scholarly journals Directed Evolution of the Methanosarcina barkeri Pyrrolysyl tRNA/aminoacyl tRNA Synthetase Pair for Rapid Evaluation of Sense Codon Reassignment Potential

2021 ◽  
Vol 22 (2) ◽  
pp. 895
Author(s):  
David G. Schwark ◽  
Margaret A. Schmitt ◽  
John D. Fisk
Keyword(s):  
Genetic Code ◽  
Directed Evolution ◽  
Trna Synthetase ◽  
Methanosarcina Barkeri ◽  
Rapid Evaluation ◽  
Codon Reassignment ◽  
Aminoacyl Trna Synthetase ◽  
E Coli ◽  
Orthogonal Pair ◽  
Sense Codon

Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber suppression to incorporate multiple copies of ncAAs in response to sense codons in Escherichia coli. Predicting which sense codons are most amenable to reassignment and which orthogonal translation machinery is best suited to each codon is challenging. This manuscript describes the directed evolution of a new, highly efficient variant of the Methanosarcina barkeri pyrrolysyl orthogonal tRNA/aaRS pair that activates and incorporates tyrosine. The evolved M. barkeri tRNA/aaRS pair reprograms the amber stop codon with 98.1 ± 3.6% efficiency in E. coli DH10B, rivaling the efficiency of the wild-type tyrosine-incorporating Methanocaldococcus jannaschii orthogonal pair. The new orthogonal pair is deployed for the rapid evaluation of sense codon reassignment potential using our previously developed fluorescence-based screen. Measurements of sense codon reassignment efficiencies with the evolved M. barkeri machinery are compared with related measurements employing the M. jannaschii orthogonal pair system. Importantly, we observe different patterns of sense codon reassignment efficiency for the M. jannaschii tyrosyl and M. barkeri pyrrolysyl systems, suggesting that particular codons will be better suited to reassignment by different orthogonal pairs. A broad evaluation of sense codon reassignment efficiencies to tyrosine with the M. barkeri system will highlight the most promising positions at which the M. barkeri orthogonal pair may infiltrate the E. coli genetic code.

scholarly journals A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes

2016 ◽  
Author(s):  
Stefanie Mühlhausen ◽  
Peggy Findeisen ◽  
Uwe Plessmann ◽  
Henning Urlaub ◽  
Martin Kollmar
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Amino Acid Sequences ◽  
Codon Reassignment ◽  
Proteomics Data ◽  
Pachysolen Tannophilus ◽  
Sequence Comparisons ◽  
Codon Capture ◽  
Cellular Translation ◽  
Sense Codon

AbstractThe genetic code is the universal cellular translation table to convert nucleotide into amino acid sequences. Changes to sense codons are expected to be highly detrimental. However, reassignments of single or multiple codons in mitochondria and nuclear genomes demonstrated that the code can evolve. Still, alterations of nuclear genetic codes are extremely rare leaving hypotheses to explain these variations, such as the ‘codon capture’, the ‘genome streamlining’ and the ‘ambiguous intermediate’ theory, in strong debate. Here, we report on a novel sense codon reassignment inPachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons we show that inPachysolenCUG codons are translated as alanine and not as the universal leucine. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by atRNA loss driven codon reassignmentmechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is outside the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects.

Directed evolution of an improved aminoacyl‐tRNA synthetase for incorporation of L‐3,4‐dihydroxyphenylalanine (L‐DOPA)

2021 ◽  
Author(s):  
Ross Thyer ◽  
Simon d’Oelsnitz ◽  
Molly S. Blevins ◽  
Dustin R. Klein ◽  
Jennifer S. Brodbelt ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase

scholarly journals Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 255 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yaroslav Andreev
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Nonsense Suppression ◽  
Expression Vectors ◽  
Site Specific ◽  
Suppressor Trna ◽  
E Coli ◽  
Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

scholarly journals Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 546 ◽  
Author(s):  
David Schwark ◽  
Margaret Schmitt ◽  
John Fisk
Keyword(s):  
Stop Codon ◽  
Release Factor ◽  
Codon Reassignment ◽  
Methanocaldococcus Jannaschii ◽  
E Coli ◽  
Amber Stop Codon ◽  
Orthogonal Pair ◽  
Amber Codon ◽  
Quantitative Contribution ◽  
Stop Codon Reassignment

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.

scholarly journals Discovery of a Novel Class of Boron-Based Antibacterials with Activity against Gram-Negative Bacteria

2013 ◽  
Vol 57 (3) ◽  
pp. 1394-1403 ◽  
Author(s):  
Vincent Hernandez ◽  
Thibaut Crépin ◽  
Andrés Palencia ◽  
Stephen Cusack ◽  
Tsutomu Akama ◽  
...  
Keyword(s):  
Resistance Mechanisms ◽  
Trna Synthetase ◽  
Gram Negative Bacteria ◽  
Aminoacyl Trna Synthetase ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Drug Classes ◽  
Medical Need ◽  
Infection Models

ABSTRACTGram-negative bacteria cause approximately 70% of the infections in intensive care units. A growing number of bacterial isolates responsible for these infections are resistant to currently available antibiotics and to many in development. Most agents under development are modifications of existing drug classes, which only partially overcome existing resistance mechanisms. Therefore, new classes of Gram-negative antibacterials with truly novel modes of action are needed to circumvent these existing resistance mechanisms. We have previously identified a new a way to inhibit an aminoacyl-tRNA synthetase, leucyl-tRNA synthetase (LeuRS), in fungi via the oxaborole tRNA trapping (OBORT) mechanism. Herein, we show how we have modified the OBORT mechanism using a structure-guided approach to develop a new boron-based antibiotic class, the aminomethylbenzoxaboroles, which inhibit bacterial leucyl-tRNA synthetase and have activity against Gram-negative bacteria by largely evading the main efflux mechanisms inEscherichia coliandPseudomonas aeruginosa. The lead analogue, AN3365, is active against Gram-negative bacteria, includingEnterobacteriaceaebearing NDM-1 and KPC carbapenemases, as well asP. aeruginosa. This novel boron-based antibacterial, AN3365, has good mouse pharmacokinetics and was efficacious againstE. coliandP. aeruginosain murine thigh infection models, which suggest that this novel class of antibacterials has the potential to address this unmet medical need.

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