# An archaeal genetic code with all TAG codons as pyrrolysine

Veronika Kivenson, Samantha Peters, Guillaume Borrel, Aleksandr Kivenson, Leah Roe, Noah Hamlish, Khaled Fadhlaoui, Alanna Schepartz, Simonetta Gribaldo, Robert Hettich, et al.

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## To cite this version:

Veronika Kivenson, Samantha Peters, Guillaume Borrel, Aleksandr Kivenson, Leah Roe, et al. An archaeal genetic code with all TAG codons as pyrrolysine. 2025. pasteur-05373021

HAL Id: pasteur-05373021

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An archaeal genetic code with all TAG codons as pyrrolysine

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Veronika Kivenson¹, Samantha L. Peters², Guillaume Borrel³, Aleksandr Kivenson⁴, Leah T.

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Roe⁵, Noah X. Hamlish⁶, Khaled Fadhlaoui⁷,⁸, Alanna Schepartz⁵,⁶,⁹,¹⁰, Simonetta Gribaldo³,

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Robert L. Hettich², Jillian F. Banfield¹,¹¹,¹²,¹³,¹⁴

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Corresponding authors

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Correspondence to kivenson@berkeley.edu and jbanfield@berkeley.edu

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Affiliations

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1. Innovative Genomics Institute, University of California, Berkeley, CA, USA

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2. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA

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3. Institut Pasteur, Université Paris Cité, Unit Evolutionary Biology of the Microbial Cell, Paris,

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France

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4. Independent Researcher, New York, NY, USA

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5. Department of Chemistry, University of California Berkeley, Berkeley, CA, USA

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6. Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA,

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USA

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7. Université Clermont Auvergne, CNRS, Laboratoire Microorganismes : Génome et

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Environnement, Clermont-Ferrand, France

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8. Université Clermont Auvergne, UMR 454 MEDIS UCA-INRAE, Clermont-Ferrand, France

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9. Chan Zuckerberg Biohub, San Francisco, CA, USA

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10. Arc Institute, Palo Alto, CA, USA

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11. Earth and Planetary Science, University of California, Berkeley, CA, USA

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12. Department of Environmental Science Policy, and Management, University of California,

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Berkeley, Berkeley, CA, USA

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13. Earth and Environmental Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA,

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USA

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14. Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
70 without Pyl machinery use TAG as a stop codon, and those with the Pyl machinery are believed to use TAG as a stop codon, except in the very few enzymes in which Pyl is known to occur.
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72 Additionally, Pyl has been hypothesized to occur within proteins when internal TAG codons were observed during *in silico* analysis(22, 33, 34). However, incorporation has been experimentally confirmed only in five proteins (mono-, di-, tri-methylamine methyltransferases,
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74 tRNA guanylyltransferase, and PylB) (10, 18, 35, 36). The archaeon *Methanosarcina acetivorans* has five additional proteins presumed to contain Pyl because their detected molecular masses are consistent with TAG readthrough(18). The authors suggest that TAG may not be selectively reassigned in *M. acetivorans*(18), but genome-wide incorporation of Pyl at this codon was not established.
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81 We asked if some archaea broadly incorporate Pyl into proteins using an alternative genetic code, and whether these genomes would yield biotechnologically relevant PylRS/tRNA<sup>Pyl</sup> pairs.
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83 Our analysis revealed that organisms within two major groups of Archaea contain multiple genes with internal in-frame TAG codons. Using global and targeted proteomics, we analyzed two such Archaea, a human gut and a marine isolate, and confirmed in both cases the genome-wide incorporation of Pyl at TAG codons, including within multiple enzyme classes. On this basis we propose the existence of a previously unrecognized genetic code, currently confined to archaea, and designate it as Genetic Code 34. We confirmed that five PylRS/tRNA<sup>Pyl</sup> pairs predicted by our analysis efficiently introduce Pyl analogs into proteins in *E. coli*. This work confirms that Genetic Code 34 organisms can add distinct “parts” to the genetic code expansion toolbox and facilitate the synthesis of new-to-nature heteropolymers. We find that the distribution of Genetic Code 34 among archaea is not monophyletic, which is consistent with multiple independent code transitions and hints that codon reassignment to Pyl could be facile. We infer features indicative of recoding that occurred recently vs. in the distant past and propose a series of steps that may lead to genetic code transition.
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## Results

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### Unusual phylogenetic distribution of Pyl

100 To investigate the phylogenetic distribution of archaea with Pyl machinery (Pyl-encoding archaea; Pyl archaea), we augmented a collection of genomes from all families known to incorporate Pyl, together with additional taxa that we determined carry the Pyl machinery (Table S1-S3). The majority of Pyl archaea belong to the Halobacteriota (Methanosarcinia, Archaeoglobi and Methanonatronarchaia) and Thermoplasmatota (Methanomassiliicocci) phyla, with few others within the phyla of Asgardarcheota, Thermoproteota, and Hydrothermarchaeota (Fig. 1, Table S3, S5). Notably, organisms that encode Pyl machinery have a discontinuous phylogenetic distribution (Fig. 1), sometimes even at the genus level. For example, the Pyl pathway is encoded and actively utilized by a single member of the Archaeoglobus (Ca. Methanoglobus hypatiae LCB24)(24). The proteins from this genome share 98.6% average
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amino acid identity (AAI) with Archaeoglobi LCB024-003(24), yet LCB024-003 does not
encode Pyl. In a similar way, Pyl is limited to a single family within the Asgardarcheota and
Hydrothermarchaeota phyla, and in the Ca. Methanomethylicus genus, only two of five archaea
encode Pyl.

Given the intermixing of closely related archaea that do and do not encode the Pyl machinery, we compared the phylogenies of the Pyl machinery (Fig. 2) to that of the organisms (Fig. 1). Consistent with prior suggestions(37, 38), discordance between the tree structures provides evidence for lateral transfer of the Pyl genes. For example, MSBL1 archaea (Hadarchaeota) likely gained their Pyl machinery through horizontal gene transfer from Methanonatronarchaeia, and "*Ca*. Methanoglobus" from a Thermoproteota.

## TAG frequency varies among Pyl archaea

Given that organisms almost always interpret TAG as a stop codon(39), we reasoned that there
may be detrimental consequences associated with the acquisition of a Pyl machinery, such as the
erroneous incorporation of Pyl and readthrough of the normal gene end. It is also possible that
TAG codons possess a dual meaning in organisms that carry a Pyl machinery, however there
exist no validated mechanisms for deciphering a dual-message TAG codon. Initial studies
suggested that the presence of an RNA stem loop immediately downstream of the TAG codon (a
Pyl insertion sequence, or PYLIS) is required to interpret a TAG codon as Pyl and not as
stop(16, 17). However, the presence of a PYLIS was later shown to have no effect on levels of
Pyl incorporation in E. coli(40) and no PYLIS or PYLIS-like structures could be detected in a
subset of known Pyl-containing methylamine methyltransferase genes in archaea(18, 19).
Absence of regulatory sequences is consistent with evolutionary expectations because
maintaining additional contextual requirements like a PYLIS for recoding would be inefficient,
and unlike the SECIS-binding protein used for recognition of the SECIS, no counterpart
"PYLIS-binding protein" is required when the Pyl system is introduced in E. coli.

One strategy that would avoid or minimize the need to decipher a dual-message TAG codon is
simply to eliminate its use as a stop codon. In fact, it has long been thought that low TAG
frequency (≤ 5%) represents a hallmark of archaea that encode a Pyl machinery(19, 20), yet a
few outliers have been reported(22, 25, 29). We calculated the percentage of genes with a TAG
codon in the genomes of Pyl archaea, and find that the TAG frequencies are always high (>15%)
among Pyl archaea that are intermixed phylogenetically with genomes that lack the Pyl
machinery (Fig. 1, Table S5). Thus, we conclude that low TAG frequency is not a core feature
across Pyl archaea. The discrepancy from expected TAG content in many Pyl archaea highlights
the need to determine the distribution of TAG codons and investigate how they are used.

## TAG is recoded genome-wide as a sense codon
To determine how TAG is used by Pyl archaea, we first predicted the genes in each organism using Genetic Code 11, which is standard for bacteria and archaea. We then re-predicted all TAG codons as sense codons and classified the resulting genes into four categories (Fig. 3, Supplementary Fig. 1). Category 1 genes contain one or more TAG codons in which the sequences before and after the TAG codon possess >30% sequence identity to a known protein (see Methods). In addition, readthrough of the TAG codon in Category 1 genes extends the protein sequence by >15 amino acids (aa). Based on this analysis, we predict that genes in Category 1 interpret the TAG as an internal sense codon. Category 2 genes were characterized by short extensions of ≤15 aa, and had no overlap with sense codon(s) of an adjacent gene. This scenario was originally thought to be the main outcome of TAG readthrough (19). Genes in Category 3 were characterized by an extension of >15 aa after TAG readthrough (sequence extension lacks similarity with known genes) and/or a short (<60 nt) gene overlap and/or in-frame gene fusion. For Category 2 and 3, it is not possible to predict whether TAG is a stop or sense codon based on sequence analysis alone. For Category 4, readthrough led to a large (>60 nt) out-of-frame overlap. In this case, there is no support for TAG reassignment, as large gene overlaps are rare in prokaryotes (41). The prevalence of Category 1 and absence or extreme rarity of Category 4 cases in a genome may be an indicator of genome-wide TAG reassignment.

Notably, Pyl archaea from eight genera (Methanomethylophilus, Methanogranum, Methanoplasma, Methanimicrococcus, Methanosarcina, Methanohalobium, Methanohalophilus, Methanococcoides, and JDFR-19) have numerous Category 1 TAG genes (Fig. 4, Table S6). In fact, in a subset of the Thermoplasmatota and Euryarchaeota, most TAG codons are found within Category 1 genes and there are very few, or no, Category 4 genes. This pattern is consistent with reliance only on TGA and TAA as stop codons. We infer that in these genomes, TAG is completely recoded as a sense codon (Pyl). Thus, we sought experimental evidence to confirm that these archaea interpret the TAG codon as Pyl.

## Proteomic validation of Genetic Code 34

To test for recoding of TAG, we chose two isolates from different lineages and environments with genomes that are predicted to contain a large fraction of Category 1 genes and used mass spectrometry to identify proteolytic peptides and their subsequent fragmentation whose exact masses confirmed the presence of a Pyl residue. *Methanococcoides burtonii* is a psychrophilic Halobacteriota from an Antarctic lake(42) and *Methanomethylophilus alvi* (formerly *M. alvus*) is a Thermoplasmatota from the human gut(27, 43). To increase the probability of detection of the specific peptides of interest, we used both trypsin and endoproteinase Glu-C, which cleave between different amino acids and thus increased the number of peptides suitable for analysis. Pyl-containing tryptic or GluC-fragments will be observed if and only if two criteria are met: the Pyl-containing protein is sufficiently abundant, and the Pyl-containing fragment(s) possesses the appropriate mass and charge to be detected. Although comprehensive detection of all peptides is
not possible due to variable abundance, searching specifically for peptides of interest based on their predicted masses (targeted peptide detection) greatly improved our ability to identify Pyl-containing peptides (see *Methods*). Peptides with the mass-to-charge ratio (m/z) expected for a Pyl-containing tryptic or GluC-product were manually validated by analysis of the MS/MS spectra.

For both *M. burtonii* and *M. alvi*, the proteomic data confirm genome-wide recoding of TAG as a sense codon (Table S7, S8). The detection of ≥2 unique peptide matches in the global tryptic or Glu-C datasets was used to confirm protein expression (Table S7, S8). Notably, for all proteins that were expressed and predicted to contain Pyl, and for which a proteolytic peptide (>6aa and <50aa) spanning the region predicted to contain Pyl was detected, the presence of Pyl was confirmed. For *M. burtonii*, peptides containing Pyl were detected in 27 of 39 expressed Pyl proteins. Similarly, for *M. alvi*, Pyl was detected in 12 of the 15 expressed Pyl proteins.

Expressed proteins in which Pyl was not detected had low sequence coverage and/or had a Pyl-containing proteolytic peptide that was too short or too long for detection. As an additional confirmation of Pyl incorporation at TAG codons, additional de-novo-assisted database searches were conducted to enable identification of any of the standard amino acids at the TAG codon.

We did not detect any peptides with another amino acid in the TAG position in the *de novo*-assisted database searches. Pyl was detected in 24 additional proteins that had low levels of expression (<2 unique peptides matches in the global datasets). In total, Pyl was detected in 54 proteins not previously shown to contain Pyl (Table S9).

Examples of proteins found to contain Pyl in *M. burtonii* include 12,18-didecarboxysiroheme deacetylase, thiouridine synthase, DNA helicase, histidine tRNA synthetase, isocitrate dehydrogenase, epimerase, glycosyltransferase, DNA polymerase, and multiple hypothetical proteins (Fig. 5A, Supplementary Fig. 2A, Table S8). Pyl proteins in *M. alvi* include transacetylase-like protein, a methyltransferase, kinase, acetyltransferase, hypothetical and DUF-containing proteins, and a AAA family associated protein (Fig. 5B, Table S7). Pyl was also identified in trimethylamine methyltransferase (Supplementary Fig. 2B), as previously reported (10, 36). Thus, we conclude that *M. burtonii* and *M. alvi* archaea have adopted a nonstandard genetic code, which we propose as Genetic Code 34, with 62 sense codons encoding 21 amino acids, and only two stop codons. It is probable that the other Pyl-encoding archaea for which we detected evidence for numerous internal TAG codons (Category 1) also use Genetic Code 34.

## Over a thousand proteins are predicted to contain Pyl

After establishing that select archaea use TAG as a dedicated sense codon, we predicted 1903 Pyl-containing proteins in genomes inferred to use Code 34 (Table S10-12). This finding increases the number of predicted Pyl proteins by two orders of magnitude. Of the 1903 proteins, Codetta(44) predicted that 360 contain TAG within the gene based on alignment of profile
hidden Markov models (HMMs) of conserved proteins (Table S13). The other cases involve N-terminal or C-terminal extensions beyond the TAG codon that do not align with known protein sequences (HMMs), or fusions with subsequent genes in the same reading frame. The largest number of predicted Pyl proteins of a single organism, 191 in total, occurs in JDFR-19 (Methermicoccaceae; Table S14). This archaeon is from crustal fluids collected from oceanic crust near the Juan de Fuca Ridge(45), and is a recoding outlier with a 13% TAG frequency.

Pyl occurs in numerous protein families, and all complete genomes with Code 34 encode one or more of the methylamine methyltransferases (Table S3). Notably, these methyltransferases are also the only Pyl-containing proteins that archaea have in common, suggesting that this function alone drove the evolution of the nonstandard genetic code. The proposed function of Pyl in these proteins is to activate and orient the methylamine for methyl transfer to a cognate corrinoid protein, via the reactivity of the electrophilic imine bond(10, 36). Outside of the methyltransferases, nearly all proteins found to incorporate Pyl have a limited phylogenetic range, such that Pyl is in a protein within a single genome or genus, as a singleton.

The position of Pyl is maintained in a GAF domain-containing / PAS family protein in three genera of the Methanosarcinaceae family (Supplementary Fig. 3). In methylcobalamin:CoM methyltransferase (MtaA), Pyl is in a different position in the amino acid sequence in organisms belonging to two genera (positions 90 and 289 in Methanosarcina and Methanohalophilus respectively) but both positions are predicted to be near the opening of the binding cavity in the folded protein (Supplementary Fig. 4).

It remains to be established what (if any) role Pyl plays in the enzymology of these Pyl-containing proteins. However, the lack of phylogenetic consistency in Pyl distribution in proteins apart from the methylamine methyltransferases is consistent with Pyl occurrence due to a neutral by-product of this adaptation, as described for Pyl in tRNA-His guanylyltransferase(35). In general, the functions of proteins that incorporate Pyl are not consistent within or between evolutionary clades, further supporting the lack of selection for Pyl variants.

Pyl occurs in many proteins involved in energy metabolism and DNA processing, including citrate synthase, isocitrate dehydrogenase, aminoacyl-tRNA synthetases, DNA polymerases, and helicases. Additionally, transposases and CRISPR Cas proteins contain Pyl. Pyl was previously predicted in transposases and the Cas1 and Cas3 proteins(22, 38, 46). Here, we observed Pyl in Cas1 via targeted proteomic analysis (Table S7), and identified 12 transposase families with Pyl, with some occurring in many copies per genome (Table S10). Pyl is also in Cas2, Cas4, Cas8e, Cas10, and a Cas1 casposon (Table S10, S15) but these do not co-occur in the same locus and Pyl incorporation for each of these is generally limited to a single genome.
269 Archaeal TnpB and IscB proteins are predicted to contain Pyl (Table S10, S15). TnpB and IscB
270 are RNA-guided nucleases that are the likely ancestors of Cas12 and Cas9, respectively(47, 48).
271 The location of the Pyl residue is not conserved in IscB homologs, but is close to the
272 heteroduplex of the guide RNA and target DNA, in either the bridge helix or the HNH
273 endonuclease domain (Fig. 6). The Pyl-containing nucleases identified in this study represent a
274 diverse and overlooked set of potential gene-editing proteins. In studies of IscB and TnpB
275 nucleases, the Pyl-containing variants are always either mis-annotated as fragments, hypothetical
276 proteins, or missed altogether (Supplementary Fig. 5).

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278 PylRS/tRNA<sup>Pyl</sup> pairs from Genetic Code 34 organisms support genetic code expansion
279 (GCE) in <i>E. coli</i>

280
281 PylRS/tRNA<sup>Pyl</sup> pairs have long enabled genetic code expansion (GCE), yet their widespread use
282 has been hindered by unpredictable stop codon suppression. Genetic Code 34 organisms appear
283 to have naturally overcome this challenge through their PylRS/tRNA<sup>Pyl</sup> systems. The high
284 incidence of TAG codon(s) encoding Pyl within genes of some Genetic Code 34 archaeal
285 genomes suggests a robust system for Pyl incorporation that may be leveraged for the site-
286 specific incorporation of unnatural monomers into ribosomal products. Indeed the
287 PylRS/tRNA<sup>Pyl</sup> pairs from <i>M. mazei</i>, <i>M. barkeri</i>, and <i>M. alvi</i> are already widely used for GCE in
288 bacterial, yeast, and mammalian cells(49). Here, we tested whether PylRS/tRNA<sup>Pyl</sup> pairs from
289 Genetic Code 34 archaea support the incorporation of two widely used analogs of Pyl (1), the α-
290 amino acid Boc-Lys (2) and the α-hydroxy acid HO-Boc-Lys (3) (<b>Fig. 8A</b>), into a model protein,
291 super folder green fluorescent protein (sfGFP)(50), containing an in-frame TAG codon located at
292 position 3 (sfGFP-3TAG). At least one representative sequence from each recoded genus was
293 evaluated. All in all, 8 PylRS/tRNA<sup>Pyl</sup> pairs were tested for their ability to support genetic code
294 expansion in <i>E. coli</i>, using the PylRS/tRNA<sup>Pyl</sup> pair from <i>M. alvi</i> as a benchmark for current GCE
295 efficiency levels. Initial screenings evaluated whether organisms supplemented with Boc-Lys (2)
296 or HO-Boc-Lys (3) developed higher levels of sfGFP fluorescence (F<sub>528</sub>) as a function of cell
297 growth (OD<sub>600</sub>) over 20 hours (h) (<b>Fig. 8B</b>, <b>Supplementary Fig. 6</b>). When BL21(DE3) <i>E. coli</i>
298 were supplemented with Boc-Lys (2), the PylRS/tRNA<sup>Pyl</sup> pairs from <i>Methanomethylophilus alvi</i>
299 and <i>Methanohalophilus levihalophilus</i> developed substantial levels of sfGFP compared to non-
300 supplemented growths, while the PylRS/tRNA<sup>Pyl</sup> pair from <i>Methanococcocoides LMO-1</i>
301 supported moderate sfGFP expression. SDS-PAGE analysis of sfGFP-3TAG proteins expressed
302 in BL21(DE3) <i>E. coli</i> supplemented with either 1 mM of Boc-Lys (2) or HO-Boc-Lys (3)
303 indicates that the major protein product has a molecular mass of approximately 27 kDa, the
304 expected molecular weight of sfGFP (<b>Fig. 8C</b>, <b>Supplementary Fig. 7</b>). LC-MS analysis of intact
305 sfGFP-3TAG purified from cells expressing the PylRS/tRNA<sup>Pyl</sup> pair from <i>Methanomethylophilus
306 alvi</i> and <i>Methanohalophilus levihalophilus</i>, confirmed the introduction of Boc-Lys (2) or HO-
307 Boc-Lys (3) into sfGFP-3TAG (<b>Fig. 8D</b>). The activity of the PylRS/tRNA<sup>Pyl</sup> pair from
308 *Methanohalophilus levihalophilus* is sufficient to support robust genetic code expansion with α-
309 amino acid and α-hydroxy acid Pyl analogs in *E. coli*.

310
311 A surprising result was the greater effectiveness of some PylRS/tRNA<sup>Pyl</sup> pairs for incorporation
312 of α-hydroxy acid HO-Boc-Lys (3) compared to the α-amino acid Boc-Lys (2). In particular, the
313 PylRS/tRNA<sup>Pyl</sup> pairs from Methanomicrobia JDFR-19 and *Methanogranum* 1D supported sfGFP
314 expression only in the presence of HO-Boc-Lys (3). This finding is important because, unlike
315 Boc-Lys (2), HO-Boc-Lys (3) generates an ester linkage within the protein backbone. Site-
316 specific ester linkages can be exploited to post-translationally edit the protein backbone to
317 introduce backbone elements that cannot otherwise be introduced, such as β<sup>2</sup>-amino acid
318 linkages(51) and reactive 1,3-diketones(52). LC-MS analysis of intact sfGFP-3TAG purified
319 from large-scale *E. coli* growths expressing PylRS/tRNA<sup>Pyl</sup> pairs from JDFR 19 and
320 *Methanogranum* 1 as well as *Methanohalobium* bin 14 and supplemented with α-hydroxy acid
321 HO-Boc-Lys (3) showed unambiguous evidence for the ester linkage (**Fig. 8C, Supplementary
322 Fig. 8**). The selectivity for an α-hydroxy acid substrate by the PylRS/tRNA<sup>Pyl</sup> pairs identified
323 here avoids well documented problems(53, 54) associated with metabolic conversion of α-
324 hydroxy acids into α-amino acids that would circumvent post-translational backbone editing(51,
325 52).

326

327 **Clues to the pathway to genome-wide TAG reassignment**

328 Based on the pattern of distribution and the functions of TAG codons in archaea that do, and do
329 not incorporate Pyl, we suggest that the adoption of Genetic Code 34 may follow several steps
330 **(Supplementary Fig. 9)**.

331
332 First, the Pyl system may be acquired in some archaea via lateral transfer of a gene cassette and
333 is associated primarily or exclusively with methylamine methyltransferases. While unequivocal
334 evidence for the origin of the Pyl system is lacking, lateral gene transfer is implied in a subset of
335 archaea due to the incongruence between protein and organismal phylogenies (Fig. 2). This
336 scenario leads to the question of how TAG codons are interpreted by organisms following
337 acquisition of the Pyl system. We did not find the PYLIS motif, riboswitches, or other conserved
338 motifs flanking TAG codons of genes encoding Pyl-containing proteins, except for a small
339 subset of the methylamine methyltransferases where the PYLIS motif was present (Table S16).
340 Since the PYLIS is not required for Pyl incorporation and does not occur consistently at the
341 methylamine methyltransferases or other Pyl-containing proteins, the Pyl machinery may
342 introduce Pyl at normal TAG stop positions. There may be an unknown means by which TAG is
343 interpreted independently for each gene, but there is no evidence for this process at present. It is
344 possible that there is transcriptional regulation of the Pyl machinery, potentially linked to
345 methylamine availability. This is supported by observations that a non-recoded Pyl archaean(55)
346 and a bacterium(20) turn off the Pyl system when the organism is grown on a substrate other
347 than trimethylamine, but the regulatory mechanism was not determined. The ability to turn off
348 the Pyl machinery would eliminate unintended TAG readthrough, except when methylamines are
349 available.

350
351 It has been suggested that Pyl incorporation at stop positions would not be highly detrimental
352 and protein extension would be limited by backup stop codons. Adjacent stop codons would lead
353 to only short extensions, as observed for Category 2 genes in the preceding section, and non-
354 functional proteins would be degraded(11). Additionally, release factor 1 (RF1; recognizes TAG
355 and TAA) is present in all complete and near-complete archaeal genomes examined and may
356 compete with the Pyl tRNA for the TAG codon, reducing the instances of Pyl incorporation at
357 stop positions. This situation is analogous to the 'ambiguous intermediate' evolutionary model for
358 recoding proposed for Candidate phyla radiation (CPR) bacteria and eukaryotes(7, 56, 57).
359

360
361 Based on the frequency of TAG codons, Category 1 and Category 4 genes, we delineated three
362 groups of genomes (Group A, B and C) which reflect different stages of the transition to Code 34
363 (Fig. 4, Supplementary Fig. 9, Table S6). Group A genomes generally have high TAG%, many
364 Category 4 genes (all >7%) and few Category 1 genes (<5%). Group C genomes primarily have
365 opposite characteristics, while Group B genomes have similar TAG% as Group C but the
366 number of Category 1 and 4 genes is similar to that of Group A (Fig. 4, Supplementary Fig.
367 10). Several factors may explain these patterns: 1) the period of time since the acquisition of the
368 Pyl machinery, 2) the level of dependence on this machinery (for methylamine utilization) and 3)
369 environmental conditions. The combination of extent of dependence and environmental
370 conditions may drive the transition to Code 34.

