Valorization of Biomasses from Energy Crops for the Discovery of Novel Thermophilic Glycoside Hydrolases through Metagenomic Analysis.

The increasing interest for environmentally friendly technologies is driving the transition from fossil-based economy to bioeconomy. A key enabler for circular bioeconomy is to valorize renewable biomasses as feedstock to extract high value-added chemicals. Within this transition the discovery and the use of robust biocatalysts to replace toxic chemical catalysts play a significant role as technology drivers. To meet both the demands, we performed microbial enrichments on two energy crops, used as low-cost feed for extremophilic consortia. A culture-dependent approach coupled to metagenomic analysis led to the discovery of more than 300 glycoside hydrolases and to characterize a new α-glucosidase from an unknown hyperthermophilic archaeon. Aglu1 demonstrated to be the most active archaeal GH31 on 4Np-α-Glc and it showed unexpected specificity vs. kojibiose, revealing to be a promising candidate for biotechnological applications such as the liquefaction/saccharification of starch.

Carnitine is a pharmacological allosteric chaperone of the human lysosomal α-glucosidase.

Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient's enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.

Xyloglucan Oligosaccharides Hydrolysis by Exo-Acting Glycoside Hydrolases from Hyperthermophilic Microorganism Saccharolobus solfataricus.

In the field of biocatalysis and the development of a bio-based economy, hemicellulases have attracted great interest for various applications in industrial processes. However, the study of the catalytic activity of the lignocellulose-degrading enzymes needs to be improved to achieve the efficient hydrolysis of plant biomasses. In this framework, hemicellulases from hyperthermophilic archaea show interesting features as biocatalysts and provide many advantages in industrial applications thanks to their stability in the harsh conditions encountered during the pretreatment process. However, the hemicellulases from archaea are less studied compared to their bacterial counterpart, and the activity of most of them has been barely tested on natural substrates. Here, we investigated the hydrolysis of xyloglucan oligosaccharides from two different plants by using, both synergistically and individually, three glycoside hydrolases from Saccharolobus solfataricus: a GH1 ß-gluco-/ß-galactosidase, a α-fucosidase belonging to GH29, and a α-xylosidase from GH31. The results showed that the three enzymes were able to release monosaccharides from xyloglucan oligosaccharides after incubation at 65 °C. The concerted actions of ß-gluco-/ß-galactosidase and the α-xylosidase on both xyloglucan oligosaccharides have been observed, while the α-fucosidase was capable of releasing all α-linked fucose units from xyloglucan from apple pomace, representing the first GH29 enzyme belonging to subfamily A that is active on xyloglucan.

Transcript Regulation of the Recoded Archaeal α-l-Fucosidase In Vivo.

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed "recoding". In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for -1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed -1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed.

Spatial Metagenomics of Three Geothermal Sites in Pisciarelli Hot Spring Focusing on the Biochemical Resources of the Microbial Consortia.

Terrestrial hot springs are of great interest to the general public and to scientists alike due to their unique and extreme conditions. These have been sought out by geochemists, astrobiologists, and microbiologists around the globe who are interested in their chemical properties, which provide a strong selective pressure on local microorganisms. Drivers of microbial community composition in these springs include temperature, pH, in-situ chemistry, and biogeography. Microbes in these communities have evolved strategies to thrive in these conditions by converting hot spring chemicals and organic matter into cellular energy. Following our previous metagenomic analysis of Pisciarelli hot springs (Naples, Italy), we report here the comparative metagenomic study of three novel sites, formed in Pisciarelli as result of recent geothermal activity. This study adds comprehensive information about phylogenetic diversity within Pisciarelli hot springs by peeking into possible mechanisms of adaptation to biogeochemical cycles, and high applicative potential of the entire set of genes involved in the carbohydrate metabolism in this environment (CAZome). This site is an excellent model for the study of biodiversity on Earth and biosignature identification, and for the study of the origin and limits of life.

GlcNAc De-N-Acetylase from the Hyperthermophilic Archaeon Sulfolobus solfataricus.

Sulfolobus solfataricus is an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group of Sulfolobales, N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of the N-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood in Archaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening of S. solfataricus extracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified in Archaea Mass spectrometry analysis of the purified activity showed a protein encoded by the sso2901 gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed in S. solfataricus extracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream of sso2901 (sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism in S. solfataricusIMPORTANCE In this study, a preliminary screening of cellular extracts of S. solfataricus allowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism of Archaea.

Identification of a novel esterase from the thermophilic bacterium Geobacillus thermodenitrificans NG80-2.

