Improving the level of the cytidine biosynthesis in E. coli through atmospheric room temperature plasma mutagenesis and metabolic engineering.

This article focuses on the development of a genetically engineered *Escherichia coli* strain for enhanced cytidine production, a key precursor in antiviral and antitumor drug synthesis. Using atmospheric room temperature plasma (ARTP) mutagenesis combined with high-throughput screening, the researchers obtained a mutant strain (E. coli NXBG-11-A379) with relieved uridine monophosphate (UMP) feedback inhibition and good genetic stability. Further metabolic engineering via CRISPR/Cas9-mediated knockout of the udk and rihA genes, which are involved in cytidine catabolism, led to the recombinant strain E. coli NXBG-13, achieving a cytidine titer of 15.7 g/L, yield of 0.164 g/g glucose, and productivity of 0.327 g/L/h in a 5-liter bioreactor. Transcriptome analysis revealed significant upregulation of genes in the pentose phosphate pathway and de novo cytidine biosynthesis, providing insights into the metabolic changes underlying improved cytidine accumulation.