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Direct charging of tRNACUA with pyrrolysine in vitro and in vivo
... For the construction of the Vac-Split-CFP strain, the Superfolder self-assembling Split-GFP fragments designed by Cabantous and coworkers [35,36] and optimized by our team [22] were used. In this system, the 11 beta strands of the Superfolder GFP are separated into two ) were respectively mutated into Trp and Thr to obtain the CFP fragment ( ). ...
... As for Sec, Pyl active site residues appear to be crucial for the activity of enzymes involved in methanogenesis [35]. Pyrrolysyl-tRNA Pyl is synthesized by direct pyrrolysylation of an amber-suppressor tRNA Pyl by the class IIc PylRS [36]. Pyl incorporation has become the first known example to date of direct aminoacylation of a tRNA with a non-standard proteinogenic aa. ...
The aminoacyl-tRNA synthetases (aaRSs) are responsible for the formation of aminoacyl-tRNAs used by the translational machinery. The subcellular relocalization of these proteins is often associated with non-canonical functions. The yeast S. cerevisiae contains a multisynthetasic complex, called AME complex, composed of the methionyl- and glutamyl-tRNA synthetases (MRS and ERS) and the cofactor Arc1. Even if the complex has been described as exclusively cytosolic, the two aaRSs can relocate in different subcellular compartments. These proteins are thus referred as cytosolic or organellar echoforms. Moreover, Arc1 interacts with vacuolar lipids in vitro, suggesting a vacuolar localization in vivo. To visualize mitochondrial and vacuolar (vace) echoforms of cytosolic proteins, we engineered two epifluorescence microscopy tools. We identified a new mitochondrial echoform for two aaRSs, as well as vacuolar echoforms for all the cytosolic aaRSs tested and the AME components. A potential implication of vaceMRS in TORC1 inhibition was also observed.
... Sec is then co-translationally inserted into proteins at a TGA stop codon with the help of special elongation factor (SelB) and a stem-loop Sec insertion sequence element (SECIS) (Forchhammer et al., 1989). Pyl is directly attached to pyrrolysyl tRNA (tRNA pyl ) by pyrrolysyl-tRNA synthetase (PylRS) and incorporated into proteins at a TAG stop codon (Blight et al., 2004;Polycarpo et al., 2004). Inspired by this natural expansion of the genetic code, researchers have developed a site-specific UAA incorporation technique (also called genetic Fig. 1. ...
... The discovery of the PylRS/tRNA pyl pair in methanogenic archaea, in particular, has helped rapidly advance this field (Wan et al., 2014). PylRS/ tRNA pyl is a naturally occurring orthogonal pair that functions efficiently in diverse living organisms from bacteria (Blight et al., 2004) and yeast (Hancock et al., 2010) to mice (Han et al., 2017). PylRS has a flexible amino acid binding pocket that is easily modified to accommodate a wide range of UAAs with diverse chemical structures. ...
Labeling of a protein with a specific dye or tag at defined positions is a critical step in tracing the subtle behavior of the protein and assessing its cellular function. Over the last decade, many strategies have been developed to achieve selective labeling of proteins in living cells. In particular, the site-specific unnatural amino acid (UAA) incorporation technique has gained increasing attention since it enables attachment of various organic probes to a specific position of a protein in a more precise way. In this review, we describe how the UAA incorporation technique has expanded our ability to achieve site-specific labeling and visualization of target proteins for functional analyses in live cells.
... Therefore, expanding the repertoire of amino acids in translation is useful for developing novel protein functions (reviewed in Wang et al., 2006;Liu and Schultz, 2010). ''Orthogonal'' pairs of an engineered aminoacyl-tRNA synthetase and tRNA, including bacterial and archaeal pairs of tyrosyl-tRNA synthetase (TyrRS) and tRNA Tyr (CUA) (Wang and Schultz, 2001;Wang et al., 2002;Chin et al., 2002Chin et al., , 2003Kiga et al., 2002;Sakamoto et al., 2002) and archaeal pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNA Pyl (CUA) for the 22nd amino acid, pyrrolysine (Blight et al., 2004;Polycarpo et al., 2004), have enabled the site-specific incorporation of non-natural amino acids into proteins in response to the amber (UAG) codon (reviewed in Wang et al., 2006;Liu and Schultz, 2010;Wan et al., 2014;Chin, 2014;Crnkovi c et al., 2016;Brabham and Fascione, 2017;Chin, 2017;Wang, 2017;Vargas-Rodriguez et al., 2018;Tharp et al., 2018). Chemically reactive functional groups, such as alkene, alkyne, azide, and diazirine groups, on non-natural amino acids allow the post-translational labeling of proteins with detection probes, polymers, drugs, and UV crosslinkers (reviewed in Lang and Chin, 2014aChin, , 2014bElliott et al., 2014;Nguyen et al., 2018). ...
... PylRS consists of two domains, the N-terminal tRNA binding domain and the C-terminal catalytic domain (Herring et al., 2007;Yanagisawa et al., 2008a;Jiang and Krzycki, 2012;Suzuki et al., 2017). Pyrrolysine is site specifically incorporated into a specific UAG site of methylamine methyltransferases with M. barkeri PylRS (Blight et al., 2004;Polycarpo et al., 2004). Thus, the PylRSs from M. barkeri, Methanosarcina mazei, and D. hafniense have been used for the incorporation of non-standard amino acids into proteins (Ambrogelly et al., 2007;Neumann et al., 2008;Mukai et al., 2008;Yanagisawa et al., 2008b;Chen et al., 2009;Nguyen et al., 2009;Katayama et al., 2012). ...
Pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids. Among 17 non-natural lysine derivatives, Nɛ-(benzyloxycarbonyl)lysine (ZLys) and 10 ortho/meta/para-substituted ZLys derivatives were efficiently ligated to tRNAPyl and were incorporated into proteins by PylRS(Y306A/Y384F). We determined crystal structures of 14 non-natural lysine derivatives bound to the PylRS(Y306A/Y384F) catalytic fragment. The meta- and para-substituted ZLys derivatives are snugly accommodated in the productive mode. In contrast, ZLys and the unsubstituted or ortho-substituted ZLys derivatives exhibited an alternative binding mode in addition to the productive mode. PylRS(Y306A/Y384F) displayed a high aminoacylation rate for ZLys, indicating that the double-binding mode minimally affects aminoacylation. These precise substrate recognition mechanisms by PylRS(Y306A/Y384F) may facilitate the structure-based design of novel non-natural amino acids.
... 29 The PylRS/tRNA Pyl pair functions in the natural context of the common 20 amino acids, and this orthogonality is retained when the PylRS/tRNA Pyl pair is transferred from archaea to other organisms. Indeed, in E. coli or mammalian cells, the PylRS/tRNA Pyl pair from either M. barkeri or M. mazei does not cross-react with endogenous aaRSs or tRNAs, 30 providing several advantages over the previous systems for UAA mutagenesis. First, no directed evolution is needed for the tRNA, because the tRNA Pyl is already a perfect substrate for its cognate PylRS, but not any endogenous aaRSs. ...
... The first application of the PylRS/tRNA Pyl system in genetic code expansion in E. coli (with pyrrolysine) was reported in 2004. 30 The encoding of an UAA by PylRS/ tRNA Pyl was subsequently demonstrated in E. coli 32,33 and in mammalian cells. 34 Surprisingly, wild-type PylRS exhibits an extraordinarily broad substrate spectrum for many lysine analogues, 34−39 including UAAs for bioconjugation reactions 35,36 and photo-cross-linking. 39 To genetically encode other UAAs that are not substrates of the wild-type PylRS, libraries of PylRS mutants were constructed and passed through multiple rounds of positive and negative genetic selections. ...
Expanding the genetic code to enable the incorporation of unnatural amino acids into proteins in biological systems provides a powerful tool to study protein structure and function. While this technology has been mostly developed and applied in bacterial and mammalian cells, it recently expanded into animals, including worms, fruit flies, zebrafish, and mice. In this review, we highlight recent advances toward the methodology development of genetic code expansion in animal model organisms. We further illustrate the applications, including proteomic labeling in fruit flies and mice, and optical control of protein function in mice and zebrafish. We summarize the challenges of unnatural amino acid mutagenesis in animals, and the promising directions towards broad application of this emerging technology.
