Dataset Information

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli.

ABSTRACT: The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to L-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize L-homoarginine and L-N(6)-(1-iminoethyl)lysine (L-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA(Pyl) CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host's ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to L-homoarginine or L-NIL. The assignment of AGG to L-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.

SUBMITTER: Mukai T

PROVIDER: S-EPMC4652775 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli.

Mukai Takahito T Yamaguchi Atsushi A Ohtake Kazumasa K Takahashi Mihoko M Hayashi Akiko A Iraha Fumie F Kira Satoshi S Yanagisawa Tatsuo T Yokoyama Shigeyuki S Hoshi Hiroko H Kobayashi Takatsugu T Sakamoto Kensaku K

Nucleic acids research 20150803 16

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to L-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize L-homoarginine and L-N(6)-(1-iminoethyl)lysine (L-NIL). When this PylRS variant or HarRS was expressed ...[more]

PMID: 26240376

Similar Datasets

Project description:BackgroundEscherichia coli is one of the most important hosts for production of recombinant proteins in biopharmaceutical industry. However, when selecting a suitable production strain, it is often not considered that a lot of different sub-species exist, which can differ in their genotypes and phenotypes. Another important development step is the scale-up of bioprocesses with the particular challenge that heterogeneities and gradients occur at production scale. These in turn can affect the production organism and can have negative impact on the process and the product quality. Therefore, researchers developed scale-down reactors, which are used to mimic manufacturing conditions in laboratory scale. The main objectives of this study were to determine the extent to which scale-related process inhomogeneities affect the misincorporation of non-canonical amino acids into the recombinant target protein, which is an important quality attribute, and whether strain specific properties may have an impact.ResultsWe investigated two industrially relevant E. coli strains, BL21(DE3) and HMS174(DE3), which produced an antigen binding fragment (Fab). The cells were cultivated in high cell density fed-batch mode at laboratory scale and under scale-down conditions. We demonstrated that the two host strains differ significantly with respect to norleucine misincorporation into the target protein, especially under heterogeneous cultivation conditions in the scale-down reactor. No norleucine misincorporation was observed in E. coli BL21(DE3) for either cultivation condition. In contrast, norleucine incorporation into HMS174(DE3) was already detectable in the reference process and increased dramatically in scale-down experiments. Norleucine incorporation was not random and certain positions were preferred over others, even though only a single codon exists. Differences in biomass and Fab production between the strains during scale-down cultivations could be observed as well.ConclusionsThis study has shown that E. coli BL21(DE3) is much more robust to scale-up effects in terms of norleucine misincorporation than the K12 strain tested. In this respect, BL21(DE3) enables better transferability of results at different scales, simplifies process implementation at production scale, and helps to meet regulatory quality guidelines defined for biopharmaceutical manufacturing.

Dataset Information

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli.

Publications

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets