Review Article | Open Access
Xian Fu, Yijian Huang, Yue Shen, "Improving the Efficiency and Orthogonality of Genetic Code Expansion", BioDesign Research, vol. 2022, Article ID 9896125, 13 pages, 2022. https://doi.org/10.34133/2022/9896125
Improving the Efficiency and Orthogonality of Genetic Code Expansion
Abstract
The site-specific incorporation of the noncanonical amino acid (ncAA) into proteins via genetic code expansion (GCE) has enabled the development of new and powerful ways to learn, regulate, and evolve biological functions in vivo. However, cellular biosynthesis of ncAA-containing proteins with high efficiency and fidelity is a formidable challenge. In this review, we summarize up-to-date progress towards improving the efficiency and orthogonality of GCE and enhancing intracellular compatibility of introduced translation machinery in the living cells by creation and optimization of orthogonal translation components, constructing genomically recoded organism (GRO), utilization of unnatural base pairs (UBP) and quadruplet codons (four-base codons), and spatial separation of orthogonal translation.
1. Introduction
Genetically incorporating ncAA with diverse functional groups into a protein of interest (POI) by GCE technology is a powerful method to manipulate protein functions and enables many applications, including the development of new drugs, biopolymers, and novel probes, as well as investigation of protein posttranslational modifications (PTMs). To date, over 200 ncAAs have been cotranslationally incorporated into POI in living cells by GCE approaches [1]. Correct incorporation of ncAA by GCE requires orthogonal translational systems. As aminoacyl-tRNA synthetase and tRNA (aaRS/tRNA) pair plays the central role in ensuring accurate genetic code interpretation by attaching the appropriate amino acid onto its corresponding tRNAs, creating orthogonal aaRS/tRNA pairs is required for specific ncAA incorporation. The orthogonality means the introduced aaRS/tRNA pair should not cross-react with endogenous aaRS/tRNA pairs (Figure 1). The anticodon of orthogonal tRNA is then engineered to pair with a “blank” codon that is not assigned to a canonical amino acid. The amber stop codon (TAG) is most often used. Finally, the amino acid binding pocket of orthogonal aaRS is modified to recognize the desired ncAAs and reject canonical amino acids selectively. Thus, by introducing the orthogonal aaRS/tRNA pair and hijacking the host translational apparatus, ncAAs can be site-specifically incorporated into proteins in response to the amber stop codon in vivo (Figure 1). With orthogonal aaRS/tRNA pairs, reprogramming the genetic code for ncAA insertion is now possible in bacteria and eukaryotic cells and animals [2, 3].
Despite the rapid progress of our ability to genetically incorporate diverse ncAAs via GCE, two inherent problems of ncAA incorporation in living cells, including ambiguous decoding due to the lack of a specific blank codon and low incorporation efficiency, need to be addressed to ensure further practical and sophisticated applications (e.g., the mass and accurate production of ncAA-containing proteins, especially in eukaryotes). Since each of the 64 triplet codons is utilized in every organism to synthesize natural proteins, the codon chosen for ncAA incorporation by GCE has on two definitions in the same cell, which may cause serious problems. Taking the TAG-directed ncAA incorporation as an example, ambiguous decoding of the amber codon causes two issues. First, introducing orthogonal aaRS/tRNACUA pair gives rise to global suppression of all TAG codons, resulting in impaired physiological function [4]. Second, release factors compete with the introduced aaRS/tRNA pair, resulting in a portion of polypeptide synthesis terminating at the amber codon [5] (Figure 1). In addition, the introduced aaRS/tRNA pair often does not perfectly fit with the endogenous translational machinery. For instance, the orthogonal tRNA might interact with the elongation factor poorly and the endogenous ribosome might function as a suboptimal decoder for the ncAA-charged tRNA [3, 6]. Thus, it is pivotal to improve the efficiency of GCE by systematically optimizing different steps involved in the biosynthesis of ncAA-containing protein (Figure 1).
In this review, we provide essential background on aforementioned problems of ncAA incorporation in living cells and focus primarily on recent efforts and achievements overcoming many challenges for genetically encoding noncanonical biopolymers. We also compared distinct strategies for improving the orthogonality and efficiency of GCE, including the advantages and disadvantages, the improved efficiency, and different methods utilized in these studies. This review does not cover the advances in GCE utilizing in vitro protein synthesis [7] and many exciting applications of GCE [8].
