Hijacking a bacterial ABC transporter for genetic code expansion.

The incorporation of non-canonical amino acids (ncAAs) using engineered aminoacyl-tRNA synthetases (aaRSs) has emerged as a powerful methodology to expand the chemical repertoire of proteins. However, the low efficiencies of typical aaRS variants limit the incorporation of ncAAs to only one or a few sites within a protein chain, hindering the design of protein-based polymers (PBPs) in which multi-site ncAA incorporation can be used to impart new properties and functions. Here, we determined the substrate specificities of 11 recently developed high-performance aaRS variants and identified those that enable an efficient multi-site incorporation of 15 different aromatic ncAAs. We used these aaRS variants to produce libraries of two temperature-responsive PBPs—elastin- and resilin-like polypeptides (ELPs and RLPs, respectively)—that bear multiple instances of each ncAA. We show that incorporating such aromatic ncAAs into the protein structure of ELPs and RLPs can affect their temperature responsiveness, secondary structure, and self-assembly propensity, yielding new and diverse families of ELPs and RLPs, each from a single DNA template. Finally, using a molecular model, we demonstrate that the temperature-responsive behavior of RLPs is strongly affected by both the hydrophobicity and the size of the unnatural aromatic side-chain. The ability to efficiently incorporate multiple instances of diverse ncAAs alongside the 20 natural amino acids can help to elucidate the effect of ncAA incorporation on these and many other PBPs, with the aim of designing additional precise and chemically diverse polymers with new or improved properties.

Supplementing translation with noncanonical amino acids (ncAAs) can yield protein sequences with new-to-nature functions but existing ncAA incorporation strategies suffer from low efficiency and context dependence. We uncover codon usage as a previously unrecognized contributor to efficient genetic code expansion using non-native codons. Relying only on conventional Escherichia coli strains with native ribosomes, we develop a plasmid-based codon compression strategy that minimizes context dependence and improves ncAA incorporation at quadruplet codons. We confirm that this strategy is compatible with all known genetic code expansion resources, which allowed us to identify 12 mutually orthogonal transfer RNA (tRNA)-synthetase pairs. Enabled by these findings, we evolved and optimized five tRNA-synthetase pairs to incorporate a broad repertoire of ncAAs at orthogonal quadruplet codons. Lastly, we extend these resources to an in vivo biosynthesis platform that can readily create >100 new-to-nature peptide macrocycles bearing up to three unique ncAAs. Our approach will accelerate innovations in multiplexed genetic code expansion and the discovery of chemically diverse biomolecules.

The pyrrolysyl-tRNA synthetase/tRNAPyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNAPyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNAPyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.

The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.

Supplementing translation with non-canonical amino acids (ncAAs) can yield protein sequences with new-to-nature functions, but existing ncAA incorporation strategies suffer from low efficiency and context dependence. We uncover codon usage as a previously unrecognized contributor to efficient genetic code expansion using non-native codons. Relying only on conventionalE. colistrains with native ribosomes, we develop a novel plasmid-based codon compression strategy that minimizes context dependence and improves ncAA incorporation at quadruplet codons. We confirm that this strategy is compatible with all known genetic code expansion resources, which allows us to identify 12 mutually orthogonal tRNA–synthetase pairs. Enabled by these findings, we evolve and optimize five tRNA–synthetase pairs to incorporate a broad repertoire of ncAAs at orthogonal quadruplet codons. Finally, we extend these resources to anin vivobiosynthesis platform that can readily create >100 new-to-nature peptide macrocycles bearing up to three unique ncAAs. Given the generality of our approach and streamlined resources, our findings will accelerate innovations in multiplexed genetic code expansion and enable the discovery of chemically diverse biomolecules for researcher-defined applications.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications.

The emergence of antimicrobial resistance is a global health concern and calls for the development of novel antibiotic agents. Antimicrobial peptides seem to be promising candidates due to their diverse sources, mechanisms of action, and physicochemical characteristics, as well as the relatively low emergence of resistance. The incorporation of noncanonical amino acids into antimicrobial peptides could effectively improve their physicochemical and pharmacological diversity. Recently, various antimicrobial peptides variants with improved or novel properties have been produced by the incorporation of single and multiple distinct noncanonical amino acids. In this review, we summarize strategies for the incorporation of noncanonical amino acids into antimicrobial peptides, as well as their features and suitabilities. Recent applications of noncanonical amino acid incorporation into antimicrobial peptides are also presented. Finally, we discuss the related challenges and prospects.

