Metabolic reprogramming and biosensor-assisted mutagenesis screening for high-level production of L-arginine in Escherichia coli.

L-arginine is a value-added amino acid with promising applications in the pharmaceutical and nutraceutical industries. Further unleashing the potential of microbial cell factories to make L-arginine production more competitive remains challenging due to the sophisticated intracellular interaction networks and the insufficient knowledge of global metabolic regulation. Here, we combined multilevel rational metabolic engineering with biosensor-assisted mutagenesis screening to exploit the L-arginine production potential of Escherichia coli. First, multiple metabolic pathways were systematically reprogrammed to redirect the metabolic flux into L-arginine synthesis, including the L-arginine biosynthesis, TCA cycle, and L-arginine export. Specifically, a toggle switch responding to special cellular physiological conditions was designed to dynamically control the expression of sucA and pull more carbon flux from the TCA cycle toward L-arginine biosynthesis. Subsequently, a biosensor-assisted high-throughput screening platform was designed and applied to further exploit the L-arginine production potential. The best-engineered ARG28 strain produced 132 g/L L-arginine in a 5-L bioreactor with a yield of 0.51 g/g glucose and productivity of 2.75 g/(L ⋅ h), which were the highest values reported so far. Through whole genome sequencing and reverse engineering, Frc frameshift mutant, PqiB A78P mutant, and RpoB P564T mutant were revealed for enhancing the L-arginine biosynthesis. Our study exhibited the power of coupling rational metabolic reprogramming and biosensor-assisted mutagenesis screening to unleash the cellular potential for value-added metabolite production.