Improved arginine production in Escherichia coli by harnessing the intracellular citrulline

L-arginine is a high-value amino acid with widely utilized in the food, feed, and pharmaceutical industries. However, its large-scale biosynthesis remains limited by the low efficiency of current microbial strains. In this study, intracellular citrulline accumulation in Escherichia coli-Arg4 was enhanced by 2.45-, 1.90-, and 1.94-fold through supplementation with monosodium glutamate, monosodium aspartate, and glutamine hydrochloride, respectively. Correspondingly, L-arginine titers increased by 47.85 %, 21.18 %, and 10.66 %. Metabolic flux analysis and transcriptomic profiling indicated that exogenous ammonia donors redirected flux through critical metabolic nodes, including oxaloacetate, α-ketoglutarate, and citrulline, thus increasing precursor availability and enhancing L-arginine biosynthesis. Based on these findings, eight key gene targets, such as gdhA, ppc, icd, aspC, glnA, pyrF, gltA, and argF were identified for pathway optimization. Promoter engineering was subsequently employed to modulate their expression, and heterologous gdhA from Salmonella enterica and glnA from Bacillus subtilis were introduced. Consequently, an optimized strain, E. coli-Arg10, was constructed. Following process optimization in a 1000-L fermenter, the titer, yield and productivity of E. coli-Arg10 was achieved 108.33 g/L, 0.54 g/g, and of 2.26 g/L/h, respectively. These results highlight a scalable and efficient approach for microbial L-arginine production.