371
372 Group A organisms do not appear to fully recode TAG, as indicated by the low number of
373 Category 1 genes. Those archaea belong to genera in which the Pyl machinery is patchy and
374 sometimes rare, which indicates recent acquisitions and/or weak dependence on the Pyl
375 machinery. Non-methanogenic archaea (all belonging to Group A) may have an intermittent
376 dependence on Pyl machinery as they do not depend exclusively on methylamines(29, 58, 59)
377 and may regulate the Pyl machinery, expressing it only when methylamines are available (Fig. 4;
378 Supplementary Fig. 10). In addition, in case of acquisition of the Pyl-machinery through
379 horizontal gene transfer, the initially high level of TAG stop codons (indicated by both high
380 readthrough would lead to large out-of-frame gene overlaps. A first phase of the code transition
381 may thus be the progressive reduction of TAG stop codons to limit potentially deleterious
382 readthrough. This process may be favored in primarily obligate methylotrophic or methyl-
383 reducing methanogens that use methylamines (thus Pyl occurs in dedicated methyltransferases).
384 Some of these obligate methyl- utilizing organisms live in environments where methylamine is
385 highly available, such as saline environments where methylamines are produced from
386 osmoprotectant degradation and the animal gut where methylamine precursors are abundant in
387 certain diets (60). Group B genomes are all from methanogens living in these conditions, and
388 may be at the end of the TAG stop codon reduction process, before the adoption of Code 34
389 (Fig. 4; Supplementary Fig. 10C).
390
391 Group C genomes have fully recoded TAG to a sense codon. The constant requirement for
392 pyrrolysine in methylamine methyltransferases may result in a loss of Pyl regulation.
393 Independent of this, the availability of pyrrolysyl-tRNA synthetase and Pyl tRNA may lead to
394 insertion of TAG in many genes, including those essential for function. The insertion of TAG in
395 essential genes would require that the Pyl machinery become constitutively expressed, even if
396 the cells grow on compounds other than methylamines. Presence of TAG in essential genes and
397 constitutive expression of the Pyl machinery would establish Code 34. This proposed mechanism
398 for the emergence of Code 34 in methanogens may explain why it is not observed in bacteria, as
399 they are generally more versatile and never methanogens. Thus, this is an instance of a metabolic
400 process driving the adoption of an unusual genetic code. Group C organisms are also almost only
401 gut-dwelling and halophilic methanogens (Fig. 4; Supplementary Fig. 10C). They have a
402 majority of Category 1 genes and have thus fully transitioned to Genetic Code 34. However,
403 pyrrolysine is bulky, chemically reactive, and the ring nitrogen may introduce a positive charge
404 into proteins at physiological pH, potentially making its incorporation unfavorable(38). Thus,
405 most mutations that introduce Pyl into organisms with Pyl machinery would be selected against.
406 This is potentially why TAG frequency is generally low in Group C genomes, and Pyl is orders
407 of magnitude less abundant in any proteome than the next rarest amino acid (Fig. 4; Table S17).
408 Two Methermicoccaceae genomes represent an exception regarding the low TAG% for Group C
409 genomes, and have up to four Pyl within a single protein. Given that it takes time for the
410 proteome to explore the potential value of Pyl incorporation and identify proteins in which Pyl is
411 either neutral or beneficial, we suspect that JDFR-19 underwent code transition long ago.
412

413 We anticipate that TAG re-coding is a one way street, especially for archaea that have fully
414 adopted Code 34. Multiple families of proteins needed for key cellular processes contain Pyl.
415 The absence or loss of function due to misfolding / premature truncation at the TAG codon of
416 these genes would interrupt central metabolic and housekeeping processes. The loss of the Pyl
417 system in recoded genomes would not only eliminate the ability to use methylamines, but also
418 impede processes such as DNA, lipid and protein synthesis.
419

420 The barriers to code reversal and the sporadic distribution of Code 34 suggests multiple
421 independent code transitions. Thus, we infer that transition to Code 34 is facile and may be more
422 easily gained than lost. Ancestral character reconstruction indicates that Code 34 may have
423 emerged independently five times (Fig. 4). Interestingly, this parallels inferences of ease of code
424 switching in some phages(6) and contrasts with the monophyletic pattern of bacterial recoding(2,
425 7).
426

426
In summary, we find that the Archaea, previously not known to use more than a single genetic code, contains clades in which a genome-wide code switch has occurred such that the TAG stop codon is interpreted as pyrrolysine. We also claim that for the Group C genomes, a translation table interpreting all TAG codons as coding for Pyl should be employed for correct protein prediction. We propose an evolutionary path to code switch and provide evidence that this code transition is relatively facile, occurring in many unrelated lineages. Finally, we demonstrate the biotechnological value of the pyrrolysine incorporation machinery from recoded genomes.

## Methods

### Identification and annotation of Pyl- encoding Archaeal genomes

Genomes encoding Pyl were identified based on the presence of cellular machinery required for production and incorporation of this amino acid. A database of 14257 archaeal genomes from Genbank with more than 60% completeness and less than 5% contamination (as estimated by CheckM(61) was assembled in April 2024. For these genomes, Prodigal(62) v.2.6.3 was used to predict genes using default parameters (single genome mode, translation table 11), and hmmer(63, 64) v.3.3.1 was used via hmmscan against the NCBI-fam database(65) v.13. The HMM gene annotation used to identify the Pyl pathway is as follows: TIGR03912 (PylSn), TIGR02367 (PylSc), TIGR03910 (PylB), TIGR03909 (PylC) and TIGR0391 (PylD), and Pyl-containing methyltransferases (MtmB, MtbB and MttB) using manually curated HMM profiles. For *pylT* a HMM profile was constructed using the few previously identified *pylT* genes. Sequences were recovered together with a 10 bp flanking region. They were aligned with known *pylT*, checked for the presence of an aligned CTA (CUA) and trimmed to remove 5' and 3' extra bases. Secondary structure was checked using RNAfold (66) and an additional trimming was performed, if needed. Sequences of *pylT* are provided with and without the CCA modification of the tail (Table S4). Information on genomes identified as encoding Pyl contain at least one of the Pyl-specific genes and the detail on the presence/absence of these markers are listed in Table S2 and Table S3, respectively.

### Taxonomic distribution and evolution of the Pyl machinery and Code 34 in Archaea

The reference species tree of Archaea was built from a concatenation of the sequences of 36 universally conserved proteins from the phylosift dataset(67) together with sequences of L30 and S4 ribosomal proteins and of the RNA polymerase subunit A and B. Proteins were retrieved from the selected proteomes with HMM search(63, 64) (hmmer v3.3.2 [http://hmmer.org/]). They were aligned with MAFFT (v7.453) with the accuracy-oriented methods, L-INS-i(68), trimmed with BMGE (v2) with the Blosum30 parameter(69), and concatenated. A maximum likelihood tree was built with IQ-TREE (v2.0.6)(70) using the LG+F+R10 model. This tree was used as a guide tree to run the Posterior Mean-Site Frequency (PMSF) model under the LG + C60 + F +
467 G4 model implemented in IQ-TREE(71) using a 1,000-replicate, ultra-fast bootstrap. Genome
468 taxonomy was determined using GTDBTk 2.1.1(72). Taxonomy, TAG categories and TAG%
469 were mapped on the species tree using the ITOL server(73).

470
471 PylBCDS proteins from archaea and bacteria were separately aligned using MAFF(68) and
472 trimmed with BMGE(69) using the above mentionned parameters, and then concatenated. A
473 maximum likelihood tree was built with IQ-TREE (v2.0.6)(70) using the LG+R9.
474
475 Presence/absence of PylSn and its fusion were mapped on the three, together with taxonomy
476 using ITOL server(73). Horizontal gene transfers were predicted by comparing PylBCDS
topology with the reference species tree topology.
477
478
479 Ancestral presence and emergence of Code 34 in archaea was determined using GLOOME (74).
480 The input tree of archaea comprises 428 taxa covering the phylogenetic diversity of the domain
481 and includes archaea inferred to use Code 34. This species tree was built as described above.

482 Quantification of ORF TAG frequency

483
484 To quantify the mean TAG frequency of archaeal taxa, Pyl-encoding genomes were dereplicated
485 for each phylum with dRep(75) v.3.4.1 with default parameters. Then, Prodigal(62) v2.6.3 was
486 used to predict ORFs using default parameters and the -d option used for nucleotide output. The
487 Python script "find_tag_end.py" was used to retrieve all genes with an in-frame TAG codon. The
488 TAG frequency was calculated as the total number of predicted ORFs containing an in-frame
489 TAG codon, divided by the total number of predicted ORFs.

490
491 Identification and quantification of genes by TAG codon category

492
493 To quantify TAG codon categories, we first extracted genes with in-frame TAG codons. NCBI
494 genome coding sequence (CDS) files were used for gene identification. Prokka (76) V.1.14.5
495 was used for gene identification for genomes without NCBI CDS files. Each TAG codon was
496 treated as a sense codon, and sequences containing TAG were extended until the nearest in-
497 frame TAA or TGA stop codon. The resulting genes were then translated and annotated using the
498 UniProt- SwissProt database version 2023_05 and the Uniprot Archaeal and Bacterial Reference
499 Proteome databases(77) version 2023_05 (November 8, 2023 release). TAG- containing genes
500 were then grouped into one of the four designated categories, based on the location of the TAG
501 codon: (1) Internal (2) Short extension (3) Long extension / short overlap / fusion and (4) Large
502 out-of-frame overlap. Each gene was assigned to only a single category, using the lowest
503 category number for which criteria were satisfied. Genes that were truncated due to being located
504 at the end of a contig were excluded when extension length could not be determined. Category 1
505 (Internal) genes contain an internal TAG codon, and encode proteins for which the amino acid
506 sequence before and after the TAG codon has ≥30% sequence identity with sequences from the
UniProt databases. Additionally, for Category 1, the gene must also encode a protein that is >15 amino acids longer than if TAG was a stop. For Category 2 (short extension), readthrough of TAG results in a protein sequence extension of ≤15 aa. Sequences overlapping an adjacent gene by ≥1 sense codon were excluded from Category 2. The Category 3 designation was assigned for genes if TAG readthrough resulted in (a) gene overlap of ≤60 nt with an adjacent gene, and / or (b) genes encoding a protein with >15 aa extension and/or (c) gene fusion (in-frame overlap) with an adjacent gene. Finally, TAG readthrough in Category 4 genes results in overlap of an adjacent gene by >60 nt, including overlap of an entire gene in a different reading frame or oriented in the opposite direction.

## Prediction of Pyl-containing proteins

We initially used the genetic code prediction tool, Codetta(44), to identify Pyl proteins. By default, Codetta does not detect the recoding of TAG to Pyl. First, Codetta completes a six-frame translation of a genome nucleotide sequence that is aligned to profile HMMs. The software then recognizes recoding events when a codon has a specific nonstandard interpretation based on profile HMM alignments. For Codetta to complete this task, there must be a primary meaning that can be deduced from the alignments, for example, Codetta detected reassignment of AGG (a canonical arginine codon) to methionine(78). When TAG is recoded to Pyl, TAG occurs in place of different codons such that there is no consensus for which codon it replaces. Thus, Codetta does not determine a single amino acid (re)interpretation for TAG. Identification of known Pyl proteins may be enabled with Codetta, but Pyl detection in this case is limited exclusively to methylamine methyltransferases because they are the only proteins with Pyl-containing profile HMMs that are available for Codetta. However, the alignment output produced by Codetta includes gene matches (profile HMMs) for each codon, including TAG. Using this Codetta alignment output, we identified Pyl-containing proteins when TAG is internal in a gene in a Code 34 genome. We implemented the alignment step using the Codetta v.2.0 codettaalign.py command with a compatible profile HMM database prepared with the hmmer(63, 64) v.3.1 hmmbuild command (--enone option) for the PGAP(65) v.15 database, which includes TIGRFAM(79) v.15.0 entries.

