Upgrading aminoacyl-tRNA synthetases for genetic code expansion

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Synthesis of proteins with non-canonical amino acids via genetic code expansion is at the forefront of synthetic biology. Progress in this field has enabled site-specific incorporation of over 200 chemically and structurally diverse amino acids into proteins in an increasing number of organisms. This has been facilitated by our ability to repurpose aminoacyl-tRNA synthetases to attach non-canonical amino acids to engineered tRNAs. Current efforts in the field focus on overcoming existing limitations to the simultaneous incorporation of multiple non-canonical amino acids or amino acids that differ from the l-α-amino acid structure (e.g. d-amino acid or β-amino acid). Here, we summarize the progress and challenges in developing more selective and efficient 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.

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