Reassigning Sense Codon AGA to Encode Noncanonical Amino Acids in <i>Escherichia coli</i>

Wang, Yiyan; Tsao, Meng-Lin

doi:10.1002/cbic.201600448

Cited by 7 publications

(6 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A third strategy to further expand the genetic code is to reassign one of the 61 sense codons that normally encode a canonical amino acid. , This strategy is particularly challenging due to competition with endogenous aminoacyl-tRNAs for suppression of sense codons . Because of this competition, sense codon reassignment most often affords a heterogeneous product containing a mixture of the canonical and noncanonical amino acids. ,− …”

Section: Introductionmentioning

confidence: 99%

Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

et al. 2021

View full text Add to dashboard Cite

We recently described an orthogonal initiator tRNA (itRNA Ty2 ) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here we report that a mutant of itRNA Ty2 (itRNA Ty2 AUA) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing itRNA Ty2 AUA, UAU can function as a dual-use codon that selectively encodes ncAAs at the initiating position and tyrosine at elongating positions. Using itRNA Ty2 AUA, in conjunction with its cognate tyrosyl-tRNA synthetase and two mutually orthogonal pyrrolysyl-tRNA synthetases, we demonstrate that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. Furthermore, by engineering the substrate specificity of one of the pyrrolysyl-tRNA synthetases, we developed a triply orthogonal system that enables simultaneous reassignment of UAU, UAG, and UAA to produce proteins containing three distinct ncAAs at precisely defined sites. To showcase the utility of this system, we produced proteins containing two or three ncAAs, with unique bioorthogonal functional groups, and demonstrate that these proteins can be separately modified with multiple fluorescent probes..

show abstract

Section: Introductionmentioning

confidence: 99%

Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

et al. 2021

View full text Add to dashboard Cite

show abstract

“…We selected the arginine AGG codon for initial evaluation of this directed evolution workflow because AGG has been the most common target for sense codon reassignment using variants of both the orthogonal pyrrolysyl and M. jannaschii tyrosyl tRNA/aaRS pairs ( Krishnakumar et al, 2013 ; Zeng et al, 2014 ; Lee et al, 2015 ; Mukai et al, 2015 ; Wang and Tsao, 2016 ). Zeng et al reported approximately 90% efficient reassignment using a variant of the Methanosarcina pyrrolysyl tRNA/aaRS pair and media in which the concentration of arginine was controlled ( Zeng et al, 2014 ).…”

Section: Resultsmentioning

confidence: 99%

“…Nonsense suppression technology has been improved through genome modifications, enabling the elimination of the release factor that typically competes to read the amber stop codon as a termination signal ( Mukai et al, 2010 ; Johnson et al, 2011 ; Lajoie et al, 2013 ). Amino acid-specific reassignment has been updated by breaking the degeneracy of the genetic code to enable the reassignment of individual sense codons ( Kwon et al, 2003 ; Bohlke and Budisa, 2014 ; Zeng et al, 2014 ; Lee et al, 2015 ; Mukai et al, 2015 ; Ho et al, 2016 ; Kwon and Choi, 2016 ; Wang and Tsao, 2016 ). Improvements in both methods have focused primarily on genetic additions and deletions rather than on functional improvements of the evolved orthogonal translation components central to both technologies.…”

Section: Introductionmentioning

confidence: 99%

Directed Evolution Pipeline for the Improvement of Orthogonal Translation Machinery for Genetic Code Expansion at Sense Codons

et al. 2022

View full text Add to dashboard Cite

The expansion of the genetic code beyond a single type of noncanonical amino acid (ncAA) is hindered by inefficient machinery for reassigning the meaning of sense codons. A major obstacle to using directed evolution to improve the efficiency of sense codon reassignment is that fractional sense codon reassignments lead to heterogeneous mixtures of full-length proteins with either a ncAA or a natural amino acid incorporated in response to the targeted codon. In stop codon suppression systems, missed incorporations lead to truncated proteins; improvements in activity may be inferred from increased protein yields or the production of downstream reporters. In sense codon reassignment, the heterogeneous proteins produced greatly complicate the development of screens for variants of the orthogonal machinery with improved activity. We describe the use of a previously-reported fluorescence-based screen for sense codon reassignment as the first step in a directed evolution workflow to improve the incorporation of a ncAA in response to the Arg AGG sense codon. We first screened a library with diversity introduced into both the orthogonal Methanocaldococcus jannaschii tyrosyl tRNA anticodon loop and the cognate aminoacyl tRNA synthetase (aaRS) anticodon binding domain for variants that improved incorporation of tyrosine in response to the AGG codon. The most efficient variants produced fluorescent proteins at levels indistinguishable from the E. coli translation machinery decoding tyrosine codons. Mutations to the M. jannaschii aaRS that were found to improve tyrosine incorporation were transplanted onto a M. jannaschii aaRS evolved for the incorporation of para-azidophenylalanine. Improved ncAA incorporation was evident using fluorescence- and mass-based reporters. The described workflow is generalizable and should enable the rapid tailoring of orthogonal machinery capable of activating diverse ncAAs to any sense codon target. We evaluated the selection based improvements of the orthogonal pair in a host genomically engineered for reduced target codon competition. Using this particular system for evaluation of arginine AGG codon reassignment, however, E. coli strains with genomes engineered to remove competing tRNAs did not outperform a standard laboratory E. coli strain in sense codon reassignment.

show abstract

“…MjTyrRS is lacking most of the non-conserved domain that binds to the anticodon loop of its cognate tRNA Tyr (Steer and Schimmel, 1999 ; Kobayashi et al, 2003 ). As a result it has some promiscuity toward tRNA's anticodon allowing it to be used not only for amber suppression, but also reassignment of opal, ochre , sense codons (AGA/U) (Wang and Tsao, 2016 ; Vargas-Rodriguez et al, 2018 ) as well as unnatural codons (A NaM C) (Fischer et al, 2020 ). Surprisingly, an engineered initiator tRNA fMet harboring one mutation in the acceptor stem (A72G) and two mutations in the anticodon nucleotides (A35U, U36A) is a substrate of the MjTyrRS for ncAA acylation (Tharp et al, 2019 ).…”

Section: Genetic Encoding Of Ncaasmentioning

confidence: 99%

Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides

Cui

Johnston

Alexandrov

2020

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Composition Crude extract (>500 proteins) + necessary factors 36 purified proteins+ tRNAs + ribosomes + necessary factors Energy source Versatile energy sources including variants that take advantage of cellular metabolism Creatine phosphate/creatine kinase ATP regeneration system Flexibility Partial control of translational machinery Full control of translational machinery Linear template Unstable, requires special strains, or lysate treatment Stable Peptide synthesis Unstable, requires special strains, or lysate treatment Stable Applications Small scale protein expression Peptide expression for mRNA display Large scale protein biomanufacturing Isotopically labeled peptides (MS standard) Biological process prototyping Translation factor characterization Compatibility with selection systems Ribosome display mRNA display Microbead display (in vitro compartmentation) cDNA display Amber suppression Frequently used Seldom used Initiation reassignment Seldom used Limited amino acid structures Commonly used A wide range of ncAAs (Figure 5) Elongator codon reassignment Less used Commonly used (residue-specific manner) A wide range of ncAAs (Figure 6)

show abstract

Reassigning Sense Codon AGA to Encode Noncanonical Amino Acids in Escherichia coli

Cited by 7 publications

References 46 publications

Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

Directed Evolution Pipeline for the Improvement of Orthogonal Translation Machinery for Genetic Code Expansion at Sense Codons

Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides

Contact Info

Product

Resources

About