Abstract
In Escherichia coli, cofactor imbalance serves as a crucial limiting factor in cytidine biosynthesis, with nicotinamide adenine dinucleotide phosphate (NADPH) insufficiency representing the principal metabolic barrier. To overcome this limitation, an integrated engineering strategy targeting the enhancement of NADPH metabolism was implemented. Via CRISPR-Cas9-mediated multiplex genomic editing and strong constitutive promoter replacement, three NADPH-regenerating modules were concurrently enhanced: the membrane-bound transhydrogenase (pntAB), the oxidative pentose phosphate pathway (zwf-encoded glucose-6-phosphate dehydrogenase), and the decarboxylation shunt (gnd-encoded 6-phosphogluconate dehydrogenase). After 54-hour fermentation in 500 mL shake flasks, the cytidine titer of the engineered strain NXBG-20 reached 7.83 g/L, representing a 9.10-fold increase compared to the start strain. Systematic multi-omics profiling revealed that the metabolic network had undergone substantial alterations. These alterations were characterized by the redirection of glycolytic flux towards nucleotide precursor substances and the enhancement of ribose-5-phosphate biosynthesis. This engineering approach not only establishes a novel microbial platform for cytidine bioproduction but also provides mechanistic insights into cofactor-driven metabolic flux control.