Multistep construction of metabolically engineered Escherichia coli for enhanced cytidine biosynthesis

Abstract As a kind of pyrimidine nucleoside, cytidine has been widely recognized as an important precursor for the production of some antiviral and antitumor drugs. In this study, the systematic construction of metabolically engineered Escherichia coli strain for cytidine over-production was investigated, where the regulation of downstream pathway from uridine monophosphate (UMP) toward cytidine was focused on. Firstly, the engineered strain CR016 obtained by deleting the cytidine catabolism-related genes in E. coli MG1655 showed a significantly slower degradation rate of cytidine. Further modifications were carried out to amplify the metabolic flux from UMP to cytidine. The resulted strain CR023(pTrc03) began to accumulate 352.59 mg/L of cytidine in shake flask culture for 36 h. After optimizing the supply of three precursors, the final engineered strain CR023(pTrc03/pBAD021) produced 704.19 mg/L of cytidine with a yield of 0.15 g/g glucose, upon the supplementation with 2 mM glutamate. A fed-batch fermentation was then performed in a 5-L reactor to increase cytidine titer by 11.13-fold to 7.84 g/L in 48 h. The multistep metabolic engineering strategies presented here enabled a stepwise increase in the production of cytidine.