... Structural and biochemical work on Pyl biosynthesis was recently reviewed 89 . Pyl is then ligated onto tRNA Pyl by PylRS 90,91 . The CUA anticodon of tRNA Pyl reads the UAG codon and reassigns the meaning of UAG from stop to Pyl (FIG. ...
... The Pyl-decoding trait exists in 25 anaerobic bacteria from the Firmicutes phylum (in classes Clostridia and Negativicutes) and the δ-Proteobacteria class. Early work in the field showed that E. coli expressed Pylcontaining proteins with a recombinant Pyl-decoding system, indicating that bacteria are capable of decoding UAG codons as Pyl 90,103 . However, there has been little work to show whether Pyl is actively decoded in bacteria in which the Pyl trait naturally occurs. ...
The genetic code, initially thought to be universal and immutable, is now known to contain many variations, including biased codon usage, codon reassignment, ambiguous decoding and recoding. As a result of recent advances in the areas of genome sequencing, biochemistry, bioinformatics and structural biology, our understanding of genetic code flexibility has advanced substantially in the past decade. In this Review, we highlight the prevalence, evolution and mechanistic basis of genetic code variations in microorganisms, and we discuss how this flexibility of the genetic code affects microbial physiology.
... In "Protein 2", an alkalilabile cleavage site (AllocLys-OH) is introduced at the position specified by the opal codon UGA, during translation with tRNA Pyl and an engineered pyrrolysyl-tRNA synthetase (PylRS) that can charge the tRNA with AllocLys-OH (Yamaguchi et al., 2018;Yanagisawa et al., 2008). Since PylRS does not recognize the anticodon moiety of tRNA Pyl (Ambrogelly et al., 2007;Krishnakumar et al., 2013;Zeng et al., 2014), we changed the anticodon moiety of tRNA Pyl from 5' CUA 3' (corresponding to the amber codon UAG; Blight et al., 2004;Polycarpo et al., 2004;Srinivasan et al., 2002) to 5' CAU 3' (corresponding to the opal codon UGA). Accordingly, our system enables the dual introduction of pBpa and AllocLys-OH to the amber and opal codons, respectively, in mammalian cells. ...
Genetic code expansion enables site‐specific photo‐crosslinking by introducing photo‐reactive non‐canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein–protein interactions and is applicable in mammalian cells. However, the identification of the crosslinked region still remains challenging. Here, we developed a new method to identify the crosslinked region by pre‐installing a site‐specific cleavage site, an α‐hydroxy acid (Nε‐allyloxycarbonyl‐α‐hydroxyl‐l‐lysine acid, AllocLys‐OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α‐hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N‐terminus or C‐terminus, the crosslinked site is located within the target protein. A series of AllocLys‐OH introductions narrows down the crosslinked region. By applying this method, we identified the crosslinked regions in lysosomal‐associated membrane protein type 2A (LAMP2A), a receptor of chaperone‐mediated autophagy, in mammalian cells. The results suggested that at least two interfaces are involved in the homophilic interaction, which requires a trimeric or higher oligomeric assembly of adjacent LAMP2A molecules. Thus, the combination of site‐specific crosslinking and site‐specific cleavage promises to be useful for revealing binding interfaces and protein complex geometries.
... Expanding the genetic code with non-canonical amino acids is useful for developing novel structures and functions of proteins [reviewed in 1,2]. Site-specific incorporation of non-canonical amino acids into proteins in response to specified (e.g., UAG) codons has been achieved by pairs of an engineered aminoacyl-tRNA synthetase (aaRS) and tRNA, including the pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNA Pyl (CUA) [2,3,4,5,6,7,8,9,10]. The PylRS•tRNA Pyl pair was first found in methanogenic archaea, including Methanosarcina barkeri [11,12], and in bacteria, including Desulfitobacterium hafniense [13,14]. ...
Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including Ne-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS•tRNAPyl pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of Ne-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with pEtZLys up to 578% of that with TCO*Lys, at high enzyme concentrations in the cell-free protein synthesis.
... The identity of the UAG encoded amino acid, subsequently named pyrrolysine, was determined from the 1.55 Å crystal structure of the M. barkeri MtmB which showed density consistent with a lysine residue in amide linkage through its εnitrogen to (4R, 5R)-4-methyl-pyrroline-5-carboxylate 23,25,26 located at the center of the TIM barrel fold comprising each subunit. The characterization of synthetic pyrrolysine 25 and the biosynthetic pathway for pyrrolysine 27 , as well as the discovery that tRNA Pyl is aminoacylated with pyrrolysine by pyrrolysyl-tRNA synthetase 28,29 reinforced the conclusion that the pyrrolysine is the 22nd genetically-encoded amino acid found in nature. ...
The 22nd genetically encoded amino acid, pyrrolysine, plays a unique role in the key step in the growth of methanogens on mono-, di-, and tri-methylamines by activating the methyl group of these substrates for transfer to a corrinoid cofactor. Previous crystal structures of the Methanosarcina barkeri monomethylamine methyltransferase elucidated the structure of pyrrolysine and provide insight into its role in monomethylamine activation. Herein, we report the second structure of a pyrrolysine-containing protein, the M. barkeri trimethylamine methyltransferase MttB, and its structure bound to sulfite, a substrate analog of trimethylamine. We also report the structure of MttB in complex with its cognate corrinoid protein MttC, which specifically receives the methyl group from the pyrrolysine-activated trimethylamine substrate during methanogenesis. Together these structures provide key insights into the role of pyrrolysine in methyl group transfer from trimethylamine to the corrinoid cofactor in MttC. Structures of Methanosarcina barkeri trimethylamine methyltransferase (MttB) with its substrates reveal the role of pyrrolysine in methyl group transfer from trimethylamine to the corrinoid cofactor in MttC.
... Pyrrolysyl-tRNA synthetase (PylRS) ressembles a calssical class II aaRS and belongs to this class (Blight et al., 2004). However, tRNA Pyl displays particularities since its V-loop is composed of only 3 nt, its anticodon binding stem is extended to six base-pairs instead of five, its D-loop harbors 30 31 only 5 nt, its T-loop lacks the canonical T C motif and finally its junction between the D-stemloop and the acceptor stem is ensured only by one base (Srinivasan et al., 2002). ...
Aminoacyl-tRNAs (aa-tRNA) play a central role in protein synthesis but can also be rerouted to other biological pathways. In bacteria, aa-tRNA transferases (AAT) having a DUF2156 domain catalyze the aminoacid (aa) transfer onto glycerolipids to improve their drug resistance or virulence. Such a mechanism was never described in eukaryotes. My thesis work revealed that numerous fungi species have DUF2156/AAT enzymes that aminoacylate sterols. Thus, ergosteryl-aspartate and ergosteryl-glycine are the founding members of a new class of lipids, namely aminoacylated sterols (AS). Moreover, we also identified a specific hydrolase that removes the aa from several AS. Considering the central role of sterols, we propose that AS might participate to cell surface remodeling, trafficking, resistance to stresses and/or pathogenicity.
... Pyrrolysine (1, Pyl, Fig. 1) is the 22 nd naturally occurring, proteinogenic amino acid that is encoded in the genomes of certain anaerobic archaea and bacteria (1). In these organisms, Pyl is installed into polypeptides through the combined actions of the pyrrolysyl-tRNA synthetase (PylRS) and pyrrolysine tRNA (tRNA Pyl ) (2,3). PylRS specifically recognizes free Pyl and attaches the amino acid to the 3'-hydroxyl of tRNA Pyl (4). ...
