Improved arginine production in Escherichia coli by harnessing the intracellular citrulline
Sheng Qi, Sheng Yang He, Guangjie Liang, Gang Meng, Chunguang Zhao, Aiying Wei, Lining Gou, Jia Liu, Xiaomin Li, Jing Wu, Li Liu
Abstract
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. • The engineered strain E. coli -Arg10 achieved a titer of 108.3 g/L, a yield of 0.54 g/g, and a productivity of 2.26 g/L/h in pilot-scale fermentation, representing the highest reported values to date. • The integrated physiological characterization and multi-omics profiling were conducted to elucidate how intracellular citrulline-pool affect L-arginine biosynthesis. • Through integrated multi-omics analysis, eight key genes involved in L-arginine biosynthesis in E. coli were identified. • An ammonia donor-precursor synergistic enhancement strategy was employed in this study to systematically rewire the cellular metabolic network.