Uridine and cytidine transport in Escherichia coli B and transport-deficient mutants. Leung K K,Visser D W The Journal of biological chemistry Three mutants of Escherichia coli B which are defective in components of the transport system for uridine and uracil were isolated and utilized to study the mechanism of uridine transport. Mutant U- was isolated from a culture resistant to 77 micronM 5-fluorouracil. Mutant U-UR-, isolated from a culture of mutant U-, is resistant to 770 micronM 5-fluorouracil and 750 micronM adenosine. Mutant NUC- is resistant to 80 micronM showdomycin and has been reported previously. The characteristics of uridine transport by E. coli B and the mutants provide data supporting the following conclusions. The transport of adenosine, deoxyadenosine, guanosine, deoxyguanosine, adenine, or guanine by mutant U- and mutant U-UR- is identical with that in the parental strain. Uridine is transported by E. coli B as intact uridine. In addition, extracellular uridine is also rapidly cleaved to uracil and the ribose moiety. The latter is transported into the cells, whereas uracil appears in the medium and is transported by a separate uracil transport system. The entry of the ribose moiety of uridine is fast relative to the uracil and uridine transport processes. The Km values and the inhibitory effects of heterologous nucleosides for the transport of uridine and the ribose moiety of uridine are similar. Studies of cytidine uptake in the parental and mutant strains provide evidence that cytidine is transported by two independent systems, one of which is the same as that involved in the transport of intact uridine. Uridine inhibits but is not transported by the other system for cytidine transport. Evidence for the above conclusions was based on comparisons of the characteristics of [2-14C]uridine, [U-14C]uridine, and [2-14C]cytidine transport using E. coli B and the three transport mutants under conditions which measure initial rates. The nature of the inhibitory effects of heterologous nucleosides on the uridine transport processes and identification of extracellular components from radioactive uridine provides supportive data for the conclusions.

Computer-Based Design of a Cell Factory for High-Yield Cytidine Production. ACS synthetic biology Pyrimidine ribonucleotide de novo biosynthesis pathway (PRdnBP) is an important pathway to produce pyrimidine nucleosides. We attempted to systematically investigate PRdnBP in with genome-scale metabolic models and utilized the models to guide strain design. The balance of central carbon metabolism and PRdnBP affected the production of cytidine from glucose. Using Bayesian metabolic flux analysis, the effect of modified PRdnBP on the metabolic network was analyzed. The acetate overflow became coupled with PRdnBP flux, while they were originally independent under oxygen-sufficient conditions. The coupling between cytidine production and acetate secretion in the modified strain was weakened by deletion, which resulted in further improving the efficient accumulation of cytidine. In total, 1.28 g/L of cytidine with a yield of 0.26 g/g glucose was produced. The yield of cytidine produced by is higher than previous reports. Our strategy provides an effective attempt to find metabolic bottlenecks in genetically engineered bacteria by using flux coupling analysis. 10.1021/acssynbio.2c00431

Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15. Fang Haitian,Xie Xixian,Xu Qingyang,Zhang Chenglin,Chen Ning Biotechnology letters Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-α-D-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735 mg cytidine/l using glucose as substrate, which was approx. 128 % higher than the cytidine production by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2 %) slightly and increased glucose uptake (72 %). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli. 10.1007/s10529-012-1068-3