The pyrrolysyl-tRNA synthetase (PylRS) facilitates the co-translational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain highly active and orthogonal in bacteria and eukaryotes. In this study, we use biochemical and in vivo UAG-readthrough assays to characterize the aminoacylation efficiency and substrate spectrum of a ΔPylSn class PylRS from the archaeon Ca. Methanomethylophilus alvus. We show that, compared to the full-length enzyme from Methanosarcina mazei, the Ca. M. alvus PylRS displays reduced aminoacylation efficiency, but an expanded amino acid substrate spectrum. To gain insight into the evolution of ΔPylSn enzymes, we performed molecular phylogeny using 156 PylRS and 105 tRNAPyl sequences from diverse anaerobic archaea and bacteria. This analysis suggests that the PylRS•tRNAPyl pair diverged before the evolution of the three domains of life, placing an early limit on the evolution of the Pyl-decoding trait. Furthermore, our results document the co-evolutionary history of PylRS and tRNAPyl and reveal the emergence of tRNAPyl sequences with unique A73 and U73 discriminator bases. The orthogonality of these tRNAPyl species with the more common G73-containing tRNAPyl will enable future efforts to engineer PylRS systems for further genetic code expansion.
... N-Methylated amino acids do not only play a role as free bioactives or in peptide drugs, but they may also be co-translationally incorporated into proteins at specific locations by codon engineering (Hoesl and Budisa, 2012). For example, translational amber stop codons have been re-coded using an evolved pyrrolysyl-tRNA synthetase-pylT pair (Blight et al., 2004) to incorporate metanitrophenylacetate-photocaged Ne-L-lysine residues. Upon photolysis in vivo, the labelled proteins were converted to proteins with monomethylated lysine residues (Wang et al., 2010). ...
The growing world needs commodity amino acids such as L‐glutamate and L‐lysine for use as food and feed, and specialty amino acids for dedicated applications. To meet the supply a paradigm shift regarding their production is required. On the one hand, the use of sustainable and cheap raw materials is necessary to sustain low production cost and decrease detrimental effects of sugar‐based feedstock on soil health and food security caused by competing uses of crops in the feed and food industries. On the other hand, the biotechnological methods to produce functionalized amino acids need to be developed further, and titres enhanced to become competitive with chemical synthesis methods. In the current review, we present successful strain mutagenesis and rational metabolic engineering examples leading to the construction of recombinant bacterial strains for the production of amino acids such as L‐glutamate, L‐lysine, L‐threonine and their derivatives from methanol as sole carbon source. In addition, the fermentative routes for bioproduction of N‐methylated amino acids are highlighted, with focus on three strategies: partial transfer of methylamine catabolism, S‐adenosyl‐L‐methionine dependent alkylation and reductive methylamination of 2‐oxoacids.
... On the other hand, the structural diversity of ncAAs is limited by currently available Tyr and pyrro-Lys tRNA synthetases in SCS and the nonspecificity of endogenous aaRS in SPI. 49,53,354 The discovery of novel orthogonal aminoacyl-tRNA synthetase/tRNA pairs and the development of the corresponding aaRS is continuously expanding the application of ncAA-based protein engineering. 48,351−355 However, conventional negative and positive selections comprise mutagenesis libraries with 5−7 randomized positions covering only marginal fractions of the vast sequence space formed by up to 30 active site residues of aaRS. ...
Improvement in intrinsic enzymatic features is in many instances a prerequisite for the scalable applicability of many industrially important biocatalysts. To this end, various strategies of chemical modification of enzymes are maturing and now considered as a distinct way to improve biocatalytic properties. Traditional chemical modification methods utilize reactivities of amine, carboxylic, thiol and other side chains originating from canonical amino acids. On the other hand, noncanonical amino acid- mediated ‘click’ (bioorthogoal) chemistry and dehydroalanine (Dha)-mediated modifications have emerged as an alternate and promising ways to modify enzymes for functional enhancement. This review discusses the applications of various chemical modification tools that have been directed towards the improvement of functional properties and/or stability of diverse array of biocatalysts.
... Sifarchaeia encode a complete Pyl encoding system including all three Pyl biosynthesis proteins (PylB, PylC, PylD) and a pyrrolysyl-tRNA synthetase (PylS) to charge the pyrrolysine tRNA (tRNApyl, pylT) (Fig. 4a) 50 . Unlike selenocysteine (Fig. 3b), no specific proteins or insertion sequences are required for the tRNApyl insertion, which has been proposed to directly compete with the translation termination release factor for UAG codons (Fig. 4b) 51 . ...
Asgardarchaeota have been proposed as the closest living relatives to eukaryotes, and a total of 72 metagenome-assembled genomes (MAGs) representing six primary lineages in this archaeal phylum have thus far been described. These organisms are predicted to be fermentative heterotrophs contributing to carbon cycling in sediment ecosystems. Here, we double the genomic catalogue of Asgardarchaeota by obtaining 71 MAGs from a range of habitats around the globe, including the deep subsurface, brackish shallow lakes, and geothermal spring sediments. Phylogenomic inferences followed by taxonomic rank normalisation confirmed previously established Asgardarchaeota classes and revealed four additional lineages, two of which were consistently recovered as monophyletic classes. We therefore propose the names Candidatus Sifarchaeia class nov. and Ca. Jordarchaeia class nov., derived from the gods Sif and Jord in Norse mythology. Metabolic inference suggests that both classes represent hetero-organotrophic acetogens, which also have the ability to utilise methyl groups such as methylated amines, with acetate as the probable end product in remnants of a methanogen-derived core metabolism. This inferred mode of energy conservation is predicted to be enhanced by genetic code expansions, i.e., stop codon recoding, allowing the incorporation of the rare 21st and 22nd amino acids selenocysteine (Sec) and pyrrolysine (Pyl). We found Sec recoding in Jordarchaeia and all other Asgardarchaeota classes, which likely benefit from increased catalytic activities of Sec-containing enzymes. Pyl recoding, on the other hand, is restricted to Sifarchaeia in the Asgardarchaeota, making it the first reported non-methanogenic archaeal lineage with an inferred complete Pyl machinery, likely providing members of this class with an efficient mechanism for methylamine utilisation. Furthermore, we identified enzymes for the biosynthesis of ester-type lipids, characteristic of bacteria and eukaryotes, in both newly described classes, supporting the hypothesis that mixed ether-ester lipids are a shared feature among Asgardarchaeota.
... This residue appears to be crucial for the enzyme activity. Pyrrolysyl-tRNA Pyl is made by direct attachment of Pyl to this amber suppressor tRNA Pyl(CUA) by the class IIc PylRS (Blight et al., 2004). The structure of tRNA Pyl differs from the classical tRNA structure since it displays a D-loop with only 5 nucleotides, an AC-stem with 6 bp, and a TΨC-loop without these three residues sequence. ...
Aminoacyl-tRNA synthetases (aaRSs) are essential and ubiquitous enzymes catalyzing formation of aminoacyl- tRNAs (aa-tRNAs) during protein synthesis. However, aaRSs are not limited to aa-tRNAs formation. Indeed, they evolved to form multl-protein complexes that acquired additional functions. S. cerevisiae contains the simplest eukaryotic multi-synthetase complex which is formed by the association of methionyl-tRNA synthetase (MetRS) and glutamyl-tRNA synthetase (GluRS) to the cytosolic anchoring protein Arc1.This complex (named AME) is highly dynamic depending on the nutritional conditions that the cells are facing, and the two associated aaRSs harbor additional functions that are essential for cell survival. In this PhD thesis, I have studied three different aspect of theyeast MetRS: (i) I created a new bifluorescent reporter to quantify endogenous mismethionylation mediated by the yeast MetRS. I also (ii) characterized a new truncated yeast MetRS isoform produced in vivo, and (iii) I analysed the relative importance of Arc1for cell surviving during the diauxic shift from fermentation to respiration.
... On the other hand, the structural diversity of ncAAs is limited by currently available Tyr and pyrro-Lys tRNA synthetases in SCS and the nonspecificity of endogenous aaRS in SPI. 49,53,354 The discovery of novel orthogonal aminoacyl-tRNA synthetase/tRNA pairs and the development of the corresponding aaRS is continuously expanding the application of ncAA-based protein engineering. 48,351−355 However, conventional negative and positive selections comprise mutagenesis libraries with 5−7 randomized positions covering only marginal fractions of the vast sequence space formed by up to 30 active site residues of aaRS. ...
