Upgrading aminoacyl-tRNA synthetases for genetic code expansion
Introduction
All living organisms synthesize proteins using the same set of twenty canonical amino acids (AAs) according to the rules of the genetic code. Over the past two decades, efforts to change these rules have resulted in the co-translational incorporation of numerous structurally diverse non-canonical AAs (ncAAs) into proteins by codon reassignment. Genetic code expansion (GCE) is now possible in a wide range of model organisms, which has facilitated investigation of protein structure and function in various biological processes, as well as development of therapeutics and the biocontainment of genetically modified organisms.
Expanding the genetic code entails altering two fundamental steps in translation: synthesis of aminoacyl-tRNAs (AA-tRNAs), and codon-anticodon pairing in the ribosome [1••]. This is achieved by repurposing an aminoacyl-tRNA synthetase (aaRS) to ligate the desired ncAA to a dedicated tRNA capable of inserting the ncAA at a defined codon (Figure 1a). For correct in vivo incorporation of ncAAs, engineered aaRS•tRNA pairs should not cross-react with the host aaRSs and tRNAs (i.e. they should be orthogonal), a requirement often met by relying on aaRS•tRNA pairs from organisms belonging to different domains of life.
Although over 200 ncAAs have been incorporated into proteins, engineered orthogonal aaRSs (o-aaRSs) usually have lower catalytic activity than their parental aaRSs, yet they maintain their AA polyspecificity (Figure 1b). These characteristics have hampered the development of more sophisticated applications (e.g. mass production of ncAA-containing proteins, multi-site incorporation of an ncAA, simultaneous incorporation of various ncAAs into a protein, or sense codon reassignment). Here we review fundamental aspects of aaRS specificity and efficiency and discuss recent advances in the development and discovery of superior engineered aaRSs that can contribute to overcoming the current limitations of GCE.
Section snippets
Fundamentals of aaRS substrate specificity
Each canonical AA has its cognate aaRS. This ensures accurate genetic code interpretation by specific pairing of AAs with tRNAs with the correct anticodon (Figure 1a). aaRSs select their cognate tRNAs through a defined set of nucleotides known as identity elements [2]. The majority of these elements are located in the anticodon loop and the acceptor stem of the tRNA, but the specific positions are unique to each tRNA for a particular AA [2]. Specific interactions with the anticodon are
AaRS•tRNA pairs for GCE applications
Over two-thirds of all ncAAs incorporated to date are mediated by the wild-type or engineered Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS)•tRNATyr pair or the Methanosarcina PylRS•tRNAPyl pair (Table 1). These pairs are now standard tools for GCE applications because of their robust orthogonality in Escherichia coli and the tolerance of both aaRSs to anticodon mutations in their tRNAs. These features simplify the engineering of PylRS and TyrRS for a desired ncAA by in vivo
Efficiency of o-aaRS•tRNA pairs
The lower catalytic activity of evolved o-aaRSs results in modest concentrations of ncAA-tRNAs. This impedes successful incorporation of ncAAs into multiple positions within one protein, and prevents efficient competition with endogenous AA-tRNAs for reassignment of sense codons. High-quality libraries of diverse aaRS variants are paramount to obtaining highly active and ncAA-specific aaRSs (Figure 1c). Oligo-directed libraries depend on the availability of high-resolution crystal structures of
Polyspecificity of o-aaRS•tRNA pairs
As described above, many aaRSs are polyspecific (i.e. they can charge multiple ncAAs), which can be exploited to incorporate ncAAs without altering the active site of the enzyme. Wild-type PylRS has a remarkably large AA binding site that accommodates >20 lysine derivatives [34•] (Table 1). This inherent polyspecificity is also displayed by engineered o-aaRSs. For example, one engineered PylRS mutant was shown to acylate at least 15 different phenylalanine (Phe) analogs [35]. The ncAA
Acylation of amino acids with non-canonical backbones
Recent characterization and engineering of translation apparatus components have shown that genetic incorporation of ncAAs with structures beyond the l-α structure is possible in vitro [40•,41]. These efforts have relied on the synthesis of ncAA-tRNAs using alternative in vitro aminoacylation techniques such as flexizyme [42]. Although wild-type aaRSs were known to acylate l-β-AAs and d-α-AAs [43,44] and can facilitate the incorporation l-β-Phe in vivo [45], the recombinant protein yield was
Codon reassignment and development of mutually orthogonal o-aaRS•tRNA pairs
One of the current challenges in GCE is the incorporation of multiple different ncAAs into a single protein. This task faces two difficulties: the need for mutually orthogonal o-aaRS•tRNA pairs (Table 1, [47, 48, 49]) and emancipation of more codons that can be reassigned with ncAAs. While UAG-codon and rare sense codon recoded strains [50, 51, 52, 53] improve the incorporation of individual ncAAs in response to reassigned codons, additional efforts are needed to make these strains amenable to
Outlook
Discovery of new orthogonal aaRS•tRNA pairs through bioinformatics analyses combined with innovative techniques for directed evolution allows selection of o-aaRSs variants with high catalytic efficiency and specificity. This will advance the application of orthogonal translation systems in cutting-edge research, such as biocontainment of synthetic organisms with rewired genomes [53,57,63] or expanded genetic alphabets [61••], production of avirulent, yet fully infectious viruses for effective
Conflict of interest statement
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank Kyle Hoffman, Hui Si Kwok, Anna Merkuryev, Takahito Mukai and Noah Reynolds for critical reading of the manuscript. Research in the Söll laboratory is supported by grants from the US National Institutes of Health (NIH) (R35GM122560), the US National Science Foundation (CHE-1740549) and from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the Department of Energy (DE-FG02-98ER20311) to D.S.