A secondary annotation for ORFs was performed by modifying the protein prediction software, Prodigal, to include TAG as a sense codon in the archaeal genetic code. The modified version of Prodigal is available at https://github.com/VeronikaKivenson/Prodigal and can be invoked using the -g 34 (translation table 34) option. Geneious was used to visualize and overlay the standard genetic code annotation and the TAG readthrough annotation. Sequences with in-frame TAG codon(s) were searched against Blast to identify protein matches. Searches for PYLIS structures were performed with Infernal(80) v.1.1.4 using covariance model(s) via cmsearch with the RFAM(81) v.14.10 database. Regions of 200 nt flanking TAG-containing genes were examined
546 to determine if matches to the RFAM database were present. Prediction and annotation of Pyl-
547 containing proteins was performed using the Bridges-2 computing resource(82).

548
549 *Structural prediction of Pyl- containing proteins*
550
551 Structures for homologous proteins were obtained from RCSB PDB(83). Structure 4AY8 was
552 used for MtaA(84) and structure 7UTN was used for IscB(48). The Visual Molecular Dynamics
553 software package(85–87) was used for visualization.

554
555 *Anaerobic cultivation*
556

557 *Methanomethylophilus alvi* (formerly *M. alvus*) strain Mx-05<sup>T</sup> (JCM 31474<sup>T</sup>) was grown as
558 previously described(88), with methanol and ruminal fluid. *Methanococcoides burtonii* strain
559 ACE-M<sup>T</sup> (DSM 6242<sup>T</sup>) was obtained from Deutsche Sammlung für Mikroorganismen und
560 Zellkulturen (DSMZ) and grown on DSMZ Methanococcoides medium 141c (with 5 mM
561 trimethylamine hydrochloride), with the following modifications: instead of Wolin's mineral
562 solution and Wolin's vitamin solution, ATCC trace mineral supplement (MD-TMS) and ATCC
563 Vitamin Supplement (MD-VS) were used. *M. burtonii* was grown at room temperature in 15 mL
564 Hungate tubes with a headspace of 85% N<sub>2</sub>, 10% CO<sub>2</sub>, 5% H<sub>2</sub> gas in Anaerobe Systems
565 anaerobic chamber (AS-150).

566
567 *Sample preparation for LC-MS/MS*
568

569 20 mg cell pellets were resuspended in 100 mM ammonium bicarbonate, pH 8.0 and subjected to
570 mechanical lysis by bead beating with 0.15 mm zirconium oxide beads for five min
571 (Geno/Grinder 2010; SPEX). Resulting cell lysates were adjusted to 4% sodium dodecyl sulfate
572 (SDS)/10 mM dithiothreitol (DTT), incubated at 90°C for 10 min to denature and reduce
573 proteins, and pre-cleared by centrifugation at 21,000 x g. Samples were adjusted to 30 mM
574 iodoacetamide (Sigma) and incubated in the dark at room temperature for 20 min to alkylate
575 cysteine residues. The alkylated proteins were further cleaned and prepared for proteolytic
576 digestion via the protein aggregation capture (PAC) method(89). In brief, 300 µg of hydrophobic
577 magnetic beads (1 micron, SpeedBead Magnetic Carboxylate; GE Healthcare UK) and
578 acetonitrile (ACN) were added to protein samples for a final concentration of 80% ACN to
579 induce protein aggregation on the beads. Samples were incubated for 20 min at room
580 temperature. The aggregated proteins were placed on a magnet and the supernatant was removed.
581 The pelleted beads were subjected to a wash with 1 mL of ACN, followed by 1 mL of 70%
582 ethanol. The final wash buffer was removed from tubes and the beads were resuspended in 200
583 µL of 100 mM ammonium bicarbonate for in-solution proteolytic digestion. Protein amounts
584 were quantified by corrected absorbance (Scopes) at 205 nm (NanoDrop OneC; Thermo Fisher).
585 After protein quantitation, samples were digested using either MS-grade GluC Endoproteinase or
586 trypsin (Thermo Scientific) with a 1:75 protease:protein (wt:wt) ratio at 37°C, shaking overnight
587 at 600 rpm. An additional round of protease was added for a second 3-h digestion period at
588 37°C, shaking at 600 rpm. The resulting tryptic peptides were filtered on 10 kDa MWCO filter
589 plate (AcroPrep Advance, Omega 10 K MWCO) at 1,500 × g and adjusted to 1% formic acid
590 before quantification by NanoDrop OneC.

591
592 LC-MS/MS
593
594 3 µg of peptide solution were analyzed by automated 1D LC-MS/MS analysis using a Vanquish
595 ultra-HPLC (uHPLC) system plumbed directly in-line with a QExactive- Plus mass spectrometer
596 (Thermo Scientific) with an in-house-pulled 100 µm inner diameter nanospray emitter (packed to
597 15 cm with 1.7 µm Kinetex C18 reverse-phase resin (Phenomenex)). Peptides were loaded,
598 desalted, and separated by uHPLC under the following conditions: sample injection followed by
599 100% solvent A (98% H2O, 2% acetonitrile, 0.1% formic acid) from 0 to 30 min to load and
desalt, a linear gradient from 0 to 30% solvent B (70% acetonitrile, 30% water, 0.1% formic
600 acid) from 30 to 220 min for separation, a column wash from 0 to 100% solvent B from 220 to
601 255 min, and 100% solvent A from 255 to 275 min for column re-equilibration. Eluting peptides
602 were analyzed with the following MS settings for global searches: positive mode, data-dependent
603 acquisition, top-20 method; mass range 400–1500 m/z; MS and MS/MS resolution 70 and
604 17.5 K, respectively; MS/MS loop count 20; isolation window 1.8 m/z; isolation offset 0.3 m/z;
605 charge state exclusion of unassigned, +1, +6–8 charges.
606
607

608 For targeted searches, parallel reaction monitoring inclusions lists were constructed to detect
609 peptides of interest in the predicted pyrrolysine containing proteins. An in-silico digest of the
610 proteins was conducted with UniPept(90) using either GluC or trypsin as the protease with a
611 minimum peptide length of six amino acids. Based on the predicted peptides from the *in silico*
612 digest, peptides were selected for the PRM measurements if they were classified as any of the
613 following peptide types: (1) the first proteolytic peptide in the protein, (2) the proteolytic peptide
614 containing the pyrrolysine residue, (3) the first proteolytic peptide downstream of the
615 pyrrolysine-containing peptide, and (4) the last proteolytic peptide in the sequence. For the
616 peptides of interest, the PRM inclusion list included the monoisotopic precursor m/z of the
617 peptide including a static modification of carbamidomethylation (+57.021) of cysteine and a
618 charge state (z) of +2. No variable modifications of oxidation (+15.995) of methionine,
619 deamidation of (+0.984) of asparagine and glutamine, or methyl loss (-14.016) for pyrrolysine
620 were considered for the inclusion list. A maximum of 50 entries were monitored for each PRM
621 measurement. The same LC-MS/MS method was used as the global measurements.
622
623 Proteomics data analysis
624

622
623 Proteomics data analysis
624
Global database searches were conducted with Proteome Discoverer 3.0 (Comet/Percolator)(91, 92) against custom organism specific protein databases constructed from all predicted proteins in the respective, as well as common mass spectrometry contaminants. Protein sequences were included with the predicted pyrrolysine residue represented with “X” as well as the sequence version that terminated in a stop codon. For all database searches, the peptide mass tolerance was set at 20 ppm. Accepted modifications included static modifications of carbamidomethylation (+57.021) of cysteine residues and pyrrolysine (+237.148) of “X” residues. Database searches were performed with variable modifications of oxidation (+15.995) of methionine, deamidation of (+0.984) of asparagine and glutamine residues, and a methyl loss (-14.016) for pyrrolysine (X) residues, and a maximum of three variable modifications allowed per peptide. Semi-proteolytic digestion of peptides was allowed for either trypsin or GluC depending on the sample, and a 1% peptide-level FDR threshold was applied.

Preliminary confirmation of pyrrolysine expression was based on (1) a unique peptide match in the global datasets that passed the 1% peptide-level FDR threshold for the pyrrolysine containing proteins or (2) a detected precursor mass in the PRM measurement that was within 10ppm of the monoisotopic precursor m/z of the peptides of interest. Peptides were considered unique if the amino acid sequence was not predicted in any other protein for the respective organism. To more confidently confirm pyrrolysine expression, MS/MS spectra from all peptides of pyrrolysine-containing proteins were manually inspected and validated. A MS/MS spectrum of a peptide was considered for evidence of pyrrolysine expression if each of following criteria was met: (1) Precursor ion (MS1) mass error <10 ppm, (2) Fragmentation ion (MS/MS) mass error <0.02 Da and (3) Location of the fragment ion along the peptide sequence, with either direct sequencing of the fragment ending in pyrrolysine or fragment ions that were longer than the pyrrolysine-containing fragment.

In addition to the database searches, we searched the data using a de novo-assisted database search workflow using PEAKS StudioX(93) to recognize any of the standard amino acids at the TAG=pyrrolysine/stop position (represented as “X” in the database). This workflow will recognize the 20 standard amino acids if they were detected at the Pyl position in the MS/MS spectrum. The same modifications as previous database searches were included and the parent and fragment ion mass error tolerances were set to ± 10 ppm and ±0.02 Da, respectively. De novo only parameters were left at default settings with average local confidence (ALC) scores of >80% and de novo sequence tags displayed if at least six amino acids were shared with the database sequence.

Construction of plasmids encoding PyIRS/tRNA<sup>Pyl</sup> pairs

Plasmids encoding the PyIRS/tRNA<sup>Pyl</sup> pairs evaluated in Fig. 8 were derivatives of the previously reported pMEGA vector (parent backbone Addgene Plasmid #200225). pMEGA
plasmids containing each PylRS homolog (Table S18) were ordered as circular, dsDNA
plasmids from Twist Bioscience. Each one was then modified to eliminate the M. alvi pylT gene
and replace it with the one corresponding to each PylRS homolog (Table S18). Briefly, each
circular pMEGA vector obtained from Twist was linearized using oligonucleotides P1 and P2
(Table S18). PCR using the Q5® High-Fidelity 2X Master Mix (NEB M0492S) was performed
for 30 cycles according to manufacturer protocols. The PCR reaction was subject to a PCR clean
up with the QIAquick PCR Purification Kit (Qiagen, catalog # 28104). The concentration of
purified, linearized products was determined by absorbance at 260 nm using a NanoDrop ND-
1000 Spectrophotometer. Next, 33.3 ng of each purified, linearized pMEGA plasmid was
combined with 100 ng of a gBlock containing the appropriate pylT sequence (Table S18) in a 20
µL Gibson Assembly reaction using HiFi DNA Assembly Master Mix (NEB, catalog #E2621L).
The reaction mixture was incubated at 50 °C for 1 h to generate a family of circular pMEGA
vectors containing the coding sequence for the appropriate pylT.

The circularized pMega plasmids from the previous step were then transformed into NEB 5-
alpha competent E. coli (NEB, catalog # C2987H) as follows. Frozen stocks of cells were thawed
on ice for 10 min. Upon thawing, the entirety of the previous Gibson Assembly reaction was
added to cells and incubated on ice for 30 min. Cells incubated with plasmid were then subjected
to heat shock at 42 °C for 30 sec and placed on ice for 2 min. 350 µL of Super Optimal broth
with Catabolite repression (S.O.C.) outgrowth medium (NEB, catalog # B9020S) was added to
cells and cells were incubated at 37 °C for 1 h with shaking at 220 rpm. Agar plates containing
spectinomycin were inoculated with 50 µL of transformed cells and grown overnight at 37°C. 3
single colonies per construct were picked and inoculated into liquid cultures containing 5 mL LB
+ spectinomycin and grown for 16 h at 37°C. Pure plasmid was isolated from 5 mL cultures
using Qiaprep Spin Miniprep Kit (Qiagen, catalog # 27106) and sequences were confirmed by
whole plasmid sequencing with Primordium Labs. The new pMEGA plasmids were double
transformed with a pET22b-3TAG-GFP into BL21(DE3) cells following the transformation
protocol detailed below.

Transformation protocol

BL21(DE3) E. coli (NEB, catalog # C2987H and C2527H, respectively) were transformed in
accordance with manufacturer protocols with some modifications as follows. Frozen stocks were
thawed on ice. Upon thawing, 100 ng of each relevant plasmid was added. After a 30 min
incubation on ice, cells were heat-shocked for 30 sec at 42 °C and allowed to recover on ice for 2
min. Following, 350 µL of Super Optimal broth with Catabolite repression (S.O.C.) was
immediately added and cells were allowed to recover at 37°C with shaking for 1 h before plating
50 µL on LB agar plates containing the appropriate antibiotics. Plates were incubated overnight
at 37 °C.

