Systems metabolic engineering of a plasmid-free Escherichia coli cell factory for high-level cytidine production

Cytidine is an important precursor for nucleoside pharmaceuticals, but its fermentative production is constrained by competing degradation, poor selectivity of pyrimidine flux, insufficient precursor supply, and energy-redox imbalance. In this study, a plasmid-free E. coli cell factory was constructed through systems metabolic engineering. Sequential deletion of cytidine degradation, salvage, and precursor-consuming pathways enabled initial cytidine accumulation. The conversion from UMP to CMP was strengthened by introducing feedback-deregulated pyrHD93A and pyrGD160E, E162A, E168Kcgl mutants and nudG, while the heterologous expression of PHM8sce improved CMP-to-cytidine conversion selectivity. Scaffold-based assembly of PyrF, PyrHD93A, NudG, and PHM8sce using CipA/CipB further improved local pathway channeling and reduced uridine formation by 98.57%. To reinforce upstream flux, de novo UMP biosynthesis was reconstructed with the B. subtilis pyr operon, PRPP synthesis was enhanced, and nitrogen-donor and pyrimidine-ring precursor supply was improved through glnA, ppc, and aspC expression. Finally, an ATP-responsive ydaO motif regulatory module was used to dynamically control respiratory-chain and oxygen-utilization modules, coordinating ATP supply with NAD+ regeneration. The final strain cyt 23 produced 96.20 g/L cytidine in 50 h in a 5 L fed-batch bioreactor, with a glucose conversion yield of 0.38 g/g and productivity of 1.924 g/(L·h), representing the highest reported titer, yield and productivity for microbial cytidine production to date. This work provides an integrated strategy for high-level microbial cytidine production and nucleoside cell-factory development.