References (81)
- et al.
Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure
EMBO J
(1990) - et al.
Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center
Bioorg Med Chem
(2013) - et al.
Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli
Nucleic Acids Res
(2015) - et al.
Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome
Nature
(2010) - et al.
Design, synthesis, and testing toward a 57-codon genome
Science
(2016) - et al.
Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair
J Am Chem Soc
(2010) - et al.
Breaking the degeneracy of the genetic code
J Am Chem Soc
(2003) - et al.
Rewriting the genetic code
Annu Rev Microbiol
(2017) - et al.
Universal rules and idiosyncratic features in tRNA identity
Nucleic Acids Res
(1998) - et al.
Pyrrolysyl-tRNA synthetase-tRNAPyl structure reveals the molecular basis of orthogonality
Nature
(2009)
Quality control and infiltration of translation by amino acids outside of the genetic code
Annu Rev Genet
Quality control despite mistranslation caused by an ambiguous genetic code
Proc Natl Acad Sci U S A
Biosynthesis by Escherichia coli of active altered proteins containing selenium instead of sulfur
Biochim Biophys Acta
In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons
Nat Neurosci
Expanding the genetic code of Salmonella with non-canonical amino acids
Sci Rep
Development of an unnatural amino acid incorporation system in the actinobacterial natural product producer Streptomyces venezuelae ATCC 15439
ACS Synth Biol
Genetic incorporation of unnatural amino acids into proteins in Mycobacterium tuberculosis
PLoS ONE
An expanded eukaryotic genetic code
Science
Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase
Nat Chem Biol
Expanded genetic code technologies for incorporating modified lysine at multiple sites
Chembiochem
De novo enzyme design using Rosetta3
PLoS ONE
Directed evolution of protein catalysts
Annu Rev Biochem
Progress toward the evolution of an organism with an expanded genetic code
Proc Natl Acad Sci U S A
Genomes by design
Nat Rev Genet
Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids
Nat Biotechnol
Methods for the directed evolution of proteins
Nat Rev Genet
Development of potent in vivo mutagenesis plasmids with broad mutational spectra
Nat Commun
Continuous directed evolution of aminoacyl-tRNA synthetases
Nat Chem Biol
Photoactivatable mussel-based underwater adhesive proteins by an expanded genetic code
Chembiochem
Evolving tRNASec for efficient canonical incorporation of selenocysteine
J Am Chem Soc
Expanding the genetic code of Escherichia coli with phosphoserine
Science
Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog
Nat Chem Biol
Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET
Nat Chem
Exploring the substrate range of wild-type aminoacyl-tRNA synthetases
Chembiochem
Engineering the elongation factor Tu for efficient selenoprotein synthesis
Nucleic Acids Res
Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment
Nucleic Acids Res
Effects of heterologous tRNA modifications on the production of proteins containing noncanonical amino acids
Bioengineering (Basel)
Directed evolution of heterologous tRNAs leads to reduced dependence on post-transcriptional modifications
ACS Synth Biol
Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing
Nat Methods
Designing logical codon reassignment — Expanding the chemistry in biology
Chem Sci
Cited by (82)
Site-specific protein labeling strategies for super-resolution microscopy
2024, Current Opinion in Chemical BiologyExpanding the chemical repertoire of protein-based polymers for drug-delivery applications
2022, Advanced Drug Delivery ReviewsCitation Excerpt :Apart from very specific cases where the uAA is a natural substrate for one of the imported aaRSs·tRNA pairs (such as pyrrolysine, the natural substrate of the PylRS), the AA-binding pocket of the aaRS must undergo mutagenesis to accommodate the desired uAA. This process entails the production of a library of aaRS mutants and the application of both negative and positive selection to the diversified aaRS population, so as to eliminate non-orthogonal mutants and enrich for the best-performing variants, respectively [92,95]. First-generation aaRS variants typically permit the incorporation of one or a few uAAs per protein [95,102], while advanced protein evolution techniques [103,104] and computationally guided library designs [105,106] have recently enabled the selection of more efficient aaRS variants, capable of multi-site uAA incorporation and high-yield protein production.
Photocatalytic Reductive Olefin Hydrodifluoroalkylation Enabled by Tertiary Amine Reductants Compatible with Complex Systems
2022, Journal of Organic ChemistryWhole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria
2022, Journal of Biological ChemistryChemical insights into flexizyme-mediated tRNA acylation
2022, Cell Chemical Biology