Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase

Beyer, Jenna N.; Hosseinzadeh, Parisa; Gottfried-Lee, Ilana; Fossen, Elise M. Van; Zhu, Phillip; Bednar, Riley M.; Karplus, P.A.; Mehl, Ryan A.; Cooley, Richard B.

doi:10.1016/j.jmb.2020.06.014

Cited by 25 publications

(45 citation statements)

References 39 publications

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“…One strain-dependent complication of GCE is misincorporation of an endogenous ⍺-amino acid in place of the desired ncAA. [42][43][44] Many factors can influence the misincorporation rate, including inefficient transport of the ncAA into cells, imperfect specificity of the aaRS enzyme for the ncAA over natural ⍺-amino acids, differences in EF-Tu-mediated delivery of the mis-acylated tRNA to the ribosome, and slower rates of accommodation, elongation, or translocation of mis-acylated tRNAs. 43,64,65 Misincorporation at an amber stop codon can also arise from near-cognate suppression, in which natural (and correctly acylated) tRNAs pair imperfectly with a UAG codon and direct the incorporation of one or more natural ⍺-amino acids in place of the ncAA.…”

Section: Resultsmentioning

confidence: 99%

“…[39][40][41] While these strains can improve expression yields, genomically recoded organisms that lack RF1 suffer from fitness defects as well as higher levels of misincorporation events when utilizing suboptimal aaRS/tRNA pairs. [42][43][44][45] Cell-free translation systems offer the opportunity to omit RF1 as well as tune the individual levels of near-cognate tRNAs to potentially increase incorporation fidelity, but these systems are significantly more costly than cellular bioproduction. 46,47 Further challenges arise when high-yield expression of the target protein in E. coli demands low temperatures.…”

Section: Introductionmentioning

confidence: 99%

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Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

Santiago¹,

Shah

et al. 2021

Preprint

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We report that the recently introduced commercial strain of V. natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 non-canonical L-amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than 10-fold higher than those achieved using two different genomically recoded E. coli strains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.ΔA.opt and C321.ΔA.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of mis-incorporated natural L-amino acids at the UAG-programmed position, especially in cases where the ncAA is an imperfect substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve specific activity of highly desirable but imperfect ncAA substrates.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

Santiago¹,

Shah

et al. 2021

Preprint

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“…The interest in site-specifically installed ncAAs has fueled continuous efforts to create and optimize OTSs. Even for the commonly catalytically superior Mj TyrRS systems, enzyme and host strain engineering efforts continue to improve target production and amber suppression background [ 63 ]. For PylRS, the accumulated literature allowed us to test if previously reported mutations also act on the Bta-specific enzyme created herein.…”

Section: Discussionmentioning

confidence: 99%

“…It focuses on the combined mutagenesis of first-shell and spatially distant residues, the latter for example belonging to the tRNA binding domain [ 34 , 36 , 37 ]. In this light, combination with computational approaches should lead to aaRS variants with specificities towards synthetic substrates and activity levels comparable to the wild-type aaRS [ 63 ]. This will significantly expand the chemical space and scope of orthogonal translation with numerous aromatic ncAAs useful in biophysics, cell and synthetic biology [ 53 , 64 ].…”

Section: Discussionmentioning

confidence: 99%

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

et al. 2020

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In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as β-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation

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“…To prevent near‐cognate amber codon suppression, orthogonal AARS‐tRNA pair should have high efficiency and fidelity. Researchers screened active site residues of 3‐nitroY AARS using Rosetta software and selected two residues (C70 and S158) to randomize by saturation mutagenesis [34] . The positive clone A7 (containing C70T and S158H) was obtained after several rounds of selection.…”

Section: Approaches To Expand the Noncanonical Amino Acid Repertoirementioning

confidence: 99%

Noncanonical Amino Acids in Synthetic Biosafety and Post‐translational Modification Studies

Gang

Park

2020

ChemBioChem

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The incorporation of noncanonical amino acids (ncAAs) has been extensively studied because of its broad applicability. In the past decades, various in vitro and in vivo ncAA incorporation approaches have been developed to generate synthetic recombinant proteins. Herein, we discuss the methodologies for ncAA incorporation, and their use in diverse research areas, such as in synthetic biosafety and for studies of post‐translational modifications.

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Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase

Cited by 25 publications

References 39 publications

Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

Noncanonical Amino Acids in Synthetic Biosafety and Post‐translational Modification Studies

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