in a 5‐L fermentor, a titer of 57.13 g/L β‐alanine was achieved at 80 h. This is the highest titer of β‐alanine production ever reported using non‐inducible engineered E. coli . This metabolic modification strategy for optimal carbon flux distribution developed in this work could also be used for the production of various metabolic products.