704
705 Expression assays of sfGFP-3TAG containing Boc-Lys or HO-Boc-Lys

706
707 Starter E. coli cultures were grown overnight in 5 mL of LB Miller (AmericanBio, Catalog #AB01201) in 15 mL culture tubes supplemented with carbomycin and spectinomycin at 37°C.
708
709 The next morning, 500 µL of the saturated overnight culture was added to 50 mL LB in a 250 mL baffled flask with carbomycin and spectinomycin and grown at 37 °C to an OD₉₀₀ of 0.6.
710
711 Prior to cultures reaching OD600 0.6, the appropriate amount of each monomer (stored as 25 mM stocks) were allotted into the wells of a black, clear bottom, 96-well plate (1 mM final concentration). Once cultures reached an OD₉₀₀ of 0.6 (roughly 3 h), protein expression was induced by addition of 1 mM IPTG. The culture was transferred into the wells of the plate to a total volume of 200 µL in each well. A Breathe-Easy® sealing membrane (Sigma Z380059) was placed over the 96-well plate and the plate was loaded into a BioTek Synergy HTX microplate reader with no lid. OD₉₀₀ and F₅₂₈ values (λex = 485 nm) were measured every 10 min for 20 h.
712
713
714
715
716
717
718 The plate was maintained at 37°C and was shaken during this time. Points represented in Fig. 8B represent three biological replicates where three random colonies were picked on a plate from a single transformation.
719
720
721

722 Expression and Purification sfGFP-3TAG containing Boc-Lys or HO-Boc-Lys

723
724 Starter cultures of 5 mL of Miller's LB Broth (AmericanBio, catalog # AB01201) supplemented with carbomycin and spectinomycin were inoculated with a single colony of BL21(DE3) E. coli cells harboring pET22b-sfGFP-3TAG-6xHis alongside pMEGA-JDFR19-PylRS-pylT, pMEGA-JDFR19-PylRSCTD-pylT, pMEGA-B74G9-PylRSCTD-pylT, pMEGA-LMO1-PylRS-pylT, pMEGA-Bin14-PylRS-PylRS-pylT, pMEGA-1D-PylRS-pylT, pMEGA-Levihalophilus-PylRS-pylT, or pMEGA-MaPylRS and grown overnight for 16 h at 37°C with shaking at 220 rpm until the culture was saturated. The starter culture was used to inoculate a 100 mL expression culture in a 1:100 dilution of Miller's LB Broth supplemented with 1 mM Boc-Lys (Combi-Blocks, CAS # 2418-95-3) or 1 mM HO-Boc-Lys (Accela, CAS # 111223-31-5) with carbomycin and spectinomycin. The expression culture was grown at 37°C with shaking at 220 rpm to an OD₉₀₀ of 0.6 at which point it was induced with 1 mM IPTG and grown for 16 h under the same conditions. The expression culture was harvested by centrifugation at 4,300 x g at 4 °C for 30 min. The resulting cell pellet was suspended in 10 mL of Lysis Buffer (50 mM sodium phosphate pH 6.8 and 300 mM NaCl) containing half a tablet of cOmplete, mini EDTA-free ULTRA protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The cell suspension was disrupted by sonication on ice (Branson Sonifier 250, 5 cycles of 30 sec pulse at 50% duty cycle and microtip limit of 5 followed by 30 sec pause). The cell lysate was cleared by centrifugation at 10,000xg at 4 °C for 20 min. TALON® Metal Affinity Resin (1 mL) (Takara Biosciences, catalog # 635504) was equilibrated with Lysis Buffer, added to the cleared cell lysate, and incubated on a rotisserie at 4 °C for 1 h. The TALON® resin-lysate mixture was then passed through a gravity flow Poly-Prep Chromatography Column (Bio-Rad Laboratories, Hercules,
745 CA). Non-specifically bound proteins were removed by washing the TALON® resin with 10 mL
746 of Lysis Buffer. The 6xHis-tagged protein was eluted by washing the TALON® resin with 2 mL
747 of Elution Buffer (50 mM sodium phosphate pH 6.8 and 250 mM imidazole). The purified
748 protein was quantified using absorbance at 280 nm, snap frozen as single-use aliquots, and stored
749 at -80 °C. Protein yields are reported in **Table S19**.

750
751 *Intact Protein LC-MS for GCE*
752
753 LC-MS analysis of all protein samples were performed on an Agilent 1290 Infinity II HPLC
754 connected to an Agilent 6530B QTOF AJS-ESI. The mobile phase for LC-MS was water and
755 acetonitrile with 0.1% (v/v) formic acid at a flow rate of 0.4 mL/min. Each protein sample was
756 injected onto an Poroshell 300SB-C8 column (2.1 x 75 mm, 5 µM, room temp, Agilent) and
757 separated using a linear gradient from 5% acetonitrile for 0 to 2 min and ramping to 95%
758 acetonitrile over 7.5 min, and then washing with 95% acetonitrile for 2 min. The following
759 parameters were used during acquisition: Fragmentor voltage 225 V, gas temperature 300 °C,
760 drying gas flow 10 L/min, sheath gas temperature 350 °C, sheath gas flow 11 L/min, nebulizer
761 pressure 35 psi, skimmer voltage 65 V, Vcap 5000 V, 1 spectra/s.

762
763 **Data Availability and Code Availability**
764 Proteomic data from this study have been deposited into the ProteomeXchange repository with
765 accession numbers: ProteomeXchange-PXD053523; MassIVE-MSV000095193. Reviewers can
766 use the FTP download link with the MassIVE accession number to access the files. We will
767 provide login information to reviewers upon submission and release this data publicly upon
768 publication. The modified version of Prodigal with Genetic Code 34 that has TAG reassigned to
769 Pyl and the python script (find_tag_end.py) can be found on Zenodo (94).

770
771 **Acknowledgments**
772 We thank Jean-François Brugere, Johnathan Rylee, and Petar Penev for helpful discussions. SG
773 wishes to thank the Miller Institute at UC Berkeley for a Visiting Professorship.

774
775 **Funding**
776 This work was supported by a Tory Burch Foundation Fellowship to VK at the Innovative
777 Genomics Institute. This work used Bridges-2 at the Pittsburgh Supercomputing Center through
778 allocation BIO230230 to VK, from the Advanced Cyberinfrastructure Coordination Ecosystem:
779 Services & Support (ACCESS) program, which is supported by National Science Foundation
780 grants #2138259, #2138286, #2138307, #2137603, and #2138296. LTR was supported by the
781 NSF Graduate Research Fellowship Program (DGE-1752814). Experimental validation of PylRS
782 activity for α-amino and α-hydroxy acids was supported by the NSF Center for Genetically
783 Encoded Materials (C-GEM; CHE 2002182). Funding was provided by the Chan-Zuckerberg
784 Initiative Foundation, Technology Enabled Biological Carbon Capture and Sequestration
785 (CZIF2022-007203) and National Science Foundation, Collaborative Research: TRTech-PGR

786 Track (2334028) to JFB. GB was supported by Agence Nationale de la Recherche (ANR-19-

787 CE02-0005). Proteome work done at ORNL was supported by a U.S. Department of Energy

788 Genome Sciences Program project (ERKPA50).

789
790 Author contributions
791 V.K. and J.F.B. conceived and designed the study. V.K. performed codon analysis, identification
792 of Pyl proteins, and anaerobic cultivation of *M. burtonii*. Experimental validation of PyIRS
793 activity for α-amino and α-hydroxy acids was performed by N.X.H. and L.T.R. with oversight by
794 A.S. Proteomics experiments and data analysis was conducted by S.L.P with oversight by R.L.H.

795 Phylogenetic analysis and discussion was conducted by G.B. and S.G. Structural prediction of
796 Pyl-containing proteins and bioinformatics software development was performed by A.K.

797 Anaerobic cultivation of *M. alvus* was performed by K.F. with oversight by G.B. The manuscript
798 was written by V.K. and J.F.B. with input from all authors. All authors read and approved the
799 manuscript.

800
801 Ethics declarations
802 Competing interests
803 The Regents of the University of California have a patent pending related to this work on which

804 V.K., L.T.R., N.X.H., A.S. and J.F.B. are inventors.
<figure><img src="image_3.png" /><figcaption>**Fig. 1:** Distribution of the Pyl machinery in the Archaea is paraphyletic, with the Pyl machinery variably present among representatives of the same family (pie charts). The range of TAG frequency in Pyl genomes (salmon violins) illustrates that low TAG frequency is not a hallmark of Pyl archaea, in contrast to previous reports. Grey violins correspond to genomes without Pyl. The highest TAG% values for Thermoproteia (up to 66.9%), Bathyarchaeia-other (up to 52.2%) and Nitrososphaeria-other (up to 57.1%) are not shown. The “+” symbols indicate that Pyl genomes represent less than 5% of the genomes in the displayed lineage. The total number of genomes per lineage displayed in the tree is indicated between brackets next to the lineage name.</figcaption></figure>
<figure><img src="image_4.png" /></figure>

815

816

817 **Fig. 2:** Maximum likelihood phylogeny of the PylBCDS enzymes. Stars on the branch indicate the status of PylSn (missing, fused with PylSc or separated from PylSn). 1,241 positions, ultrafast bootstrap values >90% are displayed.
818
819
Categories
of TAG genes

(1) Internal

<figure><img src="image_5.png" /></figure>

(2) Short extension

<figure><img src="image_6.png" /></figure>

(3) Long extension / short overlap / fusion

<figure><img src="image_7.png" /></figure>

(4) Large out-of-frame overlap

<figure><img src="image_8.png" /><figcaption>**Fig. 3:** TAG-containing genes in Pyl-encoding archaea are sorted into one of four categories based on the result of readthrough of the TAG codon. Category 1 (internal) genes contain an internal TAG codon, and encode proteins for which the amino acid sequence before and after the TAG codon has >30% sequence identity with sequences from the UniProt databases. Category 2 includes short extensions, when sequence extension after TAG results in a ≤15 aa extension and no sense codon overlap. Category 3 includes genes with extension past TAG resulting in an in-frame fusion, a short overlap with an adjacent gene, or a long extension. Large-out-of-frame overlaps (>60 nt) are assigned to Category 4.</figcaption></figure>

820
821
822
823
824
825
826
827
828
829

**Fig. 3:** TAG-containing genes in Pyl-encoding archaea are sorted into one of four categories based on the result of readthrough of the TAG codon. Category 1 (internal) genes contain an internal TAG codon, and encode proteins for which the amino acid sequence before and after the TAG codon has >30% sequence identity with sequences from the UniProt databases. Category 2 includes short extensions, when sequence extension after TAG results in a ≤15 aa extension and no sense codon overlap. Category 3 includes genes with extension past TAG resulting in an in-frame fusion, a short overlap with an adjacent gene, or a long extension. Large-out-of-frame overlaps (>60 nt) are assigned to Category 4.
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Phylogenetic tree showing evidence for the sporadic distribution of Genetic Code 34 in archaea. Genomes inferred to be fully recoded (black boxes, Group C) have a large fraction of internal TAG codons (Category 1) and very few cases where TAG readthrough leads to large out-of-frame gene overlaps (Category 4). In Group A genomes, TAG content is generally high (>15%), while Group B genomes typically have 5 - 15% TAG content and internal TAG in genes in addition to the methylamine methyltransferases. Within Group C, the TAG content is variable, probably in part due to time since code transition. Tree constructed using 40 universally conserved proteins. 9816 positions, ultrafast bootstrap values >90% are displayed.</figcaption></figure>
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 5** Proteomic coverage confirms Pyl residues and extensions past the TAG codons for proteins expressed by **a**, *M. burtonii* and **b**, *M. alvi*. Coverage based on enzymatic cleavage using</figcaption></figure>
842 Trypsin is shown in blue and Glu-C is shown in green. The TAG location is shown with a
843 vertical red line and Pyrrolysine is denoted by the one-letter designation, "O".
844
845

<figure><img src="image_12.png" /><figcaption>**Fig. 6:** Pyl residues may play a role in the binding of IscB homologs to genetic material. The positions of the Pyl residue in each of five putative homologs of IscB from different organisms (a, d, Methanosarcina lacustris sp., b, Methanonatronarchaeum sp. Amet6_2, c, Candidatus Methanohalarchaeum thermophilum, e, Methanohalobium evestigatum) are marked in orange on the bridge helix a, and HNH endonuclease domain b,c,d,e, of IscB (PDB 7UTN). Although these positions are not conserved, they are all located near the paired guide RNA (yellow) and target DNA (pink) strands.</figcaption></figure>
A

