Key enzyme optimization and multi-node metabolic flux regulation drive l-arginine production in Escherichia coli

Microbial production of l-arginine is often constrained by tight metabolic regulation and insufficient precursor supply. Here a plasmid-free, non-auxotrophic Escherichia coli strain for high-level production of l-arginine was rationally engineered, based on our previous constructed strain G0 with 12.4 g/L l-arginine production in flask. Glucose metabolism and glutamate/aspartate uptake were initially enhanced, with subsequent semi-rational engineering of key enzymes, ornithine acetyltransferase (OAT) and argininosuccinate synthase (ASS), to promote ATP synthesis. OAT was firstly rational engineered by introducing amide group for the residues near substrate-binding pocket to stabilize oxyanion transition states, with achieving that variant Y386Q showed K_m/k_cat at 6.58 mM^-1 min^-1, 2 times higher than that of wild type. Variant Y332L of ASS was novelty fused with argininosuccinate lyase via GGGGS linker for ASS activity measurement, which helped improve l-arginine titer to 17.5 g/L. With further studies by screening of rate-limiting nodes on the genome-scale level based on sRNA strategy, aspartate and glutamate pathways were synergistically enhanced, along with utilizing carbon dioxide recycling for carbamoyl phosphate synthesis to drive ammonia donor supply. The obtained final plasmid-free and non-auxotrophic strain G16 produced 21.1 g/L l-arginine in flask, 76.6% higher than that of original strain G0. In 5 L fermenter, 125.6 g/L l-arginine was produced by fed-batch fermentation, with a yield of 0.53 g/g glucose. This study underscores that the convergence of mechanistic enzyme redesign and systems-level pathway optimization is critical to unlocking high-efficient amino acid production, offering a transferable blueprint for rational strain engineering in industrial biotechnology.