Structural robustness affects the engineerability of aminoacyl-tRNA synthetases for genetic code expansion

AbstractThe ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The E. coli derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs has been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the lower structural robustness of EcTyrRS relative to MjTyrRS. We show that engineered TyrRS mutants in general exhibit significantly lower thermostability relative to their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homolog from a thermophilic bacteria, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.

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Structural Basis for Genetic-Code Expansion with Various Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

SSRN Electronic Journal ◽

10.2139/ssrn.3244022 ◽

2018 ◽

Cited By ~ 1

Author(s):

Tatsuo Yanagisawa ◽

Mitsuo Kuratani ◽

Eiko Seki ◽

Nobumasa Hino ◽

Kensaku Sakamoto ◽

...

Keyword(s):

Genetic Code ◽

Trna Synthetase ◽

Structural Basis ◽

Genetic Code Expansion

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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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Structural basis of nonnatural amino acid recognition by an engineered aminoacyl-tRNA synthetase for genetic code expansion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0407039102 ◽

2005 ◽

Vol 102 (5) ◽

pp. 1366-1371 ◽

Cited By ~ 29

Author(s):

T. Kobayashi ◽

K. Sakamoto ◽

T. Takimura ◽

R. Sekine ◽

V. P. Kelly ◽

...

Keyword(s):

Amino Acid ◽

Genetic Code ◽

Trna Synthetase ◽

Structural Basis ◽

Aminoacyl Trna Synthetase ◽

Genetic Code Expansion ◽

Nonnatural Amino Acid ◽

Amino Acid Recognition

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The Role of Orthogonality in Genetic Code Expansion

Life ◽

10.3390/life9030058 ◽

2019 ◽

Vol 9 (3) ◽

pp. 58 ◽

Cited By ~ 6

Author(s):

Pol Arranz-Gibert ◽

Jaymin R. Patel ◽

Farren J. Isaacs

Keyword(s):

Gene Expression ◽

Amino Acids ◽

Genetic Code ◽

Cross Reactivity ◽

Elongation Factors ◽

Translational Machinery ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

Genetic Code Expansion

The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.

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Correction for Kobayashi et al., Structural basis of nonnatural amino acid recognition by an engineered aminoacyl-tRNA synthetase for genetic code expansion, PNAS 2005 102:1366-1371

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0505010102 ◽

2005 ◽

Vol 102 (29) ◽

pp. 10405-10405

Keyword(s):

Amino Acid ◽

Genetic Code ◽

Trna Synthetase ◽

Structural Basis ◽

Aminoacyl Trna Synthetase ◽

Genetic Code Expansion ◽

Nonnatural Amino Acid ◽

Amino Acid Recognition

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High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

10.1101/2021.07.13.452272 ◽

2021 ◽

Author(s):

Jessica T. Stieglitz ◽

James A. Van Deventer

Keyword(s):

Genetic Code ◽

High Throughput ◽

Trna Synthetase ◽

Noncanonical Amino Acids ◽

Trna Synthetases ◽

E Coli ◽

Biological Studies ◽

Screening Strategies ◽

Genetic Code Expansion ◽

Translation Systems

Protein expression with genetically encoded noncanonical amino acids (ncAAs) benefits a broad range of applications, from the discovery of biological therapeutics to fundamental biological studies. A major factor limiting the use of ncAAs is the lack of orthogonal translation systems (OTSs) that support efficient genetic code expansion at repurposed stop codons. Aminoacyl-tRNA synthetases (aaRSs) have been extensively evolved in E. coli but are not always orthogonal in eukaryotes. In this work, we use a yeast display-based ncAA incorporation reporter platform with fluorescence-activated cell sorting (FACS) to screen libraries of aaRSs in high throughput for 1) incorporation of ncAAs not previously encoded in yeast; 2) improvement of the performance of an existing aaRS; 3) highly selective OTSs capable of discriminating between closely related ncAA analogs; and 4) OTSs exhibiting enhanced polyspecificity to support translation with structurally diverse sets of ncAAs. The number of previously undiscovered aaRS variants we report in this work more than doubles the total number of translationally active aaRSs available for genetic code manipulation in yeast. The success of myriad screening strategies has important implications related to the fundamental properties and evolvability of aaRSs. Furthermore, access to OTSs with diverse activities and specific/polyspecific properties are invaluable for a range of applications within chemical biology, synthetic biology, and protein engineering.

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