The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chemical diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chemical modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and critical evaluation of the applications, recent advances, and technical breakthroughs in biocatalysis for three approaches: (i) chemical modification of cAAs, (ii) incorporation of ncAAs, and (iii) chemical modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.
... Decoding of in-frame amber stop codons (UAG), generally referred to as amber suppression, enables the translational incorporation of non-canonical amino acids (ncAAs) into target proteins in vitro and in vivo (1,2). The pyrrolysyl-tRNA synthetase (PylRS, encoded by PylS)/tRNA Pyl CUA (PylT, encoded by PylT) pair from Methanosarcina species is one of the most commonly used orthogonal translation systems (OTSs) to incorporate ncAAs at amber stop codons in bacteria (3)(4)(5), yeast (6), mammalian cells (7)(8)(9) and animals (10)(11)(12)(13). This expansion of the genetic code allows site-specific introduction of unique moieties into proteins including bioorthogonal handles for chemical conjugation (14) and photocrosslinkers (15,16) to rationally probe and control protein structure, dynamics, and function in living 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.
... Hodarchaeia encode a complete Pyl encoding system including all three Pyl biosynthesis proteins (PylB, PylC, PylD) and a pyrrolysyl-tRNA synthetase (PylS) to charge the pyrrolysine tRNA (tRNApyl, pylT) ( Fig. 4a) [45]. Unlike selenocysteine (Fig. 3b), no specific proteins or insertion sequences are required for the tRNApyl insertion, which has been proposed to directly compete with the translation termination release factor for UAG codons (Fig. 4b) [46]. ...
Asgardarchaeota have been proposed as the closest living relatives to eukaryotes, and a total of 72 metagenome-assembled genomes (MAGs) representing six primary lineages in this archaeal phylum have thus far been described. These organisms are predicted to be fermentative organoheterotrophs contributing to carbon cycling in sediment ecosystems. Here, we double the genomic catalogue of Asgardarchaeota by obtaining 71 MAGs from a range of habitats around the globe, including deep subsurface, shallow lake, and geothermal spring sediments. Phylogenomic inferences followed by taxonomic rank normalisation confirmed previously established Asgardarchaeota classes and revealed four novel lineages, two of which were consistently recovered as monophyletic classes. We therefore propose the names Candidatus Hodarchaeia class nov. and Cand. Jordarchaeia class nov., derived from the gods Hod and Jord in Norse mythology. Metabolic inference suggests that both novel classes represent methylotrophic acetogens, encoding the transfer of methyl groups, such as methylated amines, to coenzyme M with acetate as the end product in remnants of a methanogen-derived core metabolism. This inferred mode of energy conservation is predicted to be enhanced by genetic code expansions, i.e. recoding, allowing the incorporation of the rare 21st and 22nd amino acids selenocysteine (Sec) and pyrrolysine (Pyl). We found Sec recoding in Jordarchaeia and all other Asgardarchaeota classes, which likely benefit from increased catalytic activities of Sec-containing enzymes. Pyl recoding on the other hand is restricted to Hodarchaeia in the Asgardarchaeota, making it the first reported non-methanogenic lineage with an inferred complete Pyl machinery, likely providing this class with an efficient mechanism for methylamine utilisation. Furthermore, we identified enzymes for the biosynthesis of ester-type lipids, characteristic of Bacteria and Eukaryotes, in both novel classes, supporting the hypothesis that mixed ether-ester lipids are a shared feature among Asgardarchaeota.
... Free Pyl is synthesized inside the cell from two free Lys residues with the help of three enzymes encoded by pylB, pylC, and pylD (Gaston et al. 2011). The Pyl residue is then ligated to the tRNA Pyl by the PylRS (Blight et al. 2004). The UAG codon is read by the CUA anticodon of tRNA Pyl to reassign its meaning from a stop codon to Pyl. ...
The genetic code, once believed to be universal and immutable, is now known to contain many variations and is not quite universal. The basis for genome recoding strategy is genetic code variation that can be harnessed to improve cellular properties. Thus, genome recoding is a promising strategy for the enhancement of genome flexibility, allowing for novel functions that are not commonly documented in the organism in its natural environment. Here, the basic concept of genetic code and associated mechanisms for the generation of genetic codon variants, including biased codon usage, codon reassignment, and ambiguous decoding, are extensively discussed. Knowledge of the concept of natural genetic code expansion is also detailed. The generation of recoded organisms and associated mechanisms with basic targeting components, including aminoacyl-tRNA synthetase–tRNA pairs, elongation factor EF-Tu and ribosomes, are highlighted for a comprehensive understanding of this concept. The research associated with the generation of diverse recoded organisms is also discussed. The success of genome recoding in diverse multicellular organisms offers a platform for expanding protein chemistry at the biochemical level with non-canonical amino acids, genetically isolating the synthetic organisms from the natural ones, and fighting viruses, including SARS-CoV2, through the creation of attenuated viruses. In conclusion, genome recoding can offer diverse applications for improving cellular properties in the genome-recoded organisms.
... Near the methyltransferase gene cluster, genes encoding an amber suppressor tRNA, tRNA Pyl , and a novel class II aaRS, PylRS, were also found (pylT and pylS, respectively) [37]. Using chemically synthesized Pyl [9] acylation of Pyl to tRNA Pyl by PylRS was demonstrated [38,39]. These results established Pyl to be the 22nd co-translationally installed AA. ...
Within the broad field of synthetic biology, genetic code expansion (GCE) techniques
enable creation of proteins with an expanded set of amino acids. This may be invaluable
for applications in therapeutics, bioremediation, and biocatalysis. Central to GCE are
aminoacyl-tRNA synthetases (aaRSs) as they link a non-canonical amino acid (ncAA)
to their cognate tRNA, allowing ncAA incorporation into proteins on the ribosome.
The ncAA-acylating aaRSs and their tRNAs should not cross-react with 20 natural
aaRSs and tRNAs in the host, i.e., they need to function as an orthogonal translating system.
All current orthogonal aaRS•tRNA pairs have been engineered from naturally
occurring molecules to change the aaRS’s amino acid specificity or assign the tRNA
to a liberated codon of choice. Here we discuss the importance of orthogonality in
GCE, laboratory techniques employed to create designer aaRSs and tRNAs, and provide
an overview of orthogonal aaRS•tRNA pairs for GCE purposes.
... As for Sec, Pyl active site residues appear to be crucial for the activity of enzymes involved in methanogenesis [35]. Pyrrolysyl-tRNA Pyl is synthesized by direct pyrrolysylation of an amber-suppressor tRNA Pyl by the class IIc PylRS [36]. Pyl incorporation has become the first known example to date of direct aminoacylation of a tRNA with a non-standard proteinogenic aa. ...
... Free Pyl is synthesized inside the cell from two free Lys residues with the help of three enzymes encoded by pylB, pylC, and pylD (Gaston et al. 2011). The Pyl residue is then ligated to the tRNA Pyl by the PylRS (Blight et al. 2004). The UAG codon is read by the CUA anticodon of tRNA Pyl to reassign its meaning from a stop codon to Pyl. ...
Plants are very susceptible to pathogens and every year, 25% of crop loss is caused by various types of pathogens including viruses. Many different strategies are being used for developing resistance against virus infection, including RNA silencing, and the genome editing including CRISPR-Cas-9 but these may produce variants/recombinants and could cause the problems for future crops. Another promising approach named as genome recoding or rewriting would be a better potential tool for controlling viral infections in plants. It relies on the concepts of replacement of synonymous codons, change in codon bias, codon pair bias and dinucleotide content. Recoding of the genome does not alter the amino acid sequences but it affects the expression level and translation efficiency. In the present report, the concept of synonymous codons, the basics of genome recoding and the possible strategies to generate genome recoded organisms are provided in details. Viral attenuation has been achieved by consideration of dinucleotide bias and codon pair bias manipulations and used in the synthesis of vaccines against various types of pathogenic bacteria and viruses. The idea of the future scope of genome recoding for developing virus-resistant plants and their challenges for the same are also comprehensively discussed. Although genome recoding is not yet tested on plants, however it could be very helpful in controlling plant viral diseases. So, it is a novel emerging area of research for developing viral resistant plants and thus would help in minimizing the agricultural losses in the near future.