In the framework of the discovery of new thermophilic enzymes of potential biotechnological interest, we embarked in the characterization of a new thermophilic esterase from the thermophilic bacterium Geobacillus thermodenitrificans. The phylogenetic analysis of the GTNG_0744 esterase indicated that the sequence belongs to the enterochelin/enterobactin esterase group, which have never been recognized as a family in the lipases/esterase classification. These enzymes catalyze the last step in the acquisition of environmental Fe3+ through siderophore hydrolysis. In silico analysis revealed, for the first time, that the machinery for the uptake of siderophores is present in G. thermodenitrificans. The purified recombinant enzyme, EstGtA3, showed different substrate specificity from known enterochelin/enterobactin esterases, recognizing short chain esters with a higher specificity constant for 4-NP caprylate. The enzyme does not require cofactors for its activity, is active in the pH range 7.0-8.5, has highest activity at 60 °C and is 100% stable when incubated for 16 h at 55 °C. DTT, ß-mercaptoethanol and Triton X-100 have an activating effect on the enzymatic activity. Organic solvents have in general a negative effect on the enzyme, but n-hexane is a strong activator up to 150, making EstGtA3 a good candidate for applications in biotechnology.

Introducing transgalactosylation activity into a family 42 ß-galactosidase.

Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases-mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a ß-galactosidase from Alicyclobacillus acidocaldarius in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases.

Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes.

BACKGROUND: The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-ß-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a ß-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-ß-D-xylosidic linkages of xylans and the hydrolysis of (1-4)-ß-D-xylans to remove successive D-xylose residues from the non-reducing termini. RESULTS: We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. CONCLUSION: Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.

The identification and molecular characterization of the first archaeal bifunctional exo-ß-glucosidase/N-acetyl-ß-glucosaminidase demonstrate that family GH116 is made of three functionally distinct subfamilies.

BACKGROUND: ß-N-acetylhexosaminidases, which are involved in a variety of biological processes including energy metabolism, cell proliferation, signal transduction and in pathogen-related inflammation and autoimmune diseases, are widely distributed in Bacteria and Eukaryotes, but only few examples have been found in Archaea so far. However, N-acetylgluco- and galactosamine are commonly found in the extracellular storage polymers and in the glycans decorating abundantly expressed glycoproteins from different Crenarchaeota Sulfolobus sp., suggesting that ß-N-acetylglucosaminidase activities could be involved in the modification/recycling of these cellular components. METHODS: A thermophilic ß-N-acetylglucosaminidase was purified from cellular extracts of S. solfataricus, strain P2, identified by mass spectrometry, and cloned and expressed in E. coli. Glycosidase assays on different strains of S. solfataricus, steady state kinetic constants, substrate specificity analysis, and the sensitivity to two inhibitors of the recombinant enzyme were also reported. RESULTS: A new ß-N-acetylglucosaminidase from S. solfataricus was unequivocally identified as the product of gene sso3039. The detailed enzymatic characterization demonstrates that this enzyme is a bifunctional ß-glucosidase/ß-N-acetylglucosaminidase belonging to family GH116 of the carbohydrate active enzyme (CAZy) classification. CONCLUSIONS: This study allowed us to propose that family GH116 is composed of three subfamilies, which show distinct substrate specificities and inhibitor sensitivities. GENERAL SIGNIFICANCE: The characterization of SSO3039 allows, for the first time in Archaea, the identification of an enzyme involved in the metabolism ß-N-acetylhexosaminide, an essential component of glycoproteins in this domain of life, and substantially increases our knowledge on the functional role and phylogenetic relationships amongst the GH116 CAZy family members.

RNA editing and modifications of RNAs might have favoured the evolution of the triplet genetic code from an ennuplet code.

Here we suggest that the origin of the genetic code, that is to say, the birth of first mRNAs has been triggered by means of a widespread modification of all RNAs (proto-mRNAs and proto-tRNAs), as today observed in the RNA editing and in post-transcriptional modifications of RNAs, which are considered as fossils of this evolutionary stage of the genetic code origin. We consider also that other mechanisms, such as the trans-translation and ribosome frameshifting, could have favoured the transition from an ennuplet code to a triplet code. Therefore, according to our hypothesis all these mechanisms would be reflexive of this period of the evolutionary history of the genetic code.

Pharmacological enhancement of α-glucosidase by the allosteric chaperone N-acetylcysteine.