<figure><img src="image_13.png" /></figure>

1

<figure><img src="image_14.png" /></figure>

2

<figure><img src="image_15.png" /></figure>

3

B

<figure><img src="image_16.png" /></figure>

Organism

A M. alvi

B M. levihalophilus

C Methanococcoides LMO1

D JDFR19

E Methanogranum 1D

F Methanohalobium bin 14

G JDFR 19 PyISc only

H Archaeon B74 G9 PyISc only

I Methanimicrococcus sp isolate lab 288p3 bin 1112

J Methanoplasma Gsp477 bin

C

MW (kDa)

<figure><img src="image_17.png" /></figure>

D

sfGFP-3TAG

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>
**Fig. 7.** PylRS variants from organisms that utilize Genetic Code 34 support the incorporation of non-canonical α-amino and α-hydroxy acids into the model protein sfGFP. **a,** Chemical structures of three established PylRS substrates: pyrrolysine (Pyl, **1**) the natural substrate for PylRS; Boc-Lysine **2** (BocK), a synthetic α-amino acid substrate, and HO-Boc-Lysine **3** (BocK-HO), an α-hydroxy acid substrate. **b,** Shown is the F<sub>528</sub> signal normalized to OD<sub>600</sub> for bacterial cultures of BL21(DE3) cells doubly transformed with a pMega plasmid harboring the PylRS/tRNA<sup>Pyl</sup> from candidate organisms A-J and a pET22A reporter plasmid containing sfGFP with an in-frame amber codon at position 3. Cells were grown to OD<sub>600</sub> = ~0.6, supplemented with 1 mM of the indicated PylRS substrate, and sfGFP expression was induced with 1 mM IPTG. F<sub>528</sub>/OD<sub>600</sub> signal was acquired for over a 20 h time frame and the final signal at T = 20 h is shown here. We note the presence of separately encoded PylSc and PylSn domains of the PylRS protein in the JDFR19 genus. As prior studies have found PylRS enzymes naturally lacking PylSn to be active, we also evaluated 2 JDFR19 PylRS homologs with only their PylSc domain. **c,** SDS-PAGE of purified sfGFP-3TAG constructs in the presence of either 1 mM **2** or **3** and the corresponding PylRS/tRNA<sup>Pyl</sup> pair. **d,** Intact MS of sfGFP-3TAG expressed in the presence of a control PylRS/tRNA<sup>Pyl</sup> pair (from *M. alvi*) or in the presence of the best performing candidate PylRS/tRNA<sup>Pyl</sup> pair (*M. levihalophilus*) as determined by expression tests in panel B. sfGFP was expressed in the presence of 1 mM **2** or **3.**
Steps for evaluating TAG codon use

Step 1: Prediction with Genetic Code 11

<figure><img src="image_22.png" /></figure>

Step 2: Re-prediction of all TAG codons as sense codons

<figure><img src="image_23.png" /></figure>

Step 3: Classification of the results into four categories

<figure><img src="image_24.png" /><figcaption>Fig. S1: Steps for assigning TAG codon into different categories. Genes in blue contain a TAG codon, while other genes are shown in yellow.</figcaption></figure>
881

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /><figcaption>**Fig. S2:** MS/MS spectrum of two Pyl-containing proteins. The blue peaks represent observed b-ions and the yellow-peaks represent observed y-ions. Black peaks were not identified as b- or as y-ions. Observed b- and y-ions are indicated in the peptide sequence ladder as well as in the MS/MS spectrum **a**, (Upper panel) MS/MS spectrum of Pyl-containing tryptic peptide of *M. burtonii* deacetylase from the global DDA measurements. **b**, (Lower panel) MS/MS spectrum of Pyl-containing tryptic peptide for *M. burtonii* trimethylamine methyltransferase 2 from the global DDA measurements.</figcaption></figure>

890
<figure><img src="image_27.png" /></figure>

891

892

893

**Fig. S3:** Alignment of protein (GAF domain-containing / PAS family protein); Pyl (O) is at position 269.
<figure><img src="image_28.png" /><figcaption>**Fig. S4:** Pyl residues without a conserved sequence position in related genes may still play similar roles due to their spatial proximity in the folded proteins. The positions of the Pyl residue in homologs of methylcobalamin:CoM methyltransferase (MtaA) from Methanosarcina sp. (orange) and Methanohalophilus sp. (magenta) are marked on the structure of MtaA (PDB 4AY7). Both positions are near the opening of the binding cavity (yellow, shown in complex with Zn²⁺ and coenzyme M). The actual positions may be even closer together due to potential differences in the structures of the two homologs.</figcaption></figure>

894

895

896

897

898

899

900

901

902

903
Error types from misinterpreting TAG as a stop codon

<figure><img src="image_29.png" /><figcaption>**Fig. S5:** Different types of misannotation for Pyl proteins resulting from misinterpretation of TAG as a stop codon.</figcaption></figure>

905

906

907

908
<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /><figcaption>**Fig. S6:** Time-dependent changes in 528 nm emission (F<sub>528</sub>) and cell density (OD<sub>600</sub>) of BL21(DE3) *E. coli* harboring a pMega plasmid expressing the indicated PylRS/tRNA<sup>Pyl</sup> pair and a reporter plasmid encoding sfGFP-3TAG and grown in the presence of 1 mM BocK (monomer 2) or α-HO-BocK (monomer 3). Growth and expression conditions are described further in Supplementary Information.</figcaption></figure>
<figure><img src="image_41.png" /></figure>

<table><tr><td>Lane</td><td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td><td>11</td><td>12</td></tr><tr><td>Substrate</td><td>2</td><td>2</td><td>2</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td></tr><tr><td>Organism</td><td>A</td><td>B</td><td>C</td><td>A</td><td>D</td><td>B</td><td>E</td><td>F</td><td>C</td><td>G</td><td>H</td></tr></table>

A = M. alvi

B = Methanococcoides LMO 1

C = Methanohalophilus levihalophilus

D = JDFR 19 full-length

E = Methanohalbium bin 14

F = Methanogranum 1D

G = JDFR 19 C-term only

H = Archaeon B74 G9 C-term only

915
916 Fig. S7: Uncropped SDS-PAGE gel of lysates from BL21(DE3) cells transformed with a pMega
917 plasmid encoding the indicated PylRS/tRNA<sup>Pyl</sup> pair and a reporter plasmid encoding sfGFP-
918 3TAG.
<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>
920 **Fig. S8:** LC-MS characterization of sfGFP-3TAG produced in BL21(DE3) cells supplemented
921 with either Boc-Lys (monomer 2) or α-HO-Boc-Lys (monomer 3). Shown are the deconvoluted
922 mass spectra of purified sfGFP-3TAG isolated from BL21(DE3) *E. coli* transformed with a
923 pMega plasmid encoding the indicated PylRS/tRNA<sup>Pyl</sup> pair and a reporter plasmid encoding
924 sfGFP-3TAG.
Steps to adoption of Genetic Code 34

<figure><img src="image_49.png" /><figcaption>**Figure S9.** A schematic representation of the proposed mechanism for the evolution of genome-wide TAG recoding. Organisms that have recently acquired the Pyl cassette still retain their prior genomic distribution of TAG, with frequent use of TAG as a stop codon and little to no incorporation of TAG into genes (column 1). The presence of the Pyl cassette exerts a strong evolutionary pressure against the use of TAG as a stop codon, leading to a large decrease in TAG frequency. At the same time, TAG coding for Pyl begins to appear inside genes, potentially through neutral mutation (column 2). Finally, organisms well-adapted to the Pyl cassette no longer use TAG as a stop codon but their TAG frequency increases despite this due to the accumulation of TAG coding for Pyl inside genes (column 3).</figcaption></figure>

926
927
928
929
930
931
932
933
934
935
936
937
938
<figure><img src="image_50.png" /><figcaption>**Figure S10.** The relationship between TAG% and Category 1 TAG genes (A) and Category 4 TAG genes (B) and the environment type and methanogen or non-methanogen status of the organisms (C). Saline environments (env. saline) correspond to hypersaline lakes and marine environments.</figcaption></figure>
<table>
  <tbody>
    <tr>
      <td>944</td>
      <td>SUPPLEMENTARY TABLES</td>
    </tr>
    <tr>
      <td>945</td>
      <td></td>
    </tr>
    <tr>
      <td>946</td>
      <td></td>
    </tr>
    <tr>
      <td>947</td>
      <td>EDITED Kivenson SI combined for submission</td>
    </tr>
    <tr>
      <td>948</td>
      <td></td>
    </tr>
    <tr>
      <td>949</td>
      <td>Table S1: List of previously reported archaeal groups reported to encode Pyl and corresponding references.</td>
    </tr>
    <tr>
      <td>950</td>
      <td></td>
    </tr>
    <tr>
      <td>951</td>
      <td></td>
    </tr>
    <tr>
      <td>952</td>
      <td>Table S2: List of Pyl genomes including their accession numbers and estimated percent completeness and contamination.</td>
    </tr>
    <tr>
      <td>953</td>
      <td></td>
    </tr>
    <tr>
      <td>954</td>
      <td></td>
    </tr>
    <tr>
      <td>955</td>
      <td>Table S3: Pyl machinery and methylamine methyltransferase presence/absence, and estimated completeness and contaminaton of genomes.</td>
    </tr>
    <tr>
      <td>956</td>
      <td></td>
    </tr>
    <tr>
      <td>957</td>
      <td></td>
    </tr>
    <tr>
      <td>958</td>
      <td>Table S4: Pyl tRNA sequences from archaeal genomes.</td>
    </tr>
    <tr>
      <td>959</td>
      <td></td>
    </tr>
    <tr>
      <td>960</td>
      <td>Table S5: Taxonomic summary of Pyl taxa and their taxon number, and genomes encoding Pyl machinery by taxonomic group.</td>
    </tr>
    <tr>
      <td>961</td>
      <td></td>
    </tr>
    <tr>
      <td>962</td>
      <td></td>
    </tr>
    <tr>
      <td>963</td>
      <td>Table S6: Quantification of genes with TAG codons by designated codon categories and group designation.</td>
    </tr>
    <tr>
      <td>964</td>
      <td></td>
    </tr>
    <tr>
      <td>965</td>
      <td></td>
    </tr>
    <tr>
      <td>966</td>
      <td>Table S7: Compilation of the results from both the global and targeted measurements of expressed proteins from M. alvi, showing coverage of Pyl-containing proteins.</td>
    </tr>
    <tr>
      <td>967</td>
      <td></td>
    </tr>
    <tr>
      <td>968</td>
      <td></td>
    </tr>
    <tr>
      <td>969</td>
      <td>Table S8: Compilation of the results from both the global and targeted measurements of expressed proteins from M. burtonii, showing coverage of Pyl-containing proteins.</td>
    </tr>
    <tr>
      <td>970</td>
      <td></td>
    </tr>
    <tr>
      <td>971</td>
      <td></td>
    </tr>
    <tr>
      <td>972</td>
      <td>Table S9: Summary of proteins found to contain Pyl and confirmed by proteomics for M. alvi and M. burtonii (not previously reported to have Pyl) with AA and nucleotide sequence data.</td>
    </tr>
    <tr>
      <td>973</td>
      <td></td>
    </tr>
    <tr>
      <td>974</td>
      <td></td>
    </tr>
    <tr>
      <td>975</td>
      <td>Table S10: List of putative Pyl-containing proteins predicted from genomes with Code 34.</td>
    </tr>
    <tr>
      <td>976</td>
      <td></td>
    </tr>
    <tr>
      <td>977</td>
      <td>Table S11: Nucleotide sequence and coordinates of putative Pyl-containing proteins identified using manual curation for select genomes.</td>
    </tr>
    <tr>
      <td>978</td>
      <td></td>
    </tr>
    <tr>
      <td>979</td>
      <td></td>
    </tr>
    <tr>
      <td>980</td>
      <td>Table S12: Putative Pyl-containing proteins identified using Prodigal (code 34) for select genomes.</td>
    </tr>
    <tr>
      <td>981</td>
      <td></td>
    </tr>
    <tr>
      <td>982</td>
      <td></td>
    </tr>
  </tbody>
</table>
983 **Table_S13:** Putative Pyl-containing proteins predicted via TAG codon alignment within HMM
984 models for conserved proteins using Codetta.

985 **Table_S14:** List of putative Pyl proteins from the JDFR-19 genome.