... As for Sec, Pyl active site residues appear to be crucial for the activity of enzymes involved in methanogenesis [35]. Pyrrolysyl-tRNA Pyl is synthesized by direct pyrrolysylation of an amber-suppressor tRNA Pyl by the class IIc PylRS [36]. Pyl incorporation has become the first known example to date of direct aminoacylation of a tRNA with a non-standard proteinogenic aa. ...
The aminoacylation reaction is one of most extensively studied cellular processes. The so-called “canonical” reaction is carried out by direct charging of an amino acid (aa) onto its corresponding transfer RNA (tRNA) by the cognate aminoacyl-tRNA synthetase (aaRS), and the canonical usage of the aminoacylated tRNA (aa-tRNA) is to translate a messenger RNA codon in a translating ribosome. However, four out of the 22 genetically-encoded aa are made “noncanonically” through a two-step or indirect route that usually compensate for a missing aaRS. Additionally, from the 22 proteinogenic aa, 13 are noncanonically used, by serving as substrates for the tRNA- or aa-tRNA-dependent synthesis of other cellular components. These nontranslational processes range from lipid aminoacylation, and heme, aa, antibiotic and peptidoglycan synthesis to protein degradation. This chapter focuses on these noncanonical usages of aa-tRNAs and the ways of generating them, and also highlights the strategies that cells have evolved to balance the use of aa-tRNAs between protein synthesis and synthesis of other cellular components.
... As in the case of selenocysteine, the special chemical properties of pyrrolysine are used in the methyltransferase active site, via a proposed methylammonium adduct that activates methylamines (Krzycki 2004). Unlike selenocysteine, pyrrolysine exists as a free metabolite and is biosynthesized by three enzymes (PylB, C and D) and it is charged by its unique synthetase, pyrrolysyl-tRNA synthetase, PylS, directly onto the tRNA Pyl (Blight et al. 2004) ( Figure 1D). Based upon the structure of its catalytic core, PylRS is classified as a Class II enzyme, although it possesses a unique mechanism of tRNA recognition. ...
The aminoacyl-tRNA synthetases are an essential and universally distributed family of enzymes that play a critical role in protein synthesis, pairing tRNAs with their cognate amino acids according to the genetic code. Synthetases help to ensure accurate translation of the genetic code by using both highly accurate cognate substrate recognition and stringent proofreading of non-cognate products. While alterations in the quality control mechanisms of synthetases are generally detrimental to cellular viability, recent studies suggest that in some instances such changes facilitate adaption to stress conditions. Beyond their central role in translation, synthetases are also emerging as keys players in an increasing number of other cellular processes, with far reaching consequences in health and disease. The biochemical versatility of the synthetases has also proven pivotal in efforts to expand the genetic code, further emphasizing the wide-ranging roles of the aminoacyl-tRNA synthetase family in synthetic and natural biology.
... Many other orthogonal pairs such as S. cerevisiae phenylalanyl-tRNA synthetase (Sc PheRS)-tRNA Phe (Furter 1998;Kwon et al. 2003Kwon et al. , 2006Kwon and Choi 2016), Sc tryptophanyl-tRNA synthetase (Sc TrpRS)-tRNA Trp (Blight et al. 2004;Neumann et al. 2008;Yanagisawa et al. 2008;Zeng et al. 2014;Fan et al. 2015) and Ec TyrRS-tRNA Tyr (Iraha et al. 2010;Italia et al. 2017) have been discovered and utilized efficiently in various bacteria for inserting ncAAs. ...
The genetic code consists of codons that code for 20 canonical amino acids and 3 termination signals and is an indispensable element of life. It controls all cellular activities through expression of genes. Advances in the genetic engineering techniques have enabled the incorporation of novel non-canonical amino acids (ncAAs) into proteins to confer distinct physical and chemical features. Currently, over 150 ncAAs have been site-specifically introduced into proteins with high efficiency using an orthogonal tRNA synthetase pair in various organisms. In this chapter, genetic code engineering and its advances, challenges and future opportunities have been explored towards biological, biomedical, therapeutic, industrial and biotechnological applications.
... The most reliable and well-established methodology to genetically encode NAAs in E. coli is based on the application of amber stop codon, UAG, the most rarely used stop codon [125,126], recognized by the suppressor tRNA ( Figure 5). The OTS is usually derived from tyrosyl-tRNA synthetase (TyrRS)/tRNATyr from Methanocaldococcus jannaschii [127], pyrrolysine-RS/tRNAPyl pair from Methanosarcina species [128][129][130], and others [118]. Recently the overall productivity of genetic code expansion in E. coli has been greatly improved due to tremendous progress in evolving more efficient orthogonal components and reducing or eliminating the negative effect of release factor 1, RF1, responsible for translation termination by binding to amber codon [131]. ...
Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.
... Pyrrolysin and selenocysteine are natural proteinogenic non-canonical amino acids that are not encoded by a sense codon. Pylspecific tRNA Pyl reads the UAG stop codon, whereas Secspecific tRNA Sec reads the UGA codon (61)(62)(63)(64). The Pyl trait is restricted to several microbes, mostly methanogenic archaea, which encode a tRNA Pyr (pylT) and the dedicated aa-tRNA synthetase (pylS). ...
During canonical translation, the ribosome moves along an mRNA from the start to the stop codon in exact steps of one codon at a time. The collinearity of the mRNA and the protein sequence is essential for the quality of the cellular proteome. Spontaneous errors in decoding or translocation are rare and result in a deficient protein. However, dedicated recoding signals in the mRNA can reprogram the ribosome to read the message in alternative ways. This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, -1 ribosome frameshifting and translational bypassing. Recoding events provide insights into alternative modes of ribosome dynamics that are potentially applicable to other non-canonical modes of prokaryotic and eukaryotic translation.
... These methods normally reassign the meaning of a stop codon, usually UAG, to the direct incorporation of an additional, non-canonical amino acid (ncAA). The most commonly used orthogonal pairs include the archaeal enzymes tyrosyl-tRNA syntetase (TyrRS) [13], the pyrrolysyl-tRNA synthetase (PylRS) [16,17], and the phosphoseryl-tRNA synthetase (SepRS) [18]. Balasuriya et al. used the phosphoserine system to produce highly active human kinases with programmed phosphorylation from facile Escherichia coli expression systems [7]. ...
Synthetic biology is a broad and emerging discipline that capitalizes on recent advances in molecular biology, genetics, protein and RNA engineering as well as omics technologies. Together these technologies have transformed our ability to reveal the biology of the cell and the molecular basis of disease. This Special Issue on “Synthetic RNA and DNA Programming” features original research articles and reviews, highlighting novel aspects of basic molecular biology and the molecular mechanisms of disease that were uncovered by the application and development of novel synthetic biology-driven approaches.
... In order to expand the repertoire and diversity of NAAs, several additional orthogonal pairs have been adapted for application in E. coli. These pairs include some derived from archaea, such as a lysine-RS/tRNA Lys pair from Pyrococcus horikoshii [63], a glutamine-RS/tRNA Glu pair from Methanosarcina mazei [64], pyrrolysine-RS/tRNA Pyl pair from Methanosarcina species [65][66][67]; as well as a heterologous pair consisting of a leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum and a mutant tRNA Leu derived from Halobacterium sp. [34], and pair consisting of P. horikoshii proline-RS and three engineered suppressors tRNA Pro from Archaeoglobus fulgidus [68]. ...
More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.
... The discovery of the pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA, tRNA Pyl , from Methanosarcina barkeri, which are capable of charging the 22nd natural amino acid, pyrrolysine, into proteins in response to an amber codon in methanogens, has unleashed a new era of protein engineering [1,2]. Since this discovery, the PylRS and its many variants have been obtained through design and/or evolution to charge a large number of non-natural amino acids, including more than 100 pyrrolysine and phenylalanine analogs, site-specifically into proteins in Escherichia coli as well as in mammalian cells for production of engineered proteins with enhanced properties [3,4]. ...
The orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and their variants have provided powerful tools for expanding the genetic code to allow for engineering of proteins with augmented structure and function not present in Nature. To expedite the discovery of novel pyrrolysyl-tRNA synthetase (PylRS) variants that can charge non-natural amino acids into proteins site-specifically, herein we report a streamlined protocol for rapid construction of the pyrrolysyl-tRNA synthetase library, selection of the functional PylRS mutants using fluorescence-activated cell sorting, and subsequent validation of the selected PylRS mutants through direct expression of the fluorescent protein reporter using a single bacterial strain. We expect that this protocol should be generally applicable to rapid identification of the functional PylRS mutants for charging a wide range of non-natural amino acids into proteins.
... Several aaRS/tRNA pairs have been developed to incorporate more than 200 different ncAAs in different species. For recombinant expression therapeutic proteins in E. coli, the most widely used aaRS/ tRNA pairs are the Archean TyrRS/tRNA Tyr pair from Methanocaldococcus jannaschii (MjTyrRS/tRNA Tyr ) [1][2][3][4] and the Archean pyrrolysine aaRS/tRNA Pyl pair (Mb/MmPylRS/tRNA Pyl ) [5][6][7][8][9]. The MjTyrRS/tRNA Tyr pair was the first genetic code expansion system developed for site-specific ncAAs incorporation in living cells in 2000 [1] and has now been widely used in prokaryotic genetic code expansion. ...
In nature, a limited, conservative set of amino acids are utilized to synthesize proteins. Genetic code expansion technique reassigns codons and incorporates noncanonical amino acids (ncAAs) through orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The past decade has witnessed the rapid growth in diversity and scope for therapeutic applications of this technology. Here, we provided an update on the recent progress using genetic code expansion in the following areas: antibody-drug conjugates (ADCs), bispecific antibodies (BsAb), immunotherapies, long-lasting protein therapeutics, biosynthesized peptides, engineered viruses and cells, as well as other therapeutic related applications, where the technique was used to elucidate the mechanisms of biotherapeutics and drug targets.
... Starting off these results, rapid process optimization for Alk by using 10 mM Alk instead of 30 mM Plk readily boosted maximum eGFP fluorescence intensities beyond the results obtained for Plk-eGFP, possibly reflecting a higher PylRS affinity for Alk in comparison to Plk. Nevertheless, both concentrations found for the optima (10 mM for Alk and 30 mM for Plk) were far beyond commonly applied uAA concentration, typically in the range of 1 mM [27][28][29][30], although higher uAA concentrations have already been linked to increased target protein concentration as estimated after expression and purification [31][32][33]. However, high uAA concentrationsas demonstrated for Alk and Plk hereare not cost-effective for large scale expression and are therefore still the limiting factor in genetic code expansion technology. ...
Background
Genetic code expansion has developed into an elegant tool to incorporate unnatural amino acids (uAA) at predefined sites in the protein backbone in response to an amber codon. However, recombinant production and yield of uAA comprising proteins are challenged due to the additional translation machinery required for uAA incorporation.
Results
We developed a microtiter plate-based high-throughput monitoring system (HTMS) to study and optimize uAA integration in the model protein enhanced green fluorescence protein (eGFP). Two uAA, propargyl-L-lysine (Plk) and (S)-2-amino-6-((2-azidoethoxy) carbonylamino) hexanoic acid (Alk), were incorporated at the same site into eGFP co-expressing the native PylRS/tRNAPylCUA pair originating from Methanosarcina barkeri in E. coli. The site-specific uAA functionalization was confirmed by LC-MS/MS analysis. uAA-eGFP production and biomass growth in parallelized E. coli cultivations was correlated to (i) uAA concentration and the (ii) time of uAA addition to the expression medium as well as to induction parameters including the (iii) time and (iv) amount of IPTG supplementation. The online measurements of the HTMS were consolidated by end point-detection using standard enzyme-linked immunosorbent procedures.
Conclusion
The developed HTMS is powerful tool for parallelized and rapid screening. In light of uAA integration, future applications may include parallelized screening of different PylRS/tRNAPylCUA pairs as well as further optimization of culture conditions.
Electronic supplementary material
The online version of this article (doi:10.1186/s13036-016-0031-6) contains supplementary material, which is available to authorized users.
... Methylornithine synthase (PylB) converts L-lysine to (3R)- 3-methyl-D-ornithine, which in turn is ligated with a second molecule of L-lysine to produce (2R, 3R)-3-methy- lornithyl-N 6 lysine, catalysed by (2R,3R)-3-methylornithyl- N 6 -lysine synthase (PylC); pyrrolysine synthase (PylD) converts (2R,3R)-3-methylornithyl-N 6 -lysine to pyrroly- sine [53] . Pyrrolysine-tRNA ligase (PylS) catalyses the aminocylation of tRNA (CUA) which itself is encoded by pylT [54]. The operon organization is conserved across the Methanomassiliicoccales (Fig. 6), suggesting pyrrolysine use is important for members of this order. ...
Methane emissions from agriculture represent around 9 % of global anthropogenic greenhouse emissions. The single largest source of this methane is animal enteric fermentation, predominantly from ruminant livestock where it is produced mainly in their fermentative forestomach (or reticulo-rumen) by a group of archaea known as methanogens. In order to reduce methane emissions from ruminants, it is necessary to understand the role of methanogenic archaea in the rumen, and to identify their distinguishing characteristics that can be used to develop methane mitigation technologies. To gain insights into the role of methylotrophic methanogens in the rumen environment, the genome of a methanogenic archaeon has been sequenced. This isolate, strain ISO4-H5, was isolated from the ovine rumen and belongs to the order Methanomassiliicoccales. Genomic analysis suggests ISO4-H5 is an obligate hydrogen-dependent methylotrophic methanogen, able to use methanol and methylamines as substrates for methanogenesis. Like other organisms within this order, ISO4-H5 does not possess genes required for the first six steps of hydrogenotrophic methanogenesis. Comparison between the genomes of different members of the order Methanomassiliicoccales revealed strong conservation in energy metabolism, particularly in genes of the methylotrophic methanogenesis pathway, as well as in the biosynthesis and use of pyrrolysine. Unlike members of Methanomassiliicoccales from human sources, ISO4-H5 does not contain the genes required for production of coenzyme M, and so likely requires external coenzyme M to survive.
Electronic supplementary material
The online version of this article (doi:10.1186/s40793-016-0183-5) contains supplementary material, which is available to authorized users.
... The 22nd amino acid Pyl, instead, to date was found only in archaea of the order Methanosarcinales and some Gram-positive bacteria such as Desulfitobacterium hafniense [21] , Desulfotomaculum acetoxidans, and Acetohalobium arabaticum [22] where it is primarily incorporated in methylamine methyltransferases in response to the amber stop codon (UAG). In contrast to Sec incorporation, Pyl insertion does not require a complex re-coding machinery: Pyl is charged directly onto a dedicated amber-suppressor tRNA Pyl CUA by a cognate pyrrolysyl-tRNA synthetase (PylRS) [23,24] . Although a pyrrolysine insertion sequence (PYLIS) was found in Pyl-containing genes of Methanosarcina acetivorans where it enhances the level of amber suppression, it seems generally not essential for Pyl incorporation [25,26] . ...
