Metabolic flux reprogramming and protein engineering drive efficient l-arginine biosynthesis.
l-arginine is widely used in food, feed, pharmaceutical, and cosmetic industries. However, its industrial-scale biosynthesis is limited by insufficient coordination between metabolic regulation, pathway engineering, and fermentation optimization. In this study, an enzyme-constrained model (ec_iML1515) was used to identify 11 gene targets affecting l-arginine production. Based on these targets, metabolic reprogramming was performed in strain Arg4 to rebalance precursor pools (oxaloacetate, aspartate, and citrulline), generating strain Arg10 with an l-arginine titer of 87.24 g/L. Subsequently, the rate-limiting enzyme argininosuccinate synthetase (ArgG) was engineered to the optimal mutant ArgGY131F/K132R and genomically integrated to construct the strain Arg11, increasing the l-arginine titer to 94.80 g/L while reducing aspartate accumulation 7.6-fold to 1.1 g/L. Finally, after the optimization of fermentation temperature and pH, the l-arginine titer, yield, and productivity of strain Arg11 were 114.18 g/L, 0.57 g/g, and 2.27 g/L/h, respectively, in a 3-m3 fermenter, achieving the best performance reported to date.