2. Orthogonal Codons for ncAA Incorporation
An ideal GCE requires a blank codon assigned explicitly to a ncAA. To fulfill the potential of GCE applications in living cells, creating chassis with orthogonal codons is highly desired. Here, we discussed several fundamental strategies to avoid ambiguous decoding by codon compression, creating additional codons, including quadruplet codons and UBPs, and codon reassignment of tagged mRNA by using orthogonally translating organelle (Figure 2). In addition, we also compared the advantages and disadvantages for these different strategies (Table 1).
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2.1. Codon Compression by Genome Synthesis
As the genetic code is degenerate, construction of a GRO in which synonymous codons replace all target codons in the whole genomes is a fundamental way to generate blank codons (Figure 2(a)). Development of MAGE and conjugative assembly genome engineering (CAGE) methods allows genome-wide codon replacement [9]. A GRO designated as C321.ΔA, in which all known TAG codons in E. coli MG1655 have been substituted by TAA, was constructed in 2013 [5]. By removing the RF1, the amber stop codon in the C321.ΔA strain is completely assigned to ncAAs by expressing UAG-reading aaRS/tRNA pairs [5]. Similarly, the design of the ongoing synthetic yeast genome project includes TAG/TAA stop-codon swaps in all sixteen chromosomes [10]. The reassignment of sense codons to ncAAs could also be implemented by genome-wide substitution of a subclass of sense codons by their synonymous codons, followed by removing the corresponding tRNAs. Since sense codons are much higher frequent than stop codons in the genome, synthesizing entire recoded genomes is preferred over multiple-site editing such as MAGE and CRISPR–Cas9, which would likely introduce off-target mutations [5, 11]. Serine and leucine codons are chosen for codon compression in E. coli as the anticodon of tRNASer and tRNALeu is not recognized as a tRNA identity element. Thus, the introduction of blank codon decoding tRNAs would not crosstalk with endogenous aaRSs. An ambitious project that is aimed at synthesizing 57-codon E. coli genome in which total seven codons (six sense codons and amber stop codon) were substituted for synonymous alternatives has been partially completed, highlighting the feasibility of drastically changing the genetic code [12]. Recently developed methods such as replicon excision-enhanced recombination (REXER) and genome stepwise interchange synthesis (GENESIS) facilitate the replacement of genomes with synthetic DNA [13], leading to the construction of syn61, the first entirely synthesized E. coli with a 61-codon genome [14]. A subsequent work shows that replacing two serine codons and TAG codon enables elimination of cognate tRNAs and RF1 in a single strain. The three blank codons can be assigned to three distinct ncAAs in the optimized syn61 strain expressing three mutually orthogonal aaRS/tRNA pairs [15] (Figure 2(a)).
2.2. Quadruplet Codons and Orthogonal Ribosome
In principle, a total of 256 quadruplet codons could be potentially explored for GCE. Quadruplet codon suppression by tRNA with a nucleotide extension in the anticodon was observed in nature [16]. Inspired by the naturally occurring +1 frameshift suppressors, quadruplet codon-mediated GCE has been developed for ncAA incorporation [17, 18]. In response to quadruplet codons, GCE confronts two major problems: (i) the natural ribosome decodes quadruplet codons poorly and (ii) expression of quadruplet codon-reading tRNAs would lead to proteome-wide misincorporation. By assessing structure-guided libraries in the decoding center, an orthogonal ribosome termed ribo-Q1 has been developed that efficiently translates quadruplet codons on its cognate mRNA termed O-mRNA (Figure 2(b)) [19]. The O-mRNA contains modified Shine-Dalgarno (SD) sequence and therefore is not recognized by native ribosomes. As ribo-Q1 was derived from ribo-X, an engineered ribosome that efficiently decodes TAG codon [20], both amber stop codon and quadruplet codons could serve as blank codons for ribo-Q1-mediated incorporation of multiple ncAAs on an O-mRNA [19, 21]. A recent study significantly improved the efficiency of orthogonal ribosome-mediated translation by optimizing O-mRNA sequences using thermodynamic models and algorithms, which resulted in simultaneous incorporation of four different ncAAs into a single protein directed by four different quadruplet codons [22].
