Systems engineering of Escherichia colifor high-level l-alanine production

l-alanine is a critical natural amino acid that is widely used in the food, pharmaceutical, and personal care industries. However, low titer and long fermentation period limits the industrial viability of l-alanine production. In this study, we engineered a microbial cell factory for efficient l-alanine synthesis using a variety of metabolic engineering strategies. Initially, l-alanine was biosynthesized through the heterologous introduction of alanine dehydrogenase from Geobacillus stearothermophilus. Thereafter, the l-alanine biosynthetic pathway and transport module were modified to improve its titers. Furthermore, a dynamic response system was constructed based on the l-alanine-driven PAla-7promoter to regulate the tolerance target BetTexpression and enhance the tolerance of Escherichia colito high l-alanine concentrations. Finally, the resultant strain E. coliW-PAla-7-BetT could produce 134.5 g/L of l-alanine in 24 h in a 5-L bioreactor, with 5.6 g/L/h of productivity, which is the highest productivity reported to date. Our findings may help in constructing high-performance microbial cell factories for the production of l-alanine and other valuable chemicals.