Over the past decade, genetic encoding of non-canonical amino acids (ncAAs) into proteins has emerged as a powerful tool in protein engineering. Recent progresses focus mostly on the incorporation of two or more ncAAs by using stop codon suppression methodologies, but little work has been done to recode one or more sense codons. The first part of this thesis describes follow up experiments to test whether an heterologous methionyl-tRNA synthetase (MetRS)/tRNAMet pair from an archeaon behaves orthogonal in Escherichia coli for the simultaneous incorporation of the methionine (Met) analogues azidohomoalanine (Aha) and ethionine (Eth) in response to the starting and internal sense codons, respectively. We determined that this was not the case. Therefore, other established orthogonal pairs were tested, of which the Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS) pair was found as the most promising candidate. Next, a new selection system based on amber stop codon suppression in the pfkA (phosphofructokinase I) gene was developed and optimized to screen an MmPylRS library. Upon screening, a mutant that was able to incorporate Met in response to stop codons was isolated. Further optimization of this system will allow the incorporation of Met analogues into targeted locations. In the second part of this thesis, the site-specific incorporation of ncAAs by amber codon suppression was explored to generate multiple sites for protein bioconjugation and immobilization. The lipase from Thermoanaerobacter thermohydrosulfuricus (TTL) was used as model protein because lipases are one of the most versatile biocatalysts employed in the industry. Two E. coli strains lacking release factor I (RF1) were evaluated for their suppression efficiencies at single and multiple amber codons in comparison with a standard expression strain. High incorporation of N-Propargyl-Lysine (Plk) was achieved at a specific permissive position on the TTL surface using a RF1-deficient strain, whereas suppression at multiple positions was suboptimal and dependent on the orthogonal pair, ncAA and expression vector employed. Incorporation of the ncAA Plk into TTL endowed the enzyme with an alkyne group for bioconjugation without impairing its activity. The alkyne group was then selectively and efficiently conjugated to azide-biotin via copper-catalyzed azide-alkyne cycloaddition (CuAAC), with retention of enzymatic activity. Attempts to achieve direct immobilization of biotinyl-TTL on Strep-Tactin® beads as well as alkynyl-TTL on azide-agarose beads, although unsuccessful, provided some insights that will guide further optimization endeavors. In summary, this work describes the first efforts towards the development of a genetic selection system for the reassignment of the Met sense N-terminal and internal codons to two different ncAAs in E. coli. It also provides insights into the potential use of site-specific incorporation of ncAAs into proteins and biocatalysts for applications such as bioconjugation and immobilization.
... The tRNA dedicated for genetically encoding the 22nd natural amino acid, pyrrolysine (Pyl), at the UAG amber codon, is a naturally occurring suppressor tRNA (Srinivasan et al., 2002). It was originally discovered in Methanosarcina barkeri where it is aminoacylated by its cognate pyrrolysyl-tRNA synthetase (PylRS) with Pyl (Blight et al., 2004;Hao et al., 2002;Polycarpo et al., 2004). Pyl-tRNA Pyl is then used as a substrate for ribosomal synthesis of Pyl-containing methyltransferases that are critical for the growth on methylamines of Methanosarcina species (Krzycki, 2005). ...
The utility of ribosomal incorporation of unnatural amino acids (AAs) in vivo is generally restricted by low efficiencies, even with the most widely used suppressor tRNAPyl. Because of the difficulties of studying incorporation in vivo, almost nothing is known about the limiting steps after tRNA charging. Here, we measured the kinetics of all subsequent steps using a purified E. coli translation system. Dipeptide formation from initiator fMet-tRNAfMet and tRNAPyl charged with allylglycine or methylserine displayed unexpectedly sluggish biphasic kinetics, ∼30-fold slower than for native substrates. The amplitude of the fast phases increased with increasing EF-Tu concentration, allowing measurement of Kd values of EF-Tu binding, both of which were ∼25-fold weaker than normal. However, binding could be increased ∼30-fold by lowering temperature. The fast phase rates were limited by the surprisingly ∼10-fold less efficient binding of EF-Tu:GTP:AA-tRNAPyl ternary complex to the ribosomes, not GTP hydrolysis or dipeptide formation. Furthermore, processivity was unexpectedly impaired as ∼40% of the dipeptidyl-tRNAPyl could not be elongated to tripeptide. Dipeptide formation was slow enough that termination due to misreading the UAG codon by non-cognate RF2 became very significant. This new understanding provides a framework for improving unnatural AA incorporation by amber suppression. This article is protected by copyright. All rights reserved
... We recently reported the direct biosynthesis of spin-labeled proteins in live Escherichia coli cells, overcoming the aforementioned limitations of in vitro spin labeling (Schmidt, Borbas, Drescher, & Summerer, 2014). This technique relies on the genetic encoding of the spin-labeled amino acid SLK-1 (Fig. 1A) in response to the amber stop codon (TAG) (Liu & Schultz, 2010) by an evolved, orthogonal tRNA Pyl /pyrrolysyl-tRNA-synthetase (PylRS) pair (Blight et al., 2004;Chen et al., 2009;Fekner, Li, Lee, & Chan, 2009;Gautier et al., 2010;Hoppmann et al., 2014;Li et al., 2013;Luo et al., 2014;Plass et al., 2012;Polycarpo et al., 2006;Pott, Schmidt, & Summerer, 2014;Schmidt & Summerer, 2013;Wan et al., 2010;Wan, Tharp, & Liu, 2014;Yanagisawa et al., 2008). This enables the cotranslational incorporation of SLK-1 at single and multiple userdefined sites of proteins with high efficiency and fidelity. ...
Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful approach to study the structure, dynamics, and interactions of proteins. The genetic encoding of the noncanonical amino acid spin-labeled lysine 1 (SLK-1) eliminates the need for any chemical labeling steps in SDSL-EPR studies and enables the investigation of native, endogenous proteins with minimal structural perturbation, and without the need to create unique reactive sites for chemical labeling. We report detailed experimental procedures for the efficient synthesis of SLK-1, the expression and purification of SLK-1-containing proteins under conditions that ensure maximal integrity of the nitroxide radical moiety, and procedures for intramolecular EPR distance measurements in proteins by double electron-electron resonance.
Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS·tRNAPyl pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of Nε-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with pEtZLys up to 57 ± 8% of that with TCO*Lys at high enzyme concentrations in the cell-free protein synthesis.
The cotranslational incorporation of pyrrolysine (Pyl), the 22nd proteinogenic amino acid, into proteins in response to the UAG stop codon represents an outstanding example of natural genetic code expansion. Genetic encoding of Pyl is conducted by the pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA, tRNAPyl. Owing to the high tolerance of PylRS toward diverse amino acid substrates and great orthogonality in various model organisms, the PylRS/tRNAPyl-derived pairs are ideal for genetic code expansion to insert noncanonical amino acids (ncAAs) into proteins of interest. Since the discovery of cellular components involved in the biosynthesis and genetic encoding of Pyl, synthetic biologists have been enthusiastic about engineering PylRS/tRNAPyl-derived pairs to rewrite the genetic code of living cells. Recently, considerable progress has been made in understanding the molecular phylogeny, biochemical properties, and structural features of the PylRS/tRNAPyl pair, guiding its further engineering and optimization. In this review, we cover the basic and updated knowledge of the PylRS/tRNAPyl pair's unique characteristics that make it an outstanding tool for reprogramming the genetic code. In addition, we summarize the recent efforts to create efficient and (mutually) orthogonal PylRS/tRNAPyl-derived pairs for incorporation of diverse ncAAs by genome mining, rational design, and advanced directed evolution methods.
Much recent interest has focused on developing proteins for human use, such as in medicine. However, natural proteins are made up of only a limited number of canonical amino acids with limited functionalities, and this makes the discovery of variants with some functions difficult. The ability to recombinantly express proteins containing non-canonical amino acids (ncAAs) with properties selected to impart the protein with desired properties is expected to dramatically improve the discovery of proteins with different functions. Perhaps the most straightforward approach to such an expansion of the genetic code is through expansion of the genetic alphabet, so that new codon/anticodon pairs can be created to assign to ncAAs. In this review, I briefly summarize more than 20 years of effort leading ultimately to the discovery of synthetic nucleotides that pair to form an unnatural base pair, which when incorporated into DNA, is stably maintained, transcribed and used to translate proteins in Escherichia coli . In addition to discussing wide ranging conceptual implications, I also describe ongoing efforts at the pharmaceutical company Sanofi to employ the resulting ‘semi-synthetic organisms' or SSOs, for the production of next-generation protein therapeutics.