987 **Table_S15:** List and sequences of CRISPR-Cas, IscB, TnpB, and casposon proteins with Pyl
988 residues.

990 **Table S16:** Search parameters and sequence information for PYLIS detection.

991 **Table_S17:** Codon usage quantification for the JDFR-19 genome.

993 **Table_S18:** Gene fragments, oligos, and plasmids used in *in vivo* GCE studies.

996 **Table_S19:** sfGFP yield from expression and purification sfGFP-3TAG containing Boc-Lys or
997 HO-Boc-Lys.

999
1000
1001

References

1002 1. F. H. Crick, The origin of the genetic code. *J. Mol. Biol.* **38**, 367–379 (1968).
1003 2. J. M. Bové, Molecular features of mollicutes. *Clin. Infect. Dis.* **17 Suppl 1**, S10–31 (1993).
1004 3. K. C. Wrighton, B. C. Thomas, I. Sharon, C. S. Miller, C. J. Castelle, N. C. VerBerkmoes, M. J. Wilkins, R. L. Hettich, M. S. Lipton, K. H. Williams, P. E. Long, J. F. Banfield, Fermentation, hydrogen, and sulfur metabolism in multiple uncultivated bacterial phyla. *Science* **337**, 1661–1665 (2012).

1008 4. N. N. Ivanova, P. Schwientek, H. J. Tripp, C. Rinke, A. Pati, M. Huntemann, A. Visel, T. Woyke, N. C. Kyrpides, E. M. Rubin, Stop codon reassignments in the wild. *Science* **344**, 909–913 (2014).

1011 5. S. L. Peters, A. L. Borges, R. J. Giannone, M. J. Morowitz, J. F. Banfield, R. L. Hettich, Experimental validation that human microbiome phages use alternative genetic coding. *Nat. Commun.* **13**, 5710 (2022).

1014 6. A. L. Borges, Y. C. Lou, R. Sachdeva, B. Al-Shayeb, P. I. Penev, A. L. Jaffe, S. Lei, J. M. Santini, J. F. Banfield, Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes. *Nat Microbiol* **7**, 918–927 (2022).

1017 7. J. H. Campbell, P. O'Donoghue, A. G. Campbell, P. Schwientek, A. Sczyrba, T. Woyke, D. Söll, M. Podar, UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota. *Proceedings of the National Academy of Sciences* **110**, 5540–5545 (2013).

1021 8. F. Zinoni, A. Birkmann, T. C. Stadtman, A. Böck, Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli. *Proc. Natl. Acad. Sci. U. S. A.* **83**, 4650–4654 (1986).

1024 9. G. Srinivasan, C. M. James, J. A. Krzycki, Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. *Science* **296**, 1459–1462 (2002).

1026 10. B. Hao, W. Gong, T. K. Ferguson, C. M. James, J. A. Krzycki, M. K. Chan, A new UAG-encoded residue in the structure of a methanogen methyltransferase. *Science* **296**, 1462–1466 (2002).

1029 11. M. A. Gaston, R. Jiang, J. A. Krzycki, Functional context, biosynthesis, and genetic encoding of pyrrolysine. *Curr. Opin. Microbiol.* **14**, 342–349 (2011).

1031 12. M. Rother, J. A. Krzycki, Selenocysteine, pyrrolysine, and the unique energy metabolism of methanogenic archaea. *Archaea* **2010** (2010).

1033 13. M. A. Gaston, L. Zhang, K. B. Green-Church, J. A. Krzycki, The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine. *Nature* **471**, 647–650 (2011).
1035 14. D. G. Longstaff, S. K. Blight, L. Zhang, K. B. Green-Church, J. A. Krzycki, In vivo contextual requirements for UAG translation as pyrrolysine. *Mol. Microbiol.* **63**, 229–241 (2007).

1038 15. D. D. Young, P. G. Schultz, Playing with the Molecules of Life. *ACS Chem. Biol.* **13**, 854–870 (2018).

1040 16. O. Namy, J.-P. Rousset, S. Napthine, I. Brierley, Reprogrammed genetic decoding in cellular gene expression. *Mol. Cell* **13**, 157–168 (2004).

1042 17. A. Théobald-Dietrich, R. Giegé, J. Rudinger-Thirion, Evidence for the existence in mRNAs of a hairpin element responsible for ribosome dependent pyrrolysine insertion into proteins. *Biochimie* **87**, 813–817 (2005).

1045 18. P. O'Donoghue, L. Prat, M. Kucklick, J. G. Schäfer, K. Riedel, J. Rinehart, D. Söll, I. U. Heinemann, Reducing the genetic code induces massive rearrangement of the proteome. *Proceedings of the National Academy of Sciences* **111**, 17206–17211 (2014).

1048 19. Y. Zhang, P. V. Baranov, J. F. Atkins, V. N. Gladyshev, Pyrrolysine and selenocysteine use dissimilar decoding strategies. *J. Biol. Chem.* **280**, 20740–20751 (2005).

1050 20. L. Prat, I. U. Heinemann, H. R. Aerni, J. Rinehart, P. O'Donoghue, D. Söll, Carbon source-dependent expansion of the genetic code in bacteria. *Proceedings of the National Academy of Sciences* **109**, 21070–21075 (2012).

1053 21. V. Kivenson, B. G. Paul, D. L. Valentine, An Ecological Basis for Dual Genetic Code Expansion in Marine Deltaproteobacteria. *Front. Microbiol.* **12**, 680620 (2021).

1055 22. G. Borrel, N. Parisot, H. M. B. Harris, E. Peyretaillade, N. Gaci, W. Tottey, O. Bardot, K. Raymann, S. Gribaldo, P. Peyret, P. W. O'Toole, J.-F. Brugère, Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine. *BMC Genomics* **15**, 679 (2014).

1060 23. T. Mukai, A. Crnković, T. Umehara, N. N. Ivanova, N. C. Kyrpides, D. Söll, RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea. *MBio* **8** (2017).

1062 24. M. M. Lynes, Z. J. Jay, A. J. Kohtz, R. Hatzenpichler, Methylotrophic methanogenesis in the Archaeoglobi revealed by cultivation of Ca. Methanoglobus hypatiae from a Yellowstone hot spring. *ISME J.* **18**, wrae026 (2024).

1065 25. Y. Guan, M. F. Haroon, I. Alam, J. G. Ferry, U. Stingl, Single-cell genomics reveals pyrrolysine-encoding potential in members of uncultivated archaeal candidate division MSBL1. *Environ. Microbiol. Rep.* **9**, 404–410 (2017).

1068 26. D. Y. Sorokin, K. S. Makarova, B. Abbas, M. Ferrer, P. N. Golyshin, E. A. Galinski, S. Ciordia, M. C. Mena, A. Y. Merkel, Y. I. Wolf, M. C. M. van Loosdrecht, E. V. Koonin, Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the
1071 evolutionary origin of methanogenesis. *Nature Microbiology* **2**, 1–11 (2017).

1072 G. Borrel, H. M. B. Harris, W. Tottey, A. Mihajlovski, N. Parisot, E. Peyretaillade, P.
1073 Peyret, S. Gribaldo, P. W. O’Toole, J.-F. Brugère, Genome sequence of “Candidatus
1074 Methanomethylophilus alvus” Mx1201, a methanogenic archaeon from the human gut
1075 belonging to a seventh order of methanogens. *J. Bacteriol.* **194**, 6944–6945 (2012).

1076 Lang Kristina, Schuldes Jörg, Klingl Andreas, Poehlein Anja, Daniel Rolf, Brune Andreas,
1077 New Mode of Energy Metabolism in the Seventh Order of Methanogens as Revealed by
1078 Comparative Genome Analysis of “Candidatus Methanoplasma termitum.” *Appl. Environ.
1079 Microbiol.* **81**, 1338–1352 (2015).

1080 J. Sun, P. N. Evans, E. J. Gagen, B. J. Woodcroft, B. P. Hedlund, T. Woyke, P. Hugenholtz,
1081 C. Rinke, Recoding of stop codons expands the metabolic potential of two novel
1082 Asgardarchaeota lineages. *ISME Communications* **1**, 1–14 (2021).

1083 L.-T. Guo, K. Amikura, H.-K. Jiang, T. Mukai, X. Fu, Y.-S. Wang, P. O’Donoghue, D.
1084 Söll, J. M. Tharp, Ancestral archaea expanded the genetic code with pyrrolysine. *J. Biol.
1085 Chem.* **298**, 102521 (2022).

1086 B. A. Berghuis, F. B. Yu, F. Schulz, P. C. Blainey, T. Woyke, S. R. Quake,
1087 Hydrogenotrophic methanogenesis in archaeal phylum Verstraetearchaeota reveals the
1088 shared ancestry of all methanogens. *Proceedings of the National Academy of Sciences* **116**,
1089 5037–5044 (2019).

1090 S. P. Gilmore, J. K. Henske, J. A. Sexton, K. V. Solomon, S. Seppälä, J. I. Yoo, L. M.
1091 Huyett, A. Pressman, J. Z. Cogan, V. Kivenson, X. Peng, Y. Tan, D. L. Valentine, M. A.
1092 O’Malley, Genomic analysis of methanogenic archaea reveals a shift towards energy
1093 conservation. *BMC Genomics* **18**, 639 (2017).

1094 B. N. Chaudhuri, T. O. Yeates, A computational method to predict genetically encoded rare
1095 amino acids in proteins. *Genome Biol.* **6**, R79 (2005).

1096 C. Theil Have, S. Zambach, H. Christiansen, Effects of using coding potential, sequence
1097 conservation and mRNA structure conservation for predicting pyrrolysine containing genes.
1098 *BMC Bioinformatics* **14**, 118 (2013).

1099 I. U. Heinemann, P. O’Donoghue, C. Madinger, J. Benner, L. Randau, C. J. Noren, D. Söll,
1100 The appearance of pyrrolysine in tRNAHis guanylyltransferase by neutral evolution. *Proc.
1101 Natl. Acad. Sci. U. S. A.* **106**, 21103–21108 (2009).

1102 J. Li, P. T. Kang, R. Jiang, J. Y. Lee, J. A. Soares, J. A. Krzycki, M. K. Chan, Insights into
1103 pyrrolysine function from structures of a trimethylamine methyltransferase and its corrinoid
1104 protein complex. *Commun Biol* **6**, 54 (2023).

1105 J.-F. Brugère, J. F. Atkins, P. W. O’Toole, G. Borrel, Pyrrolysine in archaea: a 22nd amino
1106 acid encoded through a genetic code expansion. *Emerg Top Life Sci* **2**, 607–618 (2018).
1107 38. J. A. Krzycki, "Translation of UAG as Pyrrolysine" in *Recoding: Expansion of Decoding Rules Enriches Gene Expression*, J. F. Atkins, R. F. Gesteland, Eds. (Springer New York, New York, NY, 2010), pp. 53–77.

1110 39. E. V. Koonin, A. S. Novozhilov, Origin and Evolution of the Universal Genetic Code. *Annu. Rev. Genet.* **51**, 45–62 (2017).

1112 40. O. Namy, Y. Zhou, S. Gundlapalli, C. R. Polycarpo, A. Denise, J.-P. Rousset, D. Söll, A. Ambrogelly, Adding pyrrolysine to the Escherichia coli genetic code. *FEBS Lett.* **581**, 5282–5288 (2007).

1115 41. B. W. Wright, M. P. Molloy, P. R. Jaschke, Overlapping genes in natural and engineered genomes. *Nat. Rev. Genet.* **23**, 154–168 (2022).

1117 42. M. A. Allen, F. M. Lauro, T. J. Williams, D. Burg, K. S. Siddiqui, D. De Francisci, K. W. Y. Chong, O. Pilak, H. H. Chew, M. Z. De Maere, L. Ting, M. Katrib, C. Ng, K. R. Sowers, M. Y. Galperin, I. J. Anderson, N. Ivanova, E. Dalin, M. Martinez, A. Lapidus, L. Hauser, M. Land, T. Thomas, R. Cavicchioli, The genome sequence of the psychrophilic archaeon, Methanococcoides burtonii: the role of genome evolution in cold adaptation. *ISME J.* **3**, 1012–1035 (2009).

1123 43. G. Borrel, K. Fadhlaoui, W. Ben Hania, N. Gaci, G. Pehau-Arnaudet, P. P. Chaudhary, P. Vandekerckove, N. Ballet, M. Alric, P. W. O'Toole, M.-L. Fardeau, B. Ollivier, J.-F. Brugère, Methanomethylophilus alvi gen. nov., sp. nov., a Novel Hydrogenotrophic Methyl-Reducing Methanogenic Archaea of the Order Methanomassiliicoccales Isolated from the Human Gut and Proposal of the Novel Family Methanomethylophilaceae fam. nov. *Microorganisms* **11**, 2794 (2023).