As mentioned above, engineering the anti-SD sequence in the 16S rRNA together with the complementary SD sequence in O-mRNA could generate orthogonal 30S subunits that specifically translate O-mRNA (Figure 2(b)). However, the orthogonality relying on the small subunit is limited as the association between large subunits and the native, and orthogonal 30S subunits are stochastic. This limitation could be addressed by the creation of an orthogonal ribosome with tethered subunits (Ribo-T), which could work independently from the native ribosomes that are responsible for biosynthesis of endogenous proteins [23, 24] (Figure 2(b)). A Ribo-T variant with improved properties was recently evolved and could be harnessed to produce a green fluorescent protein that contains ncAAs at multiple sites [25].
2.3. Creating Additional Codons Using the Unnatural Base Pair
In addition to quadruplet codons, developing synthetic nucleotides that pair to form an UBP is another powerful way to create orthogonal codons for GCE. Pioneering studies have identified and developed several UBPs formed by noncovalent interactions, including hydrogen bonds and hydrophobic and packing interactions [26]. Some of these UBPs could be efficiently amplified by PCR [27, 28], selectively transcribed to RNA [29], and utilized to incorporate ncAAs via in vitro translation [30, 31]. A landmark work created a semisynthetic organism (SSO) bearing an expanded genetic alphabet. The SSO is able to intake and retain one class of UBP (dTPT3/dNaM and d5SICS/dNaM pairs) in a plasmid [32]. By optimizing the transporter to uptake the synthetic triphosphates and exploring the CRISPR-Cas9 system to prevent UBP loss, the SSO could stably retain the expanded genetic alphabet over 100 cell divisions [33]. To demonstrate that the dNaM/dTPT3 pair could be used as additional codons to retrieve increased genetic information, a further study showed that DNA containing dNaM (dX) and dTPT3 (dY) could be used as other codons to transcribe into mRNAs and tRNAs containing unnatural codons and anticodons, respectively. These unnatural codons could direct site-specific ncAA incorporation [34] (Figure 2(c)). Using a similar strategy, the unnatural codon-directed ncAA incorporation was demonstrated in eukaryotic cells [35]. In theory, the numbers of available codons would increase by 152 via expanding the genetic alphabet from four letters to six letters. A recent study that systematically screened the unnatural functional codon in SSOs identified additional unnatural codons that could efficiently produce ncAA-containing proteins [36]. By utilizing mutually orthogonal aaRS/tRNA pairs and tRNASer (AYC) with endogenous seryl-tRNA synthetase, the SSO could incorporate two distinct ncAAs and serine into a POI in response to three different unnatural codons [36].
2.4. Orthogonally Translating Organelles
The blank codons are not required if the orthogonal translation could decode a specific codon only for the gene of interest in a spatially confined microenvironment. Phase separation is recently recognized as a common mechanism for accumulating high local concentrations of biomacromolecules such as proteins and RNAs [37, 38]. Inspired by phase separation, membrane-less orthogonal translating organelles targeting to microtubule plus-ends have been created that consists of an mRNA-targeting system (mRNA::ms2 fusion), a PylRS/tRNAPyl pair derivative, and the assembler that brings tagged mRNA into proximity of the orthogonal aaRS/tRNA pair (Figure 2(d)) [39]. Thus, cellular ribosomes near the organelle function with highly concentrated PylRS/tRNAPyl pair to translate the ms2-tagged mRNAs and produce ncAA-containing proteins [39]. The following study developed several orthogonal translating organelles that support GCE on surfaces of plasma, endoplasmic reticulum, Golgi, and mitochondrial membranes [40]. Remarkably, these dual film-like organelles compromising spatially orthogonal aaRS/tRNA pairs and tagged mRNA could use the same TAG codon for distinct ncAA incorporation, generating a eukaryotic cell with two expanded genetic codes [40].
3. Improving the Efficiency of ncAA Incorporation by GCE
Many factors may affect the efficiency of GCE, including the cellular concentration of ncAA [41], the expression level of introduced aaRS/tRNA pair in heterogeneous cells [42], codon context effects [43], the ligation efficiency of ncAA to a dedicated tRNA catalyzed by the corresponding aaRS [44], the compatibility of ncAA-tRNA with the elongation factor [45, 46], the efficiency of tRNA decoding by codon-anticodon pairing and peptide bond formation in the ribosome [47], and the competition effect from release factors that terminate polypeptide formation [48]. Increasing evidence suggests that many of the factors mentioned above are interrelated and have combined effects. Thus, efficient ncAA incorporation in the living cell requires optimization of expression and activity of the introduced aaRS/tRNA pair and systematically engineering many parts of the translational apparatus involved in many steps of protein synthesis. In this section, we summarized a series of efforts to improve the efficiency of ncAA incorporation by engineering of distinct translation components including aaRS/tRNA pairs, the elongation factor, the release factors, and the ribosome, with a particular focus on the relevant technologies and the improved efficiency (Table 2).