This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.
The standard genetic code is a common language used by almost all organisms to translate nucleotide sequences from DNA and RNA into the amino acid sequences of proteins. However, the standard code is not used in all cases, and some organisms and organelles use variant codes.
• The genetic code is a basic grammar for translating genetic information into amino acid sequence of protein. Most organisms use the same genetic code, so‐called standard genetic code, assuming that all domains of life evolved from a single organism.
• Transfer RNA (tRNA)is an adaptor molecule that links the genetic code with its corresponding amino acid via codon‐anticodon interaction.
• A wide variety of chemical modifications in the tRNA anticodons play an important role in accurate protein synthesis by restriction, expansion and alteration of codon recognition.
• Natural instances of genetic code variation in several organisms and organelles violated a concept that the genetic code is frozen, but proposed it has continued to evolve.
• Recoding is an alternative usage of genetic code in some special biological contexts. including programmed ribosome frameshifting, stop‐codon read‐through, insertion of nonstandard amino acids and translational bypassing.
Natural organisms use a four-letter genetic alphabet that makes available 64 triplet codons, of which 61 are sense codons used to encode proteins with the 20 canonical amino acids. We have shown that the unnatural nucleotides dNaM and dTPT3 can pair to form an unnatural base pair (UBP) and allow for the creation of semisynthetic organisms (SSOs) with additional sense codons. Here, we report a systematic analysis of the unnatural codons. We identify nine unnatural codons that can produce unnatural protein with nearly complete incorporation of an encoded noncanonical amino acid (ncAA). We also show that at least three of the codons are orthogonal and can be simultaneously decoded in the SSO, affording the first 67-codon organism. The ability to incorporate multiple, different ncAAs site specifically into a protein should now allow the development of proteins with novel activities, and possibly even SSOs with new forms and functions. Systematic characterization of codons using the unnatural base pair dNaM·dTPT3 leads to the discovery of nine new functional codon–anticodon pairs, three of which are shown to be orthogonally decoded by ribosomes and allow incorporation of up to three noncanonical amino acids in Escherichia coli.
The emergence of robust methods to expand the genetic code allows incorporation of non-canonical amino acids into the polypeptide chain of proteins, thus making it possible to introduce unnatural chemical functionalities in enzymes. In this Perspective, we show how this powerful methodology is used to create enzymes with improved and novel, even new-to-nature, catalytic activities. We provide an overview of the current state of the art, and discuss the potential benefits of developing and using enzymes with genetically encoded non-canonical amino acids compared with enzymes containing only canonical amino acids. Genetic incorporation of unnatural amino acids into proteins broadens the possibilities of enzyme design. This Perspective discusses the exciting opportunities for biocatalysis offered by this method — such as new-to-nature catalytic activities — and potential benefits over classical enzyme engineering.
Supporting Information to Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression
Supporting Information to Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression
Large efforts have been devoted to the genetic code engineering in the past decade, aiming for unnatural amino acid mutagenesis. Recently, an increasing number of studies were reported to employ quadruplet codons to encode unnatural amino acids. We and others have demonstrated that the quadruplet decoding efficiency could be significantly enhanced by an extensive engineering of tRNAs bearing an extra nucleotide in their anticodon loops. In this work, we report the identification of tRNA mutants derived from directed evolution to efficiently decode a UAGA quadruplet codon in mammalian cells. Intriguingly, the trend of quadruplet codon decoding efficiency among the tested tRNA variants in mammalian cells was largely the same as that in E. coli. We subsequently demonstrate the utility of quadruplet codon decoding by the construction of the first HIV-1 mutant that lacks any in-frame amber nonsense codons and can be precisely activated by the decoding of a genomically embedded UAGA codon with an unnatural amino acid. Such conditionally activatable HIV-1 mutant can likely facilitate both fundamental investigations of HIV-1 as well as vaccine developments. The use of quadruplet codon, instead of an amber nonsense codon, to control HIV-1 replication has the advantage in that the correction of a frameshift caused by a quadruplet codon is much less likely than the reversion of an amber codon back into a sense codon in HIV-1.
In Escherichia coli, conventional amber and ochre stop codons can be separately targeted by engineered amber-suppressing Methanocaldococcus jannaschii tyrosyl-tRNA synthetase-tRNAPyl and ochre-suppressing Methanosarcina maezi pyrrolysyl-tRNA synthetase-tRNAPyl pairs for coding two different noncanonical amino acids in one protein gene. Here, we describe the application of this approach to produce a protein with two distinct chemical functionalites which can be selectively labeled with two fluorescent dyes.
The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNACys is first acylated with phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNACys by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, Euryarchaeota, TACK, and Asgard) and two groups of bacteria (“Candidatus Parcubacteria” and Chloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNACys species with cysteine in vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS⋅SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and Chloroflexi bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.
The superfamily of G protein-coupled receptors (GPCRs) mediates a wide range of physiological responses and serves as an important category of drug targets. Earlier biochemical and biophysical studies have shown that GPCRs exist temporally in an ensemble of interchanging conformations. Single-molecule techniques are ideally suited to understand the dynamic signaling and conformational complexity of G protein-coupled receptors (GPCRs). Here, we review the progress in single-molecule studies on GPCRs. We introduce the fundamental technical aspects of single-molecule fluorescence. We also survey the methodologies for labeling GPCRs with biophysical probes, particularly fluorescent dyes, and highlight the relevant chemical biology innovations that can be instrumental for studying GPCRs. Finally, we illustrate how the optical techniques and the labeling schemes have been combined to investigate GPCR signaling and dynamics at the single-molecule level.
The essential feature of the genetic code is the strict one-to-one correspondence between codons and amino acids. The canonical code consists of three stop codons and 61 sense codons that encode 20% of the amino acid repertoire observed in nature. It was originally designated as immutable and universal due to its conservation in most organisms, but sequencing of genes from the human mitochondrial genomes revealed deviations in codon assignments. Since then, alternative codes have been reported in both nuclear and mitochondrial genomes and genetic code engineering has become an important research field. Here, we review the most recent concepts arising from the study of natural non-standard genetic codes with special emphasis on codon re-assignment strategies that are relevant to engineering genetic code in the laboratory. Recent tools for synthetic biology and current attempts to engineer new codes for incorporation of non-standard amino acids are also reviewed in this article.
Understanding the interactions that drive the fidelity of the genetic code and the limits to which modifications can be made without breaking the translational system has practical implications for understanding the molecular mechanisms of evolution as well as expanding the set of encodable amino acids, particularly those with chemistries not provided by Nature. Because 61 sense codons encode 20 amino acids, reassigning the meaning of sense codons provides an avenue for biosynthetic modification of proteins, furthering both fundamental and applied biochemical research. We developed a simple screen that exploits the absolute requirement for fluorescence of an active site tyrosine in green fluorescent protein (GFP) to probe the pliability of the degeneracy of the genetic code. Our screen monitors the restoration of the fluorophore of GFP by incorporation of a tyrosine in response to a sense codon typically assigned another meaning in the genetic code. We evaluated sense codon reassignment at 4 of the 21 sense codons read through wobble interactions in E. coli using the Methanocaldococcus jannaschii (M. jannaschii) orthogonal tRNA/aminoacyl tRNA synthetase pair originally developed and commonly used for amber stop codon suppression. By changing only the anticodon of the orthogonal tRNA, sense codon reassignment efficiencies between 1% (Phe UUU) and 6% (Lys AAG) were achieved. Each of the orthogonal tRNAs preferentially decoded the codon traditionally read via a wobble interaction in E. coli with the exception of the orthogonal tRNA with an AUG anticodon, which incorporated tyrosine in response to both the His CAU and His CAC codons with approximately equal frequency. We applied our screen in a high throughput manner to evaluate a 109 member combined tRNA/aminoacyl tRNA synthetase library to identify improved sense codon reassigning variants for the Lys AAG codon. A single, rapid screen with the ability to broadly evaluate reassignable codons will facilitate identification and improvement of the combinations of sense codons and orthogonal pairs that display efficient reassignment.