RRC ID 35762
Author Mukai T, Hoshi H, Ohtake K, Takahashi M, Yamaguchi A, Hayashi A, Yokoyama S, Sakamoto K.
Title Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon.
Journal Sci Rep
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.
Volume 5
Pages 9699
Published 2015-5-18
DOI 10.1038/srep09699
PII srep09699
PMID 25982672
PMC PMC4434889
MeSH Codon, Terminator* Escherichia coli / genetics* Escherichia coli / metabolism* Escherichia coli Proteins / genetics Escherichia coli Proteins / metabolism Gene Knockout Techniques Genetic Engineering Genome, Bacterial Genomics Peptide Termination Factors / genetics Peptide Termination Factors / metabolism Protein Biosynthesis / genetics* Protein Processing, Post-Translational Recombinant Proteins
IF 3.998
Times Cited 53
DNA material B95. delta A (RDB13711) B95. delta A delta fabR (RDB13712).