Replacement Of 2 '-Deoxycytidine By 2 '-Deoxycytidine Analogues In The E. Coli Genome

Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences Of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'-hydroxyinethyl-2'-deoxyuridine (5hmU) in the Escherichia :coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'-deoxycytidine (dC) with 5-hydroxyinethyl-2'-deoxycytidine (5hmC) in the E. coli genome and approximately 71% replacement in plasmids: We further engineered the glucose metabolic pathway to transform the ShmC into glucosyl-5-hydroxymethyl-2'-deoxycytidine (5-gmC) and achieved 20% 5-gmC in the genome and 45% 5-gmC in plasmic DNA.