Elucidation and Elimination of Acetyl Phosphate‐Driven Catalytic Promiscuity of ArgF/ArgI Enables High‐Level L‐Arginine Production in Escherichia coli

ABSTRACT

‐Arginine is a critical amino acid widely used in pharmaceutical, food, and feed industries, yet improving its microbial production yield remains challenging due to unresolved metabolic constraints. In this study, we engineered Escherichia coli for ‐arginine production and uncovered a previously unrecognized metabolic bypass that diverts carbon and nitrogen flux. Deleting ptsG reduced overflow metabolism and improved conversion efficiency, but caused slow growth during 5‐L bioreactor fermentation. Applying a glucose–glycerol co‐feeding strategy restored growth while unexpectedly triggered massive accumulation of an unknown byproduct. Through LC‐MS and in vitro enzymatic assays, we identified the byproduct as N ^δ ‐acetylornithine ( ^δ NAO) and elucidated its formation mechanism: high carbon flux drives accumulation of acetyl phosphate (Ac‐P), which is promiscuously used by ornithine carbamoyltransferases ArgF/ArgI to acetylate ‐ornithine instead of their native substrate carbamoyl phosphate. This side reaction creates a non‐canonical bypass that diverts flux away from ‐arginine. By deleting eutD (an Ac‐P‐forming gene) and switching to dissolved‐oxygen‐controlled glycerol feeding, ^δ NAO accumulation was suppressed while maintaining main‐pathway flux, achieving an ‐arginine titer of 65.13 g/L with a conversion yield of 47.7% in a 5‐L bioreactor. Our work reveals that overflow metabolism combined with enzyme catalytic promiscuity can generate hidden byproduct routes, and provides a new mechanistic basis and engineering strategy for improving ‐arginine fermentation by rationally eliminating such bypasses.