Fine-Tuning l -Arginine Biosynthesis and Bidirectional Dynamic Control Overcome Metabolic Bottlenecks for High-Titer l -Ornithine Production in Escherichia coli

Abstract

l-Ornithine is a versatile amino acid with broad applications in the pharmaceutical, nutraceutical, and food industries. However, its efficient microbial production is constrained by a critical trade-off: excessive blockage of the downstream l-arginine pathway severely impairs cell growth, while intracellular l-ornithine accumulation causes metabolic stress. Here, we report an integrated metabolic engineering strategy in Escherichia coli that resolves these challenges through three key innovations. First, instead of complete pathway disruption, a fine-tuned attenuation of l-arginine biosynthesis was implemented to balance cellular growth with l-ornithine production. Second, a bidirectional dynamic control system was established to simultaneously repress the competing l-proline pathway and facilitate l-ornithine export, thereby coordinating intracellular synthesis with extracellular secretion. Third, fed-batch fermentation identified N-acetyl-l-ornithine accumulation as a major metabolic bottleneck, which was effectively alleviated by introducing an alternative deacetylation route to bypass feedback inhibition. The final engineered strain produced 65.55 g/L l-ornithine with a yield of 0.40 g/g glucose in a 5 L fed-batch fermentation, the highest de novo l-ornithine titer reported in E. coli to date. This work provides a generalizable framework for balancing growth, metabolic flux, and product secretion in amino acid biomanufacturing.