Pompe disease (PD) is a metabolic myopathy due to the deficiency of the lysosomal enzyme α-glucosidase (GAA). The only approved treatment for this disorder, enzyme replacement with recombinant human GAA (rhGAA), has shown limited therapeutic efficacy in some PD patients. Pharmacological chaperone therapy (PCT), either alone or in combination with enzyme replacement, has been proposed as an alternative therapeutic strategy. However, the chaperones identified so far also are active site-directed molecules and potential inhibitors of target enzymes. We demonstrated that N-acetylcysteine (NAC) is a novel allosteric chaperone for GAA. NAC improved the stability of rhGAA as a function of pH and temperature without disrupting its catalytic activity. A computational analysis of NAC-GAA interactions confirmed that NAC does not interact with GAA catalytic domain. NAC enhanced the residual activity of mutated GAA in cultured PD fibroblasts and in COS7 cells overexpressing mutated GAA. NAC also enhanced rhGAA efficacy in PD fibroblasts. In cells incubated with NAC and rhGAA, GAA activities were 3.7-8.7-fold higher than those obtained in cells treated with rhGAA alone. In a PD mouse model the combination of NAC and rhGAA resulted in better correction of enzyme activity in liver, heart, diaphragm and gastrocnemia, compared to rhGAA alone.

Archaea as a Model System for Molecular Biology and Biotechnology.

Archaea represents the third domain of life, displaying a closer relationship with eukaryotes than bacteria. These microorganisms are valuable model systems for molecular biology and biotechnology. In fact, nowadays, methanogens, halophiles, thermophilic euryarchaeota, and crenarchaeota are the four groups of archaea for which genetic systems have been well established, making them suitable as model systems and allowing for the increasing study of archaeal genes' functions. Furthermore, thermophiles are used to explore several aspects of archaeal biology, such as stress responses, DNA replication and repair, transcription, translation and its regulation mechanisms, CRISPR systems, and carbon and energy metabolism. Extremophilic archaea also represent a valuable source of new biomolecules for biological and biotechnological applications, and there is growing interest in the development of engineered strains. In this review, we report on some of the most important aspects of the use of archaea as a model system for genetic evolution, the development of genetic tools, and their application for the elucidation of the basal molecular mechanisms in this domain of life. Furthermore, an overview on the discovery of new enzymes of biotechnological interest from archaea thriving in extreme environments is reported.

Glycoside hydrolases from (hyper)thermophilic archaea: structure, function, and applications.

(Hyper)thermophilic archaeal glycosidases are enzymes that catalyze the hydrolysis of glycosidic bonds to break down complex sugars and polysaccharides at high temperatures. These enzymes have an unique structure that allows them to remain stable and functional in extreme environments such as hot springs and hydrothermal vents. This review provides an overview of the current knowledge and milestones on the structures and functions of (hyper)thermophilic archaeal glycosidases and their potential applications in various fields. In particular, this review focuses on the structural characteristics of these enzymes and how these features relate to their catalytic activity by discussing different types of (hyper)thermophilic archaeal glycosidases, including ß-glucosidases, chitinase, cellulases and α-amylases, describing their molecular structures, active sites, and mechanisms of action, including their role in the hydrolysis of carbohydrates. By providing a comprehensive overview of (hyper)thermophilic archaeal glycosidases, this review aims to stimulate further research into these fascinating enzymes.

Novel GH109 enzymes for bioconversion of group A red blood cells to the universal donor group O.

Glycoside hydrolases (GHs) have been employed for industrial and biotechnological purposes and often play an important role in new applications. The red blood cell (RBC) antigen system depends on the composition of oligosaccharides on the surface of erythrocytes, thus defining the ABO blood type classification. Incorrect blood transfusions may lead to fatal consequences, making the availability of the correct blood group critical. In this regard, it has been demonstrated that some GHs may be helpful in the conversion of groups A and B blood types to produce group O universal donor blood. GHs belonging to the GH109 family are of particular interest for this application due to their ability to convert blood from group A to group O. This work describes the biochemical characterisation of three novel GH109 enzymes (NAg68, NAg69 and NAg71) and the exploration of their ability to produce enzymatically converted RBCs (ECO-RBC). The three enzymes showed superior specificity on pNP-α-N-acetylgalactosamine compared to previously reported GH109 enzymes. These novel enzymes were able to act on purified antigen-A trisaccharides and produce ECO-RBC from human donor blood. NAg71 converted type A RBC to group O with increased efficiency in the presence of dextran compared to a commercially available GH109, previously used for this application.

Glycosynthases as tools for the production of glycan analogs of natural products.

Oligo-, polysaccharides, and glycoconjugates are a relevant part of the bioactive components of the natural products exploited in therapeutics, diagnostics, food additives, and biomaterials. Glycans are directly involved in important biological processes, such as immunostimulation, anti-inflammatory, antioxidant, and chemoprotectant actions and/or are crucial for their activity, by modulating target recognition, stability, and pharmacokinetics. On the other hand, carbohydrate extracts used for functional studies are rather heterogeneous and lack structural information because of their intrinsic complexity hampering purification and characterization. Therefore, methods for glycoside synthesis and modification are urgently needed. Recently, glycosynthases, engineered glycoside hydrolases with no hydrolytic activity that synthesize glycans in quantitative yields, were introduced. Here we will illustrate how the glycosynthases described so far might be exploited for the production of glycan analogs of natural products and their enormous potential in this field.