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3.1. Development and Optimization of Orthogonal aaRS/tRNA Pairs
Many orthogonal aaRS/tRNA pairs have been discovered and engineered for GCE. These pairs are often derived from phylogenetically distant organisms compared to the host of interest as the diverged identity elements of these orthogonal tRNAs prevent them from being recognized by endogenous aaRSs. For instance, Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS)/MjtRNATyr pair is orthogonal in Escherichia coli (E. coli) [49]. The derivatives of the E. coli EcTyrRS/EctRNATyr pair and leucyl-tRNA synthetase (EcLeuRS)/EctRNALeu pair could be utilized to incorporate various ncAAs in eukaryotic cells [50, 51]. The derivatives of pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for GCE. They have been extensively engineered as the PylRS/tRNAPyl pair is orthogonal in both bacteria and eukaryotic cells [2, 52]. Recent work created a series of chimeric aaRS/tRNA pairs that are orthogonal in both prokaryotic and eukaryotic cells by rationally transplanting the critical sequences from the PylRS/tRNAPyl pair into other canonical aaRS/tRNA pairs [53]. An integrated pipeline for discovering new orthogonal pairs was reported, which consists of both computational analysis and experimental validation [54].
Despite the successful development of many orthogonal aaRS/tRNA pairs, the mass production of ncAA-containing polypeptide remains a challenge. One major problem is the poor aminoacylation efficiency of modified orthogonal aaRSs. The structure-guided directed evolution strategy could create and improve the selectivity of orthogonal aaRS towards a designed ncAA, which relies on generating a library of mutants in the amino acid binding site of the orthogonal aaRS followed by successive rounds of positive selection (tolerance of antibiotics or fluorescence intensity) and negative selection (production of toxic proteins) [55]. Although the traditional strategy has been successfully applied at engineering aaRS for ncAA insertion, it is time-consuming and requires prior structural knowledge. To overcome these challenges, advanced methods have been developed to evolve aaRS/tRNA pairs. For instance, phage-assisted continuous evolution (PACE) and a simplified version termed phage-assisted noncontinuous evolution (PANCE) were used to generate highly active and selective aaRS variants by coupling random mutagenesis and delicate selection [56, 57]. Importantly, PACE and PANCE do not require the determined structure of aaRS to design the mutant library and thus have great potential for further evolving and improving orthogonal pairs. Multiplex automated genome engineering (MAGE) enables the generation of large libraries of chromosomal aaRSs by simultaneously mutagenizing different loci and producing polypeptide with 30 ncAA residues [58]. A recent study developed an integrated system called phage- and robotics-assisted near-continuous evolution (PRANCE) to optimize orthogonal aaRS/tRNA pairs, which offers several advantages, including scalability to high-throughput molecular experiments, a substantial reduction in reagents, and real-time feedback control [59].
tRNA is not a simple “generic adaptor.” Its optimization is often required for efficient and accurate biosynthesis of proteins containing ncAA. As many developed orthogonal tRNAs such as EctRNATyr and tRNAPyl lack intrinsic A- and B-box elements that are important for RNA polymerase III-mediated transcription, heterologous expression of these tRNAs is problematic in eukaryotic cells. Improvement of cellular concentration of orthogonal tRNAs by promoter optimization [60, 61] and increase of tRNA copy number [62] are useful to enhance the production of ncAA-containing peptides and proteins. As tRNA has evolved to function with the ribosome and other translation factors in each organism [63], the introduced orthogonal tRNA might not function very well in heterologous hosts. Optimized tRNAs could be developed by enhancing their compatibility with translational apparatus via rational design and directed-evolution strategies [64–66]. In addition, tRNA modification is known to affect codon-anticodon pairing in the ribosome. However, the effect of tRNA modification on the performance of GCE remains poorly understood. Interestingly, the incorporation efficiency of O-phosphoserine (Sep) is affected by deletion and overexpression of some posttranscriptional modification enzymes in E. coli [67], highlighting the importance of tRNA modifications for genetically encoding ncAA in proteins.
