title: Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon.
abstract: Escherichia coli is a widely used host organism for recombinant technology, and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific molecule recognizing them. The resulting strain B-95.ΔA grew as vigorously as BL21(DE3) in rich medium at 25-42°C, and its derivative B-95.ΔAΔfabR was better adapted to low temperatures and minimal media than B-95.ΔA. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.ΔA strains showed superior productivities for hirudin molecules sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin containing multiple azido groups.
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Takahito
authors.initials: T
authors.lastname: Mukai
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hoshi
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Ohtake
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Mihoko
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamaguchi
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Hayashi
authors.affiliation: 1] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
mesh_terms: D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D005818:Genetic Engineering; D016680:Genome, Bacterial; D023281:Genomics; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins
chemical_list: D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D011994:Recombinant Proteins; C489005:prfA protein, E coli