title: Replacement of 2'-Deoxycytidine by 2'-Deoxycytidine Analogues in the E. coli Genome.
abstract: 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'-hydroxymethyl-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-hydroxymethyl-2'-deoxycytidine (5hmC) in the E. coli genome and approximately 71% replacement in plasmids. We further engineered the glucose metabolic pathway to transform the 5hmC into glucosyl-5-hydroxymethyl-2'-deoxycytidine (5-gmC) and achieved 20% 5-gmC in the genome and 45% 5-gmC in plasmid DNA.
authors.affiliation: The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Angad P
authors.initials: AP
authors.lastname: Mehta
authors.affiliation: The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Han
authors.initials: H
authors.lastname: Li
authors.affiliation: The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Sean A
authors.initials: SA
authors.lastname: Reed
authors.affiliation: The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Lubica
authors.initials: L
authors.lastname: Supekova
authors.affiliation: The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Tsotne
authors.initials: T
authors.lastname: Javahishvili
authors.affiliation: The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
mesh_terms: D017122:Bacteriophage T4 / Q000235:genetics; D003841:Deoxycytidine / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics; D005818:Genetic Engineering; D016680:Genome, Bacterial / Q000235:genetics*
chemical_list: D003841:Deoxycytidine