Coordinated Metabolic Strategies for Green and Efficient Orotic Acid Biomanufacturing in Escherichia coli

Abstract Orotic acid (OA) is an intermediate in de novo pyrimidine biosynthesis and a bio-based chemical of interest for nutraceutical, animal nutrition, and biomedical applications. Efficient fermentative OA production, however, remains constrained by carbon loss, insufficient nitrogen assimilation, product-associated stress, and energy-redox imbalance during high-titer production. This study constructed a high-performance Escherichia coli cell factory by coordinating product transport, carbon-nitrogen flux, and energy-redox metabolism. Deletion of the inward OA transporter dctA improved extracellular OA accumulation, while the introduction of a nonoxidative glycolysis (NOG) module, acetyl-phosphate recycling, and byproduct-pathway attenuation reduced overflow carbon loss. Nitrogen assimilation was strengthened by tuning heterologous ammonium transport and balancing NADPH supply. Adenylate metabolism reconstruction and microaerobically responsive expression of vgb and cyoABCDE increased intracellular ATP levels and promoted the NADH/NAD+ redox balance, supporting OA biosynthesis during mid-to-late-stage fed-batch fermentation. OA24 produced 214.2 g/L OA in 42 h in a 5-L bioreactor, with an apparent glucose-based yield of 0.70 g/g glucose and productivity of 5.10 g/L/h; relative to the initial strain Ora21, the yield and productivity increased by 20.7% and 34.2%, respectively. This work provides a carbon-efficient and high-intensity biomanufacturing strategy for OA and related pyrimidine chemicals.