3.2. Engineering of Elongation Factor
Although the formation efficiency of ncAA-tRNA could be significantly enhanced by optimization of orthogonal aaRS/tRNA pair, efficient delivery of charged tRNA by an elongation factor (e.g., EF-Tu, the GTP-bound form of elongation factor in bacteria) to the ribosome remains as another challenge (Figure 1). EF-Tu recognizes both the acceptor helix of tRNA and amino acid moiety [68] and weakly binds negatively charged amino acids [1, 69]. Thus, EF-Tu often needs to be engineered to improve the incorporation efficiency of ncAAs with negative charges or bulky side chains. For instance, repurposing the substrate binding pocket of EF-Tu enables enhanced production of proteins containing ncAAs such as Sep, selenocysteine, and phenylalanine analogs, which are poorly incorporated by the wild-type EF-Tu [70–72]. Notably, the removal of EF-Sep21, a highly selective EF-Tu variant for Sep, abolishes the ability to genetically encode O-phosphoserine, highlighting the critical role of EF-Sep21 in incorporating negatively charged Sep [73].
3.3. Elimination and Engineering of Release Factors
Amber codon is commonly used for site-specific ncAA incorporation by GCE. Amber codon together with TAA (ochre stop) is recognized by release factor 1 (RF1), and RF2 recognizes TGA (opal stop) as well as ochre codon in E. coli. Thus, the amber codon-directed ncAA insertion by the orthogonal aaRS/tRNA pair competes with RF1-mediated translation termination, resulting in decreased production of ncAA-containing proteins (Figure 1). To eliminate the competition effect of RF1 and increase the efficiency of ncAA incorporation, many studies focused on the removal of RF1, a translation component that was thought to be essential [74, 75]. By expressing amber suppressor tRNA together with a few essential genes undergoing the stop codon swap (TAG>TAA), the RF1 could be deleted, and the amber codon was reassigned to encode ncAA [76]. Studies also found that RF1 is dispensable in various E. coli strains containing RF2 protein with higher activity than RF2(Thr246) in the K-12 strain [77–79]. Later construction of a GRO, in which the ochre codon replaces all instances of the amber codon, allows for the removal of RF1 and complete reassignment of TAG codon [5]. Alternatively, synonymous replacement of TAG in 95 genes of E. coli BL21(DE3) allows elimination of RF1, generating a robust growth strain [80]. Taking advantage of the RF1 knockout strain, efficient incorporation of ncAA at multiple UAG sites and improved phosphoserine and selenocysteine insertion were demonstrated [79, 81, 82]. The multisite incorporation of ncAA via cell-free translation system was also achieved by utilizing cell extracts of GRO lacking RF1 [83].
Unlike bacteria with separate RF1 and RF2, eukaryotic cells rely on a single omnipotent release factor (eRF1), an essential protein for cell viability, to recognize all stop codons [84]. Therefore, it is not feasible to improve the efficiency of ncAA in eukaryotic systems by simply eliminating eRF1. Inspired by findings that stop codons are reassigned invariant code organisms, tuning the specificity of eRF1 for stop codon recognition might be possible. For instance, TAG and TAA encode glutamine while translation termination relies on TGA in Tetrahymena and Paramecium [85, 86]. Biochemical and genetic studies identified several key residues in the N-terminal domain of eRF1 that are responsible for stop codon recognition [87–90]. Interestingly, the introduction of TASNIKS and YCF motifs from Tetrahymena eRF1 into eRF1 of Saccharomyces cerevisiae (S. cerevisiae) resulted in increased readthrough for amber codon [91]. Another study reported that ectopic overexpression of eRF1 mutant (E55D) enhances TAG-directed ncAA incorporation in mammalian cells [92]. Structural analyses also revealed the molecular basis of eRF1 recognition for stop codons [93, 94], which have implications for future structure-guided eRF1 engineering to enhance ncAA insertion by amber suppression.
3.4. Ribosome Engineering
A variety of L-α-amino acids with different noncanonical side chains has been genetically encoded in proteins [95], and certain types of N-methyl [96], D-α- [97], and β-amino acids [98] could be inserted into peptides by in vitro translation, indicating the substrate plasticity of native ribosomes. However, ribosome engineering is required to expand the substrate scope of translation, especially for those ncAAs with altered backbones. Utilization of ribosome mutants with modifications in the peptidyl transferase center (PTC), which catalyzes ribosome peptide bond formation and peptide release [99], could improve in vitro translation of dipeptides analogues and D- and β-amino acids [100, 101]. A recent study demonstrated cellular biosynthesis of a β-amino acid-containing DHFR by using E. coli cells expressing ribosome mutants together with wild-type EF-Tu and phenylalanyl-tRNA synthetase in the absence of phenylalanine [102]. Studies reported that ribosome mutants selected by randomization of 530 loop in 16S ribosomal RNAs (rRNA) and PACE-assisted directed evolution of 16S rRNA could enhance the efficiency of ncAA incorporation in living cells [20, 103].
