Multilevel Metabolic Engineering of Probiotic Escherichia coli Nissle 1917 for Antibiotic-Free Production of β-Alanine

β-Alanine is one of the most valuable three-carbon compounds. In this work, for enhanced β-alanine biosynthesis by probiotic Escherichia coli Nissle 1917 (EcN), multilevel engineering strategies were employed, including the introduction of heterologous key enzymes, dual-pathway reinforcement of the precursor L-aspartate (L-Asp) supply, dynamic modulation of the competing pathways, rewiring of the central metabolism, and engineering the transport systems. A final antibiotic-free strain was constructed, which exhibited robust biosynthesis throughout the fermentation process and produced 60.55 g/L β-alanine with a yield of 0.30 g/g glucose in a 5-L bioreactor, representing the highest level of β-alanine production achieved to date using probiotic E. coli . Notably, it is the first time the function of TdcC (proton-driven threonine/serine symporter), SstT (sodium-driven serine/threonine symporter), SdaC (proton-driven serine symporter), and RhaT (proton-driven rhamnose symporter) has been examined in β-alanine accumulation. This study demonstrated the practicality of EcN as a microbial cell factory for β-alanine production and provided an effective synthetic biology strategy for the production of other L-Asp-derived compounds.