Translational recoding in archaea.

Translational recoding includes a group of events occurring during gene translation, namely stop codon readthrough, programmed ±1 frameshifting, and ribosome bypassing, which have been found in organisms from all domains of life. They serve to regulate protein expression at translational level and represent a relatively less known exception to the traditional central 'dogma' of biology that information flows as DNA→RNA→protein and that it is stored in a co-linear way between the 5'→3' of nucleic acids and N→C-terminal of polypeptides. In archaea, in which translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, only one case of programmed -1 frameshifting has been reported so far and further examples, although promising, have not been confirmed yet. We here summarize the current state-of-the-art of this field that, especially in archaea, has relevant implications for the physiology of life in extreme environments and for the origin of life.

A new archaeal beta-glycosidase from Sulfolobus solfataricus: seeding a novel retaining beta-glycan-specific glycoside hydrolase family along with the human non-lysosomal glucosylceramidase GBA2.

Carbohydrate active enzymes (CAZymes) are a large class of enzymes, which build and breakdown the complex carbohydrates of the cell. On the basis of their amino acid sequences they are classified in families and clans that show conserved catalytic mechanism, structure, and active site residues, but may vary in substrate specificity. We report here the identification and the detailed molecular characterization of a novel glycoside hydrolase encoded from the gene sso1353 of the hyperthermophilic archaeon Sulfolobus solfataricus. This enzyme hydrolyzes aryl beta-gluco- and beta-xylosides and the observation of transxylosylation reactions products demonstrates that SSO1353 operates via a retaining reaction mechanism. The catalytic nucleophile (Glu-335) was identified through trapping of the 2-deoxy-2-fluoroglucosyl enzyme intermediate and subsequent peptide mapping, while the general acid/base was identified as Asp-462 through detailed mechanistic analysis of a mutant at that position, including azide rescue experiments. SSO1353 has detectable homologs of unknown specificity among Archaea, Bacteria, and Eukarya and shows distant similarity to the non-lysosomal bile acid beta-glucosidase GBA2 also known as glucocerebrosidase. On the basis of our findings we propose that SSO1353 and its homologs are classified in a new CAZy family, named GH116, which so far includes beta-glucosidases (EC 3.2.1.21), beta-xylosidases (EC 3.2.1.37), and glucocerebrosidases (EC 3.2.1.45) as known enzyme activities.

A novel alpha-D-galactosynthase from Thermotoga maritima converts beta-D-galactopyranosyl azide to alpha-galacto-oligosaccharides.

The large-scale production of oligosaccharides is a daunting task, hampering the study of the role of glycans in vivo and the testing of the efficacy of novel glycan-based drugs. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, are becoming important chemo-enzymatic tools for the production of oligosaccharides. However, while ß-glycosynthase can be produced with a rather well-established technology, examples of α-glycosynthases are thus far limited only to enzymes from glycoside hydrolase 29 (GH29), GH31 and GH95 families. α-L-Fucosynthases from GH29 use convenient glycosyl azide derivatives as a strategic alternative to glycosyl fluoride donors. However, the general applicability of this method to other α-glycosynthases is not trivial and remains to be confirmed. Here, ß-D-galactopyranosyl azide was converted to α-galacto-oligosaccharides with good yields and high regioselectivity, catalyzed by a novel α-galactosynthase based on the GH36 α-galactosidase from the hyperthermophilic bacterium Thermotoga maritima. These results open a new avenue to the practical synthesis of biologically interesting α-galacto-oligosaccharides and demonstrate more widespread use of ß-glycosyl-azide as donors, confirming their utility to expand the repertoire of glycosynthases.

Programmed Deviations of Ribosomes From Standard Decoding in Archaea.

Genetic code decoding, initially considered to be universal and immutable, is now known to be flexible. In fact, in specific genes, ribosomes deviate from the standard translational rules in a programmed way, a phenomenon globally termed recoding. Translational recoding, which has been found in all domains of life, includes a group of events occurring during gene translation, namely stop codon readthrough, programmed ± 1 frameshifting, and ribosome bypassing. These events regulate protein expression at translational level and their mechanisms are well known and characterized in viruses, bacteria and eukaryotes. In this review we summarize the current state-of-the-art of recoding in the third domain of life. In Archaea, it was demonstrated and extensively studied that translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, and only one case of programmed -1 frameshifting has been reported so far in Saccharolobus solfataricus P2. However, further putative events of translational recoding have been hypothesized in other archaeal species, but not extensively studied and confirmed yet. Although this phenomenon could have some implication for the physiology and adaptation of life in extreme environments, this field is still underexplored and genes whose expression could be regulated by recoding are still poorly characterized. The study of these recoding episodes in Archaea is urgently needed.