4. Conclusion and Future Perspectives
The development of strategies to create orthogonal codons, optimize orthogonal translation components, and expand the substrate diversity of translation will enable and facilitate the cellular production and directed evolution of novel biopolymers. However, many challenges remain, including creating various functional chassis containing orthogonal codons and developing super active and orthogonal translation systems that can efficiently incorporate ncAAs into any POI regardless of context effects. In particular, efficient production of ncAA-containing proteins in eukaryotic cells is more challenging than that in the E. coli system since many approaches to creating blank codons are not easily transferred to eukaryotes and the development process of additional orthogonal aaRS/tRNA pairs is slow.
To promote future GCE application in eukaryotes, we propose yeasts, including S. cerevisiae and Pichia pastoris, could serve as powerful model systems since they are widely used in both high-through screening assays (e.g., yeast display) and production of recombinant POI for medical and industrial applications [104]. The ongoing synthetic yeast, Sc2.0, would open up new opportunities for GCE applications by creating an ideal eukaryotic chassis with the orthogonal codon [105]. Due to the TAG/TAA stop-codon swaps in the final Sc2.0 strain, it would be possible to safely engineer eRF1 for abolishing its recognition to the amber stop codon without causing unwanted global amber suppression. Thus, successful engineering of the eRF1 in Sc2.0 would ultimately generate a synthetic eukaryotic organism bearing an orthogonal codon for GCE. Furthermore, we think the cost reduction of genome writing and testing tools as well as the rapid development of technologies for yeast genome engineering would allow the reassignment of more blank codons, including stop and sense codons, to ncAAs by genome recoding of the synthetic yeast. Another key feature of Sc2.0 is the synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system, which enables inducible whole-genome rearrangement by the Cre recombinase [106]. We think the utilization of the in-built SCRaMbLE system of Sc2.0 would be very powerful to generate cell populations with massive genomic diversity; the mutant strains showing improved GCE performance could be obtained by high-throughput selection method such as fluorescence-activated cell sorters (FACS). A previous work showed the disruption of the non-sense-mediated decay pathway by deleting the UPF1 gene in S. cerevisiae could enhance the ncAA incorporation in response to the amber stop codon [107], highlighting the promise of genomic background modification to optimize GCE by SCRaMbLE.
To further create and optimize the orthogonal aaRS/tRNA pairs in various chassis in higher eukaryotes, continued efforts on the development of PylRS/tRNAPyl pair are worth adhering as this pair is orthogonal in both bacteria and eukaryotic cells. We recently disclosed all PylRS enzymes encoded in 524 archaeal genomes by mining the archaeal clusters of orthologous genes (arCOGs) [108]. We envision these PylRS/tRNAPyl pairs could be explored and engineered to enrich the toolbox for GCE. Taking the advantages of advanced methods for the directed evolution of biomacromolecules such as PACE, MAGE, and PRANCE, the efficiency and substrate specificity of PylRS/tRNAPyl pair candidates could be initially optimized in model organisms (e.g., E. coli and S. cerevisiae), followed by transplanting them into other eukaryotic cells. Scientists working with different eukaryotic chassis could then utilize their own expertise to further improve the expression level and intracellular compatibility of the introduced PylRS/tRNAPyl pairs. This concerted effort would provide guidance to optimize the process of developing super active and orthogonal aaRS/tRNA pairs for ncAA incorporation and lay a solid foundation for many exciting GCE applications in eukaryotes.
Conflicts of Interest
The authors declare that they have no conflicts of interest regarding the publication of this article.
Authors’ Contributions
X.F. and Y.S. planned the manuscript. X.F. wrote the manuscript. All authors read and revised the manuscript.
Acknowledgments
The writing of this manuscript was supported by the National Key Research and Development Program of China (No. 2018YFA0900100), National Natural Science Foundation of China (No. 31901029 to X.F. and No. 31800078 to Y.S.), Natural Science Foundation of Guangdong Province, China (No. 2021A1515010995 to X.F.), and Guangdong Provincial Key Laboratory of Genome Read and Write (No. 2017B030301011).
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