Genetic code expansion is a powerful approach for advancing critical fields such as biological therapeutic discovery. However, the machinery for genetically encoding noncanonical amino acids (ncAAs) is only available in limited plasmid formats, constraining potential applications. In extreme cases, the introduction of two separate plasmids, one containing an orthogonal translation system (OTS) to facilitate ncAA incorporation and a second for expressing a ncAA-containing protein of interest, is not possible due to a lack of the available selection markers. One strategy to circumvent this challenge is to express the OTS and protein of interest from a single vector. For what we believe is the first time in yeast, we describe here several sets of single plasmid systems (SPSs) for performing genetic code manipulation and compare the ncAA incorporation capabilities of these plasmids against the capabilities of previously described dual plasmid systems (DPSs). For both dual fluorescent protein reporters and yeast display reporters tested with multiple OTSs and ncAAs, measured ncAA incorporation efficiencies with SPSs were determined to be equal to efficiencies determined with DPSs. Click chemistry on yeast cells displaying ncAA-containing proteins was also shown to be feasible in both formats, although differences in reactivity between formats suggest the need for caution when using such approaches. Additionally, we investigated whether these reporters would support the separation of yeast strains known to exhibit distinct ncAA incorporation efficiencies. Model sorts conducted with mixtures of two strains transformed with the same SPS or DPS both led to the enrichment of a strain known to support a higher efficiency ncAA incorporation, suggesting that these reporters will be suitable for conducting screens for strains exhibiting enhanced ncAA incorporation efficiencies. Overall, our results confirm that SPSs are well behaved in yeast and provide a convenient alternative to DPSs. SPSs are expected to be invaluable for conducting high-throughput investigations of the effects of genetic or genomic changes on ncAA incorporation efficiency and, more fundamentally, the eukaryotic translation apparatus.

Genetic code expansion (GCE) can enable the site-selective incorporation of non-canonical amino acids (ncAAs) into proteins. GCE has advanced tremendously in the last decade and can be used to create biorthogonal handles, monitor and control proteins inside cells, study post-translational modifications, and engineer new protein functions. Since establishing our laboratory, our research has focused on applications of GCE in protein and enzyme engineering using aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. This topic has been reviewed extensively, leaving little doubt that GCE is a powerful tool for engineering proteins and enzymes. Therefore, for this young faculty issue, we wanted to provide a more technical look into the methods we use and the challenges we think about in our laboratory. Since starting the laboratory, we have successfully engineered over a dozen novel aaRS/tRNA pairs tailored for various GCE applications. However, we acknowledge that the field can pose challenges even for experts. Thus, herein, we provide a review of methodologies in ncAA incorporation with some practical commentary and a focus on challenges, emerging solutions, and exciting developments.

A facile method for high level dual expression of recombinant and congener protein in a single expression system

Site-specific incorporation of noncanonical amino acids (ncAAs) can be realized by genetic code expansion (GCE) technology. Different orthogonal tRNA synthetase/tRNA (RS/tRNA) pairs have been developed to introduce a ncAA at the desired site, delivering a wide variety of functionalities that can be installed into selected proteins. Cytoplasmic expression of RS/tRNA pairs can cause a problem with background ncAA incorporation into host proteins. The application of orthogonally translating organelles (OTOs), inspired by the concept of phase separation, provides a solution for this issue in mammalian cells, allowing site-specific and protein-selective ncAA incorporation. So far, only Methanosarcina mazei (Mm) pyrrolysyl-tRNA synthetase (PylRS) has been used within OTOs, limiting the method’s potential. Here, we explored the implementation of four other widely used orthogonal RS/tRNA pairs with OTOs, which, to our surprise, were unsuccessful in generating mRNA-selective GCE. Next, we tested several experimental solutions and developed a new chimeric phenylalanyl-RS/tRNA pair that enables ncAA incorporation in OTOs in a site-specific and protein-selective manner. Our work reveals unaccounted design constraints in the spatial engineering of enzyme functions using designer organelles and presents a strategy to overcome those in vivo. We then discuss current limitations and future directions of in-cell engineering in general and protein engineering using GCE specifically.

The expansion of the genetic code has become a valuable tool for molecular biology, biochemistry, and biotechnology. The pyrrolysyl-tRNA synthetase (PylRS) variants with their cognate tRNAPyl derived from methanogenic archaea of the genus Methanosarcina are the most popular tools for ribosomally mediated site-specific and proteome-wide statistical incorporation of noncanonical amino acids (ncAAs) into proteins. The incorporation of ncAAs can be used for numerous biotechnological and even therapeutically relevant applications. Here we present a protocol of engineering PylRS for novel substrates with unique chemical functionalities. These functional groups can act as intrinsic probes, especially in complex biological environments such as mammalian cells, tissues, and even whole animals.