1129 44. Y. Shulgina, S. R. Eddy, Codetta: predicting the genetic code from nucleotide sequence. *Bioinformatics* **39** (2023).

1131 45. S. P. Jungbluth, J. P. Amend, M. S. Rappé, Metagenome sequencing and 98 microbial genomes from Juan de Fuca Ridge flank subsurface fluids. *Scientific Data* **4**, 1–11 (2017).

1133 46. Y. Li, S. C. Leahy, J. Jeyanathan, G. Henderson, F. Cox, E. Altermann, W. J. Kelly, S. C. Lambie, P. H. Janssen, J. Rakonjac, G. T. Attwood, The complete genome sequence of the methanogenic archaeon ISO4-H5 provides insights into the methylotrophic lifestyle of a ruminal representative of the Methanomassiliicoccales. *Stand. Genomic Sci.* **11**, 59 (2016).

1137 47. V. V. Kapitonov, K. S. Makarova, E. V. Koonin, ISC, a novel group of bacterial and Archaeal DNA transposons that encode Cas9 homologs. *J. Bacteriol.* **198**, 797–807 (2015).

1139 48. G. Schuler, C. Hu, A. Ke, Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9. *Science* **376**, 1476–1481 (2022).

1141 49. N. G. Koch, N. Budisa, Evolution of pyrrolysyl-tRNA synthetase: From methanogenesis to genetic code expansion. *Chem. Rev.*, doi: 10.1021/acs.chemrev.4c00031 (2024).
1143 50. J.-D. Pédelacq, S. Cabantous, T. Tran, T. C. Terwilliger, G. S. Waldo, Engineering and characterization of a superfolder green fluorescent protein. *Nat. Biotechnol.* **24**, 79–88 (2006).

1146 51. L. T. Roe, C. K. Schissel, T. L. Dover, B. Shah, N. X. Hamlish, S. Zheng, D. A. Dilworth, N. Wong, Z. Zhang, A. Chatterjee, M. B. Francis, S. J. Miller, A. Schepartz, Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β²-peptide linkages, *bioRxiv* (2023)p. 2023.10.03.560714.

1150 52. C. Schissel, H. Roberts-Mataric, I. Garcia, H. Kang, M. Francis, A. Schepartz, Ribosomal Synthesis of Ketone-containing Peptide Backbone via O to C Acyl Shift, *ChemRxiv* (2024). https://doi.org/10.26434/chemrxiv-2024-bkzp3.

1153 53. J. Guo, J. Wang, J. C. Anderson, P. G. Schultz, Addition of an alpha-hydroxy acid to the genetic code of bacteria. *Angew. Chem. Int. Ed Engl.* **47**, 722–725 (2008).

1155 54. R. Fricke, C. V. Swenson, L. T. Roe, N. X. Hamlish, B. Shah, Z. Zhang, E. Ficaretta, O. Ad, S. Smaga, C. L. Gee, A. Chatterjee, A. Schepartz, Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo. *Nat. Chem.* **15**, 960–971 (2023).

1159 55. M. Ferrer, D. Y. Sorokin, Y. I. Wolf, S. Ciordia, M. C. Mena, R. Bargiela, E. V. Koonin, K. S. Makarova, Proteomic analysis of Methanonatronarchaeum thermophilum AMET1, a representative of a putative new class of Euryarchaeota, “Methanonatronarchaeia.” *Genes (Basel)* **9** (2018).

1163 56. D. W. Schultz, M. Yarus, Transfer RNA mutation and the malleability of the genetic code. *J. Mol. Biol.* **235**, 1377–1380 (1994).

1165 57. S. E. Massey, G. Moura, P. Beltrão, R. Almeida, J. R. Garey, M. F. Tuite, M. A. S. Santos, Comparative evolutionary genomics unveils the molecular mechanism of reassignment of the CTG codon in Candida spp. *Genome Res.* **13**, 544–557 (2003).

1168 58. Z. Zhou, Y. Liu, W. Xu, J. Pan, Z.-H. Luo, M. Li, Genome- and community-level interaction insights into carbon utilization and element cycling functions of Hydrothermarchaeota in hydrothermal sediment. *mSystems* **5** (2020).

1171 59. Z. Zhou, J. Pan, F. Wang, J.-D. Gu, M. Li, Bathyarchaeota: globally distributed metabolic generalists in anoxic environments. *FEMS Microbiol. Rev.* **42**, 639–655 (2018).

1173 60. C. M. Thomas, E. Desmond-Le Quéméner, S. Gribaldo, G. Borrel, Factors shaping the abundance and diversity of the gut archaeome across the animal kingdom. *Nat. Commun.* **13**, 3358 (2022).

1176 61. D. H. Parks, M. Imelfort, C. T. Skennerton, P. Hugenholtz, G. W. Tyson, CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. *Genome Res.* **25**, 1043–1055 (2015).
1179 62. D. Hyatt, G.-L. Chen, P. F. Locascio, M. L. Land, F. W. Larimer, L. J. Hauser, Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11, 119 (2010).

1182 63. R. D. Finn, J. Clements, S. R. Eddy, HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 39, W29-37 (2011).

1184 64. S. R. Eddy, Accelerated Profile HMM Searches. PLoS Comput. Biol. 7, e1002195 (2011).

1185 65. W. Li, K. R. O'Neill, D. H. Haft, M. DiCuccio, V. Chetvernin, A. Badretdin, G. Coulouris, F. Chitsaz, M. K. Derbyshire, A. S. Durkin, N. R. Gonzales, M. Gwadz, C. J. Lanczycki, J. S. Song, N. Thanki, J. Wang, R. A. Yamashita, M. Yang, C. Zheng, A. Marchler-Bauer, F. Thibaud-Nissen, RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation. Nucleic Acids Res. 49, D1020-D1028 (2021).

1190 66. A. R. Gruber, R. Lorenz, S. H. Bernhart, R. Neuböck, I. L. Hofacker, The Vienna RNA websuite. Nucleic Acids Res. 36, W70-4 (2008).

1192 67. A. E. Darling, G. Jospin, E. Lowe, F. A. Matsen IV, H. M. Bik, J. A. Eisen, PhyloSift: phylogenetic analysis of genomes and metagenomes. PeerJ 2, e243 (2014).

1194 68. K. Katoh, D. M. Standley, MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772-780 (2013).

1196 69. A. Criscuolo, S. Gribaldo, BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments. BMC Evol. Biol. 10, 210 (2010).

1199 70. B. Q. Minh, H. A. Schmidt, O. Chernomor, D. Schrempf, M. D. Woodhams, A. von Haeseler, R. Lanfear, IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol. Evol. 37, 1530-1534 (2020).

1202 71. H.-C. Wang, B. Q. Minh, E. Susko, A. J. Roger, Modeling Site Heterogeneity with Posterior Mean Site Frequency Profiles Accelerates Accurate Phylogenomic Estimation. Syst. Biol. 67, 216-235 (2018).

1205 72. P.-A. Chaumeil, A. J. Mussig, P. Hugenholtz, D. H. Parks, GTDB-Tk v2: memory friendly classification with the genome taxonomy database. Bioinformatics 38, 5315-5316 (2022).

1207 73. I. Letunic, P. Bork, Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res., doi: 10.1093/nar/gkae268 (2024).

1209 74. O. Cohen, H. Ashkenazy, F. Belinky, D. Huchon, T. Pupko, GLOOME: gain loss mapping engine. Bioinformatics 26, 2914-2915 (2010).

1211 75. M. R. Olm, C. T. Brown, B. Brooks, J. F. Banfield, dRep: a tool for fast and accurate genomic comparisons that enables improved genome recovery from metagenomes through de-replication. ISME J. 11, 2864-2868 (2017).
1214 76. T. Seemann, Prokka: rapid prokaryotic genome annotation. *Bioinformatics* **30**, 2068–2069
1215 (2014).

1216 77. UniProt Consortium, UniProt: the universal protein knowledgebase in 2021. *Nucleic Acids
Res.* **49**, D480–D489 (2021).

1218 78. Y. Shulgina, S. R. Eddy, A computational screen for alternative genetic codes in over
1219 250,000 genomes. *Elife* **10** (2021).

1220 79. D. H. Haft, J. D. Selengut, R. A. Richter, D. Harkins, M. K. Basu, E. Beck, TIGRFAMs and
1221 Genome Properties in 2013. *Nucleic Acids Res.* **41**, D387–95 (2013).

1222 80. E. P. Nawrocki, S. R. Eddy, Infernal 1.1: 100-fold faster RNA homology searches.
1223 *Bioinformatics* **29**, 2933–2935 (2013).

1224 81. I. Kalvari, E. P. Nawrocki, N. Ontiveros-Palacios, J. Argasinska, K. Lamkiewicz, M. Marz,
1225 S. Griffiths-Jones, C. Toffano-Nioche, D. Gautheret, Z. Weinberg, E. Rivas, S. R. Eddy, R.
1226 D. Finn, A. Bateman, A. I. Petrov, Rfam 14: expanded coverage of metagenomic, viral and
1227 microRNA families. *Nucleic Acids Res.* **49**, D192–D200 (2021).

1228 82. S. T. Brown, P. Buitrago, E. Hanna, S. Sanielevici, R. Scibek, N. A. Nystrom, “Bridges-2:
1229 A platform for rapidly-evolving and data intensive research” in *Practice and Experience in
1230 Advanced Research Computing* (ACM, New York, NY, USA, 2021;
1231 https://dl.acm.org/doi/abs/10.1145/3437359.3465593).

1232 83. H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N.
1233 Shindyalov, P. E. Bourne, The Protein Data Bank. *Nucleic Acids Res.* **28**, 235–242 (2000).

1234 84. A. Hoeppner, F. Thomas, A. Rueppel, R. Hensel, W. Blankenfeldt, P. Bayer, A. Faust,
1235 Structure of the corrinoid:coenzyme M methyltransferase MtaA from Methanosarcina
1236 mazei. *Acta Crystallogr. D Biol. Crystallogr.* **68**, 1549–1557 (2012).

1237 85. W. Humphrey, A. Dalke, K. Schulten, VMD: visual molecular dynamics. *J. Mol. Graph.*
1238 **14**, 33–8, 27–8 (1996).

1239 86. J. E. Stone, “An efficient library for parallel ray tracing and animation,” thesis, Missouri
1240 University of Science and Technology (1998).

1241 87. A. Varshney, F. Brooks, W. Wright, Computing smooth molecular surfaces. *IEEE Comput.
Graph. Appl.* **14**, 19–25 (1994).

1243 88. A. Hocher, G. Borrel, K. Fadhlaoui, J.-F. Brugère, S. Gribaldo, T. Warnecke, Growth
1244 temperature and chromatinization in archaea. *Nat Microbiol* **7**, 1932–1942 (2022).

1245 89. T. S. Batth, M. X. Tollenaere, P. Rüther, A. Gonzalez-Franquesa, B. S. Prabhakar, S.
1246 Bekker-Jensen, A. S. Deshmukh, J. V. Olsen, Protein Aggregation Capture on
1247 Microparticles Enables Multipurpose Proteomics Sample Preparation. *Mol. Cell.*
1248 *Proteomics* **18**, 1027–1035 (2019).
1249 90. B. Mesuere, F. Van der Jeugt, T. Willems, T. Naessens, B. Devreese, L. Martens, P. Dawyndt, High-throughput metaproteomics data analysis with Unipept: A tutorial. *J. Proteomics* **171**, 11–22 (2018).

1252 91. J. K. Eng, T. A. Jahan, M. R. Hoopmann, Comet: an open-source MS/MS sequence database search tool. *Proteomics* **13**, 22–24 (2013).

1254 92. M. The, M. J. MacCoss, W. S. Noble, L. Käll, Fast and Accurate Protein False Discovery Rates on Large-Scale Proteomics Data Sets with Percolator 3.0. *J. Am. Soc. Mass Spectrom.* **27**, 1719–1727 (2016).

1257 93. N. H. Tran, R. Qiao, L. Xin, X. Chen, C. Liu, X. Zhang, B. Shan, A. Ghodsi, M. Li, Deep learning enables de novo peptide sequencing from data-independent-acquisition mass spectrometry. *Nat. Methods* **16**, 63–66 (2019).

1260 94. V. Kivenson, *An Archaeal Genetic Code with All TAG Codons as Pyrrolysine* (Zenodo, 2025; http://dx.doi.org/10.5281/zenodo.15478078).

1262